A geometric morphometric analysis of wing variations in shape and size of the blue bottle fly, Calliphora vicina (Diptera: Calliphoridae).

A geometric morphometric analysis was performed on the right wing of adult Calliphora vicina (Robineau-Desvoidy) collected across 4 altitudinal levels in Sicily. The objective of this study was to assess differences in shape and centroid size (CS) between females and males and across elevations. The wings analyzed in this study were removed from adults of C. vicina collected with baited traps at 20, 700, 1,153, and 1,552; for this study, 19 landmarks were identified in each wing. The coordinates of the landmarks were aligned and superimposed to prevent variations due to position, orientation, and scale; they were then scaled to the same CS and recentered. CS and Procrustes differences were, respectively, used to assess variations in size and shape. Significant differences were observed in wing shape between males and females but not between all altitudinal levels. Female wings were found to be significantly larger than males (P < 0.01). Wings of flies collected at the highest altitudinal level resulted in significantly larger wings than those collected at lower altitudes (P < 0.001), with CS values ranging from 12.1 to 14.1. Variation in wing shape can impact thermal regulation, and therefore, oxygen content, temperature, atmospheric pressure, and solar radiation can have an effect on an insect's body and activity levels. At high elevations and lower temperatures, larger wings could mean less energy expenditure when flying to increase body temperature.

High Temperature Limits of Survival and Oviposition of Phormia regina (Meigen) and Lucilia sericata (Meigen).

The temperature dependent development rates of blow flies allow blow flies to be used as biological clocks in forensic death investigations. However, the upper thermal limits of adult survival and oviposition, both required for producing larvae, remains largely unknown. Therefore, in this study we examined the impact of a range of temperatures between 37 °C and 44 °C on the likelihood of survival and egg-laying behavior of two species of medicolegal forensic importance, Lucilia sericata (Meigen) and Phormia regina (Meigen) (Diptera: Calliphoridae). To quantify the upper temperature limits of survival, adult fly colonies were exposed to 37 °C, 41 °C, 42 °C, 43 °C, and 44 °C for 24 h. Similarly for oviposition trials, adults of both species were exposed to 40 °C, 42 °C, and 43 °C with P. regina oviposition also observed at 41 °C. Trials lasted for 24 h with oviposition substrate replenished at the 12 h mark. A yes/no determination on egg deposition was made, eggs were counted, and a yes/no determination was made on egg hatch. Survival did not differ by species (p = 0.096). Overall, survival decreased with increasing temperatures, with ~100% at 37 °C, ~50% at 41 °C, ~37% at 42 °C, ~15% at 43 °C and 0% at 44 °C. Lucilia sericata laid eggs capable of hatch up to 43 °C, while Phormia regina egg-hatch was observed up to 41 °C. These results indicate a greater thermal tolerance of adult survival than for egg deposition and successful egg hatch, which supports previous experiments indicating blow flies stop laying eggs at sub-lethal temperatures. Furthermore, these data indicate that adult blow flies may find remains at or near time of death but may delay egg deposition until temperatures drop below an acceptable threshold.

Biodiversity of Forensically Relevant Blowflies (Diptera: Calliphoridae) at the Anthropology Research Facility in Knoxville, Tennessee, USA.

Understanding the biodiversity and distribution of forensically relevant blowflies (Diptera: Calliphoridae) in a region can aid in legal investigations when insects are associated with remains. For this purpose, we conducted a 14-month-long blowfly survey at the Anthropology Research Facility (ARF) of the University of Tennessee in Knoxville, Tennessee. Traps baited with pork kidney were deployed for 24 h twice a month throughout the study. A total of 3180 adult blowflies were collected, comprising 13 species from 7 genera. Phormia regina (Meigen) and Lucilia coeruleiviridis (Macquart) were the predominant species collected from this survey, with collections representing 65.9% and 20.6%of total flies captured, respectively. In addition to relative abundance, we investigated blowfly community composition, species abundance, richness, and diversity by season. One state record was identified, with adult Protophormia terraenovae (Robineau-Desvoidy) being collected for the first time in Tennessee. Additionally, an earlier record of Chrysomya megacephala (Fabricius) in Tennessee was noted. These findings can be used to aid in legal investigations in the area and surrounding areas where work is limited, as well as to provide information on which forensically relevant species should be the subject of future research in the area.

