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In the medico-legal application of forensic entomology, estimating the time of death is critical and traditionally relies on changes in observable traits of carrion feeding insect larvae. Traits such as size, weight, and morphology can be used to predict the insect specimen age and help define the minimum time since death. The blowfly Phormia regina Meigen (Diptera: Calliphoridae) is a key forensic insect, yet age estimation for older maggots in this and other carrion-feeding species is particularly challenging due to the limited morphological changes in the late-stage larvae. To enhance age-estimation precision, we employed transcriptomic profiling on blowfly maggots, aiming to identify genes as markers for time of death estimation. Our study characterized maggot development, reinforcing that weight and behavior cannot precisely determine age between 100 and 130 hours at 27.5 °C. We built a chromosomal scale annotated genome, establishing a reliable database for uncovering transcriptomic signatures during larval development. Applying differential gene expression analyses, weighted gene co-expression network analysis, and the generalized linear model, we identified nine candidate genes (y5078, y5076, agt2, ech1, dhb4, asm, gabd, acohc, ivd) that delineate the age of otherwise indeterminate maggots. This research introduces a molecular approach to address a longstanding problem in forensic entomology and promises to increase precision in determining the time of death at a crime scene.

Flies, especially those from the Calliphoridae family, play a crucial role in decomposition and are the first to colonize a cadaver. Firstly, accurate species identification is a prerequisite for entomological evidence-based calculation of postmortem interval (PMI). While morphological criteria for identifying the species of adult blow flies exist, there are either absent or inadequate keys for younger stages. In all phases of blow fly development, molecular identification offers a quick and accurate procedure. It is widely known that mitochondrial cytochrome oxidase subunit I has the capacity for molecular identification but is ineffective in certain species. This study was conducted to assess the effectiveness of the cytochrome oxidase 1 gene in the identification of seventeen different species of calliphorid flies involving four genera, Calliphora , Chrysomya , Lucilia , and Hemipyrellia . In West Bengal, 2,977 blow fly specimens were gathered from four distinct geo-climatic zones. COI barcodes were able to confirm morphological identification through low K2P intraspecific genetic divergences (0% to 1%) and moderate to high K2P interspecific genetic divergences (0.39% to 12.29%). The Neighbour-Joining (NJ) analysis demonstrated well-supported reciprocal monophyly among the species. The species grouping was in agreement with morphological and molecular identifications. The four delimitation methods, BIN, ASAP, PTP, and GMYC, used for species identification produced similar results and facilitated the proper identification of species. Therefore, it can be concluded that COI barcodes are a highly successful alternative for the molecular identification of blow flies, facilitating forensic cases and biodiversity research in India.

The oriental latrine fly, Chrysomya megacephala (Diptera: Calliphoridae), is a medically important synanthropic blow fly species characterized by its necrophagy and coprophagy, often observed near carrion and animal feces. Notably, C. megacephala always arrives at carcass earlier than other species. To elucidate the underlying mechanisms behind the host choice in C. megacephala , we present the chromosome-scale genome assembly for this species. The genome size is 816.79 Mb, with a contig N50 of 1.60 Mb. The Hi-C data were anchored to six chromosomes, accounting for 99.93% of the draft assembled genome. Comparative genomic analysis revealed significant expansions in pathways of ligand-gated ion channel activity, passive transmembrane transporter activity, and protein methyltransferase activity, which may be closely associated with host localization and oviposition. After identifying 69 odor-binding proteins (OBPs) in the assembled genome, phylogenetic analysis showed that DmelOBP99b and CmegOBP99b exhibited high homology. Transcriptome analysis demonstrated that the relative expression of CmegOBP99b was consistently the highest during the metamorphosis, and RT-qPCR further confirmed the similar results. Additionally, CmegOBP99b exhibited a strong binding affinity to DMDS (dimethyl disulfide) as determined by molecular docking. To determine the protein expression level of CmegOBP99b in various body parts, we prepared recombinant CmegOBP99b protein and anti- CmegOBP99b polyclonal antibodies. Western blot analysis showed that CmegOBP99b was significantly expressed in the female’s head compared to other parts, which is consistent with RT-qPCR results. Therefore, CmegOBP99b may be the primary odor-binding protein responsible for olfactory recognition and the behavioral coordination of C. megacephala . This study not only provides valuable insights into the molecular mechanisms of oviposition localization in C. megacephala but also facilitates further research into the genetic diversity and phylogeny of the Calliphoridae family.

Myiasis is a parasitic infestation of soft vertebrate tissues by larval stages of Diptera. We briefly described the lesion-causing genus Cordylobia Grünberg (Diptera: Calliphoridae). Three Polish travelers to Uganda, Gambia, and Senegal returned with furuncular myiasis. To identify the third-instar larvae removed from their skin, we examined the morphological features of the 3 specimens and sequenced a 5’ barcoding fragment of the cytochrome c oxidase subunit I gene (COI-5P). One larva was identified as C. rodhaini Gedoelst, and 2 larvae were identified as C. anthropophaga (Blanchard). We were the first to submit the COI-5P of C. rodhaini to GenBank and the Barcode of Life Database. This is the first record of the importation of C. anthropophaga and the second record of the importation of C. rodhaini to Poland.

