First record of Diamesus osculans (Vigors, 1825) (Coleoptera: Silphidae) colonization on a human corpse.

A partially skeletonized human corpse was found in bushes in Selangor, Malaysia in June 2020. Entomological evidence was collected during the autopsy and sent to the Department of Medical Microbiology and Parasitology, Faculty of Medicine, Universiti Teknologi MARA (UiTM) for minimum postmortem interval (PMImin) analysis. Standard protocols were applied when processing preserved and live insect specimens of both larval and pupal stages. Entomological evidence revealed that the corpse was colonized by Chrysomya nigripes Aubertin, 1932 (Diptera: Calliphoridae) and Diamesus osculans (Vigors, 1825) (Coleoptera: Silphidae). Chrysomya nigripes was chosen as the PMImin indicator as this fly species is an earlier colonizer compared to D. osculans beetle larvae which their presence is the indicative of late stage of decomposition. For the present case, the pupae of C. nigripes were the oldest insect evidence collected and based on the available developmental data, the estimated minimum PMI was established between 9 and 12 days. It is noteworthy to highlight that this is the first record of D. osculans colonization on a human corpse.

Effects of population variations and temperature on Chrysomya megacephala (Diptera: Calliphoridae) development: implications for estimating the postmortem interval.

Forensic entomology requires knowledge of the developmental rates of the species that colonize a body after death to estimate the postmortem interval (PMI). These developmental rates may vary depending not only on the species but also on the geographic location due to population differences. Therefore, the objectives of this work were to determine the developmental duration of the forensically important fly Chrysomya megacephala under constant controlled and field condition temperatures and to compare these results, through a meta-analysis, with data reported by other authors on populations from different localities. For this, C. megacephala colonies were established in the laboratory, and the duration of the life cycle was studied at two controlled temperatures (25 °C and 27 °C) and field conditions (27.5 ± 3.2 °C). Analysis of variance was performed to determine differences in developmental time and larval length between constant laboratory temperatures and field conditions. A generalized linear model was performed with predictor variables extracted from the literature (diet, relative humidity, latitude, longitude) to evaluate the effect of population variation on developmental times. The results showed significant differences in developmental times between 25 and 27 °C. As expected, the complete life cycle of C. megacephala was shorter at 27 °C. Finally, the meta-analysis suggested differences between the developmental times of different populations, based on temperature and geographic location. The results of this study provide fundamental developmental data to use C. megacephala in PMI estimations. Finally, we suggest that, when making expert reports, information from local populations should be used to determine a more accurate and reliable PMI.

Development of Phormia regina at seven constant temperatures for minimum postmortem interval estimation.

Phormia regina (Meigen, 1826) (Diptera: Calliphoridae) can colonize carcasses quickly, and its immature stages are reliable entomological evidence for the estimation of the minimum postmortem interval (PMImin). There are discrepancies in the developmental data from previous studies on P. regina, and the related PMImin indicators need to be refined. We investigated the accuracy of forensic entomological evidence using development durations, growth accumulated degree hours, and larval body length variations of P. regina at seven constant temperatures ranging from 16 to 34 °C. We also established development models such as the isomorphen diagram, thermal summation model, isomegalen diagram, and body length simulation equation to assist with PMImin estimation. The developmental duration of P. regina from egg to adult at 16, 19, 22, 25, 28, 31, and 34 °C was 840.8 ± 42.8 h, 580.1 ± 10.1 h, 390.4 ± 8.7 h, 316.8 ± 9.4 h, 291.4 ± 21.2 h, 238.4 ± 2.8 h, and 222.5 ± 5.2 h, respectively. The lower threshold temperature TL was 9.97 ± 0.50 °C, while the thermal constant K was 5052.7 ± 229 degree days. The lower developmental thresholds, intrinsic optimum temperature, and upper lethal developmental threshold obtained by the Optim SSI models were 13.15, 21.20, and 36.86 °C, respectively. This study aims to provide developmental models for P. regina aimed at common case-site temperatures in the northern provinces of China, which can be used for accurate PMImin estimation.

Characterization of insect stains produced by Dermestes maculatus De Greer (Coleoptera: Dermestidae) resulting from interactions with human blood.