Technical note: A technique to mount Sarcophagidae and Calliphoridae (Diptera) larvae for forensic identification using geometric morphometrics.

Geometric morphometric analyses use landmark coordinates from images of specific areas on specimens. Therefore, to minimize differences arising from human preparation, preparation of the specimen is crucial. The application of geometric morphometrics has been used in entomology for identifying adult dipteran species, however, applying this technique to larval specimens requires the production of standard protocols which would allow specimens to be compared equally. This proposed protocol consists of a two-step process: 1. using 15% potassium hydroxide (KOH) to clear the specimen, followed by rinsing the specimen with 80% ethanol; 2. removal and spreading of the mouthhooks on a slide for imaging and analysis. The goal of this work is to standardize specimen preparation, which would reduce human error and facilitate use of geometric morphometrics to identify dipteran larvae.

An Analysis of Forensically Important Insects Associated with Human Remains in Indiana, U.S.A.

Research documenting insect colonization of human remains is limited in North America, and currently nonexistent for the American Midwest. Such research is essential for forensic entomologists to identify species of research interest in a region. In this study, we collected insects from human remains in 24 cases across Indiana from June 2016 through September 2018. We analyzed species composition across scene type and season. Eight species of blow flies were collected as larvae from human remains, with Phormia regina and Lucilia sericata as the two predominant colonizers. Phormia regina was the most numerous species collected from outdoor scenes (73.6% of total collections) while L. sericata was the most numerous from the indoor scenes (60.4% of total collections). With scene types pooled, Calliphora vicina and Cochliomya macellaria were the predominant species in the fall (55.6% and 42.2%, respectively); P. regina was the dominant colonizer in the spring (68.6%); and P. regina and L. sericata were the predominant colonizers (46.5% and 44.4%, respectively) in the summer. In addition to these findings, we confirmed the first record of Lucilia cuprina colonizing human remains in Indiana having collected this species from three cases. A single adult Chrysomya megacephala was collected from an indoor scene in southern Indiana, which represents its second collection in the state. Beetles belonging to the families Staphylinidae, Silphidae, Histeridae, Cleridae, Trogidae, Dermestdae, and Nitidulidae were also collected from two outdoor scenes. This study provides important baseline data for forensic entomologists in Indiana, as well as surrounding states with similar environments.

Analysis of Alternative Food Sources for Rearing Entomological Evidence.

Forensic entomology is a well-established tool for evaluating the death, or abuse, of a person. Insect evidence provides valuable information as related to time of colonization and movement of remains from one location to another. Blow flies (Diptera: Calliphoridae) are commonly found on human remains throughout most stages of decomposition, and when entomological evidence is collected these taxa tend to be the most numerous. However, very few crime laboratories across the country have collection and rearing protocols for these forensically important insects. A lack of knowledge in collection techniques and limited access to an appropriate food source are the main reasons for absence in adequate collection and rearing protocols. Thus, when crime scene investigators or pathologists collect insects, they are often mishandled (e.g., placed into containers with no air holes, no food, or a food source that is not sustainable for their development). To address this issue, we analyzed easily accessible and cost-efficient alternative food sources for blow flies; specifically, tuna and wet cat food compared to beef liver (control). Survivorship and development were examined for each food source using the blow flies Phormia regina (Meigen) (Diptera: Calliphoridae) and Cochliomyia macellaria (Fabricus) (Diptera: Calliphoridae). These findings provide an overview of possible alternatives that could be used as a sustainable food source in crime laboratories when immediate action from a forensic entomologist cannot be obtained.

