Introduction

Burying beetles (Nicrophorus spp., Silphidae: Coleoptera) are known for their complex brood care behavior, which involves the search and burying of small vertebrate carcasses, the preparation of the buried carrion for their offspring, and elaborate care during the time when the larvae are feeding at the carcass. Mate finding is mediated by volatiles emitted by males (Pukowski 1933; Bartlett 1987; Eggert and Müller 1989). Various ecological implications of male pheromonal advertising in burying beetles have been studied (Eggert 1992; Trumbo and Egggert 1994; Beeler et al. 1999, 2002; Walling et al. 2009), however, research on the chemical nature of the male pheromones in Nicrophorus is still scarce. To date, male pheromone constituents of only two species are known. The first components identified were ethyl 4-methylheptanoate and geranylacetone, emitted by N. vespilloides (Haberer et al. 2008). A behaviorally active pheromone component, emitted by N. humator males, was identified to be methyl 4-methyloctanoate, whereas the role of the second component, isovaleric acid, found in several but not all samples of this species, remains to be clarified (Haberer et al. 2011). As calling Nicrophorus males also attract conspecific males (Müller and Eggert 1987), the corresponding chemical signals are considered aggregation pheromones rather than sex pheromones. Cross-attraction of congeneric beetles via long-range pheromones is a phenomenon also observed in Nicrophorus males (Müller and Eggert 1987; Haberer et al. 2011). This kairomonal response is thought to be adaptive in the context of interspecific competition (Trumbo and Bloch 2002) for rare breeding resources.

Nicrophorus males can employ different behavioral tactics to find a mate (Eggert 1992): Since adults of both sexes are attracted to carcasses (Kalinová et al. 2009), one option is to search for a carcass suitable for reproduction, on which a conspecific female may already be present. If that is not the case, males will start to release pheromones to attract females, since males cannot utilize the carcass on their own (Pukowski 1933). An alternative mate-finding tactic for males involves calling for females without having located a carcass (Müller and Eggert 1987). On a carcass, the attractiveness of male pheromones may be synergistically enhanced by odors emanating from the substrate itself; however, calling without a breeding resource is likely to contribute significantly to reproductive success (Eggert and Müller 1989; Eggert 1992; Beeler et al. 1999; Müller et al. 2007; Walling et al. 2009).

The aim of our study was to establish an initial inventory of volatiles released by calling males of several Nicrophorus species. This was accomplished by collecting headspace volatiles from beetles showing calling behavior by using solid phase microextraction (SPME) and analyzing the samples by gas chromatography coupled with mass spectrometry (GC/MS). The study includes five North American and five Central European species (Table 1) as well as one species of the sister genus of Nicrophorus: the Asian Ptomascopus morio. Unlike burying beetles, P. morio exhibits only rudimentary brood care behavior and is considered a facultative brood parasite of Nicrophorus concolor (Trumbo et al. 2001; Suzuki and Nagano 2005). Mate-calling behavior, however, is similar in Nicrophorus spp. and P. morio (unpublished observations). At a species-specific time of the day, males typically perch at elevated locations, extend their hind legs, raise their abdomen, and release volatiles for about one to 3hrs. Diurnal and crepuscular species like N. defodiens, N. interruptus, N. pustulatus, N. tomentosus, N. vespilloides, and N. vespillo exhibit this calling behavior in the late afternoon and in the evening, at the end of their daily activity period, in contrast to strictly nocturnal species such as N. humator and N. orbicollis, which typically signal later, during the night (Haberer, unpublished observations; Müller and Eggert 1987; Beeler et al. 1999; Mulrey et al. 2015).

Table 1 Species included in the headspace analyses. N = number of individuals of which volatiles have been collected
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Methods and Materials

Beetles, Origin and Rearing

Beetles used in this study were either caught in the wild or were laboratory-reared (Table 1). Individuals were maintained separately at 20 °C under a 16:8 h light:dark regime in transparent polystyrene containers (10x10x6cm) filled with moist peat and fed decapitated mealworms twice weekly.