Insect stains produced by adult Dermestes maculatus were characterized during interactions with human blood. Beetles were offered wet or dried blood positioned on ceramic tiles under laboratory conditions. Despite a life history strategy geared toward consumption of dried food stuffs, adult beetles interacted with wet blood more frequently than dried and produced more insect stains after ingesting wet blood. Most (> 95%) of the insect stains produced were the result of fecal elimination. These stains varied in morphologies but were consistently tan/light, black/grey, or red in color; were round to amorphous in shape; and frequently possessed tails. Tailed stains typically were tadpole-shaped or long and tapering from the stain body, yielding Ltl/Lb ratios greater than 1. Tails were the result of beetle locomotion while defecating. Human blood was detected in defecatory stains when using ABA Hematrace® lateral flow assays. When beetles interacted with dried blood, the bloodstains were most often modified due to physical disruption rather than feeding activity. This yielded flaking or dislodgement of the original stains. Within a forensic context, it is unknown whether D. maculatus interacts with any type of bloodstains at a crime scene.

Development of Megaselia scalaris at constant temperatures and its significance in estimating the time of death.

Megaselia scalaris (Schmitz, 1938) (Diptera: Phoridae) is a common insect in forensic science that is frequently found in indoor cases, particularly on corpses in closed environments. Although this species is useful for estimating the minimum postmortem interval (PMImin) in the absence of Calliphoridae, there is a lack of data on its development in China. Herein, we studied the development of M. scalaris exposed to seven constant temperatures ranging from 16 to 34 °C. The mean (± SD) developmental durations of M. scalaris from egg to adult stage at 16, 19, 22, 25, 28, 31, and 34 °C were 1486.9 ± 75.3, 823.7 ± 42.8, 448.2 ± 59.8, 417.7 ± 19.7, 297.2 ± 27.3, 272.9 ± 10.4, and 253.0 ± 5.0 h, respectively. The mean (± SE) lower developmental threshold temperature (TL) and the thermal summation constant (K) were determined by a linear model as 12.69 ± 0.3 °C and 4965.8 ± 227.9-degree hours, respectively. A nonlinear model estimated the lower developmental threshold temperature, intrinsic optimum temperature, and upper lethal developmental threshold temperature as 14.58, 21.00, and 34.15 °C, respectively. We established three development models to estimate the age of the immature insect, namely the isomegalen diagram, isomorphen diagram, and thermal summation model. In addition, a regression analysis of the relationship between body length and total development time from hatching to pupariation was performed. Our findings provide a basis for applications of M. scalaris in PMImin estimations.

Bionomics, reproductive traits and assessment of forensic relevance of Peckia (Peckia) chrysostoma (Wiedemann, 1830) (Diptera: Sarcophagidae).

Peckia (Peckia) chrysostoma (Wiedemann, 1830) (Diptera: Sarcophagidae) is a colonizer of cadavers in the Neotropical Region. Nevertheless, data on development for the P. (P.) chrysostoma (e.g., instar duration) and behavioral strategies used by the species for locating and colonizing a corpse are scant. We aimed to explore bionomic and reproductive aspects of the flesh fly P. (P.) chrysostoma, and in this article we: (a) provide quantitative data on the life cycle of P. (P.) chrysostoma; (b) present bionomic measurements (length and weight) of larvae and pupae; (c) describe intrauterine egg and larvae development; and (d) analyze the ovo/larviposition behavior by gravid females. Females showed ovaries with discernible eggs and larvae between 8 and 10 days (x̅ = 23.3 eggs/female). This study reports the first observation of egg deposition, an atypical behavior for the species. The average development time for immature stages was 22.24 h and 21.36 h for 1st and 2nd respectively, and 3rd showed an average development time of 80.47 h. Pupa had the longest duration (x̅ = 295.69 h). A direct increase was observed in weight (P < 0.05) and length (P < 0.05) throughout time. The average survival time of males and females is approximately 30 days. This study expands the knowledge on P. (P.) chrysostoma, such as facultative ovoviviparity under laboratory conditions and the life cycle, which may benefit future studies for accuracy in entomology-based estimation of minimum post-mortem interval (min PMI).

