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.

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.

Female choice in the red mason bee, Osmia rufa (L.) (Megachilidae).

Females are often thought to use several cues and more than one modality in selection of a mate, possibly because they offer complementary information on a mate's suitability. In the red mason bee, Osmia rufa, we investigated the criteria a female uses to choose a mating partner. We hypothesized that the female uses male thorax vibrations and size as signs of male viability and male odor for kin discrimination and assessment of genetic relatedness. We therefore compared males that had been accepted by a female for copulation with those rejected, in terms of their size, their immediate precopulatory vibrations (using laser vibrometry), the genetic relatedness of unmated and mated pairs (using microsatellite markers) and emitted volatiles (using chemical analyses). Females showed a preference for intermediate-sized males that were slightly larger than the modal male size. Furthermore, male precopulatory vibration burst duration was significantly longer in males accepted for copulation compared with rejected males. Vibrations may indicate vigor and assure that males selected by females are metabolically active and healthy. Females preferentially copulated with males that were genetically more closely related, possibly to avoid outbreeding depression. Volatiles of the cuticular surface differed significantly between accepted and rejected males in the relative amounts of certain hydrocarbons, although the relationship between male odor and female preference was complex. Females may therefore also use differences in odor bouquet to select among males. Our investigations show that O. rufa females appear to use multiple cues in selecting a male. Future investigations are needed to demonstrate whether odor plays a role in kin recognition and how the multiple cues are integrated in mate choice by females.

Divergence in male sexual odor signal and genetics across populations of the red mason bee, Osmia bicornis, in Europe.

In some insect species, females may base their choice for a suitable mate on male odor. In the red mason bee, Osmia bicornis, female choice is based on a male's odor bouquet as well as its thorax vibrations, and its relatedness to the female, a putative form of optimal outbreeding. Interestingly, O. bicornis can be found as two distinct color morphs in Europe, which are thought to represent subspecies and between which we hypothesize that female discrimination may be particularly marked. Here we investigated (i) if these two colors morphs do indeed represent distinct, reproductively differentiated populations, (ii) how odor bouquets of male O. bicornis vary within and between populations, and (iii) whether variation in male odor correlates with genetic distance, which might represent a cue by which females could optimally outbreed. Using GC and GC-MS analysis of male odors and microsatellite analysis of males and females from 9 populations, we show that, in Denmark, an area of subspecies sympatry, the two color morphs at any one site do not differ, either in odor bouquet or in population genetic differentiation. Yet populations across Europe are distinct in their odor profile as well as being genetically differentiated. Odor differences do not, however, mirror genetic differentiation between populations. We hypothesize that populations from Germany, England and Denmark may be under sexual selection through female choice for local odor profiles, which are not related to color morph though which could ultimately lead to population divergence and speciation.

The effect of temperature on male mating signals and female choice in the red mason bee, Osmia bicornis (L.).

Climate change and the resulting changes in air temperature are known to have a major influence on most animals, especially poikilothermic insects, because they depend on the high enough temperatures to function. Previous studies have shown that various signals can be affected by changes in temperature. However, research into the effect of temperature on mating signals and subsequently communication between mates and on female choice is still rare. In the red mason bee, Osmia bicornis, which emerges early in spring and is therefore subject to extensive temperature changes, females choose suitable males based on, among other criteria, their thorax vibrations and odor. So far there has been no research into the effect of temperature changes on these signals. We therefore investigated whether the environmental temperature has an influence on the male's mating signals by measuring vibrations using a laser vibrometer and collecting male and female odor at different temperatures. Furthermore, we performed behavioral experiments in order to show whether there is an effect of temperature-induced changes and female choice. Our results showed that rejected males differed in their vibrations between the two temperature settings but accepted ones did not. Temperature changes therefore seem to have a stronger effect on those males that are rejected by the females, whereas the accepted males are the ones that can produce desirable signals despite temperature fluctuations. Furthermore, we found that the differences in odor profiles were greater between temperature settings than between the sexes and that females change their preference for odor with temperature. We conclude that temperature strongly influences the male mating signals and therefore may have a major impact on sexual selection in this species. This is an important aspect to consider, not only in future studies on mating behavior, but also in view of our ever raising temperatures.

The role of vibrations in population divergence in the red mason bee, Osmia bicornis.

Differences in female preference for certain male characteristics can be a driving force for population divergence and speciation [1-4]. During precopulation, females of the red mason bee, Osmia bicornis, choose suitable males based on, among other criteria, their thoracic vibrations [5]. These vibrations are thought to be a signal of a male's fitness with females choosing the strongest males that can vibrate for the longest time [5]. The precise role of such vibrational signals, however, has not been determined by bioassays, and the vibrations might also play a role in species recognition [6]. There are two main subspecies of O. bicornis in Europe distinguishable only by a single morphological trait [7] (Figure S1). We therefore developed a new bioassay allowing us to impose the vibrations of one live male onto another in order to discern possible selective mate choice by females from O. bicornis originating from different regions of Europe. Females showed strong preference for males from their own region, and male vibrations were the main signal involved in this choice. Thus, vibrational signals encode not only fitness but also information about the region of origin indicating that divergence exists between the different European O. bicornis populations, which might ultimately lead to speciation. These results provide new insights into the scope of vibrational communication in bees, a group previously considered to rely predominantly on chemical signals [8, 9]. Our newly developed method should shed further light on many exciting questions concerning vibrational communication in bees and other animal taxa.