First Observations of Initial Blow Fly (Diptera: Calliphoridae) Activity on Lava Fields and in Subterranean Environments in Sicily in Cool Temperatures.

In criminal cases involving the recovery of human remains, as well as in cases of myiasis and pest management, the expertise of a forensic entomologist has been requested more and more frequently in Sicily. Recently, research on the insect species of forensic interest in Sicily has been investigated. The aim of this research was to raise awareness of this discipline and to build a Sicilian entomofauna checklist during cool temperatures. The predominant species observed in this study was Calliphora vicina (Robineau-Desvoidy) (Diptera, Calliphoridae), the first to colonize the carcasses; Lucilia spp. (Diptera, Calliphoridae) were also present. We wanted to explore the potential insect activity on decaying matter both on lava fields, exposed to sunlight, and in dark subterraneous environments. No activity was observed in our dark environment for 20 d; then, C. vicina was observed on one of the carcasses, laying eggs in complete darkness at a temperature of 6.4°C. Larval development under these conditions was delayed and the mortality rate was high. This preliminary trial allowed us to improve our experimental design and helped map new sites where we can expand our research to collect new data on insect distribution and their activity in caves.

Evaluation of bait traps as a means to predict initial blow fly (Diptera: Calliphoridae) communities associated with decomposing swine remains in New Jersey, USA.

Information about blow fly (Diptera: Calliphoridae) species distributions can be valuable for criminal investigations, with regards to determining movement of remains from one location to another and time of colonization estimates, making these data extremely useful. Past work has been conducted on initial species community structure across New Jersey, USA using traps baited with beef liver; however, if these same species frequent vertebrate carrion remains unclear. In order to evaluate these data, piglet carcasses were placed out once every two weeks for a year in New Brunswick, New Jersey, USA. The same methods were implemented as those used for traps baited with beef liver, with length of collections being based on ADD values. Seven calliphorid species, Calliphora vicina (Robineau-Desvoidy), Lucilia sericata (Meigen), Lucilia coeruleiviridis (Macquart), Phormia regina (Meigen), Pollenia pediculata Macquart, Pollenia rudis (F.) and Protophormia terraenovae (Robineau-Desvoidy) were collected from the carcasses. During this experiment L. sericata, L. coeruleiviridis and P. regina were the dominant adult blow flies captured, totaling 38.2%, 29.2% and 29.2% respectively of all adults caught. All three species colonized the carcasses as well, although not all were dominant colonizers. C. vicina was recorded ovipositing in December, while the piglet was submerged in approximately 5cm of snow. All species that totaled at least 1% of the total collection (adults captured and larvae reared) were the same across baited traps and carcasses. This study supports the use of beef liver baits for surveying forensically important blow flies and the application of such information to forensic investigations.

Death eaters respond to the dark mark of decomposition day and night: observations of initial insect activity on piglet carcasses.

Some insect taxa can be of critical importance for criminal investigations because they can be used to assist with a time since death determination. Blow flies (Diptera: Calliphoridae) often are the initial colonizers of a carcass, usually arriving within minutes to hours after carcass exposure during the day. Other insects, such as coleopterans and hymenopterans, can arrive to a carcass during early colonization and affect blow fly development. However, the extent of these interactions remains unclear. This study analyzed the initial 6 h after a piglet carcass was placed out in two locations (rural and urban) in diurnal and nocturnal conditions with continuous video recording and hourly observations. Four piglets were placed out every 2 weeks over the summer of 2014. Initial blow fly arrivals to the carcasses were only recorded during diurnal conditions, and a checklist of orders associated with each environment (time and location) was created. During diurnal conditions, initial blow fly arrival times in rural environments were significantly faster than those in urban, arriving as quickly as 23 s after exposure. These observations also included a novel interaction with Vespidae, which to the best of our knowledge has not been seen in the literature before. This experiment provides baseline data on early insect colonization in two environments in New Jersey, and lends insight into insect interactions that could affect initial colonization.