The results of our study reveal new insights into the proximate causes of the extreme dependence of N. orbicollis offspring on parental care, and the variation in offspring dependence among species. We found that starvation tolerance of larvae varied among species, but did not appear to be related to dependence on parental care. Newly hatched N. orbicollis were generally able to self-feed, but the capacity for utilizing different types of food was more limited than in the more independent species, N. pustulatus and N. vespilloides. Dependent N. orbicollis gained less weight when self-feeding than nutritionally independent N. pustulatus. In addition, our study revealed that even a highly processed liquefied carrion meal is not sufficient to secure larval survival in N. orbicollis; however, oral secretions of parents mixed into a purée of baby mice prolong the survival of larval N. orbicollis without parents, but not long enough for most larvae to pupate. Finally, we revealed that three hours of post-hatching care was sufficient to achieve a significant increase in the survival and final mass of the larvae of the most dependent species, N. orbicollis. Our results highlight key characteristics of offspring and parental traits that augment our understanding of offspring dependence on parental care. Below, we elaborate on the wider implications of these results.
The results of our first experiment make it unlikely that starvation tolerance is related to high levels of offspring dependence, but is instead more likely associated with variation in growth rate. Here, we tested whether larvae of the three species, N. orbicollis, N. pustulatus, and N. vespilloides, differ in their tolerance to starvation in the absence of parents. Combined with information on egg investment, represented by mass at hatching (see Fig. 3), the level of starvation tolerance could provide information on whether larvae are fast or slow metabolisers, or on the parental investment in egg composition, and could thus be related to the marked offspring dependence on parental provisioning in N. orbicollis. As expected, we found that offspring of the more independent species, N. pustulatus, were most tolerant to starvation and survived the longest in the absence of food. Surprisingly, however, the highly dependent larvae of N. orbicollis starved to death later than larvae of N. vespilloides, which show an intermediate dependence on parental care . Here, hatchlings of N. vespilloides were the heaviest, followed by hatchlings of N. orbicollis, and then N. pustulatus, the lightest of the three species (but see ). Given their low mass at hatching, it is even more striking that most of the larval N. pustulatus were still alive when larvae of the other two species had all starved to death, suggesting that N. pustulatus are slow metabolisers. Generally, starvation resistance tends to increase with body size and larger energy stores, despite the greater absolute energy needs of larger individuals . However, larval N. vespilloides are not only the heaviest at hatching, but also have the highest growth rate of the three species during the first 48 h with full care . Faster growth rates are usually associated with a greater need for food and higher metabolic rates, making fast-growing individuals, such as N. vespilloides, more vulnerable to starvation when resources are limited .
The aim of our second experiment was to investigate whether hatchlings of N. orbicollis are able to self-feed, or whether traits necessary for self-feeding only develop at a later larval stage compared to the more independent species, which might explain strong offspring dependency on parental care. Larvae of passalid beetles, for example, differ in their ability to feed themselves and to construct feeding tunnels, and in their dependency on parental care . Here, we found that newly hatched larvae of N. orbicollis were generally able to self-feed and gain weight when reared on baby mice, but not on prepared carcasses that parents usually use as a food resource for their offspring in nature. In contrast, no clear pattern was found in the two more independent species as larvae also increased in mass when provided with parent-prepared carcasses. It might not be surprising that larvae gain more weight on pieces of baby mice than, for example, on prepared carrion. First, baby mice are younger and probably have a higher water content, but fewer hard body parts than adult mice, making them more easily accessible for larvae. Second, pieces of baby mice are certainly fresher than the parent-prepared carrion. Further, the larvae of different species obviously differ in their ability to access and process different types of vertebrate carrion, which could be related to quantitative or qualitative differences in the oral digestive enzymes of larvae. It may be that the digestive system of young N. orbicollis hatchlings has evolved to rely more on pre-digested food from parents at the beginning, and later on, the slightly older larvae become able to consume and assimilate solid food on their own.
Alternatively, larval ability to self-feed might depend on species-specific characteristics of the mandibles. It is conceivable that mouthparts of N. orbicollis larvae may develop and sclerotize at a slower rate than the mouthparts of the other two species in our study, resulting in less robust mandibles that do not allow larvae to self-feed initially. Pukowski  observed that hatchlings and recently moulted larvae of N. vespillo are unable to self-feed, and ascribed this to their unsclerotized mouthparts. Only after five to six hours, were larvae observed to self-feed . Thus, differences in the sclerotization rate of mandibles could contribute to the variation in self-feeding and offspring dependence on parental care. In species with obligatory parental care, such as N. orbicollis, selection on mandible sclerotization rate or other traits, such as the production of digestive enzymes that could facilitate nutritional independence of offspring, may be relaxed as parents assume a greater share of the services related to food intake. As the expression and maintenance of these traits is generally costly , traits related to self-feeding may only be expressed later in life when parents withdraw from providing parental care and offspring need to become independent. Generally, as soon as offspring traits are no longer in use because parents take over the tasks that secure offspring survival by providing parental care, a reduction in the relevant offspring traits is expected. This reduction, in turn, further drives the evolution of increased offspring dependency on parental care. For example, first instar neonates of wood-feeding Cryptocercus cockroaches, which exhibit elaborate biparental care, completely lack eyes and have a pale and thin cuticle . The hindgut symbionts that help larvae to metabolise and digest wood are not fully established until the third larval instar . Consequently, until that time, nymphs depend on their parents for nutrition and symbiont transfer . Like Cryptocercus, first instar larvae of wood-feeding Salganea have a pale and transparent cuticle, and their eyes are present, but considerably reduced . Larvae feed on parental oral fluids and are somewhat less dependent than larval Cryptocercus, but more dependent than Panesthia neonates that are well developed and show no interactions with parents . In these three genera, the developmental characteristics of neonates appear to parallel a gradient of dependence on parental care .
