De Novo Genome Assembly of Phormia regina (Diptera: Calliphoridae)

poster abstractPhormia regina (Meigen), commonly known as the black blow fly, is a dipteran that belongs to the family Calliphoridae (blow flies). Calliphorids play an important role in various research fields like ecology, medical studies, veterinary and forensic sciences. P. regina is one of the common forensically relevant insects in North America and is typically used to assist in estimating post-mortem intervals (PMI). To better understand the roles it plays in the numerous research fields, we aim to re-construct its genome using next generation sequencing technologies. We are specifically focusing on generating a reference genome by de novo assembly then use the genomic data to identify genetic markers (microsatellites, single nucleotide polymorphisms) that contribute to intra- and inter-population variation with regards to geographic location. DNA was extracted from five adult male and female flies and was sequenced using the Illumina HiSeq2000 sequencing platform. More than 250 million high quality reads were produced from each sex. These reads were used in the de novo genome assembly of the female, male and combined sexes. The assembled draft genomes produced approximately 251,115 contigs, 306,273 contigs, and 325,664 contigs respectively. The assembled genome sizes totaled to ~524 Mbp and ~508 Mbp for the female and male flies, respectively. Compared to the estimated genome sizes from a previous study of 529 Mbp for females and 517 Mbp for males, we can conclude that a majority of the genome sequence (~99%) is included in the assembly. Gene prediction and annotation of the draft genomes are currently in progress. The draft reference genomes assembled from this study will provide an important resource for analyzing genetic basis of variations between and among blow fly species, which will ultimately facilitate ongoing studies in various areas of research that utilize blow flies as study models. It will also be a source where reliable genomic data can be readily available and used in downstream analysis to increase the understanding of the genetic, molecular and cellular processes of blow flies

Comparative genomics of the sheep blow fly Lucilia cuprina

poster abstractInsects employ different adaptive strategies in response to selective pressures, such as competition for limited resources. Carrion insects provide the ideal case to study these fundamental processes of adaptive evolution due to the intense selective pressures placed on developing larvae with limited food resources, their widespread and abundant distributions, and the presence of geographically distinct populations with specialized adaptations. One adaptation is facultative ectoparasitism, where the insect strikes a healthy animal and feeds on the living flesh, providing a developmental advantage over competitor fly species, but causing significant harm to the host. Lucilia species, which hybridize in the wild and form geographically distinct subpopulations in other regions, are diverging, meaning that we can observe and quantify early biological adaptive processes that govern speciation as they are occurring over hundreds, instead of millions, of years. The draft genome of a North American male Lucilia cuprina fly (carrion breeder) was assembled using a combination of short and long read sequences. This genome is compared to an existing Australian draft genome (ectoparasite) by elucidating genomic structure in key adaptive processes (i.e. immune system evasion) via high-throughput re-sequencing of parasitic specimens, gene prediction and annotation. The carcass colonized by or animal parasitized by both species, with some geographic overlap, provides a semi-controlled environment within the larger context of the ecosystem to sample a large number of individuals with similar life history strategies, allowing for direct comparative studies to elucidate the correlation between structure and function in the genomes of carrion flies – allowing us to understand biological adaptation and speciation

Population Genetics of the blow fly Phormia regina (Meigen)

poster abstractBlow flies (Diptera: Calliphoridae) are a family of flies which are associated with the decomposition of organic matter, and the many species are widely distributed across the globe inhabiting almost every ecosystem and continent, with the exception being Antarctica. For many years, biologists have been interested in these metamorphic insects with much attention in recent years with their predictable life cycle and subsequent application in death investigations. Phormia regina (Meigen) is a primary colonizer of carrion and is widely distributed across the United States and can be found throughout the year. Recently, molecular techniques have been applied to a number of blow fly species, initially for identification purposes to corroborate morphological taxonomy but also as an alternative to morphology when specimens are damaged. Even more recently, population structure of these flies have been investigated, however these studies have primarily focused on pest species which infest living livestock and directly affect the agricultural economy. Here, we build upon previous analysis with AFLP markers on P. regina to determine the population structure of this integral species by using tetra-nucleotide microsatellite markers. From the recently constructed draft genome of P. regina, several thousand tetra-nucleotide (four-base repeating sequences of DNA) were identified. Due to the high A-T content of insect DNA, repeats consisting wholly of adenine and thymine bases were discarded. From the remaining tetra-repeats, 14 were chosen for initial analysis to determine their polymorphic nature; from here six were selected for genotypic analysis. More than 200 different P. regina individuals belonging to 27 populations collected from across the contiguous United States have been genotyped across these six tetra-nucleotide loci. Five loci possess ≥30 different alleles, while the sixth appears to be fairly conserved with eight different alleles. Heterozygosity exhibited between these populations is uniform (0.5-0.8) while inbred lab colonies exhibit an increase in homozygosity from their ancestral population. Further analyses are to be performed in order to determine the population structure and geography-genetic relationships of these flies

