Distinguishing Four Calliphoridae Species (Diptera) from Jamaica Using the Cephalopharyngeal Skeleton: Application to Forensic Investigations.

Flies of the family Calliphoridae, commonly called blow flies, are important in the decomposition process. Knowledge on their succession pattern on corpses, species identification and the duration of their life cycle stages can be useful in forensic investigations especially when estimating the post-mortem interval. We performed linear-based morphometrics on the cephalopharyngeal skeleton of four blow fly species found in Jamaica to distinguish species and determine larval development stage. We collected eggs from pigs' heads used as bait in the field and conducted rearing exercises in the laboratory. We used the internal skeletonized structure, the cephalopharyngeal skeleton, to develop a practical and efficient method for species identification. For the first instar, we found species can be differentiated using all the measurements analysed in the study. We found that the mouth hook length may be useful in distinguishing larvae in the second instar. For the larvae in the third instar, the whole length of the skeleton, from mouth hook to length of the dorsal cornue, may be useful for separating species. We provide information on the cephalopharyngeal skeleton of Lucilia lucigerens (James), a blow fly species endemic to Jamaica, for the first time. Our work provides relevant information that could be utilized for species identification and life stage determination if fly evidence is to be incorporated in forensic investigations in Jamaica.

Size does matter: intraspecific geometric morphometric analysis of wings of the blowfly Chrysomya albiceps (Diptera: Calliphoridae).

Blowflies have forensic, sanitary and veterinary importance, as well as being pollinators, parasitoids and ecological bioindicators. There is still little work with real data and from experiments assessing the relationship between blowflies' morphologic features and environmental and demographic factors. The present work tests whether the variation, in the shape and size, of Chrysomya albiceps (Wiedemann, 1819) wings is influenced by the following factors: 1) time; 2) temperature; 3) sex and; 4) different types of carcasses (pig, dog/cat and whale). Male and female wings from four different sites collected in six different years were used to obtain wing size and shape of C. albiceps. Analyses between wing shape and the variables tested had low explanatory power, even though they had statistical support. However, it was possible to identify differences in wing shape between males and females, with good returns in sex identification. The comparison between wing size and the variables tested showed that wing size has a negative relationship with temperature, significant differences between sexes, slight variation over time and no influence by carcass types. Furthermore, wing size influenced wing shape. Understanding population-specific characteristics of C. albiceps provide important insights about how the species reacts under specific conditions.

An X-ray micro-computer tomography study of the Malpighian tubules of the Blue Bottle Blow Fly (Calliphora vomitoria) Diptera: Calliphoridae.

Malpighian tubules are the insect equivalent of mammalian kidneys and normally drain into the gut at the junction between the mid and hind gut. The Malpighian tubules of the fruit fly Drosophila melanogaster are increasingly being used as a model for studying human renal tract development, histology, nephrolithiasis and urolithiasis. In the present study we report when using X-ray micro-computer tomography techniques, the larval, intrapuparial and adult stages of the larger Calliphora vomitoria can contain large amounts of calcium-rich concretions which are tightly packed in the lumen of both anterior Malpighian tubules. We show that it is feasible to utilise these calcium-rich concretions as a form of marking agent to delineate the various developmental stages of the Malpighian tubules including the crucial phase when the Malpighian tubules reconnect with the hind gut. In the majority of cases during the intrapuparial period the ureters of the Malpighian tubules did not start to re-canalise and thus reconnect with the developing hind gut until the 7th day of the 10-11 day. Just prior to ecdysis, virtually all the radio-opaque concretions in the Malpighian tubules had emptied into the hind gut and had then been completely excreted by the time the imago emerged from its puparium. In contrast, we show that in flies developing from larvae previously stained by ingesting Rhodamine B, a known substrate for both the Multi Xenobiotic Resistance and Multi Drug Resistant membrane transport systems, the efficiency with which these calcium-rich concretions are excreted by the imago as it emerges from its intrapuparial period can be significantly impaired. Therefore, it might be useful to include C. vomitoria as a model when studying renal tract development and urolithiasis using X-ray micro-computer tomography.

