title: PeerJ description: Articles published in PeerJ link: https://peerj.com/articles/index.rss3?journal=peerj&month=2017-05 creator: info@peerj.com PeerJ errorsTo: info@peerj.com PeerJ language: en title: Genome-wide analysis suggests high level of microsynteny and purifying selection affect the evolution of EIN3/EIL family in Rosaceae link: https://peerj.com/articles/3400 last-modified: 2017-05-31 description: The ethylene-insensitive3/ethylene-insensitive3-like (EIN3/EIL) proteins are a type of nuclear-localized protein with DNA-binding activity in plants. Although the EIN3/EIL gene family has been studied in several plant species, little is known about comprehensive study of the EIN3/EIL gene family in Rosaceae. In this study, ten, five, four, and five EIN3/EIL genes were identified in the genomes of pear (Pyrus bretschneideri), mei (Prunus mume), peach (Prunus persica) and strawberry (Fragaria vesca), respectively. Twenty-eight chromosomal segments of EIL/EIN3 gene family were found in four Rosaceae species, and these segments could form seven orthologous or paralogous groups based on interspecies or intraspecies gene colinearity (microsynteny) analysis. Moreover, the highly conserved regions of microsynteny were found in four Rosaceae species. Subsequently it was found that both whole genome duplication and tandem duplication events significantly contributed to the EIL/EIN3 gene family expansion. Gene expression analysis of the EIL/EIN3 genes in the pear revealed subfunctionalization for several PbEIL genes derived from whole genome duplication. It is noteworthy that according to environmental selection pressure analysis, the strong purifying selection should dominate the maintenance of the EIL/EIN3 gene family in four Rosaceae species. These results provided useful information on Rosaceae EIL/EIN3 genes, as well as insights into the evolution of this gene family in four Rosaceae species. Furthermore, high level of microsynteny in the four Rosaceae plants suggested that a large-scale genome duplication event in the EIL/EIN3 gene family was predated to speciation. creator: Yunpeng Cao creator: Yahui Han creator: Dandan Meng creator: Dahui Li creator: Qing Jin creator: Yi Lin creator: Yongping Cai uri: https://doi.org/10.7717/peerj.3400 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Cao et al. title: Seed morphometric characteristics of European species of Elatine (Elatinaceae) link: https://peerj.com/articles/3399 last-modified: 2017-05-31 description: Elatine L. contains ca. 25 small, herbaceous, annual species distributed in ephemeral waters in both hemispheres. All species are amphibious and characterized by a high degree of morphological variability. The importance of seed morphology in Elatine taxonomy has been emphasized by many authors. The degree of seed curvature and seed coat reticulation have been traditionally considered very important in recognizing individual species of this genus. Seed morphometric characteristics of 10 Elatine species, including all European native taxa, are provided on the basis of material from two or three populations of each species. A total of 24–50 seeds were studied from each population, altogether 1,260 images were used for the morphometric study. In total, six parameters were measured from SEM pictures: object surface area, profile specific perimeter (object circuit), rectangle of the object (a) length, rectangle of the object (b) width, angle of the seed curvature, and number of pits in the seed coat counted in the middle row. Our study shows that the range of morphological variation of seeds in European species of Elatine is great, both between the species and the populations. Discrimination analysis showed that all six traits significantly differentiate the populations studied (λ = 0.001, p < 0.001), and the greatest contributions were “number of pits”, “rectangle_a”, and “the angle curvature”. Multidimensional scaling based on a correlation matrix of Mahalanobis distance of the six features studied revealed the greatest similarity between the three populations of E. alsinastrum, E. macropoda, and E. hexandra. Regarding interspecific differences, a Kruskal–Wallis tests showed that, in many cases, lack of statistically significant differences between species relative to the studied seed traits. If distinction of species is only based on seeds, especially if only a few seeds are evaluated, the following species pairs can be easily confused: E. alsinastrum and E. orthosperma, E. hexandra and E. macropoda, E. campylosperma and E. hydropiper, as well and E. gussonei and E. hungarica. We found no diversity in seed coat micromorphology within pits that could have potential taxonomic importance. An identification key and descriptions of species are provided on the basis of seeds traits. creator: Agnieszka Popiela creator: Andrzej Łysko creator: Bożenna Białecka creator: Magdalena Marta Bihun creator: Gábor Sramkó creator: Waldemar Staroń creator: Anetta Wieczorek creator: Attila Molnár V. uri: https://doi.org/10.7717/peerj.3399 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Popiela et al. title: Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa) link: https://peerj.com/articles/3398 last-modified: 2017-05-31 description: BackgroundThe veined rapa whelk Rapana venosa is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in R. venosa. For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes in R. venosa for use as internal controls for qRT-PCR.MethodsIn this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1α (EF-1α), α-actin (ACT), cytochrome c oxidase subunit 1 (COX1), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1α subcomplex subunit 7 (NDUFA7), 60S ribosomal protein L5 (RL5), 60S ribosomal protein L28 (RL28), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-tubulin (TUBB), 40S ribosomal protein S25 (RS25), 40S ribosomal protein S8 (RS8), ubiquitin-conjugating enzyme E2 (UBE2), histone H3 (HH3), and peptidyl-prolyl cis-trans isomerase A (PPIA). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms.ResultsOf the 13 candidate genes tested, we found that EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. For the normalization of a single specific tissue, we suggested that EF-1α and NDUFA7 are the best combination in gonad, as well as COX1 and RL28 for intestine, EF-1α and RL5 for kidney, EF-1α and COX1 for gill, EF-1α and RL28 for Leiblein and mantle, EF-1α, RL5, and NDUFA7 for liver, GAPDH, PPIA, and RL28 for hemocyte. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured, and COX1 and RL5 were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization, PPIA, RS25, and RL28 for stage 1, RL5 and RL28 for stage 2 and 5, RL28 and NDUFA7 for stage 3, and PPIA and TUBB for stage 4.DiscussionOur results are instrumental for the selection of appropriately validated housekeeping genes for use as internal controls for gene expression studies in adult tissues or larval development of R. venosa in the future. creator: Hao Song creator: Xin Dang creator: Yuan-qiu He creator: Tao Zhang creator: Hai-yan Wang uri: https://doi.org/10.7717/peerj.3398 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Song et al. title: Transcriptome analysis reveals the regulation of brassinosteroids on petal growth in Gerbera hybrida link: https://peerj.com/articles/3382 last-modified: 2017-05-31 description: Gerbera hybrida is a cut-flower crop of global importance, and an understanding of the mechanisms underlying petal development is vital for the continued commercial development of this plant species. Brassinosteroids (BRs), a class of phytohormones, are known to play a major role in cell expansion, but their effect on petal growth in G. hybrida is largely unexplored. In this study, we found that the brassinolide (BL), the most active BR, promotes petal growth by lengthening cells in the middle and basal regions of petals, and that this effect on petal growth was greater than that of gibberellin (GA). The RNA-seq (high-throughput cDNA sequencing) technique was employed to investigate the regulatory mechanisms by which BRs control petal growth. A global transcriptome analysis of the response to BRs in petals was conducted and target genes regulated by BR were identified. These differentially expressed genes (DEGs) include various transcription factors (TFs) that were activated during the early stage (0.5 h) of BL treatment, as well as cell wall proteins whose expression was regulated at a late stage (10 h). BR-responsive DEGs are involved in multiple plant hormone signal pathways, hormone biosynthesis and biotic and abiotic stress responses, showing that the regulation of petal growth by BRs is a complex network of processes. Thus, our study provides new insights at the transcriptional level into the molecular mechanisms of BR regulation of petal growth in G. hybrida. creator: Gan Huang creator: Meixiang Han creator: Wei Yao creator: Yaqin Wang uri: https://doi.org/10.7717/peerj.3382 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Huang et al. title: Quercetin reduces hydroxyurea induced cytotoxicity in immortalized mouse aortic endothelial cells link: https://peerj.com/articles/3376 last-modified: 2017-05-31 description: BackgroundChronic inflammation is a characteristic of sickle cell disease (SCD), and is invariably associated with vascular endothelial injury. Hydroxyurea (HU), a naturally cytotoxic chemotherapeutic agent, is the only FDA drug approved for SCD, and is therefore naturally cytotoxic. Quercetin (QCT) is a dietary flavonoid found ubiquitously in plants and foods that have anti-oxidative and anti-inflammatory characteristics. Our hypothesis is that dietary QCT will decrease cytotoxic effects of lipopolysaccharide (LPS) and HU induced vascular cell damage.MethodsLipopolysaccharide (LPS) was used to induce inflammation in immortalized mouse aortic endothelial cells (iMAECs), providing an in vitro model of inflamed endothelial cells. The cells were exposed to LPS throughout the entire experiment. Interventions included treating the LPS exposed cells with QCT, HU, or QCT + HU over 50 hours. The 50-hour period included 24 hours of varying treatments, followed by two hours of hypoxic exposure and then 24 hours under normal aerobic exposure.ResultsLDH level was significantly higher for LPS treated versus untreated cells (P = 0.0004). LPS plus 30 micromole QCT reduced the LDH (p = 0.1, trend), whereas LPS plus 100 micromoles HU, significantly increased LDH (p = 0.0004). However, LPS plus treatment with 30 micromoles QCT/100 micromoles HU, significantly reduced LDH, compared with HU alone (p = 0.0002).DiscussionThese results suggest that quercetin may be effective against vascular endothelial cell damage for iMAECs in vitro. In particular, it shows promise in preventing HU-induced cytotoxicity, surprisingly found from these results. This latter finding is important, and should be given more consideration, since HU is the only FDA-approved drug for treating sickle cell patients, and its use is rapidly increasing. creator: Zachary M. Kiser creator: Monica D.M. McGee creator: Racquel J. Wright creator: Alexander Quarshie creator: Gale W. Newman creator: Karen R. Randall creator: Jonathan K. Stiles creator: Adel Driss creator: Jacqueline M. Hibbert uri: https://doi.org/10.7717/peerj.3376 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Kiser et al. title: Pond dyes are Culex mosquito oviposition attractants link: https://peerj.com/articles/3361 last-modified: 2017-05-31 description: BackgroundBritish mosquito population distribution, abundance, species composition and potential for mosquito disease transmission are intimately linked to the physical environment. The presence of ponds and water storage can significantly increase the density of particular mosquito species in the garden. Culex pipiens is the mosquito most commonly found in UK gardens and a potential vector of West Nile Virus WNV, although the current risk of transmission is low. However any factors that significantly change the distribution and population of C. pipiens are likely to impact subsequent risk of disease transmission. Pond dyes are used to control algal growth and improve aesthetics of still water reflecting surrounding planting. However, it is well documented that females of some species of mosquito prefer to lay eggs in dark water and/or containers of different colours and we predict that dyed ponds will be attractive to Culex mosquitoes.MethodsBlack pond dye was used in oviposition choice tests using wild-caught gravid C. pipiens. Larvae from wild-caught C. pipiens were also reared in the pond dye to determine whether it had any impact on survival. An emergence trap caught any adults that emerged from the water. Water butts (80 L) were positioned around university glasshouses and woodland and treated with black pond dye or left undyed. Weekly sampling over a six month period through summer and autumn was performed to quantified numbers of larvae and pupae in each treatment and habitat.ResultsGravid female Culex mosquitoes preferred to lay eggs in dyed water. This was highly significant in tests conducted under laboratory conditions and in a semi-field choice test. Despite this, survivorship in black dyed water was significantly reduced compared to undyed water. Seasonal analysis of wild larval and pupal numbers in two habitats with and without dye showed no impact of dye but a significant impact of season and habitat. Mosquitoes were more successful, with significantly higher numbers of pupae, in the habitat where they had vegetation cover and shade.DiscussionOur study has raised some interesting possibilities; one is that where used, pond dyes may be encouraging mosquitoes to breed in gardens in close proximity to people. Considering the concerns over potential future spread of disease in urban environments, this as well as shading of ponds and water butts, should inform future advice over reducing mosquito breeding and spread. creator: Natali Ortiz Perea creator: Amanda Callaghan uri: https://doi.org/10.7717/peerj.3361 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Ortiz Perea and Callaghan title: Alien vs. predator: bacterial challenge alters coral microbiomes unless controlled by Halobacteriovorax predators link: https://peerj.com/articles/3315 last-modified: 2017-05-31 description: Coral microbiomes are known to play important roles in organismal health, response to environmental stress, and resistance to disease. The coral microbiome contains diverse assemblages of resident bacteria, ranging from defensive and metabolic symbionts to opportunistic bacteria that may turn harmful in compromised hosts. However, little is known about how these bacterial interactions influence the mechanism and controls of overall structure, stability, and function of the microbiome. We sought to test how coral microbiome dynamics were affected by interactions between two bacteria: Vibrio coralliilyticus, a known temperature-dependent pathogen of some corals, and Halobacteriovorax, a unique bacterial predator of Vibrio and other gram-negative bacteria. We challenged reef-building coral with V. coralliilyticus in the presence or absence of Halobacteriovorax predators, and monitored microbial community dynamics with 16S rRNA gene profiling time-series. Vibrio coralliilyticus inoculation increased the mean relative abundance of Vibrios by greater than 35% from the 4 to 8 hour time point, but not in the 24 & 32 hour time points. However, strong secondary effects of the Vibrio challenge were also observed for the rest of the microbiome such as increased richness (observed species), and reduced stability (increased beta-diversity). Moreover, after the transient increase in Vibrios, two lineages of bacteria (Rhodobacterales and Cytophagales) increased in coral tissues, suggesting that V. coralliilyticus challenge opens niche space for these known opportunists. Rhodobacterales increased from 6.99% (±0.05 SEM) to a maximum mean relative abundance of 48.75% (±0.14 SEM) in the final time point and Cytophagales from <0.001% to 3.656%. Halobacteriovorax predators are commonly present at low-abundance on coral surfaces. Based on the keystone role of predators in many ecosystems, we hypothesized that Halobacteriovorax predators might help protect corals by consuming foreign or “alien” gram negative bacteria. Halobacteriovorax inoculation also altered the microbiome but to a lesser degree than V. coralliilyticus, and Halobacteriovorax were never detected after inoculation. Simultaneous challenge with both V. coralliilyticus and predatory Halobacteriovorax eliminated the increase in V. coralliilyticus, ameliorated changes to the rest of the coral microbiome, and prevented the secondary blooms of opportunistic Rhodobacterales and Cytophagales seen in the V. coralliilyticus challenge. These data suggest that, under certain circumstances, host-associated bacterial predators may mitigate the ability of other bacteria to destabilize the microbiome. creator: Rory M. Welsh creator: Stephanie M. Rosales creator: Jesse R. Zaneveld creator: Jérôme P. Payet creator: Ryan McMinds creator: Steven L. Hubbs creator: Rebecca L. Vega Thurber uri: https://doi.org/10.7717/peerj.3315 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Welsh et al. title: Assessing the utility of an institutional publications officer: a pilot assessment link: https://peerj.com/articles/3294 last-modified: 2017-05-31 description: BackgroundThe scholarly publication landscape is changing rapidly. We investigated whether the introduction of an institutional publications officer might help facilitate better knowledge of publication topics and related resources, and effectively support researchers to publish.MethodsIn September 2015, a purpose-built survey about researchers’ knowledge and perceptions of publication practices was administered at five Ottawa area research institutions. Subsequently, we publicly announced a newly hired publications officer (KDC) who then began conducting outreach at two of the institutions. Specifically, the publications officer gave presentations, held one-to-one consultations, developed electronic newsletter content, and generated and maintained a webpage of resources. In March 2016, we re-surveyed our participants regarding their knowledge and perceptions of publishing. Mean scores to the perception questions, and the percent of correct responses to the knowledge questions, pre and post survey, were computed for each item. The difference between these means or calculated percentages was then examined across the survey measures.Results82 participants completed both surveys. Of this group, 29 indicated that they had exposure to the publications officer, while the remaining 53 indicated they did not. Interaction with the publications officer led to improvements in half of the knowledge items (7/14 variables). While improvements in knowledge of publishing were also found among those who reported not to have interacted with the publications officer (9/14), these effects were often smaller in magnitude. Scores for some publication knowledge variables actually decreased between the pre and post survey (3/14). Effects for researchers’ perceptions of publishing increased for 5/6 variables in the group that interacted with the publications officer.DiscussionThis pilot provides initial indication that, in a short timeframe, introducing an institutional publications officer may improve knowledge and perceptions surrounding publishing. This study is limited by its modest sample size and temporal relationship between the introduction of the publications officer and changes in knowledge and perceptions. A randomized trial examining the publications officer as an effective intervention is needed. creator: Kelly D. Cobey creator: James Galipeau creator: Larissa Shamseer creator: David Moher uri: https://doi.org/10.7717/peerj.3294 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Cobey et al. title: Difference on cone size preferences between two coniferous species by Great Spotted Woodpecker (Dendrocopos major) link: https://peerj.com/articles/3288 last-modified: 2017-05-31 description: The number of species that specialize in pre-dispersal seed predation is relatively small. Examples of specialized pre-dispersal seed predators adapted to feeding on closed cones include vertebrate species like Crossbills, Squirrels, Nutcrackers and Woodpeckers. Seed predation selects against certain phenotypic features of cones and favors another phenotypic features. In this study, we document preferences of the Great Spotted Woodpecker (Dendrocopos major) for specific traits in the cones of Norway spruce (Picea abies) and Scots pine (Pinus sylvestris). We found that the Great Spotted Woodpecker prefers to feed on medium sized Norway spruce cones. The results suggest a disruptive selection that favors the extreme cone lengths in Norway spruce. In Scots pine, the woodpeckers avoided cones with large apophyses. Further, the selectivity for the specific characteristics of the cones is probably related to the configuration of the anvil, a place at which woodpeckers extract seeds from the cones. We think that the Great Spotted Woodpecker preferences in relation to the morphological characteristics of cones are a key to the design of the anvil in order to maximize the use of it as a tool for processing cones of both the Norway spruce and the Scots pine. creator: Łukasz Dylewski creator: Reuven Yosef creator: Łukasz Myczko uri: https://doi.org/10.7717/peerj.3288 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Dylewski et al. title: Largest baleen whale mass mortality during strong El Niño event is likely related to harmful toxic algal bloom link: https://peerj.com/articles/3123 last-modified: 2017-05-31 description: While large mass mortality events (MMEs) are well known for toothed whales, they have been rare in baleen whales due to their less gregarious behavior. Although in most cases the cause of mortality has not been conclusively identified, some baleen whale mortality events have been linked to bio-oceanographic conditions, such as harmful algal blooms (HABs). In Southern Chile, HABs can be triggered by the ocean–atmosphere phenomenon El Niño. The frequency of the strongest El Niño events is increasing due to climate change. In March 2015, by far the largest reported mass mortality of baleen whales took place in a gulf in Southern Chile. Here, we show that the synchronous death of at least 343, primarily sei whales can be attributed to HABs during a building El Niño. Although considered an oceanic species, the sei whales died while feeding near to shore in previously unknown large aggregations. This provides evidence of new feeding grounds for the species. The combination of older and newer remains of whales in the same area indicate that MMEs have occurred more than once in recent years. Large HABs and reports of marine mammal MMEs along the Northeast Pacific coast may indicate similar processes in both hemispheres. Increasing MMEs through HABs may become a serious concern in the conservation of endangered whale species. creator: Verena Häussermann creator: Carolina S. Gutstein creator: Michael Bedington creator: David Cassis creator: Carlos Olavarria creator: Andrew C. Dale creator: Ana M. Valenzuela-Toro creator: Maria Jose Perez-Alvarez creator: Hector H. Sepúlveda creator: Kaitlin M. McConnell creator: Fanny E. Horwitz creator: Günter Försterra uri: https://doi.org/10.7717/peerj.3123 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Häussermann et al. title: Patterns of bleaching and mortality following widespread warming events in 2014 and 2015 at the Hanauma Bay Nature Preserve, Hawai‘i link: https://peerj.com/articles/3355 last-modified: 2017-05-30 description: Drastic increases in global carbon emissions in the past century have led to elevated sea surface temperatures that negatively affect coral reef organisms. Worldwide coral bleaching-related mortality is increasing and data has shown even isolated and protected reefs are vulnerable to the effects of global climate change. In 2014 and 2015, coral reefs in the main Hawaiian Islands (MHI) suffered up to 90% bleaching, with higher than 50% subsequent mortality in some areas. The location and severity of bleaching and mortality was strongly influenced by the spatial and temporal patterns of elevated seawater temperatures. The main objective of this research was to understand the spatial extent of bleaching mortality in Hanauma Bay Nature Preserve (HBNP), O‘ahu, Hawai‘i to gain a baseline understanding of the physical processes that influence localized bleaching dynamics. Surveys at HBNP in October 2015 and January 2016 revealed extensive bleaching (47%) and high levels of coral mortality (9.8%). Bleaching was highly variable among the four HBNP sectors and ranged from a low of ∼31% in the central bay at Channel (CH) to a high of 57% in the area most frequented by visitors (Keyhole; KH). The highest levels of bleaching occurred in two sectors with different circulation patterns: KH experienced comparatively low circulation velocity and a low temperature increase while Witches Brew (WB) and Backdoors (BD) experienced higher circulation velocity and higher temperature increase. Cumulative mortality was highest at WB (5.0%) and at BD (2.9%) although WB circulation velocity is significantly higher. HBNP is minimally impacted by local factors that can lead to decline such as high fishing pressure or sedimentation although human use is high. Despite the lack of these influences, high coral mortality occurred. Visitor impacts are strikingly different in the two sectors that experienced the highest mortality evidenced by the differences in coral cover associated with visitor use however, coral mortality was similar. These results suggest that elevated temperature was more influential in coral bleaching and the associated mortality than high circulation or visitor use. creator: Ku‘ulei S. Rodgers creator: Keisha D. Bahr creator: Paul L. Jokiel creator: Angela Richards Donà uri: https://doi.org/10.7717/peerj.3355 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Rodgers et al. title: Comparative transcriptome analysis of isonuclear-alloplasmic lines unmask key transcription factor genes and metabolic pathways involved in sterility of maize CMS-C link: https://peerj.com/articles/3408 last-modified: 2017-05-30 description: Although C-type cytoplasmic male sterility (CMS-C) is one of the most attractive tools for maize hybrid seed production, the detailed regulation network of the male sterility remains unclear. In order to identify the CMS-C sterility associated genes and/or pathways, the comparison of the transcriptomes between the CMS-C line C48-2 and its isonuclear-alloplasmic maintainer line N48-2 at pollen mother cell stage (PS), an early development stage of microspore, and mononuclear stage (MS), an abortive stage of microspore, were analyzed. 2,069 differentially expressed genes (DEGs) between the two stages were detected and thought to be essential for the spikelet development of N48-2. 453 of the 2,069 DEGs were differentially expressed at MS stage between the two lines and thought to be participated in the process or the causes of microspore abortion. Among the 453 DEGs, 385 (84.99%) genes were down-regulated and only 68 (15.01%) genes were up-regulated in C48-2 at MS stage. The dramatic decreased expression of the four DEGs encoding MYB transcription factors and the DEGs involved in “polyamine metabolic process”, “Cutin, suberine and wax biosynthesis”, “Fatty acid elongation”, “Biosynthesis of unsaturated fatty acids” and “Proline metabolism” might play an important role in the sterility of C48-2. This study will point out some directions for detailed molecular analysis and better understanding of sterility of CMS-C in maize. creator: Chuan Li creator: Zhuofan Zhao creator: Yongming Liu creator: Bing Liang creator: Shuxian Guan creator: Hai Lan creator: Jing Wang creator: Yanli Lu creator: Moju Cao uri: https://doi.org/10.7717/peerj.3408 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Li et al. title: MitoSuite: a graphical tool for human mitochondrial genome profiling in massive parallel sequencing link: https://peerj.com/articles/3406 last-modified: 2017-05-30 description: Recent rapid advances in high-throughput, next-generation sequencing (NGS) technologies have promoted mitochondrial genome studies in the fields of human evolution, medical genetics, and forensic casework. However, scientists unfamiliar with computer programming often find it difficult to handle the massive volumes of data that are generated by NGS. To address this limitation, we developed MitoSuite, a user-friendly graphical tool for analysis of data from high-throughput sequencing of the human mitochondrial genome. MitoSuite generates a visual report on NGS data with simple mouse operations. Moreover, it analyzes high-coverage sequencing data but runs on a stand-alone computer, without the need for file upload. Therefore, MitoSuite offers outstanding usability for handling massive NGS data, and is ideal for evolutionary, clinical, and forensic studies on the human mitochondrial genome variations. It is freely available for download from the website https://mitosuite.com. creator: Koji Ishiya creator: Shintaroh Ueda uri: https://doi.org/10.7717/peerj.3406 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Ishiya and Ueda title: How to be a great dad: parental care in a flock of greater flamingo (Phoenicopterus roseus) link: https://peerj.com/articles/3404 last-modified: 2017-05-30 description: In the last years, studies on captive greater flamingos have increased. Research on zoo animals is important to improve the knowledge on these species and to improve their ex-situ and in-situ conservation. The aim of the present study was to investigate the parental behaviour of a captive colony of greater flamingo hosted at Parco Natura Viva, an Italian zoological garden, to improve the knowledge on this species in zoos. In particular, the present study investigated and compared the parental care of females and males in 35 breeding pairs of greater flamingos. For each pair, we collected durations of parental care behaviour of both females and males, recording their position in relation to the nest (near the nest, on the nest, away from the nest) and individual and social behaviours performed. First, both partners were involved in parental care and displayed species-specific behaviours reported in the wild. The main results were that males spent more time than females on the nest (P = 0.010) and near it (P = 0.0001) and were more aggressive toward other flamingos than females, both when sitting on the nest (P = 0.003) and when near the nest (P = 0.0003). Therefore, male flamingos seem to be more involved in incubation duties and nest protection than females. This kind of research is important not only to expand the knowledge on bird species such as flamingos, but also to improve their husbandry and breeding in controlled environment. Indeed, understanding animal behaviour allows us to gain insights into their individual and social needs, addressing potential animal welfare issues. creator: Camillo Sandri creator: Vittoria Vallarin creator: Carolina Sammarini creator: Barbara Regaiolli creator: Alessandra Piccirillo creator: Caterina Spiezio uri: https://doi.org/10.7717/peerj.3404 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Sandri et al. title: The presumed oldest flying insect: more likely a myriapod? link: https://peerj.com/articles/3402 last-modified: 2017-05-30 description: The early fossil record of insects is scarce, with only few finds in the Devonian. All these finds appear problematic and controversial, partly due to incomplete preservation and challenging interpretation of many structures. We provide details of one of these important forms, Rhyniognatha hirsti from the famous Rhynie Chert Lagerstätte with up-to-date 3D imaging techniques. The fossil has been interpreted as the remains of one of the earliest flying insects. The specimen mainly preserves the remains of the head. The structures of the mandibles have been used as a main argument for an interpretation as an insect, but these are in fact less easy to interpret. New observed structures include the remains of a head capsule and an additional pair of mouth parts. Structures formerly suggested to represent remains of the head capsule or apodemes are more likely to be representing glands of ectodermal origin. The newly observed structures do not support an interpretation as an insect. Instead they make the interpretation as a myriapod more likely, possibly as a centipede. Centipede remains from the Rhynie Chert are known from scutigeromorphs. We therefore point out that R. hirsti could be interpreted as an early centipede. creator: Carolin Haug creator: Joachim T. Haug uri: https://doi.org/10.7717/peerj.3402 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Haug and Haug title: The South-American distribution and southernmost record of Biomphalaria peregrina—a potential intermediate host of schistosomiasis link: https://peerj.com/articles/3401 last-modified: 2017-05-30 description: Schistosomiasis remains a major parasitic disease, endemic in large parts of South America. Five neotropical species of Biomphalaria have been found to act as intermediate hosts of Schistosoma mansoni in natural populations, while others have been shown to be susceptible in experimental infections, although not found infected in the field. Among these potential intermediate hosts, Biomphalaria peregrina represents the most widespread species in South America, with confirmed occurrence records from Venezuela to northern Patagonia. In this study, we report the southernmost record for the species at the Pinturas River, in southern Patagonia, which finding implies a southward reassessment of the limit for the known species of this genus. The identities of the individuals from this population were confirmed through morphological examination, and by means of two mitochondrial genes, cytochrome oxidase subunit I (COI) and 16S-rRNA. With both markers, phylogenetic analyses were conducted in order to compare the genetic background of individuals from the Pinturas River with previously genetically characterized strains of B. peregrina from various South-American locations. In addition, we produced a potential distribution model of B. peregrina in South America and identified the environmental variables that best predict that distribution. The model was estimated through a maximum entropy algorithm and run with occurrence points obtained from several sources, including the scientific literature and international databases, along with climatic and hydrographic variables. Different phylogenetic analyses with either the COI or 16S-rRNA sequences did not conflict, but rather gave very similar topological organizations. Two major groups were identified, with sequences from the Pinturas River grouping together with haplotypes from subtropical and temperate regions. The model developed had a satisfactory performance for the study area. We observed that the areas with higher habitat suitability were found to be mainly linked to subtropical and temperate regions of South America between 15° and 45° south latitude, with different moderate- and low-suitability areas outside this range. We also identified the coldest temperatures as the main predictors of the potential distribution of this snail. Susceptibility surveys would be required to evaluate if southern populations of B. peregrina still retain their potential as intermediate hosts of S. mansoni. creator: Alejandra Rumi creator: Roberto Eugenio Vogler creator: Ariel Aníbal Beltramino uri: https://doi.org/10.7717/peerj.3401 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Rumi et al. title: The shoaling behavior of two cyprinid species in conspecific and heterospecific groups link: https://peerj.com/articles/3397 last-modified: 2017-05-30 description: Mixed-species shoals of fish are frequently found in the field; however, little is known about individual-level interactions within these groups. We examined the collective motion of two cyprinid species (Chinese bream, Parabramis pekinensis, and qingbo, Spinibarbus sinensis) that occupy partially overlapping habitats but differ in social behavior (high vs low aggressiveness) and preferred flow regime (slow vs fast water velocity). We extracted measures of collective motion from video recordings of eight replicate groups of four individuals of either Chinese bream or qingbo (conspecific group) or two Chinese bream plus two qingbo (heterospecific group). Chinese bream in conspecific groups showed lower percent time moving and mean swimming speed but a similar speed while moving as compared to the qingbo conspecific groups. However, the difference in mean swimming speed and percent time moving vanished in the heterospecific group as Chinese bream elevated their swimming activity to coordinate with qingbo. This finding suggests that the two species may share similar interaction rules regarding shoaling behavior. The conspecific groups of qingbo exhibited a greater distance between group members than Chinese bream, suggesting a difference in cohesion. However, the inter-individual distances of all fish were similar in the heterospecific group. Qingbo in the heterospecific group swam more frequently at the front compared to Chinese bream, possibly due to their higher activity level. We also measured the startle response to an artificial stimulus and found that there was no significant difference among groups. In conclusion, the present study demonstrated that in the heterospecific groups, Chinese bream elevated their percent time moving while qingbo decreased their inter-individual distance to achieve consistent collective movement; thus, the two species showed similar behavior in the mixed-species group. creator: Zhong-Hua Tang creator: Hui Wu creator: Qing Huang creator: Lu Kuang creator: Shi-Jian Fu uri: https://doi.org/10.7717/peerj.3397 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Tang et al. title: Character strengths, social anxiety, and physiological stress reactivity link: https://peerj.com/articles/3396 last-modified: 2017-05-30 description: In this paper, the effects of character strengths on the physiological reactivity to social anxiety induced by the Trier Social Stress Task were reported. On the basis of their scores in the Chinese Virtues Questionnaire, 30 college students were assigned to either high- (n = 15) or low-character-strength (n = 15) groups. Their psychological stress and physiological data across three laboratory stages (namely, baseline, stress exposure, and post-stress) were collected. Results indicated that individuals with high character strengths exhibited rapid cardiovascular recovery from baseline to post-stress even if high- and low-character-strength groups showed similar patterns of cardiovascular arousal in response to the stress at baseline and stress exposure. These results prove that character strengths are stress-defense factors that allow for psychological and physiological adaptation to stress. creator: Tingting Li creator: Wenjie Duan creator: Pengfei Guo uri: