Indiana University-Purdue University Indianapolis (IUPUI) / Population genetic structure is difficult to assess in blow flies (Diptera: Calliphoridae) due to high connectivity and genetic diversity of subpopulations. Previous studies revealed high relatedness among individuals within wild samples of blow fly populations, however broad geographic structure was absent. The aim of this research was to determine if blow fly genetic structure exists at a fine spatiotemporal resolution and, if so, to elucidate the influence of environmental factors and resource availability on fly genetics. Specifically, blow fly population genetic patterns were tested against a null hypothesis that flies adhere to a patchy population model with high genetic diversity (i.e. no structure) and high resource availability. Samples of the black blow fly, Phormia regina Meigen (Diptera: Calliphoridae), were collected at six urban parks in Indiana, USA (=urban) in 2016 and 2017 (N = 14 and 16 timepoints, respectively). Additional sampling in different ecoregions was performed to determine if trends observed at a high-resolution scale were also present at a broad geographic scale. Therefore, P. regina were also collected at four sites within two national parks (the Great Smoky Mountains and Yellowstone National Parks) over a three-day period. Randomly selected females (N = 10) from each sample underwent the following analyses: 1) gut DNA extraction, 2) molecular analysis at 6 microsatellite loci, 3) vertebrate-specific 12S and 16S rRNA sequencing, and, 4) vertebrate fecal metabolite screening. Flies from the national parks and a comparable subset of urban data also underwent stable isotope analysis (SIA) to determine larval food source. Overall, strong seasonal population genetic structure was observed over both years in the urban environment (2016 F’ST = 0.47, 2017 F’ST 0.34), however spatial structure was lacking, as seen in previous studies (2016 F’ST = 0.04, 2017 F’ST 0.03). Weather conditions prior to and on the day of blow fly collections, interspecific competition, and resource availability greatly impacted the genetic diversity and kinship of P. regina. A total of 17 and 19 vertebrate species were detected by flies in 2016 and 2017, respectively, and many flies tested positive for vertebrate feces, suggesting that many varied resources are important for maintaining high gene flow among geographic locations. Genetic diversity was non-existent in flies collected from the Smokies (F’ST = 0.00), while very slight spatial structure existed in the Yellowstone populations (F’ST = 0.07). Environmental factors such as temperature, humidity, and wind speed were all statistically relevant in maximizing fly collections with vertebrate resources. In 720 min of total sampling time in the national parks and a subset of urban data, 28 vertebrate species were identified, and fecal resources appeared to be the most abundant in Yellowstone. Stable isotope analysis revealed a majority of larval resources in the national parks were herbivores, with a more even distribution of carnivore and herbivore carcasses present in the urban environment, which likely explains the high genetic diversity of adult flies in these regions. Overall, the null hypothesis that P. regina adheres to a patchy population model could not be rejected for the Smokies populations. However, the urban and Yellowstone populations appear to adhere to a Levins metapopulation model in which variable availability in resources leads to random bottleneck events in the local populations. Overall, environmental conditions, competition, and resource availability are all important factors influencing P. regina population genetic structure in different environments.

Forensic entomology is a well-established science linking arthropod biology and ecology to legal investigations. Specifically, immature development on a decomposing corpse may give insight into the minimum time elapsed since death. Until recently, biological variation within a single species has been overlooked when estimating colonization events. Variation in the form of phenotypic plasticity, or the ability of a single genotype to produce multiple phenotypes under alternative stresses, has been documented in genetic and ecological literature and spans across all phyla. Taking this into account, different subpopulations of forensically pertinent insect species should also possess the ability to adapt to changing environments as geographic distribution increases. Thus, plastic responses of a species to alternative stresses may be measured in biological parameters, such as development time. In this research, three geographically distinct strains of the blow fly Cochliomyia macellaria Fabricius (Diptera Calliphoridae) were reared in two distinct environments in order to measure development time, as well as pupal and adult masses. Strains exhibited genetic variance when compared to each other, and each strain exhibited variable responses across environments (phenotypic plasticity). Plasticity in the form of genotype by environment (GxE) interactions was also exhibited by C. macellaria, although consistent adherence to any single rule explaining ontogenetic trends was not apparent. This research supports the existence of intraspecific variation in a common blow fly of forensic importance. Results of this study will impact the forensic entomology community by encouraging the generation of either strain;specific developmental datasets or statistical models to minimize variation caused by genetic, environment, or GxE effects in order to compare developmental data across strains.

