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.

Human wound colonization by Lucilia eximia and Chrysomya rufifacies (Diptera: Calliphoridae): myiasis, perimortem, or postmortem colonization?

The infestation of human or animal tissues by fly larvae has been given distinctive terminology depending on the timing and location of colonization. Wounds and orifices colonized by Diptera in a living human or animal are typically referred to as myiasis. When the colonization occurs after death, it is referred to as postmortem colonization and can be used to estimate the minimum postmortem interval. What happens when the human, as in the case presented here, has a necrotic limb while the human remains alive, at least for a short period of time? The case presented here documents perimortem wound colonization by Lucilia eximia (Wiedemann) and Chrysomya rufifacies (Macquart) and the considerations for approximating development temperatures and estimating the time of colonization (TOC). This represents the first record of L. eximia in human myiasis in the United States and the first record of the co-occurrence of L. eximia and C. rufifacies in human myiasis in the United States. The TOC was estimated using both ambient and body temperature. Insect colonization before death complicates the estimation of TOC and minimum postmortem interval and illustrates the problem of temperature approximation in forensic entomology casework.

Structural and Genetic Investigation of the Egg and First-Instar Larva of an Egg-Laying Population of Blaesoxipha plinthopyga (Diptera: Sarcophagidae), a Species of Forensic Importance.

Flies in the family Sarcophagidae incubate their eggs and are known to be ovoviviparous (i.e., ovolarviparous), but a laboratory-maintained colony of Blaesoxipha plinthopyga (Wiedemann) deposited clutches of viable eggs over 10 generations. A description of the egg and first-instar larva of this species is provided along with genetic data (genome size and cytochrome oxidase I sequences). The egg is similar to previously described eggs of other Sarcophagidae but differs in the configuration of the micropyle. In the first-instar larva, the oral ridges are much more developed than has been described for other species. B. plinthopyga has forensic importance, and the present descriptive information is critical for proper case management.

A preliminary investigation of the relationship between water quality and Anopheles gambiae larval habitats in Western Cameroon.

BACKGROUND: Water quality and anopheline habitat have received increasing attention due to the possibility that challenges during larval life may translate into adult susceptibility to malaria parasite infection and/or insecticide resistance. METHODS: A preliminary study of Anopheles gambiae s.s. larval habitats in the north-west and south-west regions of Cameroon was conducted in order to detect associations between An. gambiae s.s. molecular form and 2La inversion distributions with basic water quality parameters. Water quality was measured by temperature, pH, conductivity, total dissolved solids (TDS) at seven sites in Cameroon and one site in Selinkenyi, Mali. RESULTS: Principal components and correlation analyses indicated a complex relationship between 2La polymorphism, temperature, conductivity and TDS. Cooler water sites at more inland locations yielded more S form larvae with higher 2La inversion polymorphism while warmer water sites yielded more M form larvae with rare observations of the 2La inversion. DISCUSSION: More detailed studies that take into account the population genetics but also multiple life stages, environmental data relative to these life stages and interactions with both humans and the malaria parasite may help us to understand more about how and why this successful mosquito is able to adapt and diverge, and how it can be successfully managed.

Asymmetric introgression between the M and S forms of the malaria vector, Anopheles gambiae, maintains divergence despite extensive hybridization.

The suggestion that genetic divergence can arise and/or be maintained in the face of gene flow has been contentious since first proposed. This controversy and a rarity of good examples have limited our understanding of this process. Partially reproductively isolated taxa have been highlighted as offering unique opportunities for identifying the mechanisms underlying divergence with gene flow. The African malaria vector, Anopheles gambiae s.s., is widely regarded as consisting of two sympatric forms, thought by many to represent incipient species, the M and S molecular forms. However, there has been much debate about the extent of reproductive isolation between M and S, with one view positing that divergence may have arisen and is being maintained in the presence of gene flow, and the other proposing a more advanced speciation process with little realized gene flow because of low hybrid fitness. These hypotheses have been difficult to address because hybrids are typically rare (<1%). Here, we assess samples from an area of high hybridization and demonstrate that hybrids are fit and responsible for extensive introgression. Nonetheless, we show that strong divergent selection at a subset of loci combined with highly asymmetric introgression has enabled M and S to remain genetically differentiated despite extensive gene flow. We propose that the extent of reproductive isolation between M and S varies across West Africa resulting in a 'geographic mosaic of reproductive isolation'; a finding which adds further complexity to our understanding of divergence in this taxon and which has considerable implications for transgenic control strategies.