https://doi.org/10.7717/peerj.3396 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Li et al. title: Clinical impact of human papillomavirus in laryngeal squamous cell carcinoma: a retrospective study link: https://peerj.com/articles/3395 last-modified: 2017-05-30 description: ObjectivesThe purpose of this study is to determine the prevalence and clinical impact of human papillomavirus (HPV) related laryngeal squamous cell carcinoma (LSCC).MethodsA total of 106 LSCC patients who underwent primary surgical resection with or without adjuvant radiotherapy/chemoradiotherapy were enrolled retrospectively. Tumors collected from paraffin-embedded samples were used for HPV detection by polymerase chain reaction and in situ hybridization technique. Clinicopathological parameters were recorded for analysis.ResultsThe prevalence of HPV in patients with LSCC was 13.2% in our series and 12 out of 14 (85.7%) HPV-positive tumors were HPV-16. The patients with HPV-positive tumors were older (p = 0.042), less local/regional recurrence (p = 0.037) and non-smoker (p = 0.068). There was no significant difference in the 5-year overall survival (OS) (p = 0.8056) between HPV-positive and -negative tumors. The patients with HPV-positive tumors had a better 5-year disease-specific survival (DSS) (100% vs. 84.8%, p = 0.1485), although the difference did not reach statistical significance. However, the local/regional control rate was significantly better in HPV-positive tumors than in HPV-negative tumors (100% vs. 75%, p = 0.0494).ConclusionsA low prevalence of HPV infection in our series suggests that HPV is not a major cause of LSCC. However, a 100% local/regional control rate and DSS were observed in HPV-positive tumors. This finding suggests a different tumor behavior between HPV-positive and HPV-negative LSCC. Further research with a larger sample size is necessary to confirm our observations. creator: Wei-Chih Chen creator: Hui-Ching Chuang creator: Yu-Tsai Lin creator: Chao-Cheng Huang creator: Chih-Yen Chien uri: https://doi.org/10.7717/peerj.3395 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Chen et al. title: SIX4 promotes metastasis via activation of the PI3K-AKT pathway in colorectal cancer link: https://peerj.com/articles/3394 last-modified: 2017-05-30 description: BackgroundSeveral studies report aberrant expression of sine oculis homeobox (SIX) homolog family members during cancer development and progression. SIX4 participates in organ development, such as myogenesis and neurogenesis. However, the expression and clinical implication of SIX4 in colorectal cancer (CRC) remains unclear.MethodsThe SIX4 expression levels in colorectal patients were assessed in nine different human cancer arrays and compared using patient survival data. SIX4 expression was silenced in two cell culture lines for invasion and wound healing assessment. Finally, bioinformatics assessments ascertained the pathways impacted by SIX4.ResultsSIX4 was upregulated in The Cancer Genome Atlas CRC cohort and other gene expression omnibus (GEO) cohorts. In addition, SIX4 expression significantly correlated with lymph node metastasis and advanced Tumor Node Metastasis (TNM) stages. Moreover, SIX4 overexpression was related to unfavorable prognosis in CRC patients. Silencing SIX4 inhibited CRC cell metastasis by surpressing AKT phosphorylation.DiscussionSIX4 is upregulated in CRC and can be used as a prognosis biomarker. creator: Guodong Li creator: Fuqing Hu creator: Xuelai Luo creator: Junbo Hu creator: Yongdong Feng uri: https://doi.org/10.7717/peerj.3394 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Li et al. title: The increasing prevalence of HIV/Helicobacter pylori co-infection over time, along with the evolution of antiretroviral therapy (ART) link: https://peerj.com/articles/3392 last-modified: 2017-05-30 description: Helicobacter pylori (H. pylori) is one of the most common human bacterial infections with prevalence rates between 10–80% depending upon geographical location, age and socioeconomic status. H. pylori is commonly found in patients complaining of dyspepsia and is a common cause of gastritis. During the course of their infection, people living with HIV (PLHIV) often have a variety of gastrointestinal symptoms including dyspepsia and while previous studies have reported HIV and H. pylori co-infection, there has been little data clarifying the factors influencing this. The aim of this case-control study was to document the prevalence of H. pylori co-infection within the HIV community as well as to describe endoscopic findings, gastritis topography and histology, along with patient demographic characteristics across three different periods of time during which antiretroviral therapy (ART) has evolved, from pre- highly active antiretroviral therapy (HAART) to early and modern HAART eras. These data were compared to well-matched HIV negative controls. Two hundred and twelve PLHIV were compared with 1,617 controls who underwent their first esophagogastroduodenoscopy (EGD) to investigate dyspepsia. The prevalence of H. pylori co-infection among PLHIV was significantly higher in the early (30.2%) and modern HAART period (34.4%) compared with those with coinfection from the pre-HAART period (18.2%). The higher rates seen in patients from the HAART eras were similar to those observed among HIV negative controls (38.5%). This prevalence increase among co-infected patients was in contrast to the fall in prevalence observed among controls, from 60.7% in the early period to 52.9% in the second observed period. The three PLHIV co-infected subgroups differed regarding gastritis topography, morphology and pathology. This study suggests that ART has an important impact on the endoscopic and histological features of gastritis among HIV/H. pylori co-infected individuals, raising the possibility that H. pylori-induced gastritis could be an immune restoration disease. creator: Aleksandra Radovanović Spurnić creator: Branko Brmbolić creator: Zorica Stojšić creator: Tatijana Pekmezović creator: Zoran Bukumirić creator: Miloš Korać creator: Dubravka Salemović creator: Ivana Pešić-Pavlović creator: Goran Stevanović creator: Ivana Milošević creator: Djordje Jevtović uri: https://doi.org/10.7717/peerj.3392 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Radovanović Spurnić et al. title: Calculating site-specific evolutionary rates at the amino-acid or codon level yields similar rate estimates link: https://peerj.com/articles/3391 last-modified: 2017-05-30 description: Site-specific evolutionary rates can be estimated from codon sequences or from amino-acid sequences. For codon sequences, the most popular methods use some variation of the dN∕dS ratio. For amino-acid sequences, one widely-used method is called Rate4Site, and it assigns a relative conservation score to each site in an alignment. How site-wise dN∕dS values relate to Rate4Site scores is not known. Here we elucidate the relationship between these two rate measurements. We simulate sequences with known dN∕dS, using either dN∕dS models or mutation–selection models for simulation. We then infer Rate4Site scores on the simulated alignments, and we compare those scores to either true or inferred dN∕dS values on the same alignments. We find that Rate4Site scores generally correlate well with true dN∕dS, and the correlation strengths increase in alignments with greater sequence divergence and more taxa. Moreover, Rate4Site scores correlate very well with inferred (as opposed to true) dN∕dS values, even for small alignments with little divergence. Finally, we verify this relationship between Rate4Site and dN∕dS in a variety of empirical datasets. We conclude that codon-level and amino-acid-level analysis frameworks are directly comparable and yield very similar inferences. creator: Dariya K. Sydykova creator: Claus O. Wilke uri: https://doi.org/10.7717/peerj.3391 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Sydykova and Wilke title: Modeling the cis-regulatory modules of genes expressed in developmental stages of Drosophila melanogaster link: https://peerj.com/articles/3389 last-modified: 2017-05-30 description: Because transcription is the first step in the regulation of gene expression, understanding how transcription factors bind to their DNA binding motifs has become absolutely necessary. It has been shown that the promoters of genes with similar expression profiles share common structural patterns. This paper presents an extensive study of the regulatory regions of genes expressed in 24 developmental stages of Drosophila melanogaster. It proposes the use of a combination of structural features, such as positioning of individual motifs relative to the transcription start site, orientation, pairwise distance between motifs, and presence of motifs anywhere in the promoter for predicting gene expression from structural features of promoter sequences. RNA-sequencing data was utilized to create and validate the 24 models. When genes with high-scoring promoters were compared to those identified by RNA-seq samples, 19 (79.2%) statistically significant models, a number that exceeds previous studies, were obtained. Each model yielded a set of highly informative features, which were used to search for genes with similar biological functions. creator: Yosvany López creator: Alexis Vandenbon creator: Akinao Nose creator: Kenta Nakai uri: https://doi.org/10.7717/peerj.3389 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 López et al. title: The ratio of serum Angiopoietin-1 to Angiopoietin-2 in patients with cervical cancer is a valuable diagnostic and prognostic biomarker link: https://peerj.com/articles/3387 last-modified: 2017-05-30 description: ObjectivesAngiopoietins have been found to play essential roles in tumor angiogenesis. The present study was aimed at investigating the diagnostic and prognostic values of serum angiopoietin 1 and 2 (sAng-1 and sAng-2) in cervical cancer.MethodsThe sAng-1 and sAng-2 concentrations were analyzed in 77 women with cervical cancer, 44 women with cervical intraepithelial neoplasia (CIN) and 43 women without cervical lesions by enzyme-linked immunosorbent assay. The diagnostic values of sAng-1, sAng-2 and sAng-1/sAng-2 were evaluated by receiver operating characteristic (ROC) curves. The Ang-1 and Ang-2 expression in cervical cancer tissues as well as microvessel density (MVD), were assessed by immunohistochemistry.ResultsThe concentration of sAng-2 gradually increased and the sAng-1/Ang-2 ratio was gradually decreased from normal control to CIN, then to squamous cell cancer, and the sAng-1/sAng-2 ratio was also significantly decreased in adenocarcinoma. The area under ROC curves of sAng-2 and sAng-1/sAng-2 ratio for discriminating cervical cancer from normal were 0.744 and 0.705, respectively. Decreased sAng-1/sAng-2 was significantly associated with advanced tumor stage, poor differentiation, lymph-vascular space invasion and high MVD. sAng-2 was positively correlated with the Ang-2 expression in cervix epithelia. A high sAng-1/sAng-2 ratio was associated with a longer progression-free survival and a longer overall survival in cervical cancer patients.ConclusionsThese findings suggest that sAng-2 and the sAng-1/sAng-2 ratio may be valuable diagnostic and prognostic biomarkers for cervical cancer. creator: Ping Yang creator: Na Chen creator: Dongyun Yang creator: Janet Crane creator: Shouhua Yang creator: Hangyu Wang creator: Ruiqing Dong creator: Xiaoqing Yi creator: Lisha Xie creator: Guo Jing creator: Jing Cai creator: Zehua Wang uri: https://doi.org/10.7717/peerj.3387 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Yang et al. title: Prevalence and seroepidemiology of Haemophilus parasuis in Sichuan province, China link: https://peerj.com/articles/3379 last-modified: 2017-05-30 description: Haemophilus parasuis, the causative agent of Glässer’s disease, has been reported as widespread, but little is known about its epidemiology in the Sichuan province of China. The goal of our research is to reveal the prevalence and distribution of H. parasuis in this area. Sampling and isolation were performed across Sichuan; isolates were processed using serotyping multiplex PCR (serotyping-mPCR) and agar gel diffusion (AGD) for confirmation of serovar identity. This study was carried out from January 2014 to May 2016 and 254 H. parasuis field strains were isolated from 576 clinical samples collected from pigs displaying clinical symptoms. The isolation frequency was 44.10%. Statistically very significant differences of infection incidence were found in three age groups (P < 0.01) and different seasons (P < 0.01). Serovars 5 (25.98%) and 4 (23.62%) were the most prevalent, however, non-typeable isolates accounted for nearly 7.87%. In terms of geographical distribution, serovars 5 and 4 were mostly prevalent in west and east Sichuan. The results confirmed that the combined approach was dependable and revealed the diversity and distribution of serovars in Sichuan province, which is vital for efforts aimed at developing vaccine candidates allowing for the prevention or control of H. parasuis outbreaks. creator: Zhenghao Wang creator: Qin Zhao creator: Hailin Wei creator: Xintian Wen creator: Sanjie Cao creator: Xiaobo Huang creator: Rui Wu creator: Qigui Yan creator: Yong Huang creator: Yiping Wen uri: https://doi.org/10.7717/peerj.3379 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Wang et al. title: Increasing game prices may alter farmers’ behaviours towards leopards (Panthera pardus) and other carnivores in South Africa link: https://peerj.com/articles/3369 last-modified: 2017-05-30 description: Human-carnivore conflict occurs globally, particularly in regions where large carnivores predate livestock. Retaliatory killings do occur, and although predation of livestock by carnivores happens, losses from other factors such as disease or injury can be misattributed because of landowner perceptions. Game farming for both trophy hunting and eco-tourism is becoming increasingly common in South Africa, and there has been a rapid increase in the cost of game animals (in some species as much as five-fold) between 2010 and 2015. This could result in an increase in conflict between commercial game farmers and carnivores. We conducted two questionnaire surveys of farmers in 2010 and 2015 to investigate this. We asked if there had been changes in farming practices, perceived predator activity, perceived amount of livestock and commercial game losses, and actions taken towards carnivores in a South African farming community. We found no significant change in farming types in the area or losses of livestock between the years. However, there was a significant increase in perceived commercial game losses reported, even though protection of game had increased. Actions taken towards carnivores by livestock/game farmers were also significantly more negative in 2015 compared to farmers growing crops, but there was no such difference in 2010. We suggest that these changes could be a result of the increase in game prices over that period, leading to greater financial losses when an animal is predated, which in turn could increase the likelihood of retaliatory killings of carnivores. creator: Tara J. Pirie creator: Rebecca L. Thomas creator: Mark D.E. Fellowes uri: https://doi.org/10.7717/peerj.3369 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Pirie et al. title: Rituals decrease the neural response to performance failure link: https://peerj.com/articles/3363 last-modified: 2017-05-30 description: Rituals are found in all types of performance domains, from high-stakes athletics and military to the daily morning preparations of the working family. Yet despite their ubiquity and widespread importance for humans, we know very little of ritual’s causal basis and how (if at all) they facilitate goal-directed performance. Here, in a fully pre-registered pre/post experimental design, we examine a candidate proximal mechanism, the error-related negativity (ERN), in testing the prediction that ritual modulates neural performance-monitoring. Participants completed an arbitrary ritual—novel actions repeated at home over one week—followed by an executive function task in the lab during electroencephalographic (EEG) recording. Results revealed that relative to pre rounds, participants showed a reduced ERN in the post rounds, after completing the ritual in the lab. Despite a muted ERN, there was no evidence that the reduction in neural monitoring led to performance deficit (nor a performance improvement). Generally, the findings are consistent with the longstanding view that ritual buffers against uncertainty and anxiety. Our results indicate that ritual guides goal-directed performance by regulating the brain’s response to personal failure. creator: Nicholas M. Hobson creator: Devin Bonk creator: Michael Inzlicht uri: https://doi.org/10.7717/peerj.3363 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Hobson et al. title: Impact of enzymatic digestion on bacterial community composition in CF airway samples link: https://peerj.com/articles/3362 last-modified: 2017-05-30 description: BackgroundPrevious studies have demonstrated the importance of DNA extraction methods for molecular detection of Staphylococcus, an important bacterial group in cystic fibrosis (CF). We sought to evaluate the effect of enzymatic digestion (EnzD) prior to DNA extraction on bacterial communities identified in sputum and oropharyngeal swab (OP) samples from patients with CF.MethodsDNA from 81 samples (39 sputum and 42 OP) collected from 63 patients with CF was extracted in duplicate with and without EnzD. Bacterial communities were determined by rRNA gene sequencing, and measures of alpha and beta diversity were calculated. Principal Coordinate Analysis (PCoA) was used to assess differences at the community level and Wilcoxon Signed Rank tests were used to compare relative abundance (RA) of individual genera for paired samples with and without EnzD.ResultsShannon Diversity Index (alpha-diversity) decreased in sputum and OP samples with the use of EnzD. Larger shifts in community composition were observed for OP samples (beta-diversity, measured by Morisita-Horn), whereas less change in communities was observed for sputum samples. The use of EnzD with OP swabs resulted in significant increase in RA for the genera Gemella (p < 0.01), Streptococcus (p < 0.01), and Rothia (p < 0.01). Staphylococcus (p < 0.01) was the only genus with a significant increase in RA from sputum, whereas the following genera decreased in RA with EnzD: Veillonella (p < 0.01), Granulicatella (p < 0.01), Prevotella (p < 0.01), and Gemella (p = 0.02). In OP samples, higher RA of Gram-positive taxa was associated with larger changes in microbial community composition.DiscussionWe show that the application of EnzD to CF airway samples, particularly OP swabs, results in differences in microbial communities detected by sequencing. Use of EnzD can result in large changes in bacterial community composition, and is particularly useful for detection of Staphylococcus in CF OP samples. The enhanced identification of Staphylococcus aureus is a strong indication to utilize EnzD in studies that use OP swabs to monitor CF airway communities. creator: Kayla M. Williamson creator: Brandie D. Wagner creator: Charles E. Robertson creator: Emily J. Johnson creator: Edith T. Zemanick creator: J. Kirk Harris uri: https://doi.org/10.7717/peerj.3362 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Williamson et al. title: Cucurbitacins: elucidation of their interactions with the cytoskeleton link: https://peerj.com/articles/3357 last-modified: 2017-05-30 description: Cucurbitacins, a class of toxic tetracyclic triterpenoids in Cucurbitaceae, modulate many molecular targets. Here we investigated the interactions of cucurbitacin B, E and I with cytoskeletal proteins such as microtubule and actin filaments. The effects of cucurbitacin B, E and I on microtubules and actin filaments were studied in living cells (Hela and U2OS) and in vitro using GFP markers, immunofluorescence staining and in vitro tubulin polymerization assay. Cucurbitacin B, E and I apparently affected microtubule structures in living cells and cucurbitacin E inhibited tubulin polymerization in vitro with IC50 value of 566.91 ± 113.5 µM. Cucurbitacin E did not affect the nucleation but inhibited the growth phase and steady state during microtubule assembly in vitro. In addition, cucurbitacin B, E and I all altered mitotic spindles and induced the cell cycle arrest at G2/M phase. Moreover, they all showed potent effects on actin cytoskeleton by affecting actin filaments through the depolymerization and aggregation. The interactions of cucubitacin B, E and I with microtubules and actin filaments present new insights into their modes of action. creator: Xiaojuan Wang creator: Mine Tanaka creator: Herbenya Silva Peixoto creator: Michael Wink uri: https://doi.org/10.7717/peerj.3357 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Wang et al. title: A new view on the morphology and phylogeny of eugregarines suggested by the evidence from the gregarine Ancora sagittata (Leuckart, 1860) Labbé, 1899 (Apicomplexa: Eugregarinida) link: https://peerj.com/articles/3354 last-modified: 2017-05-30 description: BackgroundGregarines are a group of early branching Apicomplexa parasitizing invertebrate animals. Despite their wide distribution and relevance to the understanding the phylogenesis of apicomplexans, gregarines remain understudied: light microscopy data are insufficient for classification, and electron microscopy and molecular data are fragmentary and overlap only partially.MethodsScanning and transmission electron microscopy, PCR, DNA cloning and sequencing (Sanger and NGS), molecular phylogenetic analyses using ribosomal RNA genes (18S (SSU), 5.8S, and 28S (LSU) ribosomal DNAs (rDNAs)).Results and DiscussionWe present the results of an ultrastructural and molecular phylogenetic study on the marine gregarine Ancora sagittata from the polychaete Capitella capitata followed by evolutionary and taxonomic synthesis of the morphological and molecular phylogenetic evidence on eugregarines. The ultrastructure of Ancora sagittata generally corresponds to that of other eugregarines, but reveals some differences in epicytic folds (crests) and attachment apparatus to gregarines in the family Lecudinidae, where Ancora sagittata has been classified. Molecular phylogenetic trees based on SSU (18S) rDNA reveal several robust clades (superfamilies) of eugregarines, including Ancoroidea superfam. nov., which comprises two families (Ancoridae fam. nov. and Polyplicariidae) and branches separately from the Lecudinidae; thus, all representatives of Ancoroidea are here officially removed from the Lecudinidae. Analysis of sequence data also points to possible cryptic species within Ancora sagittata and the inclusion of numerous environmental sequences from anoxic habitats within the Ancoroidea. LSU (28S) rDNA phylogenies, unlike the analysis of SSU rDNA alone, recover a well-supported monophyly of the gregarines involved (eugregarines), although this conclusion is currently limited by sparse taxon sampling and the presence of fast-evolving sequences in some species. Comparative morphological analyses of gregarine teguments and attachment organelles lead us to revise their terminology. The terms “longitudinal folds” and “mucron” are restricted to archigregarines, whereas the terms “epicystic crests” and “epimerite” are proposed to describe the candidate synapomorphies of eugregarines, which, consequently, are considered as a monophyletic group. Abolishing the suborders Aseptata and Septata, incorporating neogregarines into the Eugregarinida, and treating the major molecular phylogenetic lineages of eugregarines as superfamilies appear as the best way of reconciling recent morphological and molecular evidence. Accordingly, the diagnosis of the order Eugregarinida Léger, 1900 is updated. creator: Timur G. Simdyanov creator: Laure Guillou creator: Andrei Y. Diakin creator: Kirill V. Mikhailov creator: Joseph Schrével creator: Vladimir V. Aleoshin uri: https://doi.org/10.7717/peerj.3354 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Simdyanov et al. title: High or low? Comparing high and low-variability phonetic training in adult and child second language learners link: https://peerj.com/articles/3209 last-modified: 2017-05-30 description: BackgroundHigh talker variability (i.e., multiple voices in the input) has been found effective in training nonnative phonetic contrasts in adults. A small number of studies suggest that children also benefit from high-variability phonetic training with some evidence that they show greater learning (more plasticity) than adults given matched input, although results are mixed. However, no study has directly compared the effectiveness of high versus low talker variability in children.MethodsNative Greek-speaking eight-year-olds (N = 52), and adults (N = 41) were exposed to the English /i/-/ɪ/ contrast in 10 training sessions through a computerized word-learning game. Pre- and post-training tests examined discrimination of the contrast as well as lexical learning. Participants were randomly assigned to high (four talkers) or low (one talker) variability training conditions.ResultsBoth age groups improved during training, and both improved more while trained with a single talker. Results of a three-interval oddity discrimination test did not show the predicted benefit of high-variability training in either age group. Instead, children showed an effect in the reverse direction—i.e., reliably greater improvements in discrimination following single talker training, even for untrained generalization items, although the result is qualified by (accidental) differences between participant groups at pre-test. Adults showed a numeric advantage for high-variability but were inconsistent with respect to voice and word novelty. In addition, no effect of variability was found for lexical learning. There was no evidence of greater plasticity for phonetic learning in child learners.DiscussionThis paper adds to the handful of studies demonstrating that, like adults, child learners can improve their discrimination of a phonetic contrast via computerized training. There was no evidence of a benefit of training with multiple talkers, either for discrimination or word learning. The results also do not support the findings of greater plasticity in child learners found in a previous paper (Giannakopoulou, Uther & Ylinen, 2013a). We discuss these results in terms of various differences between training and test tasks used in the current work compared with previous literature. creator: Anastasia Giannakopoulou creator: Helen Brown creator: Meghan Clayards creator: Elizabeth Wonnacott uri: https://doi.org/10.7717/peerj.3209 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Giannakopoulou et al. title: Infrared thermography applied to lower limb muscles in elite soccer players with functional ankle equinus and non-equinus condition link: https://peerj.com/articles/3388 last-modified: 2017-05-25 description: Gastrocnemius-soleus equinus (GSE) is a foot-ankle complaint in which the extensibility of the gastrocnemius (G) and soleus muscles (triceps surae) and ankle are limited to a dorsiflexion beyond a neutral ankle position. The asymmetric forces of leg muscles and the associated asymmetric loading forces might promote major activation of the triceps surae, tibialis anterior, transverses abdominal and multifidus muscles. Here, we made infrared recordings of 21 sportsmen (elite professional soccer players) before activity and after 30 min of running. These recordings were used to assess temperature modifications on the gastrocnemius, tibialis anterior, and Achilles tendon in GSE and non-GSE participants. We identified significant temperature modifications among GSE and non-GSE participants for the tibialis anterior muscle (mean, minimum, and maximum temperature values). The cutaneous temperature increased as a direct consequence of muscle activity in GSE participants. IR imaging capture was reliable to muscle pattern activation for lower limb. Based on our findings, we propose that non-invasive IR evaluation is suitable for clinical evaluation of the status of these muscles. creator: David Rodríguez-Sanz creator: Marta Elena Losa-Iglesias creator: Daniel López-López creator: César Calvo-Lobo creator: Patricia Palomo-López creator: Ricardo Becerro-de-Bengoa-Vallejo uri: https://doi.org/10.7717/peerj.3388 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Rodríguez-Sanz et al. title: Seasonal dispersal and longitudinal migration in the Relict Gull Larus relictus across the Inner-Mongolian Plateau link: https://peerj.com/articles/3380 last-modified: 2017-05-25 description: The Relict Gull Larus relictus is a globally vulnerable species and one of the least known birds, so understanding its seasonal movements and migration will facilitate the development of effective conservation plans for its protection. We repeatedly satellite-tracked 11 adult Relict Gulls from the Ordos sub-population in Hongjian Nur, China, over 33 migration seasons and conducted extensive ground surveys. Relict Gulls traveled ∼800 km between Hongjian Nur in northern China to the coast of eastern China in a predominantly longitudinal migration, following a clockwise loop migration pattern. The gulls migrated faster in spring (4 ± 2 d) than in autumn (15 ± 13 d) due to a time-minimization strategy for breeding, and they showed considerable between-individual variation in the timing of the autumn migration, probably due to differences in the timing of breeding. Gulls that made at least two round trips exhibited high flexibility in spring migration timing, suggesting a stronger influence of local environment conditions over endogenous controls. There was also high route flexibility among different years, probably due to variations in meteorological or habitat conditions at stopover sites. Relict Gulls stayed for a remarkably long time (234 ± 17 d) on their major wintering grounds in Bohai Bay and Laizhou Bay, between which there were notable dispersals. Pre-breeding dispersals away from the breeding area were distinct, which seemed to be a strategy to cope with the degradation of breeding habitat at Hongjian Nur. Overwhelming lake shrinkage on the breeding ground and at stopover sites and loss of intertidal flats on the wintering grounds are regarded as the main threats to Relict Gulls. It is crucial to make protection administrations aware of the great significance of key sites along migration routes and to promote the establishment of protected areas in these regions. creator: Dongping Liu creator: Guogang Zhang creator: Hongxing Jiang creator: Lixia Chen creator: Derong Meng creator: Jun Lu uri: https://doi.org/10.7717/peerj.3380 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Liu et al. title: Differentiation of Bifidobacterium longum subspecies longum and infantis by quantitative PCR using functional gene targets link: https://peerj.com/articles/3375 last-modified: 2017-05-25 description: BackgroundMembers of the genus Bifidobacterium are abundant in the feces of babies during the exclusively-milk-diet period of life. Bifidobacterium longum is reported to be a common member of the infant fecal microbiota. However, B. longum is composed of three subspecies, two of which are represented in the bowel microbiota (B. longum subsp. longum; B. longum subsp. infantis). B. longum subspecies are not differentiated in many studies, so that their prevalence and relative abundances are not accurately known. This may largely be due to difficulty in assigning subspecies identity using DNA sequences of 16S rRNA or tuf genes that are commonly used in bacterial taxonomy.MethodsWe developed a qPCR method targeting the sialidase gene (subsp. infantis) and sugar kinase gene (subsp. longum) to differentiate the subspecies using specific primers and probes. Specificity of the primers/probes was tested by in silico, pangenomic search, and using DNA from standard cultures of bifidobacterial species. The utility of the method was further examined using DNA from feces that had been collected from infants inhabiting various geographical regions.ResultsA pangenomic search of the NCBI genomic database showed that the PCR primers/probes targeted only the respective genes of the two subspecies. The primers/probes showed total specificity when tested against DNA extracted from the gold standard strains (type cultures) of bifidobacterial species detected in infant feces. Use of the qPCR method with DNA extracted from the feces of infants of different ages, delivery method and nutrition, showed that subsp. infantis was detectable (0–32.4% prevalence) in the feces of Australian (n = 90), South-East Asian (n = 24), and Chinese babies (n = 91), but in all cases at low abundance (<0.01–4.6%) compared to subsp. longum (0.1–33.7% abundance; 21.4–100% prevalence).DiscussionOur qPCR method differentiates B. longum subspecies longum and infantis using characteristic functional genes. It can be used as an identification aid for isolates of bifidobacteria, as well as in determining prevalence and abundance of the subspecies in feces. The method should thus be useful in ecological studies of the infant gut microbiota during early life where an understanding of the ecology of bifidobacterial species may be important in developing interventions to promote infant health. creator: Blair Lawley creator: Karen Munro creator: Alan Hughes creator: Alison J. Hodgkinson creator: Colin G. Prosser creator: Dianne Lowry creator: Shao J. Zhou creator: Maria Makrides creator: Robert A. Gibson creator: Christophe Lay creator: Charmaine Chew creator: Pheng Soon Lee creator: Khai Hong Wong creator: Gerald W. Tannock uri: https://doi.org/10.7717/peerj.3375 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Lawley et al. title: The role of kelp crabs as consumers in bull kelp forests—evidence from laboratory feeding trials and field enclosures link: https://peerj.com/articles/3372 last-modified: 2017-05-25 description: The Northern kelp crab (Pugettia producta) and the graceful kelp crab (Pugettia gracilis) are common primary consumers in bull kelp beds near the San Juan Islands (Salish Sea, NE Pacific). In this system, urchins (often considered the most voracious herbivores exerting top-down control on kelp beds) tend to remain sedentary because of the high availability of detrital macroalgae, but the extent to which kelp crabs consume kelp (and other food options) is largely unknown. I conducted four types of laboratory feeding experiments to evaluate kelp crab feeding patterns: (1) feeding electivity between bull kelp (Nereocystis luetkeana) and seven species of co-occurring local macroalgae; (2) feeding electivity on aged vs. fresh bull kelp; (3) feeding preference between N. luetkeana and small snails (Lacuna sp.); and (4) scaling of feeding rate with body size in P. producta and P. gracilis. In choice experiments, P. producta consumed greater mass of N. luetkeana than of other macroalgal species offered and elected to eat fresh bull kelp over aged. However, P. producta also consumed snails (Lacuna sp.), indicating more generalized feeding than previously suspected. Feeding rates for P. producta exceeded the expected 3∕4 scaling rule of metabolic rates, indicating that larger P. producta may have a disproportionately large impact on bull kelp. A subtidal field experiment, designed to assess the influence of consumers on juvenile bull kelp net tissue gain, found that only fully enclosed (protected) bull kelp increased in wet mass and blade length. Herbivory by kelp crabs, among other consumers, is likely to play a previously unrecognized role in mediating the growth and survival of this annual kelp species within the Salish Sea. creator: Katie Dobkowski uri: https://doi.org/10.7717/peerj.3372 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Dobkowski title: Genetic effects on life-history traits in the Glanville fritillary butterfly link: https://peerj.com/articles/3371 last-modified: 2017-05-25 description: BackgroundAdaptation to local habitat conditions may lead to the natural divergence of populations in life-history traits such as body size, time of reproduction, mate signaling or dispersal capacity. Given enough time and strong enough selection pressures, populations may experience local genetic differentiation. The genetic basis of many life-history traits, and their evolution according to different environmental conditions remain however poorly understood.MethodsWe conducted an association study on the Glanville fritillary butterfly, using material from five populations along a latitudinal gradient within the Baltic Sea region, which show different degrees of habitat fragmentation. We investigated variation in 10 principal components, cofounding in total 21 life-history traits, according to two environmental types, and 33 genetic SNP markers from 15 candidate genes.ResultsWe found that nine SNPs from five genes showed strong trend for trait associations (p-values under 0.001 before correction). These associations, yet non-significant after multiple test corrections, with a total number of 1,086 tests, were consistent across the study populations. Additionally, these nine genes also showed an allele frequency difference between the populations from the northern fragmented versus the southern continuous landscape.DiscussionOur study provides further support for previously described trait associations within the Glanville fritillary butterfly species across different spatial scales. Although our results alone are inconclusive, they are concordant with previous studies that identified these associations to be related to climatic changes or habitat fragmentation within the Åland population. creator: Anne Duplouy creator: Swee C. Wong creator: Jukka Corander creator: Rainer Lehtonen creator: Ilkka Hanski uri: https://doi.org/10.7717/peerj.3371 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Duplouy et al. title: A molecular phylogeny of the spiny lobster Panulirus homarus highlights a separately evolving lineage from the Southwest Indian Ocean link: https://peerj.com/articles/3356 last-modified: 2017-05-25 description: Accurate species description in the marine environment is critical for estimating biodiversity and identifying genetically distinct stocks. Analysis of molecular data can potentially improve species delimitations because they are easily generated and independent, and yield consistent results with high statistical power. We used classical phylogenetic (maximum likelihood and Bayesian inference) and coalescent-based methods (divergence dating with fossil calibrations and coalescent-based species delimitation) to resolve the phylogeny of the spiny lobster Panulirus homarus subspecies complex in the Indo-West Pacific. Analyses of mitochondrial data and combined nuclear and mitochondrial data recovered Panulirus homarus homarus and Panulirus homarus rubellus as separately evolving lineages, while the nuclear data trees were unresolved. Divergence dating analysis also identified Panulirus homarus homarus and Panulirus