Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum
The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF4− and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPxGl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPxGl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPxGl; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles.
Chaudhari et al. (2017) Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum. PeerJ 5:e3128 https://doi.org/10.7717/peerj.3128
The first juvenile specimen of Manchurochelys manchoukuoensis from the Early Cretaceous Jehol Biota
A small juvenile turtle is described from the Early Cretaceous Jehol Biota, shedding light on the juvenile morphology and ontogeny of Manchurochelys manchoukuoensis. Several juvenile features are uncovered, such as a small and circular carapace (less than half of the adult), wide vertebral scales, and lateral carapacial fontanelles. In contrast to the adult morphology, which has an oval carapace, closed lateral fontanelles, and longer vertebrals 2–4, the juvenile of M. manchoukuoensis is more comparable to that of Sinemys lens, except for earlier occurrence of the well-ossified carapace of the latter. Differs from Changmachelys bohlini, and Ordosemys liaoxiensis, in which the circular carapace is relatively independent of ontogenetic age, and the lateral fontanelles are only closed in adult stage of O. liaoxiensis. Therefore, the trajectory of ontogenetic change appears to be highly diversified in the sinemydids.
Shao et al. (2017) The first juvenile specimen of Manchurochelys manchoukuoensis from the Early Cretaceous Jehol Biota. PeerJ 5:e3274 https://doi.org/10.7717/peerj.3274
Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses
Background Cell membrane permeabilization by pulsed electromagnetic fields (PEMF) is a novel contactless method which results in effects similar to conventional electroporation. The non-invasiveness of the methodology, independence from the biological object homogeneity and electrical conductance introduce high flexibility and potential applicability of the PEMF in biomedicine, food processing, and biotechnology. The inferior effectiveness of the PEMF permeabilization compared to standard electroporation and the lack of clear description of the induced transmembrane transport are currently of major concern. Methods The PEMF permeabilization experiments have been performed using a 5.5 T, 1.2 J pulse generator with a multilayer inductor as an applicator. We investigated the feasibility to increase membrane permeability of Chinese Hamster Ovary (CHO) cells using short microsecond (15 µs) pulse bursts (100 or 200 pulses) at low frequency (1 Hz) and high dB/dt (>106 T/s). The effectiveness of the treatment was evaluated by fluorescence microscopy and flow cytometry using two different fluorescent dyes: propidium iodide (PI) and YO-PRO®-1 (YP). The results were compared to conventional electroporation (single pulse, 1.2 kV/cm, 100 µs), i.e., positive control. Results The proposed PEMF protocols (both for 100 and 200 pulses) resulted in increased number of permeable cells (70 ± 11% for PI and 67 ± 9% for YP). Both cell permeabilization assays also showed a significant (8 ± 2% for PI and 35 ± 14% for YP) increase in fluorescence intensity indicating membrane permeabilization. The survival was not affected. Discussion The obtained results demonstrate the potential of PEMF as a contactless treatment for achieving reversible permeabilization of biological cells. Similar to electroporation, the PEMF permeabilization efficacy is influenced by pulse parameters in a dose-dependent manner.
Novickij et al. (2017) Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses. PeerJ 5:e3267 https://doi.org/10.7717/peerj.3267
Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus
Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR in Amorphophallus.
Wang et al. (2017) Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus. PeerJ 5:e3260 https://doi.org/10.7717/peerj.3260
Forelimb bone curvature in terrestrial and arboreal mammals
It has recently been proposed that the caudal curvature (concave caudal side) observed in the radioulna of terrestrial quadrupeds is an adaptation to the habitual action of the triceps muscle which causes cranial bending strains (compression on cranial side). The caudal curvature is proposed to be adaptive because longitudinal loading induces caudal bending strains (increased compression on the caudal side), and these opposing bending strains counteract each other leaving the radioulna less strained. If this is true for terrestrial quadrupeds, where triceps is required for habitual elbow extension, then we might expect that in arboreal species, where brachialis is habitually required to maintain elbow flexion, the radioulna should instead be cranially curved. This study measures sagittal curvature of the ulna in a range of terrestrial and arboreal primates and marsupials, and finds that their ulnae are curved in opposite directions in these two locomotor categories. This study also examines sagittal curvature in the humerus in the same species, and finds differences that can be attributed to similar adaptations: the bone is curved to counter the habitual muscle action required by the animal’s lifestyle, the difference being mainly in the distal part of the humerus, where arboreal animals tend have a cranial concavity, thought to be in response the carpal and digital muscles that pull cranially on the distal humerus.