New Distribution Record for Lucilia cuprina (Diptera: Calliphoridae) in Indiana, United States

Determining range expansion for insect species is vital in order to evaluate their impact on new ecosystems and communities. This is particularly important for species which could be potentially harmful to humans or domestic animals. Lucilia cuprina Wiedemann (Diptera: Calliphoridae) can act as a facultative ectoparasite and has an extensive history as the primary inducer of sheep-strike in Australia, New Zealand, and Africa. We present here the first record of this species in Indiana, United States. Lucilia cuprina's range expansion northward in the United States may be indicative of changing environmental conditions conducive to the proliferation of this species into historically cooler climates. The presence of this species could significantly impact forensic death investigations utilizing dipteran larvae to estimate a minimum postmortem interval. If range expansion of this species is not taken into account by a forensic entomologist (especially if L. cuprina is not known previously in their region), an inaccurate minimum postmortem interval (PMIMIN) estimation may be made, given the differences in development times for both species. Therefore, the range expansion of this fly could have large impacts for many different entomological disciplines

Estimation of the number of contributors of theoretical mixture profiles based on allele counting: Does increasing the number of loci increase success rate of estimates?

DNA mixtures are more frequently encountered in casework due to increased kit sensitivity, protocols with increased cycle number, and requests for low copy number DNA samples to be tested. Generally, the first step in mixture interpretation is determining the number of contributors, with the most common approach of maximum allele count. Although there are previous studies regarding the accuracy of this approach, none have evaluated the accuracy with the newly expanded U.S. core STR loci. In this work, 4,976,355 theoretical mixture combinations were generated with the PowerPlex® Fusion 6C system which includes 23 autosomal STR loci and three Y-STR loci. The number of contributors could be correctly assumed for 100% two-person and 99.99% three-person mixtures, whereas, four-, five-, and six-person mixtures were correctly assumed in 89.7%, 57.3%, and 7.8% of mixtures, respectively. Y-STR analysis showed the 3 Y-STR markers are only accurate for two-person male mixtures (96.7%). This work demonstrates that maximum allele count using the expanded U.S. core loci is not much improved from previous smaller panels, reiterating that this method is not as accurate beyond three contributors

Female Blow Flies As Vertebrate Resource Indicators

Rapid vertebrate diversity evaluation is invaluable for monitoring changing ecosystems worldwide. Wild blow flies naturally recover DNA and chemical signatures from animal carcasses and feces. We demonstrate the power of blow flies as biodiversity monitors through sampling of flies in three environments with varying human influences: Indianapolis, IN and two national parks (the Great Smoky Mountains and Yellowstone). Dissected fly guts underwent vertebrate DNA sequencing (12S and 16S rRNA genes) and fecal metabolite screening. Integrated Nested Laplace Approximation (INLA) was used to determine the most important abiotic factor influencing fly-derived vertebrate richness. In 720 min total sampling time, 28 vertebrate species were identified, with 42% of flies containing vertebrate resources: 23% DNA, 5% feces, and 14% contained both. The species of blow fly used was not important for vertebrate DNA recovery, however the use of female flies versus male flies directly influenced DNA detection. Temperature was statistically relevant across environments in maximizing vertebrate detection (mean = 0.098, sd = 0.048). This method will empower ecologists to test vertebrate community ecology theories previously out of reach due practical challenges associated with traditional sampling

