Burying Beetle Parents Adaptively Manipulate Information Broadcast from a Microbial Community.

AbstractMicrobial volatiles provide essential information for animals, which compete to detect, respond to, and perhaps control this information. Burying beetle parents have the opportunity to influence microbially derived semiochemicals, because they monopolize a small carcass for their family, repairing feeding holes and applying exudates that alter the microbial community. To study adaptive manipulation of microbial cues, we integrated mechanistic and functional approaches. We contrasted gas chromatography-mass spectrometry (GC-MS) volatile profiles from carcasses that were or were not prepared by a resident pair of Nicrophorus orbicollis. Methyl thiocyanate (MeSCN), the primary attractant for burying beetles seeking a fresh carcass, was reduced 20-fold by carcass preparation, while dimethyl trisulfide (DMTS), which deters breeding beetles, was increased 20-fold. These results suggest that parental care serves to make previously public information more private (crypsis, MeSCN) and to disinform rivals with a deterrent (DMTS). Functional tests in the field demonstrated that carcass preparation reduced discovery and use by congeners (threefold) as well as by dipteran rivals. Because microbes and their chemicals influence nearly every aspect of animal ecology, animal manipulation of microbial cues may be as widespread as manipulation of their own signals.

Microbiome-assisted carrion preservation aids larval development in a burying beetle.

The ability to feed on a wide range of diets has enabled insects to diversify and colonize specialized niches. Carrion, for example, is highly susceptible to microbial decomposers, but is kept palatable several days after an animal's death by carrion-feeding insects. Here we show that the burying beetle Nicrophorus vespilloides preserves carrion by preventing the microbial succession associated with carrion decomposition, thus ensuring a high-quality resource for their developing larvae. Beetle-tended carcasses showed no signs of degradation and hosted a microbial community containing the beetles' gut microbiota, including the yeast Yarrowia In contrast, untended carcasses showed visual and olfactory signs of putrefaction, and their microbial community consisted of endogenous and soil-originating microbial decomposers. This regulation of the carcass' bacterial and fungal community and transcriptomic profile was associated with lower concentrations of putrescine and cadaverine (toxic polyamines associated with carcass putrefaction) and altered levels of proteases, lipases, and free amino acids. Beetle-tended carcasses develop a biofilm-like matrix housing the yeast, which, when experimentally removed, leads to reduced larval growth. Thus, tended carcasses hosted a mutualistic microbial community that promotes optimal larval development, likely through symbiont-mediated extraintestinal digestion and detoxification of carrion nutrients. The adaptive preservation of carrion coordinated by the beetles and their symbionts demonstrates a specialized resource-management strategy through which insects modify their habitats to enhance fitness.

The Impact of Environmental Factors on the Efficacy of Chemical Communication in the Burying Beetle (Coleoptera: Silphidae).

There is growing evidence that a wide range of insect sex pheromones are condition dependent and play a fundamental role in mate choice. However, the effectiveness of pheromonal communication might not only depend on internal factors of the sender, but also on attributes of the microhabitat, in which the signaler chooses to emit its chemical signal. For example, the degree of anthropogenic land use might affect how successful the signal is transmitted, as land use has been shown to affect animal communities and the complexity of biotic interactions. To test the hypothesis that parameters of the microenvironment determine males' ability to attract females via their sex pheromone, we used the burying beetle Nicrophorus vespilloides Herbst (Coleoptera: Silphidae) as our model system. We exposed 144 males across differently managed forest stands and analyzed the impact of 29 environmental parameters. Our data revealed that human land use intensity had no effect on a male's attractiveness. However, the harvested tree biomass positively affected the proportion of competitors attracted. Furthermore, we found that soil characteristics were important factors determining the amount and body size of females a male was able to attract. Consequently, we present evidence that the environmental context of a signaling male influences the effectiveness of chemical signaling either because it affects the transmission process or the prevailing abundance of potential signal receivers. Thus, our results demonstrate that males need to make careful decisions about the location where they emit their pheromone, as this choice of microhabitat has an impact on their fitness.

Manipulation of parental nutritional condition reveals competition among family members.

