Transcriptomic profiling of different developmental stages reveals parasitic strategies of Wohlfahrtia magnifica, a myiasis-causing flesh fly.

BACKGROUND: Wohlfahrtia magnifica is an obligatory parasite that causes myiasis in several warm-blooded vertebrates. Adult females deposit the first-stage larvae directly onto wounds or natural body orifices (e.g., genitalia) of the host, from where they quickly colonize the host tissue and feed on it for development. The infestation of W. magnifica can lead to health issues, welfare concerns, and substantial economic losses. To date, little is known about the molecular mechanisms of the W. magnifica-causing myiasis. RESULTS: In this study, we collected parasitic-stage larvae of W. magnifica from wounds of naturally infested Bactrian camels, as well as pupae and adult flies reared in vitro from the wound-collected larvae, for investigating the gene expression profiles of the different developmental stages of W. magnifica, with a particular focus on examining gene families closely related to the parasitism of the wound-collected larvae. As key proteins related to the parasite-host interaction, 2049 excretory/secretory (ES) proteins were identified in W. magnifica through the integration of multiple bioinformatics approaches. Functional analysis indicates that these ES proteins are primarily involved in cuticle development, peptidase activity, immune response, and metabolic processes. The global investigation of gene expression at different developmental stages using pairwise comparisons and weighted correlation network analysis (WGCNA) showed that the upregulated genes during second-stage larvae were related to cuticle development, peptidase activity, and RNA transcription and translation; during third-stage larvae to peptidase inhibitor activity and nutrient reservoir activity; during pupae to cell and tissue morphogenesis and cell and tissue development; and during adult flies to signal perception, many of them involved in light perception, and adult behavior, e.g., feeding, mating, and locomotion. Specifically, the expression level analysis of the likely parasitism-related genes in parasitic wound-collected larvae revealed a significant upregulation of 88 peptidase genes (including 47 serine peptidase genes), 110 cuticle protein genes, and 21 heat shock protein (hsp) genes. Interestingly, the expression of 2 antimicrobial peptide (AMP) genes, including 1 defensin and 1 diptericin, was also upregulated in the parasitic larvae. CONCLUSIONS: We identified ES proteins in W. magnifica and investigated their functional distribution. In addition, gene expression profiles at different developmental stages of W. magnifica were examined. Specifically, we focused on gene families closely related to parasitism of wound-collected larvae. These findings shed light on the molecular mechanisms underlying the life cycle of the myiasis-causing fly, especially during the parasitic larval stages, and provide guidance for the development of control measures against W. magnifica.

Molecular identification and functional analysis of chitinase genes reveal their importance in the metamorphosis of Sarcophaga peregrina (Diptera: Sarcophagidae).

Chitinases play a crucial role in insect metamorphosis by facilitating chitin degradation. Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a typical holometabolous insect and an important hygiene pest that causes myiasis in humans and other mammals and acts as a vector for various parasitic agents, including bacteria, viruses, and parasites. Enhancing the understanding of the metamorphosis in this species has significance for vector control. In this study, we identified a total of 12 chitinase genes in S. peregrina using bioinformatic analysis methods. Based on transcriptome data, SpIDGF2 and SpCht10 were selected for further functional investigation. The down-regulation of these genes by RNA interference led to developmental delays, disruptions in molting, and differences in cuticle composition during the pupal stage. These findings underscore the pivotal role of chitinase genes in the metamorphic process and offer valuable insights for effective control strategies.

Effects of heat tolerance on the gut microbiota of Sarcophaga peregrina (Diptera: Sarcophagidae) and impacts on the life history traits.

