Evaluating the impact of hot water killing larvae on gene expression using the transformer gene in Cochliomyia macellaria (Diptera: Calliphoridae).

In forensic entomology, determining the age of a larva from a body to estimate time since death is commonly performed through the measurement of a physical trait. Gene expression has been studied as an alternative age estimation approach, but the storage conditions required for these studies are different than those used in forensic entomological casework. Studies analyzing gene expression prioritize the preservation of RNA, which requires fresh tissue and ultra-cold storage. Casework, in contrast, utilizes hot water killing specimens that may not be analyzed for a long period after collection. In the current study, the impact of hot water killing on gene expression was assessed for larval samples of the forensically important blow fly, Cochliomyia macellaria. Successful amplification of the sex-determining gene, transformer, was tested across larvae ranging in size from 3.22 to 16.85 mm in length after storage times of 1-2 weeks, 4-5 weeks, and 8-9 weeks at 4°C in RNAlater. Larvae hot water killed were processed in tandem with larvae stored live to allow for a direct assessment of the impact of boiling on gene expression. As expected, the transformer gene was successfully amplified in all larvae stored live. For the hot water-killed larvae, the success rate was only slightly lower, with 3 out of 75 larvae not generating a sex-specific band pattern. The results show gene expression can be used for hot water-killed samples, though future work across different genes, species, and extending to quantitative gene expression methods is needed.

Case report on Cochliomyia hominivorax (Coquerel, 1858) and Co. macellaria (Fabricius, 1775) (Calliphoridae, Diptera) myiasis prior to death: Implications on postmortem interval estimation.

The most important contribution of forensic entomology is the estimation of the postmortem interval (PMI) or time since death. The forensic entomologist assumes that the biological clock of necrophagous insects that develop by feeding on cadaver tissues begins when the victim's biological clock is turned off. However, tissues can be invaded while the host is still alive (called myiasis), so the period of activity of necrophagous insects would not be indicative of PMI. The aim of this work was, through the presentation of a case report, to highlight the importance of expertise in the identification of necrophagous species associated with a corpse and their type of relationship in order to avoid an erroneous estimation of PMI. The corpse of a woman, missing for 14 days, was found outdoors in a small river 15 cm deep. Her corpse had numerous lesions infested with dipteran larvae that were collected during autopsy. Entomological evidence included second and third instar larvae of Cochliomyia hominivorax and Co. macellaria. The obligate parasitic nature of Co. hominivorax as a primary producer of myiasis and secondary of Co. macellaria allowed us to establish the time when the victim was still alive and from there to estimate the PMI.

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.

In vitro evaluation of the association of medicinal larvae (Insecta, Diptera, Calliphoridae) and topical agents conventionally used for the treatment of wounds.

Wound healing is a complex process involving multiple biochemical and cellular events and represents a neglected public health issue. As a consequence, millions of people worldwide suffer from chronic wounds. The search for new treatment alternatives is therefore an important issue. In the context of wound healing, Maggot Debridement Therapy (MDT) is an inexpensive treatment with few contraindications and very promising results. This study aimed to evaluate the in vitro feasibility and implications of larvae (= MDT) use when combined with topical agents as a strategy to identify additive or synergistic combinations. The weight and survival rate of Cochliomyia macellaria (Fabricius) (Insecta, Diptera, Calliphoridae) larvae reared in an in vitro wound with either honey, hydrogel, 10% papain gel, essential fatty acids (EFA), collagenase, or silver sulfadiazine were evaluated after 24, 48 and 72 h. Hydrogel (for weight: 24 and 72 h; for survival: 24 h) and 10% papain gel (for weight: 48 h; for survival: 48 and 72 h) demonstrated the least interference in larval weight gain and survival rate, when compared to the control group. The results obtained in this study showed that the combined use of larvae and hydrogel or 10% papain gel may be promising to maximize the tissue repair. Honey, EFA and collagenase could be used to prepare the bed of the dry wounds to receive the MDT.

Quantifying pteridines in the heads of blow flies (Diptera: Calliphoridae): Application for forensic entomology.

In forensic cases involving entomological evidence, establishing the postcolonization interval (post-CI) is a critical component of the investigation. Traditional methods of estimating the post-CI rely on estimating the age of immature blow flies (Diptera: Calliphoridae) collected from remains. However, in cases of delayed discovery (e.g., when remains are located indoors), these insects may have completed their development and be present in the environment as adults. Adult fly collections are often ignored in cases of advanced decomposition because of a presumed little relevance to the investigation; herein we present information on how these insects can be of value. In this study we applied an age-grading technique to estimate the age of adults of Chrysomya megacephala (Fabricius), Cochliomyia macellaria (Fabricius), and Phormia regina (Meigen), based on the temperature-dependent accumulation of pteridines in the compound eyes, when reared at temperatures ranging from 5 to 35°C. Age could be estimated for all species*sex*rearing temperature combinations (mean r2±SE: 0.90±0.01) for all but P. regina reared at 5.4°C. These models can be used to increase the precision of post-CI estimates for remains found indoors, and the high r2 values of 22 of the 24 regression equations indicates that this is a valid method for estimating the age of adult blow flies at temperatures ≥15°C.

Factors Affecting Species Identifications of Blow Fly Pupae Based upon Chemical Profiles and Multivariate Statistics.