Parental care is thought to be costly, as it consumes time and energy. Such costs might be reduced in animal parents that raise their young on valuable food sources such as dung or carcasses, as parents are able to invest in self-maintenance by feeding from the same resource. However, this might lower the nutritional value for other family members and, as a consequence, food competition might arise. To promote our understanding of the outcome of such competition, we manipulated the necessity of parents to feed from the resource. Using a full factorial design, we paired food-deprived or well-fed males with food-deprived or well-fed females of burying beetles, which are known to raise their young on vertebrate cadavers. We found that food-deprived parents consumed more of the carrion than those that were well-fed and this had a negative impact on other family members. However, the outcome of the competition depended on the sex of the parents, with females suffering when males fed more and offspring suffering when females fed more. Thus, family life involves selfish elements, as both parents remove resources for the purpose of self-maintenance. However, females show altruistic aspects, as they appear to restrict their food consumption for the benefit of their offspring when paired with a food-deprived male. Interestingly, males extend their stay with the brood when having faced food scarcity prior to reproduction, presumably to replenish their energy reserves. Our study therefore reveals that breeding on shared resources can promote family living, but also results in competition.

Offspring dependence on parental care and the role of parental transfer of oral fluids in burying beetles.

BACKGROUND: Immature stages of many animals can forage and feed on their own, whereas others depend on their parents' assistance to obtain or process food. But how does such dependency evolve, and which offspring and parental traits are involved? Burying beetles (Nicrophorus) provide extensive biparental care, including food provisioning to their offspring. Interestingly, there is substantial variation in the reliance of offspring on post-hatching care among species. Here, we examine the proximate mechanisms underlying offspring dependence, focusing on the larvae of N. orbicollis, which are not able to survive in the absence of parents. We specifically asked whether the high offspring dependence is caused by (1) a low starvation tolerance, (2) a low ability to self-feed or (3) the need to obtain parental oral fluids. Finally, we determined how much care (i.e. duration of care) they require to be able to survive. RESULTS: We demonstrate that N. orbicollis larvae are not characterized by a lower starvation tolerance than larvae of the more independent species. Hatchlings of N. orbicollis are generally able to self-feed, but the efficiency depends on the kind of food presented and differs from the more independent species. Further, we show that even when providing highly dependent N. orbicollis larvae with easy ingestible liquefied mice carrion, only few of them survived to pupation. However, adding parental oral fluids significantly increased their survival rate. Finally, we demonstrate that survival and growth of dependent N. orbicollis larvae is increased greatly by only a few hours of parental care. CONCLUSIONS: Considering the fact that larvae of other burying beetle species are able to survive in the absence of care, the high dependence of N. orbicollis larvae is puzzling. Even though they have not lost the ability to self-feed, an easily digestible, liquefied carrion meal is not sufficient to ensure their survival. However, our results indicate that the transfer of parental oral fluids is an essential component of care. In the majority of mammals, offspring rely on the exchange of fluids (i.e. milk) to survive, and our findings suggest that even in subsocial insects, such as burying beetles, parental fluids can significantly affect offspring survival.

Pheromones Regulating Reproduction in Subsocial Beetles: Insights with References to Eusocial Insects.

Beetles have evolved diverse strategies to cope with environmental challenges. Although parents of the vast majority of beetle species do not take care of their offspring, there are some species, in which parents provide elaborate post-hatching care and remain temporarily associated with their offspring to defend them from competitors or to provision them with food. Usually, socially induced reproductive "control" is a core feature of eusocial societies, but here we highlight that already in small family groups, socially induced reproductive regulation can play a fundamental role. By discussing the family life of burying beetles, we illustrate the mechanisms behind such a reproductive "control" and show that - similar to eusocial insects - pheromones can be an important regulating factor. However, apart from burying beetles, our knowledge of pheromones or other signals mediating reproductive regulation is surprisingly rudimentary for social beetles. More data are required to broaden our currently patchy picture.

Function of bacterial community dynamics in the formation of cadaveric semiochemicals during in situ carcass decomposition.