BACKGROUND: Heat tolerance is a distinct abiotic factor affecting the distribution and abundance of insects. Gut microbiota can contribute to host fitness, thereby increasing resistance to abiotic stress conditions. In this study, Sarcophaga peregrina is closely associated with human life in ecological habits and shows remarkable adaptability to daily and seasonal temperature fluctuations. To date, the role of gut microbiota in S. peregrina response to heat stress and its influence on the host phenotypic variability remain poorly studied. METHODS: We exposed S. peregrina to heat stress at 40 °C for 3 h every day throughout the developmental stages from newly hatched larva to adult, after which gut DNA was extracted from third-instar larvae, early pupal stage, late pupal stage, and newly emerged adults, respectively. Then, 16S rRNA microbial community analyses were performed. RESULTS: Firstly, we analyzed whether heat stress could have an impact on the life history traits of S. peregrina and showed that the growth rate of larvae was higher and the developmental time was significantly shorter after heat stress. We then proposed the role of the gut microbiota in the heat tolerance of S. peregrina, which indicated that the bacterial abundance and community structure changed significantly after heat tolerance. In particular, the relative abundance of Wohlfahrtiimonas and Ignatzschineria was higher in the third-instar larval larvae; the former increased and the latter decreased significantly after heat stress. To further explore the effect of disturbing the microbial community on thermotolerant phenotype, newly hatched larvae were fed with amikacin under heat stress, which indicated that the larval length and the whole developmental cycle was significantly shorter. CONCLUSION: This study indicated that Wohlfahrtiimonas and Ignatzschineria should play an important role in the post-feeding stage under heat stress, but further study is still needed. In general, heat tolerance can affect the gut microbial community structure, which in turn affects the fitness of the host.

Genome-wide analysis of long noncoding RNAs and their association in regulating the metamorphosis of the Sarcophaga peregrina (Diptera: Sarcophagidae).

BACKGROUND: The flesh fly, Sarcophaga peregrina (Diptera: Sarcophagidae), is an important hygiene pest, that causes myiasis in humans and other mammals, typically livestock, and as a vector for various parasitic agents, including bacteria, viruses, and parasites. The role of long non-coding RNAs (lncRNAs) in regulating gene expression during metamorphosis of the flesh fly has not been well established. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we performed genome-wide identification and characterization of lncRNAs from the early pupal stage (1-days pupae), mid-term pupal stage (5-days pupae), and late pupal stage (9-days pupae) of S. peregrina by RNA-seq, and a total of 6921 lncRNAs transcripts were identified. RT-qPCR and enrichment analyses revealed the differentially expressed lncRNAs (DE lncRNAs) that might be associated with insect metamorphosis development. Furthermore, functional analysis revealed that the DE lncRNA (SP_lnc5000) could potentially be involved in regulating the metamorphosis of S. peregrina. RNA interference of SP_lnc5000 caused reduced expression of metamorphosis-related genes in 20-hydroxyecdysone (20E) signaling (Br-c, Ftz-F1), cuticle tanning pathway (TH, DOPA), and chitin related pathway (Cht5). Injection of dsSP_lnc5000 in 3rd instar larvae of S. peregrina resulted in deformed pupae, stagnation of pupal-adult metamorphosis, and a decrease in development time of pupal, pupariation rates and eclosion rates. Hematoxylin-eosin staining (H&E), scanning electron microscope (SEM) observation and cuticle hydrocarbons (CHCs) analysis indicated that SP_lnc5000 had crucial roles in the metamorphosis developmental by modulating pupal cuticular development. CONCLUSIONS/SIGNIFICANCE: We established that the lncRNA SP_lnc5000 potentially regulates the metamorphosis of S. peregrina by putatively affecting the structure and composition of the pupal cuticle. This study enhances our understanding of lncRNAs as regulators of metamorphosis in S. peregrina, and provide valuable insights into the identification of potential targets for vector control and the development of effective strategies for controlling the spread of myiasis and parasitic diseases.

Multimethod combination for age estimation of Sarcophaga peregrina (Diptera: Sarcophagidae) with implications for estimation of the postmortem interval.

Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a forensically important flesh fly with potential value for estimating the minimum postmortem interval (PMImin). Here, the developmental patterns of S. peregrina were investigated at 5 constant temperatures (15-35 °C). Morphological changes at different developmental stages of the pupa were observed at 4 constant temperatures (15-30 °C) by removing the puparium and staining the pupa with hematoxylin and eosin. Furthermore, differentially expressed genes (DEGs) were analyzed at 25 °C in the intrapuparial period to estimate the age of S. peregrina during the intrapuparial stage. S. peregrina completed development from larviposition to adult eclosion at 15 °C, 20 °C, 25 °C, and 30 °C; the developmental durations were 1090.3 ± 30.6 h, 566.6 ± 21.9 h, 404.6 ± 13.01 h, and 280.3 ± 4.5 h, respectively, while the development could not be completed at 35 °C. The intrapuparial period of S. peregrina was divided into 12 sub-stages on the basis of the overall external morphological changes; 6 sub-stages on the basis of individual morphological structures such as the compound eyes, antennae, thorax, legs, wings, and abdomen; and 10 sub-stages on the basis of internal morphological changes detected using histological analysis. The period of each sub-stage or structure that appeared was determined. Moreover, we found that 6 genes (NDUFS2, CPAMD8, NDUFV2, Hsp27, Hsp23, and TPP) with differential expression can be used for the precise age estimation of S. peregrina during the intrapuparial period. This study provided basic developmental data for the use of S. peregrina in PMImin estimation, and we successfully estimated PMImin in a real forensic case by using a multimethod combination.

Genomic insights into evolution and control of Wohlfahrtia magnifica, a widely distributed myiasis-causing fly of warm-blooded vertebrates.

Wohlfahrtia magnifica is a pest fly species, invading livestock in many European, African and Asian countries, and causing heavy agroeconomic losses. In the life cycle of this obligatory parasite, adult flies infect the host by depositing the first-stage larvae into body cavities or open wounds. The feeding larvae cause severe (skin) tissue damage and potentially fatal infections if untreated. Despite serious health detriments and agroeconomic concerns, genomic resources for understanding the biology of W. magnifica have so far been lacking. Here, we present a complete genome assembly from a single adult female W. magnifica using a Low-DNA Input workflow for PacBio HiFi library preparation. The de novo assembled genome is 753.99 Mb in length, with a scaffold N50 of 5.00 Mb, consisting of 16,718 predicted protein-encoding genes. Comparative genomic analysis revealed that W. magnifica has the closest phylogenetic relationship to Sarcophaga bullata followed by Lucilia cuprina. Evolutionary analysis of gene families showed expansions of 173 gene families in W. magnifica that were enriched for gene ontology (GO) categories related to immunity, insecticide-resistance mechanisms, heat stress response and cuticle development. In addition, 45 positively selected genes displaying various functions were identified. This new genomic resource contributes to the evolutionary and comparative analysis of dipterous flies and an in-depth understanding of many aspects of W. magnifica biology. Furthermore, it will facilitate the development of novel tools for controlling W. magnifica infection in livestock.

What's in a child's ear? A case of otomyiasis by Sarcophaga argyrostoma (Diptera, Sarcophagidae).

A clinical report of otomyiasis in a 1-year-old girl is reported. A III instar larva of Sarcophaga sp. was microscopically identified and Sarcophaga (Liopygia) argyrostoma (Diptera, Sarcophagidae) was suspected. A molecular method targeting a fragment of the cox1 gene was used to confirm the identity of the specimen. Although myiases are not frequent manifestations in otolaryngology, they should arouse the attention of doctors, social workers and parents dealing with disabled people, the elderly and children. This contribution also highlights the need of combining microscopy and molecular tools to achieve a correct and reliable identification of the specimen/s.

A review of morphological characters for the identification of three common European species of Sarcophaga s. str. (Diptera: Sarcophagidae), with an emphasis on female terminalia.

