Methods for the optimal preservation of blow fly intra-puparial forms for morphological analysis in forensic casework.

Accurate minimum post-mortem interval (minPMI) estimations often rely on a precise age determination of insect developmental stages, which is significantly influenced by environmental temperature. An optimal preservation of the entomological samples collected at crime scenes is pivotal for a reliable aging of immature insect samples. For blow flies (Diptera: Calliphoridae), the most widely used insect indicators in forensic investigations, an appropriate preservation of tissues is particularly important in the case of puparial samples because aging methods for intra-puparial forms usually depend on morphological analyses; however, although informative soft tissues and structures could be discoloured and/or distorted if they are not properly fixed, there is a lack of studies to assess different methods for the optimal preservation of intra-puparial forms collected in forensic investigations. The present study compares three preservation methods for intra-puparial forms of the blow fly Calliphora vicina Robineau-Desvoidy, 1830: (i) direct immersion into 80% ethanol, (ii) puncturing of the puparium and hot water killing (HWK) prior to preservation in 80% ethanol, and (iii) HWK without puncturing before preservation in 80% ethanol. External and internal morphological analyses of intra-puparial forms of different ages were conducted to assess the quality of preservation. The results indicate that direct immersion in ethanol led to poor preservation, affecting both external and internal tissues. Both methods with HWK resulted in a better preservation, but puncturing resulted, in some cases, in physical damage of the specimens. HWK without puncturing emerged as the optimal preservation method, consistently yielding high preservation scores for both external and internal morphological analyses. These findings have practical implications for forensic practitioners and emphasise the need for updating some published guidelines and protocols in forensic entomology.

Application of biomolecular techniques on tsetse fly puparia for species identification at larvipostion sites.

Puparia are commonly found in tsetse fly larviposition sites during studies on larval ecology. This chitinous shell is representative of past or ongoing exploitation of these sites by tsetse flies. The morphological characteristics of the puparium are not sufficiently distinctive to allow identification of the species. This study explores the applicability of biomolecular techniques on empty puparia for tsetse fly species identification. Five techniques were compared for DNA extraction from tsetse fly puparia, 1/Chelex® 100 Resin, 2/CTAB, 3/Livak's protocol, 4/DEB + proteinase K and 5/QIAamp® DNA Mini kit, using two homogenisation methods (manual and automated). Using a combination of two primer pairs, Chelex, CTAB, and DEB + K proved the most efficient on fresh puparia with 90, 85, and 70% samples identified, respectively. Shifting from fresh to one- to nine-month-old puparia, the Chelex method gave the best result allowing species identification on puparia up to seven months old. The subsequent testing of the Chelex extraction protocol identified 152 (60%) of 252 field-collected puparia samples at species level. The results show that reliable genetic identification of tsetse flies species can be performed from empty puparia, what can prove of great interest for future ecological studies on larviposition sites. The Chelex technique was the most efficient for DNA extraction, though the age-limit of the samples stood at seven months, beyond which DNA degradation probably compromises the genetic analysis.

The optimal post-eclosion interval while estimating the post-mortem interval based on an empty puparium.

The puparium is the hardened exoskeleton of the last larval instar of a fly, inside which a prepupa, a pupa and a pharate adult fly successively develop. Empty puparia are frequently collected at death scenes, especially in cases with a long post mortem interval (PMI). Although we are not able to estimate the interval between the eclosion of an adult fly and the collection of an empty puparium (i.e. the post-eclosion interval (PEI)), empty puparia may still provide valuable evidence about the minimum PMI. However, because of the unknown PEI, it is impossible to determine the time when the fly emerged, and thus when the retrospective calculation of the minimum PMI should start. In this study, the estimation of PMI (or minimum PMI) for empty puparia of Protophormia terraenovae Rob.-Desv. (Calliphoridae) and Stearibia nigriceps Meig. (Piophilidae) was simulated, to gain insight into the changes in estimates, when different PEIs and different temperature conditions were assumed. The simulations showed that the PEI (in a range of 0-90 days) had no effect on the PMI (or minimum PMI) when the puparium was collected in winter or early spring (December-April). In late spring, summer, or autumn (May-November) the PMI (or minimum PMI) increased with the PEI. The increase in PMI was large in the summer months, and surprisingly small in the autumn months, frequently smaller than the PEI used in the estimation. The shortest PMI was always obtained with a PEI of 0, indicating that the true minimum PMI is always estimated using a PEI of 0. When the puparium was collected during spring, simulations indicated that oviposition had occurred in the previous year, while in summer the previous-year oviposition has been indicated by the simulations only when longer PEIs had been assumed. These findings should guide estimation of the PMI (or minimum PMI) based on an empty puparium.