The decomposition of dead mammalian tissue involves a complex temporal succession of epinecrotic bacteria. Microbial activity may release different cadaveric volatile organic compounds which in turn attract other key players of carcass decomposition such as scavenger insects. To elucidate the dynamics and potential functions of epinecrotic bacteria on carcasses, we monitored bacterial communities developing on still-born piglets incubated in different forest ecosystems by combining high-throughput Illumina 16S rRNA sequencing with gas chromatography-mass spectrometry of volatiles. Our results show that the community structure of epinecrotic bacteria and the types of cadaveric volatile compounds released over the time course of decomposition are driven by deterministic rather than stochastic processes. Individual cadaveric volatile organic compounds were correlated with specific taxa during the first stages of decomposition which are dominated by bacteria. Through best-fitting multiple linear regression models, the synthesis of acetic acid, indole and phenol could be linked to the activity of Enterobacteriaceae, Tissierellaceae and Xanthomonadaceae, respectively. These conclusions are also commensurate with the metabolism described for the dominant taxa identified for these families. The predictable nature of in situ synthesis of cadaveric volatile organic compounds by epinecrotic bacteria provides a new basis for future chemical ecology and forensic studies.

Variation in sex pheromone emission does not reflect immunocompetence but affects attractiveness of male burying beetles-a combination of laboratory and field experiments.

Life history theory predicts a trade-off between male sexual trait expression and immunocompetence. Using burying beetles, Nicrophorus vespilloides, as a model, we investigated the relationship between male immune function, sex pheromone emission, and attractiveness under field conditions. In the first experiment, we tested whether there is a positive correlation between immune capacity, sex pheromone characteristics (quantity, relative composition, and time invested in pheromone emission), and male attractiveness. As a measurement of immune capacity, we used an individual's encapsulation ability against a novel antigen. In the second experiment, we specifically examined whether a trade-off between chemical trait expression and immune function existed. To this end, we challenged the immune system and measured the subsequent investment in sex pheromone emission and the attractiveness of the male under field conditions. We found that a male's immunocompetence was neither related to the emission of the male's sex pheromone nor to its attractiveness in the field. Furthermore, none of the immune-challenge treatments affected the subsequent investment in pheromone emission or number of females attracted. However, we showed that the same males that emitted a high quantity of their sex pheromone in the laboratory were able to attract more females in the field. Our data suggest that the chemical signal is not a reliable predictor of a male's immunocompetence but rather is a general important fitness-related trait, with a higher emission of the sex pheromone measured in the laboratory directly affecting the attractiveness of a male under field conditions.

Beyond Cuticular Hydrocarbons: Chemically Mediated Mate Recognition in the Subsocial Burying Beetle Nicrophorus vespilloides.

Burying beetles have fascinated scientists for centuries due to their elaborate form of biparental care that includes the burial and defense of a vertebrate carcass, as well as the subsequent feeding of the larvae. However, besides extensive research on burying beetles, one fundamental question has yet to be answered: what cues do males use to discriminate between the sexes? Here, we show in the burying beetle Nicrophorus vespilloides that cuticular lipids trigger male mating behavior. Previous chemical analyses have revealed sex differences in cuticular hydrocarbon (CHC) composition; however, in the current study, fractionated-guided bioassay showed that cuticular lipids, other than CHCs, elicit copulation. Chemical analyses of the behaviorally active fraction revealed 17 compounds, mainly aldehydes and fatty acid esters, with small quantitative but no qualitative differences between the sexes. Supplementation of males with hexadecanal, the compound contributing most to the statistical separation of the chemical profiles of males and females, did not trigger copulation attempts by males. Therefore, a possible explanation is that the whole profile of polar lipids mediates sex recognition in N. vespilloides.

Beyond species recognition: somatic state affects long-distance sex pheromone communication.

Long-range sex pheromones have been subjected to substantial research with a particular focus on their biosynthesis, peripheral perception, central processing and the resulting orientation behaviour of perceivers. Fundamental to the research on sex attractants was the assumption that they primarily coordinate species recognition. However, especially when they are produced by the less limiting sex (usually males), the evolution of heightened condition dependence might be expected and long-range sex pheromones might, therefore, also inform about a signaller's quality. Here we provide, to our knowledge, the first comprehensive study of the role of a male's long-range pheromone in mate choice that combines chemical analyses, video observations and field experiments with a multifactorial manipulation of males' condition. We show that the emission of the long-distance sex pheromone of the burying beetle, Nicrophorus vespilloides is highly condition-dependent and reliably reflects nutritional state, age, body size and parasite load--key components of an individual's somatic state. Both, the quantity and ratio of the pheromone components were affected but the time invested in pheromone emission was largely unaffected by a male's condition. Moreover, the variation in pheromone emission caused by the variation in condition had a strong effect on the attractiveness of males in the field, with males in better nutritional condition, of older age, larger body size and bearing less parasites being more attractive. That a single pheromone is influenced by so many aspects of the somatic state and causes such variation in a male's attractiveness under field conditions was hitherto unknown and highlights the need to integrate indicator models of sexual selection into pheromone research.

