title: PeerJ description: Articles published in PeerJ link: https://peerj.com/articles/index.rss3?journal=peerj&page=1033 creator: info@peerj.com PeerJ errorsTo: info@peerj.com PeerJ language: en title: Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams link: https://peerj.com/articles/9973 last-modified: 2020-10-27 description: Glacier-fed streams (GFS) are harsh ecosystems dominated by microbial life organized in benthic biofilms, yet the biodiversity and ecosystem functions provided by these communities remain under-appreciated. To better understand the microbial processes and communities contributing to GFS ecosystems, it is necessary to leverage high throughput sequencing. Low biomass and high inorganic particle load in GFS sediment samples may affect nucleic acid extraction efficiency using extraction methods tailored to other extreme environments such as deep-sea sediments. Here, we benchmarked the utility and efficacy of four extraction protocols, including an up-scaled phenol-chloroform protocol. We found that established protocols for comparable sample types consistently failed to yield sufficient high-quality DNA, delineating the extreme character of GFS. The methods differed in the success of downstream applications such as library preparation and sequencing. An adapted phenol-chloroform-based extraction method resulted in higher yields and better recovered the expected taxonomic profile and abundance of reconstructed genomes when compared to commercially-available methods. Affordable and straight-forward, this method consistently recapitulated the abundance and genomes of a mock community, including eukaryotes. Moreover, by increasing the amount of input sediment, the protocol is readily adjustable to the microbial load of the processed samples without compromising protocol efficiency. Our study provides a first systematic and extensive analysis of the different options for extraction of nucleic acids from glacier-fed streams for high-throughput sequencing applications, which may be applied to other extreme environments. creator: Susheel Bhanu Busi creator: Paraskevi Pramateftaki creator: Jade Brandani creator: Stilianos Fodelianakis creator: Hannes Peter creator: Rashi Halder creator: Paul Wilmes creator: Tom J. Battin uri: https://doi.org/10.7717/peerj.9973 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2020 Busi et al. title: Vertical distribution of polycyclic aromatic hydrocarbons in the brackish sea water column: ex situ experiment link: https://peerj.com/articles/10087 last-modified: 2020-10-27 description: BackgroundOil spills can cause severe damage within a marine ecosystem. Following a spill, the soluble fraction of polycyclic aromatic hydrocarbons is rapidly released into the water column. These remain dissolved in seawater over an extended period of time, even should the insoluble fraction be removed. The vertical distribution of the aromatic hydrocarbon component and how these become transferred is poorly understood in brackish waters. This study examines the vertical distribution of polycyclic aromatic hydrocarbons having been released from a controlled film of spilled oil onto the surface of brackish water.MethodsThe study was undertaken under controlled conditions so as to minimize the variability of environmental factors such as temperature and hydrodynamics. The distribution of polycyclic aromatic hydrocarbons was measured in the dissolved and suspended phases throughout the 1 m water column with different intensity of water sampling: 1, 2, 4, 7, 72, 120, 336, 504 and 984 h.ResultsThe total concentration of polycyclic aromatic hydrocarbons ranged from 19.01 to 214.85 ng L–1 in the dissolved phase and from 5.14 to 63.92 ng L–1 in the suspended phase. These hydrocarbons were released immediately following a controlled spill attaining 214.9 ng L–1 in the dissolved phase and 54.4 ng L–1 in the suspended phase near the cylinder bottom after 1–2 h. The 2–3 ring polycyclic aromatic hydrocarbons dominated in the dissolved phase (60–80%), whereas the greater amount of 4–6 ring polycyclic aromatic hydrocarbons (55–90%) occurred in the suspended phase. A relatively low negative correlation (rS = –0.41) was determined between the concentration of phenanthrene and suspended matter, whereas a high negative correlation (r =  − 0.79) was found between the concentration of pyrene and suspended matter. Despite the differences in the relationships between the concentration ratio and amount of suspended matter the obtained regressions allow roughly to predict the concentration of polycyclic aromatic hydrocarbons. creator: Zilvinas Kryzevicius creator: Kristina Mickuviene creator: Martynas Bucas creator: Monika Vilkiene creator: Audrone Zukauskaite uri: https://doi.org/10.7717/peerj.10087 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2020 Kryzevicius et al. title: Effects of different drying methods on smears of canine blood and effusion fluid link: https://peerj.com/articles/10092 last-modified: 2020-10-27 description: BackgroundGlass slide preparations from a variety of specimens (blood, masses, effusions) are commonly made as part of the diagnostic work-up, however the effects of various drying methods in veterinary practice and diagnostic laboratory settings is not clear.ObjectiveCompare the effects of four drying methods on results of microscopic examination of canine blood smears and direct smears of pleural or peritoneal effusion fluid.MethodsTwelve canine blood samples (6 from healthy dogs, 6 from sick dogs) and 6 canine peritoneal or pleural effusion samples. Four smears were prepared from each of the 18 samples and dried using the following methods: air-dry, hair dryer with or without heat, and heat block at 58 °C. Observers, blinded to the drying method, independently reviewed the slides microscopically, using a scoring system to evaluate cell morphology and (for blood smears) echinocyte numbers; scoring results were analyzed statistically.ResultsFor blood smears, several comparisons showed more adverse effects on morphology using the heat block method than for one or more other drying methods. For effusion fluid smears, RBCs dried with the heat block or air-dry methods had more poorly preserved morphology than RBCs dried by the hair dryer method without heat.Conclusions and clinical relevanceThe results (1) indicate that different drying methods had a significant effect, (2) support using a hair dryer without heat for both blood smears and effusion fluid smears, and (3) discourage using a 58 °C heat block. creator: Fiamma G. De Witte creator: Aimee Hebrard creator: Carolyn N. Grimes creator: Kristin Owens creator: Deanna M. Schaefer creator: Xiaojuan Zhu creator: Michael M. Fry uri: https://doi.org/10.7717/peerj.10092 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2020 De Witte et al. title: The osteology and phylogenetic position of the loricatan (Archosauria: Pseudosuchia) Heptasuchus clarki, from the ?Mid-Upper Triassic, southeastern Big Horn Mountains, Central Wyoming (USA) link: https://peerj.com/articles/10101 last-modified: 2020-10-27 description: Loricatan pseudosuchians (known as “rauisuchians”) typically consist of poorly understood fragmentary remains known worldwide from the Middle Triassic to the end of the Triassic Period. Renewed interest and the discovery of more complete specimens recently revolutionized our understanding of the relationships of archosaurs, the origin of Crocodylomorpha, and the paleobiology of these animals. However, there are still few loricatans known from the Middle to early portion of the Late Triassic and the forms that occur during this time are largely known from southern Pangea or Europe. Heptasuchus clarki was the first formally recognized North American “rauisuchian” and was collected from a poorly sampled and disparately fossiliferous sequence of Triassic strata in North America. Exposed along the trend of the Casper Arch flanking the southeastern Big Horn Mountains, the type locality of Heptasuchus clarki occurs within a sequence of red beds above the Alcova Limestone and Crow Mountain formations within the Chugwater Group. The age of the type locality is poorly constrained to the Middle—early Late Triassic and is likely similar to or just older than that of the Popo Agie Formation assemblage from the western portion of Wyoming. The holotype consists of associated cranial elements found in situ, and the referred specimens consist of crania and postcrania. Thus, about 30% of the osteology of the taxon is preserved. All of the pseudosuchian elements collected at the locality appear to belong to Heptasuchus clarki and the taxon is not a chimera as previously hypothesized. Heptasuchus clarki is distinct from all other archosaurs by the presence of large, posteriorly directed flanges on the parabasisphenoid and a distinct, orbit-overhanging postfrontal. Our phylogenetic hypothesis posits a sister-taxon relationship between Heptasuchus clarki and the Ladinian-aged Batrachotomus kupferzellensis from current-day Germany within Loricata. These two taxa share a number of apomorphies from across the skull and their phylogenetic position further supports ‘rauisuchian’ paraphyly. A minimum of three individuals of Heptasuchus are present at the type locality suggesting that a group of individuals died together, similar to other aggregations of loricatans (e.g., Heptasuchus, Batrachotomus, Decuriasuchus, Postosuchus). creator: Sterling J. Nesbitt creator: John M. Zawiskie creator: Robert M. Dawley uri: https://doi.org/10.7717/peerj.10101 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2020 Nesbitt et al. title: Novel targets for engineering Physostegia chlorotic mottle and tomato brown rugose fruit virus-resistant tomatoes: in silico prediction of tomato microRNA targets link: https://peerj.com/articles/10096 last-modified: 2020-10-27 description: BackgroundPhysostegia chlorotic mottle virus (PhCMoV; genus: Alphanucleorhabdovirus, family: Rhabdoviridae) and tomato brown rugose fruit virus (ToBRFV; genus: Tobamovirus, family: Virgaviridae) are newly emerging plant viruses that have a dramatic effect on tomato production. Among various known virus-control strategies, RNAi-mediated defence has shown the potential to protect plants against various pathogens including viral infections. Micro(mi)RNAs play a major role in RNAi-mediated defence.MethodsUsing in silico analyses, we investigated the possibility of tomato-encoded miRNAs (TomiRNA) to target PhCMoV and ToBRFV genomes using five different algorithms, i.e., miRanda, RNAhybrid, RNA22, Tapirhybrid and psRNATarget.ResultsThe results revealed that 14 loci on PhCMoV and 10 loci on ToBRFV can be targeted by the TomiRNAs based on the prediction of at least three algorithms. Interestingly, one TomiRNA, miR6026, can target open reading frames from both viruses, i.e., the phosphoprotein encoding gene of PhCMoV, and the two replicase components of ToBRFV. There are currently no commercially available PhCMoV- or ToBRFV-resistant tomato varieties, therefore the predicted data provide useful information for the development of PhCMoV- and ToBFRV-resistant tomato plants. creator: Yahya Zakaria Abdou Gaafar creator: Heiko Ziebell uri: https://doi.org/10.7717/peerj.10096 license: https://creativecommons.org/licenses/by-nc/4.0 rights: ©2020 Gaafar and Ziebell title: The Bacteria Genome Pipeline (BAGEP): an automated, scalable workflow for bacteria genomes with Snakemake link: https://peerj.com/articles/10121 last-modified: 2020-10-27 description: Next generation sequencing technologies are becoming more accessible and affordable over the years, with entire genome sequences of several pathogens being deciphered in few hours. However, there is the need to analyze multiple genomes within a short time, in order to provide critical information about a pathogen of interest such as drug resistance, mutations and genetic relationship of isolates in an outbreak setting. Many pipelines that currently do this are stand-alone workflows and require huge computational requirements to analyze multiple genomes. We present an automated and scalable pipeline called BAGEP for monomorphic bacteria that performs quality control on FASTQ paired end files, scan reads for contaminants using a taxonomic classifier, maps reads to a reference genome of choice for variant detection, detects antimicrobial resistant (AMR) genes, constructs a phylogenetic tree from core genome alignments and provide interactive short nucleotide polymorphism (SNP) visualization across core genomes in the data set. The objective of our research was to create an easy-to-use pipeline from existing bioinformatics tools that can be deployed on a personal computer. The pipeline was built on the Snakemake framework and utilizes existing tools for each processing step: fastp for quality trimming, snippy for variant calling, Centrifuge for taxonomic classification, Abricate for AMR gene detection, snippy-core for generating whole and core genome alignments, IQ-TREE for phylogenetic tree construction and vcfR for an interactive heatmap visualization which shows SNPs at specific locations across the genomes. BAGEP was successfully tested and validated with Mycobacterium tuberculosis (n = 20) and Salmonella enterica serovar Typhi (n = 20) genomes which are about 4.4 million and 4.8 million base pairs, respectively. Running these test data on a 8 GB RAM, 2.5 GHz quad core laptop took 122 and 61 minutes on respective data sets to complete the analysis. BAGEP is a fast, calls accurate SNPs and an easy to run pipeline that can be executed on a mid-range laptop; it is freely available on: https://github.com/idolawoye/BAGEP. creator: Idowu B. Olawoye creator: Simon D.W. Frost creator: Christian T. Happi uri: https://doi.org/10.7717/peerj.10121 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2020 Olawoye et al. title: The size of signal detection and emission organs in a synchronous firefly: sexual dimorphism, allometry and assortative mating link: https://peerj.com/articles/10127 last-modified: 2020-10-27 description: The size of the organs responsible for emitting and detecting sexual communication signals is a likely target for selection. Communication via bioluminescent signals in synchronous fireflies is a promising model to test hypotheses regarding differences between males and females in the effect of the size of signal emission and detection organs on fitness components. Synchronous firefly species congregate in large numbers during the mating season, displaying bioluminescent signals aimed at potential mates during relatively short nightly periods. Operational sex ratios are male-biased and, thus, the so-called typical sex roles (indiscriminate males and choosy females) are expected to evolve. We studied the synchronous firefly Photinus palaciosi, a species that during the mating season congregates in forests of central Mexico offering a magnificent natural show that attracts numerous tourists. P. palaciosi females have reduced wings (brachyptery) and cannot fly. Our field study tested the