Introduction

Carrion beetles (Coleoptera: Silphidae) are part of a large group (ca. 210 species worldwide) of scavengers that aid in the break down and recycling of organisms back into the ecosystem. The genus Nicrophorus Fabricius (Nicrophorinae), also called burying beetles, is well known for biparental care of its offspring, which is believed to have developed evolutionarily from an intense competition within scavengers for unpredictable and discrete food resources. Burying beetles specialize in small vertebrate carcasses. Males attract females using pheromone released on a carcass suitable for reproduction. When a female arrives, the beetles move the carcass to a suitable burying spot. During burial, the beetles excavate soil out from underneath the carcass, strip it of hair or feathers and mould it into a brood ball coated with oral and anal secretions to preserve and control the decay of the carcass. The pair mates repeatedly during the burial. A female will lay around 10-30 eggs depending on the carcass size. The parents feed hatched larvae by regurgitated partially digested carcass. Formation of a suitable brood ball requires fresh carcasses, which are not yet occupied by other scavenger species. This depends critically on an ability of the burying beetle to find a carcass as soon as possible after an animal death. Nicrophorinae, using sensitive chemoreceptors on their antennae, can recognize a dead animal within an hour and can locate the carcass from a distance of up to several kilometres away.

As soon as an animal dies decomposition begins. The breaking tissues emit volatiles attracting the burying beetle to the source. It is not known, which compounds mediate the carcass' attractiveness for different scavenger species though the volatiles of many different decomposing carcasses have been studied. The goals of this project were to determine the composition of volatiles emitted by fresh rodent carcasses (laboratory mice, Mus musculus) and to identify the infochemicals mediating the carcass attractiveness for Nicrophorus vespillo (Linnaeus) and Nicrophorus vespilloides Herbst, the two most abundant Nicrophorinae species in Central Europe. The target of this study was to increase our knowledge of the infochemicals and behaviour of these ecologically important beetles and acquire a theoretical basis for development of a suitable burying beetles monitoring technique.

Material and Methods

Beetles
The individuals used for experiments were wild-caught (Prague, Suchdol area, N 50� 08' E 014� 21') during April - September, 2006 and 2007. Pit-fall traps were used.

Chemicals Commercially available standards of dimethyl sulfide (DMS), dimethyl disulfide (DMDS), methyl thiolacetate (MeSAc), dimethyl trisulfide (DMTS) and dimethyl tetrasulfide (DMQS) were used.

Electroantennography (EAG)
In EAG experiments, the dose-response relationships of MeSH, MeSAc, DMS, DMDS and DMTS were determined in both N. vespillo and N. vespilloides. In these experiments, isolated male antennae connected between two Ag/AgCl glass microelectrodes filled with Ringer solution were used. The reference electrode was connected with the antennal base, with the recording one positioned to make contact with the sensory epithelium on the last antennomere surface.

Solid-phase micro-extraction (SPME) sample collection
SPME experiments were performed at ambient temperature. A mouse carcass (laboratory mice, Mus musculus) was placed on a glass rectangular plate (10 cm � 10 cm) and covered with an oval glass cover lid (10 cm dia, 7 cm height). The centre of the glass lid protrudes up to form a standard glass screw joint (8 mm dia). The joint was closed using a corresponding plastic cap with a PTFE septum. The SPME holder with CAR/PDMS fibre (Supelco, previously desorbed for 5 min in GC injection port heated to 200 �C) was inserted through the PTFE septum into the atmosphere surrounding the mouse carcass and the fibre was exposed for 15 min and immediately GC�GC-TOFMS analysed. 10 mouse carcasses (Mus musculus) were used. During the first 24 h, SPME samplings were performed at every hour. Later on, samplings were repeated at longer intervals.

Gas chromatography with electroantennographic detection (SPME-GC-EAD)
The SPME-GC-EAD was used to determine whether and which compounds emanating from dead mice (1-3 days old) are perceived by burying beetles N. vespilloides or N. vespillo. In these experiments, isolated male antennae were used as biological detectors (EAD) along with FID.

Two dimensional gas chromatography with time of flight mass spectrometric detection (GC�GC-TOFMS)
The analyses of the carcass volatiles were performed using a LECO Pegasus 4D instrument (LECO Corp., St. Joseph, MI, USA). The compounds were identified based on comparison of their MS spectra and Kov�ts indices together with commercially available standards.

Y-Olfactometer
The attractivity of carcasses and synthetic standards of DMS, DMDS and DMTS, were tested in a laboratory olfactometer placed in dimly illuminated fumed space. The olfactometer consisted of two glass Y-parts (4 cm dia, 30 cm length) connected together by their arms. The 7 cm long arms were replaceable to aid control of contaminations. The olfactometer was connected by a round-shaped arena (35 cm dia, 15 cm height) into which the beetles were released. The tested stimuli were placed in one olfactometer arm (randomly selected), with the other arm remaining empty. The charcoal cleaned humidified air (100 ml/s) was blown through both arms into the arena.

For further experimental details see our paper in Naturwissenschaften.