homarus rubellus as two distinct clades which diverged from a common ancestor during the Oligocene, approximately 26 million years ago. Species delimitation using coalescent-based methods corroborated these findings. A long pelagic larval life stage and the influence of ocean currents on post-larval settlement patterns suggest that a parapatric mode of speciation drives evolution in this subspecies complex. In combination, the results indicate that Panulirus homarus rubellus from the Southwest Indian Ocean is a separately evolving lineage and possibly a separate species. creator: Sohana P. Singh creator: Johan C. Groeneveld creator: Abdulaziz Al-Marzouqi creator: Sandi Willows-Munro uri: https://doi.org/10.7717/peerj.3356 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Singh et al. title: Interactions of 172 plant extracts with human organic anion transporter 1 (SLC22A6) and 3 (SLC22A8): a study on herb-drug interactions link: https://peerj.com/articles/3333 last-modified: 2017-05-25 description: BackgroundHerb-drug interactions (HDIs) resulting from concomitant use of herbal products with clinical drugs may cause adverse reactions. Organic anion transporter 1 (OAT1) and 3 (OAT3) are highly expressed in the kidney and play a key role in the renal elimination of substrate drugs. So far, little is known about the herbal extracts that could modulate OAT1 and OAT3 activities.MethodsHEK293 cells stably expressing human OAT1 (HEK-OAT1) and OAT3 (HEK-OAT3) were established and characterized. One hundred seventy-two extracts from 37 medicinal and economic plants were prepared. An initial concentration of 5 µg/ml for each extract was used to evaluate their effects on 6-carboxylfluorescein (6-CF) uptake in HEK-OAT1 and HEK-OAT3 cells. Concentration-dependent inhibition studies were conducted for those extracts with more than 50% inhibition to OAT1 and OAT3. The extract of Juncus effusus, a well-known traditional Chinese medicine, was assessed for its effect on the in vivo pharmacokinetic parameters of furosemide, a diuretic drug which is a known substrate of both OAT1 and OAT3.ResultsMore than 30% of the plant extracts at the concentration of 5 µg/ml showed strong inhibitory effect on the 6-CF uptake mediated by OAT1 (61 extracts) and OAT3 (55 extracts). Among them, three extracts for OAT1 and fourteen extracts for OAT3 were identified as strong inhibitors with IC50 values being <5 µg/ml. Juncus effusus showed a strong inhibition to OAT3 in vitro, and markedly altered the in vivo pharmacokinetic parameters of furosemide in rats.ConclusionThe present study identified the potential interactions of medicinal and economic plants with human OAT1 and OAT3, which is helpful to predict and to avoid potential OAT1- and OAT3-mediated HDIs. creator: Hang Lu creator: Zhiqiang Lu creator: Xue Li creator: Gentao Li creator: Yilin Qiao creator: Robert P. Borris creator: Youcai Zhang uri: https://doi.org/10.7717/peerj.3333 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Lu et al. title: Invasive lionfish had no measurable effect on prey fish community structure across the Belizean Barrier Reef link: https://peerj.com/articles/3270 last-modified: 2017-05-25 description: Invasive lionfish are assumed to significantly affect Caribbean reef fish communities. However, evidence of lionfish effects on native reef fishes is based on uncontrolled observational studies or small-scale, unrepresentative experiments, with findings ranging from no effect to large effects on prey density and richness. Moreover, whether lionfish affect populations and communities of native reef fishes at larger, management-relevant scales is unknown. The purpose of this study was to assess the effects of lionfish on coral reef prey fish communities in a natural complex reef system. We quantified lionfish and the density, richness, and composition of native prey fishes (0–10 cm total length) at sixteen reefs along ∼250 km of the Belize Barrier Reef from 2009 to 2013. Lionfish invaded our study sites during this four-year longitudinal study, thus our sampling included fish community structure before and after our sites were invaded, i.e., we employed a modified BACI design. We found no evidence that lionfish measurably affected the density, richness, or composition of prey fishes. It is possible that higher lionfish densities are necessary to detect an effect of lionfish on prey populations at this relatively large spatial scale. Alternatively, negative effects of lionfish on prey could be small, essentially undetectable, and ecologically insignificant at our study sites. Other factors that influence the dynamics of reef fish populations including reef complexity, resource availability, recruitment, predation, and fishing could swamp any effects of lionfish on prey populations. creator: Serena Hackerott creator: Abel Valdivia creator: Courtney E. Cox creator: Nyssa J. Silbiger creator: John F. Bruno uri: https://doi.org/10.7717/peerj.3270 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Hackerott et al. title: Isolation and characterization of microsatellite markers for Sturnira parvidens and cross-species amplification in Sturnira species link: https://peerj.com/articles/3367 last-modified: 2017-05-24 description: BackgroundSturnira is one of the most species-rich genera in the Neotropics, and it is found from Mexico and the Lesser Antilles to Argentina. This genus forms a well-supported monophyletic clade with at least twenty-one recognized species, as well as several others under taxonomic review. Sturnira parvidens is a widespread frugivorous bat of the deciduous forests of the Neotropics, is highly abundant, and is a major component in fruit dispersal to regenerate ecosystems.MethodsWe used a technique based on Illumina paired-end sequencing of a library highly enriched for microsatellite repeats to develop loci for S. parvidens. We analyzed millions of resulting reads with specialized software to extract those reads that contained di-, tri-, tetra-, penta-, and hexanucleotide microsatellites.ResultsWe selected and tested 14 polymorphic (di, tri, and tetra) microsatellites. All markers were genotyped on 26 different individuals from distinct locations of the distributional area of S. parvidens. We observed medium—high genetic variation across most loci, but only 12 were functionally polymorphic. Levels of expected heterozygosity across all markers were high to medium (mean HE = 0.79, mean HO = 0.72). We examined ascertainment bias in twelve bats of the genus, obtaining null/monomorphic/polymorphic amplifications.DiscussionThe Illumina paired-end sequencing system is capable of identifying massive numbers of microsatellite loci, while expending little time, reducing costs, and providing a large amount of data. The described polymorphic loci for S. parvidens in particular, and for the genus in general, could be suitable for further genetic analysis, including taxonomic inconsistencies, parentage/relatedness analysis, and population genetics assessments. creator: Edgar G. Gutiérrez creator: Giovani Hernández Canchola creator: Livia S. León Paniagua creator: Norberto Martínez Méndez creator: Jorge Ortega uri: https://doi.org/10.7717/peerj.3367 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Gutiérrez et al. title: Restoring lepidopteran diversity in a tropical dry forest: relative importance of restoration treatment, tree identity and predator pressure link: https://peerj.com/articles/3344 last-modified: 2017-05-24 description: Tropical dry forests (TDFs) have been widely transformed by human activities worldwide and the ecosystem services they provide are diminishing. There has been an urgent call for conservation and restoration of the degraded lands previously occupied by TDFs. Restoration experiences aim to recover species diversity and ecological functions. Different restoration strategies have been used to maximize plant performance including weeding, planting or using artificial mulching. In this investigation, we evaluated whether different restoration practices influence animal arrival and the reestablishment of biotic interactions. We particularly evaluated lepidopteran larvae diversity and caterpillar predation on plants established under different restoration treatments (mulching, weeding and control) in the Pacific West Coast of México. This study corroborated the importance of plant host identity for lepidopteran presence in a particular area. Lepidopteran diversity and herbivory rates were not affected by the restoration treatment but they were related to tree species. In contrast, caterpillar predation marks were affected by restoration treatment, with a greater number of predation marks in control plots, while caterpillar predation marks among plant species were not significantly different. This study highlights the importance of considering the introduction of high plant species diversity when planning TDF restoration to maximize lepidopteran diversity and ecosystem functioning. creator: Lizet Solis-Gabriel creator: Wendy Mendoza-Arroyo creator: Karina Boege creator: Ek del-Val uri: https://doi.org/10.7717/peerj.3344 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Solis-Gabriel et al. title: A novel genus and cryptic species harboured within the monotypic freshwater crayfish genus Tenuibranchiurus Riek, 1951 (Decapoda: Parastacidae) link: https://peerj.com/articles/3310 last-modified: 2017-05-24 description: Identifying species groups is an important yet difficult task, with there being no single accepted definition as to what constitutes a species, nor a set of criteria by which they should be delineated. Employing the General Lineage Concept somewhat circumvents these issues, as this concept allows multiple concordant lines of evidence to be used as support for species delimitation, where a species is defined as any independently evolving lineage. Genetically diverse groups have previously been identified within the monotypic parastacid genus Tenuibranchiurus Riek, 1951, but no further investigation of this diversity has previously been undertaken. Analysis of two mitochondrial DNA gene regions has previously identified two highly divergent groups within this taxon, representing populations from Queensland (Qld) and New South Wales (NSW), respectively. Additional testing within this study of both mitochondrial and nuclear DNA through species discovery analyses identified genetically diverse groups within these regions, which were further supported by lineage validation methods. The degree of genetic differentiation between Qld and NSW populations supports the recognition of two genera; with Qld retaining the original genus name Tenuibranchiurus, and NSW designated as Gen. nov. until a formal description is completed. Concordance between the species discovery and lineage validation methods supports the presence of six species within Tenuibranchiurus and two within Gen. nov. The recognition of additional species removes the monotypy of the genus, and the methods used can improve species identification within groups of organisms with taxonomic problems and cryptic diversity. creator: Kathryn L. Dawkins creator: James M. Furse creator: Clyde H. Wild creator: Jane M. Hughes uri: https://doi.org/10.7717/peerj.3310 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Dawkins et al. title: Eumelanin and pheomelanin are predominant pigments in bumblebee (Apidae: Bombus) pubescence link: https://peerj.com/articles/3300 last-modified: 2017-05-24 description: BackgroundBumblebees (Hymenoptera: Apidae: Bombus) are well known for their important inter- and intra-specific variation in hair (or pubescence) color patterns, but the chemical nature of the pigments associated with these patterns is not fully understood. For example, though melanization is believed to provide darker colors, it still unknown which types of melanin are responsible for each color, and no conclusive data are available for the lighter colors, including white.MethodsBy using dispersive Raman spectroscopy analysis on 12 species/subspecies of bumblebees from seven subgenera, we tested the hypothesis that eumelanin and pheomelanin, the two main melanin types occurring in animals, are largely responsible for bumblebee pubescence coloration.ResultsEumelanin and pheomelanin occur in bumblebee pubescence. Black pigmentation is due to prevalent eumelanin, with visible signals of additional pheomelanin, while the yellow, orange, red and brown hairs clearly include pheomelanin. On the other hand, white hairs reward very weak Raman signals, suggesting that they are depigmented. Additional non-melanic pigments in yellow hair cannot be excluded but need other techniques to be detected. Raman spectra were more similar across similarly colored hairs, with no apparent effect of phylogeny and both melanin types appeared to be already used at the beginning of bumblebee radiation.DiscussionWe suggest that the two main melanin forms, at variable amounts and/or vibrational states, are sufficient in giving almost the whole color range of bumblebee pubescence, allowing these insects to use a single precursor instead of synthesizing a variety of chemically different pigments. This would agree with commonly seen color interchanges between body segments across Bombus species. creator: Carlo Polidori creator: Alberto Jorge creator: Concepción Ornosa uri: https://doi.org/10.7717/peerj.3300 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Polidori et al. title: The first reported ceratopsid dinosaur from eastern North America (Owl Creek Formation, Upper Cretaceous, Mississippi, USA) link: https://peerj.com/articles/3342 last-modified: 2017-05-23 description: Ceratopsids (“horned dinosaurs”) are known from western North America and Asia, a distribution reflecting an inferred subaerial link between the two landmasses during the Late Cretaceous. However, this clade was previously unknown from eastern North America, presumably due to limited outcrop of the appropriate age and depositional environment as well as the separation of eastern and western North America by the Western Interior Seaway during much of the Late Cretaceous. A dentary tooth from the Owl Creek Formation (late Maastrichtian) of Union County, Mississippi, represents the first reported occurrence of Ceratopsidae from eastern North America. This tooth shows a combination of features typical of Ceratopsidae, including a double root and a prominent, blade-like carina. Based on the age of the fossil, we hypothesize that it is consistent with a dispersal of ceratopsids into eastern North America during the very latest Cretaceous, presumably after the two halves of North America were reunited following the retreat of the Western Interior Seaway. creator: Andrew A. Farke creator: George E. Phillips uri: https://doi.org/10.7717/peerj.3342 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Farke and Phillips title: Clonidine or remifentanil for adequate surgical conditions in patients undergoing endoscopic sinus surgery: a randomized study link: https://peerj.com/articles/3370 last-modified: 2017-05-23 description: BackgroundDeliberate hypotension is one way to achieve a bloodless surgical field in endoscopic sinus surgery (ESS). We compared two anaesthesia regimens to induce deliberate hypotension and attempted to determine the most efficient one.MethodsFifty-nine patients undergoing ESS were minimized into two groups. In the CLO group, patients received I.V. sufentanil 0.15 µg/kg together with I.V. clonidine 2–3 µg/kg. In the REMI group, patients received remifentanil at a rate of up to 1 µg/kg/min. Fromme scores were collected 15 min after the incision and at the end of the procedure. Mean arterial pressure readings (MAP), heart rate readings, time to eyes opening, time to extubation, pain scores, analgesic requirements, and oxygen needs were collected and compared.ResultsThere were no significant differences in Fromme scores between the two groups. The averaged MAP from 15 min to the end of the procedure was significantly lower in the REMI group; these patients also received more ephedrine. Significantly fewer patients in the CLO group needed oxygen therapy to keep their Pulse Oximeter Oxygen Saturation within 3% of their preoperative values. Patients in this group also needed less piritramide in the recovery room, and their pain scores were lower at discharge from the recovery room.DiscussionAlthough both anaesthesia regimens offered a similar quality of surgical field, this study suggests that clonidine had a better average safety profile. Furthermore, patients who received this regimen required fewer painkillers immediately after surgery. creator: Laurent Bairy creator: Marie Vanderstichelen creator: Jacques Jamart creator: Edith Collard uri: https://doi.org/10.7717/peerj.3370 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Bairy et al. title: Within outlying mean indexes: refining the OMI analysis for the realized niche decomposition link: https://peerj.com/articles/3364 last-modified: 2017-05-23 description: The ecological niche concept has regained interest under environmental change (e.g., climate change, eutrophication, and habitat destruction), especially to study the impacts on niche shift and conservatism. Here, we propose the within outlying mean indexes (WitOMI), which refine the outlying mean index (OMI) analysis by using its properties in combination with the K-select analysis species marginality decomposition. The purpose is to decompose the ecological niche into subniches associated with the experimental design, i.e., taking into account temporal and/or spatial subsets. WitOMI emphasize the habitat conditions that contribute (1) to the definition of species’ niches using all available conditions and, at the same time, (2) to the delineation of species’ subniches according to given subsets of dates or sites. The latter aspect allows addressing niche dynamics by highlighting the influence of atypical habitat conditions on species at a given time and/or space. Then, (3) the biological constraint exerted on the species subniche becomes observable within Euclidean space as the difference between the existing fundamental subniche and the realized subniche. We illustrate the decomposition of published OMI analyses, using spatial and temporal examples. The species assemblage’s subniches are comparable to the same environmental gradient, producing a more accurate and precise description of the assemblage niche distribution under environmental change. The WitOMI calculations are available in the open-access R package “subniche.” creator: Stéphane Karasiewicz creator: Sylvain Dolédec creator: Sébastien Lefebvre uri: https://doi.org/10.7717/peerj.3364 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Karasiewicz et al. title: Storm effects on intertidal invertebrates: increased beta diversity of few individuals and species link: https://peerj.com/articles/3360 last-modified: 2017-05-23 description: Climate change is predicted to lead to more extreme weather events, including changes to storm frequency, intensity and location. Yet the ecological responses to storms are incompletely understood for sandy shorelines, the globe’s longest land-ocean interface. Here we document how storms of different magnitude impacted the invertebrate assemblages on a tidal flat in Brazil. We specifically tested the relationships between wave energy and spatial heterogeneity, both for habitat properties (habitat heterogeneity) and fauna (β-diversity), predicting that larger storms redistribute sediments and hence lead to spatially less variable faunal assemblages. The sediment matrix tended to become less heterogeneous across the flat after high-energy wave events, whereas β-diversity increased after storms. This higher β-diversity was primarily driven by species losses. Significantly fewer species at a significantly lower density occurred within days to weeks after storms. Negative density and biomass responses to storm events were most prominent in crustaceans. Invertebrate assemblages appeared to recover within a short time (weeks to months) after storms, highlighting that most species typical of sedimentary shorelines are, to some degree, resilient to short-term changes in wave energy. Given that storm frequency and intensity are predicted to change in the coming decades, identifying properties that determine resilience and recovery of ecosystems constitute a research priority for sedimentary shorelines and beyond. creator: Guilherme N. Corte creator: Thomas A. Schlacher creator: Helio H. Checon creator: Carlos A.M. Barboza creator: Eduardo Siegle creator: Ross A. Coleman creator: Antonia Cecília Z. Amaral uri: https://doi.org/10.7717/peerj.3360 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Corte et al. title: Factors affecting genotyping success in giant panda fecal samples link: https://peerj.com/articles/3358 last-modified: 2017-05-23 description: Fecal samples play an important role in giant panda conservation studies. Optimal preservation conditions and choice of microsatellites for giant panda fecal samples have not been established. In this study, we evaluated the effect of four factors (namely, storage type (ethanol (EtOH), EtOH −20 °C, 2-step storage medium, DMSO/EDTA/Tris/salt buffer (DETs) and frozen at −20 °C), storage time (one, three and six months), fragment length, and repeat motif of microsatellite loci) on the success rate of microsatellite amplification, allelic dropout (ADO) and false allele (FA) rates from giant panda fecal samples. Amplification success and ADO rates differed between the storage types. Freezing was inferior to the other four storage methods based on the lowest average amplification success and the highest ADO rates (P < 0.05). The highest microsatellite amplification success was obtained from either EtOH or the 2-step storage medium at three storage time points. Storage time had a negative effect on the average amplification of microsatellites and samples stored in EtOH and the 2-step storage medium were more stable than the other three storage types. We only detected the effect of repeat motif on ADO and FA rates. The lower ADO and FA rates were obtained from tri- and tetra-nucleotide loci. We suggest that freezing should not be used for giant panda fecal preservation in microsatellite studies, and EtOH and the 2-step storage medium should be chosen on priority for long-term storage. We recommend candidate microsatellite loci with longer repeat motif to ensure greater genotyping success for giant panda fecal studies. creator: Ying Zhu creator: Hong-Yi Liu creator: Hai-Qiong Yang creator: Yu-Dong Li creator: He-Min Zhang uri: https://doi.org/10.7717/peerj.3358 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Zhu et al. title: The metabolic profile of a rat model of chronic kidney disease link: https://peerj.com/articles/3352 last-modified: 2017-05-23 description: BackgroundThe kidney is always subjected to high metabolic demand. The aim of this study was to characterize metabolic profiles of a rat model of chronic kidney disease (CKD) with cardiorenal syndrome (CRS) induced by prolonged hypertension.MethodsWe used inbred male Dahl salt-sensitive (DS) rats fed an 8% NaCl diet from six weeks of age (high-salt; HS group) or a 0.3% NaCl diet as controls (low-salt; LS group). We analyzed function, pathology, metabolome, and the gene expression related to energy metabolism of the kidney.ResultsDS rats with a high-salt diet showed hypertension at 11 weeks of age and elevated serum levels of creatinine and blood urea nitrogen with heart failure at 21 weeks of age. The fibrotic area in the kidneys increased at 21 weeks of age. In addition, gene expression related to mitochondrial function was largely decreased. The levels of citrate and isocitrate increased and the gene expression of alpha-ketoglutaratedehydrogenase and succinyl-CoA synthetase decreased; these are enzymes that metabolize citrate and isocitrate, respectively. In addition, the levels of succinate and acetyl Co-A, both of which are metabolites of the tricarboxylic acid (TCA) cycle, decreased.ConclusionsDS rats fed a high-salt diet were deemed a suitable model of CKD with CRS. Gene expression and metabolites related to energy metabolism and mitochondria in the kidney significantly changed in DS rats with hypertension in accordance with the progression of renal injury. creator: Yohei Tanada creator: Junji Okuda creator: Takao Kato creator: Eri Minamino-Muta creator: Ichijiro Murata creator: Tomoyoshi Soga creator: Tetsuo Shioi creator: Takeshi Kimura uri: https://doi.org/10.7717/peerj.3352 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Tanada et al. title: Construct validity and test–retest reliability of the International Fitness Scale (IFIS) in Colombian children and adolescents aged 9–17.9 years: the FUPRECOL study link: https://peerj.com/articles/3351 last-modified: 2017-05-23 description: BackgroundThere is a lack of instruments and studies written in Spanish evaluating physical fitness, impeding the determination of the current status of this important health indicator in the Latin population, especially in Colombia. The aim of the study was two-fold: to examine the validity of the International Fitness Scale (IFIS) with a population-based sample of schoolchildren from Bogota, Colombia and to examine the reliability of the IFIS with children and adolescents from Engativa, Colombia.MethodsThe sample comprised 1,873 Colombian youths (54.5% girls) aged 9–17.9 years. We measured their adiposity markers (waist-to-height ratio, skinfold thickness, percentage of body fat and body mass index), blood pressure, lipids profile, fasting glucose, and physical fitness level (self-reported and measured). A validated cardiometabolic risk index score was also used. An age- and sex-matched subsample of 229 schoolchildren who were not originally included in the sample completed the IFIS twice for reliability purposes.ResultsOur data suggest that both measured and self-reported overall physical fitness levels were inversely associated with percentage of body fat indicators and the cardiometabolic risk index score. Overall, schoolchildren who self-reported “good” or “very good” fitness had better measured fitness levels than those who reported “very poor/poor” fitness (all p < 0.001). The test-retest reliability of the IFIS items was also good, with an average weighted kappa of 0.811.DiscussionOur findings suggest that self-reported fitness, as assessed by the IFIS, is a valid, reliable, and health-related measure. Furthermore, it can be a good alternative for future use in large studies with Latin schoolchildren from Colombia. creator: Robinson Ramírez-Vélez creator: Sandra Milena Cruz-Salazar creator: Myriam Martínez creator: Eduardo L. Cadore creator: Alicia M. Alonso-Martinez creator: Jorge E. Correa-Bautista creator: Mikel Izquierdo creator: Francisco B. Ortega creator: Antonio García-Hermoso uri: https://doi.org/10.7717/peerj.3351 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Ramírez-Vélez et al. title: Effects of arginine vasopressin on the urine proteome in rats link: https://peerj.com/articles/3350 last-modified: 2017-05-23 description: Biomarkers are the measurable changes associated with a physiological or pathophysiological process. The content of urine frequently changes because it is not controlled by homeostatic mechanisms, and these alterations can be a source of biomarkers. However, urine is affected by many factors. In this study, vasoconstrictor and antidiuretic arginine vasopressin (AVP) were infused into rats using an osmotic pump. The rats’ urinary proteome after one week of infusion was analyzed by label-free LC-MS/MS. A total of 408 proteins were identified; among these proteins, eight and 10 proteins had significantly altered expression in the low and high dose groups, respectively, compared with the control group using the one-way ANOVA analysis followed by post hoc analysis with the least significant difference (LSD) test or Dunnett’s T3 test. Three differential proteins were described in prior studies as related to AVP physiological processes, and nine differential proteins are known disease biomarkers. Sixteen of the 17 differential proteins have human orthologs. These results suggest that we should consider the effects of AVP on urinary proteins in future urinary disease biomarker researches. The study data provide clues regarding underlying mechanisms associated with AVP for future physiological researches on AVP. This study provide a sensitive changes associated with AVP. However, the limitation of this result is that the candidate biomarkers should be further verified and filtered. Large clinical samples must be examined to verify the differential proteins identified in this study before these proteins are used as biomarkers for pathological AVP increased diseases, such as syndrome of inappropriate antidiuretic hormone secretion (SIADH). creator: Manxia An creator: Yanying Ni creator: Xundou Li creator: Youhe Gao uri: https://doi.org/10.7717/peerj.3350 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 An et al. title: Effectiveness of coral relocation as a mitigation strategy in Kāne‘ohe Bay, Hawai‘i link: https://peerj.com/articles/3346 last-modified: 2017-05-23 description: Coral reef restoration and management techniques are in ever-increasing demand due to the global decline of coral reefs in the last several decades. Coral relocation has been established as an appropriate restoration technique in select cases, particularly where corals are scheduled for destruction. However, continued long-term monitoring of recovery of transplanted corals is seldom sustained. Removal of coral from a navigation channel and relocation to a similar nearby dredged site occurred in 2005. Coral recovery at the donor site and changes in fish populations at the receiving site were tracked periodically over the following decade. Coral regrowth at the donor site was rapid until a recent bleaching event reduced coral cover by more than half. The transplant of mature colonies increased spatial complexity at the receiving site, immediately increasing fish biomass, abundance, and species that was maintained throughout subsequent surveys. Our research indicates that unlike the majority of historical accounts of coral relocation in the Pacific, corals transplanted into wave-protected areas with similar conditions as the original site can have high survival rates. Data on long-term monitoring of coral transplants in diverse environments is central in developing management and mitigation strategies. creator: Ku’ulei S. Rodgers creator: Koi Lorance creator: Angela Richards Donà creator: Yuko Stender creator: Claire Lager creator: Paul L. Jokiel uri: https://doi.org/10.7717/peerj.3346 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Rodgers et al. title: A retrospective analysis of postoperative hypokalemia in pituitary adenomas after transsphenoidal surgery link: https://peerj.com/articles/3337 last-modified: 2017-05-23 description: BackgroundPituitary adenoma is one of the most common intracranial neoplasms, and its primary treatment is endoscopic endonasal transsphenoidal tumorectomy. Postoperative hypokalemia in these patients is a common complication, and is associated with morbidity and mortality. This study aimed to analyze the etiopathology of postoperative hypokalemia in pituitary adenomas after endoscopic transsphenoidal surgery.Methods and MaterialsThis retrospective study included 181 pituitary adenomas confirmed by histopathology. Unconditional logistic regression analysis was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Repeated measures ANOVA was used to analyze change in serum potassium levels at different time points.ResultsMultiple Logistic regression analysis revealed that only ACTH-pituitary adenoma (OR = 4.92, 95% CI [1.18–20.48], P = 0.029) had a significant association with postoperative hypokalemia. Moreover, the overall mean serum potassium concentration was significantly lower in the ACTH versus the non-ACTH group (3.34 mmol/L vs. 3.79 mmol/L, P = 0.001). Postoperative hypokalemia was predominantly found in patients with ACTH-pituitary adenoma (P = 0.033).ConclusionsACTH-pituitary adenomas may be an independent factor related postoperative hypokalemia in patients despite conventional potassium supplementation in the immediate postoperative period. creator: Lili You creator: Wenpeng Li creator: Tang Chen creator: Dongfang Tang creator: Jinliang You creator: Xianfeng Zhang uri: https://doi.org/10.7717/peerj.3337 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 You et al. title: Sports training enhances visuo-spatial cognition regardless of open-closed typology link: https://peerj.com/articles/3336 last-modified: 2017-05-23 description: The aim of this study was to investigate the effects of open and closed sport participation on visuo-spatial attention and memory performance among young adults. Forty-eight young adults—16 open-skill athletes, 16 closed-skill athletes, and 16 non-athletes controls—were recruited for the study. Both behavioral performance and event-related potential (ERP) measurement were assessed when participants performed non-delayed and delayed match-to-sample task that tested visuo-spatial attention and memory processing. Results demonstrated that regardless of training typology, the athlete groups exhibited shorter reaction times in both the visuo-spatial attention and memory conditions than the control group with no existence of speed-accuracy trade-off. Similarly, a larger P3 amplitudes were observed in both athlete groups than in the control group for the visuo-spatial memory condition. These findings suggest that sports training, regardless of typology, are associated with superior visuo-spatial attention and memory performance, and more efficient neural resource allocation in memory processing. creator: Ting-Yu Chueh creator: Chung-Ju Huang creator: Shu-Shih Hsieh creator: Kuan-Fu Chen creator: Yu-Kai Chang creator: Tsung-Min Hung uri: https://doi.org/10.7717/peerj.3336 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Chueh et al. title: pathVar: a new method for pathway-based interpretation of gene expression variability link: https://peerj.com/articles/3334 last-modified: 2017-05-23 description: Identifying the pathways that control a cellular phenotype is the first step to building a mechanistic model. Recent examples in developmental biology, cancer genomics, and neurological disease have demonstrated how changes in the variability of gene expression can highlight important genes that are under different degrees of regulatory control. Simple statistical tests exist to identify differentially-variable genes; however, methods for investigating how changes in gene expression variability in the context of pathways and gene sets are under-explored. Here we present pathVar, a new method that provides functional interpretation of gene expression variability changes at the level of pathways and gene sets. pathVar is based on a multinomial exact test, or an asymptotic Chi-squared test as a more computationally-efficient alternative. The method can be used for gene expression studies from any technology platform in all biological settings either with a single phenotypic group, or two-group comparisons. To demonstrate its utility, we applied the method to a diverse set of diseases, species and samples. Results from pathVar are benchmarked against analyses based on average expression and two methods of GSEA, and demonstrate that analyses using both statistics are useful for understanding transcriptional regulation. We also provide recommendations for the choice of variability statistic that have been informed through analyses on simulations and real data. Based on the datasets selected, we show how pathVar can be used to gain insight into expression variability of single cell versus bulk samples, different stem cell populations, and cancer versus normal tissue comparisons. creator: Laurence de Torrente creator: Samuel Zimmerman creator: Deanne Taylor creator: Yu Hasegawa creator: Christine A. Wells creator: Jessica C. Mar uri: https://doi.org/10.7717/peerj.3334 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 de Torrente et al. title: Comparison in antioxidant and antitumor activities of pine polyphenols and its seven biotransformation extracts by fungi link: https://peerj.com/articles/3264 last-modified: 2017-05-23 description: Microbial transformation can strengthen the antioxidant and antitumor activities of polyphenols. Polyphenols contents, antioxidant and antitumor activities of pine polyphenols and its biotransformation extracts by Aspergillus niger, Aspergillus oryzae, Aspergillus carbonarius, Aspergillus candidus, Trichodermas viride, Mucor wutungkiao and Rhizopus sp were studied. Significant differences were noted in antioxidant and antitumor activities. The highest antioxidant activities in Trolox equivalent antioxidant capacity (TEAC), DPPH radical scavenging activity, superoxide anion radical scavenging activity, hydroxyl radical scavenging activity, reducing power assay and antitumor activity against LoVo cells were biotransformation extract of Aspergillus carbonarius (BAC), biotransformation extract of Mucor wutungkiao (BMW), biotransformation extract of Aspergillus carbonarius (BAC), biotransformation extract of Aspergillus niger (BAN), biotransformation extract of Aspergillus oryzae (BAO) and BMW, respectively. Correlation analysis found that antioxidant and antitumor activities were associated with polyphenols contents and types of free radicals and tumors. A. carbonarius can make polyphenol oxidation, hydroxylation and methylation, and form new polyphenols. In conclusion, A. carbonarius, A. niger and M. wutungkiao are valuable microorganisms used for polyphenols biotransformation and enhance the antioxidant and antitumor activities of polyphenols. creator: Hui Li creator: Zhenyu Wang uri: https://doi.org/10.7717/peerj.3264 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Li and Wang title: A mixed methods study of multiple health behaviors among individuals with stroke link: https://peerj.com/articles/3210 last-modified: 2017-05-23 description: BackgroundIndividuals with stroke often have multiple cardiovascular risk factors that necessitate promoting engagement in multiple health behaviors. However, observational studies of individuals with stroke have typically focused on promoting a single health behavior. Thus, there is a poor understanding of linkages between healthy behaviors and the circumstances in which factors, such as stroke impairments, may influence a single or multiple health behaviors.MethodsWe conducted a mixed methods convergent parallel study of 25 individuals with stroke to examine the relationships between stroke impairments and physical activity, sleep, and nutrition. Our goal was to gain further insight into possible strategies to promote multiple health behaviors among individuals with stroke. This study focused on physical activity, sleep, and nutrition because of their importance in achieving energy balance, maintaining a healthy weight, and reducing cardiovascular risks. Qualitative and quantitative data were collected concurrently, with the former being prioritized over the