Temporal population genetic structure of Phormia regina (Diptera: Calliphoridae).

The genetic structure of forensically important blow fly (Brauer & Bergenstamm) (Diptera: Calliphoridae) populations has remained elusive despite high relatedness within wild-caught samples. This research aimed to determine if the implementation of a high-resolution spatiotemporal sampling design would reveal latent genetic structure among blow fly populations and to elucidate any environmental impacts on observed patterns of genetic structure. Adult females of the black blow fly, Phormia regina (Meigen) (Diptera: Calliphoridae), were collected from 9 urban parks in Indiana, USA over 3 yr and genotyped at 6 polymorphic microsatellite loci. The data analysis involved 3 clustering methods: principal coordinate analysis (PCoA), discriminant analysis of principal components (DAPC), and STRUCTURE. While the PCoA did not uncover any discernible clustering patterns, the DAPC and STRUCTURE analyses yielded significant results, with 9 and 4 genetic clusters, respectively. Visualization of the STRUCTURE bar plot revealed N = 11 temporal demarcations indicating barriers to gene flow. An analysis of molecular variance of these STRUCTURE-inferred populations supported strong temporally driven genetic differentiation (FST = 0.048, F'ST = 0.664) relative to geographic differentiation (FST = 0.009, F'ST = 0.241). Integrated Nested Laplace Approximation and Boosted Regression Tree analyses revealed that collection timepoint and 4 main abiotic factors (temperature, humidity, precipitation, and wind speed) were associated with the genetic subdivisions observed for P. regina. A complex interplay between environmental conditions, the unique reproductive strategies of the blow fly, and the extensive dispersal abilities of these organisms likely drives the strong genetic structure of P. regina in the Midwestern US.

Insects as Chemical Sensors: Detection of Chemical Warfare Agent Simulants and Hydrolysis Products in the Blow Fly Using LC-MS/MS.

In this work, blow flies were investigated as environmental chemical sample collectors following a chemical warfare attack (CWA). Blow flies sample the environment as they search for water and food sources and can be trapped from kilometers away using baited traps. Three species of blow flies were exposed to CWA simulants to determine the persistence and detectability of these compounds under varying environmental conditions. A liquid chromatography mass spectrometry (LC-MS/MS) method was developed to detect CWA simulants and hydrolysis products from fly guts. Flies were exposed to the CWA simulants dimethyl methylphosphonate and diethyl phosphoramidate as well as the pesticide dichlorvos, followed by treatment-dependent temperature and humidity conditions. Flies were sacrificed at intervals within a 14 day postexposure period. Fly guts were extracted and analyzed with the LC-MS/MS method. The amount of CWA simulant in fly guts decreased with time following exposure but were detectable 14 days following exposure, giving a long window of detectability. In addition to the analysis of CWA simulants, isopropyl methylphosphonic acid, the hydrolysis product of sarin, was also detected in blow flies 14 days post exposure. This work demonstrates the potential to obtain valuable samples from remote or access-restricted areas without risking lives.

Global population genetic structure and demographic trajectories of the black soldier fly, Hermetia illucens.

BACKGROUND: The black soldier fly (Hermetia illucens) is the most promising insect candidate for nutrient-recycling through bioconversion of organic waste into biomass, thereby improving sustainability of protein supplies for animal feed and facilitating transition to a circular economy. Contrary to conventional livestock, genetic resources of farmed insects remain poorly characterised. We present the first comprehensive population genetic characterisation of H. illucens. Based on 15 novel microsatellite markers, we genotyped and analysed 2862 individuals from 150 wild and captive populations originating from 57 countries on seven subcontinents. RESULTS: We identified 16 well-distinguished genetic clusters indicating substantial global population structure. The data revealed genetic hotspots in central South America and successive northwards range expansions within the indigenous ranges of the Americas. Colonisations and naturalisations of largely unique genetic profiles occurred on all non-native continents, either preceded by demographically independent founder events from various single sources or involving admixture scenarios. A decisive primarily admixed Polynesian bridgehead population serially colonised the entire Australasian region and its secondarily admixed descendants successively mediated invasions into Africa and Europe. Conversely, captive populations from several continents traced back to a single North American origin and exhibit considerably reduced genetic diversity, although some farmed strains carry distinct genetic signatures. We highlight genetic footprints characteristic of progressing domestication due to increasing socio-economic importance of H. illucens, and ongoing introgression between domesticated strains globally traded for large-scale farming and wild populations in some regions. CONCLUSIONS: We document the dynamic population genetic history of a cosmopolitan dipteran of South American origin shaped by striking geographic patterns. These reflect both ancient dispersal routes, and stochastic and heterogeneous anthropogenic introductions during the last century leading to pronounced diversification of worldwide structure of H. illucens. Upon the recent advent of its agronomic commercialisation, however, current human-mediated translocations of the black soldier fly largely involve genetically highly uniform domesticated strains, which meanwhile threaten the genetic integrity of differentiated unique local resources through introgression. Our in-depth reconstruction of the contemporary and historical demographic trajectories of H. illucens emphasises benchmarking potential for applied future research on this emerging model of the prospering insect-livestock sector.