Random forests for predicting species identity of forensically important blow flies (Diptera: Calliphoridae) and flesh flies (Diptera: Sarcophagidae) using geometric morphometric data: Proof of concept.

Wing shape variation has been shown to be useful for delineating forensically important fly species in two Diptera families: Calliphoridae and Sarcophagidae. Compared to DNA-based identification, the cost of geometric morphometric data acquisition and analysis is relatively much lower because the tools required are basic, and stable softwares are available. However, to date, an explicit demonstration of using wing geometric morphometric data for species identity prediction in these two families remains lacking. Here, geometric morphometric data from 19 homologous landmarks on the left wing of males from seven species of Calliphoridae (n = 55), and eight species of Sarcophagidae (n = 40) were obtained and processed using Generalized Procrustes Analysis. Allometric effect was removed by regressing centroid size (in log10 ) against the Procrustes coordinates. Subsequently, principal component analysis of the allometry-adjusted Procrustes variables was done, with the first 15 principal components used to train a random forests model for species prediction. Using a real test sample consisting of 33 male fly specimens collected around a human corpse at a crime scene, the estimated percentage of concordance between species identities predicted using the random forests model and those inferred using DNA-based identification was about 80.6% (approximate 95% confidence interval = [68.9%, 92.2%]). In contrast, baseline concordance using naive majority class prediction was 36.4%. The results provide proof of concept that geometric morphometric data has good potential to complement morphological and DNA-based identification of blow flies and flesh flies in forensic work.

Wing morphometrics for identification of forensically important blowflies (Diptera: Calliphoridae) in Iberian Peninsula.

Calliphoridae is a family of Diptera of great forensic interest because some of its species belong to the sarcosaprophagous diptera community, as their larvae are necrophagous or necrophilous. In the Iberian Peninsula there are six species that stand out, as they are the first insects to arrive at carrion: Calliphora vicina, Calliphora vomitoria, Lucilia sericata, Lucilia caesar, Chrysomya albiceps and Chrysomya megacephala. To differentiate among these blowflies, we compared the value of using traditional and geometric morphometrics by performing a morphometric analysis of the wings (shape and size). A total of 600 individuals were considered, segregated by species and sex equally. Seventeen landmarks per wing were recognized, and then centroid size and shape analyses were performed using geometric morphometrics, and size using traditional morphometrics, to identify species and sex. The results showed differences in shape among the species and it was found that landmarks 2 to 11 were the most variable, while the landmarks located at the base of the wings were relatively stable. However, according to both traditional and geometric morphometrics, no significant differences were found among species in wing size (Calliphora vicina, Chrysomya megacephala and Lucilia caesar were indistinguishable), but females were larger than males (except for the two Chrysomya species). Our results indicate that the use of geometric morphometrics to analyze wing configuration (shape) is an easy-to-use tool that helps to distinguish among common blowfly species of forensic importance.

Contrasting Responses of Wing Morphology of Three Blowfly (Diptera: Calliphoridae) Species to Competition.

Competition influences the expression of morphological, physiological, and behavioral traits and also regulates ecological and evolutionary dynamics. This study aims to identify and characterize changes in wing morphology in response to intra- and interspecific competition in three necrophagous blowfly species. Using geometric morphometry, we analyzed 3,238 wings from Lucilia sericata (Meigen, 1826), Calliphora vicina Robineau-Desvoidy, 1830, and C. vomitoria (Linnaeus, 1758) raised under cloistered and pairwise conditions. The three species reacted similarly to intraspecific competition-reducing wing size with increased competition-but displayed contrasting patterns of response to interspecific competition. Lucilia sericata displayed a directional change in wing shape in response to an interspecific competitor, while C. vicina increased the scattering of individuals across the morphospace, and C. vomitoria displayed no significant change in response to the same stimulus. Our results show that the same stimulus yields distinctive responses; thus, different competition-related strategies are expected to occur in the three species.