latter. Qualitative data was prioritized in order to develop a conceptual model of engagement in multiple health behaviors among individuals with stroke. Qualitative and quantitative data were analyzed independently and then were integrated during the inference stage to develop meta-inferences. The 25 individuals with stroke completed closed-ended questionnaires on healthy behaviors and physical function. They also participated in face-to-face focus groups and one-to-one phone interviews.ResultsWe found statistically significant and moderate correlations between hand function and healthy eating habits (r = 0.45), sleep disturbances and limitations in activities of daily living (r =  − 0.55), BMI and limitations in activities of daily living (r =  − 0.49), physical activity and limitations in activities of daily living (r = 0.41), mobility impairments and BMI (r =  − 0.41), sleep disturbances and physical activity (r =  − 0.48), sleep disturbances and BMI (r = 0.48), and physical activity and BMI (r =  − 0.45). We identified five qualitative themes: (1) Impairments: reduced autonomy, (2) Environmental forces: caregivers and information, (3) Re-evaluation: priorities and attributions, (4) Resiliency: finding motivation and solutions, and (5) Negative affectivity: stress and self-consciousness. Three meta-inferences and a conceptual model described circumstances in which factors could influence single or multiple health behaviors.DiscussionThis is the first mixed methods study of individuals with stroke to elaborate on relationships between multiple health behaviors, BMI, and physical function. A conceptual model illustrates addressing sleep disturbances, activity limitations, self-image, and emotions to promote multiple health behaviors. We discuss the relevance of the meta-inferences in designing multiple behavior change interventions for individuals with stroke. creator: Matthew Plow creator: Shirley M. Moore creator: Martha Sajatovic creator: Irene Katzan uri: https://doi.org/10.7717/peerj.3210 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Plow et al. title: A comparison of freezing-damage during isochoric and isobaric freezing of the potato link: https://peerj.com/articles/3322 last-modified: 2017-05-18 description: BackgroundFreezing is commonly used for food preservation. It is usually done under constant atmospheric pressure (isobaric). While extending the life of the produce, isobaric freezing has detrimental effects. It causes loss of food weight and changes in food quality. Using thermodynamic analysis, we have developed a theoretical model of the process of freezing in a constant volume system (isochoric). The mathematical model suggests that the detrimental effects associated with isobaric freezing may be reduced in an isochoric freezing system. To explore this hypothesis, we performed a preliminary study on the isochoric freezing of a produce with which our group has experience, the potato.MethodExperiments were performed in an isochoric freezing device we designed. The device is robust and has no moving parts. For comparison, we used a geometrically identical isobaric freezing device. Following freezing and thawing, the samples were weighed, examined with colorimetry, and examined with microscopy.ResultsIt was found that potatoes frozen to −5 °C in an isochoric system experienced no weight loss and limited enzymatic browning. In contrast the −5 °C isobaric frozen potato experienced substantial weight loss and substantial enzymatic browning. Microscopic analysis shows that the structural integrity of the potato is maintained after freezing in the isochoric system and impaired after freezing in the isobaric system.DiscussionTissue damage during isobaric freezing is caused by the increase in extracellular osmolality and the mechanical damage by ice crystals. Our thermodynamic analysis predicts that during isochoric freezing the intracellular osmolality remains comparable to the extracellular osmolality and that isochoric systems can be designed to eliminate the mechanical damage by ice. The results of this preliminary study seem to confirm the theoretical predictions.ConclusionThis is a preliminary exploratory study on isochoric freezing of food. We have shown that the quality of a food product preserved by isochoric freezing is better than the quality of food preserved to the same temperature in isobaric conditions. Obviously, more extensive research remains to be done to extend this study to lower freezing temperatures and other food items. creator: Chenang Lyu creator: Gabriel Nastase creator: Gideon Ukpai creator: Alexandru Serban creator: Boris Rubinsky uri: https://doi.org/10.7717/peerj.3322 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Lyu et al. title: StrainSeeker: fast identification of bacterial strains from raw sequencing reads using user-provided guide trees link: https://peerj.com/articles/3353 last-modified: 2017-05-18 description: BackgroundFast, accurate and high-throughput identification of bacterial isolates is in great demand. The present work was conducted to investigate the possibility of identifying isolates from unassembled next-generation sequencing reads using custom-made guide trees.ResultsA tool named StrainSeeker was developed that constructs a list of specific k-mers for each node of any given Newick-format tree and enables the identification of bacterial isolates in 1–2 min. It uses a novel algorithm, which analyses the observed and expected fractions of node-specific k-mers to test the presence of each node in the sample. This allows StrainSeeker to determine where the isolate branches off the guide tree and assign it to a clade whereas other tools assign each read to a reference genome. Using a dataset of 100 Escherichia coli isolates, we demonstrate that StrainSeeker can predict the clades of E. coli with 92% accuracy and correct tree branch assignment with 98% accuracy. Twenty-five thousand Illumina HiSeq reads are sufficient for identification of the strain.ConclusionStrainSeeker is a software program that identifies bacterial isolates by assigning them to nodes or leaves of a custom-made guide tree. StrainSeeker’s web interface and pre-computed guide trees are available at http://bioinfo.ut.ee/strainseeker. Source code is stored at GitHub: https://github.com/bioinfo-ut/StrainSeeker. creator: Märt Roosaare creator: Mihkel Vaher creator: Lauris Kaplinski creator: Märt Möls creator: Reidar Andreson creator: Maarja Lepamets creator: Triinu Kõressaar creator: Paul Naaber creator: Siiri Kõljalg creator: Maido Remm uri: https://doi.org/10.7717/peerj.3353 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Roosaare et al. title: Exoskeleton may influence the internal body temperatures of Neotropical dung beetles (Col. Scarabaeinae) link: https://peerj.com/articles/3349 last-modified: 2017-05-18 description: The insect exoskeleton is a multifunctional coat with a continuum of mechanical and structural properties constituting the barrier between electromagnetic waves and the internal body parts. This paper examines the ability of beetle exoskeleton to regulate internal body temperature considering its thermal permeability or isolation to simulated solar irradiance and infrared radiation. Seven Neotropical species of dung beetles (Coleoptera, Scarabaeinae) differing in colour, surface sculptures, size, sexual dimorphism, period of activity, guild category and altitudinal distribution were studied. Specimens were repeatedly subjected to heating trials under simulated solar irradiance and infrared radiation using a halogen neodymium bulb light with a balanced daylight spectrum and a ceramic infrared heat emitter. The volume of exoskeleton and its weight per volume unit were significantly more important for the heating rate at the beginning of the heating process than for the asymptotic maximum temperature reached at the end of the trials: larger beetles with relatively thicker exoskeletons heated more slowly. The source of radiation greatly influences the asymptotic temperature reached, but has a negligible effect in determining the rate of heat gain by beetles: they reached higher temperatures under artificial sunlight than under infrared radiation. Interspecific differences were negligible in the heating rate but had a large magnitude effect on the asymptotic temperature, only detectable under simulated sun irradiance. The fact that sun irradiance is differentially absorbed dorsally and transformed into heat among species opens the possibility that differences in dorsal exoskeleton would facilitate the heat gain under restrictive environmental temperatures below the preferred ones. The findings provided by this study support the important role played by the exoskeleton in the heating process of beetles, a cuticle able to act passively in the thermal control of body temperature without implying energetic costs and metabolic changes. creator: Valentina Amore creator: Malva I.M. Hernández creator: Luis M. Carrascal creator: Jorge M. Lobo uri: https://doi.org/10.7717/peerj.3349 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Amore et al. title: Atorvastatin alters the expression of genes related to bile acid metabolism and circadian clock in livers of mice link: https://peerj.com/articles/3348 last-modified: 2017-05-18 description: AimAtorvastatin is a HMG-CoA reductase inhibitor used for hyperlipidemia. Atorvastatin is generally safe but may induce cholestasis. The present study aimed to examine the effects of atorvastatin on hepatic gene expression related to bile acid metabolism and homeostasis, as well as the expression of circadian clock genes in livers of mice.MethodsAdult male mice were given atorvastatin (10, 30, and 100 mg/kg, po) daily for 30 days, and blood biochemistry, histopathology, and gene expression were examined.ResultsRepeated administration of atorvastatin did not affect animal body weight gain or liver weights. Serum enzyme activities were in the normal range. Histologically, the high dose of atorvastatin produced scattered swollen hepatocytes, foci of feathery-like degeneration, together with increased expression of Egr-1 and metallothionein-1. Atorvastatin increased the expression of Cyp7a1 in the liver, along with FXR and SHP. In contract, atorvastatin decreased the expression of bile acid transporters Ntcp, Bsep, Ostα, and Ostβ. The most dramatic change was the 30-fold induction of Cyp7a1. Because Cyp7a1 is a circadian clock-controlled gene, we further examined the effect of atorvastatin on clock gene expression. Atorvastatin increased the expression of clock core master genes Bmal1 and Npas2, decreased the expression of clock feedback genes Per2, Per3, and the clock targeted genes Dbp and Tef, whereas it had no effect on Cry1 and Nr1d1 expression.ConclusionRepeated administration of atorvastatin affects bile acid metabolism and markedly increases the expression of the bile acid synthesis rate-limiting enzyme gene Cyp7a1, together with alterations in the expression of circadian clock genes. creator: Wen-Kai Li creator: Huan Li creator: Yuan-Fu Lu creator: Ying-Ying Li creator: Zidong Donna Fu creator: Jie Liu uri: https://doi.org/10.7717/peerj.3348 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Li et al. title: Predictive ability of the Mini Nutritional Assessment Short Form (MNA-SF) in a free-living elderly population: a cross-sectional study link: https://peerj.com/articles/3345 last-modified: 2017-05-18 description: BackgroundVarious scales have been used to perform a quick and first level nutritional assessment, and the MNA is one of the most used and recommended by experts in the elderly in all areas. This scale has a short form, the MNA-SF, revised and validated in 2009, which has two versions: the BMI-MNA-SF contains the first six items of the full scale including Body Mass Index while the CC-MNA-SF includes Calf Circumference instead of BMI.ObjectiveTo evaluate the predictive ability for nutritional status of the two versions of the MNA-SF against the MNA in free-living elderly in the province of Valencia.MethodsCross-sectional study of 660 free-living elderly in the province of Valencia selected in 12 community centres using stratified sampling by blocks. Inclusion criteria: being aged 65 or over, living at home, having functional autonomy, residing in the province of study for more than one year, regularly attending community centres and voluntarily wanting to take part.ResultsOf the 660 subjects studied, 319 were men (48.3%) and 341 (51.7%) women with a mean age of 74.3 years (SD = 6.6). In terms of nutritional assessment, using the BMI-MNA-SF and the CC-MNA-SF we found that 26.5% and 26.2% were at risk of malnutrition and 0.9% and 1.5% were malnourished respectively. With the full MNA, 23.3% were at risk of malnutrition. Spearman’s rank correlation coefficients indicate a high association between the full MNA score and the MNA-SFs scores (BMI-MNA-SF: ρ = 0.78p < 0.001; CC-MNA-SF: ρ = 0.78p < 0.001). In addition we obtained a very high correlation between the two MNA-SFs (ρ = 0.96p < 0.001). We evaluated the agreement between the full MNA and the MNA-SFs classification in three nutritional categories (normal nutritional status, risk of malnutrition, malnutrition) with Cohen’s kappa coefficients (BMI-MNA-SF: κ = 0.54p < 0.001; CC-MNA-SF: κ = 0.52p < 0.001). These values indicate moderate agreement with the full MNA. There is very good agreement between the BMI-MNA-SF and CC-MNA-SF (κ = 0.88p < 0.001). In order to determine the ability of both MNA-SFs to identify subjects not requiring any nutritional intervention, we considered the dichotomised categorisation of the full MNA and the MNA-SFs as “normal nutritional status” vs. “malnutrition and risk of malnutrition” Areas under the ROC curves using MNA as the gold standard indicate moderately high prognostic accuracy (BMI-MNA-SF: AUC = 0.88p < 0.001; CC-MNA-SF: AUC = 0.87 p < 0.001). Both versions of the MNA-SF showed similar sensitivity, specificity and diagnostic effectiveness (BMI-MNA-SF: 73.4%, 86.6%, 83.5%; CC-MNA-SF 73.4%, 86.2%, 83.2%).ConclusionsIn its two versions the MNA-SF presents useful predictive ability against the MNA. The advantage of the CC-MNA-SF is that using it requires fewer resources and less time in primary care, although always the characteristics of the population must take into account to make the right decision based on the MNA-SF scales. creator: Raimunda Montejano Lozoya creator: Nieves Martínez-Alzamora creator: Gonzalo Clemente Marín creator: Silamani J.A. Guirao-Goris creator: Rosa María Ferrer-Diego uri: https://doi.org/10.7717/peerj.3345 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Montejano Lozoya et al. title: Comparative transcriptome analyses of a late-maturing mandarin mutant and its original cultivar reveals gene expression profiling associated with citrus fruit maturation link: https://peerj.com/articles/3343 last-modified: 2017-05-18 description: Characteristics of late maturity in fruit are good agronomic traits for extending the harvest period and marketing time. However, underlying molecular basis of the late-maturing mechanism in fruit is largely unknown. In this study, RNA sequencing (RNA-Seq) technology was used to identify differentially expressed genes (DEGs) related to late-maturing characteristics from a late-maturing mutant ‘Huawan Wuzishatangju’ (HWWZSTJ) (Citrus reticulata Blanco) and its original line ‘Wuzishatangju’ (WZSTJ). A total of approximately 17.0 Gb and 84.2 M paried-end reads were obtained. DEGs were significantly enriched in the pathway of photosynthesis, phenylpropanoid biosynthesis, carotenoid biosynthesis, chlorophyll and abscisic acid (ABA) metabolism. Thirteen candidate transcripts related to chlorophyll metabolism, carotenoid biosynthesis and ABA metabolism were analyzed using real-time quantitative PCR (qPCR) at all fruit maturing stages of HWWZSTJ and WZSTJ. Chlorophyllase (CLH) and divinyl reductase (DVR) from chlorophyll metabolism, phytoene synthase (PSY) and capsanthin/capsorubin synthase (CCS) from carotenoid biosynthesis, and abscisic acid 8′-hydroxylase (AB1) and 9-cis-epoxycarotenoid dioxygenase (NCED1) from ABA metabolism were cloned and analyzed. The expression pattern of NCED1 indicated its role in the late-maturing characteristics of HWWZSTJ. There were 270 consecutive bases missing in HWWZSTJ in comparison with full-length sequences of NCED1 cDNA from WZSTJ. Those results suggested that NCED1 might play an important role in the late maturity of HWWZSTJ. This study provides new information on complex process that results in the late maturity of Citrus fruit at the transcriptional level. creator: Lu Wang creator: Qingzhu Hua creator: Yuewen Ma creator: Guibing Hu creator: Yonghua Qin uri: https://doi.org/10.7717/peerj.3343 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Wang et al. title: Lid opening and conformational stability of T1 Lipase is mediated by increasing chain length polar solvents link: https://peerj.com/articles/3341 last-modified: 2017-05-18 description: The dynamics and conformational landscape of proteins in organic solvents are events of potential interest in nonaqueous process catalysis. Conformational changes, folding transitions, and stability often correspond to structural rearrangements that alter contacts between solvent molecules and amino acid residues. However, in nonaqueous enzymology, organic solvents limit stability and further application of proteins. In the present study, molecular dynamics (MD) of a thermostable Geobacillus zalihae T1 lipase was performed in different chain length polar organic solvents (methanol, ethanol, propanol, butanol, and pentanol) and water mixture systems to a concentration of 50%. On the basis of the MD results, the structural deviations of the backbone atoms elucidated the dynamic effects of water/organic solvent mixtures on the equilibrium state of the protein simulations in decreasing solvent polarity. The results show that the solvent mixture gives rise to deviations in enzyme structure from the native one simulated in water. The drop in the flexibility in H2O, MtOH, EtOH and PrOH simulation mixtures shows that greater motions of residues were influenced in BtOH and PtOH simulation mixtures. Comparing the root mean square fluctuations value with the accessible solvent area (SASA) for every residue showed an almost correspondingly high SASA value of residues to high flexibility and low SASA value to low flexibility. The study further revealed that the organic solvents influenced the formation of more hydrogen bonds in MtOH, EtOH and PrOH and thus, it is assumed that increased intraprotein hydrogen bonding is ultimately correlated to the stability of the protein. However, the solvent accessibility analysis showed that in all solvent systems, hydrophobic residues were exposed and polar residues tended to be buried away from the solvent. Distance variation of the tetrahedral intermediate packing of the active pocket was not conserved in organic solvent systems, which could lead to weaknesses in the catalytic H-bond network and most likely a drop in catalytic activity. The conformational variation of the lid domain caused by the solvent molecules influenced its gradual opening. Formation of additional hydrogen bonds and hydrophobic interactions indicates that the contribution of the cooperative network of interactions could retain the stability of the protein in some solvent systems. Time-correlated atomic motions were used to characterize the correlations between the motions of the atoms from atomic coordinates. The resulting cross-correlation map revealed that the organic solvent mixtures performed functional, concerted, correlated motions in regions of residues of the lid domain to other residues. These observations suggest that varying lengths of polar organic solvents play a significant role in introducing dynamic conformational diversity in proteins in a decreasing order of polarity. creator: Jonathan Maiangwa creator: Mohd Shukuri Mohamad Ali creator: Abu Bakar Salleh creator: Raja Noor Zaliha Raja Abd Rahman creator: Yahaya M. Normi creator: Fairolniza Mohd Shariff creator: Thean Chor Leow uri: https://doi.org/10.7717/peerj.3341 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Maiangwa et al. title: Effects of knee sleeves on coordination of lower-limb segments in healthy adults during level walking and one-leg hopping link: https://peerj.com/articles/3340 last-modified: 2017-05-18 description: The evaluation of multisegment coordination is important in gaining a better understanding of the gait and physical activities in humans. Therefore, this study aims to verify whether the use of knee sleeves affects the coordination of lower-limb segments during level walking and one-leg hopping. Eleven healthy male adults participated in this study. They were asked to walk 10 m on a level ground and perform one-leg hops with and without a knee sleeve. The segment angles and the response velocities of the thigh, shank, and foot were measured and calculated by using a motion analysis system. The phases between the segment angle and the velocity were then calculated. Moreover, the continuous relative phase (CRP) was calculated as the phase of the distal segment subtracted from the phase of the proximal segment and denoted as CRPTS (thigh–shank), CRPSF (shank–foot), and CRPTF (thigh–foot). The root mean square (RMS) values were used to evaluate the in-phase or out-of-phase states, while the standard deviation (SD) values were utilized to evaluate the variability in the stance and swing phases during level walking and in the preflight, flight, and landing phases during one-leg hopping. The walking velocity and the flight time improved when the knee sleeve was worn (p < 0.05). The segment angles of the thigh and shank also changed when the knee sleeve was worn during level walking and one-leg hopping. The RMS values of CRPTS and CRPSF in the stance phase and the RMS values of CRPSF in the preflight and landing phases changed (p < 0.05 in all cases). Moreover, the SD values of CRPTS in the landing phase and the SD values of CRPSF in the preflight and landing phases increased (p < 0.05 in all cases). These results indicated that wearing a knee sleeve caused changes in segment kinematics and coordination. creator: Chang-Yong Ko creator: Yunhee Chang creator: Bora Jeong creator: Sungjae Kang creator: Jeicheong Ryu creator: Gyoosuk Kim uri: https://doi.org/10.7717/peerj.3340 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Ko et al. title: Mitochondrial complex I deficiency leads to the retardation of early embryonic development in Ndufs4 knockout mice link: https://peerj.com/articles/3339 last-modified: 2017-05-18 description: BackgroundThe NDUFS4 gene encodes an 18-kD subunit of mitochondria complex I, and mutations in this gene lead to the development of a severe neurodegenerative disease called Leigh syndrome (LS) in humans. To investigate the disease phenotypes and molecular mechanisms of Leigh syndrome, the Ndufs4 knockout (KO) mouse has been widely used as a novel animal model. Because the homozygotes cannot survive beyond child-bearing age, whether Ndufs4 and mitochondrial complex I influence early embryonic development remains unknown. In our study, we attempted to investigate embryonic development in Ndufs4 KO mice, which can be regarded as a Leigh disease model and were created through the CRISPR (clustered regularly interspaced short palindromic repeat) and Cas9 (CRISPR associated)-mediated genome editing system.MethodsWe first designed a single guide RNA (sgRNA) targeting exon 2 of Ndufs4 to delete the NDUFS4 protein in mouse embryos to mimic Leigh syndrome. Then, we described the phenotypes of our mouse model by forced swimming and the open-field test as well as by assessing other behavioral characteristics. Intracytoplasmic sperm injection (ICSI) was performed to obtain KO embryos to test the influence of NDUFS4 deletion on early embryonic development.ResultsIn this study, we first generated Ndufs4 KO mice with physical and behavioral phenotypes similar to Leigh syndrome using the CRISPR/Cas9 system. The low developmental rate of KO embryos that were derived from knockout gametes indicated that the absence of NDUFS4 impaired the development of preimplantation embryos.DiscussionIn this paper, we first obtained Ndufs4 KO mice that could mimic Leigh syndrome using the CRISPR/Cas9 system. Then, we identified the role of NDUFS4 in early embryonic development, shedding light on its roles in the respiratory chain and fertility. Our model provides a useful tool with which to investigate the function of Ndufs4. Although the pathological mechanisms of the disease need to be discovered, it helps to understand the pathogenesis of NDUFS4 deficiency in mice and its effects on human diseases. creator: Mei Wang creator: Ya-Ping Huang creator: Han Wu creator: Ke Song creator: Cong Wan creator: A-Ni Chi creator: Ya-Mei Xiao creator: Xiao-Yang Zhao uri: https://doi.org/10.7717/peerj.3339 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Wang et al. title: A genome-wide assessment of stages of elevational parapatry in Bornean passerine birds reveals no introgression: implications for processes and patterns of speciation link: https://peerj.com/articles/3335 last-modified: 2017-05-18 description: Topographically complex regions often contain the close juxtaposition of closely related species along elevational gradients. The evolutionary causes of these elevational replacements, and thus the origin and maintenance of a large portion of species diversity along elevational gradients, are usually unclear because ecological differentiation along a gradient or secondary contact following allopatric diversification can produce the same pattern. We used reduced representation genomic sequencing to assess genetic relationships and gene flow between three parapatric pairs of closely related songbird taxa (Arachnothera spiderhunters, Chloropsis leafbirds, and Enicurus forktails) along an elevational gradient in Borneo. Each taxon pair presents a different elevational range distribution across the island, yet results were uniform: little or no gene flow was detected in any pairwise comparisons. These results are congruent with an allopatric “species-pump” model for generation of species diversity and elevational parapatry of congeners on Borneo, rather than in situ generation of species by “ecological speciation” along an elevational gradient. creator: Robert G. Moyle creator: Joseph D. Manthey creator: Peter A. Hosner creator: Mustafa Rahman creator: Maklarin Lakim creator: Frederick H. Sheldon uri: https://doi.org/10.7717/peerj.3335 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Moyle et al. title: Ranking of critical species to preserve the functionality of mutualistic networks using the k-core decomposition link: https://peerj.com/articles/3321 last-modified: 2017-05-18 description: BackgroundNetwork analysis has become a relevant approach to analyze cascading species extinctions resulting from perturbations on mutualistic interactions as a result of environmental change. In this context, it is essential to be able to point out key species, whose stability would prevent cascading extinctions, and the consequent loss of ecosystem function. In this study, we aim to explain how the k-core decomposition sheds light on the understanding the robustness of bipartite mutualistic networks.MethodsWe defined three k-magnitudes based on the k-core decomposition: k-radius, k-degree, and k-risk. The first one, k-radius, quantifies the distance from a node to the innermost shell of the partner guild, while k-degree provides a measure of centrality in the k-shell based decomposition. k-risk is a way to measure the vulnerability of a network to the loss of a particular species. Using these magnitudes we analyzed 89 mutualistic networks involving plant pollinators or seed dispersers. Two static extinction procedures were implemented in which k-degree and k-risk were compared against other commonly used ranking indexes, as for example MusRank, explained in detail in Material and Methods.ResultsWhen extinctions take place in both guilds, k-risk is the best ranking index if the goal is to identify the key species to preserve the giant component. When species are removed only in the primary class and cascading extinctions are measured in the secondary class, the most effective ranking index to identify the key species to preserve the giant component is k-degree. However, MusRank index was more effective when the goal is to identify the key species to preserve the greatest species richness in the second class.DiscussionThe k-core decomposition offers a new topological view of the structure of mutualistic networks. The new k-radius, k-degree and k-risk magnitudes take advantage of its properties and provide new insight into the structure of mutualistic networks. The k-risk and k-degree ranking indexes are especially effective approaches to identify key species to preserve when conservation practitioners focus on the preservation of ecosystem functionality over species richness. creator: Javier García-Algarra creator: Juan Manuel Pastor creator: José María Iriondo creator: Javier Galeano uri: https://doi.org/10.7717/peerj.3321 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 García-Algarra et al. title: Divergent evolutionary histories of DNA markers in a Hawaiian population of the coral Montipora capitata link: https://peerj.com/articles/3319 last-modified: 2017-05-18 description: We investigated intra- and inter-colony sequence variation in a population of the dominant Hawaiian coral Montipora capitata by analyzing marker gene and genomic data. Ribosomal ITS1 regions showed evidence of a reticulate history among the colonies, suggesting incomplete rDNA repeat homogenization. Analysis of the mitochondrial genome identified a major (M. capitata) and a minor (M. flabellata) haplotype in single polyp-derived sperm bundle DNA with some colonies containing 2–3 different mtDNA haplotypes. In contrast, Pax-C and newly identified single-copy nuclear genes showed either no sequence differences or minor variations in SNP frequencies segregating among the colonies. Our data suggest past mitochondrial introgression in M. capitata, whereas nuclear single-copy loci show limited variation, highlighting the divergent evolutionary histories of these coral DNA markers. creator: Hollie M. Putnam creator: Diane K. Adams creator: Ehud Zelzion creator: Nicole E. Wagner creator: Huan Qiu creator: Tali Mass creator: Paul G. Falkowski creator: Ruth D. Gates creator: Debashish Bhattacharya uri: https://doi.org/10.7717/peerj.3319 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Putnam et al. title: Transcriptome analyses provide insights into the difference of alkaloids biosynthesis in the Chinese goldthread (Coptis chinensis Franch.) from different biotopes link: https://peerj.com/articles/3303 last-modified: 2017-05-18 description: Coptis chinensis Franch., the Chinese goldthread (‘Weilian’ in Chinese), one of the most important medicinal plants from the family Ranunculaceae, and its rhizome has been widely used in Traditional Chinese Medicine for centuries. Here, we analyzed the chemical components and the transcriptome of the Chinese goldthread from three biotopes, including Zhenping, Zunyi and Shizhu. We built comprehensive, high-quality de novo transcriptome assemblies of the Chinese goldthread from short-read RNA-Sequencing data, obtaining 155,710 transcripts and 56,071 unigenes. More than 98.39% and 95.97% of core eukaryotic genes were found in the transcripts and unigenes respectively, indicating that this unigene set capture the majority of the coding genes. A total of 520,462, 493,718, and 507,247 heterozygous SNPs were identified in the three accessions from Zhenping, Zunyi, and Shizhu respectively, indicating high polymorphism in coding regions of the Chinese goldthread (∼1%). Chemical analyses of the rhizome identified six major components, including berberine, palmatine, coptisine, epiberberine, columbamine, and jatrorrhizine. Berberine has the highest concentrations, followed by coptisine, palmatine, and epiberberine sequentially for all the three accessions. The drug quality of the accession from Shizhu may be the highest among these accessions. Differential analyses of the transcriptome identified four pivotal candidate enzymes, including aspartate aminotransferaseprotein, polyphenol oxidase, primary-amine oxidase, and tyrosine decarboxylase, were significantly differentially expressed and may be responsible for the difference of alkaloids contents in the accessions from different biotopes. creator: Hanting Chen creator: Cao Deng creator: Hu Nie creator: Gang Fan creator: Yang He uri: https://doi.org/10.7717/peerj.3303 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Chen et al. title: Body composition estimation from selected slices: equations computed from a new semi-automatic thresholding method developed on whole-body CT scans link: https://peerj.com/articles/3302 last-modified: 2017-05-18 description: BackgroundEstimating volumes and masses of total body components is important for the study and treatment monitoring of nutrition and nutrition-related disorders, cancer, joint replacement, energy-expenditure and exercise physiology. While several equations have been offered for estimating total body components from MRI slices, no reliable and tested method exists for CT scans. For the first time, body composition data was derived from 41 high-resolution whole-body CT scans. From these data, we defined equations for estimating volumes and masses of total body AT and LT from corresponding tissue areas measured in selected CT scan slices.MethodsWe present a new semi-automatic approach to defining the density cutoff between adipose tissue (AT) and lean tissue (LT) in such material. An intra-class correlation coefficient (ICC) was used to validate the method. The equations for estimating the whole-body composition volume and mass from areas measured in selected slices were modeled with ordinary least squares (OLS) linear regressions and support vector machine regression (SVMR).Results and DiscussionThe best predictive equation for total body AT volume was based on the AT area of a single slice located between the 4th and 5th lumbar vertebrae (L4-L5) and produced lower prediction errors (|PE| = 1.86 liters, %PE = 8.77) than previous equations also based on CT scans. The LT area of the mid-thigh provided the lowest prediction errors (|PE| = 2.52 liters, %PE = 7.08) for estimating whole-body LT volume. We also present equations to predict total body AT and LT masses from a slice located at L4-L5 that resulted in reduced error compared with the previously published equations based on CT scans. The multislice SVMR predictor gave the theoretical upper limit for prediction precision of volumes and cross-validated the results. creator: Alizé Lacoste Jeanson creator: Ján Dupej creator: Chiara Villa creator: Jaroslav Brůžek uri: https://doi.org/10.7717/peerj.3302 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Lacoste Jeanson et al. title: Toxicity, repellency and flushing out in Triatoma infestans (Hemiptera: Reduviidae) exposed to the repellents DEET and IR3535 link: https://peerj.com/articles/3292 last-modified: 2017-05-18 description: DEET and IR3535 are insect repellents present worldwide in commercial products; their efficacy has been mainly evaluated in mosquitoes. This study compares the toxicological effects and the behavioral responses induced by both repellents on the blood-sucking bug Triatoma infestans Klug (Hemiptera: Reduviidae), one of the main vectors of Chagas disease. When applied topically, the Median Lethal Dose (72 h) for DEET was 220.8 µg/insect. Using IR3535, topical application of 500 µg/insect killed no nymphs. The minimum concentration that produced repellency was the same for both compounds: 1,15 µg/cm2. The effect of a mixture DEET:IR3535 1:1 was similar to that of their pure components. Flushing out was assessed in a chamber with a shelter containing groups of ten nymphs. The repellents were aerosolized on the shelter and the number of insects leaving it was recorded for 60 min. During that time, 0.006 g/m3 of the positive control tetramethrin flushed out 76.7% of the nymphs, while 1.76 g/m3 of DEET or IR3535 flushed out 30 and 0%, respectively. The concentrations required for both compounds to produce toxicity or flushing out are too high to have any practical applications. However, they showed a promising repellency. Additional research should be done to evaluate their possible use for personal protection against T. infestans bites. creator: Mercedes M.N. Reynoso creator: Emilia A. Seccacini creator: Javier A. Calcagno creator: Eduardo N. Zerba creator: Raúl A. Alzogaray uri: https://doi.org/10.7717/peerj.3292 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Reynoso et al. title: Face recognition is similarly affected by viewpoint in school-aged children and adults link: https://peerj.com/articles/3253 last-modified: 2017-05-18 description: There is an ongoing debate on the question when face processing abilities mature. One aspect that has been part of this debate is the ability to recognize faces in and across different viewpoints. Here, we tested 128 participants consisting of school-age children (ages, 5–10 years) and adults (ages, 19–37 years) in two experiments to investigate the effects of different viewpoints (including front, three-quarter, profile view) on face recognition during development. Furthermore, we compared recognition performance for faces to that of another object category (cars). In the first experiment (n = 88) we tested if the pattern of performance for faces presented in different viewpoints is similar in school-aged children and adults. Participants completed a two-alternative-forced-choice (2AFC) memory task comprising images of both faces and cars in front, three-quarter and profile view, which were presented in the same viewpoint during learning and testing. In the second experiment (n = 40) we tested if face recognition is similarly affected by viewpoint changes in children and adults. In this experiment the 2AFC memory task included a change of viewpoint between learning and testing. While in both experiments we found higher recognition performance for faces with increasing age, the overall pattern of both viewpoint and viewpoint-change-effects and also the difference between view-change- and no-change-conditions was similar across age groups. In contrast to faces, no viewpoint effects were observed in cars (experiment 1), viewpoint change effects, however, were similar for cars and faces (experiment 2). In sum, our results suggest early maturity of the ability to recognize faces in and across different viewpoints. creator: Marisa Nordt creator: Sarah Weigelt uri: https://doi.org/10.7717/peerj.3253 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Nordt and Weigelt title: Lygistorrhinidae (Diptera: Bibionomorpha: Sciaroidea) in early Eocene Cambay amber link: https://peerj.com/articles/3313 last-modified: 2017-05-17 description: One new genus and three new species of Lygistorrhinidae in early Eocene Cambay amber from India are described, which