Damien Charabidze

Repellent effect of some household products on fly attraction to cadavers

The most common task of a forensic entomologist is to determine an accurate minimum post-mortem interval (PMI) using necrophagous fly larvae found on carrion. More often, blowflies (Diptera: Calliphoridae) are the first insects to detect the cadaver and, if the circumstances are favourable, to leave eggs on the body. However, several studies reveal that products such as gas or paint found on the cadaver induce a delay in the colonisation of the body, leading to an under-estimate of the PMI. Six common household products (gas, mosquito citronella repellent, perfume, bleach, hydrochloric acid and soda) were added to dead rats (Rattus norvegicus) in a field (Lille Forensic Institute, France). The presence of necrophagous flies was checked at regular intervals during 1 month. This experiment was repeated at the same period for four consecutive years. Results clearly showed the repellent effect of three of the six tested substances: gas (petroleum spirit), perfume and mosquito citronella repellent, which resulted in a mean delay of several days in the appearance of the first Dipteran species. Experiments were then carried out in controlled conditions in order to confirm previous observations. An olfactometer was specially designed to observe the behaviour of female Calliphora vicina (Diptera: Calliphoridae) in response to mice (Mus musculus) cadaver odour stimuli combined with household products. Dead mouse odour was a strong attractive stimulus for most of the tested individuals. Furthermore, it was noticed that the presence of mosquito citronella repellent, perfume, hydrochloric acid and paradichlorobenzene produced a significant repellent effect on female flies. All these results together confirm the repellent effect of some household products on flies and the necessity for forensic entomologists to consider this hypothesis when estimating the PMI.

Effect of body length and temperature on the crawling speed of Protophormia terraenovae larvae (Robineau-Desvoidy) (Diptera Calliphoridae)

This work investigates variation in the crawling speed of Protophormia terraenovae larvae (Robineau-Desvoidy) (Diptera Calliphoridae) as a function of body length and ambient temperature. A video-tracking system was used to follow the movement of larvae in an experimental arena, and to estimate their average crawling speed. Recordings were carried out at various temperatures using larvae of different developmental stages. As temperature increased, the larvae crawled at a faster speed. Furthermore, speed increased as a function of body length. We present a general allometric relationship to express the average crawling speed of larvae as a combined function of temperature and body length, expressed in the following equation: speed (cm/min)=5.45 x log[length (mm)]+0.66 x temperature (degrees C)-12.8. These results are particularly useful in forensic entomology for estimating the time that elapsed following the larvas departure from the corpse. This information can provide more accuracy for post-mortem interval estimations and a better comprehension of larvae behaviour and displacement.

Discontinuous foraging behavior of necrophagous Lucilia sericata (Meigen 1826) (Diptera Calliphoridae) larvae.

Larvae of the necrophagous Blowfly Lucilia sericata (Diptera Calliphoridae) live on vertebrate cadavers. Although continuously feeding was previously assumed for this species, we hypothesized that larvae do not feed constantly. According to this hypothesis, their crop should not always be full, which should be reflected in crop surfaces. We dissected and measured the crops of larvae of the same age and bred in the same conditions. Crop surfaces of 117 larvae just removed from the food ranged from 0 to 16.6 mm(2) (mean=5.325±2.84 mm(2)). The distribution of these crop surfaces indicates a continuous variation of satiation/feeding activity in the population. Starving experiments showed a quite long digestive process. After 90 min of starving, the decrease in crop surfaces became obvious, but 150 min were necessary to observe more than a half of the population with an empty crop (less than 2 mm(2)). No more differences were observed after 150, 180 and 240 min of starving. We finally used starved larvae to observe the kinetic of food absorption and the duration of the food-intake phase. Our results indicates that larvae can ingest faster than they digest. After 5 min spent in the food, 70% of the larvae had a crop surface larger or equal to 8 mm(2). We observed for the first time an over-feeding of the larvae, with high crop surfaces overrepresented compared to larvae never starved (control). Together, these results indicate that larvae do not feed continuously, and regulate their foraging behavior. We propose that the foraging behavior of the larvae creates a permanent movement inside the larval masses. This turnover/scramble competition may be linked to the larval-mass effect, i.e. the local temperature increase observed in large necrophagous larvae aggregates.

