Sarcophagidae Research Articles

Anchored phylogenomics and revised classification of the Miltogramminae (Diptera: Sarcophagidae)

AbstractThe Miltogramminae (Diptera: Sarcophagidae) includes ~600 species across >40 genera, which constitute ~20% of global Sarcophagidae. While molecular phylogenetic hypotheses have been produced for this group, critical problems persist, including the presence of paraphyletic genera, uncertain relationships between genera, a bias of sampling towards Palaearctic taxa, and low support for many branches. The present study remedies these issues through the application of Anchored Hybrid Enrichment (AHE) to a sample including ~60% of the currently recognised genera (16% of known species) representing all biogeographic regions except the Neotropical. An alignment of 1,281 concatenated loci was analysed with maximum likelihood (RAxML, IQ‐TREE), Bayesian inference (ExaBayes) and coalescent‐based approaches (ASTRAL, SVDquartets), which resulted in highly supported and concordant topologies, providing unprecedented insight into the relationships of this subfamily of flesh flies, allowing a major update to miltogrammine classification. The AHE phylogenetic hypothesis supports the monophyly of a large proportion of genera. The monophyly of Metopia Meigen is restored by synonymy with Aenigmetopia Malloch, syn.n. To achieve monophyly of Miltogramma Meigen, eight species are transferred from Pterella Robineau‐Desvoidy. The genus Pterella is shown to be paraphyletic in its current circumscription, and to restore generic monophyly Pterella is restricted to contain only Pt. grisea (Meigen). Erioprocta Enderlein, stat.rev., is resurrected. The genus Senotainia Macquart is reconstructed as paraphyletic. The monotypic genus Metopodia Brauer & Bergenstamm is synonymised with Taxigramma Macquart, syn.n. In light of our phylogenetic hypotheses, a new Miltogramminae tribal classification is proposed, composed of six tribes.

Time measurement in insect photoperiodism: external and internal coincidence

The identity and nature of the photoperiodic photoreceptors are now quite well known, as is the nature of the endocrine regulation of the resulting diapauses. The central problem of time measurement—how the photoperiodic clock differentiates long from short days—however, is still obscure, known only from whole-animal experiments and abstract models, although it is clearly a function of the insect circadian system. This review describes some of these experiments in terms of oscillator entrainment and two widely applicable photoperiodic clock models, external and internal coincidence, mainly using data from experiments on flesh flies (Sarcophaga spp) and the parasitic wasp, Nasonia vitripennis.

Analysis of the Influence of Changing and Fixed Temperatures on the Growth and Pteridine Content in the Head of Adults Sarcophaga crassipalpis (Diptera: Sarcophagidae).

Flesh flies (Diptera: Sarcophagidae) are regarded as significant in medical and veterinary entomology, and their development models can be utilized as considerable markers to ascertain the minimum postmortem interval (PMImin). In this research, we explored the growth cycle and larval body length of Sarcophaga crassipalpis Macquart 1839 (Diptera: Sarcophagidae) reared under variable temperatures ranging from 15.7 to 31.1 °C, with an average of 24.55 °C and relative humidity ranges from 31.4 to 82.8% and at six fixed temperatures of 15, 20, 25, 30, 32, and then 35 °C. Moreover, pteridine from the head was used to assess adult age grading. Our results allowed us to provide three development models: the isomorphen chart, the isomegalen chart, and the thermal summation models. The time taken for S. crassipalpis to complete its development from larviposition to adult emergence at constant temperatures of 15, 20, 25, 30, 32, and 35 °C was 1256.3 ± 124.2, 698.6 ± 15.1, 481.8 ± 35.7, 366.0 ± 13.5, and 295.8 ± 20.5 h, respectively, except 35 °C, where all pupae were unable to attain adulthood. They lasted 485.8 ± 5.4 h under variable temperatures. The minimum developmental limit (D0) temperature and the thermal summation constant (K) of S. crassipalpis were 9.31 ± 0.55 °C and 7290.0 ± 388.4 degree hours, respectively. The increase in pteridine content exhibited variations across different temperatures. There was quite a considerable distinction in the pteridine contents of male and female S. crassipalpis at 15 °C (p = 0.0075) and 25 °C (p = 0.0213). At 32 °C and variable temperatures, the pteridine content between female and male S. crassipalpis was not statistically divergent. However, temperature and gender remain the main factors influencing the pteridine content in the head of S. crassipalpis. We aim to provide detailed developmental data on S. crassipalpis that can be used as a valuable resource for future research and PMI estimation.

Two new species of the 'Big Yellows' (Diptera: Sarcophagidae: Sarcophaga).