Henderson et al. (2017) Forelimb bone curvature in terrestrial and arboreal mammals. PeerJ 5:e3229 https://doi.org/10.7717/peerj.3229
An improved method for the molecular identification of single dinoflagellate cysts
Background Dinoflagellate cysts (i.e., dinocysts) are biologically and ecologically important as they can help dinoflagellate species survive harsh environments, facilitate their dispersal and serve as seeds for harmful algal blooms. In addition, dinocysts derived from some species can produce more toxins than vegetative forms, largely affecting species through their food webs and even human health. Consequently, accurate identification of dinocysts represents the first crucial step in many ecological studies. As dinocysts have limited or even no available taxonomic keys, molecular methods have become the first priority for dinocyst identification. However, molecular identification of dinocysts, particularly when using single cells, poses technical challenges. The most serious is the low success rate of PCR, especially for heterotrophic species. Methods In this study, we aim to improve the success rate of single dinocyst identification for the chosen dinocyst species (Gonyaulax spinifera, Polykrikos kofoidii, Lingulodinium polyedrum, Pyrophacus steinii, Protoperidinium leonis and Protoperidinium oblongum) distributed in the South China Sea. We worked on two major technical issues: cleaning possible PCR inhibitors attached on the cyst surface and designing new dinoflagellate-specific PCR primers to improve the success of PCR amplification. Results For the cleaning of single dinocysts separated from marine sediments, we used ultrasonic wave-based cleaning and optimized cleaning parameters. Our results showed that the optimized ultrasonic wave-based cleaning method largely improved the identification success rate and accuracy of both molecular and morphological identifications. For the molecular identification with the newly designed dinoflagellate-specific primers (18S634F-18S634R), the success ratio was as high as 86.7% for single dinocysts across multiple taxa when using the optimized ultrasonic wave-based cleaning method, and much higher than that (16.7%) based on traditional micropipette-based cleaning. Discussion The technically simple but robust method improved on in this study is expected to serve as a powerful tool in deep understanding of population dynamics of dinocysts and the causes and consequences of potential negative effects caused by dinocysts.
Gao et al. (2017) An improved method for the molecular identification of single dinoflagellate cysts. PeerJ 5:e3224 https://doi.org/10.7717/peerj.3224
The emerging contribution of social wasps to grape rot disease ecology
Grape sour (bunch) rot is a polymicrobial disease of vineyards that causes millions of dollars in lost revenue per year due to decreased quality of grapes and resultant wine. The disease is associated with damaged berries infected with a community of acetic acid bacteria, yeasts, and filamentous fungi that results in rotting berries with high amounts of undesirable volatile acidity. Many insect species cause the initial grape berry damage that can lead to this disease, but most studies have focused on the role of fruit flies in facilitating symptoms and vectoring the microorganisms of this disease complex. Like fruit flies, social wasps are abundant in vineyards where they feed on ripe berries and cause significant damage, while also dispersing yeasts involved in wine fermentation. Despite this, their possible role in disease facilitation and dispersal of grape rots has not been explored. We tested the hypothesis that the paper wasp Polistes dominulus could facilitate grape sour rot in the absence of other insect vectors. Using marker gene sequencing we characterized the bacterial and fungal community of wild-caught adults. We used a sterilized foraging arena to determine if these wasps transfer viable microorganisms when foraging. We then tested if wasps harboring their native microbial community, or those inoculated with sour rot, had an effect on grape sour rot incidence and severity using a laboratory foraging arena. We found that all wasps harbor some portion of the sour rot microbial community and that they have the ability to transfer viable microorganisms when foraging. Foraging by inoculated and uninoculated wasps led to an increase in berry rot disease symptom severity and incidence. Our results indicate that paper wasps can facilitate sour rot diseases in the absence of other vectors and that the mechanism of this facilitation may include both increasing host susceptibility and transmitting these microbial communities to the grapes. Social wasps are understudied but relevant players in the sour rot ecology of vineyards.
Madden et al. (2017) The emerging contribution of social wasps to grape rot disease ecology. PeerJ 5:e3223 https://doi.org/10.7717/peerj.3223
Density estimates of monarch butterflies overwintering in central Mexico
Given the rapid population decline and recent petition for listing of the monarch butterfly (Danaus plexippus L.) under the Endangered Species Act, an accurate estimate of the Eastern, migratory population size is needed. Because of difficulty in counting individual monarchs, the number of hectares occupied by monarchs in the overwintering area is commonly used as a proxy for population size, which is then multiplied by the density of individuals per hectare to estimate population size. There is, however, considerable variation in published estimates of overwintering density, ranging from 6.9–60.9 million ha−1. We develop a probability distribution for overwinter density of monarch butterflies from six published density estimates. The mean density among the mixture of the six published estimates was ∼27.9 million butterflies ha−1 (95% CI [2.4–80.7] million ha−1); the mixture distribution is approximately log-normal, and as such is better represented by the median (21.1 million butterflies ha−1). Based upon assumptions regarding the number of milkweed needed to support monarchs, the amount of milkweed (Asclepias spp.) lost (0.86 billion stems) in the northern US plus the amount of milkweed remaining (1.34 billion stems), we estimate >1.8 billion stems is needed to return monarchs to an average population size of 6 ha. Considerable uncertainty exists in this required amount of milkweed because of the considerable uncertainty occurring in overwinter density estimates. Nevertheless, the estimate is on the same order as other published estimates. The studies included in our synthesis differ substantially by year, location, method, and measures of precision. A better understanding of the factors influencing overwintering density across space and time would be valuable for increasing the precision of conservation recommendations.