Rehydration of forensically important larval Diptera specimens.

Established procedures for collecting and preserving evidence are essential for all forensic disciplines to be accepted in court and by the forensic community at large. Entomological evidence, such as Diptera larvae, are primarily preserved in ethanol, which can evaporate over time, resulting in the dehydration of specimens. In this study, methods used for rehydrating specimens were compared. The changes in larval specimens with respect to larval length and weight for three forensically important blow fly (Diptera: Calliphoridae) species in North America were quantified. Phormia regina (Meigen), Cochliomyia macellaria (F.), and Chrysomya rufifacies (Macquart) third-instar larvae were collected from various decomposing animals and preserved with three preservation methods (80% ethanol, 70% isopropyl alcohol, and hot-water kill then 80% ethanol). Preservative solutions were allowed to evaporate. Rehydration was attempted with either of the following: 80% ethanol, commercial trisodium phosphate substitute solution, or 0.5% trisodium phosphate solution. All three methods partially restored weight and length of specimens recorded before preservation. Analysis of variance results indicated that effects of preservation, rehydration treatment, and collection animal were different in each species. The interaction between preservative method and rehydration treatment had a significant effect on both P. regina and C. macellaria larval length and weight. In addition, there was a significant interaction effect of collection animal on larval C. macellaria measurements. No significant effect was observed in C. rufifacies larval length or weight among the preservatives or treatments. These methods could be used to establish a standard operating procedure for dealing with dehydrated larval specimens in forensic investigations.

Facultative Viviparity in a Flesh Fly (Diptera: Sarcophagidae): Forensic Implications of High Variability in Rates of Oviparity in Blaesoxipha plinthopyga (Diptera: Sarcophagidae).

Flesh flies are major primary consumers of carrion and are commonly found on human remains. Due to this latter feeding habit, their development rates can be used to provide temporal information in forensic investigations. This is usually done by referencing published flesh fly development datasets. Flesh flies are typically assumed to be strictly viviparous and datasets reporting their development rates therefore start at the first larval instar. However, an increasing number of studies has identified oviposition by flesh flies, including the forensically relevant species Blaesoxipha plinthopyga Wiedemann. To assess the impact of egg-laying behavior on casework, oviparity rates and time before larval hatching were assessed under controlled laboratory conditions that reflect common casework conditions in Harris County, Texas. We demonstrated systematic deposition of viable eggs but at a very variable rate between samples. Similarly, the duration between oviposition and larval hatching was highly variable, with some eggs taking more than a day to hatch after deposition. These results highlight the need to account for embryonic development in forensic investigations including B. plinthopyga and advocates for the re-evaluation of the assumed strict viviparity of the Sarcophagidae.

Developmental Plasticity of the Flesh Fly Blaesoxipha plinthopyga (Diptera: Sarcophagidae) on Different Substrates.