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.

The oncometabolite L-2-hydroxyglutarate is a common product of dipteran larval development.

The oncometabolite L-2-hydroxyglutarate (L-2HG) is considered an abnormal product of central carbon metabolism that is capable of disrupting chromatin architecture, mitochondrial metabolism, and cellular differentiation. Under most circumstances, mammalian tissues readily dispose of this compound, as aberrant L-2HG accumulation induces neurometabolic disorders and promotes renal cell carcinomas. Intriguingly, Drosophila melanogaster larvae were recently found to accumulate high L-2HG levels under normal growth conditions, raising the possibility that L-2HG plays a unique role in insect metabolism. Here we explore this hypothesis by analyzing L-2HG levels in 18 insect species. While L-2HG was present at low-to-moderate levels in most of these species (<100 pmol/mg; comparable to mouse liver), dipteran larvae exhibited a tendency to accumulate high L-2HG concentrations (>100 pmol/mg), with the mosquito Aedes aegypti, the blow fly Phormia regina, and three representative Drosophila species harboring concentrations that exceed 1 nmol/mg - levels comparable to those measured in mutant mice that are unable to degrade L-2HG. Overall, our findings suggest that one of the largest groups of animals on earth commonly generate high concentrations of an oncometabolite during juvenile growth, hint at a role for L-2HG in the evolution of dipteran development, and raise the possibility that L-2HG metabolism could be targeted to restrict the growth of key disease vectors and agricultural pests.

The genomes of a monogenic fly: views of primitive sex chromosomes.

The production of male and female offspring is often determined by the presence of specific sex chromosomes which control sex-specific expression, and sex chromosomes evolve through reduced recombination and specialized gene content. Here we present the genomes of Chrysomya rufifacies, a monogenic blow fly (females produce female or male offspring, exclusively) by separately sequencing and assembling each type of female and the male. The genomes (> 25X coverage) do not appear to have any sex-linked Muller F elements (typical for many Diptera) and exhibit little differentiation between groups supporting the morphological assessments of C. rufifacies homomorphic chromosomes. Males in this species are associated with a unimodal coverage distribution while females exhibit bimodal coverage distributions, suggesting a potential difference in genomic architecture. The presence of the individual-sex draft genomes herein provides new clues regarding the origination and evolution of the diverse sex-determining mechanisms observed within Diptera. Additional genomic analysis of sex chromosomes and sex-determining genes of other blow flies will allow a refined evolutionary understanding of how flies with a typical X/Y heterogametic amphogeny (male and female offspring in similar ratios) sex determination systems evolved into one with a dominant factor that results in single sex progeny in a chromosomally monomorphic system.

Improved transgenic sexing strains for genetic control of the Australian sheep blow fly Lucilia cuprina using embryo-specific gene promoters.

For genetic approaches for controlling insect pests such as the sterile insect technique (SIT), it is advantageous to release only males as females are ineffective as control agents and they consume about 50% of the diet. Here we developed tetracycline-repressible Lucilia cuprina transgenic strains in which adult females were fully fertile and viable on a diet that lacked tetracycline and all of their female offspring died at the embryo stage. The transgenic strains are an improvement over the strains we developed previously, which had the disadvantage that adult females on diet without tetracycline were sterile and died prematurely. This was possibly due to the low level expression of the effector gene in ovaries. In the strains developed in this study, the early promoters from L. cuprina nullo or Cochliomyia macellaria CG14427 genes were used to drive the tetracycline transactivator (tTA) expression in the early embryo. In the absence of tetracycline, tTA activates expression of the proapoptotic gene Lshid which contains a female-specific intron. Consequently, only females produce active HID protein and die at the embryo stage. Crossing the tTA-expressing driver lines with an RFPex reporter line confirmed that there was no expression of the effector gene in the ovary. These new embryonic L. cuprina transgenic sexing strains hold great promise for genetic control programs and the system reported here might also be transferable to other major calliphorid livestock pests such as the New World screwworm, Cochliomyia hominivorax.