Population genetic patterns and changes in allele frequency within blow flies can provide valuable insight into population structure, diversification, dispersal, gene flow, and population assignments. The population genetic structure of the oriental latrine blow fly, Chrysomya megacephala (Fabricius), was investigated using amplified fragment length polymorphisms (AFLP). The goal was to validate the use of this technique to create genetic profile data and determine it is feasibility for inferring postmortem relocation of corpses. The AFLP technique generated 590 polymorphic loci for C. megacephala . Analysis of molecular variation (AMOVA) found significantly high genetic variation within individuals of all fly populations, with little variation among populations from different geographic locations. STRUCTURE and principal coordinate analyses (PCoA) revealed no population structure based on geography, with weak correlation between genetic and geographic distances, and moderate temporal differentiation was noted among C. megacephala samples. Across the entire data set, the mean relative relatedness coefficients were positive, suggesting that flies arriving at the same bait (carcass) share nonrandom proportions of alleles and comprise of closely related individuals. Genetic assignment of C. megacephala flies to a putative source population resulted in a 90.81% success rate, indicating the possibility of using these flies to connect sites between which a corpse had been moved even in the absence of overall geographic population structure.

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.

An aberrant bot fly specimen was removed from the scalp of a patient 3 wk after returning from Belize. The specimen showed little resemblance to the typical human bot fly larva, Dermatobia hominis, prompting a molecular identification using cytochrome oxidase I and II (COI and COII, respectively) mitochondrial DNA sequence regions. A BLAST search was subsequently performed, and both our COI and COII amplicon sequences showed 99–100% match with Dermatobia hominis, despite the specimen’s clearly aberrant morphology. These findings suggest there is much wider variation in Dermatobia larval morphology than previously known or perhaps that there are new or cryptic species within this group.

Background Flystrike (cutaneous myiasis) is caused by blowfly larvae of the genus Lucilia . This disease is a major obstacle to sustainable global sheep and wool production. Flystrike control relies primarily on breech modification surgery (mulesing) and insecticidal treatment; however, control is constantly compromised by the emergence and spread of insecticide-resistance. Preventing the spread of resistance is severely hindered by a limited understanding of genetic variation, structure and gene flow within and among Lucilia populations. Australia is one of the world’s largest producers of sheep and wool products, where Lucilia cuprina dorsalis is the major cause of flystrike. Results Here, we collected 2,034 Lucilia cuprina dorsalis individuals among 86 populations from sheep-grazing regions across the continent. Each fly was genetically characterised at 20,000 loci using DArTseq, a reduced complexity genome sequencing strategy. Three genetically distinct population clusters (i.e., Western Australia, Eastern Australia and Tasmania) were revealed through population structure analyses. Conclusions This investigation into population structure and gene flow yields significant insights into the genetic composition of diverse L. c. dorsalis populations throughout Australia. These findings will be essential for the sustainable management of flystrike on a global scale and for addressing the ongoing challenge of insecticide resistance.

Tetracyclines are broad-spectrum antibiotics widely used in agriculture, medicine, and research. However, they are associated with harmful side effects. In arthropods, parental exposure to tetracyclines has been linked to reduced health and fitness in untreated offspring. These transgenerational effects of tetracyclines could jeopardize the success of pest control programs that use tetracyclines to control gene expression. In this study, we investigated the transgenerational effects of 2 tetracyclines, doxycycline (DOX) and anhydrotetracycline (ATC), in the blowfly Lucilia cuprina, a significant pest of sheep. To simulate the rearing conditions of a transgenic male-only release program, blowflies were reared on standard diet alone, or standard diet plus DOX or ATC, for 3 generations, and then reared for an additional fourth generation on standard diet alone. We used behavioral assays, 16S amplicon sequencing, and mRNA sequencing to determine how DOX and ATC influenced male sexual competitiveness, microbiome composition, and gene expression in the third and fourth generations. We found that 3 generations of DOX treatment led to lower sexual competitiveness in both third- and fourth-generation males. In addition, DOX and ATC shifted the composition of the blowfly microbiome and altered the expression of numerous mitochondria- and immunity-related genes in both generations. Our study supports an emerging body of evidence that tetracyclines exert not only direct but also transgenerational effects, and sheds light on the transcriptional and microbial responses to antibiotic exposure and removal. Our findings emphasize the need for pest control programs that use tetracyclines to evaluate the long-term effects of these antibiotics.

Abstract Recent studies on oestroidean Diptera (Brachycera) are providing a comprehensive and nuanced understanding of the evolutionary history of this remarkably diverse clade of holometabolous insects. The Oestroidea, which includes formidable pests such as various blowflies, botflies, and flesh flies that infest livestock, pets and humans, are mostly composed of beneficial species that act as scavengers or parasitoids on various pest insects. In our research, we used genomic methods to elucidate the phylogenetic position of Nesodexia corsicana Villeneuve, 1911 (Diptera: Calliphoridae), a mysterious oestroid species endemic to Corsica and characterized by distinctive morphological features that have puzzled taxonomists for years. Contrary to initial hypotheses, our results place Nesodexia Villeneuve, 1911 within the Calliphoridae subfamily Rhinophorinae, a small lineage of terrestrial isopod parasitoids. Through detailed morphological analysis of adults of both sexes and eggs, we uncovered significant insights consistent with our phylogenomic reconstruction. The unique morphological features of the species, coupled with its restricted and fragmented habitat, highlight its potential conservation importance. We delineated the area of occupancy for N. corsicana and assessed its “threatened” category using specific IUCN Red List criteria. In addition, we mapped the available habitat within its range and determined potential key biodiversity areas (KBA) triggered by N. corsicana. New potential KBAs are only partially covered by the Corsican Regional Park. Finally, we mapped the distribution of habitats on the island to assess the potential distribution of the species beyond its currently known geographic range. Graphical Abstract Graphical Abstract