The genome of the stable fly, Stomoxys calcitrans, reveals potential mechanisms underlying reproduction, host interactions, and novel targets for pest control.

BACKGROUND: The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies. RESULTS: This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways. CONCLUSIONS: The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.

Pigs vs people: the use of pigs as analogues for humans in forensic entomology and taphonomy research.

Most studies of decomposition in forensic entomology and taphonomy have used non-human cadavers. Following the recommendation of using domestic pig cadavers as analogues for humans in forensic entomology in the 1980s, pigs became the most frequently used model cadavers in forensic sciences. They have shaped our understanding of how large vertebrate cadavers decompose in, for example, various environments, seasons and after various ante- or postmortem cadaver modifications. They have also been used to demonstrate the feasibility of several new or well-established forensic techniques. The advent of outdoor human taphonomy facilities enabled experimental comparisons of decomposition between pig and human cadavers. Recent comparisons challenged the pig-as-analogue claim in entomology and taphonomy research. In this review, we discuss in a broad methodological context the advantages and disadvantages of pig and human cadavers for forensic research and rebut the critique of pigs as analogues for humans. We conclude that experiments using human cadaver analogues (i.e. pig carcasses) are easier to replicate and more practical for controlling confounding factors than studies based solely on humans and, therefore, are likely to remain our primary epistemic source of forensic knowledge for the immediate future. We supplement these considerations with new guidelines for model cadaver choice in forensic science research.

Identifying similar transcripts in a related organism from de Bruijn graphs of RNA-Seq data, with applications to the study of salt and waterlogging tolerance in Melilotus.

BACKGROUND: A popular strategy to study alternative splicing in non-model organisms starts from sequencing the entire transcriptome, then assembling the reads by using de novo transcriptome assembly algorithms to obtain predicted transcripts. A similarity search algorithm is then applied to a related organism to infer possible function of these predicted transcripts. While some of these predictions may be inaccurate and transcripts with low coverage are often missed, we observe that it is possible to obtain a more complete set of transcripts to facilitate possible functional assignments by starting the search from the intermediate de Bruijn graph that contains all branching possibilities. RESULTS: We develop an algorithm to extract similar transcripts in a related organism by starting the search from the de Bruijn graph that represents the transcriptome instead of from predicted transcripts. We show that our algorithm is able to recover more similar transcripts than existing algorithms, with large improvements in obtaining longer transcripts and a finer resolution of isoforms. We apply our algorithm to study salt and waterlogging tolerance in two Melilotus species by constructing new RNA-Seq libraries. CONCLUSIONS: We have developed an algorithm to identify paths in the de Bruijn graph that correspond to similar transcripts in a related organism directly. Our strategy bypasses the transcript prediction step in RNA-Seq data and makes use of support from evolutionary information.

The upper thermal tolerance of the secondary screwworm, Cochliomyia macellaria Fabricius (Diptera: Calliphoridae).

Determining the thermal tolerance of an organism is important when assessing its activity time and survival rate in a given environment. However, thermal tolerance is not a static trait and may be influenced by a number of environmental and organismal factors. We report the results of three experiments investigating the effects of environmental temperature, exposure duration, age, sex, and nutrient availability on the upper thermal tolerance of the adult secondary screwworm, Cochliomyia macellaria. The probability of knockdown and survival was determined using a static method for different environmental temperatures (22, 40, 42, 44, or 45 °C), exposure durations (1, 2, 4, or 6 h), and nutrient availabilities (no food or water, water only, or both food and water) for both sexes and two age classes (young = 7-9 days post pupal emergence, old = 10-12 days post pupal emergence). In general, environmental temperature and exposure duration had the greatest effects on both the probability of knockdown and survival. As temperature or duration increased, the probability of knockdown increased while the probability of survival decreased. The availability of nutrients (water only or food and water) increased thermal tolerance at moderate temperatures (42 and 44 °C), but had no effect at 45 °C. Female flies were more thermally tolerant than males, regardless of nutrient availability. And age exhibited negligible effects on the probabilities of knockdown or survival, regardless of nutrient availability. These data show multiple environmental factors affected the thermal tolerance of C. macellaria. Thus, such aspects of basic thermal biology should feature more prominently in applied fields using blow flies, including but not limited to forensic entomology, disease ecology, and pollination ecology.