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.

Amplified fragment length polymorphism analysis supports the valid separate species status of Lucilia caesar and L. illustris (Diptera: Calliphoridae).

Common DNA-based species determination methods fail to distinguish some blow flies in the forensically and medically important genus Lucilia Robineau-Desvoidy. This is a practical problem, and it has also been interpreted as casting doubt on the validity of some morphologically defined species. An example is Lucilia illustris and L. caesar, which co-occur in Europe whilst only L. illustris has been collected in North America. Reports that these species shared both mitochondrial and nuclear gene sequences, along with claims that diagnostic morphological characters are difficult to interpret, were used to question their separate species status. We report here that amplified fragment length polymorphism profiles strongly support the validity of both species based on both assignment and phylogenetic analysis, and that traditional identification criteria based on male and female genital morphology are more reliable than has been claimed.

Ubiquity of polystyrene digestion and biodegradation within yellow mealworms, larvae of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae).

Academics researchers and "citizen scientists" from 22 countries confirmed that yellow mealworms, the larvae of Tenebrio molitor Linnaeus, can survive by eating polystyrene (PS) foam. More detailed assessments of this capability for mealworms were carried out by12 sources: five from the USA, six from China, and one from Northern Ireland. All of these mealworms digested PS foam. PS mass decreased and depolymerization was observed, with appearance of lower molecular weight residuals and functional groups indicative of oxidative transformations in extracts from the frass (insect excrement). An addition of gentamycin (30 mg g-1), a bactericidal antibiotic, inhibited depolymerization, implicating the gut microbiome in the biodegradation process. Microbial community analyses demonstrated significant taxonomic shifts for mealworms fed diets of PS plus bran and PS alone. The results indicate that mealworms from diverse locations eat and metabolize PS and support the hypothesis that this capacity is independent of the geographic origin of the mealworms, and is likely ubiquitous to members of this species.

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.

Chemical Assay for the Detection of Vertebrate Fecal Metabolites in Adult Blow Flies (Diptera: Calliphoridae).

Filth flies are commonly implicated in pathogen transmission routes due to their affinity for vertebrate waste and their synanthropic associations. However, solidifying the link between flies and infected feces in the wild can be difficult, as interpretations made solely from microbial culturing or sequencing methods may represent an incomplete picture of pathogen acquisition. We present an analytical assay using high performance liquid chromatography tandem mass spectrometry (HPLC MS/MS) to detect vertebrate fecal metabolites (urobilinoids) in adult blow fly guts. Proof of concept experiments consisted of controlled feeding in which flies were grouped into three treatments (unfed, exposure to beef liver tissue, and exposure to canine feces; N = 20/treatment) using the black blow fly Phormia regina Meigen (Diptera: Calliphoridae). It was revealed that only feces-related samples exhibited peaks with an m/z of 591 and MS/MS spectra consistent with urobilinoids. These peaks were not seen for beef liver tissue, flies exposed to beef liver tissue, or unfed flies. Samples taken directly from beef liver tissue and from feces of several animals were also tested. To test this assay in wild flies, 216 flies were additionally analyzed to determine whether they had ingested vertebrate feces. About 13% of the wild flies exhibited these same peaks, providing a baseline measure of blow flies collected in urban and residential areas consuming feces from the environment. Overall, this assay can be used for P. regina collected in an applied setting and its integration with microbial culturing and sequencing methods will help to improve its use.

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.

Effects of microbial DNA on human DNA profiles generated using the PowerPlex® 16 HS system.

Most crime scenes are not sterile and therefore may be contaminated with environmental DNA, especially if a decomposing body is found. Collecting biological evidence from this individual will yield DNA samples mixed with microbial DNA. This also becomes important if postmortem swabs are collected from sexually assaulted victims. Although genotyping kits undergo validation tests, including bacterial screens, they do not account for the diverse microbial load during decomposition. We investigated the effect of spiking human DNA samples with known concentrations of DNA from 17 microbe species associated with decomposition on DNA profiles produced using the Promega PowerPlex® HS system. Two species, Bacillus subtilis and Mycobacterium smegmatis, produced an extraneous allele at the TPOX locus. When repeated with the PowerPlex® Fusion kit, the extra allele no longer amplified with these two species. This experiment demonstrates that caution should be exhibited if microbial load is high and the PowerPlex® 16HS system is used.