The subgenus Sarcophaga Meigen, 1824 (s. str.) currently comprises over 30 species distributed in the West Palearctic Region, the identification of which is normally based on characters of the male terminalia. Females of the three closely-related species Sarcophaga (Sarcophaga) carnaria (Linnaeus, 1758), S. (S.) subvicina Rohdendorf, 1937 and S. (S.) variegata (Scopoli, 1763), which are especially widespread and abundant in NW Europe, are considered morphologically indistinguishable by most authors. However, a few authors have proposed keys to separate females of these three species based on external and internal characters of the terminalia. Following a preliminary molecular identification using DNA barcode sequences (COI, cytochrome c oxidase subunit I), we herein revise the morphological characters used to differentiate female S. carnaria, S. subvicina and S. variegata in existing identification keys as well as search for additional diagnostic characters. Our results suggest that only one previously-proposed female character, namely the length to width ratio of abdominal sternite 7, can be used to separate S. subvicina from the other two species (Mann-Whitney U test: p < 0.0001), at least in a majority of cases. Other characters, such as the degree of sclerotisation and setation of tergite 8, show a high degree of overlap that does not allow to reliably separate females of these three species. Nevertheless, we propose a combination of characters that should allow the separation of female S. carnaria from female S. variegata in most cases. An additional analysis of males of the same species showed that the distribution of pruinosity and setation on syntergosternite 7+8, a character mentioned in a previously-published key, is also not reliable for identification.

Integrative taxonomy reveals remarkable diversity in Australian Protomiltogramma (Diptera: Sarcophagidae).

Protomiltogramma Townsend is the largest and most diverse genus of miltogrammine flesh flies in Australia. However, no comprehensive taxonomic work had been completed on the Australian members of this genus in almost a century. This study presents the first taxonomic revision of all Australian species of Protomiltogramma (Sarcophagidae: Miltogramminae), completed using an integrative approach combining molecular and morphological data. Eight new species endemic to Australia are described: P. dalbiensis sp. n., P. grandis sp. n., P. incana sp. n., P. kapnos sp. n., P. nigrisensa sp. n., P. popularis sp. n., P. rubra sp. n. and P. subtilis sp. n. In addition, P. australis Malloch, 1930 is synonymised with P. cincta Townsend, 1916, syn. n. and P. mallochi Verves, 1987 is synonymised with P. laticeps Malloch, 1930, syn. n. Molecular phylogenetics is used to place the Australian Protomiltogramma among the miltogrammine genera of the world.

A phylotranscriptomic framework for flesh fly evolution (Diptera, Calyptratae, Sarcophagidae).

The Sarcophagidae (flesh flies) comprise a large and widely distributed radiation within the Calyptratae (Diptera). Larval feeding habits are ecologically diverse and include sarcosaprophagy, coprophagy, herbivory, invertebrate and vertebrate predation, and kleptoparasitism. To elucidate the geographic origin and evolution of flesh fly life-history, we inferred a backbone phylogeny based on transcriptomic data from 26 sarcophagid species covering all three subfamilies plus 15 outgroups. The phylogeny was inferred using maximum parsimony and maximum likelihood methods based on a series of supermatrices, one set with overall information content improved by MARE (2290 loci), one set with 100% gene coverage for all included species (587 loci), and the last set including mitochondrial and nuclear genes (589 loci) and additional taxa. In order to obtain a more detailed hypothesis, we utilized the supertree approach to combine results from the present study with previously published hypotheses. This resulted supertree covers 84 of the one hundred currently recognized sarcophagid genera and formed the basis for the ancestral state reconstructions. The monophyletic Sarcophagidae is well-supported as sister to {Mystacinobiidae + Oestridae}, and relationships at the subfamily level are inferred as {Sarcophaginae, (Paramacronychiinae + Miltogramminae)}. The Sarcophagidae and each subfamily originated in the Americas, with Sarcophaginae diversifying mainly in the Neotropics, whereas the major radiation of both Miltogramminae and Paramacronychiinae occurred in the Palaearctic. Sarcosaprophagy is reconstructed as the ancestral larval feeding habit of the family Sarcophagidae and each subfamily. The ancestral sarcophagid larva probably utilized dead invertebrates as food, and the food spectrum expanded together with the diversification of breeding strategies. Particularly, kleptoparasitism in Miltogramminae is derived from sarcosaprophagy and may be seen as having derived from the breeding biology of 'lower' miltogrammines, the larvae of which feed on buried vertebrate carrion.