Volatile Collection

Headspace sampling was accomplished using solid phase micro extraction (SPME) fibers (Supelco, coating: polydimethylsiloxane/divinylbenzene, Sigma-Aldrich). Volatiles were collected in the plastic containers in which beetles were housed. An SPME fiber was inserted into the container through a perforated lid and mounted close to the beetles. Under laboratory conditions, three main phases of male behavior can be observed during their active time of the day (Eggert 1992). First, the beetles appear at the surface of the substrate, run around and repeatedly attempt to fly. Subsequently, the males choose an elevated site, remain there more or less stationary and show the typical calling behavior: head down, hind legs extended, and abdomen extended and raised. The last abdominal segment, otherwise mostly concealed within the penultimate segment, is extruded and may appear wet on the surface. After this calling phase, which typically lasts 1–3 h, the beetles may move around again on the surface before disappearing into the peat, where they remain inactive until the next calling period on the following day. To identify volatiles emitted only during calling, headspace samples obtained from calling beetles were compared to samples taken before or after calling.

Chemical Analysis

The SPME-fiber, loaded with volatiles, was inserted at the injection port of a gas chromatograph (HP series 6890, Hewlett Packard, Palo Alto, California) equipped with a DB-5 column (30 m × 0.25 mm × 0.25 μm, J & W, Folsom, California), coupled to a quadrupole mass spectrometer (HP series 5973, Hewlett Packard, Palo Alto, California), which was run at 70 eV. The injection temperature was 250 °C, and the column temperature was ramped from 50° to 250 °C by 10 °C/min with a final hold of 5 min at 250 °C. Chemical structures of target compounds were inferred from diagnostic fragments and compared to those reported in the literature. Retention times, retention indices, and fragmentation patterns of the substances detected were compared to those of authentic reference compounds: ethyl (9Z)-hexadecenoate (Tokyo Chemical Industry Co., Japan), (2E,6Z)-dodecadienal, ethyl tetradecanoate, geranylacetone, n-heptadecane, isovaleric acid (Fluka GmbH, Switzerland), and methyl geranate (Chemos GmbH, Germany). Racemates of 4-methylheptanoic acid and 4-methyloctanoic acid were synthesized according to Joung et al. (1998) and esterified according to laboratory standards - the plotted 70 eV mass spectrum of the new isoamyl 4-methylheptanoate is shown in the Supplement, along with its interpretation and a comment. 3-Methylundecan-2-one was synthesized according to Midgley and Thomas (1987); fuscumyl acetate was synthesized from geranylacetone according to Li et al. (2003); 3-methyl-2-oxopentanoic acid was obtained by acidification of sodium 3-methyl-2-oxopentanoate (Aldrich, Gillingham, Dorset, UK) with acetic acid; (9Z)-octadecenal and hexadecanal were synthesized by oxidation of the corresponding alcohols (Roth, Germany) according to Corey and Suggs (1975). (2E)-Dodecenal was provided by Prof. Wittko Francke, Univ. Hamburg. Methyl (2E,6E)-farnesoate and (E,E)-farnesylacetone were tentatively identified by comparing their mass spectra with reference data reported in the Wiley data base of mass spectra (Wiley & Sons Inc., 6th edition). Structures of 3-methyldodecan-2-one and 3-methyltridecan-2-one were identifed according to their mass spectra, which were very similar to that of 3-methylundecan-2-one, which we had identified by means of a synthetic reference compound.

Results

Volatiles Detected in the Headspaces of Calling Beetles and Proportions in Released Blends

Volatiles emitted by males of Nicrophorus spp. comprise 5 alkyl 4-methylalkanoates, 5 terpenoids, 7 saturated and unsaturated acetogenins, and 2 compounds that may be related to amino acids (Table 2). P. morio males do not produce any of the compounds detected in Nicrophorus spp. but emit three 3-methylalkane-2-ones (Table 2), among which 3-methylundecan-2-one is quantitatively dominant (Fig. 1i).