Forensic entomologists rely on laboratory growth data to estimate the time of colonization on human remains thus extrapolating a minimum postmortem interval (PMI) if assumptions are satisfied. The flesh fly Blaesoxipha plinthopyga (Wiedemann) is one species that occurs in casework in Idaho, Texas, and central California. Because of the few laboratory studies on the development of this fly, the following study was conducted to determine if different substrates impact immature development of the species. In this study, flies were reared on different substrates that are likely to be encountered at indoor and outdoor scenes (Wet Sand, Dry Sand, Clothes [Polyester fibers], and Carpet [Polypropylene fibers]) to determine the influence of substrate on larval, intrapuparial, and total immature development times at 25°C, 50% RH, and 14:10 (L:D) h cycle. The results revealed that substrate significantly affected minimum immature development times without affecting the sexes differently; though a female bias in sex ratio was observed consistently. Average minimum larval developmental times were 160-179 h with a significantly faster development in Carpet than in Clothes. Similarly, average minimum intrapuparial developmental times were 331-352 h; fastest on Carpet and the slowest in Dry Sand. For this species, it may be important to consider the substrates encountered at a death scene as they may affect the development of B. plinthopyga (Wiedemann) in casework by up to 29 h at 25°C and 50% humidity. These effects will also be important to consider when planning future development studies with the species.

Unexpected Human Fatality Associated with Bed Bug (Hemiptera: Cimicidae) Heat Treatment.

Thermal insect treatments can be an effective method of controlling cryptic insect pests, particularly bed bugs, in residential settings. The treatments require high heat over a sustained timeframe. Residents are not present during treatment but return after completion. An unexpected death was investigated following heat treatment for bed bugs in the home of an elderly female, found with a postmortem axillary temperature of 106.2°F (41.2°C) approximately 8 h after returning home. The cause of death was classified as hyperthermia with a contributory cause of hypertensive cardiovascular disease. The manner of death was an accident. The decedent had medical history and medications that could have impaired her ability to dissipate heat. Furthermore, the air conditioning inside the residence was not functional following treatment. Recommendations including education regarding heat stroke and treatment, re-entry timeframes, and/or temperature thresholds are suggested to reduce the risk of heat-related injury or death in vulnerable populations.

Black soldier fly (Diptera: Stratiomyidae) larvae reduce Escherichia coli in dairy manure.

Escherichia coli labeled with a green fluorescent protein was inoculated into sterile dairy manure at 7.0 log cfu/g. Approximately 125 black soldier fly larvae were placed in manure inoculated and homogenized with E. coli. Manure inoculated with E. coli but without black soldier fly larvae served as the control. For the first experiment, larvae were introduced into 50, 75, 100, or 125 g sterilized dairy manure inoculated and homogenized with E. coli and stored 72 h at 27 degrees C. Black soldier fly larvae significantly reduced E. coli counts in all treatments. However, varying the amount of manure provided the black soldier fly larvae significantly affected their weight gain and their ability to reduce E. coli populations present. For the second experiment, larvae were introduced into 50 g manure inoculated with E. coli and stored for 72 h at 23, 27, 31, or 35 degrees C. Minimal bacterial growth was recorded in the control held at 35 degrees C and was excluded from the analysis. Black soldier fly larvae significantly reduced E. coli counts in manure held at remaining temperatures. Accordingly, temperature significantly influenced the ability of black soldier fly larvae to develop and reduce E. coli counts with greatest suppression occurring at 27 degrees C.

Comparing Species Composition of Passive Trapping of Adult Flies with Larval Collections from the Body during Scene-Based Medicolegal Death Investigations.

Collection of insects at the scene is one of the most important aspects of forensic entomology and proper collection is one of the biggest challenges for any investigator. Adult flies are highly mobile and ubiquitous at scenes, yet their link to the body and the time of colonization (TOC) and post-mortem interval (PMI) estimates is not well established. Collection of adults is widely recommended for casework but has yet to be rigorously evaluated during medicolegal death investigations for its value to the investigation. In this study, sticky card traps and immature collections were compared for 22 cases investigated by the Harris County Institute of Forensic Sciences, Houston, TX, USA. Cases included all manner of death classifications and a range of decomposition stages from indoor and outdoor scenes. Overall, the two methods successfully collected at least one species in common only 65% of the time, with at least one species unique to one of the methods 95% of the time. These results suggest that rearing of immature specimens collected from the body should be emphasized during training to ensure specimens directly associated with the colonization of the body can be identified using adult stages if necessary.