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.

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.

Survey of the Genetic Diversity of Forensically Important Chrysomya (Diptera: Calliphoridae) from Egypt.

Minimum postmortem interval estimations of a corpse using blow fly larvae in medicolegal investigations require correct identification and the application of appropriate developmental data of the identified fly species. Species identification of forensically relevant blow flies could be very difficult and time consuming when specimens are damaged or in the event of morphologically indistinguishable immature stages, which are most common at crime scenes. In response to this, an alternative, accurate determination of species may depend on sequencing and molecular techniques for identification. Chrysomyinae specimens (n = 158) belonging to three forensically important species [Chrysomya albiceps (Wiedemann), Chrysomya megacephala (F.), and Chrysomya marginalis (Wiedemann)] (Diptera: Calliphoridae) were collected from four locations in Egypt (Giza, Dayrout, Minya, and North Sinai) and sequenced across the mitochondrial cytochrome oxidase subunit I (COI) gene. Phylogenetic analyses using neighbor-joining, maximum likelihood and maximum parsimony methods resulted in the same topological structure and confirmed DNA based identification of all specimens. Interspecific divergence between pairs of species was 5.3% (C. marginalis-C. megacephala), 7% (C. albiceps-C. megacephala), and 8% (C. albiceps-C. marginalis). These divergences are sufficient to confirm the utility of cytochrome oxidase subunit I gene in the molecular identification of these flies in Egypt. Importantly, the maximum intraspecific divergence among individuals within a species was <1% and the least nucleotide divergence between species used for phylogenetic analysis was 3.6%. This study highlights the need for thorough and diverse sampling to capture all of the possible genetic diversity if DNA barcoding is to be used for molecular identification.

Verification of AFLP kinship methods of entomological evidence by sequencing.

Kinship analysis allows the determination of sibship based on the individuals' genetic profile. In a recent empirical study, amplified fragment length polymorphism (AFLP) analysis was proposed as a test to determine kinship between Phormia regina individuals useful in inferring postmortem transport of a corpse. In order to validate this technique, mitochondrial DNA gene cytochrome oxidase II was sequenced for all individuals used in the previous study. Then, the relatedness coefficient based on AFLP profiles was determined for the pairs of individuals that had different haplotypes, and thus could not be full siblings, to determine a conservative false positive error rate of this proposed test. A majority, 96%, of pair wise comparisons of individuals with different haplotypes had relatedness coefficients <0.41 supporting the conclusion that AFLP analysis for full sibship is a valid and robust technique and thus useful for the detection of postmortem movement of a corpse.

Evaluation of the IrisPlex DNA-based eye color prediction assay in a United States population.

DNA phenotyping is a rapidly developing area of research in forensic biology. Externally visible characteristics (EVCs) can be determined based on genotype data, specifically based on single nucleotide polymorphisms (SNPs). These SNPs are chosen based on their association with genes related to the phenotypic expression of interest, with known examples in eye, hair, and skin color traits. DNA phenotyping has forensic importance when unknown biological samples at a crime scene do not result in a criminal database hit; a phenotypic profile of the sample can therefore be used to develop investigational leads. IrisPlex, an eye color prediction assay, has previously shown high prediction rates for blue and brown eye color in a Dutch European population. The objective of this work was to evaluate its utility in a North American population. We evaluated six SNPs included in the IrisPlex assay in population sample collected from a USA college campus. We used a quantitative method of eye color classification based on (RGB) color components of digital photographs of the eye taken from each study volunteer so that each eye was placed in one of three eye color categories: brown, intermediate, or blue. Objective color classification was shown to correlate with basic human visual determination making it a feasible option for use in future prediction assay development. Using these samples and various models, the maximum prediction accuracies of the IrisPlex system after allele frequency adjustment was 58% and 95% brown and blue eye color predictions, respectively, and 11% for intermediate eye colors. Future developments should include incorporation of additional informative SNPs, specifically related to the intermediate eye color, and we recommend the use of a Bayesian approach as a prediction model as likelihood ratios can be determined for reporting purposes.