Aggregation in an heterospecific population of blowfly larvae: social behaviour is impacted by species-specific thermal requirements and settlement order.

Larvae of several blowfly species grow on carcasses and actively aggregate together. They face harsh developmental conditions resulting in a strong pressure to reduce development time: this is achieved either through thermoregulation or aggregation. We investigate how these two developmental strategies are modulated within heterospecific groups. In a first experiment, larvae of two species with different thermal requirements were deposited simultaneously on a thermal gradient. This resulted in the formation of two monospecific groups, each located at the species-specific thermal preferendum. However, when Calliphora vomitoria (Linnaeus) larvae were placed first, the later arriving Lucilia sericata (Meigen) larvae attracted the whole group to its own thermal preferendum. In the reverse experiment, half of the replicates resulted in single dense heterospecific groups observed at temperatures ranging from C. vomitoria to L. sericata preferendum. The other half of the replicates resulted in loose groups spread out on the thermal gradient. These results highlight the emergence of collective decisions ranging from thermal optimization to heterospecific aggregation at suboptimal temperatures. They demonstrate that species settlement order strongly affects self-organization processes and mixed-species group formation. We conclude that thermal optimization and heterospecific niche construction are two developmental strategies of carrion fly larvae. This article is part of the theme issue 'Mixed-species groups and aggregations: shaping ecological and behavioural patterns and processes'.

Synomones in necrophagous larvae of the blow flies Lucilia sericata and Calliphora vomitoria.

Chemical signals are widespread in insects, but those resulting in interspecific communication (i.e., synomones) remain understudied. Here, we analysed chemicals left on substrates by two species of blow fly larvae, Lucilia sericata (Meigen) and Calliphora vomitoria (Linneaus) (Diptera: Calliphoridae), which can aggregate together on carrion. Using solid-phase microextraction and dynamic headspace analysis, we identified six compounds common to both species: the decanoic, tetradecanoic, pentadecanoic, hexadecanoic and octadecanoic acids, and the 2-ethylhexyl salicylate. We then tested the behavioural effects of the decanoic and pentadecanoic acids using binary-choice experiments, along with the (Z)-9-tricosene, a pheromone found in many arthropods. The time spent by a larva and its average crawling speed were measured in two sides of an arena, where only one contained a compound at 0.25 or 25 µg/µl. No effect was observed when testing the decanoic acid. The pentadecanoic acid only reduced the speed of C. vomitoria larvae at 25 µg/µl. Finally, L. sericata larvae spent less time in the side containing the (Z)-9-tricosene at 0.25 µg/µl, whereas C. vomitoria spent more time and crawled faster in this side at 25 µg/µl. Although these results did not directly evidence synomones, they suggest that the (Z)-9-tricosene could regulate larval aggregations on carrion.

Experimental Evidence of Bone Lesions Due to Larder Beetle Dermestes maculatus (Coleoptera: Dermestidae).

Dermestid beetles (Coleoptera: Dermestidae) are necrophagous insects feeding on mummified carcasses. After six to seven molts, the larvae stop feeding and dig pupation chambers to hide and safely evolve into adults. Such pupation chambers have already been observed on archaeological mammals' bones, but the attribution and interpretation of these osteological lesions lack experimental evidence in a forensic context. To observe whether dermestid larvae dig pupation chambers in human bones, 20 or 40 Dermestes maculatus (De Geer, 1774) larvae were placed in a dermestarium with different types of bones varying in species (Bos taurus or human), age (adult or immature), and preservation method (fresh or dry). Our results show that dermestid larvae caused multiple lesions, including larval mandible traces on cortical bone, cortical perforations, drilling of pupation chambers, destruction of the trabecular network, and the perforation of cartilage. Bone destruction was mainly observed on aged dry bones, while fresh bones only exhibited soft tissue and superficial cartilage lesions. According to these results, pupation chambers could indicate the simultaneous presence of several corpses at different decomposition stages, or the addition of new corpses while others were already skeletonized. These conclusions are particularly important in the case of mass graves, where chronology is sometimes difficult to establish.