Museomics and phylogenomics with protein-encoding ultraconserved elements illuminate the evolution of life history and phallic morphology of flesh flies (Diptera: Sarcophagidae).

BACKGROUND: The common name of the Flesh flies (Sarcophagidae) usually relates them with organisms feeding on decomposing organic matter, although the biology of one of the largest radiations among insects also includes predation, coprophagy, and even kleptoparasitism. The question of whether the ancestor of all sarcophagids was a predator or a decomposer, or in association to which host have sarcophagids evolved, has thus always piqued the curiosity of flesh fly specialists. Such curiosity has often been hindered by both the impossibility of having a well-supported phylogeny of Sarcophagidae and its sister group to trace live habits and the scarcity of information on the biology of the group. Using a phylogenomic dataset of protein-encoding ultraconserved elements from representatives of all three subfamilies of Sarcophagidae as ingroup and a large Calyptratae outgroup, a robust phylogenetic framework and timescale are generated to understand flesh fly systematics and the evolution of their life histories. RESULTS: The evolutionary history for Sarcophagidae reconstructed here differs considerably from previous hypotheses. Within subfamily Sarcophaginae, a group of predatory flies, including genera Lepidodexia and Boettcheria, emerged as sister-group to the rest of Sarcophaginae. The genera Oxysarcodexia, Ravinia, and Tricharaea, long considered archaic and early-branching coprophagous and sarcosaprophagous lineages, were found nested well within the Sarcophaginae as sister-group to the sarcosaprophagous Microcerella. Predation on invertebrates is suggested as the ancestral and dominant strategy throughout the early evolution of flesh flies. Several transitions from predation to sarcosaprophagy and coprophagy occur across the sarcophagid phylogenetic tree, in contrast with almost no transitions from sarcosaprophagy or coprophagy to predatory habits. Regarding the morphological evolution of flesh flies, there might be a concerted evolution of male genitalia traits, such as the phallotrema position and the juxta, or the vesica and the folding of the phallotrema. One diversification rate shift was inferred in the evolution of sarcophagids, which is related to the origin of genus Sarcophaga. CONCLUSIONS: This study has a significant impact on understanding sarcophagid evolution and highlights the importance of having a robust phylogenetic framework to reconstruct the ancestral character state of biological and morphological characters. I discuss the evolution of life histories of the family in relation to their hosts or substrates and outline how sarcosaprophagy, coprophagy, and kleptoparasitism behavior on various hosts may have evolved from predation on invertebrates. This study provides a phylogenetic framework for further physiological and comparative genomic work between predatory, sarcosaprophagous, coprophagous, and kleptoparasitic lineages, which could also have significant implications for the evolution of diverse life histories in other Diptera.

DNA-based and taxonomic identification of forensically important Sarcophagidae (Diptera) in southeastern Spain.

Studying dipterans at the scene of a death can provide essential information for interpreting the evidence and help to reconstruct the events happened to a corpse in the past. Molecular tools have been employed for identification at specific levels in the cases of cryptic species or poorly conserved specimens. Identification of specimens is essential in forensic entomology since each species has a specific growth rate, which determines the calculation of the minimum post mortem interval (minPMI). In addition, phylogeographic reconstruction within a species can help to differentiate the haplotypes from a geographic area, thereby helping to clarify the possible relocation of a corpse. The morphological identification of Sarcophagidae species is often difficult, especially for the females. This is an important Diptera family since some of its species are among the first to reach a corpse, especially in warm areas. In this study, we compared the sarcophagids found in human corpses in forensic cases in Alicante (southeast of Spain) with specimens collected from baited traps in the same area and surrounding provinces. In total, 189 specimens were collected, comprising 72 from forensic cases and 117 from baited traps. Molecular identification was conducted by sequencing the cox1 mitochondrial gene and analyzing the sequences using ABGD, GMYC, and BIN species delimitation methods. The median joining algorithm in the PopART program was used to construct phylogeographic networks. Eight species in the family Sarcophagidae were identified. The most widely collected species were Sarcophaga argyrostoma and Sarcophaga tibialis. The haplotype networks obtained for these species did not indicate a clear geographic distribution of haplotypes. The S. argyrostoma samples from Alcoy were clearly isolated. The results demonstrated that this method is useful for identifying Sarcophagidae samples in forensic investigations and it can be employed for minPMI estimation.