A divide-and-conquer algorithm for large-scale de novo transcriptome assembly through combining small assemblies from existing algorithms.

BACKGROUND: While the continued development of high-throughput sequencing has facilitated studies of entire transcriptomes in non-model organisms, the incorporation of an increasing amount of RNA-Seq libraries has made de novo transcriptome assembly difficult. Although algorithms that can assemble a large amount of RNA-Seq data are available, they are generally very memory-intensive and can only be used to construct small assemblies. RESULTS: We develop a divide-and-conquer strategy that allows these algorithms to be utilized, by subdividing a large RNA-Seq data set into small libraries. Each individual library is assembled independently by an existing algorithm, and a merging algorithm is developed to combine these assemblies by picking a subset of high quality transcripts to form a large transcriptome. When compared to existing algorithms that return a single assembly directly, this strategy achieves comparable or increased accuracy as memory-efficient algorithms that can be used to process a large amount of RNA-Seq data, and comparable or decreased accuracy as memory-intensive algorithms that can only be used to construct small assemblies. CONCLUSIONS: Our divide-and-conquer strategy allows memory-intensive de novo transcriptome assembly algorithms to be utilized to construct large assemblies.

A scalable and memory-efficient algorithm for de novo transcriptome assembly of non-model organisms.

BACKGROUND: With increased availability of de novo assembly algorithms, it is feasible to study entire transcriptomes of non-model organisms. While algorithms are available that are specifically designed for performing transcriptome assembly from high-throughput sequencing data, they are very memory-intensive, limiting their applications to small data sets with few libraries. RESULTS: We develop a transcriptome assembly algorithm that recovers alternatively spliced isoforms and expression levels while utilizing as many RNA-Seq libraries as possible that contain hundreds of gigabases of data. New techniques are developed so that computations can be performed on a computing cluster with moderate amount of physical memory. CONCLUSIONS: Our strategy minimizes memory consumption while simultaneously obtaining comparable or improved accuracy over existing algorithms. It provides support for incremental updates of assemblies when new libraries become available.

Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.

Intrapopulation genome size variation in D. melanogaster reflects life history variation and plasticity.

We determined female genome sizes using flow cytometry for 211 Drosophila melanogaster sequenced inbred strains from the Drosophila Genetic Reference Panel, and found significant conspecific and intrapopulation variation in genome size. We also compared several life history traits for 25 lines with large and 25 lines with small genomes in three thermal environments, and found that genome size as well as genome size by temperature interactions significantly correlated with survival to pupation and adulthood, time to pupation, female pupal mass, and female eclosion rates. Genome size accounted for up to 23% of the variation in developmental phenotypes, but the contribution of genome size to variation in life history traits was plastic and varied according to the thermal environment. Expression data implicate differences in metabolism that correspond to genome size variation. These results indicate that significant genome size variation exists within D. melanogaster and this variation may impact the evolutionary ecology of the species. Genome size variation accounts for a significant portion of life history variation in an environmentally dependent manner, suggesting that potential fitness effects associated with genome size variation also depend on environmental conditions.

Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines.

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

Heuristic pairwise alignment of de Bruijn graphs to facilitate simultaneous transcript discovery in related organisms from RNA-Seq data.

BACKGROUND: The advance of high-throughput sequencing has made it possible to obtain new transcriptomes and study splicing mechanisms in non-model organisms. In these studies, there is often a need to investigate the transcriptomes of two related organisms at the same time in order to find the similarities and differences between them. The traditional approach to address this problem is to perform de novo transcriptome assemblies to obtain predicted transcripts for these organisms independently and then employ similarity comparison algorithms to study them. RESULTS: Instead of obtaining predicted transcripts for these organisms separately from the intermediate de Bruijn graph structures employed by de novo transcriptome assembly algorithms, we develop an algorithm to allow direct comparisons between paths in two de Bruijn graphs by first enumerating short paths in both graphs, and iteratively extending paths in one graph that have high similarity to paths in the other graph to obtain longer corresponding paths between the two graphs. These paths represent predicted transcripts that are present in both organisms. CONCLUSIONS: Our approach generalizes the pairwise sequence alignment problem to allow the input to be non-linear structures, and provides a heuristic to reliably recover similar paths from the two structures. Our algorithm allows detailed investigation of the similarities and differences in alternative splicing between the two organisms at both the sequence and structure levels, even in the absence of reference transcriptomes or a closely related model organism.