Internal morphological changes during metamorphosis in the sheep nasal bot fly, Oestrus ovis.

During the larval stage, oestrid flies (Diptera: Oestridae) are obligate parasites, whereas during the adult stage they are free-living and do not feed. Like other cyclorrhaphous flies, oestrids undergo metamorphosis inside an opaque puparium, formed by the contracted and hardened cuticle of the third-instar larva. The present study documents the internal morphological changes taking place during metamorphosis of the sheep nasal bot fly, Oestrus ovis L., using non-invasive, micro-CT-based virtual histology and provides quantitative data of volumetric changes in specific organs. Virtual histological sections allowed visualisation of the progression and completion of the apolyses, which delimit the different intra-puparial stages, and the connection to the tracheal system of a large gas bubble, which plays an essential role during early metamorphosis. Overall, our results show that the sequence of morphological and volumetric changes in tissues and organs is similar to those found in other cyclorrhaphous flies, but they also reveal developmental differences that result in an adult vestigial digestive tract. Future studies could develop non-invasive, reliable methods for aging the intra-puparial forms of different oestrid species of veterinary importance, based on both qualitative and quantitative markers, thus improving our knowledge of their development and the efficiency of control strategies.

Six new record species of whiteflies (Hemiptera: Aleyrodidae) infesting Morus alba in China.

To determine the species of whiteflies occurring on mulberry, Morus alba L. (Rosales: Moraceae) in China, we collected samples in more than 87 sites in 16 provinces of China from 2008 to 2011. In total, 10 species, representing seven genera of the subfamily Aleyrodinae, were identified. Of these, six species are newly recorded on mulberry in China, namely, Aleuroclava ficicola Takahashi, Aleuroclava gordoniae (Takahashi), Aleurotrachelus camelliae (Kuwana), Bemisia afer (Priesner & Hosny), Bemisia tabaci Gennadius, and Pealius machili Takahashi. Information on the taxonomy, distribution, and host plants of the whitefly species found on mulberry in China, along with a brief description and illustrations of each species are provided.

Combining spectrum and machine learning algorithms to predict the weathering time of empty puparia of Sarcophaga peregrine (Diptera: Sarcophagidae).

The weathering time of empty puparia could be important in predicting the minimum postmortem interval (PMImin). As corpse decomposition progresses to the skeletal stage, empty puparia often remain the sole evidence of fly activity at the scene. In this study, we used empty puparia of Sarcophaga peregrina (Diptera: Sarcophagidae) collected at ten different time points between January 2019 and February 2023 as our samples. Initially, we used the scanning electron microscope (SEM) to observe the surface of the empty puparia, but it was challenging to identify significant markers to estimate weathering time. We then utilized attenuated total internal reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to detect the puparia spectrogram. Absorption peaks were observed at 1064 cm-1, 1236 cm-1, 1381 cm-1, 1538 cm-1, 1636 cm-1, 2852 cm-1, 2920 cm-1. Three machine learning models were used to regress the spectral data after dimensionality reduction using principal component analysis (PCA). Among them, eXtreme Gradient Boosting regression (XGBR) showed the best performance in the wavenumber range of 1800-600 cm-1, with a mean absolute error (MAE) of 1.20. This study highlights the value of refining these techniques for forensic applications involving entomological specimens and underscores the considerable potential of combining FTIR and machine learning in forensic practice.

Analysis and comparison of machine learning methods for species identification utilizing ATR-FTIR spectroscopy.

Accurate identification of insect species holds paramount significance in diverse fields as it facilitates a comprehensive understanding of their ecological habits, distribution range, and impact on both the environment and humans. While morphological characteristics have traditionally been employed for species identification, the utilization of empty pupariums for this purpose remains relatively limited. In this study, ATR-FTIR was employed to acquire spectral information from empty pupariums of five fly species, subjecting the data to spectral pre-processing to obtain average spectra for preliminary analysis. Subsequently, PCA and OPLS-DA were utilized for clustering and classification. Notably, two wavebands (3000-2800 cm-1 and 1800-1300 cm-1) were found to be significant in distinguishing A. grahami. Further, we established three machine learning models, including SVM, KNN, and RF, to analyze spectra from different waveband groups. The biological fingerprint region (1800-1300 cm-1) demonstrated a substantial advantage in identifying empty puparium species. Remarkably, the SVM model exhibited an impressive accuracy of 100 % in identifying all five fly species. This study represents the first instance of employing infrared spectroscopy and machine learning methods for identifying insect species using empty pupariums, providing a robust research foundation for future investigations in this area.