Comparative analysis of the mitochondrial genomes of flesh flies and their evolutionary implication.

Flesh flies (Diptera: Sarcophagidae) include a large and widely distributed rapid radiation within the Calyptratae. They are vital for the ecosystem, as well as economic, forensic, and evolutionary studies, because of their extremely diverse habits as larvae. Phylogenetic studies of Sarcophagidae have been reaching convergence, which leads the opportunity to elucidate the evolution of these fast-evolving insects from the perspective of mitochondrial genome. Complete mitochondrial genomes of eight species were sequenced, and comparative mitochondrial genomic analysis between subfamilies were conducted. Mitochondrial genomes of these flesh flies are conserved in gene content with gene arrangement, same as the inferred ancestral insect, and the nucleotide composition is highly biased towards A + T like other flesh flies. The evolutionary rates of Sarcophagidae vary considerably across subfamilies, with that of Miltogramminae higher than the other two subfamilies. Phylogenetic analysis strongly supports monophyly of Sarcophagidae and each subfamily, with subfamily-level relationship inferred as (Sarcophaginae, (Miltogramminae, Paramacronychiinae)). The main topological inconsistency of all reconstructions is the relationship within Miltogramminae and Sarcophaga, which might be caused by their rapid evolution. Our study indicates that the mitochondrial genomes of flesh flies are highly conserved, and they are practically useful for phylogenetic inference of calyptrates.

Chromosome-level de novo genome assembly of Sarcophaga peregrina provides insights into the evolutionary adaptation of flesh flies.

Sarcophaga peregrina is considered to be of great ecological, medical and forensic significance, and has unusual biological characteristics such as an ovoviviparous reproductive pattern and adaptation to feed on carrion. The availability of a high-quality genome will help to further reveal the mechanisms underlying these charcateristics. Here we present a de novo-assembled genome at chromosome scale for S. peregrina. The final assembled genome was 560.31 Mb with contig N50 of 3.84 Mb. Hi-C scaffolding reliably anchored six pseudochromosomes, accounting for 97.76% of the assembled genome. Moreover, 45.70% of repeat elements were identified in the genome. A total of 14,476 protein-coding genes were functionally annotated, accounting for 92.14% of all predicted genes. Phylogenetic analysis indicated that S. peregrina and S. bullata diverged ~ 7.14 million years ago. Comparative genomic analysis revealed expanded and positively selected genes related to biological features that aid in clarifying its ovoviviparous reproduction and carrion-feeding adaptations, such as lipid metabolism, olfactory receptor activity, antioxidant enzymes, proteolysis and serine-type endopeptidase activity. Protein-coding genes associated with ovoviparity, such as yolk proteins, transferrin and acid sphingomyelinase, were identified. This study provides a valuable genomic resource for S. peregrina, and sheds insight into further revealing the underlying molecular mechanisms of adaptive evolution.

Development of Sarcophaga dux (diptera: Sarcophagidae) at constant temperatures and differential gene expression for age estimation of the pupae.