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.

Establishment of a multiplex PCR-CE assay for the simultaneous and rapid analysis of age markers for Calliphora vicina pupae.

Insects, especially blow flies, are forensically relevant to determine the minimal postmortem interval (PMImin), based on the fact that they are usually the first colonisers of a body. By estimating the age of immature blow flies, interferences can be made about the time since death. Whilst morphological parameters are valuable for age estimation of blow fly larvae, gene expression profiling is more applicable for blow fly pupae. Here, the age-dependent changes in the gene expression levels during the development are analysed. 28 temperature-independent markers have already been described for the age estimation of pupae of the forensically important blow fly Calliphora vicina and are analysed by RT-qPCR. To allow simultaneous analysis of these age markers, a multiplex assay was developed in the present study. After reverse transcription, the markers are analysed simultaneously in an endpoint PCR and subsequently separated by capillary electrophoresis (CE). This method is highly attractive due to its quick and easy procedure and interpretation. The present age prediction tool was adapted and validated. The multiplex PCR assay reproduced the same expression profiles as the RT-qPCR assay based on the same markers. The statistical evaluation shows that the new assay has a lower precision but a better trueness for age determination compared to the RT-qPCR assay. Since the new assay is also qualified to estimate the age of C. vicina pupae and is practical, cost-effective and, even more importantly, time-saving, it is attractive for use in forensic casework.

Parasitic and predatory behavior of Alysia manducator (Hymenoptera: Braconidae) on blow fly larvae feeding on an adult pig carcass in the Western Cape Province of South Africa: preliminary observations and forensic implications.

Wasps are part of the entomofauna associated with vertebrate carrion. They are known to parasitize and prey on specific life stages of insect hosts such as eggs, larvae, pupae, and/or adults associated with vertebrate carrion. However, reports of parasitic behavior of wasps on carrion-associated insect life stages and their possible forensic implications are non-existent in the Western Cape Province of South Africa. This study is part of ongoing research investigating the entomofauna and their pattern of succession on an adult pig carcass in Cape Town, South Africa. During this study, the parasitic wasp Alysia manducator was noted parasitizing and preying on blow fly larvae associated with the decomposing carcass. The arrival of A. manducator coincides with the occurrence of blow fly eggs and/or larvae on the carcass. These wasps were seen in close association with the eggs and larvae of blow flies on various parts of the carcass and some wasps were seen dragging fly larvae attached to their ovipositors away from one part of the carcass to another. Some A. manducator were also observed walking over several larvae on the carcass while exhibiting a stabbing behavior presumably in search of a host for oviposition. We suggest that the observations recorded in this study are of considerable forensic importance as the dragging effect and predatory and stabbing behavior exhibited by A. manducator could potentially disrupt the feeding and development of the fly larvae on the carcass. This could subsequently alter the process of carcass decomposition and/or affect minimum post-mortem interval estimations.

An effective method for preparing histological sections of blow fly intra-puparial stages for minimum PMI estimations.

Blow flies (Diptera: Calliphoridae) are generally early colonisers of fresh cadavers, enabling the estimation of a minimum post-mortem interval (minPMI) based on an accurate aging of the oldest immature stages associated with a cadaver. In blow flies, the pupal stage and the subsequent development of the adult take place inside a protective case, the puparium, formed from the hardened and darkened cuticle of the third instar larva. Because the puparium is an opaque structure that shows virtually no external changes, qualitative analyses of the internal tissues can be very informative for determining reliable age-specific morphological markers. Those analyses can be performed using either non-invasive but expensive and not widely accessible techniques, or traditional histological methods, which are invasive as they require the serial sectioning of the sample. Histological methods are often readily available for forensic researchers and practitioners; however, the histological study of blow fly intra-puparial stages has traditionally been hampered by the poor paraffin infiltration of tissues due to the abundance of fat bodies, resulting in usually fragmented sections and the subsequent loss of relevant information. We present here an effective method for the preparation of histological sections of blow fly intra-puparial stages, maximising the paraffin infiltration while enabling the production of clean and entire sections that allow for the use of reliable age-specific morphological markers, thus improving the accuracy of minPMI estimations when access to more costly techniques is not feasible.