First Morpho-Functional Assessment of Immature Stages of Pelecocera Species (Diptera: Syrphidae) Feeding on False Truffles.

With 14 species, Pelecocera Meigen, 1822 is a scarce and small genus of hoverflies (Diptera: Syrphidae: Rhingiini) from the Holarctic Region. Apart from the finding of larvae of Pelecocera (Chamaesyrphus) japonica (Shiraki, 1956) in fungi in Japan, the larval biology of these hoverflies is virtually unknown. The early stages of all Pelecocera species are undescribed. The adults of Pelecocera (Pelecocera) tricincta Meigen, 1822 and Pelecocera (Chamaesyrphus) lugubris Perris, 1839 are found in Palearctic conifer forests with sand dunes. We here report the first morphological evidence of the immature stages of Pelecocera (P. lugubris and P. tricincta), as well as specific data on their breeding sites. Larvae of both species were collected feeding on the hypogean basidiomycete Rhizopogon luteolus Fr. & Nordholm, 1817 in Denmark in 2021. The first larval stage and second larval stage of P. tricincta, the third larval stage of P. lugubris, the anterior respiratory process, and the posterior respiratory process of the puparia of these two species were analyzed and studied using stereomicroscope and scanning electron microscope techniques. The chaetotaxy of the puparium of each species is also described and illustrated. A taxonomic diagnosis of the larvae of the genus Pelecocera is proposed to separate them from the larvae of other genera of the tribe.

Morphological studies of fly puparia (Diptera: Calliphoridae) in the soil from a Joseon Dynasty grave in Korea.

Archaeoentomological investigations were conducted on soil contents from a grave belonging to the Joseon Dynasty as part of the Urban Environment Maintenance Project (UEMP) in Cheongjin 12-16 dong (districts), Jongno-gu, Seoul, Korea, from December 01, 2008 to February 19, 2011. A total of 28 insect puparia with hard shells of the common green bottle fly Lucilia sericata were identified in the soil. Evidence suggested that the corpse was placed outside for some days instead of being buried immediately after death. This is the first report of fly puparia in soil samples from a tomb of the Joseon Dynasty during 16-17 AD in Korea. Our findings may help determine the timeframe of burial and offer archaeological insights into the funerary customs of the period.

Expression profiles of tyrosine metabolic pathway genes and functional analysis of DOPA decarboxylase in puparium tanning of Bactrocera dorsalis (Hendel).

BACKGROUND: Tanning is an important physiological process with critical roles in cuticle pigmentation and sclerotization. Previous studies have shown that insect cuticle tanning is closely associated with the tyrosine metabolism pathway, which consists of a series of enzymes. RESULTS: In this study, 24 tyrosine metabolism pathway genes were identified in the oriental fruit fly Bactrocera dorsalis (Hendel) genome. Gene expression profiles throughout 15 developmental stages of B. dorsalis were established based on our previous RNA sequencing data, and we found that 13 enzyme genes could be involved in the process of pupariation. Accordingly, a tyrosine-mediated tanning pathway during the pupariation of B. dorsalis was predicted and a critical enzyme, 3,4-dihydroxyphenylalanine (DOPA) decarboxylase (DDC), was used to explore its possible roles in formation of the puparium. First, a real-time quantitative polymerase chain reaction confirmed that BdDDC had an epidermis-specific expression pattern, and was highly expressed during larval metamorphosis in B. dorsalis. Subsequent disruption of BdDDC by feeding 5-day-old larvae with DDC inhibitor (l-α-methyl-DOPA) could lead to: (i) a significant decrease in BdDDC enzyme activity and dopamine concentration; (ii) defects in puparium pigmentation; (iii) impairment of the morphology and less thickness of the puparium; and (iv) lower pupal weight and obstacles to eclosion. CONCLUSION: This study provided a potential tyrosine metabolic pathway that was responsible for insect tanning during pupariation, and the BdDDC enzyme has been shown to have crucial roles in larval-pupal tanning of B. dorsalis. © 2021 Society of Chemical Industry.

Intra-puparial development in the hoverflies Eristalinus aeneus and Eristalis tenax (Diptera: Syrphidae).