Insights into the palaeobiology of an early Homo infant: multidisciplinary investigation of the GAR IVE hemi-mandible, Melka Kunture, Ethiopia.

Childhood is an ontogenetic stage unique to the modern human life history pattern. It enables the still dependent infants to achieve an extended rapid brain growth, slow somatic maturation, while benefitting from provisioning, transitional feeding, and protection from other group members. This tipping point in the evolution of human ontogeny likely emerged from early Homo. The GAR IVE hemi-mandible (1.8 Ma, Melka Kunture, Ethiopia) represents one of the rarely preserved early Homo infants (~ 3 years at death), recovered in a richly documented Oldowan archaeological context. Yet, based on the sole external inspection of its teeth, GAR IVE was diagnosed with a rare genetic disease-amelogenesis imperfecta (AI)-altering enamel. Since it may have impacted the child's survival, this diagnosis deserves deeper examination. Here, we reassess and refute this diagnosis and all associated interpretations, using an unprecedented multidisciplinary approach combining an in-depth analysis of GAR IVE (synchrotron imaging) and associated fauna. Some of the traits previously considered as diagnostic of AI can be better explained by normal growth or taphonomy, which calls for caution when diagnosing pathologies on fossils. We compare GAR IVE's dental development to other fossil hominins, and discuss the implications for the emergence of childhood in early Homo.

Looking Back to Move Forward: How Review Articles Could Boost Forensic Entomology.

The Locard's exchange principle (1930) holds that the perpetrator of a crime leaves traces behind that can later be sampled and used as forensic evidence [...].

Balance between larval and pupal development time in carrion blowflies.

Several studies have highlighted the impact of environmental factors such as food type or larval density on the development of blowfly larvae. We investigated how changes in development speed (due to larval density and group composition) are divided among feeding and post-feeding stages. Even if these parameters impinge only on feeding larvae, they may ultimately also affect their subsequent development, and especially metamorphosis duration. Therefore, this study analysed the effect of larval density and group composition on the rhythm of necrophagous blowfly development. Based on laboratory studies, we highlighted that Calliphora vicina individuals with a fast development during their feeding phase developed slower in the later post-feeding phase (i.e., they had a compensatory effect). Lucilia sericata, a calliphorid species also frequently found on carrion at the same time as C. vicina, showed a different developmental strategy by not making its post-larval development speed dependent on the larval development speed. Finally, while a compensatory effect may exist, variations in the development rate more often accumulate through life-stages and resulted in a larger variability for later development instars. In this respect, the inclusion of detailed development duration covering all life stages, including post-feeding, is recommended in future studies, especially those dedicated to forensic entomology.

Effect of density and species preferences on collective choices: an experimental study on maggot aggregation behaviours.

Collective decisions have been extensively studied in arthropods, but they remain poorly understood in heterospecific groups. This study was designed to (1) assess the collective behaviours of blow fly larvae (Diptera: Calliphoridae) in groups varying in density and species composition, and (2) relate them to the costs and benefits of aggregating on fresh or decomposed food. First, experiments testing conspecific groups of Lucilia sericata and Calliphora vicina larvae, two species feeding at the same time on fresh carcasses, demonstrated decreases in growth and survival on rotten beef liver compared with fresh liver. However, mixing species together reduced this adverse impact of decomposition by increasing the mass of emerged adults. Second, larval groups were observed in binary choice tests between fresh and rotten liver (i.e. optimal and sub-optimal food sources). The results showed that larvae interacted with each other and that these interactions influenced their food preferences. We observed that (1) larvae were able to collectively choose the optimal food, (2) their choice accuracy increased with larval density and (3) the presence of another species induced a reversal in larval preference towards rotten food. These results highlight the ubiquity of collective decision properties in gregarious insects. They also reveal an unexpected effect of interspecific association, suggesting the colonization of new resources through a developmental niche construction.

Benefits of heterospecific aggregation on necromass: influence of temperature, group density, and composition on fitness-related traits.