Eggert et al.  showed that 12 h of parental care resulted in a significant increase in survival and growth of larval N. vespilloides, suggesting that this was due, in part, to the opening in the carcass that is created by the parents, thereby facilitating easier access of the larvae to the carrion. In an experimental evolution study, larvae descended from beetles reared in the absence of post-hatching care became increasingly independent, a result that was attributed to the ability of larvae to self-feed more efficiently or through morphological adaptation of larval mouthparts . Although these behavioural or morphological adaptations are undoubtedly advantageous, their absence in larval N. orbicollis alone cannot explain their nutritional dependency. In our study, even an opening in the integument of a prepared carcass did not increase the efficiency of larvae to self-feed. Also, although larval N. orbicollis were able to consume small pieces of juvenile mouse carcasses, none of the larvae were able to survive more than 24 h in the absence of parents (A. Capodeanu-Nägler, pers. obs.). Even when provided with liquefied mouse carrion, most of the larvae did not survive to pupation.
One other factor that could account for the differences in self-feeding is the behaviour of larvae towards food when parents are absent. From a study on N. vespilloides, we know that larvae cooperate to penetrate the carcass when parents are absent . One precondition for cooperation between siblings is that larvae need to aggregate first. Generally, larvae seem to be attracted to one another and without another larva, larvae of the more independent species may have directly attempted to feed. However, larvae of N. orbicollis that benefit most from their parents’ care, might be selected to focus on approaching their parents instead of converging to other larvae. Thus, especially when carcass preparation indicates the presence of parents by parent-derived cues on the carcass surface, larvae might wander around and search for a parent instead of attempting to feed (A. Capodeanu-Nägler, pers. obs.). Nevertheless, behavioural observations are needed to confirm these predictions.
Having shown that highly dependent N. orbicollis larvae are able to self-feed and increase in weight when provided with small pieces of baby mice, we attempted to determine whether they could be successfully reared in the absence of parents on a diet of homogenized mouse carrion mixed with oral secretions from parental beetles. We found that larvae reared on this diet were more likely to survive to dispersal than larvae receiving the same diet but without parental secretions. Thus, oral secretions of parents are clearly beneficial to N. orbicollis larvae, and may contain important symbionts, antimicrobial compounds, enzymes, or hormones. Eggert et al.  examined the importance of symbiont transfer in N. vespilloides, but found that the positive effects of parental provisioning on larval survival and growth were not mediated by the transfer of symbionts. However, the transfer of symbionts in N. orbicollis may be more important as larvae in this species are more dependent on parental provisioning. In addition, the beetles’ anal and oral secretions contain a wide range of compounds, some of which have antimicrobial properties [49,50,51], and express a variety of immune-related genes  with several antimicrobial peptides and lysozymes that are specifically upregulated in the presence of carrion [53, 54], and that could enhance offspring survival. Finally, parents may transfer growth-regulatory proteins or hormones that are essential for survival and development of dependent offspring. Juvenile hormone III (JH III), for example, has recently been found to be transferred to larvae by trophallaxis in ants . In burying beetles, JH III plays a regulatory role in a multiple contexts [55,56,57,58,59,60,61], and parents might thus transfer some JH III when they regurgitate to larvae, which may contribute to their survival and growth (but see ).
Alternatively, oral secretions might signal the presence of parents to offspring. Carpenter ants, for instance, have been shown to exchange chemical signals by trophallaxis that help them to recognize nestmates [20, 21]. Likewise, oral secretions of burying beetles might have a signalling function that helps larvae to localize pre-digested food or initiates larval feeding. Nonetheless, despite receiving homogenized carrion mixed with oral secretions of parents, most of the larvae of N. orbicollis did not survive until dispersal. However, since we do not know the actual volume of oral fluids that parents transfer to larvae, we may have provided larvae with less than the requisite amount of oral secretions. In our last experiment, we showed that larval survival and mass of N. orbicollis increased with the duration of post-hatching care, which is not surprising as parental care usually enhances offspring fitness [7, 8]. More surprisingly, we found that survival of the highly dependent N. orbicollis larvae was significantly enhanced after only three hours of parental care. Why might such a short period of care have such a profound effect on offspring survival? For N. vespilloides, larval begging as well as parental provisioning is known to peak 24 h after hatching [32, 63]. However, we observed larvae begging and parents provisioning in the first three hours after hatching (A. Capodeanu-Nägler and M. Prang, pers. obs.). Thus, parents might provide begging larvae with enough food during these first few hours that larvae have sufficient energy to survive until they are efficient self-feeders.
In the light of the other results of our study, however, we find it more likely that the transfer of oral secretions and maybe also anal secretions might be crucial for larval survival and growth, especially in the first few hours after larval hatching. For example, if larvae are given a single dose of symbionts by the parents in the first three hours after hatching, they may be able to survive thereafter. Burying beetles are known to harbour a diverse gut microbiome including various Yarrowia-like yeasts . Yarrowia are present in both adult and larval life stages, and are possibly involved in carrion digestion and preservation . More recent studies have shown that burying beetle parents not only transfer microorganisms to larvae via oral secretions, but tightly regulate the microbiome of the carcass by applying anal and oral secretions to it, which serves not only as a nutritional resource, but also facilitates the vertical transmission of symbiotic microbiota to larvae [66,67,68]. Thus, the transfer of preservation- and digestion-related microbiota to the carcass during the first hours might enhance larval survival for the more dependent offspring of N. orbicollis after parents have been removed.