hypothesis that the male-biased operational sex ratio and the short daily mating period result in strong male-male competition that selects for males with larger lanterns and larger eyes, and against male mate choice, whereas female-female mate competition is absent and, thus, no selection on lantern or eye size is expected. Even though lantern, eye or body size do not predict the probability of being found in copula for either sex, sexual dimorphism in these features, along with allometric slopes of lantern size and assortative mating in terms of relative lantern size, support not only the hypothesis of intense sexual selection among males, but the possibility of subtle mechanisms of sexual selection among females. Trade-offs between investment in signaling (lanterns) versus detection (eyes) structures, or with pressures different from sexual selection such as those imposed by predators, are also likely to be important in shaping the evolution of sexual signaling in these fireflies. creator: Tania López-Palafox creator: Rogelio Macías-Ordóñez creator: Carlos R. Cordero uri: https://doi.org/10.7717/peerj.10127 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2020 López-Palafox et al. title: Integrated transcriptome meta-analysis of pancreatic ductal adenocarcinoma and matched adjacent pancreatic tissues link: https://peerj.com/articles/10141 last-modified: 2020-10-27 description: A comprehensive meta-analysis of publicly available gene expression microarray data obtained from human-derived pancreatic ductal adenocarcinoma (PDAC) tissues and their histologically matched adjacent tissue samples was performed to provide diagnostic and prognostic biomarkers, and molecular targets for PDAC. An integrative meta-analysis of four submissions (GSE62452, GSE15471, GSE62165, and GSE56560) containing 105 eligible tumor-adjacent tissue pairs revealed 344 differentially over-expressed and 168 repressed genes in PDAC compared to the adjacent-to-tumor samples. The validation analysis using TCGA combined GTEx data confirmed 98.24% of the identified up-regulated and 73.88% of the down-regulated protein-coding genes in PDAC. Pathway enrichment analysis showed that “ECM-receptor interaction”, “PI3K-Akt signaling pathway”, and “focal adhesion” are the most enriched KEGG pathways in PDAC. Protein-protein interaction analysis identified FN1, TIMP1, and MSLN as the most highly ranked hub genes among the DEGs. Transcription factor enrichment analysis revealed that TCF7, CTNNB1, SMAD3, and JUN are significantly activated in PDAC, while SMAD7 is inhibited. The prognostic significance of the identified and validated differentially expressed genes in PDAC was evaluated via survival analysis of TCGA Pan-Cancer pancreatic ductal adenocarcinoma data. The identified candidate prognostic biomarkers were then validated in four external validation datasets (GSE21501, GSE50827, GSE57495, and GSE71729) to further improve reliability. A total of 28 up-regulated genes were found to be significantly correlated with worse overall survival in patients with PDAC. Twenty-one of the identified prognostic genes (ITGB6, LAMC2, KRT7, SERPINB5, IGF2BP3, IL1RN, MPZL2, SFTA2, MET, LAMA3, ARNTL2, SLC2A1, LAMB3, COL17A1, EPSTI1, IL1RAP, AK4, ANXA2, S100A16, KRT19, and GPRC5A) were also found to be significantly correlated with the pathological stages of the disease. The results of this study provided promising prognostic biomarkers that have the potential to differentiate PDAC from both healthy and adjacent-to-tumor pancreatic tissues. Several novel dysregulated genes merit further study as potentially promising candidates for the development of more effective treatment strategies for PDAC. creator: Sevcan Atay uri: https://doi.org/10.7717/peerj.10141 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2020 Atay title: Developing mathematical model for diurnal dynamics of photosynthesis in Saccharum officinarum responsive to different irrigation and silicon application link: https://peerj.com/articles/10154 last-modified: 2020-10-27 description: In the dynamic era of climate change, agricultural farming systems are facing various unprecedented problems worldwide. Drought stress is one of the serious abiotic stresses that hinder the growth potential and crop productivity. Silicon (Si) can improve crop yield by enhancing the efficiency of inputs and reducing relevant losses. As a quasi-essential element and the 2nd most abundant element in the Earth’s crust, Si is utilized by plants and applied exogenously to combat drought stress and improve plant performance by increasing physiological, cellular and molecular responses. However, the physiological mechanisms that respond to water stress are still not well defined in Saccharum officinarum plants. To the best of our knowledge, the dynamics of photosynthesis responsive to different exogenous Si levels in Saccharum officinarum has not been reported to date. The current experiment was carried out to assess the protective role of Si in plant growth and photosynthetic