significantly increases our knowledge about this group in the Eocene. Lygistorrhina indica n. sp. is the oldest fossil known from this extant genus. Indorrhina sahnii n. gen. et sp. shows morphological similarities to each of the two extant genera Lygistorrhina and Asiorrhina. Palaeognoriste orientale is the third species known from a group that has only been recorded from Eocene Baltic amber before. The latter finding reveals faunal links between Cambay amber and the probably slightly younger Baltic amber, adding further evidence that faunal exchange between Europe/Asia and India took place before the formation of Cambay amber. creator: Frauke Stebner creator: Hukam Singh creator: Jes Rust creator: David A. Grimaldi uri: https://doi.org/10.7717/peerj.3313 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Stebner et al. title: Metabarcoding monitoring analysis: the pros and cons of using co-extracted environmental DNA and RNA data to assess offshore oil production impacts on benthic communities link: https://peerj.com/articles/3347 last-modified: 2017-05-17 description: Sequencing environmental DNA (eDNA) is increasingly being used as an alternative to traditional morphological-based identification to characterize biological assemblages and monitor anthropogenic impacts in marine environments. Most studies only assess eDNA which, compared to eRNA, can persist longer in the environment after cell death. Therefore, eRNA may provide a more immediate census of the environment due to its relatively weaker stability, leading some researchers to advocate for the use of eRNA as an additional, or perhaps superior proxy for portraying ecological changes. A variety of pre-treatment techniques for screening eDNA and eRNA derived operational taxonomic units (OTUs) have been employed prior to statistical analyses, including removing singleton taxa (i.e., OTUs found only once) and discarding those not present in both eDNA and eRNA datasets. In this study, we used bacterial (16S ribosomal RNA gene) and eukaryotic (18S ribosomal RNA gene) eDNA- and eRNA-derived data from benthic communities collected at increasing distances along a transect from an oil production platform (Taranaki, New Zealand). Macro-infauna (visual classification of benthic invertebrates) and physico-chemical data were analyzed in parallel. We tested the effect of removing singleton taxa, and removing taxa not present in the eDNA and eRNA libraries from the same environmental sample (trimmed by shared OTUs), by comparing the impact of the oil production platform on alpha- and beta-diversity of the eDNA/eRNA-based biological assemblages, and by correlating these to the morphologically identified macro-faunal communities and the physico-chemical data. When trimmed by singletons, presence/absence information from eRNA data represented the best proxy to detect changes on species diversity for both bacteria and eukaryotes. However, assessment of quantitative beta-diversity from read abundance information of bacteria eRNA did not, contrary to eDNA, reveal any impact from the oil production activity. Overall, the data appeared more robust when trimmed by shared OTUs, showing a greater effect of the platform on alpha- and beta-diversity. Trimming by shared OTUs likely removes taxa derived from legacy DNA and technical artefacts introduced through reverse transcriptase, polymerase-chain-reaction and sequencing. Findings from our scoping study suggest that metabarcoding-based biomonitoring surveys should, if funds, time and expertise allow, be assessed using both eDNA and eRNA products. creator: Olivier Laroche creator: Susanna A. Wood creator: Louis A. Tremblay creator: Gavin Lear creator: Joanne I. Ellis creator: Xavier Pochon uri: https://doi.org/10.7717/peerj.3347 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Laroche et al. title: Foraging behaviour of an egg parasitoid exploiting plant volatiles induced by pentatomids: the role of adaxial and abaxial leaf surfaces link: https://peerj.com/articles/3326 last-modified: 2017-05-17 description: Several phases of herbivorous insect attack including feeding and oviposition are known to induce plant defenses. Plants emit volatiles induced by herbivores to recruit insect parasitoids as an indirect defense strategy. So far, volatiles induced by herbivore walking and their putative role in the foraging behavior of egg parasitoids have not been investigated. In this paper we studied the response of the egg parasitoid Trissolcus basalis toward volatiles emitted by Vicia faba plants as consequence of the walking activity of the host Nezara viridula. Olfactometer bioassays were carried out to evaluate wasp responses to plants in which the abaxial or the adaxial surfaces were subjected to walking or/and oviposition. Results showed that host female walking on the abaxial but not on the adaxial surface caused a repellence effect in T. basalis 24 h after plant treatment. The emission of active volatiles also occurred when the leaf was turned upside-down, indicating a specificity of stress localization. This specificity was supported by the results, which showed that oviposition combined with feeding elicit the induction of plant volatiles, attracting the parasitoid, when the attack occurred on the abaxial surface. Analyses of plant volatile blends showed significant differences between the treatments. creator: Francesca Frati creator: Antonino Cusumano creator: Eric Conti creator: Stefano Colazza creator: Ezio Peri creator: Salvatore Guarino creator: Letizia Martorana creator: Roberto Romani creator: Gianandrea Salerno uri: https://doi.org/10.7717/peerj.3326 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Frati et al. title: Plastomes of the green algae Hydrodictyon reticulatum and Pediastrum duplex (Sphaeropleales, Chlorophyceae) link: https://peerj.com/articles/3325 last-modified: 2017-05-17 description: BackgroundComparative studies of chloroplast genomes (plastomes) across the Chlorophyceae are revealing dynamic patterns of size variation, gene content, and genome rearrangements. Phylogenomic analyses are improving resolution of relationships, and uncovering novel lineages as new plastomes continue to be characterized. To gain further insight into the evolution of the chlorophyte plastome and increase the number of representative plastomes for the Sphaeropleales, this study presents two fully sequenced plastomes from the green algal family Hydrodictyaceae (Sphaeropleales, Chlorophyceae), one from Hydrodictyon reticulatum and the other from Pediastrum duplex.MethodsGenomic DNA from Hydrodictyon reticulatum and Pediastrum duplex was subjected to Illumina paired-end sequencing and the complete plastomes were assembled for each. Plastome size and gene content were characterized and compared with other plastomes from the Sphaeropleales. Homology searches using BLASTX were used to characterize introns and open reading frames (orfs) ≥ 300 bp. A phylogenetic analysis of gene order across the Sphaeropleales was performed.ResultsThe plastome of Hydrodictyon reticulatum is 225,641 bp and Pediastrum duplex is 232,554 bp. The plastome structure and gene order of H. reticulatum and P. duplex are more similar to each other than to other members of the Sphaeropleales. Numerous unique open reading frames are found in both plastomes and the plastome of P. duplex contains putative viral protein genes, not found in other Sphaeropleales plastomes. Gene order analyses support the monophyly of the Hydrodictyaceae and their sister relationship to the Neochloridaceae.DiscussionThe complete plastomes of Hydrodictyon reticulatum and Pediastrum duplex, representing the largest of the Sphaeropleales sequenced thus far, once again highlight the variability in size, architecture, gene order and content across the Chlorophyceae. Novel intron insertion sites and unique orfs indicate recent, independent invasions into each plastome, a hypothesis testable with an expanded plastome investigation within the Hydrodictyaceae. creator: Hilary A. McManus creator: Daniel J. Sanchez creator: Kenneth G. Karol uri: https://doi.org/10.7717/peerj.3325 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 McManus et al. title: Characterisation of false-positive observations in botanical surveys link: https://peerj.com/articles/3324 last-modified: 2017-05-17 description: Errors in botanical surveying are a common problem. The presence of a species is easily overlooked, leading to false-absences; while misidentifications and other mistakes lead to false-positive observations. While it is common knowledge that these errors occur, there are few data that can be used to quantify and describe these errors. Here we characterise false-positive errors for a controlled set of surveys conducted as part of a field identification test of botanical skill. Surveys were conducted at sites with a verified list of vascular plant species. The candidates were asked to list all the species they could identify in a defined botanically rich area. They were told beforehand that their final score would be the sum of the correct species they listed, but false-positive errors counted against their overall grade. The number of errors varied considerably between people, some people create a high proportion of false-positive errors, but these are scattered across all skill levels. Therefore, a person’s ability to correctly identify a large number of species is not a safeguard against the generation of false-positive errors. There was no phylogenetic pattern to falsely observed species; however, rare species are more likely to be false-positive as are species from species rich genera. Raising the threshold for the acceptance of an observation reduced false-positive observations dramatically, but at the expense of more false negative errors. False-positive errors are higher in field surveying of plants than many people may appreciate. Greater stringency is required before accepting species as present at a site, particularly for rare species. Combining multiple surveys resolves the problem, but requires a considerable increase in effort to achieve the same sensitivity as a single survey. Therefore, other methods should be used to raise the threshold for the acceptance of a species. For example, digital data input systems that can verify, feedback and inform the user are likely to reduce false-positive errors significantly. creator: Quentin J. Groom creator: Sarah J. Whild uri: https://doi.org/10.7717/peerj.3324 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Groom and Whild title: Microbiome patterns across the gastrointestinal tract of the rabbitfish Siganus fuscescens link: https://peerj.com/articles/3317 last-modified: 2017-05-17 description: Most of our knowledge regarding the biodiversity of gut microbes comes from terrestrial organisms or marine species of economic value, with less emphasis on ecologically important species. Here we investigate the bacterial composition associated with the gut of Siganus fuscescens, a rabbitfish that plays an important ecological role in coastal ecosystems by consuming seaweeds. Members of Firmicutes, Bacteroidetes and delta-Proteobacteria were among the dominant taxa across samples taken from the contents and the walls (sites) of the midgut and hindgut (location). Despite the high variability among individual fish, we observed statistically significant differences in beta-diversity between gut sites and gut locations. Some bacterial taxa low in abundance in the midgut content (e.g., Desulfovibrio) were found in greater abundances on the midgut wall and within the hindgut, suggesting that the gut may select for specific groups of environmental and/or food-associated microorganisms. In contrast, some distinct taxa present in the midgut content (e.g., Synechococcus) were noticeably reduced in the midgut wall and hindgut, and are thus likely to be representative of transient microbiota. This is the first assessment of the bacterial diversity associated with the gut of S. fuscescens and highlights the need to consider the variability across different gut locations and sites when analyzing fish gut microbiomes. creator: Shaun Nielsen creator: Jackson Wilkes Walburn creator: Adriana Vergés creator: Torsten Thomas creator: Suhelen Egan uri: https://doi.org/10.7717/peerj.3317 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Nielsen et al. title: Dietary flavonoids of Spanish youth: intakes, sources, and association with the Mediterranean diet link: https://peerj.com/articles/3304 last-modified: 2017-05-17 description: BackgroundPlant-based diets have been linked to high diet quality and reduced risk of cardiovascular diseases. The health impact of plant-based diets might be partially explained by the concomitant intake of flavonoids. Estimation of flavonoids intake in adults has been important for the development of dietary recommendations and interventions for the prevention of weight gain and its consequences. However, estimation of flavonoids intake in children and adolescents is limited.MethodsAverage daily intake and sources of flavonoids were estimated for a representative national sample of 3,534 children and young people in Spain, aged 2–24 years. The data was collected between 1998 and 2000 by 24-h recalls. The Phenol-Explorer database and the USDA database on flavonoids content were used. Adherence to the Mediterranean diet was measured by the KIDMED index.ResultsThe mean and median intakes of total flavonoids were 70.7 and 48.1 mg/day, respectively. The most abundant flavonoid class was flavan-3-ols (35.7%), with fruit being the top food source of flavonoids intake (42.8%). Total flavonoids intake was positively associated with the KIDMED index (p < 0.001).ConclusionThe results of this study provide primary information about flavonoids intake and main food sources in Spanish children, adolescents and young adults. Participants with high daily mean intake of flavonoids have higher adherence to the Mediterranean diet. creator: Rowaedh Ahmed Bawaked creator: Helmut Schröder creator: Lourdes Ribas-Barba creator: Gabriela Cárdenas creator: Luis Peña-Quintana creator: Carmen Pérez-Rodrigo creator: Montserrat Fíto creator: Lluis Serra-Majem uri: https://doi.org/10.7717/peerj.3304 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Bawaked et al. title: Preservation media, durations and cell concentrations of short-term storage affect key features of human adipose-derived mesenchymal stem cells for therapeutic application link: https://peerj.com/articles/3301 last-modified: 2017-05-17 description: BackgroundAdipose-derived mesenchymal stem cells (ADSCs) have shown great potential in the treatment of various diseases. However, the optimum short-term storage condition of ADSCs in 2∼8 °C is rarely reported. This study aimed at optimizing a short-term storage condition to ensure the viability and function of ADSCs before transplantation.MethodsPreservation media and durations of storage were evaluated by cell viability, apoptosis, adhesion ability and colony-forming unit (CFU) capacity of ADSCs. The abilities of cell proliferation and differentiation were used to optimize cell concentrations. Optimized preservation condition was evaluated by cell surface markers, cell cycle and immunosuppressive capacity.ResultsA total of 5% human serum albumin in multiple electrolytes (ME + HSA) was the optimized medium with high cell viability, low cluster rate, good adhesion ability and high CFU capacity of ADSCs. Duration of storage should be limited to 24 h to ensure the quality of ADSCs before transplantation. A concentration of 5 × 106 cells/ml was the most suitable cell concentration with low late stage apoptosis, rapid proliferation and good osteogenic and adipogenic differentiation ability. This selected condition did not change surface markers, cell cycle, indoleamine 2, 3-dioxygenase 1 (IDO1) gene expression and kynurenine (Kyn) concentration significantly.DiscussionIn this study, ME + HSA was found to be the best medium, most likely due to the supplement of HSA which could protect cells, the physiological pH (7.4) of ME and sodium gluconate ingredient in ME which could provide energy for cells. Duration should be limited to 24 h because of reduced nutrient supply and increased waste and lactic acid accumulation during prolonged storage. To keep cell proliferation and limit lactic acid accumulation, the proper cell concentration is 5× 106 cells/ml. Surface markers, cell cycle and immunosuppressive capacity did not change significantly after storage using the optimized condition, which confirmed our results that this optimized short-term storage condition of MSCs has a great potential for the application of cell therapy. creator: Fengli Zhang creator: Huaijuan Ren creator: Xiaohu Shao creator: Chao Zhuang creator: Yantian Chen creator: Nianmin Qi uri: https://doi.org/10.7717/peerj.3301 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Zhang et al. title: Among-site variability in the stochastic dynamics of East African coral reefs link: https://peerj.com/articles/3290 last-modified: 2017-05-17 description: Coral reefs are dynamic systems whose composition is highly influenced by unpredictable biotic and abiotic factors. Understanding the spatial scale at which long-term predictions of reef composition can be made will be crucial for guiding conservation efforts. Using a 22-year time series of benthic composition data from 20 reefs on the Kenyan and Tanzanian coast, we developed Bayesian vector autoregressive state-space models for reef dynamics, incorporating among-site variability, and quantified their long-term behaviour. We estimated that if there were no among-site variability, the total long-term variability would be approximately one-third of its current value. Thus, our results showed that among-site variability contributes more to long-term variability in reef composition than does temporal variability. Individual sites were more predictable than previously thought, and predictions based on current snapshots are informative about long-term properties. Our approach allowed us to identify a subset of possible climate refugia sites with high conservation value, where the long-term probability of coral cover ≤0.1 (as a proportion of benthic cover of hard substrate) was very low. Analytical results show that this probability is most strongly influenced by among-site variability and by interactions among benthic components within sites. These findings suggest that conservation initiatives might be successful at the site scale as well as the regional scale. creator: Katherine A. Allen creator: John F. Bruno creator: Fiona Chong creator: Damian Clancy creator: Tim R. McClanahan creator: Matthew Spencer creator: Kamila Żychaluk uri: https://doi.org/10.7717/peerj.3290 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Allen et al. title: Quantifying climate change impacts emphasises the importance of managing regional threats in the endangered Yellow-eyed penguin link: https://peerj.com/articles/3272 last-modified: 2017-05-16 description: Climate change is a global issue with effects that are difficult to manage at a regional scale. Yet more often than not climate factors are just some of multiple stressors affecting species on a population level. Non-climatic factors—especially those of anthropogenic origins—may play equally important roles with regard to impacts on species and are often more feasible to address. Here we assess the influence of climate change on population trends of the endangered Yellow-eyed penguin (Megadyptes antipodes) over the last 30 years, using a Bayesian model. Sea surface temperature (SST) proved to be the dominating factor influencing survival of both adult birds and fledglings. Increasing SST since the mid-1990s was accompanied by a reduction in survival rates and population decline. The population model showed that 33% of the variation in population numbers could be explained by SST alone, significantly increasing pressure on the penguin population. Consequently, the population becomes less resilient to non-climate related impacts, such as fisheries interactions, habitat degradation and human disturbance. However, the extent of the contribution of these factors to declining population trends is extremely difficult to assess principally due to the absence of quantifiable data, creating a discussion bias towards climate variables, and effectively distracting from non-climate factors that can be managed on a regional scale to ensure the viability of the population. creator: Thomas Mattern creator: Stefan Meyer creator: Ursula Ellenberg creator: David M. Houston creator: John T. Darby creator: Melanie Young creator: Yolanda van Heezik creator: Philip J. Seddon uri: https://doi.org/10.7717/peerj.3272 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Mattern et al. title: Conserved queen pheromones in bumblebees: a reply to Amsalem et al. link: https://peerj.com/articles/3332 last-modified: 2017-05-16 description: In a recent study, Amsalem, Orlova & Grozinger (2015) performed experiments with Bombus impatiens bumblebees to test the hypothesis that saturated cuticular hydrocarbons are evolutionarily conserved signals used to regulate reproductive division of labor in many Hymenopteran social insects. They concluded that the cuticular hydrocarbon pentacosane (C25), previously identified as a queen pheromone in a congeneric bumblebee, does not affect worker reproduction in B. impatiens. Here we discuss some shortcomings of Amsalem et al.’s study that make its conclusions unreliable. In particular, several confounding effects may have affected the results of both experimental manipulations in the study. Additionally, the study’s low sample sizes (mean n per treatment = 13.6, range: 4–23) give it low power, not 96–99% power as claimed, such that its conclusions may be false negatives. Inappropriate statistical tests were also used, and our reanalysis found that C25 substantially reduced and delayed worker egg laying in B. impatiens. We review the evidence that cuticular hydrocarbons act as queen pheromones, and offer some recommendations for future queen pheromone experiments. creator: Luke Holman creator: Jelle S. van Zweden creator: Ricardo C. Oliveira creator: Annette van Oystaeyen creator: Tom Wenseleers uri: https://doi.org/10.7717/peerj.3332 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Holman et al. title: Serotonergic activation during courtship and aggression in the brown anole, Anolis sagrei link: https://peerj.com/articles/3331 last-modified: 2017-05-16 description: The role of serotonin (5-hydroxytryptamine, 5-HT) in social behavior regulation is not fully understood. While 5-HT release in nuclei of the social behavior network has generally been associated with inhibition of aggressive behavior across multiple classes of vertebrates, less is known about its effects on sexual, especially non-copulatory courtship display behaviors. Furthermore, most research has examined effects at 5-HT release sites, while studies examining the behavioral relevance of source cell populations have generated contradictory findings. This study utilized immunohistochemistry to examine the colocalization of 5-HT with Fos, an immediate early gene product and marker of neural activity, in the raphe and superior reticular nuclei of male brown anoles (Anolis sagrei) exposed to either aggression, courtship, or control social interactions. Supporting previous research, copulation was associated with a decrease in 5-HT activity, while a novel link between 5-HT activity and latency to non-copulatory courtship was also found. Within the aggression group, intensity and frequency of behavior were both associated with decreased 5-HT activity. An effect of social context was also seen, with anoles exposed to either courtship or aggression encounters showing decreased 5-HT activity in certain raphe and superior reticular nuclei populations compared to controls. Interestingly, context effects and behavioral effects were seen at separate brain nuclei, suggesting the presence of separate systems with distinct functional roles. creator: Jacob T. Hartline creator: Alexandra N. Smith creator: David Kabelik uri: https://doi.org/10.7717/peerj.3331 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Hartline et al. title: Phylogenetic climatic niche conservatism and evolution of climatic suitability in Neotropical Angraecinae (Vandeae, Orchidaceae) and their closest African relatives link: https://peerj.com/articles/3328 last-modified: 2017-05-16 description: In the present study we investigate the concept of phylogenetic niche conservatism (PNC) within the American species of angraecoid orchids (Campylocentrum and Dendrophylax) and their closest relatives in the Old World (Angraecum) using ecological niche modelling (ENM). The predicted niche occupancy profiles were matched with the outcomes of previous phylogenetic studies to reconstruct the evolution of climatic suitability within the orchid group studied and evaluate the role of niche differentiation in the speciation of Angraecinae. No correlation between preferred niches and taxonomic relationships within the orchid group studied was revealed. The climatic suitability of the majority of the species overlapped each other, either fully or partially. This pattern is also present in the species of other orchid genera. Our research confirms a significant level of PNC in Orchidaceae, even within taxa exhibiting a transatlantic disjunction. The analysis of the evolution of climatic suitability indicated that the adaptation to various climatic conditions is not a factor that has driven speciation within orchids studied. creator: Marta Kolanowska creator: Elżbieta Grochocka creator: Kamil Konowalik uri: https://doi.org/10.7717/peerj.3328 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Kolanowska et al. title: An exhaustive survey of regular peptide conformations using a new metric for backbone handedness (h) link: https://peerj.com/articles/3327 last-modified: 2017-05-16 description: The Ramachandran plot is important to structural biology as it describes a peptide backbone in the context of its dominant degrees of freedom—the backbone dihedral angles φ and ψ (Ramachandran, Ramakrishnan & Sasisekharan, 1963). Since its introduction, the Ramachandran plot has been a crucial tool to characterize protein backbone features. However, the conformation or twist of a backbone as a function of φ and ψ has not been completely described for both cis and trans backbones. Additionally, little intuitive understanding is available about a peptide’s conformation simply from knowing the φ and ψ values of a peptide (e.g., is the regular peptide defined by φ = ψ =  − 100°  left-handed or right-handed?). This report provides a new metric for backbone handedness (h) based on interpreting a peptide backbone as a helix with axial displacement d and angular displacement θ, both of which are derived from a peptide backbone’s internal coordinates, especially dihedral angles φ, ψ and ω. In particular, h equals sin(θ)d∕|d|, with range [−1, 1] and negative (or positive) values indicating left(or right)-handedness. The metric h is used to characterize the handedness of every region of the Ramachandran plot for both cis (ω = 0°) and trans (ω = 180°) backbones, which provides the first exhaustive survey of twist handedness in Ramachandran (φ, ψ) space. These maps fill in the ‘dead space’ within the Ramachandran plot, which are regions that are not commonly accessed by structured proteins, but which may be accessible to intrinsically disordered proteins, short peptide fragments, and protein mimics such as peptoids. Finally, building on the work of (Zacharias & Knapp, 2013), this report presents a new plot based on d and θ that serves as a universal and intuitive alternative to the Ramachandran plot. The universality arises from the fact that the co-inhabitants of such a plot include every possible peptide backbone including cis and trans backbones. The intuitiveness arises from the fact that d and θ provide, at a glance, numerous aspects of the backbone including compactness, handedness, and planarity. creator: Ranjan V. Mannige uri: https://doi.org/10.7717/peerj.3327 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Mannige title: Regression assumptions in clinical psychology research practice—a systematic review of common misconceptions link: https://peerj.com/articles/3323 last-modified: 2017-05-16 description: Misconceptions about the assumptions behind the standard linear regression model are widespread and dangerous. These lead to using linear regression when inappropriate, and to employing alternative procedures with less statistical power when unnecessary. Our systematic literature review investigated employment and reporting of assumption checks in twelve clinical psychology journals. Findings indicate that normality of the variables themselves, rather than of the errors, was wrongfully held for a necessary assumption in 4% of papers that use regression. Furthermore, 92% of all papers using linear regression were unclear about their assumption checks, violating APA-recommendations. This paper appeals for a heightened awareness for and increased transparency in the reporting of statistical assumption checking. creator: Anja F. Ernst creator: Casper J. Albers uri: https://doi.org/10.7717/peerj.3323 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Ernst and Albers title: Changes in the spatial and temporal pattern of natural forest cover on Hainan Island from the 1950s to the 2010s: implications for natural forest conservation and management link: https://peerj.com/articles/3320 last-modified: 2017-05-16 description: The study of the past, present, and future state and dynamics of the tropical natural forest cover (NFC) might help to better understand the pattern of deforestation and fragmentation as well as the influence of social and natural processes. The obtained information will support the development of effective conservation policies and strategies. In the present study, we used historical data of the road network, topography, and climatic productivity to reconstruct NFC maps of Hainan Island, China, from the 1950s to the 2010s, using the random forest algorithm. We investigated the spatial and temporal patterns of NFC change from the 1950s to the 2010s and found that it was highly dynamic in both space and time. Our data showed that grid cells with low NFC were more vulnerable to NFC decrease, suggesting that conservation actions regarding natural forests need to focus on regions with low NFC and high ecological value. We also identified the hot spots of NFC change, which provides insights into the dynamic changes of natural forests over time. creator: Siliang Lin creator: Yaozhu Jiang creator: Jiekun He creator: Guangzhi Ma creator: Yang Xu creator: Haisheng Jiang uri: https://doi.org/10.7717/peerj.3320 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Lin et al. title: Reappraisal of the extinct seal “Phoca” vitulinoides from the Neogene of the North Sea Basin, with bearing on its geological age, phylogenetic affinities, and locomotion link: https://peerj.com/articles/3316 last-modified: 2017-05-16 description: BackgroundDiscovered on the southern margin of the North Sea Basin, “Phoca” vitulinoides represents one of the best-known extinct species of Phocidae. However, little attention has been given to the species ever since its original 19th century description. Newly discovered material, including the most complete specimen of fossil Phocidae from the North Sea Basin, prompted the redescription of the species. Also, the type material of “Phoca” vitulinoides is lost.Methods“Phoca” vitulinoides is redescribed. Its phylogenetic position among Phocinae is assessed through phylogenetic analysis. Dinoflagellate cyst biostratigraphy is used to determine and reassess the geological age of the species. Myological descriptions of extant taxa are used to infer muscle attachments, and basic comparative anatomy of the gross morphology and biomechanics are applied to reconstruct locomotion.ResultsDetailed redescription of “Phoca” vitulinoides indicates relatively little affinities with the genus Phoca, but rather asks for the establishment of a new genus: Nanophoca gen. nov. Hence, “Phoca” vitulinoides is recombined into Nanophoca vitulinoides. This reassignment is confirmed by the phylogenetic analysis, grouping the genus Nanophoca and other extinct phocine taxa as stem phocines. Biostratigraphy and lithostratigraphy expand the known stratigraphic range of N. vitulinoides from the late Langhian to the late Serravallian. The osteological anatomy of N. vitulinoides indicates a relatively strong development of muscles used for fore flipper propulsion and increased flexibility for the hind flipper.DiscussionThe extended stratigraphic range of N. vitulinoides into the middle Miocene confirms relatively early diversification of Phocinae in the North Atlantic. Morphological features on the fore- and hindlimb of the species point toward an increased use of the fore flipper and greater flexibility of the hind flipper as compared to extant Phocinae, clearly indicating less derived locomotor strategies in this Miocene phocine species. Estimations of the overall body size indicate that N. vitulinoides is much smaller than Pusa, the smallest extant genus of Phocinae (and Phocidae), and than most extinct phocines. creator: Leonard Dewaele creator: Eli Amson creator: Olivier Lambert creator: Stephen Louwye uri: https://doi.org/10.7717/peerj.3316 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Dewaele et al. title: Morphology of an Early Oligocene beaver Propalaeocastor irtyshensis and the status of the genus Propalaeocastor link: https://peerj.com/articles/3311 last-modified: 2017-05-16 description: The Early to Late Oligocene Propalaeocastor is the earliest known beaver genus from Eurasia. Although many species of this genus have been described, these species are defined based on very fragmentary specimens. Propalaeocastor irtyshensis from the Early Oligocene Irtysh River Formation in northwestern Xinjiang, China is one of the earliest-known members of Propalaeocastor. This species is defined on a single maxillary fragment. We revise the diagnosis of P. irtyshensis and the genus Propalaeocastor, based on newly discovered specimens from the Irtysh River Formation. The dental morphology of P. irtyshensis is very similar to other early castorids. The caudal palatine foramen of P. irtyshensis is situated in the maxillary-palatine suture. This is a feature generally accept as diagnostic character for the castorids. On the other hand, P. irtyshensis has two upper premolars, a rudimentarily developed sciuromorph-like zygomatic plate, and a relatively large protrogomorph-like infraorbital foramen. Some previous researchers suggested that Propalaeocastor is a junior synonym of Steneofiber, while other took it as a valid genus. Our morphological comparison and phylogenetic analysis suggest that Propalaeocastor differs from Steneofiber and is a valid genus. We also suggest that Agnotocastor aubekerovi, A. coloradensis, A. galushai, A. readingi, Oligotheriomys primus, and “Steneofiber aff. dehmi” should be referred to Propalaeocastor. Propalaeocastor is the earliest and most basal beaver. The origin place of Propalaeocastor and castorids is uncertain. The Early Oligocene radiation of castorids probably is propelled by the global climate change during the Eocene-Oligocene transition. creator: Lüzhou Li creator: Qiang Li creator: Xiaoyu Lu creator: Xijun Ni uri: https://doi.org/10.7717/peerj.3311 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Li et al. title: From effective biocontrol agent to successful invader: the harlequin ladybird (Harmonia axyridis) as an example of good ideas that could go wrong link: https://peerj.com/articles/3296 last-modified: 2017-05-16 description: The use of biological control agents to control pests is an alternative to pesticides and a tool to manage invasive alien species. However, biocontrol agents can themselves become invasive species under certain conditions. The harlequin ladybird (Harmonia axyridis) is a native Asian biocontrol agent that has become a successful invader. We reviewed articles containing “Harmonia axyridis” to gather information on its presence and surveyed entomologists researching Coccinellidae around the world to investigate further insights about the current distribution, vectors of introduction, habitat use and threats this species pose. The harlequin ladybird has established populations in at least 59 countries outside its native range. Twenty six percent of the surveyed scientists considered it a potential threat to native Coccinellidae. Published studies and scientists suggest Adalia bipunctata, native to Europe, is under the highest risk of population declines. Strict policies should be incorporated to prevent its arrival to non-invaded areas and to prevent further expansion range. Managing invasive species is a key priority to prevent biodiversity loss and promote ecosystem services. creator: Morelia Camacho-Cervantes creator: Adrián Ortega-Iturriaga creator: Ek del-Val uri: https://doi.org/10.7717/peerj.3296 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Camacho-Cervantes et al. title: PLD2 regulates microtubule stability and spindle migration in mouse oocytes during meiotic division link: https://peerj.com/articles/3295 last-modified: 2017-05-16 description: Phospholipase D2 (PLD2) is involved in cytoskeletal reorganization, cell migration, cell cycle progression, transcriptional control and vesicle trafficking. There is no evidence about PLD2 function in oocytes during meiosis. Herein, we analyzed PLD2 expression and its relationship with spindle formation and positioning in mouse oocyte meiosis. High protein level of PLD2 was revealed in oocytes by Western blot, which remained consistently stable from prophase I with intact germinal vesicle (GV) up to metaphase II (MII) stage. Immunofluorescence showed that PLD2 appeared and gathered around the condensed chromosomesafter germinal vesicle breakdown (GVBD), and co-localized with spindle from pro-metaphase I (pro-MI) to metaphase I (MI) and at MII stage. During anaphase I (Ana I) to telophase I (Tel I) transition, PLD2 was concentrated in the spindle polar area but absent from the midbody. In oocytes incubated with NFOT, an allosteric and catalytic inhibitor to PLD2, the spindle was enlarged and center-positioned, microtubules were resistant to cold-induced depolymerization and, additionally, the meiotic progression was arrested at MI stage. However, spindle migration could not be totally prevented by PLD2 catalytic specific inhibitors, FIPI and 1-butanol, implying at least partially, that PLD2 effect on spindle migration needs non-catalytic domain participation. NFOT-induced defects also resulted in actin-related molecules’ distribution alteration, such as RhoA, phosphatidylinosital 4, 5- biphosphate (PIP2), phosphorylated Colifin and, consequently, unordered F-actin dynamics. Taken together, these data indicate PLD2 is required for the regulation of microtubule dynamics and spindle migration toward the cortex in mammalian oocytes during meiotic progression. creator: Xiaoyu Liu creator: Xiaoyun Liu creator: Dandan Chen creator: Xiuying Jiang creator: Wei Ma uri: https://doi.org/10.7717/peerj.3295 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Liu et al. title: Exercise and dietary program-induced weight reduction is associated with cognitive function among obese adolescents: a longitudinal study link: https://peerj.com/articles/3286 last-modified: 2017-05-16 description: ObjectiveThe present study was to determine the effect of a combined exercise and dietary program on cognitive function as well as the relationship between the program-induced weight change and cognitive function alterations.DesignThe study applies a quasi-experimental design.MethodsFifty-eight adolescents with obese status (body mass index, BMI >28 kg/m2) were assigned to either an experiment (n = 30) or control group (n = 28). Participants in the experiment group received a scheduled program with a specific exercise protocol (two sessions per day, six days per week) and diet plan for four consecutive weeks; the control group was instructed to maintain their normal school activities. The primary outcome measures were anthropometric data and flanker task performance.ResultsThe combined program led to reduced BMI with maintenance of the incongruent accuracy in the experiment group, but the incongruent accuracy decreased in the control group after the four-week period. Additionally, the change in weight status between post- and pre-test measurements was inversely correlated with the change in incongruent accuracy.ConclusionThe combined exercise and dietary program resulted in decreased weight and enhanced executive function in the obese adolescents, and the weight alteration may be considered the mediator between the intervention and executive function. creator: Chun Xie creator: Xiaochun Wang creator: Chenglin Zhou creator: Chang Xu creator: Yu-Kai Chang uri: https://doi.org/10.7717/peerj.3286 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Xie et al. title: Interplay among pain intensity, sleep disturbance and emotion in patients with non-specific low back pain link: https://peerj.com/articles/3282 last-modified: 2017-05-16 description: BackgroundLow back pain (LBP) is the most common problem worldwide. There are several negative consequences of LBP, such as sleep disorders, work leave, disability, depression, anxiety, and poor quality of life. In this study, we designed to evaluate the prevalence of sleep disturbance in patients with non-specific LBP(NSLBP), and cross-correlation among sleep disorder, anxiety, depression and pain intensity in patients with NSLBP.AimIn this study, we designed to evaluate the prevalence of sleep disturbance in patients with NSLBP, and cross-correlation among sleep disorder, anxiety, depression and pain intensity in patients with NSLBP.MethodsA cross-sectional self-assessment questionnaire survey was carried out in an outpatient clinic. Anonymous assessments were used to characterize the presence of NSLBP, PSQI, VAS, SF-36 form, ODI, BAI and BDI. Cross-correlation among the severity of NSLBP and sleep disturbance, anxiety, depression and life quality were evaluated.ResultsPatients with NSLBP have a higher incidence of sleep disorder, anxiety and depression, and higher ODI scores than healthy people without LPB (P < 0.01). NSLBP patients with sleep disorders have more severe anxiety, depression, an increased VAS score and poor daily living (P < 0.05). NSLBP patients with anxiety have declined sleep quality, poor daily living, decreased work and social skills, and increased LBP severity (P < 0.05). NSLBP patients with depression have declined sleep quality, poor daily living, decreased work and social skills (P < 0.05). Significant associations were found between the severity of NSLBP and sleep disorders, anxiety and ODI scores.ConclusionPsychological and social factors play an important role in the development of NSLBP. NSLBP leads to sleep disorders, which decrease the sleep quality and increase the unpleasant emotions and memories in return; these can exacerbate the severity of LBP, with the cycle repeating to form a vicious circle. creator: Shilabant Sen Sribastav creator: He Peiheng creator: Long Jun creator: Li Zemin creator: Wei Fuxin creator: Wang Jianru creator: Liu Hui creator: Wang Hua creator: Zheng Zhaomin uri: https://doi.org/10.7717/peerj.3282 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Sribastav et al. title: Are invasive populations characterized by a broader diet than native populations? link: https://peerj.com/articles/3250 last-modified: 2017-05-16 description: BackgroundInvasive species are among the most significant threats to biodiversity. The diet of invasive animal populations is a crucial factor that must be considered in the context of biological invasions. A broad dietary spectrum is a frequently cited characteristic of invasive species, allowing them to thrive in a wide range of environments. Therefore, empirical studies comparing diet in invasive and native populations are necessary to understand dietary requirements, dietary flexibility, and the associated impacts of invasive species.MethodsIn this study, we compared the diet of populations of the African clawed frog, Xenopus laevis in its native range, with several areas where it has become invasive. Each prey category detected in stomach contents was assigned to an ecological category, allowing a comparison of the diversity of ecological traits among the prey items in the diet of native and introduced populations. The comparison of diets was also performed using evenness as a niche breadth index on all sampled populations, and electivity as a prey selection index for three out of the six sampled populations.ResultsOur results showed that diet breadth could be either narrow or broad in invasive populations. According to diet and prey availability, zooplankton was strongly preferred in most cases. In lotic environments, zooplankton was replaced by benthic preys, such as ephemeropteran larvae.DiscussionThe relative proportions of prey with different ecological traits, and dietary variability within and between areas of occurrence, suggest that X. laevis is a generalist predator in both native and invasive populations. Shifts in the realized trophic niche are observed, and appear related to resource availability. Xenopus laevis may strongly impact aquatic ecosystems because of its near complete aquatic lifestyle and its significant consumption of key taxa for the trophic relationships in ponds. creator: Julien Courant creator: Solveig Vogt creator: Raquel Marques creator: John Measey creator: Jean Secondi creator: Rui Rebelo creator: André De Villiers creator: Flora Ihlow creator: Charlotte De Busschere creator: Thierry Backeljau creator: Dennis Rödder creator: Anthony Herrel uri: https://doi.org/10.7717/peerj.3250 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Courant et al. title: Worldwide exploration of the microbiome harbored by the cnidarian model, Exaiptasia pallida (Agassiz in Verrill, 1864) indicates a lack of bacterial association specificity at a lower taxonomic rank link: https://peerj.com/articles/3235 last-modified: 2017-05-16 description: Examination of host-microbe interactions in early diverging metazoans, such as cnidarians, is of great interest from an evolutionary perspective to understand how host-microbial consortia have evolved. To address this problem, we analyzed whether the bacterial community associated with the cosmopolitan and model sea anemone Exaiptasia pallida shows specific patterns across worldwide populations ranging from the Caribbean Sea, and the Atlantic and Pacific oceans. By comparing sequences of the V1–V3 hypervariable regions of the bacterial 16S rRNA gene, we revealed that anemones host a complex and diverse microbial community. When examined at the phylum level, bacterial diversity and abundance associated with E. pallida are broadly conserved across geographic space with samples, containing largely Proteobacteria and Bacteroides. However, the species-level makeup within these phyla differs drastically across space suggesting a high-level core microbiome with local adaptation of the constituents. Indeed, no bacterial OTU was ubiquitously found in all anemones samples. We also revealed changes in the microbial community structure after rearing anemone specimens in captivity within a period of four months. Furthermore, the variation in bacterial community assemblages across geographical locations did not correlate with the composition of microalgal Symbiodinium symbionts. Our findings contrast with the postulation that cnidarian hosts might actively select and maintain species-specific microbial communities that could have resulted from an intimate co-evolution process. The fact that E. pallida is likely an introduced species in most sampled localities suggests that this microbial turnover is a relatively rapid process. Our findings suggest that environmental settings, not host specificity, seem to dictate bacterial community structure associated with this sea anemone. More than maintaining a specific composition of bacterial species some cnidarians associate with a wide range of bacterial species as long as they provide the same physiological benefits towards the maintenance of a healthy host. The examination of the previously uncharacterized bacterial community associated with the cnidarian sea anemone model E. pallida is the first global-scale study of its kind. creator: Tanya Brown creator: Christopher Otero creator: Alejandro Grajales creator: Estefania Rodriguez creator: Mauricio Rodriguez-Lanetty uri: https://doi.org/10.7717/peerj.3235 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Brown et al. title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells link: https://peerj.com/articles/3233 last-modified: 2017-05-16 description: BackgroundTreatments that target cancer stem cells play an important role in the controlling and eliminating of tumor initiation as well as in development, progression, and chemotherapy/radiotherapy resistance. In our previous study, we cultured and harvested human laryngeal cancer stem cells (CSCs) and applied microRNA biochips to screen differentially expressed miRNAs that were related to radiation tolerance in irradiated human laryngeal CSCs. According to the predicted genes and pathways of differential miRNAs target, down-regulated expression of hsa-miR-138-2-3p under radiation was thought to play a key role in enhancing the radio-sensitivity in human laryngeal squamous cancer stem cells.MethodTo investigate the radiational enhancement of hsa-miR-138-2-3p, we transfected hsa-miR-138-2-3p mimics that were synthesized based on the sequences of hsa-miR-138-2-3p in vitrointo human laryngeal CSCs (Hep-2, M2e, and TU212 cell lines) to make hsa-miR-138-2-3p overexpressed, and the tumorous specialities of CSCs, like cell proliferation, invasion, apoptosis, cell cycle arrest, and DNA damage were evaluated by CCK-8 assay, clone formation assay, invasion assay, flow cytometry, and comet assay. Furthermore, we explored the signal transduction pathways that regulated the cancer stem cell initiation, development, invasion, apoptosis and cell cycle arrest, which were controlled by hsa-miR-138-2-3p.ResultOverexpressed hsa-miR-138-2-3p played a key role in many anti-cancer biological processes in human laryngeal CSCs: (1) it decreased laryngeal CSCs proliferation and invasion in response to radiotherapy; (2) it increased the proportion of early and late apoptosis in laryngeal CSCs after radiation, raised G1 phase arrest in laryngeal CSCs after radiation, and decreased the proportion of S stage cells of cell cycle that were related to radio-resistance in laryngeal CSCs; (3) it down-regulated the expression of β-catenin in Wnt signal pathway that was related to the tolerance of laryngeal CSCs to radiotherapy; (4) it down-regulated the expression of YAP1 in Hippo signal pathway that regulated cell proliferation, invasion and apoptosis; (5) it up-regulated the expression of p38 and JNK1 in MAPK signal pathway that was concerned to radio-sensitivity.ConclusionIn the present study, it was found that hsa-miR-138-2-3p regulated the Wnt/β-catenin pathways, the Hippo/YAP1 pathways, and the MAPK/p38/JNK1 pathways that were involved in cell proliferation, invasion, apoptosis, cell cycle arrest, radio-resistance and radio-sensitivity in laryngeal CSCs. These results will be useful for a better understanding of the cell biology of hsa-miR-138-2-3p in laryngeal CSCs, and for serving hsa-miR-138-2-3p as a promising biomarker and as a target for diagnosis and for novel anti-cancer therapies for laryngeal cancers. creator: Ying Zhu creator: Li-Yun Shi creator: Yan-Min Lei creator: Yan-Hong Bao creator: Zhao-Yang Li creator: Fei Ding creator: Gui-Ting Zhu creator: Qing-Qing Wang creator: Chang-Xin Huang uri: https://doi.org/10.7717/peerj.3233 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Zhu et al. title: Ungulates rely less on visual cues, but more on adapting movement behaviour, when searching for forage link: https://peerj.com/articles/3178 last-modified: 2017-05-16 description: Finding suitable forage patches in a heterogeneous landscape, where patches change dynamically both spatially and temporally could be challenging to large herbivores, especially if they have no a priori knowledge of the location of the patches. We tested whether three large grazing herbivores with a variety of different traits improve their efficiency when foraging at a heterogeneous habitat patch scale by using visual cues to gain a priori knowledge about potential higher value foraging patches. For each species (zebra (Equus burchelli), red hartebeest (Alcelaphus buselaphus subspecies camaa) and eland (Tragelaphus oryx)), we used step lengths and directionality of movement to infer whether they were using visual cues to find suitable forage patches at a habitat patch scale. Step lengths were significantly longer for all species when moving to non-visible patches than to visible patches, but all movements showed little directionality. Of the three species, zebra movements were the most directional. Red hartebeest had the shortest step lengths and zebra the longest. We conclude that these large grazing herbivores may not exclusively use visual cues when foraging at a habitat patch scale, but would rather adapt their movement behaviour, mainly step length, to the heterogeneity of the specific landscape. creator: Jan A. Venter creator: Herbert H.T. Prins creator: Alla Mashanova creator: Rob Slotow uri: https://doi.org/10.7717/peerj.3178 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Venter et al. title: Cells tile a flat plane by controlling geometries during morphogenesis of Pyropia thalli link: https://peerj.com/articles/3314 last-modified: 2017-05-11 description: BackgroundPyropia haitanensis thalli, which are made of a single layer of polygonal cells, are a perfect model for studying the morphogenesis of multi-celled organisms because their cell proliferation process is an excellent example of the manner in which cells control their geometry to create a two-dimensional plane.MethodsCellular geometries of thalli at different stages of growth revealed by light microscope analysis.ResultsThis study showed the cell division transect the middle of the selected paired-sides to divide the cell into two equal portions, thus resulting in cell sides ≥4 and keeping the average number of cell sides at approximately six even as the thallus continued to grow, such that more than 90% of the cells in thalli longer than 0.08 cm had 5–7 sides. However, cell division could not fully explain the distributions of intracellular angles. Results showed that cell-division-associated fast reorientation of cell sides and cell divisions together caused 60% of the inner angles of cells from longer thalli to range from 100–140°. These results indicate that cells prefer to form regular polygons.ConclusionsThis study suggests that appropriate cell-packing geometries maintained by cell division and reorientation of cell walls can keep the cells bordering each other closely, without gaps. creator: Kai Xu creator: Yan Xu creator: Dehua Ji creator: Ting Chen creator: Changsheng Chen creator: Chaotian Xie uri: https://doi.org/10.7717/peerj.3314 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Xu et al. title: Estimating the effect of burrowing shrimp on deep-sea sediment community oxygen consumption link: https://peerj.com/articles/3309 last-modified: 2017-05-11 description: Sediment community oxygen consumption (SCOC) is a proxy for organic matter processing and thus provides a useful proxy of benthic ecosystem function. Oxygen uptake in deep-sea sediments is mainly driven by bacteria, and the direct contribution of benthic macro- and mega-infauna respiration is thought to be relatively modest. However, the main contribution of infaunal organisms to benthic respiration, particularly large burrowing organisms, is likely to be indirect and mainly driven by processes such as feeding and bioturbation that stimulate bacterial metabolism and promote the chemical oxidation of reduced solutes. Here, we estimate the direct and indirect contributions of burrowing shrimp (Eucalastacus cf. torbeni) to sediment community oxygen consumption based on incubations of sediment cores from 490 m depth on the continental slope of New Zealand. Results indicate that the presence of one shrimp in the sediment is responsible for an oxygen uptake rate of about 40 µmol d−1, only 1% of which is estimated to be due to shrimp respiration. We estimate that the presence of ten burrowing shrimp m−2 of seabed would lead to an oxygen uptake comparable to current estimates of macro-infaunal community respiration on Chatham Rise based on allometric equations, and would increase total sediment community oxygen uptake by 14% compared to sediment without shrimp. Our findings suggest that oxygen consumption mediated by burrowing shrimp may be substantial in continental slope ecosystems. creator: Daniel Leduc creator: Conrad A. Pilditch uri: https://doi.org/10.7717/peerj.3309 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Leduc and Pilditch title: The density and biomass of mesozooplankton and ichthyoplankton in the Negro and the Amazon Rivers during the rainy season: the ecological importance of the confluence boundary link: https://peerj.com/articles/3308 last-modified: 2017-05-11 description: The boundary zone between two different hydrological regimes is often a biologically enriched environment with distinct planktonic communities. In the center of the Amazon River basin, muddy white water of the Amazon River meets with black water of the Negro River, creating a conspicuous visible boundary spanning over 10 km along the Amazon River. Here, we tested the hypothesis that the confluence boundary between the white and black water rivers concentrates prey and is used as a feeding habitat for consumers by investigating the density, biomass and distribution of mesozooplankton and ichthyoplankton communities across the two rivers during the rainy season. Our results show that mean mesozooplankton density (2,730 inds. m−3) and biomass (4.8 mg m−3) were higher in the black-water river compared to the white-water river (959 inds. m−3; 2.4 mg m−3); however an exceptionally high mesozooplankton density was not observed in the confluence boundary. Nonetheless we found the highest density of ichthyoplankton in the confluence boundary (9.7 inds. m−3), being up to 9-fold higher than in adjacent rivers. The confluence between white and black waters is sandwiched by both environments with low (white water) and high (black water) zooplankton concentrations and by both environments with low (white water) and high (black water) predation pressures for fish larvae, and may function as a boundary layer that offers benefits of both high prey concentrations and low predation risk. This forms a plausible explanation for the high density of ichthyoplankton in the confluence zone of black and white water rivers. creator: Ryota Nakajima creator: Elvis V. Rimachi creator: Edinaldo N. Santos-Silva creator: Laura S.F. Calixto creator: Rosseval G. Leite creator: Adi Khen creator: Tetsuo Yamane creator: Anthony I. Mazeroll creator: Jomber C. Inuma creator: Erika Y.K. Utumi creator: Akira Tanaka uri: https://doi.org/10.7717/peerj.3308 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Nakajima et al. title: Self-centeredness and selflessness: happiness correlates and mediating psychological processes link: https://peerj.com/articles/3306 last-modified: 2017-05-11 description: The main objective of this research was to test central assumptions from the Self-centeredness/Selflessness Happiness Model. According to this model, while self-centered psychological functioning induces fluctuating happiness, authentic–durable happiness results from selflessness. Distinct mediating processes are supposed to account for these relationships: afflictive affects (e.g., anger, fear, jealousy, frustration) in the case of the former, and both emotional stability and feelings of harmony in the case of the latter. We tested these hypotheses in two studies based on heterogeneous samples of citizens (n = 547). Factor analyses revealed that self-centeredness (assessed through egocentrism and materialism) and selflessness (assessed through self-transcendence and connectedness to other) were two distinct psychological constructs. Second, while self-centeredness was positively and significantly related to fluctuating happiness, selflessness was positively and significantly related to authentic–durable happiness. Finally, distinct psychological processes mediated these relationships (study 2). On one hand, the relationship between self-centeredness and fluctuating happiness was fully mediated by afflictive affects. On the other hand, emotional stability and the feeling of being in harmony partially mediated the relation between selflessness and authentic–durable happiness. creator: Michael Dambrun uri: https://doi.org/10.7717/peerj.3306 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Dambrun title: Radix Stellariae extract prevents high-fat-diet-induced obesity in C57BL/6 mice by accelerating energy metabolism link: https://peerj.com/articles/3305 last-modified: 2017-05-11 description: Stellaria dichotoma L. is widely distributed in Ningxia and surrounding areas in northwestern China. Its root, Radix Stellariae (RS), has been used in herbal formulae for treating asthenic-fever, infection, malaria, dyspepsia in children and several other symptoms. This study investigated whether the RS extract (RSE) alleviates metabolic disorders. The results indicated that RSE significantly inhibited body weight gain in high-fat (HF)-diet-fed C57BL/6 mice, reduced fasting glucose levels, and improved insulin tolerance. Moreover, RSE increased the body temperature of the mice and the expression of uncoupling proteins and peroxisome proliferator-activated receptors in the white adipose tissue. Thus, RSE alleviated metabolic disorders in HF-diet-fed C57BL/6 mice by potentially activating UCP and PPAR signaling. creator: Yin Li creator: Xin Liu creator: Yu Fan creator: Baican Yang creator: Cheng Huang uri: https://doi.org/10.7717/peerj.3305 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Li et al. title: Which psychological, social and physical environmental characteristics predict changes in physical activity and sedentary behaviors during early retirement? A longitudinal study link: https://peerj.com/articles/3242 last-modified: 2017-05-11 description: BackgroundIn the context of healthy ageing, it is necessary to identify opportunities to implement health interventions in order to develop an active lifestyle with sufficient physical activity and limited sedentary time in middle-aged and older adults. The transition to retirement is such an opportunity, as individuals tend to establish new routines at the start of retirement. Before health interventions can be developed, the psychological, social and physical environmental determinants of physical activity and sedentary behaviors during early retirement should be identified, ideally with longitudinal studies. The aim of this paper was first to examine whether psychological, social and physical environmental factors at the start of retirement predict longitudinal changes in physical activity and sedentary behaviors during the first years of retirement. Second, moderating effects of gender and educational levels were examined.MethodsThis longitudinal study was conducted in Flanders, Belgium. In total, 180 recently retired (>1 month, <2 years at baseline) adults completed a postal questionnaire twice (in 2012–2013 and two years later in 2014–2015). The validated questionnaire assessed socio-demographic information, physical activity, sedentary behaviors, and psychological, social and physical environmental characteristics. Multiple moderated hierarchic regression analyses were conducted in SPSS 22.0.ResultsHigher perceived residential density (p < 0.001) and lower aesthetics (p = 0.08) predicted an increase in active transportation (adjusted R2 = 0.18). Higher baseline self-efficacy was associated with an increase in leisure-time physical activity (p = 0.001, adjusted R2 = 0.13). A more positive perception of old age (p = 0.04) and perceiving less street connectivity (p = 0.001) were associated with an increase in screen time (adjusted R2 = 0.06). Finally, higher baseline levels of modeling from friends (p = 0.06) and lower perceived land use mix access (p = 0.09) predicted an increase in car use (adjusted R2 = 0.06). A few moderating effects, mainly of educational level, were found.DiscussionWalkability characteristics (perceived residential density) and self-efficacy at the start of retirement are the most important predictors of longitudinal changes in active transportation and leisure-time physical activity. Few moderating effects were found, so health interventions at the start of retirement focusing on self-efficacy and specific walkability characteristics could be effective to increase physical activity in recently retired adults. No firm conclusions can be drawn on the importance of the examined predictors to explain change in car use and screen time, possibly other factors like the home environment, or automatic processes and habit strength are more important to explain sedentary behaviors. creator: Delfien Van Dyck creator: Greet Cardon creator: Ilse De Bourdeaudhuij uri: https://doi.org/10.7717/peerj.3242 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Van Dyck et al. title: Psychophysical measurements in children: challenges, pitfalls, and considerations link: https://peerj.com/articles/3231 last-modified: 2017-05-11 description: Measuring sensory sensitivity is important in studying development and developmental disorders. However, with children, there is a need to balance reliable but lengthy sensory tasks with the child’s ability to maintain motivation and vigilance. We used simulations to explore the problems associated with shortening adaptive psychophysical procedures, and suggest how these problems might be addressed. We quantify how adaptive procedures with too few reversals can over-estimate thresholds, introduce substantial measurement error, and make estimates of individual thresholds less reliable. The associated measurement error also obscures group differences. Adaptive procedures with children should therefore use as many reversals as possible, to reduce the effects of both Type 1 and Type 2 errors. Differences in response consistency, resulting from lapses in attention, further increase the over-estimation of threshold. Comparisons between data from individuals who may differ in lapse rate are therefore problematic, but measures to estimate and account for lapse rates in analyses may mitigate this problem. creator: Caroline Witton creator: Joel B. Talcott creator: G. Bruce Henning uri: https://doi.org/10.7717/peerj.3231 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Witton et al. title: A new molecular diagnostic tool for surveying and monitoring Triops cancriformis populations link: https://peerj.com/articles/3228 last-modified: 2017-05-11 description: The tadpole shrimp, Triops cancriformis, is a freshwater crustacean listed as endangered in the UK and Europe living in ephemeral pools. Populations are threatened by habitat destruction due to land development for agriculture and increased urbanisation. Despite this, there is a lack of efficient methods for discovering and monitoring populations. Established macroinvertebrate monitoring methods, such as net sampling, are unsuitable given the organism’s life history, that include long lived diapausing eggs, benthic habits and ephemerally active populations. Conventional hatching methods, such as sediment incubation, are both time consuming and potentially confounded by bet-hedging hatching strategies of diapausing eggs. Here we develop a new molecular diagnostic method to detect viable egg banks of T. cancriformis, and compare its performance to two conventional monitoring methods involving diapausing egg hatching. We apply this method to a collection of pond sediments from the Wildfowl & Wetlands Trust Caerlaverock National Nature Reserve, which holds one of the two remaining British populations of T. cancriformis. DNA barcoding of isolated eggs, using newly designed species-specific primers for a large region of mtDNA, was used to estimate egg viability. These estimates were compared to those obtained by the conventional methods of sediment and isolation hatching. Our method outperformed the conventional methods, revealing six ponds holding viable T. cancriformis diapausing egg banks in Caerlaverock. Additionally, designed species-specific primers for a short region of mtDNA identified degraded, inviable eggs and were used to ascertain the levels of recent mortality within an egg bank. Together with efficient sugar flotation techniques to extract eggs from sediment samples, our molecular method proved to be a faster and more powerful alternative for assessing the viability and condition of T. cancriformis diapausing egg banks. creator: Graham S. Sellers creator: Larry R. Griffin creator: Bernd Hänfling creator: Africa Gómez uri: https://doi.org/10.7717/peerj.3228 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Sellers et al. title: Poly(ADP-ribosylation) is present in murine sciatic nerve fibers and is altered in a Charcot-Marie-Tooth-1E neurodegenerative model link: https://peerj.com/articles/3318 last-modified: 2017-05-10 description: BackgroundPoly-ADP-ribose (PAR) is a polymer synthesized by poly-ADP-ribose polymerases (PARPs) as a postranslational protein modification and catabolized mainly by poly-ADP-ribose glycohydrolase (PARG). In spite of the existence of cytoplasmic PARPs and PARG, research has been focused on nuclear PARPs and PAR, demonstrating roles in the maintenance of chromatin architecture and the participation in DNA damage responses and transcriptional regulation. We have recently detected non-nuclear PAR structurally and functionally associated to the E-cadherin rich zonula adherens and the actin cytoskeleton of VERO epithelial cells. Myelinating Schwann cells (SC) are stabilized by E-cadherin rich autotypic adherens junctions (AJ). We wondered whether PAR would map to these regions. Besides, we have demonstrated an altered microfilament pattern in peripheral nerves of Trembler-J (Tr-J) model of CMT1-E. We hypothesized that cytoplasmic PAR would accompany such modified F-actin pattern.MethodsWild-type (WT) and Tr-J mice sciatic nerves cryosections were subjected to immunohistofluorescence with anti-PAR antibodies (including antibody validation), F-actin detection with a phalloidin probe and DAPI/DNA counterstaining. Confocal image stacks were subjected to a colocalization highlighter and to semi-quantitative image analysis.ResultsWe have shown for the first time the presence of PAR in sciatic nerves. Cytoplasmic PAR colocalized with F-actin at non-compact myelin regions in WT nerves. Moreover, in Tr-J, cytoplasmic PAR was augmented in close correlation with actin. In addition, nuclear PAR was detected in WT SC and was moderately increased in Tr-J SC.DiscussionThe presence of PAR associated to non-compact myelin regions (which constitute E-cadherin rich autotypic AJ/actin anchorage regions) and the co-alterations experienced by PAR and the actin cytoskeleton in epithelium and nerves, suggest that PAR may be a constitutive component of AJ/actin anchorage regions. Is PAR stabilizing the AJ-actin complexes? This question has strong implications in structural cell biology and cell signaling networks. Moreover, if PAR played a stabilizing role, such stabilization could participate in the physiological control of axonal branching. PARP and PAR alterations exist in several neurodegenerative pathologies including Alzheimer’s, Parkinson’s and Hungtington’s diseases. Conversely, PARP inhibition decreases PAR and promotes neurite outgrowth in cortical neurons in vitro. Coherently, the PARP inhibitor XAV939 improves myelination in vitro, ex vivo and in vivo. Until now such results have been interpreted in terms of nuclear PARP activity. Our results indicate for the first time the presence of PARylation in peripheral nerve fibers, in a healthy environment. Besides, we have evidenced a PARylation increase in Tr-J, suggesting that the involvement of cytoplasmic PARPs and PARylation in normal and neurodegenerative conditions should be re-evaluated. creator: Laura I. Lafon Hughes creator: Carlos J. Romeo Cardeillac creator: Karina B. Cal Castillo creator: Salomé C. Vilchez Larrea creator: José R. Sotelo Sosa creator: Gustavo A. Folle Ungo creator: Silvia H. Fernández Villamil creator: Alejandra E. Kun González uri: https://doi.org/10.7717/peerj.3318 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Lafon Hughes et al. title: A survey of fishes associated with Hawaiian deep-water Halimeda kanaloana (Bryopsidales: Halimedaceae) and Avrainvillea sp. (Bryopsidales: Udoteaceae) meadows link: https://peerj.com/articles/3307 last-modified: 2017-05-10 description: The invasive macroalgal species Avrainvillea sp. and native species Halimeda kanaloana form expansive meadows that extend to depths of 80 m or more in the waters off of O‘ahu and Maui, respectively. Despite their wide depth distribution, comparatively little is known about the biota associated with these macroalgal species. Our primary goals were to provide baseline information on the fish fauna associated with these deep-water macroalgal meadows and to compare the abundance and diversity of fishes between the meadow interior and sandy perimeters. Because both species form structurally complex three-dimensional canopies, we hypothesized that they would support a greater abundance and diversity of fishes when compared to surrounding sandy areas. We surveyed the fish fauna associated with these meadows using visual surveys and collections made with clove-oil anesthetic. Using these techniques, we recorded a total of 49 species from 25 families for H. kanaloana meadows and surrounding sandy areas, and 28 species from 19 families for Avrainvillea sp. habitats. Percent endemism was 28.6% and 10.7%, respectively. Wrasses (Family Labridae) were the most speciose taxon in both habitats (11 and six species, respectively), followed by gobies for H. kanaloana (six species). The wrasse Oxycheilinus bimaculatus and cardinalfish Apogonichthys perdix were the most frequently-occurring species within the H. kanaloana and Avrainvillea canopies, respectively. Obligate herbivores and food-fish species were rare in both habitats. Surprisingly, the density and abundance of small epibenthic fishes were greater in open sand than in the meadow canopy. In addition, species richness was also higher in open sand for Avrainvillea sp. We hypothesize that the dense holdfasts and rhizoids present within the meadow canopy may impede benthic-dwelling or bioturbator species, which accounted for 86% and 57% of individuals collected in sand adjacent to H. kanaloana and Avrainvillea sp. habitats, respectively. Of the 65 unique species recorded in this study, 16 (25%) were detected in clove oil stations alone, illustrating the utility of clove-oil anesthetic in assessing the diversity and abundance of small-bodied epibenthic fishes. creator: Ross C. Langston creator: Heather L. Spalding uri: https://doi.org/10.7717/peerj.3307 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Langston and Spalding title: Sooty tern (Onychoprion fuscatus) survival, oil spills, shrimp fisheries, and hurricanes link: https://peerj.com/articles/3287 last-modified: 2017-05-10 description: Migratory seabirds face threats from climate change and a variety of anthropogenic disturbances. Although most seabird research has focused on the ecology of individuals at the colony, technological advances now allow researchers to track seabird movements at sea and during migration. We combined telemetry data on Onychoprion fuscatus (sooty terns) with a long-term capture-mark-recapture dataset from the Dry Tortugas National Park to map the movements at sea for this species, calculate estimates of mortality, and investigate the impact of hurricanes on a migratory seabird. Included in the latter analysis is information on the locations of recovered bands from deceased individuals wrecked by tropical storms. We present the first known map of sooty tern migration in the Atlantic Ocean. Our results indicate that the birds had minor overlaps with areas affected by the major 2010 oil spill and a major shrimp fishery. Indices of hurricane strength and occurrence are positively correlated with annual mortality and indices of numbers of wrecked birds. As climate change may lead to an increase in severity and frequency of major hurricanes, this may pose a long-term problem for this colony. creator: Ryan M. Huang creator: Oron L. Bass Jr creator: Stuart L. Pimm uri: https://doi.org/10.7717/peerj.3287 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Huang et al. title: Screening of a natural compound library identifies emodin, a natural compound from Rheum palmatum Linn that inhibits DPP4 link: https://peerj.com/articles/3283 last-modified: 2017-05-10 description: Historically, Chinese herbal medicines have been widely used in the treatment of hyperglycemia, but the mechanisms underlying their effectiveness remain largely unknown. Here, we screened a compound library primarily comprised of natural compounds extracted from herbs and marine organisms. The results showed that emodin, a natural compound from Rheum palmatum Linn, inhibited DPP4 activity with an in vitro IC50 of 5.76 µM without inhibiting either DPP8 or DPP9. A docking model revealed that emodin binds to DPP4 protein through Glu205 and Glu206, although with low affinity. Moreover, emodin treatment (3, 10 and 30 mg/kg, P.O.) in mice decreased plasma DPP4 activity in a dose-dependent manner. Our study suggests that emodin inhibits DPP4 activity and may represent a novel therapeutic for the treatment of type 2 diabetes. creator: Zhaokai Wang creator: Longhe Yang creator: Hu Fan creator: Peng Wu creator: Fang Zhang creator: Chao