Insects and associated arthropods analyzed during medicolegal death investigations in Harris County, Texas, USA: January 2013- April 2016.

The application of insect and arthropod information to medicolegal death investigations is one of the more exacting applications of entomology. Historically limited to homicide investigations, the integration of full time forensic entomology services to the medical examiner's office in Harris County has opened up the opportunity to apply entomology to a wide variety of manner of death classifications and types of scenes to make observations on a number of different geographical and species-level trends in Harris County, Texas, USA. In this study, a retrospective analysis was made of 203 forensic entomology cases analyzed during the course of medicolegal death investigations performed by the Harris County Institute of Forensic Sciences in Houston, TX, USA from January 2013 through April 2016. These cases included all manner of death classifications, stages of decomposition and a variety of different scene types that were classified into decedents transported from the hospital (typically associated with myiasis or sting allergy; 3.0%), outdoor scenes (32.0%) or indoor scenes (65.0%). Ambient scene air temperature at the time scene investigation was the only significantly different factor observed between indoor and outdoor scenes with average indoor scene temperature being slightly cooler (25.2°C) than that observed outdoors (28.0°C). Relative humidity was not found to be significantly different between scene types. Most of the indoor scenes were classified as natural (43.3%) whereas most of the outdoor scenes were classified as homicides (12.3%). All other manner of death classifications came from both indoor and outdoor scenes. Several species were found to be significantly associated with indoor scenes as indicated by a binomial test, including Blaesoxipha plinthopyga (Wiedemann) (Diptera: Sarcophagidae), all Sarcophagidae (including B. plinthopyga), Megaselia scalaris Loew (Diptera: Phoridae), Synthesiomyia nudiseta Wulp (Diptera: Muscidae) and Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae). The only species that was a significant indicator of an outdoor scene was Lucilia eximia (Wiedemann) (Diptera: Calliphoridae). All other insect species that were collected in five or more cases were collected from both indoor and outdoor scenes. A species list with month of collection and basic scene characteristics with the length of the estimated time of colonization is also presented. The data presented here provide valuable casework related species data for Harris County, TX and nearby areas on the Gulf Coast that can be used to compare to other climate regions with other species assemblages and to assist in identifying new species introductions to the area. This study also highlights the importance of potential sources of uncertainty in preparation and interpretation of forensic entomology reports from different scene types.

Is PMI the Hypothesis or the Null Hypothesis?

Over the past several decades, there have been several strident exchanges regarding whether forensic entomologists estimate the postmortem interval (PMI), minimum PMI, or something else. During that time, there has been a proliferation of terminology reflecting this concern regarding "what we do." This has been a frustrating conversation for some in the community because much of this debate appears to be centered on what assumptions are acknowledged directly and which are embedded within a list of assumptions (or ignored altogether) in the literature and in case reports. An additional component of the conversation centers on a concern that moving away from the use of certain terminology like PMI acknowledges limitations and problems that would make the application of entomology appear less useful in court-a problem for lawyers, but one that should not be problematic for scientists in the forensic entomology community, as uncertainty is part of science that should and can be presented effectively in the courtroom (e.g., population genetic concepts in forensics). Unfortunately, a consequence of the way this conversation is conducted is that even as all involved in the debate acknowledge the concerns of their colleagues, parties continue to talk past one another advocating their preferred terminology. Progress will not be made until the community recognizes that all of the terms under consideration take the form of null hypothesis statements and that thinking about "what we do" as a null hypothesis has useful legal and scientific ramifications that transcend arguments over the usage of preferred terminology.

Effects of Temperature and Tissue Type on the Development of Megaselia scalaris (Diptera: Phoridae).