Acceptance threshold theory can explain occurrence of homosexual behaviour.

Same-sex sexual behaviour (SSB) has been documented in a wide range of animals, but its evolutionary causes are not well understood. Here, we investigated SSB in the light of Reeve's acceptance threshold theory. When recognition is not error-proof, the acceptance threshold used by males to recognize potential mating partners should be flexibly adjusted to maximize the fitness pay-off between the costs of erroneously accepting males and the benefits of accepting females. By manipulating male burying beetles' search time for females and their reproductive potential, we influenced their perceived costs of making an acceptance or rejection error. As predicted, when the costs of rejecting females increased, males exhibited more permissive discrimination decisions and showed high levels of SSB; when the costs of accepting males increased, males were more restrictive and showed low levels of SSB. Our results support the idea that in animal species, in which the recognition cues of females and males overlap to a certain degree, SSB is a consequence of an adaptive discrimination strategy to avoid the costs of making rejection errors.

Size Exclusion High Performance Liquid Chromatography: Re-Discovery of a Rapid and Versatile Method for Clean-Up and Fractionation in Chemical Ecology.

Solvent extraction of bioactive molecules from glands, tissues, or whole organisms is a common first step in chemoecological studies. Co-extraction of a surplus of high boiling materials such as triacylglycerides (TAGs) and other lipids with higher molecular weight might hamper the identification of volatile or medium-volatile semiochemicals by high resolution chromatographic and spectroscopic techniques. Therefore, effective clean-up procedures are needed to separate potential semiochemicals from the accompanying materials. Size exclusion high performance liquid chromatography (SE-HPLC), a technique often disregarded by chemoecologists, has proved to be a rapid and efficient clean-up method for complex crude extracts. We demonstrated that TAGs can be baseline separated from typical semiochemicals within less than 10 min on a porous gel stationary phase based on highly cross-linked polystyrene/divinylbenzene. We applied the method as a rapid one-step clean-up procedure for the analysis of juvenile hormone III in insect hemolymph by gas chromatography-mass spectrometry. We furthermore introduced some recent application examples on insect pheromones to demonstrate that SE-HPLC is not only an effective method for the purification of crude extracts, but can as well be used as a first fractionation step for the bioassay-guided identification of behavior modifying natural products. SE-HPLC can be well operated with low-boiling solvents such as dichloromethane, and results in fraction volumes of typically less than one ml, which decreases the danger of losing volatile analytes during subsequent concentration steps.

Unearthing carrion beetles' microbiome: characterization of bacterial and fungal hindgut communities across the Silphidae.

Carrion beetles (Coleoptera, Silphidae) are well known for their behaviour of exploiting vertebrate carcasses for nutrition. While species in the subfamily Silphinae feed on large carcasses and on larvae of competing scavengers, the Nicrophorinae are unique in monopolizing, burying and defending small carrion, and providing extensive biparental care. As a first step towards investigating whether microbial symbionts may aid in carcass utilization or defence, we characterized the microbial hindgut communities of six Nicrophorinae (Nicrophorus spp.) and two Silphinae species (Oiceoptoma noveboracense and Necrophila americana) by deep ribosomal RNA amplicon sequencing. Across all species, bacteria in the family Xanthomonadaceae, related to Ignatzschineriao larvae, were consistently common, and several other taxa were present in lower abundance (Enterobacteriales, Burkholderiales, Bacilli, Clostridiales and Bacteroidales). Additionally, the Nicrophorinae showed high numbers of unusual Clostridiales, while the Silphinae were characterized by Flavobacteriales and Rhizobiales (Bartonella sp.). In addition to the complex community of bacterial symbionts, each species of carrion beetle harboured a diversity of ascomycetous yeasts closely related to Yarrowia lipolytica. Despite the high degree of consistency in microbial communities across the Silphidae--specifically within the Nicrophorinae--both the fungal symbiont phylogeny and distance-based bacterial community clustering showed higher congruence with sampling locality than host phylogeny. Thus, despite the possibility for vertical transmission via anal secretions, the distinct hindgut microbiota of the Silphidae appears to be shaped by frequent horizontal exchange or environmental uptake of symbionts. The microbial community profiles, together with information on host ecology and the metabolic potential of related microorganisms, allow us to propose hypotheses on putative roles of the symbionts in carcass degradation, detoxification and defence.