The informal name 'Big Yellows' is proposed for a possibly non-monophyletic assemblage of large-bodied, yellow-coloured species of flesh flies found in the Melanesian archipelagoes of Bismarck, Solomon and Vanuatu. The group comprises several species of Sarcophaga Meigen subgenus Sarcorohdendorfia Baranov plus the only species of Sarcophaga subgenus Chrysosarcophaga Townsend. Two new species are described from Melanesia: Sarcophaga (Sarcorohdendorfia) confusio sp. nov. (Bismarck Archipelago: New Britain, Yalom) and S. (S.) narabondxii sp. nov. (Solomon Islands: Nggele Islands). A key to the species of this group is provided, and their distribution is summarised. The subgenus Sarcorohdendorfia is diagnosed, and the presence of a single row of downward-facing setae along the ventral margin of the mesopleural anatergite is suggested as an important diagnostic feature. Sarcorohdendorfia Baranov, 1938 is proposed as a senior synonym of Omarisca Kurahashi, Tan & Leh, 2021, syn. nov., and Chrysosarcophaga whitneyi Curran, 1936 is proposed as a senior synonym of Sarcorohdendorfia imitatrix Baranov, 1938, syn. nov. Sarcophaga aureolata Pape & Kurahashi, 2000, S. pattoni Senior-White, 1924 and S. saprianovae Pape & Bänziger, 2000 are transferred from the subgenus Lioproctia Enderlein, 1928 to the subgenus Sarcorohdendorfia Baranov, 1938.

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

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. 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. 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.

In vitro antibacterial activity and synergetic effect of crude extract of the Wohlfahrtia nuba (Diptera: Sarcophagidae) flesh fly larvae

Multidrug-resistant pathogens have become ubiquitous, and effective treatment alternatives are urgently required. Maggot therapy is a promising agent that is being studied to overcome antibiotic-resistant pathogens. This study evaluated the antibacterial activity of the larvae extract of the Wohlfahrtia nuba (wiedmann) (Diptera: Sarcophagidae) flesh fly on the growth of five pathogenic bacterial species (methicillin-sensitive Staphylococcus aureus [ATCC 29213], methicillin-resistant Staphylococcus aureus [ATCC BAA-1680], Pseudomonas aeruginosa [ATCC 27853], Escherichia coli [ATCC 25922], and Salmonella typhi [ATCC 19430]) in vitro by using different techniques. Resazurin-based turbidimetric assay demonstrated that the W. nuba maggot exosecretion (ES) was potent against all the bacterial species tested, and according to the determined minimum inhibitory concentration (MIC) for each bacterium, gram-negative bacteria were more sensitive than gram-positive bacteria. Additionally, colony-forming unit assay showed that maggot ES was able to inhibit bacterial growth rate for all bacterial species tested, where the highest bacterial reduction was observed with methicillin-sensitive S. aureus (MSSA) followed by S. typhi. Moreover, maggot ES was shown to be concentration-dependent, where 100 μL of ES at 200 mg/mL was bactericidal towards methicillin-resistant S. aureus (MRSA) and P. aeruginosa compared with 100 μL at the MIC of the ES. Moreover, based on the result of agar disc diffusion assay, maggot extract was more efficient against P. aeruginosa and E. coli than the remaining reference strains tested. Furthermore, the combination between regular antibiotics with maggot ES at different concentrations indicated that ES acts synergistically with the tested antibiotics against the five bacterial models.

Sarcosaprophagous dipterans associated with differentially-decomposed substrates in Atlantic Forest environments

Sarcosaprophagous flies may show temporal variations in terms of their location and colonization in ephemeral resources, in addition to their richness and abundance changing according to the substrate age. The present study examined the influence of decomposition time on the: (i) composition of the sarcosaprophagous dipterofauna; (ii) sexual proportion of the collected specimens; and (iii) the stage of sexual maturity of female flies (Calliphoridae and Sarcophagidae). For this, we used chicken liver with different decomposition times (0h, 24h, 48h, and 76h) for the collection of adult flies. The results showed that: i) the assemblages were most diverse and abundant in more decomposed baits; ii) Calliphoridae was the most abundant taxa in all treatments; iii) the reproductive stage varied according to the age of the substrate, with high rates of mature or pregnant females in the older substrates; and iv) the number of males blowflies was higher in older baits, but it did not vary for flesh flies. Thus, this work demonstrates experimentally that the substrate age is an influencing factor in the composition of sarcosaprophagous flies’ assemblage, and it indication of the dipterans potential as primary or secondary colonizers throughout colonization of ephemeral resources.

The genome sequence of the variegated flesh fly, Sarcophaga variegata (Scopoli, 1763).