Alternative methods for the identification of species of blow fly pupae have been developed over the years that consist of the analyses of chemical profiles. However, the effect of biotic and abiotic factors that could influence the predictive manner for the tests have not been evaluated. The lipids of blowfly pupae (Cochliomyia macellaria, Lucilia cuprina, Lucilia sericata, and Phormia regina) were extracted in pentane, derivatized, and analyzed by total-vaporization solid phase microextraction gas chromatography-mass spectrometry (TV-SPME GC-MS). Peak areas for 26 compounds were analyzed. Here we evaluated one biotic factor (colonization) on four species of blow flies to determine how well a model produced from lipid profiles of colonized flies predicted the species of flies of offspring of wild-caught flies and found very good species identification following 10 generations of inbreeding. When we evaluated four abiotic factors in our fly rearing protocols (temperature, humidity, pupation substrate, and diet), we found that the ability to assign the chemical profile to the correct species was greatly reduced.

Post-Colonization Interval Estimates Using Multi-Species Calliphoridae Larval Masses and Spatially Distinct Temperature Data Sets: A Case Study.

Common forensic entomology practice has been to collect the largest Diptera larvae from a scene and use published developmental data, with temperature data from the nearest weather station, to estimate larval development time and post-colonization intervals (PCIs). To evaluate the accuracy of PCI estimates among Calliphoridae species and spatially distinct temperature sources, larval communities and ambient air temperature were collected at replicate swine carcasses (N = 6) throughout decomposition. Expected accumulated degree hours (ADH) associated with Cochliomyia macellaria and Phormia regina third instars (presence and length) were calculated using published developmental data sets. Actual ADH ranges were calculated using temperatures recorded from multiple sources at varying distances (0.90 m-7.61 km) from the study carcasses: individual temperature loggers at each carcass, a local weather station, and a regional weather station. Third instars greatly varied in length and abundance. The expected ADH range for each species successfully encompassed the average actual ADH for each temperature source, but overall under-represented the range. For both calliphorid species, weather station data were associated with more accurate PCI estimates than temperature loggers associated with each carcass. These results provide an important step towards improving entomological evidence collection and analysis techniques, and developing forensic error rates.

Identification and characterization of microRNAs in the screwworm flies Cochliomyia hominivorax and Cochliomyia macellaria (Diptera: Calliphoridae).

MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression through post-transcriptional regulation. Here, we report the identification and characterization of miRNAs in two closely related screwworm flies with different feeding habits: Cochliomyia hominivorax and Cochliomyia macellaria. The New World screwworm, C. hominivorax, is an obligatory parasite of warm-blooded vertebrates, whereas the secondary screwworm, C. macellaria, is a free-living organism that feeds on decaying organic matter. Here, the small RNA transcriptomes of adults and third-instar larvae of both species were sequenced. A total of 110 evolutionarily conserved miRNAs were identified, and 10 putative precursor miRNAs (pre-miRNAs) were predicted. The relative expression of six selected miRNAs was further investigated, including miRNAs that are related to reproduction and neural processes in other insects. Mature miRNAs were also characterized across an evolutionary time scale, suggesting that the majority of them have been conserved since the emergence of the Arthropoda [540 million years ago (Ma)], Hexapoda (488 Ma) and Brachycera (195 Ma) lineages. This study is the first report of miRNAs for screwworm flies. We also performed a comparative analysis with the hereby predicted miRNAs from the sheep blowfly, Lucilia cuprina. The results presented may advance our understanding of parasitic habits within Calliphoridae and assist further functional studies in blowflies.

Molecular phylogeny of the forensically important genus Cochliomyia (Diptera: Calliphoridae).

Cochliomyia Townsend includes several abundant and one of the most broadly distributed, blow flies in the Americas, and is of significant economic and forensic importance. For decades, Cochliomyia hominivorax (Coquerel) and Cochliomyia macellaria (Fabricius) have received attention as livestock parasites and primary indicator species in forensic entomology. However, Cochliomyia minima Shannon and Cochliomyia aldrichi Del Ponte have only been subject to basic taxonomy and faunistic studies. Here we present the first complete phylogeny of Cochliomyia including numerous specimens per species, collected from 13 localities in the Caribbean. Four genes, the mitochondrial COI and the nuclear EF-1α, 28S rRNA, and ITS2, were analyzed. While we found some differences among gene trees, a concatenated gene matrix recovered a robustly supported monophyletic Cochliomyia with Compsomyiops Townsend as its sister group and recovered the monophyly of Cochliomyia hominivorax, Cochliomyia macellaria and Cochliomyia minima. Our results support a close relationship between Cochliomyia minima and Cochliomyia aldrichi. However, we found Cochliomyia aldrichi containing Cochliomyia minima, indicating recent speciation, or issues with the taxonomy of the group. We provide basic information on habitat preference, distribution and feeding habits of Cochliomyia minima and Cochliomyia aldrichi that will be useful for future forensic studies in the Caribbean.

Larval Distribution and Behavior of Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) Relative to Other Species on Florida Black Bear (Carnivora: Ursidae) Decomposing Carcasses.

Larval interactions of dipteran species, blow flies in particular, were observed and documented daily over time and location on five black bear carcasses in Gainesville, FL, USA, from June 2002 - September 2004. Cochliomyia macellaria (Fabricius) or Chrysomya megacephala (Fabricius) larvae were collected first, after which Chrysomya rufifacies (Macquart) oviposited on the carcasses in multiple locations (i.e., neck, anus, and exposed flesh) not inhabited already by the other blow fly larvae. Within the first week of decomposition, C. rufifacies larvae grew to ≥12 mm, filling the carcasses with thousands of larvae and replacing the other calliphorid larvae either through successful food source competition or by predation. As a result, C. macellaria and C. megacephala were not collected past their third instar feeding stage. The blow fly species, C. megacephala, C. macellaria, Lucilia caeruleiviridis (Macquart), Phormia regina (Meigen), Lucilia sericata (Meigen), and C. rufifacies, completed two developmental cycles in the 88.5-kg carcass. This phenomenon might serve to complicate or prevent the calculation of an accurate postmortem interval.