The scent of offspring: chemical profiles of larvae change during development and affect parental behavior in a burying beetle.

Chemical cues and signals, especially in insects, play a pivotal role in mediating interactions between individuals. Past studies have largely focused on adult semiochemicals and have neglected those of juvenile stages. Especially in the context of parental care, the larval odor might have a profound impact on parenting behavior, guiding parents in how much resources they should allocate to the different developmental stages. However, whether ontogenetic changes occur in subsocial species and whether larval-emitted scents influence parent-offspring interactions is largely unknown. Using 3 different sampling techniques, we analyzed the cuticular and VOC profile of the 3 larval instars of the burying beetle Nicrophorus vespilloides, which is known for its elaborate parental care. We found distinct differences in the cuticular and VOC profiles across the 3 larval stages. Second-instar larvae, which receive more frequent feedings from parents than the other larval stages, released greater amounts of acetophenone, methyl geranate, and octanoic acid isopropyl ester than the first and third instar. Additionally, using a newly developed bioassay with automated video tracking, we found that adding the odor of second-instar larvae to first-instar larvae increased the number of maternal feeding trips. Our results suggest that the odor produced by larvae plays an important role in mediating parent-offspring interactions. Given these findings, burying beetles might emerge as a promising candidate for identifying a potential begging pheromone.

Revisiting the ecology and evolution of burying beetle behavior (Staphylinidae: Silphinae).

Investigating fundamental processes in biology requires the ability to ground broad questions in species-specific natural history. This is particularly true in the study of behavior because an organism's experience of the environment will influence the expression of behavior and the opportunity for selection. Here, we provide a review of the natural history and behavior of burying beetles of the genus Nicrophorus to provide the groundwork for comparative work that showcases their remarkable behavioral and ecological diversity. Burying beetles have long fascinated scientists because of their well-developed parenting behavior, exhibiting extended post-hatching care of offspring that varies extensively within and across taxa. Despite the burgeoning success of burying beetles as a model system for the study of behavioral evolution, there has not been a review of their behavior, ecology, and evolution in over 25 years. To address this gap, we leverage a developing community of researchers who have contributed to a detailed knowledge of burying beetles to highlight the utility of Nicrophorus for investigating the causes and consequences of social and behavioral evolution.

Cuticular hydrocarbons as a basis for chemosensory self-referencing in crickets: a potentially universal mechanism facilitating polyandry in insects.

Females of many species obtain benefits by mating polyandrously, and often prefer novel males over previous mates. However, how do females recognise previous mates, particularly in the face of cognitive constraints? Female crickets appear to have evolved a simple but effective solution: females imbue males with their own cuticular hydrocarbons (CHCs) at mating and utilise chemosensory self-referencing to recognise recent mates. Female CHC profiles exhibited significant additive genetic variation, demonstrating that genetically unique chemical cues are available to support chemosensory self-referencing. CHC profiles of males became more similar to those of females after mating, indicating physical transfer of CHCs between individuals during copulation. Experimental perfuming of males with female CHCs resulted in a female aversion to males bearing chemical cues similar to their own. Chemosensory self-referencing, therefore, could be a widespread mechanism by which females increase the diversity of their mating partners.

Bigger mothers are better mothers: disentangling size-related prenatal and postnatal maternal effects.