We present a genome assembly from an individual male Sarcophaga variegata (the variegated flesh fly; Arthropoda; Insecta; Diptera; Sarcophagidae). The genome sequence is 718.5 megabases in span. Most of the assembly is scaffolded into 7 chromosomal pseudomolecules including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 18.7 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,660 protein coding genes.

Simplified COI barcoding of blow, flesh, and scuttle flies encountered in medicolegal investigations.

Accurate insect identification is critical to the estimation of time of colonization (TOC) and post-mortem interval (PMI) in medicolegal death investigations. DNA testing is advantageous because it enables the identification of immature specimens that may not be identified based on morphology alone. We describe here a simplified DNA barcoding method for identifying relevant species that may be implemented by forensic genetics laboratories. A cytochrome oxidase (COI) fragment is analyzed after PCR amplification with a single primer set. The method is effective for many species commonly encountered in death investigations in the USA: members of blowfly genera Calliphora, Chrysomya, Cochliomyia, Lucilia, and Phormia; members of the flesh fly genera Blaesoxipha, Oxysarcodexia, Ravinia, and Sarcophaga; and the scuttle fly Megaselia scalaris. We tested the method on specimens with verified identifications and used it to build a collection of reference sequences from specimens collected in Harris County, Texas. We show here the correct identification of larvae, pupae, and pupal exuviae from the medicolegal casework.

Silver Ion High-Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry: A Tool for Analyzing Cuticular Hydrocarbons.

Aliphatic hydrocarbons (HCs) are usually analyzed by gas chromatography (GC) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. However, analyzing long-chain HCs by GC is difficult because of their low volatility and the risk of decomposition at high temperatures. MALDI cannot distinguish between isomeric HCs. An alternative approach based on silver ion high-performance liquid chromatography (Ag-HPLC) is shown here. The separation of HC standards and cuticular HCs was accomplished using two ChromSpher Lipids columns connected in series. A gradient elution of the analytes was optimized using mobile phases prepared from hexane (or isooctane) and acetonitrile, 2-propanol, or toluene. HCs were detected by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Good separation of the analytes according to the number of double bonds, cis/trans geometry, and position of double bonds was achieved. The retention times increased with the number of double bonds, and trans isomers eluted ahead of cis isomers. The mobile phase significantly affected the mass spectra of HCs. Depending on the mobile phase composition, deprotonated molecules, molecular ions, protonated molecules, and various solvent-related adducts of HCs were observed. The optimized Ag-HPLC/APCI-MS was applied for characterizing cuticular HCs from a flesh fly, Neobellieria bullata, and cockroach, Periplaneta americana. The method made it possible to detect a significantly higher number of HCs than previously reported for GC or MALDI-MS. Unsaturated HCs were frequently detected as isomers differing by double-bond position(s). Minor HCs with trans double bonds were found beside the prevailing cis isomers. Ag-HPLC/APCI-MS has great potential to become a new tool in chemical ecology for studying cuticular HCs.

Diversity of flesh flies (Sarcophagidae, Sarcophaginae) of pond habitats in rural areas in the Croatian part of Baranja.

The diversity of grey flesh flies (Sarcophagidae: Sarcophaginae) from the Croatian part of Baranja was studied during 2019 to 2021, resulting in 37 species, of which the following are new for the area: Raviniapernix (Harris, 1780); Sarcophaga (Het.) depressifrons Zetterstedt, 1845; S. (Het.) filia Rondani, 1860; S. (Het.) haemorrhoides Böttcher, 1913; S. (Het.) pumila Meigen, 1826; S. (Het.) vagans Meigen, 1826; S. (Lis.) dux Thomson, 1869; S. (Lis.) tuberosa Pandellé, 1896; S. (Meh.) sexpunctata (Fabricius, 1805); S. (Pan.) protuberans Pandellé, 1896; S. (Sar.) carnaria (Linnaeus, 1758); S. (Sar.) variegata (Scopoli, 1763), and S. (Pse.) spinosa Villeneuve, 1912. New locality records are provided for 25 species. Sarcophaga (Sar.) croatica Baranov, 1941 was the most abundant with 37%, followed by S. (Sar.) lehmanni Müller, 1922 (21%), and S. (Pas.) albiceps Meigen, 1826 (5%), making up 63% of all collected specimens. Most species (35) were collected in locality of Zmajevac, while the fewest (3) were collected in Bilje locality. During this study, S. (Pse.) spinosa was recorded in Croatia for the first time. Combined with previous records, 42 species of flesh flies have been recorded from Croatian Baranja, which comprise 27% of the flesh flies known to occur in Croatia. The total number of species of the family Sarcophagidae currently known in Croatia has increased to 156.