Sarcophaga dux (Diptera: Sarcophagidae) is a necrophagous flesh fly species with potential forensic value for estimating minimum postmortem interval (PMImin). The basic developmental data and precise age estimates of the pupae are significant for PMImin estimation in forensic investigations. In the present study, we investigated the development data of that species at seven constant temperatures varying from 16 °C to 34 °C, including body length changes of the larve, developmental duration and accumulated degree hours of the preadults. Several reference genes for relative quantification of the differentially expressed genes (DEGs) were firstly selected and evaluated in the pupae of different ages under different temperatures. The DEGs of the insects during the pupal period at different constant temperatures (34, 25 and 16 °C) were further analyzed for more precise age estimation. The results showed that the developmental durations of the preadults at 16, 19, 22, 25, 28, 31 and 34 °C were 1478.6 ± 18.3 h, 726.1 ± 15.8 h, 538.5 ± 0.9 h, 394.1 ± 9.5 h, 375.6 ± 10.8 h, 284.1 ± 7.3 h, and 252.5 ± 6.1 h, respectively. The developmental threshold temperature the flies was 12.27 ± 0.35 °C, and the thermal summation constant was 5341.71 ± 249.29° hours. The most reliable reference genes during the pupal period at different temperatures were found: GST1 and 18S rRNA for the 34 °C group, GST1 and RPL49 for 25 °C, and 18S rRNA and 28S rRNA for 16 °C. The four differential expression genes (Hsp60, A-alpha, ARP, and RPL8) have the potential to be used for more precise age estimation of pupal S. dux. This work provides important basic developmental data and a more precise age estimation method for pupal S. dux, and improves the value of this species for PMImin estimation in forensic investigations.

Comparative analysis of mitochondrial genomes among the subfamily Sarcophaginae (Diptera: Sarcophagidae) and phylogenetic implications.

The subfamily Sarcophaginae is extremely diverse in morphology, habit and geographical distribution, and usually considered to be of significant ecological, medical, and forensic significance. In the present study, 18 mitochondrial genomes (mitogenomes) of sarcophagid flies were first obtained. The rearrangement and orientation of genes were identical with that of ancestral insects. The degrees of compositional heterogeneity in the datasets were extremely low. Furthermore, 13 protein-coding genes were evolving under purifying selection. The phylogenic relationship of the genus-group taxa Boettcheria + (Sarcophaga + (Peckia + (Ravinia + Oxysarcodexia))) was strongly supported. Four subgenera were recovered as monophyletic (Liopygia, Liosarcophaga, Pierretia, Heteronychia) in addition to Parasarcophaga as polyphyletic. The sister-relationships between S. dux and S. aegyptiaca, S. pingi and S. kawayuensis were recovered, respectively. Moreover, the molecular phylogenetic relationships among the subgenera Helicophagella, Kozlovea, Kramerea, Pandelleisca, Phallocheira, Pseudothyrsocnema, Sinonipponia and Seniorwhitea were rarely put forward prior to this study. This study provides insight into the population genetics, molecular biology, and phylogeny for the subfamily Sarcophaginae, especially for the subgeneric classification of Sarcophaga. However, compared with the enormous species diversity of flesh flies, the available mitogenomes are still limited for recovering the phylogeny of Sarcophaginae.

Differential Gene Expression for Age Estimation of Forensically Important Sarcophaga peregrina (Diptera: Sarcophagidae) Intrapuparial.

Sarcophaga peregrina is an important flesh fly species for estimating the minimum postmortem interval (PMImin) in forensic entomology. The accurate determination of the developmental age is a crucial task for using necrophagous sarcophagids to estimate PMImin. During larval development, the age determination is straight forward by the morphological changes and variation of length, weight, and width; however, the age estimation of sarcophagid intrapuparial is more difficult due to anatomical and morphological changes not being visible. The analysis of differentially expressed genes (DEGs) during sarcophagid metamorphosis is a potential method for age estimation of intrapuparial. In the present study, real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the differential gene expression level of S. peregrina intrapuparial in different constant temperatures (35°C, 25°C, and 15°C). In addition, the appropriate reference genes of S. peregrina were selected in the intrapuparial and at different temperatures to obtain reliable and valid gene expression profiles. The results indicated that two candidate genes (18S rRNA and 28S rRNA) were the most reliable reference genes, and four DEGs (Hsp90, A-alpha, AFP, AFBP) have the potential to be used to more accuracy estimate the age of S. peregrina intrapuparial.

Identification of Forensically Important Flesh Flies Using the Cytochrome C Oxidase Subunits I and II Genes.