Table 2 Volatiles emitted by calling males of ten Nicrophorus species and Ptomascopus morio
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Fig. 1
figure 1

Proportions of headspace components collected from calling Nicrophorus spp. and Ptomascopus morio. Mean and standard error of the integrated peak areas of the total ion chromatograms. The X-axis shows relative proportions of volatiles generated by SPME-GC/MS-analysis (peak areas in ion-chromatograms, data not corrected)

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The number of volatiles released by males range from two in N. defodiens, N. humator, N. marginatus, N. vespillo, and N. vespilloides to a maximum of seven components in N. pustulatus (Table 2, Fig. 1).

Esters of 4-methyl-branched carboxylic acids were detected in eight of the ten Nicrophorus species included in this study (Table 2). Ethyl 4-methylheptanoate is released by six species, and predominant among the volatiles of N. pustulatus, N. tomentosus, and N. vespilloides (Fig. 1c, d, h). N. investigator also produces ethyl 4-methylheptanoate, along with (2E,6Z)-dodecadienal (Table 2), but information on the ratio of these compounds in the blend cannot be provided here, because the ratios strongly differed between the two specimens available. In the three-component blends obtained from two male N. marginatus (Table 2), we found primarily ethyl 4-methyloctanoate but also small amounts of ethyl 4-methylheptanoate and ethyl (9Z)-hexadecenoate. As in N. investigator, more individuals of this species must be analyzed to reliably estimate volatile proportions. In all studied Nicrophorus species, the quantitative composition of blends was highly variable among individuals (Fig. 1), especially in N. defodiens, N. tomentosus, N. vespillo, and N. vespilloides (Fig. 1a, d, g, h). However, P. morio showed little interindividual variation (Fig. 1i).

With the exception of N. humator and N. vespillo, all Nicrophorus species released acetogenins or terpenoids (Table 2). N. vespillo released isoamyl 4-methylheptanoate as a unique ester.

Discussion

Our study yielded six principal results: (1) None of the species in the survey releases a single-component signal, but blends, containing two to seven compounds. (2) The volatiles involved are chemically diverse; they represent different classes of compounds and, according to their carbon skeletons are produced via distinct biosynthetic pathways. (3) P. morio males release a blend of three 3-methylalkan-2-ones, but none of the compounds found in Nicrophorus. (4) Several of the compounds present in Nicrophorus are not unique to a single species, but occur in several species. (5) The blends of some but not all Nicrophorus species include terpenoids. (6) In most (8 of 10) of the Nicrophorus species studied, male volatiles include esters of 4-methyloctanoic acid and 4-methylheptanoic acid, which may play a pivotal role in pheromone signaling in the genus. N. defodiens and N. orbicollis do not produce such esters but terpenoids, whereas the two-component blend of N. vespilloides contains both.

The occurrence of esters of 4-methyloctanoic acid and 4-methylheptanoic acid is not restricted to Nicrophorus. These esters have also been identified as male pheromone components in several rhinoceros beetles species of the genus Oryctes (Gries et al. 1994; Hallett et al. 1995; Rochat et al. 2004; Saïd et al. 2015). The terpenoids identified here are also known as components of male pheromones of a number of other organisms. Methyl geranate (N. pustulatus) occurs in the pheromone of male stink bugs in the genus Chlorochroa (Ho and Millar 2001a, b), as does methyl (2E,6E)-farnesoate (N. pustulatus). This latter ester is also produced by male African milkweed butterflies (Schulz et al. 1993) and male African fruit fly species of the Ceratitis complex, where its biological activity has been confirmed in electrophysiological studies (Břízová et al. 2015). The nor-terpenoid geranylacetone (N. interruptus, orbicollis, pustulatus, and vespilloides), is found in many different organisms, ranging from microorganisms and plants to insects and even mammals. Fuscumyl acetate, the main volatile in N. defodiens is, along with the corresponding alcohol, a male produced pheromone component of longhorned beetles (Silk et al. 2007; Fonseca et al. 2010). Farnesylacetone (N. defodiens, orbicollis, and pustulatus) is less common, but has been identified among plant seed volatiles attractive to beetles (Adhikary et al. 2015), the dorsal gland of an antelope (Burger et al. 1981), and the androgenic glands of male crabs Carcinus maenas (Ferezou et al. 1977).