On the life cycle of Brachymeria podagrica (Fabricius, 1787) (Hymenoptera: Chalcididae) - a parasitoid of sacrophagid, calliphorid, and muscid flies.

The lifecycle of Brachymeria podagrica, a parasitic wasp with a worldwide distribution, was studied under laboratory conditions using the flesh fly, Sarcophaga dux, as a host. Two hundred parasite-free 3rd instars of S. dux were exposed for 24 h to 20 female B. podagrica. In daily intervals, maggots and later pupae were examined for developmental stages of the parasitoid. The whole pre-imaginal development at a temperature of 26 °C lasted 21 to 26 days. Three morphologically different instars, followed by a prepupal and a pupal stage, were described using light and scanning electron microscopy. In a second experiment with 100 3rd stage Sarcophaga larvae and 10 parasitoids, a total of 70 wasps emerged 20 to 25 days after exposure. Two fly larvae did not pupate and dried out, while 28 pupae contained a dry or caseous content, dead wasp imagos, or their larval stages. No fly imagines emerged from exposed groups, while all 100 unexposed larvae pupated and adults eclosed between day 12 and day 14 after the start of the experiment, while the imagoes of the parasitoids appeared 8 to 12 days later.

Identification of medically and forensically relevant flies using a decision treelearning method.

Blow flies, flesh flies, and house flies can provide excellent evidence for forensic entomologists and are also essential to the fields of public health, medicine, and animal health. In all questions, the correct identification of fly species is an important initial step. The usual methods based on morphology or even molecular approaches can reach their limits here, especially when dealing with larger numbers of specimens. Since machine learning already plays a major role in many areas of daily life, such as education, business, industry, science, and medicine, applications for the classification of insects have been reported. Here, we applied the decision tree method with wing morphometric data to construct a model for discriminating flies of three families [Calliphoridae, Sarcophagidae, Muscidae] and seven species [Chrysomya megacephala (Fabricius), Chrysomya rufifacies (Macquart), Chrysomya (Ceylonomyia) nigripes Aubertin, Lucilia cuprina (Wiedemann), Hemipyrellia ligurriens (Wiedemann), Musca domestica Linneaus, and Parasarcophaga (Liosarcophaga) dux Thomson]. One hundred percent overall accuracy was obtained at a family level, followed by 83.33% at a species level. The results of this study suggest that non-experts might utilize this identification tool. However, more species and also samples per specimens should be studied to create a model that can be applied to the different fly species in Thailand.

The genome sequence of the lesser worm flesh fly, Sarcophaga ( Sarcophaga) subvicinaRohdendorf, 1937.

We present a genome assembly from an individual male Sarcophaga subvicina (the lesser worm flesh fly; Arthropoda; Insecta; Diptera; Sarcophagidae). The genome sequence is 71 megabases in span. Most of the assembly (95.91%) is scaffolded into six chromosomal pseudomolecules, with the X sex chromosome assembled. The mitochondrial genome has also been assembled and is 16.7 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,793 protein coding genes.

Flesh Flies (Diptera: Sarcophagidae) of Forensic Importance Collected From Pig Carcasses in the Cerrado of Northeastern Brazil.

Sarcophagidae is a family of true flies frequently found colonizing carcasses and corpses as adults and/or larvae, therefore, they are of great forensic value in the medico-legal field. Thus, this study aims to inventory the sarcophagids species and describe their succession process in the decomposition of swine carcasses in the Cerrado (savanna-like vegetation), Brazilian Northeastern region. A total of 10,819 specimens were collected, belonging to 45 sarcophagid species. Of this total, one species was recently described (Helicobia neuzalmeidae Silva, Brasil, De-Souza & Carvalho-Filho), two [Blaesoxipha (Acanthodotheca) denieri (Blanchard) and Dexosarcophaga pusilla Lopes] are new records for the Cerrado biome, five [Argoravinia (Argoravinia) catiae Carvalho Filho & Esposito, B. (A.) denieri, Nephochaetopteryx orbitalis (Curran & Walley), Peckia (Sarcodexia) tridentata (Hall) and Tricharaea (Sarcophagula) ramirezi (Lopes)] are new records for the Northeast region and eight are new records for the state of Maranhão. The species A. (A.) catiae, Blaesoxipha (Gigantotheca) stallengi (Lahille), D. pusilla, H. neuzalmeidae, Lipoptilocnema misella Lopes, Oxysarcodexia bakeri (Aldrich) and Retrocitomyia retrocita (Hall) were collected for the first time in pig carcasses. Peckia (Sarcodexia) lambens (Wiedemann) and Oxysarcodexia thornax (Walker) were the most abundant species. In both collection seasons, five species were carcasses pioneers at an early stage. The majority of species (42 spp.) were collected during the dry season, primarily during the black putrefaction (39 spp.) and butyric fermentation (30 spp.) stages. The greatest number specimens (n = 6,484) were recorded during the rainy season, at the butyric fermentation (n = 2,517) and black putrefaction (n = 2,080).