Results

GC�GC-TOFMS analysis
GC�GC-TOFMS analysis disclosed that 0-30 min old mice carcasses emit volatiles that did not differ from those emitted by a living mouse. In the course of time however, traces of low molecular sulfur containing volatile organic compounds (S-VOCs) MeSH, MeSAc, DMS, DMDS, DMTS appeared. In addition, DMQS appeared in older carcasses.

Structure of main organosulfur compounds emitted by mouse carcass. Methanethiol (MeSH), Dimethyl sulfide (DMS), dimethyl disulfide (DMDS), methyl thiolacetate (MeSAc), dimethyl trisulfide (DMTS) and dimethyl tetrasulfide (DMQS).

The S-VOCs compounds were identified based on mass spectra and the identification was confirmed by full match of their mass spectra and GC�GC retention parameters with the corresponding synthetic standards. Small quantitative variations were observed in different analysis with respect to individual mouse carcasses depending on their weight, feeding history and other factors. The S-VOCs dynamics of individual compounds were variable in the course of time. MeSAc and MeSH peaked at 24 and 36 h, respectively, and then declined. DMS peaked at 48 - 96 h and then declined. On the other hand, quantity of DMS, DMDS and DMTS increased steadily during the examined period. DMQS appeared in 2 day old carcasses. In exception of the compounds described above, traces of other S-VOCs were found in older (> 3 days) carcasses, i.e. mercaptoacetic acid, methyl thiolpropanoate, methyl ethyl disulfide, 2,4-dithiapentane, thiopivalic acid and methyl (methylthio)methyl disulfide. These compounds were identified based on NIST mass spectra libraries (with spectra match >95%). Besides S-VOCs, no volatile products of the protein enzymatic/bacterial degradation like scatole, indole, amines etc. were detected in carcasses below 3 days of post mortem time. For further details see our paper in Naturwissenschaften.

GC-EAD
SPME & GC-EAD analysis of mouse carcass emanations showed that all identified S-VOCs are antennally active i.e. they are perceived by antennae of the burying beetle. SPME & GC-EAD analyses of synthetic S-VOCs elicit EAD responses at similar retention times as did the compounds emanated from the mouse carcasses.

EAG
EAG responses to carcass S-VOCs were obtained mainly from the intact surface of the last antennomere in males of both investigated species. The antennal responses to synthetic compound were dose-dependent and ranged over several orders of concentration suggesting the high probability that beetle antennae bear specific receptors for S-VOCs. Species-specific differences in dose-response curves were observed.

EAG responses curves of S-VOCs identified in volatile bouquet of mouse carcass in N. vespilloides and N. vespillo. Each dot represents a mean of 5 EAG recordings from male antennae; doses in �g/�l recalculated to nmol/�l and corrected using relative volatilies. Error bars represent SEM. Air and hexane stimuli were used as controls.

Behavioural experiments
In an empty olfactometer, where no attractive stimuli were present, the beetles released in the olfactometer arena explored it and randomly visited Y olfactometer (5 % of N. vespillo and 3 % of N. vespilloides). When an attractive stimulus was presented however, the beetle behaviour changed, they oriented upwind and within short period (t < 25 s) entered the Y entrance and selected the arm with the attractive source. Beetles that did not find Y entrance within a defined period of 2 minutes were discarded and were considered as non-responding. 12 N. vespillo individuals out of a total of 259 and 7 N. vespilloides individuals out of a total of 272 did not find the olfactometer entrance within 2 minutes and were discarded.

In N. vespillo, GLM analysis revealed non significant sex-specific and bait-specific differences (both effects ξ² < 1.99, P = 0.137) and marginally significant sex�bait interaction (ξ² = 2.60, P = 0.0501). In N. vespilloides, GLM analysis indicated non significant sex-specific differences (ξ² = 0.03, P = 0.0.387), non significant sex�bait interaction (ξ² = 0.61; P = 0.435), but significant bait-specific differences (ξ² = 4.04, P = 0.007). The Fisher test following GLM analysis aimed to determine significant differences in bait attractiveness was applied to pooled data from both sexes, since no sex specific differences were indicated by previous GLM analysis in either species investigated. In N. vespillo, the Fisher test showed that 24 h old mouse carcass and DMTS were highly attractive (P = 0.003 and 0.002, respectively) followed by DMDS and DMS (P = 0.0236 and 0.0199, respectively). In N. vespilloides, mouse carcass was also highly attractive (P = 0.0001) followed by DMS (P = 0.003) and DMTS (P = 0.0163). Responses to DMDS were below statistical significance (P = 0.0731). The described data are displayed in following graphs. Bars represent percentages of beetles selecting the baited arm of the olfactometer. Numbers within bars represent the sum of all tested beetles (beetles that found the entrance of the Y olfactometer within the defined 2 min period) and numbers in brackets represent sum of those beetles, who found successfully the baited arm (numbers in brackets). Thus for instance in N. vespilloides, 29 out of 31 tested females and 27 out of 32 tested males found the arm baited with mouse carcass. The same logic applies to all baits in the graph. Asterisks above bars represent significant differences between the respective baits (as provided by Fisher test). Graphs shows that the most attractive stimulus for both studied species was, as expected, mouse carcass. Graphs also shows that, with exception of DMDS in N. vespilloides, all synthetic compounds tested reveal significant attractiveness for both species.