Genome sequence of Phormia regina Meigen (Diptera: Calliphoridae): implications for medical, veterinary and forensic research

BACKGROUND: Blow flies (Diptera: Calliphoridae) are important medical, veterinary and forensic insects encompassing 8 % of the species diversity observed in the calyptrate insects. Few genomic resources exist to understand the diversity and evolution of this group. RESULTS: We present the hybrid (short and long reads) draft assemblies of the male and female genomes of the common North American blow fly, Phormia regina (Diptera: Calliphoridae). The 550 and 534 Mb draft assemblies contained 8312 and 9490 predicted genes in the female and male genomes, respectively; including > 93 % conserved eukaryotic genes. Putative X and Y chromosomes (21 and 14 Mb, respectively) were assembled and annotated. The P. regina genomes appear to contain few mobile genetic elements, an almost complete absence of SINEs, and most of the repetitive landscape consists of simple repetitive sequences. Candidate gene approaches were undertaken to annotate insecticide resistance, sex-determining, chemoreceptors, and antimicrobial peptides. CONCLUSIONS: This work yielded a robust, reliable reference calliphorid genome from a species located in the middle of a calliphorid phylogeny. By adding an additional blow fly genome, the ability to tease apart what might be true of general calliphorids vs. what is specific of two distinct lineages now exists. This resource will provide a strong foundation for future studies into the evolution, population structure, behavior, and physiology of all blow flies

Factors Affecting Species Identifications of Blow Fly Pupae Based upon Chemical Profiles and Multivariate Statistics

Alternative methods for the identification of species of blow fly pupae have been developed over the years that consist of the analyses of chemical profiles. However, the effect of biotic and abiotic factors that could influence the predictive manner for the tests have not been evaluated. The lipids of blowfly pupae (Cochliomyia macellaria, Lucilia cuprina, Lucilia sericata, and Phormia regina) were extracted in pentane, derivatized, and analyzed by total-vaporization solid phase microextraction gas chromatography-mass spectrometry (TV-SPME GC-MS). Peak areas for 26 compounds were analyzed. Here we evaluated one biotic factor (colonization) on four species of blow flies to determine how well a model produced from lipid profiles of colonized flies predicted the species of flies of offspring of wild-caught flies and found very good species identification following 10 generations of inbreeding. When we evaluated four abiotic factors in our fly rearing protocols (temperature, humidity, pupation substrate, and diet), we found that the ability to assign the chemical profile to the correct species was greatly reduced

Primary deficiency of interleukin-1 receptor-associated kinase (IRAK-4) presenting as fatal pseudomonas aeruginosa bacteremia in two siblings

Interleukin-1 receptor-associated kinase 4 (IRAK-4) deficiency is a primary immunodeficiency of innate immunity. This is the case of a previous healthy toddler and his sibling, who both died of fulminant sepsis due to Pseudomonas aeruginosa. Subsequent genetic analysis demonstrated IRAK-4 deficiency with compound heterozygous splice mutations. Fulminant fatal Pseudomonas aeruginosa sepsis may be the first manifestation of IRAK-4 deficiency

Blow fly stable isotopes reveal larval diet: A case study in community level anthropogenic effects

Response to human impacts on the environment are typically initiated too late to remediate negative consequences. We present the novel use of stable isotope analysis (SIA) of blow flies to determine human influences on vertebrate communities in a range of human-inhabited environments, from a pristine national park to a dense metropolitan area. The refrain “you are what you eat” applies to the dietary isotope record of all living organisms, and for carrion-breeding blow flies, this translates to the type of carcasses present in an environment. Specifically, we show that carnivore carcasses make up a large proportion of the adult fly’s prior larval diet, which contrasts to what has been reportedly previously for the wild adult fly diet (which consists of mostly herbivore resources). Additionally, we reveal the potential impact of human food on carcasses that were fed on by blow flies, underscoring the human influences on wild animal populations. Our results demonstrate that using SIA in conjunction with other methods (e.g., DNA analysis of flies) can reveal a comprehensive snapshot of the vertebrate community in a terrestrial ecosystem