Novel use of a servosphere to study apodous insects: Investigation of blow fly post-feeding larval dispersal.

Blow flies (Diptera: Calliphoridae) are arguably the most important providers of an estimate of minimum post-mortem interval in forensic investigations. They usually undergo a post-feeding dispersal from the body. While previous studies have looked at dispersal of groups of larvae, recording the dispersal activity of individual larvae has not previously been demonstrated. A servosphere was used here to record the speed, directionality and phototaxis of individual post-feeding larvae of two species of blow fly on a smooth plastic surface over time. The servosphere rotates to compensate for the movement of an insect placed at its apex, thereby enabling its unimpeded locomotion in any direction to be studied and behavioural changes to external stimuli recorded. To our knowledge, the servosphere has not previously been used to study apodous insects. The objective of our study was to compare dispersal behaviour of Calliphora vicina Robineau-Desvoidy and Protophormia terraenovae (Robineau-Desvoidy), both common primary colonisers of human and animal cadavers, but showing different post-feeding dispersal strategies. Larvae of C. vicina generally disperse from the body while those of P. terraenovae remain on or close to the body. Our aims were to study (1) changes in dispersal speed over a 1-h period; (2) changes in dispersal speed once a day for 4 days, between the end of feeding and onset of pupariation; and (3) response of dispersing larvae to light. We demonstrated that (1) the movement of three C. vicina larvae tracked for 1 continuous hour on 1 day slowed from an average of 3 to <1.7 mms-1; (2) the average speed of 20 larvae of C. vicina (4.08 mms-1) recorded for 5 min once per day over a 4-day period between onset of dispersal and pupariation was significantly greater than that of P. terraenovae (2.36 mms-1; p < 0.0001), but that speed of both species increased slightly over the 4 days; (3) the responses of larvae of C. vicina to changes in light direction from the four cardinal directions of the compass, showed that they exhibited a strong negative phototactic response within 5 s, turning to move at approximately 180° away from the new light position. While conducted to observe larval calliphorid post-feeding behaviour, the results of this proof of concept study show that apodous insects can be studied on a servosphere to produce both qualitative and quantitative data.

A preliminary study of carrion insects in Greece and their attraction to three animal baits: a forensic entomology perspective.

The current study provides data on the composition of the dipteran and coleopteran fauna attracted to three different animal baits (chicken stomach, beef liver, fish) in the urban ecosystem of Athens, Greece. An additional objective was to examine the effect of bait choice by the collected taxa. Three trap locations were established within the Agricultural University of Athens. At each location two different types of baited traps were used to capture and record the local dipteran and coleopteran biodiversity. During the sampling period, a total of 2416 dipteran and coleopteran specimens representing 43 species were collected and identified. The three most abundant dipteran taxa were Sarcophaga sp. (Diptera: Sarcophagidae), Calliphora vicina Robineau-Desvoidy, Lucilia sericata (Meigen) (Diptera: Calliphoridae). The results showed that the structure of the local insect community associated with the decomposition of animal baits depends on the bait used for sampling. This study reinforced the feasibility of using animal tissues as attractants to a wide diversity of medical, parasitological and forensically important taxa. Overall, beef liver attracted the most insects with Diptera preferring beef liver and Coleoptera preferring chicken stomach. Furthermore, this study revealed that baits of one animal tissue did not attract all species but in combination a much better understanding of this biodiversity was achieved. Finally, from a forensic entomology perspective this study highlights the lack of understanding of the value of entomological data related to case work in Greece.

New geographic location data on the occurrence and abundance of carrion insects of forensic interest.