Thogmartin et al. (2017) Density estimates of monarch butterflies overwintering in central Mexico. PeerJ 5:e3221 https://doi.org/10.7717/peerj.3221
The determinants and engagement patterns of chaperones and chauffeurs by Australian doctors in after-hours house-call services
Objectives The use of escorts (chauffeurs and chaperones) while on duty in after-hours-house-call (AHHC) is one key protective option available to doctors in the service, and has been linked to low burnout and increased satisfaction in AHHC. This study aims to explore the patterns of engagement of escorts in Australian AHHC. Method This is a questionnaire-based, electronic survey of all 300 doctors involved in AHHC through the National Home Doctor Service (NHDS), Australia’s largest providers of the service. The survey explored the doctor’s experiences over the 12-month period from October 2013 to September 2014. Results This survey received a total of 168 valid responses, giving a response rate of 56%. Nearly 61% of the doctors involved in AHHC engaged escorts (chauffeurs and chaperones). Of those doctors that engage chauffeurs, three-quarters do so “all or most times”, while only one-quarter engaged chaperones to the same degree of frequency. Hiring escorts is very popular among Brisbane (91.7%) and Sydney-based (88.2%) practitioners, but is unpopular in the City of Gold Coast (26.1%). There were moderate patronages in Adelaide (52.9%) and Melbourne Area (46.4%). Compared to males, females were less likely to drive themselves without escorts (OR 0.20; P < 0.01; CI [0.07–0.57]), but more likely to engage chauffeurs (OR 5.87; P = 0.03; CI [1.16–29.77]). Practitioners who were apprehensive were three times more likely to either engage escorts as chauffeurs (OR 3.10; P = 0.04; CI [1.05–9.15]) or as an accompanying chaperone if they self-drive (OR 3.03; P = 0.02; CI [1.16–7.89]). Conclusion More needs to be done to increase the engagement of escorts by doctors involved in the Australian AHHC, particularly given their proven benefits in the service. Future studies may be needed to fully explore the real reasons behind the significant associations identified in this study.
Ifediora (2017) The determinants and engagement patterns of chaperones and chauffeurs by Australian doctors in after-hours house-call services. PeerJ 5:e3218 https://doi.org/10.7717/peerj.3218
The importance of scale-dependent ravine characteristics on breeding-site selection by the Burrowing Parrot, Cyanoliseus patagonus
In birds, the environmental variables and intrinsic characteristics of the nest have important fitness consequences through its influence on the selection of nesting sites. However, the extent to which these variables interact with variables that operate at the landscape scale, and whether there is a hierarchy among the different scales that influences nest-site selection, is unknown. This interaction could be crucial in burrowing birds, which depend heavily on the availability of suitable nesting locations. One representative of this group is the burrowing parrot, Cyanoliseus patagonus that breeds on specific ravines and forms large breeding colonies. At a particular site, breeding aggregations require the concentration of adequate environmental elements for cavity nesting, which are provided by within ravine characteristics. Therefore, intrinsic ravine characteristics should be more important in determining nest site selection compared to landscape level characteristics. Here, we assess this hypothesis by comparing the importance of ravine characteristics operating at different scales on nest-site selection and their interrelation with reproductive success. We quantified 12 characteristics of 105 ravines in their reproductive habitat. For each ravine we quantified morphological variables, distance to resources and disturbance as well as nest number and egg production in order to compare selected and non-selected ravines and determine the interrelationship among variables in explaining ravine differences. In addition, the number of nests and egg production for each reproductive ravine was related to ravine characteristics to assess their relation to reproductive success. We found significant differences between non-reproductive and reproductive ravines in both intrinsic and extrinsic characteristics. The multidimensional environmental gradient of variation between ravines, however, shows that differences are mainly related to intrinsic morphological characteristics followed by extrinsic variables associated to human disturbance. Likewise, within reproductive ravines, intrinsic characteristics are more strongly related to the number of nests. The probability of producing eggs, however, was related only to distance to roads and human settlements. Patterns suggest that C. patagonus mainly selects nesting sites based on intrinsic morphological characteristics of ravines. Scale differences in the importance of ravine characteristics could be a consequence of the particular orography of the breeding habitat. The arrangement of resources is associated to the location of the gullies rather than to individual ravines, determining the spatial availability and disposition of resources and disturbances. Thus, nest selection is influenced by intrinsic characteristics that maximize the fitness of individuals. Scaling in nest-selection is discussed under an optimality approach that partitions patch selection based on foraging theory.
Ramirez-Herranz et al. (2017) The importance of scale-dependent ravine characteristics on breeding-site selection by the Burrowing Parrot, Cyanoliseus patagonus. PeerJ 5:e3182 https://doi.org/10.7717/peerj.3182