Blow fly (Diptera: Calliphoridae) interactions in decomposition ecology are well studied; however, the non-consumptive effects (NCE) of predators on the behavior and development of prey species have yet to be examined. The effects of these interactions and the resulting cascades in the ecosystem dynamics are important for species conservation and community structures. The resulting effects can impact the time of colonization (TOC) of remains for use in minimum post-mortem interval (mPMI) estimations. The development of the predacious blow fly, Chrysomya rufifacies (Macquart) was examined and determined to be sensitive to muscle type reared on, and not temperatures exposed to. Development time is important in forensic investigations utilizing entomological evidence to help establish a mPMI. Validation of the laboratory-based development data was done through blind TOC calculations and comparisons with known TOC times to assess errors. A range of errors was observed, depending on the stage of development of the collected flies, for all methods tested with no one method providing the most accurate estimation. The NCE of the predator blow fly on prey blow fly, Cochliomyia macellaria (Fabricius) behavior and development were observed in the laboratory. Gravid female adult attraction was significantly greater to resources with predatory larvae rather than prey larvae and oviposition occurred on in the presence of heterospecific (predatory) and conspecific larvae equally. However, the life stages necessary for predation to occur never overlapped and so these results may not be as surprising as they seem. Conversely, exposing prey larvae to predator cues through larval excretions/secretions led to larger prey larvae and faster times to pupariation when appropriate life stages overlapped. Differences in size and development times of prey larvae in the presence of predatory cues could lead to errors when estimating the mPMI. These data also partially explain the ability of C. macellaria to survive in the presence of Ch. rufifacies. Colonization of a resource with late instar Ch. rufifacies enhanced development and size of resulting larvae indicating that lag colonization, rather than being a primary colonizer, could become an alternate strategy for C. macellaria to survive the selective pressures of the predator, Ch. rufifacies. The differing effects of temperature on Ch. rufifacies and C. macellaria may also lend an advantage to C. macellaria over the predacious Ch. rufifacies in an environment with variable temperatures unlike what Ch. rufifacies is adapted for.

No description available.

<p dir="ltr"><i>Phormia regina</i> Meigen is a forensically relevant species of blow fly, common in North America and used to estimate the minimum postmortem interval in forensic casework. It is also possible to use blow flies to survey the environment for biotic and abiotic information drawn from both larval and adult stages. There are both forensic and environmental uses for genetic analysis of blow flies. Blow fly kinship is especially useful for detecting postmortem movement of a corpse or to assess abundance of carrion in a given location. To test genetic relationships between individuals, discriminatory loci such as microsatellites, or polymorphic tandemly repeated sequences of DNA are necessary. Here, we characterize novel microsatellites generated from the genome of <i>P. regina</i>. Thirty-four candidate polymorphic loci with conserved flanking regions, have been isolated. To date, seven are heterozygous and polymorphic testing in two lab populations and one wild population. The simple sequence repeats characterized here complement existing loci (N = 6) for greater discrimination for testing relationships between individual flies.</p>