The intra-puparial development of 150 pupae of Eristalinus aeneus (Scopoli, 1763) and Eristalis tenax (Linnaeus, 1758) was analyzed. Individuals were obtained from the sixth laboratory generation kept under artificial rearing conditions at the facilities of the University of Alicante (Spain). The experiment was conducted at 25 ± 1°C temperature, 50 ± 5% relative humidity, and 12:12 hr (L:D) of photoperiod. Groups of 10 pupae were collected every 6 hr over 48 hr, after that period, pupae were collected daily until the adult emergence. They were fixed in 5% formic acid and preserved in 70% ethanol. Fixed pupae were dissected and photographed. The chronology and morphological changes that take place during the intra-puparial development in both species were analyzed and compared. Five phases were observed: prepupa, before 6 hr; cryptocephalic pupa, between 6 and 24 hr; phanerocephalic pupa, between 24 and 30 hr; pharate adult, after 30 hr; and the adult imago, restricted to the very end of the development process just before adult emergence. In total, the intra-puparial development lasted 189 ± 4 hr in E. aeneus and 192 ± 3 hr in E. tenax, with the pharate adult the longest phase (some 81% of the total developmental time). These data can be used to develop accurate cold storage protocols during artificial rearing of both pollinator species, avoiding critical events during the development and increasing survival.

Effect of Irradiating Puparia of Oriental Fruit Fly (Diptera: Tephritidae) on Adult Survival and Fecundity for Sterile Insect Technique and Quarantine Purposes.

The aim of this study was to investigate the effect of gamma irradiation on survivability (adult emergence, sex ratio, adult longevity), fecundity (eggs hatchability, number of eggs produced), and morphological differences in the size of the ovary and testes of unirradiated and irradiated adults of Oriental fruit fly [Bactrocera dorsalis (Hendel)]. A dose of 100 Gy was determined as the minimum needed for inhibitory effects against B. dorsalis that would not deter adult emergence, sex ratio (male:female), and adult longevity, with 82.6 ±7.02, 1:1.09, and 107 ± 24.5, respectively. Doses from 50 to 400 Gy range did not completely prevent the adult emergence; however, the emergence decreased by increasing the radiation dose. Adult survivability significantly decreased among all the treatment groups of B. dorsalis, except for the 50 Gy and unirradiated flies. At a dose of 50 Gy, fertile females showed a significant reduction in fecundity by not producing eggs after mating with the sterile males. Meanwhile, the number of eggs laid decreased with increasing dose and no fertile egg was hatched starting at 100 Gy of irradiation. Testes and ovaries of 20-d old flies irradiated as pupae were smaller than those of control flies. In this study, 100 Gy was concluded as the minimum effective dose for the disinfestation and sterilization of B. dorsalis puparia. Results represent new findings used as a basis for sterile insect technique and quarantine programs for managing B. dorsalis, particularly in Malaysia.

Revision of the Hybobathus arx and Pelecinobaccha summa species groups (Diptera: Syrphidae).

The morphologically similar Hybobathus arx and Pelecinobaccha summa species groups are revised and distinguished from each other. Four new species are described (H. avittatus sp. nov., H. luciane sp. nov., P. alia sp. nov. and P. impostor sp. nov.), and three new combinations (H. obsoletus comb. nov., H. persimilis comb. nov., and P. cubensis comb. nov.) and several synonimies are proposed: Baccha bivittata Curran = H. arx (Fluke), B. myrtella Hull and B. nectarina Hull = H. obsoletus (Curran), B. druida Hull = H. phaeopterus (Schiner), B. pandora Hull = P. cubensis (Macquart), and B. susio Hull = P. summa (Fluke). The lectotype and paralectotypes of B. phaeoptera Schiner are also designated. The female genitalia are described for all species, and the puparium of H. luciane sp. nov. is described too. New prey records for H. luciane sp. nov. and H. phaeopterus are presented. Picture multi-optional keys were produced and links to these keys, as well as traditional dichotomous keys, are presented in this paper to identify the groups and their species.

Rearing and observation of immature stages of the hoverfly Microdon katsurai (Diptera, Syrphidae).

BACKGROUND: The hoverfly Microdon (Chymophila) katsurai Maruyama et Hironaga 2004 was speculated to be a myrmecophilous species associated with the ant Polyrhachis lamellidens based on observations of adults near the ant nest. However, there have been no reports regarding the observation of immature stages of this species in association with P. lamellidens. NEW INFORMATION: For the first time, we found three M. katsurai larvae inside a P. lamellidens nest and conducted rearing experiments on the larval M. katsurai. P. lamellidens workers did not show any inspection or attack behavior against the M. katsurai larvae under rearing conditions, suggesting that M. katsurai larvae can survive inside a P. lamellidens nest. Although no predatory behavior by the M. katsurai larvae was observed, all the M. katsurai larvae pupated and emerged in a rearing environment. The dorsal surface of the larval M. katsurai has a distinct pale green color with a uniform reticular structure. The puparium of M. katsurai shows several morphological features that are characteristic of the subgenus Chymophila. We conclude that M. katsurai is likely a myrmecophilous species that utilizes P. lamellidens as a specific host and that classification of M. katsurai based on puparium morphology is concordant with that based on adult morphology.