Field Documentation of Unusual Post-Mortem Arthropod Activity on Human Remains.

During a forensic investigation, the presence of physical marks on human remains can influence the interpretation of events related to the death of an individual. Some tissue injury on human remains can be misinterpreted as ante- or peri-mortem wounds by an investigator when in reality the markings resulted from post-mortem arthropod activity. Unusual entomological data were collected during a study examining the decomposition of a set of human remains in San Marcos, Texas. An adult female Pediodectes haldemani (Girard) (Orthoptera: Tettigoniidae) and an Armadillidium cf. vulgare (Isopoda: Armadilidiidae) were documented feeding on the remains. Both arthropods produced physical marks or artifacts on the remains that could be misinterpreted as attack, abuse, neglect, or torture. Additionally, red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae), were observed constructing structures in the mark produced by the P. haldemani feeding. These observations provide insight into the potential of post-mortem arthropod damage to human remains, which previously had not been described for these taxa, and therefore, physical artifacts on any remains found in similar circumstances may result from arthropod activity and not ante- or peri-mortem wounds.

A metagenomic assessment of the bacteria associated with Lucilia sericata and Lucilia cuprina (Diptera: Calliphoridae).

Lucilia Robineau-Desvoidy (Diptera: Calliphoridae) is a blow fly genus of forensic, medical, veterinary, and agricultural importance. This genus is also famous because of its beneficial uses in maggot debridement therapy (MDT). Although the genus is of considerable economic importance, our knowledge about microbes associated with these flies and how these bacteria are horizontally and trans-generationally transmitted is limited. In this study, we characterized bacteria associated with different life stages of Lucilia sericata (Meigen) and Lucilia cuprina (Wiedemann) and in the salivary gland of L. sericata by using 16S rDNA 454 pyrosequencing. Bacteria associated with the salivary gland of L. sericata were also characterized using light and transmission electron microscopy (TEM). Results from this study suggest that the majority of bacteria associated with these flies belong to phyla Proteobacteria, Firmicutes, and Bacteroidetes, and most bacteria are maintained intragenerationally, with a considerable degree of turnover from generation to generation. In both species, second-generation eggs exhibited the highest bacterial phylum diversity (20 % genetic distance) than other life stages. The Lucilia sister species shared the majority of their classified genera. Of the shared bacterial genera, Providencia, Ignatzschineria, Lactobacillus, Lactococcus, Vagococcus, Morganella, and Myroides were present at relatively high abundances. Lactobacillus, Proteus, Diaphorobacter, and Morganella were the dominant bacterial genera associated with a survey of the salivary gland of L. sericata. TEM analysis showed a sparse distribution of both Gram-positive and Gram-negative bacteria in the salivary gland of L. sericata. There was more evidence for horizontal transmission of bacteria than there was for trans-generational inheritance. Several pathogenic genera were either amplified or reduced by the larval feeding on decomposing liver as a resource. Overall, this study provides information on bacterial communities associated with different life stages of Lucilia and their horizontal and trans-generational transmission, which may help in the development of better vector-borne disease management and MDT methods.

A memory-efficient algorithm to obtain splicing graphs and de novo expression estimates from de Bruijn graphs of RNA-Seq data.