Deciphering the biological significance of these terpenes in Nicrophorus will require more in-depth studies. Laboratory and field tests are required to assess which of the volatiles detected in this study are actually perceived by potential recipients and modify their behavior. To date, only pheromones identified from N. vespilloides and N. humator have been tested in the field (Haberer et al. 2008; Haberer et al. 2011). However, these results must be interpreted with caution, since the stereochemical configuration of the respective main components, ethyl 4-methylheptanoate and methyl 4-methyloctanoate, has not yet been determined. This limitation applies also to the two other alkyl 4-methylcarboxylates identified in this study.

The composition of pheromone blends produced by male Nicrophorus appears to vary greatly between individuals. This raises an important issue concerning the significance of such variation: does it convey information about characteristics of the signaling individual such as size, health, reproductive history, or nutritional status to the signal recipient? A recent study on N. vespilloides revealed that such factors substantially affect amounts and proportions in the two-component blend, which significantly affect attractiveness to females (Chemnitz et al. 2015). Moreover, it would be particularly beneficial for females to distinguish between males that have located a carcass and males that have not. A carcass, buried by the monopolizing male, is olfactorily “invisible” or at least difficult to detect (Trumbo and Bloch 2002), and thus, a semiochemical advertising ownership could be a way for the male to communicate the presence of the hidden resource to the intended receiver only (however, conspecific males and congeners might be able to intercept this information, see Eggert and Müller 1989). Candidate compounds for such advertising are substances derived from carcass material that the beetles ingest during the first hours on the carcass. Isovaleric acid, emitted by males of N. humator, N. investigator, and N. interruptus may be such a candidate compound as it most probably originates from the amino acid L-leucine. Its production may, thus, depend on the access to a protein-rich diet. Similar considerations apply to 3-methyl-2-oxopentanoic acid in N. orbicollis and N. vespillo as this compound is the immediate transamination product of isoleucine (Francke and Schulz 1999). Further studies are necessary to test this hypothesis.

Several Nicrophorus species show remarkable overlap in blend constituents. Ethyl 4-methylheptanoate was detected in six of ten species from Europe and America. Presumably, this ester and esters of its homologue, 4-methyloctanoic acid, appeared early in the evolution of burying beetles. The distribution of species producing these esters across several different major branches in the phylogenetic tree of Nicrophorus (Sikes and Venables 2013) supports this hypothesis. Several species appear to have evolved additional pheromone constituents that facilitate species-specific recognition. The recent finding that breeding N. vespilloides females produce similar esters with as of yet unknown biological function (Haberer et al. 2014) also suggests a long evolutionary history of signaling with these esters in Nicrophorus. Apparently, these substances are used to convey information in very different contexts. More chemical details such as the stereochemical configurations of the compounds involved, but also fundamental ecological information about niche differentiation and mating systems are needed to understand the evolution of long-range recognition in burying beetles, especially among sympatric species that share pheromone constituents.

Although P. morio produce 3-methylalkan-2-ones that are chemically distinct from the compounds found in Nicrophorus so far, a comparison of the likely biosynthesic pathways of these ketones and that of the alkyl 4-methylcarboxylates in Nicrophorus reveals a close relationship. The biosynthesis of the 4-methylcarboxylates of Nicrophorus and of the 3-methylalkan-2-ones of P. morio involve the incorporation of propanoate, similar to the formation of the methyl group in the 4 position of 4-methylnonan-1-ol, the sex pheromone of the yellow mealworm, Tenebrio molitor (Islam et al. 1999). The 4-methylacyl-motif, formed in a propanoate - acetate - sequence as the final steps during chain formation, would directly yield 3-methylalkan-2-ones upon β-oxidation and decarboxylation of the corresponding 4-methyl-3-oxocarboxlic acids. Ptomascopus is the sister genus to Nicrophorus (Sikes and Venables 2013) and has retained some ancestral characteristics in morphology (Peck 1982) and behavior (Suzuki and Nagano 2006). Several studies on the breeding behavior of P. morio have been carried out (Trumbo et al. 2001; Suzuki et al. 2005; Nagano and Suzuki 2008), but we are just beginning to understand its mating system (Suzuki et al. 2005).