Despite a vast literature on the factors controlling adult size, few studies have investigated how maternal size affects offspring size independent of direct genetic effects, thereby separating prenatal from postnatal influences. I used a novel experimental design that combined a cross-fostering approach with phenotypic manipulation of maternal body size that allowed me to disentangle prenatal and postnatal maternal effects. Using the burying beetle Nicrophorus vespilloides as model organism, I found that a mother's body size affected egg size as well as the quality of postnatal maternal care, with larger mothers producing larger eggs and raising larger offspring than smaller females. However, with respect to the relative importance of prenatal and postnatal maternal effects on offspring growth, only the postnatal effects were important in determining offspring body size. Thus, prenatal effects can be offset by the quality of postnatal maternal care. This finding has implications for the coevolution of prenatal and postnatal maternal effects as they arise as a consequence of maternal body size. In general, my study provides evidence that there can be transgenerational phenotypic plasticity, with maternal size determining offspring size leading to a resemblance between mothers and their offspring above and beyond any direct genetic effects.

Sexual selection on cuticular hydrocarbons of male sagebrush crickets in the wild.

Cuticular hydrocarbons (CHCs) play an essential role in mate recognition in insects but the form and intensity of sexual selection on CHCs has only been evaluated in a handful of studies, and never in a natural population. We quantified sexual selection operating on CHCs in a wild population of sagebrush crickets, a species in which nuptial feeding by females imposes an unambiguous phenotypic marker on males. Multivariate selection analysis revealed a saddle-shaped fitness surface, suggesting a complex interplay between the total abundance of CHCs and specific CHC combinations in their influence on female choice. The fitness surface resulting from two axes of disruptive selection reflected a trade-off between short- and long-chained CHCs, suggesting that males may be sacrificing some level of desiccation resistance in favour of increased attractiveness. There was a significant correlation between male body size and total CHC abundance, suggesting that male CHCs provide females with a reliable cue for maximizing benefits obtained from males. Notwithstanding the conspicuousness of males' acoustic signals, our results suggest that selection imposed on males via female mating preferences may be far more complex than previously appreciated and operating in multiple sensory modalities.

Brood size, food availability, and body size affects male care decisions and offspring performance.

Parental care strategies do not only vary greatly across species, but also within species there can be substantial between- and within-individual variation in parental care behavior. To better understand the evolution of care strategies, it is crucial to determine how and when parents modify their behavior in response to internal as well as environmental factors. Here, we investigated the effect of brood size, resource size and an individual's quality on care strategies of uniparental males and examined the downstream consequences on offspring performance in the burying beetle Nicrophorus vespilloides. Burying beetles breed on small vertebrate cadavers and, on average, males invest much less in care than females. Nevertheless, we found that uniparentally caring males were responsive to their social and non-social environment and adjusted the amount as well as the type of care to the size of the brood, the size of the cadaver and their own body size. Additionally, we show that the care strategies affected offspring performance. Specifically, males that cared longer had larger and more surviving larvae. Our results add to our understanding of plastic parenting strategies by showing that even the sex that provides less care can evolve a very flexible care behavior.

Harsh nutritional environment has positive and negative consequences for family living in a burying beetle.

Harsh environmental conditions in form of low food availability for both offspring and parents alike can affect breeding behavior and success. There has been evidence that food scarce environments can induce competition between family members, and this might be intensified when parents are caring as a pair and not alone. On the other hand, it is possible that a harsh, food-poor environment could also promote cooperative behaviors within a family, leading, for example, to a higher breeding success of pairs than of single parents. We studied the influence of a harsh nutritional environment on the fitness outcome of family living in the burying beetle Nicrophorus vespilloides. These beetles use vertebrate carcasses for reproduction. We manipulated food availability on two levels: before and during breeding. We then compared the effect of these manipulations in broods with either single females or biparentally breeding males and females. We show that pairs of beetles that experienced a food-poor environment before breeding consumed a higher quantity of the carcass than well-fed pairs or single females. Nevertheless, they were more successful in raising a brood with higher larval survival compared to pairs that did not experience a food shortage before breeding. We also show that food availability during breeding and social condition had independent effects on the mass of the broods raised, with lighter broods in biparental families than in uniparental ones and on smaller carcasses. Our study thus indicates that a harsh nutritional environment can increase both cooperative as well as competitive interactions between family members. Moreover, our results suggest that it can either hamper or drive the formation of a family because parents choose to restrain reproductive investment in a current brood or are encouraged to breed in a food-poor environment, depending on former experiences and their own nutritional status.