BACKGROUND: The recent advance of high-throughput sequencing makes it feasible to study entire transcriptomes through the application of de novo sequence assembly algorithms. While a popular strategy is to first construct an intermediate de Bruijn graph structure to represent the transcriptome, an additional step is needed to construct predicted transcripts from the graph. RESULTS: Since the de Bruijn graph contains all branching possibilities, we develop a memory-efficient algorithm to recover alternative splicing information and library-specific expression information directly from the graph without prior genomic knowledge. We implement the algorithm as a postprocessing module of the Velvet assembler. We validate our algorithm by simulating the transcriptome assembly of Drosophila using its known genome, and by performing Drosophila transcriptome assembly using publicly available RNA-Seq libraries. Under a range of conditions, our algorithm recovers sequences and alternative splicing junctions with higher specificity than Oases or Trans-ABySS. CONCLUSIONS: Since our postprocessing algorithm does not consume as much memory as Velvet and is less memory-intensive than Oases, it allows biologists to assemble large libraries with limited computational resources. Our algorithm has been applied to perform transcriptome assembly of the non-model blow fly Lucilia sericata that was reported in a previous article, which shows that the assembly is of high quality and it facilitates comparison of the Lucilia sericata transcriptome to Drosophila and two mosquitoes, prediction and experimental validation of alternative splicing, investigation of differential expression among various developmental stages, and identification of transposable elements.

Developmental variation among Cochliomyia macellaria Fabricius (Diptera: Calliphoridae) populations from three ecoregions of Texas, USA.

Forensic entomologists rely on published developmental datasets to estimate the age of insects developing on human remains. Currently, these datasets only represent populations of targeted insects from specific locations. However, recent data indicate that populations can exhibit genetic variation in their development, including signatures of local adaptation demonstrated by regionally distinct plastic responses to their environments. In this study, three geographically distinct populations of the secondary screwworm, Cochliomyia macellaria Fabricius (Diptera: Calliphoridae; College Station, Longview, and San Marcos, TX, USA), a common blow fly collected from human remains in the southern USA, were reared in two distinct environments (cool 21 °C, 65 % relative humidity (RH); and warm 31 °C, 70 % RH) over 2 years (2011 and 2012) in order to determine differences in development time and mass. Significant differences in immature and pupal development time, as well as pupal mass, were shown to exist among strains derived from different populations and years. For immature development times, there was evidence of only an environmental effect on phenotype, while genotype by environment interactions was observed in pupal development times and pupal mass. College Station and San Marcos populations exhibited faster pupal development and smaller pupal sizes in the cooler environment relative to the Longview population, but showed an opposite trend in the warm environment. Rank order for College Station and Longview populations was reversed across years. Failure to take genetic variation into consideration when making such estimates can lead to unanticipated error and bias. These results indicate that genetics will have little impact on error when working with Texas genotypes of C. macellaria at ~30 °C and 70 % RH, but will have a more meaningful impact on error in postmortem interval estimates with this species in cooler, drier environments.

The potential use of bacterial community succession in forensics as described by high throughput metagenomic sequencing.

Decomposition studies of vertebrate remains primarily focus on data that can be seen with the naked eye, such as arthropod or vertebrate scavenger activity, with little regard for what might be occurring with the microorganism community. Here, we discuss the necrobiome, or community of organisms associated with the decomposition of remains, specifically, the "epinecrotic" bacterial community succession throughout decomposition of vertebrate carrion. Pyrosequencing was used to (1) detect and identify bacterial community abundance patterns that described discrete time points of the decomposition process and (2) identify bacterial taxa important for estimating physiological time, a time-temperature metric that is often commensurate with minimum post-mortem interval estimates, via thermal summation models. There were significant bacterial community structure differences in taxon richness and relative abundance patterns through the decomposition process at both phylum and family taxonomic classification levels. We found a significant negative linear relationship for overall phylum and family taxon richness as decomposition progressed. Additionally, we developed a statistical model using high throughput sequencing data of epinecrotic bacterial communities on vertebrate remains that explained 94.4 % of the time since placement of remains in the field, which was within 2-3 h of death. These bacteria taxa are potentially useful for estimating the minimum post-mortem interval. Lastly, we provide a new framework and standard operating procedure of how this novel approach of using high throughput metagenomic sequencing has remarkable potential as a new forensic tool. Documenting and identifying differences in bacterial communities is key to advancing knowledge of the carrion necrobiome and its applicability in forensic science.

The role of spatial aggregation in forensic entomology.