Necrophagous blowflies (Diptera: Calliphoridae) such as Calliphora vicina, a cold-tolerant species, and Lucilia sericata, a warm-adapted species, are pioneer carrion-breeder. Although these two species have different temperature preferences, larvae aggregate actively and often feed simultaneously on carrion. The hypothesis to be tested was that L. sericata benefits from the association with C. vicina at lower temperatures (i.e., 15 °C) and that C. vicina derives greater benefits from this association at higher temperatures (i.e., 28 °C). Therefore, both species were raised at these two constant temperatures from first instars to adults under three different conditions: monospecific low-density, monospecific high-density, and heterospecific high-density. The time until larval migration, surface area of puparia, and survival rates were determined for each condition. Differences between these fitness-related traits were found between species, temperatures, group densities, and species compositions. At 28 °C, C. vicina larvae bred in heterospecific groups migrated significantly earlier and in higher numbers than that under same density conspecific conditions, with a lower mortality rate. At 15 °C, both species benefited from high-density heterospecific associations, expressed by faster development and larger puparia. In conclusion, necrophagous larvae benefited from heterospecific aggregations at suboptimal temperatures by adapting their migration time to that of the faster species. Since temperature changes throughout the day and over the year, the beneficiary of such a collective association also changes. The costs involved and deviations to the temperature-size rule highlight the complexity of the carrion ecosystem.

The maggot, the ethologist and the forensic entomologist: Sociality and thermoregulation in necrophagous larvae.

Necrophagous insects are mostly known through forensic entomology. Indeed, experimental data investigating the effect of temperature on larval development underlies post-mortem interval estimations. However, such developmental studies rarely considered the behavior of maggots. In contrast, previous results supposed that calliphoridae larvae use behavioral strategies to optimize their development on carcasses. To test this idea, we analyzed the trade-off between thermal regulation (individual thermal preferences) and social behavior (aggregation) in Lucilia sericata larvae. The first set of experiments analyzed the behavior of third instars in response to thermal changes in their environment. The results demonstrated a clear thermoregulation behavior, supporting the assumption that larvae continuously move to reach a suitable internal temperature. The second set of experiments focused on the trade-off between thermal optimization and aggregation. The results showed a constant search for congeners and an attractiveness of aggregates, sometimes to the detriment of thermal optimization. Together, these results demonstrate a balance between behavioral thermoregulation and social strategies, two significant mechanisms for developmental optimization in necrophagous larvae. In conclusion, these findings highlights unexpected (social) strategies to cope with ephemeral resource and high selection pressure. They also raise important questions for forensic entomology.

Mixed-species aggregations in arthropods.

This review offers the first synthesis of the research on mixed-species groupings of arthropods and highlights the behavioral and evolutionary questions raised by such behavior. Mixed-species groups are commonly found in mammals and birds. Such groups are also observed in a large range of arthropod taxa independent of their level of sociality. Several examples are presented to highlight the mechanisms underlying such groupings, particularly the evidence for phylogenetic proximity between members that promotes cross-species recognition. The advantages offered by such aggregates are described and discussed. These advantages can be attributed to the increase in group size and could be identical to those of nonmixed groupings, but competition-cooperation dynamics might also be involved, and such effects may differ between homo- and heterospecific groups. We discuss three extreme cases of interspecific recognition that are likely involved in mixed-species groups as vectors for cross-species aggregation: tolerance behavior between two social species, one-way mechanism in which one species is attractive to others and two-way mechanism of mutual attraction. As shown in this review, the study of mixed-species groups offers biologists an interesting way to explore the frontiers of cooperation-competition, including the process of sympatric speciation.

Temperature: the weak point of forensic entomology.

Measuring temperature is a key factor in forensic entomology. While noting factors to consider for a posteriori temperature estimation, many studies lack detailed methods or general rules allowing their integration into insect development-time calculations. This article proposes tools for determining the adequacy of weather station temperature datasets versus the local temperature experienced by carrion breeders. The idea is to start from a local scale (i.e., the cadaver) and gradually move to larger scales: at each step, the temperature can be increased, decreased or smoothed by environmental or biological factors. While a one-size-fits-all solution is not feasible for a complex and sensitive issue such as forensic meteorology, this checklist increases the reliability of minimum post-mortem interval (PMImin) estimation and the traceability of the proposed assumption.