Braconid wasps of subfamily Alysiinae (Hymenoptera: Braconidae) as endoparasitoids of Selachops flavocinctus Wahlberg, 1844 (Diptera: Agromyzidae) in the Central Urals, Russia.

The Agromyzidae fly Selachops flavocinctus Wahlberg, 1844 as host of the Alysiinae genera Asyntactus Marshall, 1898 (Alysiini) and Protochorebus Perepechaenko, 1997 (Dacnusini) is recorded for the first time. A new species from Central Urals, Protochorebus pervushini sp. nov., is described and illustrated. The new material for Asyntactus rhogaleus Marshall, 1898 is studied. A key to Protochorebus species is provided. Description of puparium of Selachops flavocinctus and new data on its life history are published for the first time.

Micromorphology of immature stages of Sarcophaga (Liopygia) cultellata Pandellé, 1896 (Diptera: Sarcophagidae), a forensically important fly.

The Sarcophagidae are one of the most numerous groups of Diptera in the world, consisting of many species of forensic interest, whose immature stages are useful in the estimation of postmortem interval. The immature stages of some species of forensic importance still remain unknown or undescribed, like in the case of Sarcophaga (Liopygia) cultellata Pandellé, 1986, a species restricted to the Iberian Peninsula, south of France and north of Italy, which shares a ecological niche with species of the same subgenus, e.g., Sarcophaga (Liopygia) argyrostoma (Robineau-Desvoidy, 1830) and Sarcophaga (Liopygia) crassipalpis Macquart, 1839, making it necessary to lay the groundwork for a proper specific differentiation before it can be successfully applied in forensic practice. This study provides the first micromorphological description of all preimaginal stages of S. (L.) cultellata using light microscopy and scanning electron microscopy (SEM), the results of which allow the morphology of the main features to be followed during the immature life cycle. We propose a combination of features for distinguishing Liopygia from other sarcophagid subgenera, based on the current level of morphological knowledge of immature stages. S. (L.) cultellata can be differentiated from S. (L.) argyrostoma and S. (L.) crassipalpis in every immature stage by both light microscopy and SEM. The presence of tegumental warts and a fan-shaped anterior spiracle with a single row of 15-18 respiratory papillae allow distinguishing the third instar larvae of S. (L.) cultellata from other Sarcophaga species described hitherto by SEM. Identification keys based on light microscopy observations are provided, covering all the immature stages of Liopygia subgenus occurring in the Iberian Peninsula.

Forensic use of a subtropical blowfly: the first case indicating minimum postmortem interval (mPMI) in southern Brazil and first record of Sarconesia chlorogaster from a human corpse.

Southern Brazil is unique due to its subtropical climate. Here, we report on the first forensic entomology case and the first record of Sarconesia chlorogaster (Wiedemann) in a human corpse in this region. Flies' samples were collected from a body indoors at 20°C. Four species were found, but only Chrysomya albiceps (Wiedemann) and S. chlorogaster were used to estimate the minimum postmortem interval (mPMI). The mPMI was calculated using accumulated degree hour (ADH) and developmental time. The S. chlorogaster puparium collected was light in color, so we used an experiment to establish a more accurate estimate for time since initiation of pupation where we found full tanning after 3 h. Development of C. albiceps at 20°C to the end of the third instar is 7.4 days. The mPMI based on S. chlorogaster (developmental time until the third instar with no more than 3 h of pupae development) was 7.6 days.

Control of the developmental timer forDrosophila pupariation.

In the insectDrosophila, formation of the puparium marks the onset of metamorphosis and serves as a useful marker for developmental progress. The cells of the adult remain diploid and divide during the larval stage while the larval cells become polytene and do not divide. We use a high dose of gamma-irradiation (10 krad) to selectively delete the imaginal lineage from the developing larvae ofDrosophila melanogaster. We find that animals depleted of imaginal cells including those of the imaginal brain pupariate only if the larval cells are allowed to mature, demonstrating that the larval cells harbor the primary developmental timer for this process. However, proliferating imaginal cells can exert a negative influence on the timing of pupariation.