<p dir="ltr">Blow flies are a family of carrion insects that are among the first to arrive in the decomposition process. Blow flies are known to ingest carrion, feces, water, and occasionally nectar to meet nutritional requirements. These behaviors make blow flies a unique organism potentially containing genetic material from a variety of sources within one environment. Their global distribution and ease of capture makes them a strong candidate for resource monitoring and identification. While previous studies have evaluated the suitability of blow flies for vertebrate biodiversity estimates, no work has been done looking at their ability to ingest and store genetic material from plants and microbes present in water. It is also not known how long these DNA signals persist in the gut. Through DNA analysis of the blow fly gut, researchers can identify vertebrates that have recently died in an environment, what plant species are present, and what water source the insect utilized. Through lab colony (Phormia regina) feeding experiments, it was determined that at 25 ˚C and 50 % relative humidity, vertebrate and plant DNA persist in the gut for over 120 hours post-ingestion. Wild sample analysis of flies collected from Yellowstone National Park was performed to identify plant species ingested by P. regina in the wild. Following Sanger sequencing, top hits on BLASTn included Brassicales, Juglans cathyensis, and uncultured Candida. This is the first application of environmental DNA analysis techniques to insects for the purpose of plant identification. This work also attempts to characterize microbial profiles of the gut of P. regina for the purpose of water resource identification. Over a two-month collection period, samples were collected from different water resources across Indianapolis. Flies were exposed to these samples in a controlled feeding experiment, followed by sampling at 0- and 72-hours post-exposure. Gut samples were sequenced using Illumina and Operational Taxonomic Unit clustering grouped reads by sequence similarity for identification. Bacteria classes identified included Gammaproteobacteria, Bacteroidia, Flavobacteria, Alphaproteobacteria, Bacilli, Clostridia, Actinobacteria, Betaproteobacteria, and Fusobacteria. Many bacteria classes were common across water samples, although the abundance of each class changed between samples and across time. These unique microbial profiles can be used to identify water resources for potential contamination and chemical dumping. Further work is necessary to generate microbial profiles from the original water sources themselves and for generation of alpha and beta diversities. Overall, this work spans multiple fields. Species identification is important for biodiversity monitoring and environmental surveys. Utilizing blow fly derived DNA allows for detection of living and deceased vertebrates in an environment, plant life, and water quality within one sample. This work also has implications in forensic science, specifically wildlife forensics and chemical detection of clandestine laboratories and chemical weapon compounds.</p>

<div>Forensic entomology is a subdiscipline of entomology that involves the use of insect behavior and developmental data to aid in criminal investigations. Genetic data has become increasingly important to the field as there has been a push for DNA-based species identification methods of forensically relevant insects. Genetic data can also elucidate population structure and relatedness of these insects, and such knowledge can contribute to the development of more specific datasets for insects in different regions. The first study presented here investigated the phylogenetics of sister species <i>Lucilia cuprina</i> and <i>Lucilia sericata</i> to identify possible subspecies divisions and issues with DNA-based identifications in the United States. The initial aim of this study was to identify genetic differences between specimens of <i>L. cuprina</i> that preferred live versus carrion flesh. Flies collected from Indiana, USA and South Africa were sequenced and analyzed. Upon sequencing of the genes <i>COI, Period,</i> and <i>28s,</i> our results indicated that <i>L. cuprina</i> from Indiana possess a unique combination of nuclear and mitochondrial haplotypes that suggest a unique lineage, possibly indicating modern hybridization with <i>L. sericata. </i>The inability of both nuclear and mitochondrial genes to distinguish between <i>L. cuprina</i> and <i>L. sericata</i> raises questions about the capabilities of DNA-based species identifications within this genus. Additionally, the inability of these genes to distinguish between specimens that preferred live versus carrion flesh highlights a need for continued research of these behavioral differences. The second study presented here investigated the population structure and relatedness of house flies in the American southwest in relation to a civil lawsuit where neighbors of a poultry farm alleged that flies were emanating from the farm to their homes. <i>Musca domestica</i> (house fly) specimens were collected from the chicken farm and from locations in varying directions and distances from the farm. Amplified fragment length polymorphism (AFLP) analysis was performed and the data were used in a number of analyses. Population reallocation simulations generally indicated that samples from different locations were not genetically different enough from other locations to allocate to their true origin population over others. Kinship analysis showed differences in samples collected in a later season that indicate a genetic bottleneck over time. Population structure analysis indicated the presence of two intermixing genetic populations in the dataset. AMOVA revealed that the majority of genetic variation laid within, rather than among, populations. A Mantel test revealed no significant correlation between genetic and geographic distances. These results indicate that the <i>M. domestica</i> population in this region of southwestern America is large and intermixing, with no clear genetic distinctions between specimens collected at the poultry farm versus the surrounding locations. In regard to the civil lawsuit, it was not possible to conclude that the flies did not emanate from the poultry farm. In a broader perspective, these data can be utilized to develop pest management strategies in this region. Overall, the data from both studies presented here will be useful to forensic investigations, development of more specific and detailed data and identification techniques, and pest control measures.</div>