The world's forensic entomologists have much in common. They face similar research challenges, apply the same scientific methodology, study the same kinds of evidence, and access global research databases. Nevertheless, some regional heterogeneity inevitably exists. For most countries, and particularly those that have complex and diverse ecosystems, the current priority is to stimulate use of forensic entomology by establishing open access databases with time series data using standardised protocols for occurrence, abundance, distribution, niche preferences, life cycle, and identification characteristics for the key regional species that may be encountered in forensic entomology cases. Even in countries where forensic entomology is routinely used as a tool in reconstructing the history of corpses found on crime scenes in accordance with principles found in the rapidly developing body of literature, there is still much to learn about forensically useful insects. Examples of regional gaps include the taxonomy of lesser-known carrion insect species, seasonal occurrence of carrion species in the country's various geographical regions, and rates of development of the local species modeled in the many site situations of forensic interest. The first published study of carrion insects found in Athens, Greece published in this journal is an example of entomologists in a region taking the necessary first step towards establishing baseline data about native and introduced species and hence, physiological and behavioural responses to local environmental conditions, including life cycles and likelihood of occurrence or absence in the region.

Decomposition and insect succession on human cadavers in a humid, continental (Dfb) climate (Quebec, Canada).

Forensic entomology has been developing globally for decades. Despite this discipline being used in criminal investigations around the world, only a few controlled studies have been performed on human cadavers in human decomposition facilities, with the majority of these being conducted in warm and often dry climates. Therefore, the purpose of our research was to catalogue the first published data on insects associated with decomposed human bodies in a humid, continental (Dfb) climate. Specifically, our objective was to document the diversity and succession of the entomofauna associated with human cadavers throughout the decomposition process, in the Quebec province of Canada, during the summer season. Two human cadavers were studied in 2020 at the site for Research in Experimental and Social Thanatology, REST, located in Bécancour (Quebec, Canada). Insects (and other arthropods) were regularly sampled by visual observations, collection from the cadavers, and by using an entomological net and pitfall traps. Our results highlight that the decomposition process is a heterogeneous and complex process in Quebec, with cadavers showing signs of precocious desiccation/mummification. In addition, our observations confirm that the presence of superficial skin lesions accelerates the colonization of blow flies (Diptera: Calliphoridae) and, consequently, the process of decomposition. Finally, we were able to discriminate between "early colonizers" (e.g., Calliphoridae Lucilia sp. or Calliphora livida), "late colonizers" (e.g., larvae of Piophilidae or Heleomyzidae), and "non-specific colonizers." We also officially report the first observation of Cochliomyia macellaria (Diptera: Calliphoridae) in Quebec. These findings will provide new information to help medico-legal death investigations by determining the minimum time elapsed since death and the circumstances surrounding death.

Initial laboratory validation of temperature development models for Necrodes littoralis L. (Staphylinidae: Silphinae).

Development models of necrophagous insects are applied in forensic entomology for post-mortem interval estimation. Such estimates may be used as scientific evidence in legal investigations. For this reason, it is important that the models are valid and that the expert witness is aware of their limitations. Necrodes littoralis L. (Staphylinidae: Silphinae) is a necrophagous beetle species that frequently colonizes human cadavers. Temperature models of development for the Central European population of these beetles were recently published. In this article, we present results of the laboratory validation study for these models. Errors of beetle age estimation differed significantly between the models. Thermal summation models yielded the most accurate estimates, and the isomegalen diagram least accurate estimates. Errors of the beetle age estimation varied across beetle developmental stages and rearing temperatures. In general, most development models of N. littoralis were satisfactorily accurate in estimating beetle age under laboratory conditions; therefore, the study provides initial evidence to support their validity in forensic cases.

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.

Calibrating insect age at eclosion by size in a gregarious carrion beetle Thanatophilus sinuatus (Staphylinidae: Silphinae).

Recent discoveries have shown that the physiological age at eclosion of forensically useful beetles differs between males and females and between beetles of various sizes. Accordingly, it was postulated that the size and sex of the beetles at eclosion may be used to calibrate their age, which may improve the accuracy of age (and post-mortem interval) estimates in forensic entomology. In this study, we derived thermal summation models for the eclosion for the Central European population of carrion beetles Thanatophilus sinuatus (Fabricius, 1775), (Staphylinidae: Silphinae), and tested the usefulness of sex and size for the calibration of beetle age at eclosion. Although in previous developmental studies, the beetles were reared individually, we reared them in larval aggregations, since in natural conditions T. sinuatus beetles are gregarious. Weak (r2 between 5% and 13%) negative correlations were observed between the size and age of T. sinuatus males or females at eclosion, demonstrating that calibration of age by beetle size and sex may bring only minimal benefits regarding the accuracy of age estimation in this species. However, it may still be worthwhile in the case of extremely large or small beetles. Moreover, the total development times recorded in this study were much shorter than in the previous T. sinuatus study, at 14°C by about 15 days and at 26°C by about 2 days. These differences emphasise the importance of gregariousness for the development of carrion beetles, and at the same time highlight the need for the ecologically-relevant protocols of development studies in forensic entomology.