Forensically important flesh flies (Diptera: Sarcophagidae) often are not morphologically distinguishable, especially at the immature stage. In addition, female flies are quite similar in general morphology, making accurate identifications difficult. DNA-based technologies, particularly mitochondrial DNA (mtDNA), have been used for species-level identification. The cytochrome oxidase subunits I and II (COI-COII) sequences of Iranian Sarcophagidae are still unavailable in GenBank. In this study as many as 648 (540 males and 106 females) fly specimens from family Sarcophagidae, representing 10 sarcophagid species, including eight forensically important species were collected from seven locations in five Iranian provinces. Of these, 150 male specimens were identified based on both morphology of male genitalia and DNA sequencing analysis. Sequence data from the COI-COII regions for 10 flesh fly species collected in Iran were generated for the first time. Digestion of COI-COII region by restriction enzymes RsaI, EcoRV, and HinfI provided distinct restriction fragment length polymorphism profiles among the species and can serve as molecular markers for species determination. Phylogenetic analysis represented that the COI-COII sequences are helpful for delimitation of sarcophagid species and implementation in forensic entomology. However, the application of the COI-COII fragment as a species identifier requires great caution and additional species and markers should be studied to ensure accurate species identification in the future.

Molecular identification and detection of Wohlfahrtia magnifica in ovine vulvar myiasis in Gansu, China.

Traumatic myiasis causes substantial economic losses to farmers worldwide. In the present study, six flocks of sheep (2261 sheep) were investigated in Gansu, China, and 207 of 552 larvae were genetically characterized based on three genes, including cyt b, EF-1α, and white gene, by polymerase chain reaction and sequence analysis. A survey of sheep in China revealed that the prevalence of vulvar myiasis of six sheep flocks was 5.00% (11/220, Flock1), 4.85% (10/206, Flock2), 4.50% (9/200, Flock3), 5.00% (15/300, Flock4), 4.68% (15/320, Flock5), 0% (0/1015, Flock6), respectively. The sequence and phylogenetic analysis showed that only Wohlfahrtia magnifica was detected in the field samples. This is the first report of ovine vulvar myiasis caused by W. magnifica in Gansu, China. Some prophylactic measures are strongly recommended to reduce the risk of sheep acquiring traumatic myiasis in Gansu, China.

SNP Typing Using Multiplex Real-Time PCR Assay for Species Identification of Forensically Important Blowflies and Fleshflies Collected in South Korea (Diptera: Calliphoridae and Sarcophagidae).

Medicolegal entomology-a subfield of forensic entomology-is mainly used in medicolegal investigations to estimate the postmortem interval (PMI). The minimum PMI of a corpse invaded by necrophagous immature insects can be estimated because the PMI is near to or earlier than the oviposition time of the larvae that hatched and fed on the corpse. As the growth speeds of larvae differ depending on temperature and species, species-specific growth data are used to estimate the minimum PMI. While morphological identification of adult necrophagous flies can be done by a well-trained entomologist, identification of larvae is relatively difficult. Larvae can only be identified up to the family level and developmental stage by observing the posterior spiracles. For these reasons, the molecular biology method of DNA barcoding has been developed. DNA barcoding that targets the mitochondrial cytochrome c oxidase subunit I (COI) gene is commonly used. COI sequences are currently acquired using polymerase chain reaction (PCR) and Sanger sequencing, which are too time-consuming and complex for practical use in medicolegal investigations. To compensate for these limitations and facilitate the use of entomology for medicolegal investigation, we designed a multiplex real-time PCR system to identify nineteen forensically important species of Calliphoridae and Sarcophagidae flies collected in South Korea. In contrast to the Sanger nucleotide sequencing process, this technology only requires a one-step real-time PCR with melt curve analysis of amplicons generated by primers targeting species-specific single nucleotide polymorphisms (SNPs). Multiplex real-time PCR was performed for twelve species of Calliphoridae (four reactions) and for seven species of Sarcophagidae (three reactions). This assay is expected to make it easier and faster for investigating authorities to identify major species of necrophagous flies at beginning of investigation and to increase the utilization of entomological evidence in forensic investigations.