<div>Chromosomal mechanisms of sex determination vary greatly in phylogenetically closely related species, indicative of rapid evolutionary rates. Sex chromosome karyotypes are generally conserved within families; however, many species have derived sex chromosome configurations. Insects display a plethora of sex chromosome systems due to rapid diversification caused by changes in evolutionary processes within and between species. A good example of such a system are insects in the blow fly family Calliphoridae. While cytogenetic studies observe that the karyotype in blow flies is highly conserved (five pairs of autosomal chromosomes and one pair sex chromosome), there is variation in sex determining mechanisms and sex chromosome structure within closely related species in blow flies. The evolutionary history of sex chromosomes in blow fly species have not been fully explored. Therefore, the objective of this research was to characterize the sex chromosome structures in four species of blow flies and investigate the selective forces which have played a role in shaping the diverse sex chromosome system observed in blow flies. The blow fly species used in this study are Phormia regina, Lucilia cuprina, Chrysomya rufifacies and Chrysomya albiceps. Phormia regina,and Lucilia cuprina have a heteromorphic sex chromosome system and are amphogenic (females produce both male and female offspring in equal ratio). In contrast, Chrysomya rufifacies and Chrysomya albiceps, have a homomorphic sex chromosome system, are monogenic (females produce unisexual progeny), have two types of females (arrhenogenic females – male producers and thelygenic females – female producers), and sex of the offspring is determined by the maternal genotype. </div><div>To accomplish these tasks, a total of nine male and female individual draft genomes for each of the four species (including three individual draft genomes of Chrysomya rufifacies – male, and the two females) were sequenced and assembled providing genomic data to explore sex chromosome evolution in blow flies. Whole genome analysis was utilized to characterize and identify putative sex chromosomal sequences of the four blow fly species. Genomic evidence confirmed the presence of genetically differentiated sex chromosomes in P. regina and L. cuprina; and genetically undifferentiated sex chromosomes in C. rufifacies and C. albiceps. Furthermore, comparative analysis of the ancestral Dipteran sex chromosome (Muller element F in Drosophila) was determined to be X-linked in P. regina and L. cuprina contributing to sex chromosome differentiation but not sex-linked in C. rufifacies and C. albiceps. Evolutionary pressures are often quantified by the ratio of substitution rates at non-synonymous (dN) and synonymous (dS) sites. Substitution rate ratio analysis (dN/dS) of homologous genes indicated a weaker purifying selection may have contributed to the loss of sex-linked genes in Muller element F genes of the undifferentiated sex chromosome as compared to the differentiated sex chromosome system. Overall, the results presented herein greatly expands our knowledge in sex chromosome evolution within blow flies and will reinforce the study of sex chromosome evolution in other species with diverse sex chromosome systems.</div><div><br></div>

Indiana University-Purdue University Indianapolis (IUPUI) / Phormia regina (Meigen), commonly known as the black blow fly is a dipteran that belongs to the family Calliphoridae. Calliphorids play an important role in various research fields including ecology, medical studies, veterinary and forensic sciences. P. regina, a non-model organism, is one of the most common forensically relevant insects in North America and is typically used to assist in estimating postmortem intervals (PMI). To better understand the roles P. regina plays in the numerous research fields, we re-constructed its genome using next generation sequencing technologies. The focus was on generating a reference genome through de novo assembly of high-throughput short read sequences. Following assembly, genetic markers were identified in the form of microsatellites and single nucleotide polymorphisms (SNPs) to aid in future population genetic surveys of P. regina. A total 530 million 100 bp paired-end reads were obtained from five pooled male and female P. regina flies using the Illumina HiSeq2000 sequencing platform. A 524 Mbp draft genome was assembled using both sexes with 11,037 predicted genes. The draft reference genome assembled from this study provides an important resource for investigating the genetic diversity that exists between and among blow fly species; and empowers the understanding of their genetic basis in terms of adaptations, population structure and evolution. The genomic tools will facilitate the analysis of genome-wide studies using modern genomic techniques to boost a refined understanding of the evolutionary processes underlying genomic evolution between blow flies and other insect species.