A central concept in forensic entomology is that arthropod succession on carrion is predictable and can be used to estimate the postmortem interval (PMI) of human remains. However, most studies have reported significant variation in successional patterns, particularly among replicate carcasses, which has complicated estimates of PMIs. Several forensic entomology researchers have proposed that further integration of ecological and evolutionary theory in forensic entomology could help advance the application of succession data for producing PMI estimates. The purpose of this essay is to draw attention to the role of spatial aggregation of arthropods among carrion resources as a potentially important aspect to consider for understanding and predicting the assembly of arthropods on carrion over time. We review ecological literature related to spatial aggregation of arthropods among patchy and ephemeral resources, such as carrion, and when possible integrate these results with published forensic literature. We show that spatial aggregation of arthropods across resources is commonly reported and has been used to provide fundamental insight for understanding regional and local patterns of arthropod diversity and coexistence. Moreover, two suggestions are made for conducting future research. First, because intraspecific aggregation affects species frequency distributions across carcasses, data from replicate carcasses should not be combined, but rather statistically quantified to generate occurrence probabilities. Second, we identify a need for studies that tease apart the degree to which community assembly on carrion is spatially versus temporally structured, which will aid in developing mechanistic hypotheses on the ecological factors shaping community assembly on carcasses.

Structural and Genetic Investigation of the Egg and First-Instar Larva of an Egg-Laying Population of Blaesoxipha plinthopyga (Diptera: Sarcophagidae), a Species of Forensic Importance.

Flies in the family Sarcophagidae incubate their eggs and are known to be ovoviviparous (i.e., ovolarviparous), but a laboratory-maintained colony of Blaesoxipha plinthopyga (Wiedemann) deposited clutches of viable eggs over 10 generations. A description of the egg and first-instar larva of this species is provided along with genetic data (genome size and cytochrome oxidase I sequences). The egg is similar to previously described eggs of other Sarcophagidae but differs in the configuration of the micropyle. In the first-instar larva, the oral ridges are much more developed than has been described for other species. B. plinthopyga has forensic importance, and the present descriptive information is critical for proper case management.

Population-based resequencing of experimentally evolved populations reveals the genetic basis of body size variation in Drosophila melanogaster.

Body size is a classic quantitative trait with evolutionarily significant variation within many species. Locating the alleles responsible for this variation would help understand the maintenance of variation in body size in particular, as well as quantitative traits in general. However, successful genome-wide association of genotype and phenotype may require very large sample sizes if alleles have low population frequencies or modest effects. As a complementary approach, we propose that population-based resequencing of experimentally evolved populations allows for considerable power to map functional variation. Here, we use this technique to investigate the genetic basis of natural variation in body size in Drosophila melanogaster. Significant differentiation of hundreds of loci in replicate selection populations supports the hypothesis that the genetic basis of body size variation is very polygenic in D. melanogaster. Significantly differentiated variants are limited to single genes at some loci, allowing precise hypotheses to be formed regarding causal polymorphisms, while other significant regions are large and contain many genes. By using significantly associated polymorphisms as a priori candidates in follow-up studies, these data are expected to provide considerable power to determine the genetic basis of natural variation in body size.

Antitoxin MqsA helps mediate the bacterial general stress response.

Although it is well recognized that bacteria respond to environmental stress through global networks, the mechanism by which stress is relayed to the interior of the cell is poorly understood. Here we show that enigmatic toxin-antitoxin systems are vital in mediating the environmental stress response. Specifically, the antitoxin MqsA represses rpoS, which encodes the master regulator of stress. Repression of rpoS by MqsA reduces the concentration of the internal messenger 3,5-cyclic diguanylic acid, leading to increased motility and decreased biofilm formation. Furthermore, the repression of rpoS by MqsA decreases oxidative stress resistance via catalase activity. Upon oxidative stress, MqsA is rapidly degraded by Lon protease, resulting in induction of rpoS. Hence, we show that external stress alters gene regulation controlled by toxin-antitoxin systems, such that the degradation of antitoxins during stress leads to a switch from the planktonic state (high motility) to the biofilm state (low motility).