responses in Saccharum officinarum under water stress conditions. Saccharum officinarum cv. ‘GT 42’ plants were subjected to drought stress conditions (80–75%, 55–50% and 35–30% of soil moisture) after ten weeks of normal growth, followed by the soil irrigation of Si (0, 100, 300 and 500 mg L−1) for 8 weeks. The results indicated that Si addition mitigated the inhibition in Saccharum officinarum growth and photosynthesis, and improved biomass accumulation during water stress. The photosynthetic responses (photosynthesis, transpiration and stomatal conductance) were found down-regulated under water stress, and it was significantly enhanced by Si application. No phytotoxic effects were monitored even at excess (500 mg L−1). Soil irrigation of 300 mg L−1 of Si was more effective as 100 and 500 mg L−1 under water stress condition. It is concluded that the stress in Saccharum officinarum plants applied with Si was alleviated by improving plant fitness, photosynthetic capacity and biomass accumulation as compared with the control. Thus, this study offers new information towards the assessment of growth, biomass accumulation and physiological changes related to water stress with Si application in plants. creator: Krishan K. Verma creator: Kai-Chao Wu creator: Chhedi Lal Verma creator: Dong-Mei Li creator: Mukesh Kumar Malviya creator: Rajesh Kumar Singh creator: Pratiksha Singh creator: Gan-Lin Chen creator: Xiu Peng Song creator: Yang Rui Li uri: https://doi.org/10.7717/peerj.10154 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2020 Verma et al. title: Comparing sediment DNA extraction methods for assessing organic enrichment associated with marine aquaculture link: https://peerj.com/articles/10231 last-modified: 2020-10-27 description: Marine sediments contain a high diversity of micro- and macro-organisms which are important in the functioning of biogeochemical cycles. Traditionally, anthropogenic perturbation has been investigated by identifying macro-organism responses along gradients. Environmental DNA (eDNA) analyses have recently been advocated as a rapid and cost-effective approach to measuring ecological impacts and efforts are underway to incorporate eDNA tools into monitoring. Before these methods can replace or complement existing methods, robustness and repeatability of each analytical step has to be demonstrated. One area that requires further investigation is the selection of sediment DNA extraction method. Environmental DNA sediment samples were obtained along a disturbance gradient adjacent to a Chinook (Oncorhynchus tshawytscha) salmon farm in Otanerau Bay, New Zealand. DNA was extracted using four extraction kits (Qiagen DNeasy PowerSoil, Qiagen DNeasy PowerSoil Pro, Qiagen RNeasy PowerSoil Total RNA/DNA extraction/elution and Favorgen FavorPrep Soil DNA Isolation Midi Kit) and three sediment volumes (0.25, 2, and 5 g). Prokaryotic and eukaryotic communities were amplified using primers targeting the 16S and 18S ribosomal RNA genes, respectively, and were sequenced on an Illumina MiSeq. Diversity and community composition estimates were obtained from each extraction kit, as well as their relative performance in established metabarcoding biotic indices. Differences were observed in the quality and quantity of the extracted DNA amongst kits with the two Qiagen DNeasy PowerSoil kits performing best. Significant differences were observed in both prokaryotes and eukaryotes (p < 0.001) richness among kits. A small proportion of amplicon sequence variants (ASVs) were shared amongst the kits (~3%) although these shared ASVs accounted for the majority of sequence reads (prokaryotes: 59.9%, eukaryotes: 67.2%). Differences were observed in the richness and relative abundance of taxonomic classes revealed with each kit. Multivariate analysis showed that there was a significant interaction between “distance” from the farm and “kit” in explaining the composition of the communities, with the distance from the farm being a stronger determinant of community composition. Comparison of the kits against the bacterial and eukaryotic metabarcoding biotic index suggested that all kits showed similar patterns along the environmental gradient. Overall, we advocate for the use of Qiagen DNeasy PowerSoil kits for use when characterizing prokaryotic and eukaryotic eDNA from marine farm sediments. We base this conclusion on the higher DNA quality values and richness achieved with these kits compared to the other kits/amounts investigated in this study. The additional advantage of the PowerSoil Kits is that DNA extractions can be performed using an extractor robot, offering additional standardization and reproducibility of results. creator: John K. Pearman creator: Nigel B. Keeley creator: Susanna A. Wood creator: Olivier Laroche creator: Anastasija Zaiko creator: Georgia Thomson-Laing creator: Laura Biessy creator: Javier Atalah creator: Xavier Pochon uri: https://doi.org/10.7717/peerj.10231 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2020 Pearman et al.