Attractiveness of DMS, DMDS and DMTS, and 1 day old mouse carcass for burying beetles N. vespilloides and N. vespillo as measured in laboratory Y-olfactometer. Bars show percentages of responding beetles to respective stimuli. The upper numbers in bars represent number of beetles which found the odour source; the numbers in parentheses represent number of tested beetles for the respective bait. Slight differences in the responsiveness between the sexes are not statistically significant based on GLM analysis. Fisher's test for both sexes altogether: ** - P < 0.01, * - P < 0.05, ns - non significant.

Conclusions

In our experiments, we observed that the major products formed shortly after mouse death are mainly S-VOCs, specifically MeSH, MeSAc, DMS, DMSDS, DMTS and traces of DMQS. We provided evidence that majority of S-VOCs are perceived by carrion beetle's antennae and that DMS, DMSDS, DMTS are attractive in laboratory olfactometric experiments. Based on these data, we hypothesize that S-VOCs might be responsible for the attractiveness of fresh carcasses for N. vespillo and N. vespilloides.

Sex-specific differences in antennal sensitivities and attractiveness of individual compounds were observed. EAG experiments showed that the both species were similarly sensitive to MeSH and DMDS, but they differ in their perception of MeSAc, DMS and DMTS. Slight differences were also observed in the attractiveness of individual compounds in olfactometer experiments (N. vespilloides was found less responsive to DMDS). In this context, the attractiveness of DMDS in N. vespilloides needs a specific discussion. A Fisher test did not provide significant value for DMDS in this species. However in the olfactometer, DMDS elicited beetle upwind orientation and an effort to find the odour source. A non significant value in Fisher test thus indicates that N. vespilloides beetles are attracted by DMDS but are relatively less successful to correctly locate the odour source. Our data does not provide enough evidence to specify the basis for such specific inability. It is possible that with N. vespilloides DMDS alone is not enough for precise orientation to odour source and that a combination with other S-VOCs is required. In the abundant chemical ecology literature, there is a lot of evidence showing that insect orientation to host is based on mixtures of volatiles rather then on a single compound. We did not observe big differences in attractiveness of individual S-VOCs compared to fresh mouse carcasses indicating that complex stimuli are required by orienting beetles. However, the compounds tested in our olfactometric experiments were presented only in one concentration which cannot provide enough information about subtle differences in attractiveness of individual compounds. It is also necessary to keep in mind, that there are huge differences in volatilities among tested S-VOCs that were not corrected in our behavioural experiments. Thus further research is needed to specify more precisely the role of individual S-VOCs compounds in the orientation of N. vespillo and N. vespilloides beetles to fresh carcasses.

Our data show that both sexes of N. vespillo and N. vespilloides possess the ability to respond to tested S-VOCs suggests that both males and females might locate the fresh carcass based on the same cues. Our hypothesis that DMS, DMDS and DMTS are likely to be involved in carcass finding is also supported by our results from field experiments. In these experiments, the DMS, DMDS and DMTS were quite attractive when presented individually, but the attractiveness increased significantly, when DMS, DMDS and DMTS were presented in combination. The fact that a blend of DMS, DMDS and DMTS was less attractive than a 1 day old mouse carcass suggests that perhaps other S-VOCs might be also involved.

Many insect species are attracted by odorants produced by the microbial and fungal decomposition of vertebrate carcasses. Their succession on a decomposing carcass is quite predictable and quite well known. Blow flies (Diptera: Calliphoridae) and burying beetles (Silphidae: Nicrophorinae) are usually the first to colonize a carcass. Then beetle predators and scavengers, such as Histeridae, Staphylinidae, Silphidae and Scarabaeidae, move in and prey on the fly larvae as well as feed on the carcass. In spite of this general knowledge of carcass attractiveness and species succession, little is known about the perception of decaying volatiles in arthropods. In our experiments, we observed that EAG responses to S-VOCs could be recorded mainly from the surface of the last antennomere suggesting that different odours can be perceived by different part of the antenna.

In general, there has been very little research performed in the area of analysis of the volatile compounds that influence the behaviour of burying beetles. The research presented here is a small part that addresses the question of carcass detection. Future research, including field experiments is needed to understand the problem in its complexity.

Research team

Blanka Kalinov�, Michal Hoskovec, Jan R��i�ka, Hana Podskalsk�

Published in...

Kalinov� B., Podskalsk� H., R��i�ka J., Hoskovec M.:
Irresistible bouquet of death � how are burying beetles (Coleoptera: Silphidae: Nicrophorus) attracted by carcasses.
Naturwissenschaften 96: 889-899 (2009).

Podskalsk� H., R��i�ka J., Hoskovec M., ��lek M.:
Use of infochemicals to attract carrion beetles into pitfall traps.
Entomologia Experimentalis et Applicata 132: 59�64 (2009).