The scuttle fly, Megaselia scalaris (Loew) (Diptera: Phoridae), is of medical, veterinary, and forensic importance. In the case of the latter, M. scalaris is commonly associated with indoor death or neglect cases of humans or household animals, and its larvae are useful in determining time of colonization (TOC). This study is the first to examine the effects of different temperatures and tissues from two vertebrate species on the growth rate and larval length of M. scalaris A preliminary validation of these data was also conducted. Immatures of M. scalaris were reared on either bovine or porcine biceps femoris at 24 °C, 28 °C, and 32 °C. Temperature significantly impacted immature development time, including egg eclosion, eclosion to pupation, and pupation to adult emergence, to favor faster development at higher temperatures. From ovipostion to eclosion, development rate was 32.1% faster from 24 °C to 28 °C, 13.9% faster from 28 °C to 32 °C, and 45.5% faster from 24 °C to 32 °C. Development from eclosion to pupation displayed similar results with differences of 30.3% between 24 °C and 28 °C, 15.4% between 28 °C and 32 °C, and 45.2% between 24 °C and 32 °C. Development from pupation to adult emergence, likewise, displayed a 44.4% difference from 24 °C and 28 °C, 7.3% from 28 °C to 32 °C, and 51.2% from 24 °C to 32 °C. From oviposition to adult emergence, M. scalaris needed ∼32.7% more hours to complete development when reared at 24 °C than 28 °C, 8.5% when reared at 28 °C rather than 32 °C, and 38.4% more time when reared at 24 °C over 32 °C. Tissue type did not significantly impact development.A preliminary validation study was conducted in four indoor environments (two attics, a closet, and a bathroom) spanning two different buildings. Utilizing minimum and mean lengths, time of colonization estimates were underestimated in all instances. The predicted range encompassed the actual TOC for two of the four environments. On average, when using minimum length, time of colonization was underestimated by 45%, but overestimated by only 2% when using maximum development range. Data generated from this research could be useful when estimating a TOC of decomposing vertebrate remains. Future research will need to examine development for each stadium in order to increase precision of such estimates.

Effects of inorganic nitrogen enrichment on mosquitoes (Diptera: Culicidae) and the associated aquatic community in constructed treatment wetlands.

Ammonium nitrogen (NH4-N) is a significant component of municipal and agricultural wastewaters, and nitrogen reduction is an important use of constructed treatment wetlands. The effects of ammonium nitrogen enrichment on resources of larval mosquitoes, larval mosquito abundance, adult mosquito production, and the abundance of related wetland organisms were examined in 0.1-ha replicate treatment wetlands. The hypothesis of a bottom-up effect induced by ammonium addition was not supported by bacterial abundance, mean bacterial cell size, or algal biomass in the water column. There was, however, a significant negative correlation between bacterial cell length and Culex tarsalis Coquillett (Diptera: Culicidae) larval abundance 1 wk later in wetlands enriched with ammonium nitrogen. Larval mosquito (Culex spp.) abundance in southern California wetlands enriched with NH4-N (mean approximately equal to 3 mg/liter) was significantly greater than in control wetlands at ambient nitrogen levels (8.3 mg NO3-N/liter, 0.1 mg NH4-N/liter). Adult mosquito production was nine-fold greater and chironomid larvae were significantly more abundant in wetlands enriched with NH4-N than in controls but other censused taxa exhibited no significant trends. Mosquitofish, Gambusia affinis (Baird & Girard), abundance was significantly reduced in enriched wetlands, but other potential mosquito predators were not significantly affected by ammonium enrichment.

Forensic entomology of decomposing humans and their decomposing pets.

Domestic pets are commonly found in the homes of decedents whose deaths are investigated by a medical examiner or coroner. When these pets become trapped with a decomposing decedent they may resort to feeding on the body or succumb to starvation and/or dehydration and begin to decompose as well. In this case report photographic documentation of cases involving pets and decedents were examined from 2009 through the beginning of 2014. This photo review indicated that in many cases the pets were cats and dogs that were trapped with the decedent, died and were discovered in a moderate (bloat to active decay) state of decomposition. In addition three cases involving decomposing humans and their decomposing pets are described as they were processed for time of insect colonization by forensic entomological approach. Differences in timing and species colonizing the human and animal bodies were noted as was the potential for the human or animal derived specimens to contaminate one another at the scene.