The potential of forensic analysis on human bones found in riverine environment

Human remains found in aquatic contexts are frequently recovered incomplete and badly decomposed, and therefore present a challenge for medico-legal institutes as their possibilities of analysis for identification and investigation of cause and manner of death are limited. This article aims to demonstrate the potential of forensic examination and analyses (DNA, toxicology, diatoms and entomology) on a set of bones recovered from a river in Strasbourg and the possibility to trigger identification of the victim and circumstances of death despite the state of decomposition and incompleteness of remains.

Interspecific shared collective decision-making in two forensically important species

To date, the study of collective behaviour has mainly focused on intraspecific situations: the collective decision-making of mixed-species groups involving interspecific aggregation–segregation has received little attention. Here, we show that, in both conspecific and heterospecific groups, the larvae of two species (Lucilia sericata and Calliphora vomitoria, calliphorid carrion-feeding flies) were able to make a collective choice. In all groups, the choice was made within a few minutes and persisted throughout the period of the experiment. The monitoring of a focal individual within a group showed that these aggregations were governed by attractive and retentive effects of the group. Furthermore, the similarity observed between the conspecific and heterospecific groups suggested the existence of shared aggregation signals. The group size was found to have a stronger influence than the species of necrophagous larvae. These results should be viewed in relation to the well-known correlation between group size and heat generation. This study provides the first experimental examination of the dynamics of collective decision-making in mixed-species groups of invertebrates, contributing to our understanding of the cooperation–competition phenomenon in animal social groups.

Thermoregulation in gregarious dipteran larvae: evidence of species‐specific temperature selection

Due to the ephemeral nature of carcasses they grow on, necrophagous blowfly larvae should minimize the time spent on the cadaver. This could be achieved by moving to high‐temperature areas. On that basis, we theorized that larvae placed in a heterogeneous thermal environment would move to the higher temperature that speed up their development. This study was designed to (1) test the ability of necrophagous larvae to orientate in a heterogeneous thermal environment, and (2) compare the temperatures selected by the larvae of three common blowfly species: Lucilia sericata (Meigen), Calliphora vomitoria (L.) and Calliphora vicina (Robineau‐Desvoidy). For this purpose, we designed a setup we named Thermograde. It consists of a food‐supplied linear thermal gradient that allows larvae to move, feed, and grow in close‐to‐real conditions, and to choose to stay at a given temperature. For each species and replication, 80 young third instars were placed on the thermal gradient. The location of larvae was observed after 19 h, with fifteen replications per species. The larvae of each species formed aggregations that were always located at the same temperatures, which were highly species‐specific: 33.3 ± 1.52 °C for L. sericata, 29.6 ± 1.63 °C for C. vomitoria, and 22.4 ± 1.55 °C for C. vicina. According to the literature, these value allows a fast development of the larvae, but not to reach the maximum development rate. As control experiments clearly demonstrate that larval distribution was not due to differences in food quality, we hypothesized that the local temperature selection by larvae may result from a trade‐off between development quality and duration. Indeed, temperature controls not only the development rate of the larvae, but also the quality of their growth and survival rate. Finally, results raise questions regarding the way larvae moved on the gradient and located their preferential temperature.

An experimental investigation into the colonization of concealed cadavers by necrophagous blowflies.

We used seven baited boxes with different combinations of access holes and odor diffusion surfaces to study the arrival of necrophagous flies. During laboratory experiments, 30 gravid Lucilia sericata females were kept in a chamber with one of the boxes. The box with the largest odor diffusion surface (99 cm2) combined with the lowest accessibility (one 1 cm2 entrance hole) was entered least (5 ± 3.7 flies per run). In contrast, the most frequently entered box (one 9 cm2 entrance hole with no additional odor diffusion surface) caught a mean of 24.6 ± 3.4 flies per run. These results indicate that 1) L. sericata entered nearly inaccessible places and 2) both odor diffusion and accessibility impacted the number of flies caught. During field experiments, the seven boxes were placed together outdoors. The box with the most entrances (ten 9-cm2 holes) caught the most flies (55.6–99.4% of the total). Only a few flies entered the other boxes. Access to the less accessible boxes (poor odor diffusion and small entrances) was also delayed. The major conclusions of the field experiments are that 1) boxes with low accessibility took longer to be accessed; 2) larger odor diffusion surfaces were more attractive to flies; and 3) flies accessed boxes more readily through larger holes than through an equivalent surface area made up of smaller holes. With these conclusions in mind, attempts to quantify the preappearance interval or to interpret the number of flies observed in indoor forensic entomology cases should be approached with caution.