Zhang creator: Wenjie Liu creator: Min Li uri: https://doi.org/10.7717/peerj.3283 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Wang et al. title: Analysis of genomic variation in lung adenocarcinoma patients revealed the critical role of PI3K complex link: https://peerj.com/articles/3216 last-modified: 2017-05-10 description: BackgroundMolecularly targeted therapies improved survival status of some patients with lung adenocarcinoma, which accounts for 40% of all lung cancers, and in-depth study of gene alterations is important for the personalized treatment.MethodsThe legacy archive data of clinical information and genomic variations under the project TCGA Lung Adenocarcinoma were downloaded from the GDC Data Portal using R package TCGAbiolinks. The significantly aberrant copy number variants segments were figured out using GAIA. After annotation, the genes involving CNV were used to get enriched pathways. Recurrent amplifications and deletions were identified and visualized by OncoPrint. Genomic alterations in cancer, including CNV and mutations, were represented in Circos.ResultsThe significantly aberrant CNV segments were found, and the genes involved were associated with the immune system. In an analysis of 517 mutation annotated files, we highlighted 63 highly recurrent mutated genes which were associated with lung cancer signaling. These genes involved in important pathways related to cancer progression. The intersections between the genes involving in the significantly aberrant CNV and the genes harboring recurrent somatic SNP were extracted. The PI3K protein family acted as critical roles in the lung adenocarcinoma, since the components of the PI3K protein family include PIK3C2B, PIK3CA, PIK3R1 and so forth were presented in the intersections.ConclusionWe represented a comprehensive annotation of genomic alterations in lung adenocarcinoma and proposed that PI3K signaling proteins were critical for it. creator: Zhao min Deng creator: Lin Liu creator: Wen hai Qiu creator: Yong qun Zhang creator: Hong yan Zhong creator: Ping Liao creator: Yun hong Wu uri: https://doi.org/10.7717/peerj.3216 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Deng et al. title: Transcriptome sequencing and SNP detection in Phoebe chekiangensis link: https://peerj.com/articles/3193 last-modified: 2017-05-10 description: BackgroundPhoebe chekiangensis is a rare tree species that is only distributed in south-eastern China. Although this species is famous for its excellent wood properties, it has not been extensively studied at the molecular level.ResultsHere, the transcriptome of P. chekiangensis was sequenced using next-generation sequencing technology, and 75,647 transcripts with 48,011 unigenes were assembled and annotated. In addition, 162,938 putative single nucleotide polymorphisms (SNPs) were predicted and 25 were further validated using the Sanger method.ConclusionThe currently available SNP prediction software packages showed low levels of correspondence when compared. The transcriptome and SNPs will contribute to the exploration of P. chekiangensis genetic resources and the understanding of its molecular mechanisms. creator: Bing He creator: Yingang Li creator: Zhouxian Ni creator: Li-an Xu uri: https://doi.org/10.7717/peerj.3193 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 He et al. title: Cave features, seasonality and subterranean distribution of non-obligate cave dwellers link: https://peerj.com/articles/3169 last-modified: 2017-05-10 description: Seasonality impacts species distributions through changes of the environmental conditions that affect the presence of individuals at a given place. Although the dynamics of cave microclimates are well known, only a few studies have evaluated the effects of such dynamics on non-strictly cave species. Here we assessed if species exploiting subterranean environments show changes in habitat occupation related to seasonal variation of cave microclimates. We surveyed 16 caves in central Italy every month for one year. Caves were subdivided into longitudinal sectors of three meters. In each sector we measured cave morphology and microclimatic features, assessed the occurrence of eight non-troglobitic taxa (orthopterans, spiders, gastropods and amphibians), and related species distribution to environmental features and sampling periods. The occurrence of most species was related to both cave morphology and microclimatic features. The survey month was the major factor determining the presence of species in cave sectors, indicating that cave-dwelling taxa show strong seasonality in activity and distribution. For multiple species, we detected interactions between sampling period and microclimatic features, suggesting that species may associate with different microhabitats throughout the year. The richest communities were found in sites with specific microclimates (i.e., high humidity, warm temperature and low light) but seasonality for species richness was strong as well, stressing the complexity of interactions between outdoor and subterranean environments. creator: Enrico Lunghi creator: Raoul Manenti creator: Gentile Francesco Ficetola uri: https://doi.org/10.7717/peerj.3169 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Lunghi et al. title: Intervention effects of five cations and their correction on hemolytic activity of tentacle extract from the jellyfish Cyanea capillata link: https://peerj.com/articles/3338 last-modified: 2017-05-09 description: Cations have generally been reported to prevent jellyfish venom-induced hemolysis through multiple mechanisms by spectrophotometry. Little attention has been paid to the potential interaction between cations and hemoglobin, potentially influencing the antagonistic effect of cations. Here, we explored the effects of five reported cations, La3+, Mn2+, Zn2+, Cu2+ and Fe2+, on a hemolytic test system and the absorbance of hemoglobin, which was further used to measure their effects on the hemolysis of tentacle extract (TE) from the jellyfish Cyanea capillata. All the cations displayed significant dose-dependent inhibitory effects on TE-induced hemolysis with various dissociation equilibrium constant (Kd) values as follows: La3+ 1.5 mM, Mn2+ 93.2 mM, Zn2+ 38.6 mM, Cu2+ 71.9 μM and Fe2+ 32.8 mM. The transparent non-selective pore blocker La3+ did not affect the absorbance of hemoglobin, while Mn2+ reduced it slightly. Other cations, including Zn2+, Cu2+ and Fe2+, greatly decreased the absorbance with Kd values of 35.9, 77.5 and 17.6 mM, respectively. After correction, the inhibitory Kd values were 1.4 mM, 45.8 mM, 128.5 μM and 53.1 mM for La3+, Zn2+, Cu2+ and Fe2+, respectively. Mn2+ did not inhibit TE-induced hemolysis. Moreover, the inhibitory extent at the maximal given dose of all cations except La3+ was also diminished. These corrected results from spectrophotometry were further confirmed by direct erythrocyte counting under microscopy. Our results indicate that the cations, except for La3+, can interfere with the absorbance of hemoglobin, which should be corrected when their inhibitory effects on hemolysis by jellyfish venoms are examined. The variation in the inhibitory effects of cations suggests that the hemolysis by jellyfish venom is mainly attributed to the formation of non-selective cation pore complexes over other potential mechanisms, such as phospholipases A2 (PLA2), polypeptides, protease and oxidation. Blocking the pore-forming complexes may be a primary strategy to improve the in vivo damage and mortality from jellyfish stings due to hemolytic toxicity. creator: Hui Zhang creator: Qianqian Wang creator: Liang Xiao creator: Liming Zhang uri: https://doi.org/10.7717/peerj.3338 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Zhang et al. title: Divergent effect of fluoxetine on the response to physical or chemical stressors in zebrafish link: https://peerj.com/articles/3330 last-modified: 2017-05-09 description: Fluoxetine is a selective serotonin reuptake inhibitor that increases serotonin concentration in the central nervous system and modulates various systems, including the control of sympathetic outflow and the hypothalamus–pituitary–adrenal. However, it is not yet established whether fluoxetine can modulate the responses to stressors stimulants (physical or chemical) that trigger cortisol response in zebrafish. We demonstrate that fluoxetine blunts the response to physical stress, but not to chemical stress. creator: Murilo S. Abreu creator: Ana Cristina V.V. Giacomini creator: Gessi Koakoski creator: Angelo L.S. Piato creator: Leonardo J.G. Barcellos uri: https://doi.org/10.7717/peerj.3330 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Abreu et al. title: Social relationships enhance the time spent eating and intake of a novel diet in pregnant Hanwoo (Bos taurus coreanae) heifers link: https://peerj.com/articles/3329 last-modified: 2017-05-09 description: The objective of this study was to evaluate the effects of social relationships on the feed intake, eating behavior, and growth, upon exposure to a novel diet, in Hanwoo (Bos taurus coreanae) heifers during pregnancy. Twenty-four pregnant Hanwoo heifers, averaging 438 ± 27.8 kg in weight, 21 months in age, and 194 ± 8.5 days in pregnancy, were involved in a two-month (eight weeks) experiment. The heifers were randomly assigned to either the single housing group (SG; one individual per pen, n = 12), or the paired housing group (PG; two individuals per pen, n = 12). All pens were of the same size (5 × 5 m) and provided with one feed bin, which automatically recorded the individual feed intake and eating behavior. As the experiment began, the diet of the heifers was switched from a total mixed ration (TMR; 250 g/kg ryegrass straw and 750 g/kg concentrate mix) to a forage-only diet (mixed hay cubes composed of 500 g/kg alfalfa, 250 g/kg timothy, and 250 g/kg blue grass hay). The heifers were fed ad libitum twice a day. The individual feed intake and eating behavior were recorded daily throughout the experiment, and body weights (BWs) were measured every four weeks before the morning feeding. PG animals visited the feed bin 22% less often than SG. PG, however, stayed 39% longer in the feed bin and consumed 40% more feed per visit, compared with SG. Consequently, PG heifers spent 23% more time in eating and had 16% more daily dry matter intake than SG during the experiment. Average daily gain during the experimental period tended to be greater in PG than in SG. When pregnant Hanwoo heifers encountered a novel diet, social relationships (i.e., presence of a pen-mate) enhanced their time spent eating and feed intake. Social interactions, even with an unfamiliar individual, may be helpful for pregnant Hanwoo heifers cope with a diet challenge compared to solitary situation. creator: Dong-Han Shin creator: Hyun-Min Kang creator: Seongwon Seo uri: https://doi.org/10.7717/peerj.3329 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2017 Shin et al. title: Student evaluations of teaching: teaching quantitative courses can be hazardous to one’s career link: https://peerj.com/articles/3299 last-modified: 2017-05-09 description: Anonymous student evaluations of teaching (SETs) are used by colleges and universities to measure teaching effectiveness and to make decisions about faculty hiring, firing, re-appointment, promotion, tenure, and merit pay. Although numerous studies have found that SETs correlate with various teaching effectiveness irrelevant factors (TEIFs) such as subject, class size, and grading standards, it has been argued that such correlations are small and do not undermine the validity of SETs as measures of professors’ teaching effectiveness. However, previous research has generally used inappropriate parametric statistics and effect sizes to examine and to evaluate the significance of TEIFs on personnel decisions. Accordingly, we examined the influence of quantitative vs. non-quantitative courses on SET ratings and SET based personnel decisions using 14,872 publicly posted class evaluations where each evaluation represents a summary of SET ratings provided by individual students responding in each class. In total, 325,538 individual student evaluations from a US mid-size university contributed to theses class evaluations. The results demonstrate that class subject (math vs. English) is strongly associated with SET ratings, has a substantial impact on professors being labeled satisfactory vs. unsatisfactory and excellent vs. non-excellent, and the impact varies substantially depending on the criteria used to classify professors as satisfactory vs. unsatisfactory. Professors teaching quantitative courses are far more likely not to receive tenure, promotion, and/or merit pay when their performance is evaluated against common standards. creator: Bob Uttl creator: Dylan Smibert uri: https://doi.org/10.7717/peerj.3299 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Uttl and Smibert title: A public dataset of running biomechanics and the effects of running speed on lower extremity kinematics and kinetics link: https://peerj.com/articles/3298 last-modified: 2017-05-09 description: BackgroundThe goals of this study were (1) to present the set of data evaluating running biomechanics (kinematics and kinetics), including data on running habits, demographics, and levels of muscle strength and flexibility made available at Figshare (DOI: 10.6084/m9.figshare.4543435); and (2) to examine the effect of running speed on selected gait-biomechanics variables related to both running injuries and running economy.MethodsThe lower-extremity kinematics and kinetics data of 28 regular runners were collected using a three-dimensional (3D) motion-capture system and an instrumented treadmill while the subjects ran at 2.5 m/s, 3.5 m/s, and 4.5 m/s wearing standard neutral shoes.ResultsA dataset comprising raw and processed kinematics and kinetics signals pertaining to this experiment is available in various file formats. In addition, a file of metadata, including demographics, running characteristics, foot-strike patterns, and muscle strength and flexibility measurements is provided. Overall, there was an effect of running speed on most of the gait-biomechanics variables selected for this study. However, the foot-strike patterns were not affected by running speed.DiscussionSeveral applications of this dataset can be anticipated, including testing new methods of data reduction and variable selection; for educational purposes; and answering specific research questions. This last application was exemplified in the study’s second objective. creator: Reginaldo K. Fukuchi creator: Claudiane A. Fukuchi creator: Marcos Duarte uri: https://doi.org/10.7717/peerj.3298 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Fukuchi et al. title: Pollen extracts and constituent sugars increase growth of a trypanosomatid parasite of bumble bees link: https://peerj.com/articles/3297 last-modified: 2017-05-09 description: Phytochemicals produced by plants, including at flowers, function in protection against plant diseases, and have a long history of use against trypanosomatid infection. Floral nectar and pollen, the sole food sources for many species of insect pollinators, contain phytochemicals that have been shown to reduce trypanosomatid infection in bumble and honey bees when fed as isolated compounds. Nectar and pollen, however, consist of phytochemical mixtures, which can have greater antimicrobial activity than do single compounds. This study tested the hypothesis that pollen extracts would inhibit parasite growth. Extracts of six different pollens were tested for direct inhibitory activity against cell cultures of the bumble bee trypanosomatid gut parasite Crithidia bombi. Surprisingly, pollen extracts increased parasite growth rather than inhibiting it. Pollen extracts contained high concentrations of sugars, mainly the monosaccharides glucose and fructose. Experimental manipulations of growth media showed that supplemental monosaccharides (glucose and fructose) increased maximum cell density, while a common floral phytochemical (caffeic acid) with inhibitory activity against other trypanosomatids had only weak inhibitory effects on Crithidia bombi. These results indicate that, although pollen is essential for bees and other pollinators, pollen may promote growth of intestinal parasites that are uninhibited by pollen phytochemicals and, as a result, can benefit from the nutrients that pollen provides. creator: Evan C. Palmer-Young creator: Lucy Thursfield uri: https://doi.org/10.7717/peerj.3297 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Palmer-Young and Thursfield title: Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12 link: https://peerj.com/articles/3293 last-modified: 2017-05-09 description: RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, also in the E. coli K12 context. However, it did not encode a site-specific recombinase as mobile genomic islands usually do. It was then deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity. creator: María F. Azpiroz creator: Magela Laviña uri: https://doi.org/10.7717/peerj.3293 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Azpiroz and Laviña title: Toward a better understanding of the mechanisms of symbiosis: a comprehensive proteome map of a nascent insect symbiont link: https://peerj.com/articles/3291 last-modified: 2017-05-09 description: Symbiotic bacteria are common in insects and can affect various aspects of their hosts’ biology. Although the effects of insect symbionts have been clarified for various insect symbiosis models, due to the difficulty of cultivating them in vitro, there is still limited knowledge available on the molecular features that drive symbiosis. Serratia symbiotica is one of the most common symbionts found in aphids. The recent findings of free-living strains that are considered as nascent partners of aphids provide the opportunity to examine the molecular mechanisms that a symbiont can deploy at the early stages of the symbiosis (i.e., symbiotic factors). In this work, a proteomic approach was used to establish a comprehensive proteome map of the free-living S. symbiotica strain CWBI-2.3T. Most of the 720 proteins identified are related to housekeeping or primary metabolism. Of these, 76 were identified as candidate proteins possibly promoting host colonization. Our results provide strong evidence that S. symbiotica CWBI-2.3T is well-armed for invading insect host tissues, and suggest that certain molecular features usually harbored by pathogenic bacteria are no longer present. This comprehensive proteome map provides a series of candidate genes for further studies to understand the molecular cross-talk between insects and symbiotic bacteria. creator: François Renoz creator: Antoine Champagne creator: Hervé Degand creator: Anne-Marie Faber creator: Pierre Morsomme creator: Vincent Foray creator: Thierry Hance uri: https://doi.org/10.7717/peerj.3291 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Renoz et al. title: Network science meets respiratory medicine for OSAS phenotyping and severity prediction link: https://peerj.com/articles/3289 last-modified: 2017-05-09 description: Obstructive sleep apnea syndrome (OSAS) is a common clinical condition. The way that OSAS risk factors associate and converge is not a random process. As such, defining OSAS phenotypes fosters personalized patient management and population screening. In this paper, we present a network-based observational, retrospective study on a cohort of 1,371 consecutive OSAS patients and 611 non-OSAS control patients in order to explore the risk factor associations and their correlation with OSAS comorbidities. To this end, we construct the Apnea Patients Network (APN) using patient compatibility relationships according to six objective parameters: age, gender, body mass index (BMI), blood pressure (BP), neck circumference (NC) and the Epworth sleepiness score (ESS). By running targeted network clustering algorithms, we identify eight patient phenotypes and corroborate them with the co-morbidity types. Also, by employing machine learning on the uncovered phenotypes, we derive a classification tree and introduce a computational framework which render the Sleep Apnea Syndrome Score (SASScore); our OSAS score is implemented as an easy-to-use, web-based computer program which requires less than one minute for processing one individual. Our evaluation, performed on a distinct validation database with 231 consecutive patients, reveals that OSAS prediction with SASScore has a significant specificity improvement (an increase of 234%) for only 8.2% sensitivity decrease in comparison with the state-of-the-art score STOP-BANG. The fact that SASScore has bigger specificity makes it appropriate for OSAS screening and risk prediction in big, general populations. creator: Stefan Mihaicuta creator: Mihai Udrescu creator: Alexandru Topirceanu creator: Lucretia Udrescu uri: https://doi.org/10.7717/peerj.3289 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Mihaicuta et al. title: Direct evidence of megamammal-carnivore interaction decoded from bone marks in historical fossil collections from the Pampean region link: https://peerj.com/articles/3117 last-modified: 2017-05-09 description: Pleistocene South American megafauna has traditionally attracted the interest of scientists and the popular media alike. However, ecological interactions between the species that inhabited these ecosystems, such as predator-prey relationships or interspecific competition, are poorly known. To this regard, carnivore marks imprinted on the fossil bones of megamammal remains are very useful for deciphering biological activity and, hence, potential interspecific relationships among taxa. In this article, we study historical fossil collections housed in different European and Argentinean museums that were excavated during the 19th and early 20th centuries in the Pampean region, Argentina, in order to detect carnivore marks on bones of megamammals and provide crucial information on the ecological relationships between South American taxa during the Pleistocene. Our results indicate that the long bones of megafauna from the Pampean region (e.g., the Mylodontidae and Toxodontidae families) exhibit carnivore marks. Furthermore, long bones of medium-sized species and indeterminate bones also present punctures, pits, scores and fractures. Members of the large-carnivore guild, such as ursids, canids and even felids, are recognised as the main agents that inflicted the marks. We hypothesize that the analysed carnivore marks represent the last stages of megaherbivore carcass exploitation, suggesting full consumption of these animals by the same or multiple taxa in a hunting and/or scavenging scenario. Moreover, our observations provide novel insights that help further our understanding of the palaeoecological relationships of these unique communities of megamammals. creator: Karina Vanesa Chichkoyan creator: Borja Figueirido creator: Margarita Belinchón creator: José Luis Lanata creator: Anne-Marie Moigne creator: Bienvenido Martínez-Navarro uri: https://doi.org/10.7717/peerj.3117 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Chichkoyan et al. title: Running wheel training does not change neurogenesis levels or alter working memory tasks in adult rats link: https://peerj.com/articles/2976 last-modified: 2017-05-09 description: BackgroundExercise can change cellular structure and connectivity (neurogenesis or synaptogenesis), causing alterations in both behavior and working memory. The aim of this study was to evaluate the effect of exercise on working memory and hippocampal neurogenesis in adult male Wistar rats using a T-maze test.MethodsAn experimental design with two groups was developed: the experimental group (n = 12) was subject to a forced exercise program for five days, whereas the control group (n = 9) stayed in the home cage. Six to eight weeks after training, the rats’ working memory was evaluated in a T-maze test and four choice days were analyzed, taking into account alternation as a working memory indicator. Hippocampal neurogenesis was evaluated by means of immunohistochemistry of BrdU positive cells.ResultsNo differences between groups were found in the behavioral variables (alternation, preference index, time of response, time of trial or feeding), or in the levels of BrdU positive cells.DiscussionResults suggest that although exercise may have effects on brain structure, a construct such as working memory may require more complex changes in networks or connections to demonstrate a change at behavioral level. creator: Cesar A. Acevedo-Triana creator: Manuel J. Rojas creator: Fernando P. Cardenas uri: https://doi.org/10.7717/peerj.2976 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Acevedo-Triana et al. title: Anhanguera taxonomy revisited: is our understanding of Santana Group pterosaur diversity biased by poor biological and stratigraphic control? link: https://peerj.com/articles/3285 last-modified: 2017-05-04 description: BackgroundAnhanguerids comprise an important clade of pterosaurs, mostly known from dozens of three-dimensionally preserved specimens recovered from the Lower Cretaceous Romualdo Formation (northeastern Brazil). They are remarkably diverse in this sedimentary unit, with eight named species, six of them belonging to the genus Anhanguera. However, such diversity is likely overestimated, as these species have been historically diagnosed based on subtle differences, mainly based on the shape and position of the cranial crest. In spite of that, recently discovered pterosaur taxa represented by large numbers of individuals, including juveniles and adults, as well as presumed males and females, have crests of sizes and shapes that are either ontogenetically variable or sexually dimorphic.MethodsWe describe in detail the skull of one of the most complete specimens referred to Anhanguera, AMNH 22555, and use it as a case study to review the diversity of anhanguerids from the Romualdo Formation. In order to accomplish that, a geometric morphometric analysis was performed to assess size-dependent characters with respect to the premaxillary crest in the 12 most complete skulls bearing crests that are referred in, or related to, this clade, almost all of them analyzed first hand.ResultsGeometric morphometric regression of shape on centroid size was highly statistically significant (p = 0.0091) and showed that allometry accounts for 25.7% of total shape variation between skulls of different centroid sizes. Premaxillary crests are both taller and anteroposteriorly longer in larger skulls, a feature consistent with ontogenetic growth. A new diagnosis is proposed for Anhanguera, including traits that are nowadays known to be widespread within the genus, as well as ontogenetic changes. AMNH 22555 cannot be referred to “Anhanguera santanae” and, in fact, “Anhanguera santanae”, “Anhanguera araripensis”, and “Anhanguera robustus” are here considered nomina dubia.DiscussionHistorically, minor differences in crest morphology have been used in the definition of new anhanguerid species. Nowadays, this practice resulted in a considerable difficulty in referring well-preserved skulls into known taxa. When several specimens are analyzed, morphologies previously believed to be disparate are, in fact, separated by a continuum, and are thus better explained as individual or temporal variations. Stratigraphically controlled excavations on the Romualdo Formation have showed evidence for faunal turnover regarding fish communities. It is thus possible that some of the pterosaurs from this unit were not coeval, and might even represent anagenetic morphotypes. Unfortunately, amateur collecting of Romualdo Formation fossils, aimed especially at commerce, resulted in the lack of stratigraphic data of virtually all its pterosaurs and precludes testing of these further hypotheses. creator: Felipe L. Pinheiro creator: Taissa Rodrigues uri: https://doi.org/10.7717/peerj.3285 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Pinheiro and Rodrigues title: Plants from the abandoned Nacozari mine tailings: evaluation of their phytostabilization potential link: https://peerj.com/articles/3280 last-modified: 2017-05-04 description: Phytostabilization is a remediation technology that uses plants for in-situ stabilization of contamination in soils and mine tailings. The objective of this study was to identify native plant species with potential for phytostabilization of the abandoned mine tailings in Nacozari, Sonora in northern Mexico. A flora of 42 species in 16 families of angiosperms was recorded on the tailings site and the abundance of the most common perennial species was estimated. Four of the five abundant perennial species showed evidence of regeneration: the ability to reproduce and establish new seedlings. A comparison of selected physicochemical properties of the tailings in vegetated patches with adjacent barren areas suggests that pH, electrical conductivity, texture, and concentration of potentially toxic elements do not limit plant distribution. For the most abundant species, the accumulation factor for most metals was <1, with the exception of Zn in two species. A short-term experiment on adaptation revealed limited evidence for the formation of local ecotypes in Prosopis velutina and Amaranthus watsonii. Overall, the results of this study indicate that five native plant species might have potential for phytostabilization of the Nacozari tailings and that seed could be collected locally to revegetate the site. More broadly, this study provides a methodology that can be used to identify native plants and evaluate their phytostabilization potential for similar mine tailings. creator: Alina E. Santos creator: Rocio Cruz-Ortega creator: Diana Meza-Figueroa creator: Francisco M. Romero creator: Jose Jesus Sanchez-Escalante creator: Raina M. Maier creator: Julia W. Neilson creator: Luis David Alcaraz creator: Francisco E. Molina Freaner uri: https://doi.org/10.7717/peerj.3280 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Santos et al. title: Identification of demographic factors and health problems that affect the acceptance of disease and health behaviors of patients with osteoarthritis link: https://peerj.com/articles/3276 last-modified: 2017-05-04 description: IntroductionOsteoarthritis (OA) is one of the most common causes of musculoskeletal system’s ailments. In the prevention of the disease and in its comprehensive treatment, proper health-related behavior becomes an extremely important factor for maintaining an optimal health condition. The aim of the study is to assess the relationship between the reported pain and the disability level, and the health-related behaviors undertaken by OA patients as well as their acceptance of the disease.Materials/MethodsThe study group consisted of 198 patients with diagnosed OA, according to ACR criteria (1988). The method used in the study employed a Pain VAS (0-10), Health Assessment Questionnaire Disability Index (HAQ DI 0-3), Acceptance of Illness Scale (AIS 8-40) and Health and Behavior Inventory (IZZ 24-120).ResultsThe average age among respondents with OA has been 59.16 years of age (±15.87), duration of disease 5.5 years (±4.32). Pain experienced both during movement (rs = 0.319, p < 0.001) and at rest (rs = 0.382, p < 0.001) correlated positively with physical disability (HAQ DI). Studies indicated a positive linear correlation between the age and physical disability (rs = 0.200, p = 0.005). Acceptance of the disease (AIS) depends mostly on age (rs =  − 0.325, p < 0.001), on pain in motion (rp =  − 0.209, p < 0.001) and at rest (rp =  − 0.218, p < 0.001) and on the disability levels (rp =  − 0.353, p < 0.001). Analysis of the health-related behaviors (IZZ) indicates that the average severity of declared behavior is statistically significant with physical disability (HAQ DI) (p = 0.029).ConclusionsThe acceptance of illness is significantly reduced with age and progressive levels of disability as well as with the severity of pain. The progressive levels of disability and the younger age of the respondents motivate them to engage in health beneficial behavior. creator: Matylda Sierakowska creator: Izabela Wysocka-Skurska creator: Wojciech Kułak uri: https://doi.org/10.7717/peerj.3276 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Sierakowska et al. title: Oceanic adults, coastal juveniles: tracking the habitat use of whale sharks off the Pacific coast of Mexico link: https://peerj.com/articles/3271 last-modified: 2017-05-04 description: Eight whale sharks tagged with pop-up satellite archival tags off the Gulf of California, Mexico, were tracked for periods of 14–134 days. Five of these sharks were adults, with four females visually assessed to be pregnant. At least for the periods they were tracked, juveniles remained in the Gulf of California while adults moved offshore into the eastern Pacific Ocean. We propose that parturition occurs in these offshore waters. Excluding two juveniles that remained in the shallow tagging area for the duration of tracking, all sharks spent 65 ± 20.7% (SD) of their time near the surface, even over deep water, often in association with frontal zones characterized by cool-water upwelling. While these six sharks all made dives into the meso- or bathypelagic zones, with two sharks reaching the maximum depth recordable by the tags (1285.8 m), time spent at these depths represented a small proportion of the overall tracks. Most deep dives (72.7%) took place during the day, particularly during the early morning and late afternoon. Pronounced habitat differences by ontogenetic stage suggest that adult whale sharks are less likely to frequent coastal waters after the onset of maturity. creator: Dení Ramírez-Macías creator: Nuno Queiroz creator: Simon J. Pierce creator: Nicolas E. Humphries creator: David W. Sims creator: Juerg M. Brunnschweiler uri: https://doi.org/10.7717/peerj.3271 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Ramírez-Macías et al. title: Photoperiodic regime influences onset of lens opacities in a non-human primate link: https://peerj.com/articles/3258 last-modified: 2017-05-04 description: BackgroundOpacities of the lens are typical age-related phenomena which have a high influence on photoreception and consequently circadian rhythm. In mouse lemurs, a small bodied non-human primate, a high incidence (more than 50% when >seven years) of cataracts has been previously described during aging. Previous studies showed that photoperiodically induced accelerated annual rhythms alter some of mouse lemurs’ life history traits. Whether a modification of photoperiod also affects the onset of age dependent lens opacities has not been investigated so far. The aim of this study was therefore to characterise the type of opacity and the mouse lemurs’ age at its onset in two colonies with different photoperiodic regimen.MethodsTwo of the largest mouse lemur colonies in Europe were investigated: Colony 1 having a natural annual photoperiodic regime and Colony 2 with an induced accelerated annual cycle. A slit-lamp was used to determine opacities in the lens. Furthermore, a subset of all animals which showed no opacities in the lens nucleus in the first examination but developed first changes in the following examination were further examined to estimate the age at onset of opacities. In total, 387 animals were examined and 57 represented the subset for age at onset estimation.ResultsThe first and most commonly observable opacity in the lens was nuclear sclerosis. Mouse lemurs from Colony 1 showed a delayed onset of nuclear sclerosis compared to mouse lemurs from Colony 2 (4.35 ± 1.50 years vs. 2.75 ± 0.99 years). For colony 1, the chronological age was equivalent to the number of seasonal cycles experienced by the mouse lemurs. For colony 2, in which seasonal cycles were accelerated by a factor of 1.5, mouse lemurs had experienced 4.13 ± 1.50 seasonal cycles in 2.75 ± 0.99 chronological years.DiscussionOur study showed clear differences in age at the onset of nuclear sclerosis formation between lemurs kept under different photoperiodic regimes. Instead of measuring the chronological age, the number of seasonal cycles (N = four) experienced by a mouse lemur can be used to estimate the risk of beginning nuclear sclerosis formation. Ophthalmological examinations should be taken into account when animals older than 5–6 seasonal cycles are used for experiments in which unrestricted visual ability has to be ensured. This study is the first to assess and demonstrate the influence of annual photoperiod regime on the incidence of lens opacities in a non-human primate. creator: Marko Dubicanac creator: Julia Strueve creator: Nadine Mestre-Frances creator: Jean-Michel Verdier creator: Elke Zimmermann creator: Marine Joly uri: https://doi.org/10.7717/peerj.3258 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Dubicanac et al. title: Genome-wide analysis of WRKY transcription factors in wheat (Triticum aestivum L.) and differential expression under water deficit condition link: https://peerj.com/articles/3232 last-modified: 2017-05-04 description: BackgroundWRKY proteins, which comprise one of the largest transcription factor (TF) families in the plant kingdom, play crucial roles in plant development and stress responses. Despite several studies on WRKYs in wheat (Triticum aestivum L.), functional annotation information about wheat WRKYs is limited.ResultsHere, 171 TaWRKY TFs were identified from the whole wheat genome and compared with proteins from 19 other species representing nine major plant lineages. A phylogenetic analysis, coupled with gene structure analysis and motif determination, divided these TaWRKYs into seven subgroups (Group I, IIa–e, and III). Chromosomal location showed that most TaWRKY genes were enriched on four chromosomes, especially on chromosome 3B. In addition, 85 (49.7%) genes were either tandem (5) or segmental duplication (80), which suggested that though tandem duplication has contributed to the expansion of TaWRKY family, segmental duplication probably played a more pivotal role. Analysis of cis-acting elements revealed putative functions of WRKYs in wheat during development as well as under numerous biotic and abiotic stresses. Finally, the expression of TaWRKY genes in flag leaves, glumes, and lemmas under water-deficit condition were analyzed. Results showed that different TaWRKY genes preferentially express in specific tissue during the grain-filling stage.ConclusionOur results provide a more extensive insight on WRKY gene family in wheat, and also contribute to the screening of more candidate genes for further investigation on function characterization of WRKYs under various stresses. creator: Pan Ning creator: Congcong Liu creator: Jingquan Kang creator: Jinyin Lv uri: https://doi.org/10.7717/peerj.3232 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Ning et al. title: Effect of the molecular weight of water-soluble chitosan on its fat-/cholesterol-binding capacities and inhibitory activities to pancreatic lipase link: https://peerj.com/articles/3279 last-modified: 2017-05-03 description: BackgroundObesity has become a worldwide burden to public health