New collection record for Uranotaenia anhydor in southern California.

Larvae of Uranotaenia anhydor Dyar were collected from decaying vegetation within a constructed treatment wetland in western Riverside County, CA. This find extends the range of this species into the inland valleys south of the San Bernardino Mountains of southern California.

The impact of wetland vegetation drying time on abundance of mosquitoes and other invertebrates.

Vegetation management for constructed treatment wetlands often involves knocking down emergent vegetation with heavy equipment and inundating the dead vegetation after a period of drying. Such practices create favorable conditions for larval mosquitoes. We studied the relationship between length of the drying period for an emergent macrophyte, Typha sp., and the abundance of aquatic invertebrates in replicated 0.18-m3 wading pools. The mosquito, Culex tarsalis, was significantly more abundant in pools containing vegetation aged for 2 wk before inundation compared to pools containing vegetation aged 5 wk, freshly cut vegetation, or without vegetation. Potential larval mosquito food resources (particles between 2 and 61 microm in equivalent spherical diameter) in the 2-wk aging treatment did not differ significantly from the other treatments during the 5-wk experiment. The abundance of other larval culicids, nonculicine Diptera, and potential mosquito predators (i.e., Dytiscidae and Aeshnidae) did not differ significantly among the vegetation aging treatments.

Plasmodium falciparum infection rates for some Anopheles spp. from Guinea-Bissau, West Africa.

Presence of Plasmodium falciparum circumsporozoite protein (CSP) was detected by enzyme linked immunosorbent assay (ELISA) in a sample of Anopheles gambiae s.s., A. melas and A. pharoensis collected in Guinea-Bissau during October and November 2009. The percentage of P. falciparum infected samples (10.2% overall) was comparable to earlier studies from other sites in Guinea-Bissau (9.6-12.4%). The majority of the specimens collected were identified as A. gambiae which had an individual infection rate of 12.6 % across collection sites. A small number of specimens of A. coluzzii, A. coluzzii x A. gambiae hybrids, A. melas and A. pharoensis were collected and had infection rates of 4.3%, 4.1%, 11.1% and 33.3% respectively. Despite being present in low numbers in indoor collections, the exophilic feeding behaviors of A. melas (N=18) and A. pharoensis (N=6) and high infection rates observed in this survey suggest falciparum-malaria transmission potential outside of the protection of bed nets.

Chromosome inversions, genomic differentiation and speciation in the African malaria mosquito Anopheles gambiae.

The African malaria vector, Anopheles gambiae, is characterized by multiple polymorphic chromosomal inversions and has become widely studied as a system for exploring models of speciation. Near complete reproductive isolation between different inversion types, known as chromosomal forms, has led to the suggestion that A. gambiae is in early stages of speciation, with divergence evolving in the face of considerable gene flow. We compared the standard chromosomal arrangement (Savanna form) with genomes homozygous for j, b, c, and u inversions (Bamako form) in order to identify regions of genomic divergence with respect to inversion polymorphism. We found levels of divergence between the two sub-taxa within some of these inversions (2Rj and 2Rb), but at a level lower than expected and confined near the inversion breakpoints, consistent with a gene flux model. Unexpectedly, we found that the majority of diverged regions were located on the X chromosome, which contained half of all significantly diverged regions, with much of this divergence located within exons. This is surprising given that the Bamako and Savanna chromosomal forms are both within the S molecular form that is defined by a locus near centromere of X chromosome. Two X-linked genes (a heat shock protein and P450 encoding genes) involved in reproductive isolation between the M and S molecular forms of A. gambiae were also significantly diverged between the two chromosomal forms. These results suggest that genes mediating reproductive isolation are likely located on the X chromosome, as is thought to be the case for the M and S molecular forms. We conclude that genes located on the sex chromosome may be the major force driving speciation between these chromosomal forms of A. gambiae.