The genome of Roselle's flesh fly Sarcophaga ( Helicophagella) rosellei (Böttcher, 1912).

We present a genome assembly from an individual male Sarcophaga rosellei (Roselle's flesh fly; Arthropoda; Insecta; Diptera; Sarcophagidae). The genome sequence is 541 megabases in span. Most of the assembly is scaffolded into six chromosomal pseudomolecules, with the X sex chromosome assembled. The mitochondrial genome has also been assembled and is 19.5 kilobases in length. Gene annotation of this assembly on Ensembl has identified 15,437 protein coding genes.

Pupal Age Estimation of Sarcophaga peregrina (Diptera: Sarcophagidae) at Different Constant Temperatures Utilizing ATR-FTIR Spectroscopy and Cuticular Hydrocarbons

Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a forensically important flesh fly that has potential value in estimating the PMImin. The precise pupal age estimation has great implications for PMImin estimation. During larval development, the age determination is straightforward by the morphological changes and variation of length and weight, however, the pupal age estimation is more difficult due to anatomical and morphological changes not being visible. Thus, it is necessary to find new techniques and methods that can be implemented by standard experiments for accurate pupal age estimation. In this study, we first investigated the potential of attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and cuticular hydrocarbons (CHCs) for the age estimations of S. peregrina pupae at different constant temperatures (20 °C, 25 °C, and 30 °C). The orthogonal projections latent structure discrimination analysis (OPLS-DA) classification model was used to distinguish the pupae samples of different developmental ages. Then, a multivariate statistical regression model, partial least squares (PLS), was established with the spectroscopic and hydrocarbon data for pupal age estimations. We identified 37 CHCs with a carbon chain length between 11 and 35 in the pupae of S. peregrina. The results of the OPLS-DA model show a significant separation between different developmental ages of pupae (R2X > 0.928, R2Y > 0.899, Q2 > 0.863). The PLS model had a satisfactory prediction with a good fit between the actual and predicted ages of the pupae (R2 > 0.927, RMSECV < 1.268). The results demonstrate that the variation tendencies of spectroscopy and hydrocarbons were time-dependent, and ATR-FTIR and CHCs may be optimal for the age estimations of pupae of forensically important flies with implications for PMImin estimation in forensic practice.

Significance of Forensic Fly and Seasonal Variation in the Temperature of Developmental Stages in Lifecycle for Family Sarcophagidae, Sarcophaga Africa

Sarcophaga Africa, is one of the hairy maggot flesh flies which feeds on meat carrion, and dead and decaying matter of animals to the completes its life cycle which is the useful for post mortem interval (PMI). Determination in forensic investigations. The actual life cycle hours and days are calculated due to their morphological parameter of their life cycle of Sarcophaga Africa were studied in different seasons; Life cycle in rainy season was completed in 270± 1.25 hrs (11.25 ± 0.40 days), when the maximum temperature was 27.02°C and the minimum temperature was 26.4°C; in summer season when the maximum temperature was 36.6°C and the minimum temperature 33.2°C, the life cycle was completed in 220± 1.17 hrs (6.16. ± 0.10 days), while in cycle was completed in 310 ± 1.35hrs (12.91 ± 0.21 days) when the maximum and minimum winter season life cm temperatures were 27.4°C and 17.2°C respectively. The temperature is the important role to determine the developmental stages of life cycle of Sarcophaga Africa which should be considered during PMI determination. The external parameters of different stages differ from season to season. Larvae were healthy and bigger in size in rainy season but in summer were short and small sized. The size of larvae in winter season was also smaller than the size in both summer and rainy seasons.

The genome sequence of the bluish flesh fly, Sarcophaga( Robineauella) caerulescens (Zetterstedt, 1838).

We present a genome assembly from an individual male Sarcophaga caerulescens (the bluish flesh fly; Arthropoda; Insecta; Diptera; Sarcophagidae). The genome sequence is 597 megabases in span. Most of the assembly is scaffolded into seven chromosomal pseudomolecules, including the assembled X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 21.1 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,559 protein coding genes.

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 ± 30.6h, 566.6 ± 21.9h, 404.6 ± 13.01h, and 280.3 ± 4.5h, 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.