Communication in necrophagous Diptera larvae: interspecific effect of cues left behind by maggots and implications in their aggregation.

Necrophagous Calliphoridae breed in vertebrate carrion. Their larvae aggregate and form large masses of individuals. These aggregated larvae can reach adulthood faster than scattered larvae, increasing their chances of survival. Furthermore, the gathering of larvae of different species suggests possible interspecific aggregation vectors. In this context, the effect of larval ground-left cues on larvae of Calliphora vomitoria and Lucilia sericata was studied. We used video tracking to follow larvae placed in binary choice tests. We observed (1) a preference of both species for a side marked by conspecific or heterospecific larvae compared to an unmarked side, (2) a preference of L. sericata larvae for a conspecific-marked side compared to a heterospecific-marked side but only at high concentration of cues and (3) a preference of both species for the side marked by the greater number of larvae. These results demonstrate that larvae leave a mark locally which is retentive, has an interspecific range, has an effect proportional to its intensity and whose strength varies depending on the emitting species. According to the self-organization theory, this mark could enhance larval gathering and promote interspecific aggregations. While not yet demonstrated, an interspecific Allee effect could explain the interspecific association of necrophagous calliphorid larvae.

To eat or get heat: Behavioral trade-offs between thermoregulation and feeding in gregarious necrophagous larvae.

The thermoregulation behavior of Lucilia sericata larvae (Diptera: Calliphoridae), a necrophagous species that feeds on vertebrate cadavers, was investigated. These larvae require high heat incomes to develop, and can elevate temperatures by forming large aggregates. We hypothesized that L. sericata larvae should continue to feed at temperatures up to 38 °C, which can be reached inside larval masses. Thermal regulation behavior such as movement between a hot food spot and colder areas was also postulated. The hypotheses were tested by tracking for 1 h the activity of single, starved third instar larvae in a Petri dish containing 1 food spot (FS) that was heated to a constant temperature of 25 °C, 34 °C or 38 °C with an ambient temperature of 25 °C. The influence of previous conspecific activity in the food on larval behavior was also tested. The crops of larvae were dissected to monitor food content in the digestive systems. Based on relative crop measurements, larvae fed at all food temperatures, but temperature strongly affected larval behavior and kinematics. The total time spent by larvae in FS and the duration of each stay decreased at high FS temperature. Previous activity of conspecifics in the food slightly increased the time spent by larvae in FS and also decreased the average distance to FS. Therefore, necrophagous L. sericata larvae likely thermoregulate during normal feeding activities by adjusting to local fluctuations in temperature, particularly inside maggot masses. By maintaining a steady internal body temperature, larvae likely reduce their development time.

[Thermoregulation behavior in necrophagous dipteran larvae]. / Les larves de diptères nécrophages en entomologie médico-légale : une histoire de température - Prix médecine/sciences.

The forensic entomology is the use of insects to date the death. The forensic expert assessment is based on the development of necrophagous insects which are growing on the cadaver, to calculate their age and then estimate the Post-Mortem Interval. This development depends on a number of parameters like temperature, species or behavior. The French Forensic Taphonomy unit, the only expert team on the subject in France, works on the biology, physiology and ethology of the necrophagous insects. Their works are focused on thermoregulation behavior and thermal preferendum of maggot masses, aggregation phenomenon and social interaction or on food intake. These works are particularly of interest to understand the pre-social parameters and evolution strategies. More importantly, their aim is to better understand the development of necrophagous insects and, in fine, to improve the forensic expert assessment.

Use of necrophagous insects as evidence of cadaver relocation: myth or reality?

The use of insects as indicators of post-mortem displacement is discussed in many texts, courses and TV shows, and several studies addressing this issue have been published. Although the concept is widely cited, it is poorly understood, and only a few forensic cases have successfully applied such a method. The use of necrophagous insects as evidence of cadaver relocation actually involves a wide range of biological aspects. Distribution, microhabitat, phenology, behavioral ecology, and molecular analysis are among the research areas associated with this topic. This article provides the first review of the current knowledge and addresses the potential and limitations of different methods to evaluate their applicability. This work reveals numerous weaknesses and erroneous beliefs as well as many possibilities and research opportunities.