in recent decades. Given that obesity is caused by an imbalance between caloric intake and expenditure, and that dietary fat is the most important energy source of all macronutrients (by providing the most calories), a valuable strategy for obesity treatment and prevention is to block fat absorption via the gastrointestinal pathway. In this study, the fat- and cholesterol-binding capacities and the inhibition of pancreatic lipase by water-soluble chitosan (WSC) with different weight-average molecular weight (Mw) were tested and compared in vitro, in order to determine the anti-obesity effects of WSC and the influence of its Mw.MethodsIn this study, WSC with different Mw (∼1,000, ∼3,000, ∼5,000, ∼7,000 and ∼9,000 Da) were prepared by oxidative degradation assisted with microwave irradiation. A biopharmaceutical model of the digestive tract was used to determine the fat- and cholesterol-binding capacity of WSC samples. The pancreatic lipase assays were based on p-nitrophenyl derivatives.ResultsThe results showed that all of the WSC samples exhibit great fat- and cholesterol-binding capacities. Within the testing range, 1 g of WSC sample could absorb 2–8 g of peanut oil or 50–65 mg of cholesterol, which are both significantly higher than the ability of cellulose to do the same. Meanwhile, all the WSC samples were proven to be able to inhibit pancreatic lipase activity to some extent.DiscussionBased on the results, we suggest that there is a significant correlation between the binding capacity of WSC and its Mw, as WSC2 (∼3,000 Da) shows the highest fat- and cholesterol-binding capacities (7.08 g g−1 and 63.48 mg g−1, respectively), and the binding ability of WSC declines as its Mw increases or decreases from 3,000 Da. We also suggest WSC as an excellent resource in the development of functional foods against obesity for its adsorption, electrostatic binding and entrapment of cholesterol, fat, sterols and triglycerides in the diet. creator: Qiu Jin creator: Huahua Yu creator: Xueqin Wang creator: Kecheng Li creator: Pengcheng Li uri: https://doi.org/10.7717/peerj.3279 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Jin et al. title: Isolated teeth of Anhangueria  (Pterosauria: Pterodactyloidea) from the Lower Cretaceous of Lightning Ridge, New South Wales, Australia link: https://peerj.com/articles/3256 last-modified: 2017-05-03 description: The fossil record of Australian pterosaurs is sparse, consisting of only a small number of isolated and fragmentary remains from the Cretaceous of Queensland, Western Australia and Victoria. Here, we describe two isolated pterosaur teeth from the Lower Cretaceous (middle Albian) Griman Creek Formation at Lightning Ridge (New South Wales) and identify them as indeterminate members of the pterodactyloid clade Anhangueria. This represents the first formal description of pterosaur material from New South Wales. The presence of one or more anhanguerian pterosaurs at Lightning Ridge correlates with the presence of ‘ornithocheirid’ and Anhanguera-like pterosaurs from the contemporaneous Toolebuc Formation of central Queensland and the global distribution attained by ornithocheiroids during the Early Cretaceous. The morphology of the teeth and their presence in the estuarine- and lacustrine-influenced Griman Creek Formation is likely indicative of similar life habits of the tooth bearer to other members of Anhangueria. creator: Sienna A. Birch creator: Elizabeth T. Smith creator: Phil R. Bell uri: https://doi.org/10.7717/peerj.3256 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Birch et al. title: Population genetics data help to guide the conservation of palm species with small population sizes and fragmented habitats in Madagascar link: https://peerj.com/articles/3248 last-modified: 2017-05-03 description: BackgroundThe need to incorporate genetic data into conservation management decisions is increasingly recognised. However, many published studies represent a ‘gold standard’ of sampling, techniques, and analyses. Such rigour is often not possible with limited funding and resourcing available for developing plans for the increasing number of threatened species requiring conservation management. Two endemic palm species of the Itremo Massif in central Madagascar, Dypsis ambositrae and D. decipiens, are known to be threatened with extinction and conservation management for these species is a priority for the newly created protected area in the region.MethodsThe genetic diversity of these two species was studied using the relatively low-cost and rapid AFLP technique. DNA fragments generated using three primer combinations were analysed for 20 and 50 individuals of the two species, respectively, from across their ranges.ResultsGenetic diversity was relatively low for both species. The two sites where the highly restricted D. ambositrae grows were found to be genetically distinct (although overall heterozygosity was low). Despite having a much wider distribution and relatively large population, D. decipiens did not show clear geographical nor genetic groupings and had similarly low genetic heterozygosity to D. ambositrae.Discussion and RecommendationsWith so few individuals remaining in the wild and two genetically distinct subpopulations, it is recommended that both sites of D. ambositrae are conserved and that seed are collected from both for ex situ conservation and potential future reintroduction. It may be less important to focus resources on conserving or collecting ex situ material from all sites where D. decipiens is found, as the genetic diversity represented by each subpopulation is limited and increasing sampling may not protect significantly higher levels of genetic diversity. This study provides data that inform and support conservation decisions taken for both species within this region, and in the management of the newly designated Itremo Massif Protected Area, which covers most of the sites where these two species remain in the wild. creator: Lauren M. Gardiner creator: Mijoro Rakotoarinivo creator: Landy R. Rajaovelona creator: Colin Clubbe uri: https://doi.org/10.7717/peerj.3248 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Gardiner et al. title: Dynamics and genetic diversification of Escherichia coli during experimental adaptation to an anaerobic environment link: https://peerj.com/articles/3244 last-modified: 2017-05-03 description: BackgroundMany bacteria are facultative anaerobes, and can proliferate in both anoxic and oxic environments. Under anaerobic conditions, fermentation is the primary means of energy generation in contrast to respiration. Furthermore, the rates and spectra of spontaneous mutations that arise during anaerobic growth differ to those under aerobic growth. A long-term selection experiment was undertaken to investigate the genetic changes that underpin how the facultative anaerobe, Escherichia coli, adapts to anaerobic environments.MethodsTwenty-one populations of E. coli REL4536, an aerobically evolved 10,000th generation descendent of the E. coli B strain, REL606, were established from a clonal ancestral culture. These were serially sub-cultured for 2,000 generations in a defined minimal glucose medium in strict aerobic and strict anaerobic environments, as well as in a treatment that fluctuated between the two environments. The competitive fitness of the evolving lineages was assessed at approximately 0, 1,000 and 2,000 generations, in both the environment of selection and the alternative environment. Whole genome re-sequencing was performed on random colonies from all lineages after 2,000-generations. Mutations were identified relative to the ancestral genome, and based on the extent of parallelism, traits that were likely to have contributed towards adaptation were inferred.ResultsThere were increases in fitness relative to the ancestor among anaerobically evolved lineages when tested in the anaerobic environment, but no increases were found in the aerobic environment. For lineages that had evolved under the fluctuating regime, relative fitness increased significantly in the anaerobic environment, but did not increase in the aerobic environment. The aerobically-evolved lineages did not increase in fitness when tested in either the aerobic or anaerobic environments. The strictly anaerobic lineages adapted more rapidly to the anaerobic environment than did the fluctuating lineages. Two main strategies appeared to predominate during adaptation to the anaerobic environment: modification of energy generation pathways, and inactivation of non-essential functions. Fermentation pathways appeared to alter through selection for mutations in genes such as nadR, adhE, dcuS/R, and pflB. Mutations were frequently identified in genes for presumably dispensable functions such as toxin-antitoxin systems, prophages, virulence and amino acid transport. Adaptation of the fluctuating lineages to the anaerobic environments involved mutations affecting traits similar to those observed in the anaerobically evolved lineages.DiscussionThere appeared to be strong selective pressure for activities that conferred cell yield advantages during anaerobic growth, which include restoring activities that had previously been inactivated under long-term continuous aerobic evolution of the ancestor. creator: Thomas J. Finn creator: Sonal Shewaramani creator: Sinead C. Leahy creator: Peter H. Janssen creator: Christina D. Moon uri: https://doi.org/10.7717/peerj.3244 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Finn et al. title: vConTACT: an iVirus tool to classify double-stranded DNA viruses that infect Archaea and Bacteria link: https://peerj.com/articles/3243 last-modified: 2017-05-03 description: Taxonomic classification of archaeal and bacterial viruses is challenging, yet also fundamental for developing a predictive understanding of microbial ecosystems. Recent identification of hundreds of thousands of new viral genomes and genome fragments, whose hosts remain unknown, requires a paradigm shift away from traditional classification approaches and towards the use of genomes for taxonomy. Here we revisited the use of genomes and their protein content as a means for developing a viral taxonomy for bacterial and archaeal viruses. A network-based analytic was evaluated and benchmarked against authority-accepted taxonomic assignments and found to be largely concordant. Exceptions were manually examined and found to represent areas of viral genome ‘sequence space’ that are under-sampled or prone to excessive genetic exchange. While both cases are poorly resolved by genome-based taxonomic approaches, the former will improve as viral sequence space is better sampled and the latter are uncommon. Finally, given the largely robust taxonomic capabilities of this approach, we sought to enable researchers to easily and systematically classify new viruses. Thus, we established a tool, vConTACT, as an app at iVirus, where it operates as a fast, highly scalable, user-friendly app within the free and powerful CyVerse cyberinfrastructure. creator: Benjamin Bolduc creator: Ho Bin Jang creator: Guilhem Doulcier creator: Zhi-Qiang You creator: Simon Roux creator: Matthew B. Sullivan uri: https://doi.org/10.7717/peerj.3243 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Bolduc et al. title: ‘Unwilling’ versus ‘unable’: Tonkean macaques’ understanding of human goal-directed actions link: https://peerj.com/articles/3227 last-modified: 2017-05-03 description: The present study investigated the understanding of goal-directed actions in Tonkean macaques (Macaca tonkeana) using the unwilling versus unable paradigm, previously used in several species. Subjects were tested in three experimental conditions that varied according to the goal-directed actions of a human actor. In the “unwilling” condition, the actor was capable of giving the subject food but unwilling to do it; in the “unable” condition, she was willing to give food but was unable to do it because of a physical barrier; and in the “distracted” condition, she was occupied by manipulating a pebble instead of food. We report for the first time that Tonkean macaques, like capuchins, chimpanzees and human infants, behaved differently across these experimental conditions. They attempted to grasp food in the actor’s hand significantly more in the presence of an unwilling actor rather than an unable or a distracted one. Inversely, they begged significantly more facing a distracted and unable experimenter rather than an unwilling one. These results suggest that Tonkean macaques understand human goal-directed actions by predicting whether they were likely to obtain food merely based on movements, cue and motor intentions reading and understanding of physical constraints. creator: Charlotte Canteloup creator: Hélène Meunier uri: https://doi.org/10.7717/peerj.3227 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Canteloup and Meunier title: Immune responsiveness to phytohemagglutinin displays species but not sex differences in three anuran species link: https://peerj.com/articles/3181 last-modified: 2017-05-03 description: Phytohemagglutinin (PHA)-induced skin swelling response is widely used as a rough surrogate of integrative cell-mediated and innate immunity across multiple vertebrate taxa due to its simplification and feasibility. However, little is known whether there are sex and interspecific differences of immune responsiveness to PHA in ectotherms, especially for anurans. Therefore, we studied sex and species differences of PHA response in three anurans, Asiatic toads (Bufo gargarizans), Dark-spotted frogs (Pelophylax nigromaculatus) and Mongolian toads (Pseudepidalea raddei), captured in northern regions of Anhui Province (China). Footpad thickness was measured prior to (0 h) and after (6, 12, 24, 48 and 72 h) a PHA injection and normalized against saline injection in the opposite footpad. Body mass was recorded at the beginning (0 h) and end of each assay (72 h). Results showed effects of PHA assay, sex and taxa on body mass. Relative maximum swelling response (PHAmax) ranged from 18.58–29.75%, 9.77 to 20.56% and 21.97 to 31.78% and its occurrence over time was apparent 10.6–19.72 h , 7.74–14.01 h and 17.39–23.94 h postinjection for Asiatic toads, Dark-spotted frogs and Mongolian toads, respectively. Finally, the magnitude or timing of PHAmax in Dark-spotted frogs was significantly thinner and faster than in Mongolian toads, and Asiatic toads had an in-between value, not different from the other two species. The magnitude of PHAmax was significantly positively correlated with the timing of PHAmax considering individuals altogether, but not when analyzed within species. Our results indicate that male and female anuran species respond similarly to PHA antigen stimulation, but the magnitude and timing of PHAmax is species-specific. Briefly, we provide new evidence for the suitability of PHA assay in non-model anuran species with different body sizes, and exhort the need to further investigate the nature of PHA assay at the hematological and histological levels in order to extend its application in ecoimmunological studies of amphibians. creator: Zhiqiang Zhang creator: Chenchen Jin creator: Kangshan Qu creator: Enrique Caviedes-Vidal uri: https://doi.org/10.7717/peerj.3181 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Zhang et al. title: The earliest known titanosauriform sauropod dinosaur and the evolution of Brachiosauridae link: https://peerj.com/articles/3217 last-modified: 2017-05-02 description: Brachiosauridae is a clade of titanosauriform sauropod dinosaurs that includes the well-known Late Jurassic taxa Brachiosaurus and Giraffatitan. However, there is disagreement over the brachiosaurid affinities of most other taxa, and little consensus regarding the clade’s composition or inter-relationships. An unnamed partial sauropod skeleton was collected from middle–late Oxfordian (early Late Jurassic) deposits in Damparis, in the Jura department of eastern France, in 1934. Since its brief description in 1943, this specimen has been informally known in the literature as the ‘Damparis sauropod’ and ‘French Bothriospondylus’, and has been considered a brachiosaurid by most authors. If correctly identified, this would make the specimen the earliest known titanosauriform. Coupled with its relatively complete nature and the rarity of Oxfordian sauropod remains in general, this is an important specimen for understanding the early evolution of Titanosauriformes. Full preparation and description of this specimen, known from teeth, vertebrae and most of the appendicular skeleton of a single individual, recognises it as a distinct taxon: Vouivria damparisensis gen. et sp. nov. Phylogenetic analysis of a data matrix comprising 77 taxa (including all putative brachiosaurids) scored for 416 characters recovers a fairly well resolved Brachiosauridae. Vouivria is a basal brachiosaurid, confirming its status as the stratigraphically oldest known titanosauriform. Brachiosauridae consists of a paraphyletic array of Late Jurassic forms, with Europasaurus, Vouivria and Brachiosaurus recovered as successively more nested genera that lie outside of a clade comprising (Giraffatitan + Sonorasaurus) + (Lusotitan + (Cedarosaurus + Venenosaurus)). Abydosaurus forms an unresolved polytomy with the latter five taxa. The Early Cretaceous South American sauropod Padillasaurus was previously regarded as a brachiosaurid, but is here placed within Somphospondyli. A recent study contended that a number of characters used in a previous iteration of this data matrix are ‘biologically related’, and thus should be excluded from phylogenetic analysis. We demonstrate that almost all of these characters show variation between taxa, and implementation of sensitivity analyses, in which these characters are excluded, has no effect on tree topology or resolution. We argue that where there is morphological variation, this should be captured, rather than ignored. Unambiguous brachiosaurid remains are known only from the USA, western Europe and Africa, and the clade spanned the Late Jurassic through to the late Albian/early Cenomanian, with the last known occurrences all from the USA. Regardless of whether their absence from the Cretaceous of Europe, as well as other regions entirely, reflects regional extinctions and genuine absences, or sampling artefacts, brachiosaurids appear to have become globally extinct by the earliest Late Cretaceous. creator: Philip D. Mannion creator: Ronan Allain creator: Olivier Moine uri: https://doi.org/10.7717/peerj.3217 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Mannion et al. title: The use of informativity in the development of robust viromics-based examinations link: https://peerj.com/articles/3281 last-modified: 2017-05-02 description: Metagenomics-based studies have provided insight into many of the complex microbial communities responsible for maintaining life on this planet. Sequencing efforts often uncover novel genetic content; this is most evident for phage communities, in which upwards of 90% of all sequences exhibit no similarity to any sequence in current data repositories. For the small fraction that can be identified, the top BLAST hit is generally posited as being representative of a viral taxon present in the sample of origin. Homology-based classification, however, can be misleading as sequence repositories capture but a small fraction of phage diversity. Furthermore, lateral gene transfer is pervasive within phage communities. As such, the presence of a particular gene may not be indicative of the presence of a particular viral species. Rather, it is just that: an indication of the presence of a specific gene. To circumvent this limitation, we have developed a new method for the analysis of viral metagenomic datasets. BLAST hits are weighted, integrating the sequence identity and length of alignments as well as a taxonomic signal, such that each gene is evaluated with respect to its information content. Through this quantifiable metric, predictions of viral community structure can be made with confidence. As a proof-of-concept, the approach presented here was implemented and applied to seven freshwater viral metagenomes. While providing a robust method for evaluating viral metagenomic data, the tool is versatile and can easily be customized to investigations of any environment or biome. creator: Siobhan C. Watkins creator: Catherine Putonti uri: https://doi.org/10.7717/peerj.3281 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Watkins and Putonti title: Development and application of 14 microsatellite markers in the burying beetle Nicrophorus vespilloides reveals population genetic differentiation at local spatial scales link: https://peerj.com/articles/3278 last-modified: 2017-05-02 description: Burying beetles (genus Nicrophorus) are relatively rare among insects in providing sophisticated parental care. Consequently, they have become model species in research analysing social evolution, the evolution of parental care and mating systems. We used the recently published N. vespilloides genome and transcriptome to develop microsatellite markers. Specifically, we developed 14 polymorphic markers with five to 13 alleles per locus and used them to investigate levels of genetic differentiation in four south Cambridgeshire (UK) populations of N. vespilloides, separated by 21 km at most. The markers revealed significant genetic structuring among populations (global FST = 0.023) with all but one of the pairwise comparisons among populations being significant. The single exception was the comparison between the two closest populations, which are approximately 2.5 km apart. In general, the microsatellite markers showed lower observed heterozygosity than expected. We infer that there is limited dispersal between populations and potentially also some inbreeding within them and suggest that this may be due to habitat fragmentation. We discuss these results in the context of recent laboratory experiments on inbreeding and beetle flight. creator: Sonia Pascoal creator: Rebecca M. Kilner uri: https://doi.org/10.7717/peerj.3278 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Pascoal and Kilner title: Insights of the dental calculi microbiome of pre-Columbian inhabitants from Puerto Rico link: https://peerj.com/articles/3277 last-modified: 2017-05-02 description: BackgroundThe study of ancient microorganisms in mineralized dental plaque or calculi is providing insights into microbial evolution, as well as lifestyles and disease states of extinct cultures; yet, little is still known about the oral microbial community structure and function of pre-Columbian Caribbean cultures. In the present study, we investigated the dental calculi microbiome and predicted function of one of these cultures, known as the Saladoid. The Saladoids were horticulturalists that emphasized root-crop production. Fruits, as well as small marine and terrestrial animals were also part of the Saladoid diet.MethodsDental calculi samples were recovered from the archaeological site of Sorcé, in the municipal island of Vieques, Puerto Rico, characterized using 16S rRNA gene high-throughput sequencing, and compared to the microbiome of previously characterized coprolites of the same culture, as well modern plaque, saliva and stool microbiomes available from the Human Microbiome Project.ResultsActinobacteria, Proteobacteria and Firmicutes comprised the majority of the Saladoid dental calculi microbiome. The Saladoid dental calculi microbiome was distinct when compared to those of modern saliva and dental plaque, but showed the presence of common inhabitants of modern oral cavities including Streptococcus sp., Veillonella dispar and Rothia mucilaginosa. Cell motility, signal transduction and biosynthesis of other secondary metabolites may be unique features of the Saladoid microbiome.DiscussionResults suggest that the Saladoid dental calculi microbiome structure and function may possibly reflect a horticulturalist lifestyle and distinct dietary habits. Results also open the opportunity to further elucidate oral disease states in extinct Caribbean cultures and extinct indigenous cultures with similar lifestyles. creator: Tasha M. Santiago-Rodriguez creator: Yvonne Narganes-Storde creator: Luis Chanlatte-Baik creator: Gary A. Toranzos creator: Raul J. Cano uri: https://doi.org/10.7717/peerj.3277 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Santiago-Rodriguez et al. title: Transcriptome assembly and candidate genes involved in nutritional programming in the swordtail fish Xiphophorus multilineatus link: https://peerj.com/articles/3275 last-modified: 2017-05-02 description: BackgroundNutritional programming takes place in early development. Variation in the quality and/or quantity of nutrients in early development can influence long-term health and viability. However, little is known about the mechanisms of nutritional programming. The live-bearing fish Xiphophorus multilineatus has the potential to be a new model for understanding these mechanisms, given prior evidence of nutritional programming influencing behavior and juvenile growth rate. We tested the hypotheses that nutritional programming would influence behaviors involved in energy homeostasis as well gene expression in X. multilineatus.MethodsWe first examined the influence of both juvenile environment (varied in nutrition and density) and adult environment (varied in nutrition) on behaviors involved in energy acquisition and energy expenditure in adult male X. multilineatus. We also compared the behavioral responses across the genetically influenced size classes of males. Males stop growing at sexual maturity, and the size classes of can be identified based on phenotypes (adult size and pigment patterns). To study the molecular signatures of nutritional programming, we assembled a de novo transcriptome for X. multilineatus using RNA from brain, liver, skin, testis and gonad tissues, and used RNA-Seq to profile gene expression in the brains of males reared in low quality (reduced food, increased density) and high quality (increased food, decreased density) juvenile environments.ResultsWe found that both the juvenile and adult environments influenced the energy intake behavior, while only the adult environment influenced energy expenditure. In addition, there were significant interactions between the genetically influenced size classes and the environments that influenced energy intake and energy expenditure, with males from one of the four size classes (Y-II) responding in the opposite direction as compared to the other males examined. When we compared the brains of males of the Y-II size class reared in a low quality juvenile environment to males from the same size class reared in high quality juvenile environment, 131 genes were differentially expressed, including metabolism and appetite master regulator agrp gene.DiscussionOur study provides evidence for nutritional programming in X. multilineatus, with variation across size classes of males in how juvenile environment and adult diet influences behaviors involved in energy homeostasis. In addition, we provide the first transcriptome of X. multilineatus, and identify a group of candidate genes involved in nutritional programming. creator: Yuan Lu creator: Charlotte M. Klimovich creator: Kalen Z. Robeson creator: William Boswell creator: Oscar Ríos-Cardenas creator: Ronald B. Walter creator: Molly R. Morris uri: https://doi.org/10.7717/peerj.3275 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Lu et al. title: Analysis of microsatellites from the transcriptome of downy mildew pathogens and their application for characterization of Pseudoperonospora populations link: https://peerj.com/articles/3266 last-modified: 2017-05-02 description: Downy mildew pathogens affect several economically important crops worldwide but, due to their obligate nature, few genetic resources are available for genomic and population analyses. Draft genomes for emergent downy mildew pathogens such as the oomycete Pseudoperonospora cubensis, causal agent of cucurbit downy mildew, have been published and can be used to perform comparative genomic analysis and develop tools such as microsatellites to characterize pathogen population structure. We used bioinformatics to identify 2,738 microsatellites in the P. cubensis predicted transcriptome and evaluate them for transferability to the hop downy mildew pathogen, Pseudoperonospora humuli, since no draft genome is available for this species. We also compared the microsatellite repertoire of P. cubensis to that of the model organism Hyaloperonospora arabidopsidis, which causes downy mildew in Arabidopsis. Although trends in frequency of motif-type were similar, the percentage of SSRs identified from P. cubensis transcripts differed significantly from H. arabidopsidis. The majority of a subset of microsatellites selected for laboratory validation (92%) produced a product in P. cubensis isolates, and 83 microsatellites demonstrated transferability to P. humuli. Eleven microsatellites were found to be polymorphic and consistently amplified in P. cubensis isolates. Analysis of Pseudoperonospora isolates from diverse hosts and locations revealed higher diversity in P. cubensis compared to P. humuli isolates. These microsatellites will be useful in efforts to better understand relationships within Pseudoperonospora species and P. cubensis on a population level. creator: Emma C. Wallace creator: Lina M. Quesada-Ocampo uri: https://doi.org/10.7717/peerj.3266 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Wallace and Quesada-Ocampo title: High diversity and suggested endemicity of culturable Actinobacteria in an extremely oligotrophic desert oasis link: https://peerj.com/articles/3247 last-modified: 2017-05-02 description: The phylum Actinobacteria constitutes one of the largest and anciently divergent phyla within the Bacteria domain. Actinobacterial diversity has been thoroughly researched in various environments due to its unique biotechnological potential. Such studies have focused mostly on soil communities, but more recently marine and extreme environments have also been explored, finding rare taxa and demonstrating dispersal limitation and biogeographic patterns for Streptomyces. To test the distribution of Actinobacteria populations on a small scale, we chose the extremely oligotrophic and biodiverse Cuatro Cienegas Basin (CCB), an endangered oasis in the Chihuahuan desert to assess the diversity and uniqueness of Actinobacteria in the Churince System with a culture-dependent approach over a period of three years, using nine selective media. The 16S rDNA of putative Actinobacteria were sequenced using both bacteria universal and phylum-specific primer pairs. Phylogenetic reconstructions were performed to analyze OTUs clustering and taxonomic identification of the isolates in an evolutionary context, using validated type species of Streptomyces from previously phylogenies as a reference. Rarefaction analysis for total Actinobacteria and for Streptomyces isolates were performed to estimate species’ richness in the intermediate lagoon (IL) in the oligotrophic Churince system. A total of 350 morphologically and nutritionally diverse isolates were successfully cultured and characterized as members of the Phylum Actinobacteria. A total of 105 from the total isolates were successfully subcultured, processed for DNA extraction and 16S-rDNA sequenced. All strains belong to the order Actinomycetales, encompassing 11 genera of Actinobacteria; the genus Streptomyces was found to be the most abundant taxa in all the media tested throughout the 3-year sampling period. Phylogenetic analysis of our isolates and another 667 reference strains of the family Streptomycetaceae shows that our isolation effort produced 38 unique OTUs in six new monophyletic clades. This high biodiversity and uniqueness of Actinobacteria in an extreme oligotrophic environment, which has previously been reported for its diversity and endemicity, is a suggestive sign of microbial biogeography of Actinobacteria and it also represents an invaluable source of biological material for future ecological and bioprospecting studies. creator: Hector Fernando Arocha-Garza creator: Ricardo Canales-Del Castillo creator: Luis E. Eguiarte creator: Valeria Souza creator: Susana De la Torre-Zavala uri: https://doi.org/10.7717/peerj.3247 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Arocha-Garza et al. title: Porites superfusa mortality and recovery from a bleaching event at Palmyra Atoll, USA link: https://peerj.com/articles/3204 last-modified: 2017-05-02 description: BackgroundThe demography of a coral colony is not a binary trajectory of life and death. Based on the flexibility afforded by colonial organization, most reef-building corals employ a variety of dynamic survival strategies, including growth and shrinkage. The demographic flexibility affects coral size, shape and reproductive output, among other factors. It is thus critical to quantify the relative importance of key dynamics of recruitment, mortality, growth and shrinkage in changing the overall cover of coral on a reef.MethodsUsing fixed photographic quadrats, we tracked the patterns of change in the cover of one common central Pacific coral, Porites superfusa, before and after the 2009 ENSO event.ResultsCoral colonies suffered both whole and partial colony mortality, although larger colonies were more likely to survive. In subsequent years, recruitment of new colonies and regrowth of surviving colonies both contributed to the modest recovery of P. superfusa.DiscussionThis study is unique in its quantitative comparisons of coral recruitment versus regrowth during periods of areal expansion. Our data suggest that recovery is not limited simply to the long pathway of settlement, recruitment and early growth of new colonies but is accelerated by means of regrowth of already established colonies having suffered partial mortality. creator: Kathryn Anne Furby creator: Jennifer Ellen Smith creator: Stuart Adrian Sandin uri: https://doi.org/10.7717/peerj.3204 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Furby et al. title: Common and rare genetic markers of lipid variation in subjects with type 2 diabetes from the ACCORD clinical trial link: https://peerj.com/articles/3187 last-modified: 2017-05-02 description: BackgroundIndividuals with type 2 diabetes are at an increased risk of cardiovascular disease. Alterations in circulating lipid levels, total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides (TG) are heritable risk factors for cardiovascular disease. Here we conduct a genome-wide association study (GWAS) of common and rare variants to investigate associations with baseline lipid levels in 7,844 individuals with type 2 diabetes from the ACCORD clinical trial.MethodsDNA extracted from stored blood samples from ACCORD participants were genotyped using the Affymetrix Axiom Biobank 1 Genotyping Array. After quality control and genotype imputation, association of common genetic variants (CV), defined as minor allele frequency (MAF) ≥ 3%, with baseline levels of TC, LDL, HDL, and TG was tested using a linear model. Rare variant (RV) associations (MAF < 3%) were conducted using a suite of methods that collapse multiple RV within individual genes.ResultsMany statistically significant CV (p < 1 × 10−8) replicate findings in large meta-analyses in non-diabetic subjects. RV analyses also confirmed findings in other studies, whereas significant RV associations with CNOT2, HPN-AS1, and SIRPD appear to be novel (q < 0.1).DiscussionHere we present findings for the largest GWAS of lipid levels in people with type 2 diabetes to date. We identified 17 statistically significant (p < 1 × 10−8) associations of CV with lipid levels in 11 genes or chromosomal regions, all of which were previously identified in meta-analyses of mostly non-diabetic cohorts. We also identified 13 associations in 11 genes based on RV, several of which represent novel findings. creator: Skylar W. Marvel creator: Daniel M. Rotroff creator: Michael J. Wagner creator: John B. Buse creator: Tammy M. Havener creator: Howard L. McLeod creator: Alison A. Motsinger-Reif uri: https://doi.org/10.7717/peerj.3187 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Marvel et al. title: Osteology of Galeamopus pabsti sp. nov. (Sauropoda: Diplodocidae), with implications for neurocentral closure timing, and the cervico-dorsal transition in diplodocids link: https://peerj.com/articles/3179 last-modified: 2017-05-02 description: Diplodocids are among the best known sauropod dinosaurs. Numerous specimens of currently 15 accepted species belonging to ten genera have been reported from the Late Jurassic to Early Cretaceous of North and South America, Europe, and Africa. The highest diversity is known from the Upper Jurassic Morrison Formation of the western United States: a recent review recognized 12 valid, named species, and possibly three additional, yet unnamed ones. One of these is herein described in detail and referred to the genus Galeamopus. The holotype specimen of Galeamopus pabsti sp. nov., SMA 0011, is represented by material from all body parts but the tail, and was found at the Howe-Scott Quarry in the northern Bighorn Basin in Wyoming, USA. Autapomorphic features of the new species include a horizontal canal on the maxilla that connects the posterior margin of the preantorbital and the ventral margin of the antorbital fenestrae, a vertical midline groove marking the sagittal nuchal crest, the presence of a large foramen connecting the postzygapophyseal centrodiapophyseal fossa and the spinopostzygapophyseal fossa of mid- and posterior cervical vertebrae, a very robust humerus, a laterally placed, rugose tubercle on the concave proximal portion of the anterior surface of the humerus, a relatively stout radius, the absence of a distinct ambiens process on the pubis, and a distinctly concave posteroventral margin of the ascending process of the astragalus. In addition to the holotype specimen SMA 0011, the skull USNM 2673 can also be referred to Galeamopus pabsti. Histology shows that the type specimen SMA 0011 is sexually mature, although neurocentral closure was not completed at the time of death. Because SMA 0011 has highly pneumatized cervical vertebrae, the development of the lamination appears a more important indicator for individual age than neurocentral fusion patterns. SMA 0011 is one of very few sauropod specimens that preserves the cervico-dorsal transition in both vertebrae and ribs. The association of ribs with their respective vertebrae shows that the transition between cervical and dorsal vertebrae is significantly different in Galeamopus pabsti than in Diplodocus carnegii or Apatosaurus louisae, being represented by a considerable shortening of the centra from the last cervical to the first dorsal vertebra. Diplodocids show a surprisingly high diversity in the Morrison Formation. This can possibly be explained by a combination of geographical and temporal segregation, and niche partitioning. creator: Emanuel Tschopp creator: Octávio Mateus uri: https://doi.org/10.7717/peerj.3179 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2017 Tschopp and Mateus