Morphological and molecular characterization of puparia of Piophilidae species of forensic relevance.

Piophilidae are a small family of Diptera with a worldwide distribution and which are historically associated with human activities. In addition to their economic importance, piophilid larvae can also be of medical and legal relevance. Within a medicolegal context, piophilids are frequently associated with cadavers in advanced stages of decomposition, thus being potentially useful forensic indicators and they have been reported also from archaeo-funerary contexts. An accurate species identification is therefore an essential prerequisite to ensure the reliable analysis of insect material in medical, forensic and archaeological investigations. Identification of the adult piophilid flies is possible because of the availability of identification keys, in contrast immature insects, especially puparia, have been poorly investigated and described. In this paper, puparia of 11 species of forensic interest (Piophila casei, Piophila megastigmata, Parapiophila atrifrons, Parapiophila flavipes, Parapiophila vulgaris, Protopiophila litigata, Liopiophila varipes, Prochyliza nigrimana, Prochyliza xanthosoma and Stearibia nigriceps in subtribe Piophilina and Centrophlebomyia furcata in subtribe Thyreophorina) are described and a molecular analysis, based on the COI sequencing, is presented to show the potential of the molecular approach in their identification.

Sex-typing of ingested human blood meal in Anopheles stephensi mosquito based on the amelogenin gene.

Identifying the sex of human hosts of insect disease vectors, using PCR amplification of the amelogenin gene (AMEL) from the ingested blood meal is an increasingly useful technique for epidemiological studies of vector-borne diseases, as well as within the criminal justice system. Detection of DNA from ingested blood is influenced by the choice of DNA extraction method, genomic target region, type and length of PCR, and rate of degradation in the DNA samples over time. Here, we have tested two types of PCR (i.e. conventional and nested), producing differently-sized PCR products, in time-course assays targeting the human AMEL gene in Anopheles stephensi mosquitoes that were fed on human male and female blood. The fed female mosquitoes were allowed to digest at 28 °C for times ranging from 0 to 120 h. Three AMEL primer pairs were used to amplify three sequences that were 977, 539, and 106 bp for the X chromosome and 790, 355, and 112 bp for Y. We found that time since feeding had a significant negative effect on the success of PCR amplification. The shortest fragments (106 and 112 bp) were amplified for the longest time after blood feeding (up to 60 h), whereas the medium and longest loci were not amplified by conventional PCR even at 0 h. However, the nested PCR protocol, targeting the medium sequence, could detect small amounts of human DNA up to 36 h (1.5 days) after the blood meal. The shortest PCR assay standardized herein successfully detected small amounts of human DNA in female mosquitoes up to 60 h after the blood meal. This assay represents a promising tool for identifying the sex of the human host from the blood meal in field-collected female mosquitoes.

Entomology beyond research and education: 2022 student debates.

The 2022 student debates of the Entomological Society of America (ESA) happened during the Joint Annual Meeting of the Entomological Societies of America, Canada, and British Columbia in Vancouver, BC, and addressed entomological aspects beyond research and education. The Student Debates Subcommittee of the ESA Student Affairs Committee and the participating student team members communicated for 8 months and prepared for the debates. The theme of the ESA meeting in 2022 was "Entomology as inspiration: Insects through art, science, and culture". There were 2 unbiased speakers who introduced the debate topics as well as 4 teams who debated the following 2 topics: (i) Is forensic entomology viable in criminal case investigations and court cases today? and (ii) Are insects being treated ethically in scientific research? The teams prepared for about 8 months, debated their arguments, and shared their thoughts with the audience. The teams were judged by a panel and the winners were recognized at the ESA Student Awards Session during the annual meeting.