Do necrophagous blowflies (Diptera: Calliphoridae) lay their eggs in wounds?: Experimental data and implications for forensic entomology

This study was designed to examine the common belief that necrophagous blowflies lay their eggs in wounds. The egg-laying behaviour of Lucilia sericata was observed under controlled conditions on wet, artificially wounded or short-haired areas of rat cadavers. Flies laid significantly more eggs on the wet area and the area with short hair than on the dry area or area with long hair. No eggs were observed inside the wounds in any of the replicates. The effect of egg immersion (body fluids often exudes in wounds) on the survival rate of larvae was also investigated. In low water condition, an average of 72.7±7.9% of the larvae survived and they reached a mean length of 7.5±0.6mm. In contrast, submerging eggs under a high volume of water strongly affected their survival rate (25±3.7%) and development. Similar results were observed using unfrozen pig blood instead of water. These data question the information found in the literature regarding the preferential egg-laying behaviour of Calliphorids flies in wounds.

A first insight into the scanning behaviour of the presocial blow fly larvae

Aggregation of necrophagous larvae has several benefits: the sharing of salivary enzymes (exodigestion), temperature regulation, protection from predators and parasites, etc., and is well developed in blow flies (Diptera: Calliphoridae). The present study focuses on the aggregation mechanism used by the necrophagous larvae of Lucilia sericata Meigen, the common green bottle fly. The ability of single larva to detect and follow a signal (trail) created by conspecifics is investigated initially. A circular ring is drawn in a Petri dish where 20 starved larvae have crawled for a period of 30 min. A naïve (test) larva is then placed in the dish and video‐tracked. Naïve larvae are able to detect the boundaries of the larvae‐crawled area and stay preferentially within this conspecific‐marked zone. In a second step, the orientation of larvae by scanning, a dedicated, ground‐signal detection behaviour of dipteran larvae, is analyzed. Four experimental conditions are tested: control, presence of food, conspecifics, and food + conspecifics. When conspecifics have been previously present in a given area, the scanning behaviour by naïve larvae in this area decreases (both in number and frequency of scans). Accordingly, scanning by necrophagous larvae of L. sericata should be considered not only as locomotion behaviour, but also as a potential way to detect signals from conspecifics for the purpose of aggregation. The chemical composition of the attractant(s), the behavioural effects (attraction or retention) and the implication of larval signalling in the aggregation process are new fields to explore.

Use of larder beetles (Coleoptera: Dermestidae) to deflesh human jaws

We describe new experimental data for the defleshing of human bones using larder beetles (Dermestes haemorrhoidalis) (Küster, 1852). Although the ability of larder beetles to feed on vertebrate remains has been, and still is, used by taxidermists to deflesh skulls and bones, this method has never been documented from a quantitative perspective and has over time become ignored in most forensic anthropology or odontology laboratories. To promote the rational and efficient use of this method, we performed experiments to estimate the quantity of food consumed by larvae. From the 2nd instar to nymphosis, each larva consumed a mean of 0.13±0.03 g of dry beef muscle. We then used 100±50 D. haemorrhoidalis adults and 100±50 larvae to deflesh human maxillae and mandibles sampled within a forensic context (victim identification). Each sample was weighed and photographed before, during and after the experiment. According to our experiments, 20-25 days were sufficient to completely deflesh all of the samples. We concluded that a small number of larder beetles can be used to efficiently deflesh human jaws. According to this result, the use of larder beetles appears to be an inexpensive, simple and efficient way to clean mandibles and maxillae. Furthermore, this method is DNA-safe (compared to usual maceration techniques) and thus allows the samples to be used for subsequent DNA and drug analyses.