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.

The biology and ecology of Necrodes littoralis, a species of forensic interest in Europe.

Necrodes littoralis (Linnaeus, 1758) (Coleoptera: Silphidae), also known as the "shore sexton beetle," is a common silphid beetle that visits and breeds on large vertebrate cadavers. This study describes, for the first time, the involvement of N. littoralis on human corpses based on a large dataset of 154 French forensic cases. Various parameters regarding corpse location, decomposition stages, and entomofauna were extracted from each file. Compared to all of the forensic entomology cases analyzed between 1990 and 2013 (1028), N. littoralis was observed, on average, in one case out of eight; most of these cases occurred during spring and summer (73.5%). More than 90% of the cases were located outdoors, especially in woodlands, bushes, and fields. The decomposition stage of the corpse varied among cases, with more than 50% in the advanced decomposition stage, 36% in the early decomposition stage, and less than 10% in the fresh, mummified, or skeletonized stages. Regarding other necrophagous species sampled with N. littoralis, Calliphorid flies were found in 94% of the cases and Fanniidae/Muscidae in 65% of the cases. Chrysomya albiceps, a heliophilic species mostly located in the Mediterranean area, was present in 34% of the cases (only 20% in the whole dataset). The most common coleopteran species were Necrobia spp. (Coleoptera: Cleridae) and Creophilus maxillosus (Coleoptera: Staphylinidae); these beetles were observed in 27% of the cases. The over-representation of these species is likely due to similar requirements regarding the climate and decomposition stage. As N. littoralis is frequently observed and tends to become more common, we conclude that the developmental data for this species would be a precious tool for forensic entomologists in Europe.

Experimental study of Lucilia sericata (Diptera Calliphoridae) larval development on rat cadavers: Effects of climate and chemical contamination.

Household products such as bleach, gasoline or hydrochloric acid have been used to mask the presence of a cadaver or to prevent the colonization of insects. These types of chemicals affect insect development and alter the forensic entomology analysis. This study was designed to test the effects of six household products (bleach, mosquito repellent, perfume, caustic soda, insecticide and unleaded gasoline) on blowfly (Lucilia sericata, Diptera: Calliphoridae) larval development. Furthermore, the effects of climate (rain or dry conditions) on larval development were analyzed. For each replication, 100 first instars were placed on a rat cadaver on which one household product was spilled. We observed a decrease in the survival rates of the larvae but no significant effect on their development times or the adult size. The same trends were observed under rainy conditions. However, the rain altered the effects of some tested household products, especially gasoline. These results demonstrate for the first time the successful development of necrophagous larvae on chemically contaminated cadavers, and provide evidence for the range of possible effects to expect.

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.

In vitro effects of household products on Calliphoridae larvae development: implication for forensic entomology.

Several parameters can delay the first arrival of flies on a corpse and the subsequent development of the larvae. This study focuses on the development of Lucilia sericata (Diptera: Calliphoridae) (Meigen, 1826) on household chemical-contaminated substrates. bleach, perfume, hydrochloric acid, caustic soda, insecticide, mosquito repellent, and gasoline in quantities consistent with an amount that could possibly be spilled on a corpse were mixed with beef liver to simulate contaminated fleshes. Larvae were bred at 25 °C on these media until emergence. Four developmental parameters were followed: survival rates, development times, sex ratios, and adult sizes. Hydrochloric acid, insecticide, and gasoline killed all larvae. In low quantities, caustic soda and mosquito repellent increased the development time and decreased the adult size. However, high quantities of these chemicals killed all larvae. Lastly, bleach and perfume did not affect the survival rate and barely impacted the development time or adult size. These results demonstrate common household products spilled on a corpse can strongly affect the development of Calliphoridae larvae. The effects of such products should be considered in forensic entomology cases.