Biomechanical adaptations enable phoretic mite species to occupy distinct spatial niches on host burying beetles.

Niche theory predicts that ecologically similar species coexist by minimizing interspecific competition through niche partitioning. Therefore, understanding the mechanisms of niche partitioning is essential for predicting interactions and coexistence between competing organisms. Here, we study two phoretic mite species, Poecilochirus carabi and Macrocheles nataliae that coexist on the same host burying beetle Nicrophorus vespilloides and use it to 'hitchhike' between reproductive sites. Field observations revealed clear spatial partitioning between species in distinct host body parts. Poecilochirus carabi preferred the ventral side of the thorax, whereas M. nataliae were exclusively found ventrally at the hairy base of the abdomen. Experimental manipulations of mite density showed that each species preferred these body parts, largely regardless of the density of the other mite species on the host beetle. Force measurements indicated that this spatial distribution is mediated by biomechanical adaptations, because each mite species required more force to be removed from their preferred location on the beetle. While P. carabi attached with large adhesive pads to the smooth thorax cuticle, M. nataliae gripped abdominal setae with their chelicerae. Our results show that specialist biomechanical adaptations for attachment can mediate spatial niche partitioning among species sharing the same host.

Higher temperature variability in deforested mountain regions impacts the competitive advantage of nocturnal species.

Deforestation is a major contributor to biodiversity loss, yet the impact of forest loss on daily microclimate variability and its implications for species with different daily activity patterns remain poorly understood. Using a recently developed microclimate model, we investigated the effects of deforestation on the daily temperature range (DTR) in low-elevation tropical regions and high-elevation temperate regions. Our results show that deforestation substantially increases DTR in these areas, suggesting a potential impact on species interactions. To test this hypothesis, we studied the competitive interactions between nocturnal burying beetles and all-day-active blowfly maggots in forested and deforested habitats in Taiwan. We show that deforestation leads to increased DTR at higher elevations, which enhances the competitiveness of blowfly maggots during the day and leads to a higher failure rate of carcass burial by the beetles at night. Thus, deforestation-induced temperature variability not only modulates exploitative competition between species with different daily activity patterns, but also likely exacerbates the negative impacts of climate change on nocturnal organisms. In order to limit potential adverse effects on species interactions and their ecological functions, our study highlights the need to protect forests, especially in areas where deforestation can greatly alter temperature variability.

Spatiotemporal niche partitioning in a specious silphid community (Coleoptera: Silphidae Nicrophorus).

Resource niche partitioning mediates the coexistence of similar species by reducing the chance of competitive encounters. For co-occurring species that share an ephemeral resource, contrasting activity in space and time may facilitate their persistence. Burying beetles (Silphidae: Nicrophorus) depend entirely on small vertebrate carcasses to reproduce. Given the unpredictability of this resource, and its value to congeners and other scavenger species, burying beetles likely endure intense competition to secure a carcass. Here, contrasting spatial and temporal niche patterns are explored as resource allocation strategies among five sympatric species of burying beetles (N. americanus, N. marginatus, N. pustulatus, N. orbicollis, and N. tomentosus). Specifically, the space-use and daily activity patterns are measured, at a fine scale, across species pairs to extrapolate contrasting niche-use patterns within a nicrophorine-rich grassland community in North-Central Oklahoma, USA. The results of this study reveal an important interplay between space-use and daily temporal activity in mediating the scramble competition associated with carrion resources. Where spatial or temporal overlap between burying beetle species is high, direct competition is mediated along an alternative niche dimension. For instance, N. americanus and N. orbicollis, a species dyad thought to be in direct competition, do overlap temporally but were found to have segregated space-use patterns. Our findings provide key insights into the competitive interactions within a necrophilous community and further inform our broader understanding of the spatial and temporal resource dimensions that drive the ecological niche.

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.

State-dependent cooperation in burying beetles: parents adjust their contribution towards care based on both their own and their partner's size.

Handicapping experiments on species with biparental care show that a focal parent increases its contribution when its partner is handicapped. Such results are interpreted as evidence for negotiation, whereby each parent adjusts its amount of care to that of its partner. However, it is currently unclear whether the focal parent responds to a change in its handicapped partner's behaviour or state. To address this gap, we conducted an experiment on the burying beetle Nicrophorus vespilloides where we first generated different-sized males and females by varying the duration of larval development. We then used a 2 × 2 factorial design in which a small or large male was paired with a small or large female. Small females provided less direct care (food provisioning and interactions with larvae) than large females, and both males and females provided less direct care when paired with a small partner. Thus, the focal parent adjusted its contribution towards care based on both its own state and that of its partner. There was also evidence for negotiation between the two parents as the focal parent adjusted its contribution based on the amount of care by its partner. However, there was no evidence that negotiation accounted for how the focal parent responded to its partner's size. Our results have important implications for our understanding of biparental cooperation as they show that each parent adjusts its contribution not only based on the amount of care provided by its partner but also based on its own state and its partner's state.

Seasonal Patterns of Resource Use Within Natural Populations of Burying Beetles.

For organisms in temperate environments, seasonal variation in resource availability and weather conditions exert fluctuating selection pressures on survival and fitness, resulting in diverse adaptive responses. By manipulating resource availability on a local spatial scale, we studied seasonal patterns of resource use within natural populations of burying beetles Nicrophorus vespilloides in a Norfolk woodland. Burying beetles are necrophagous insects that breed on vertebrate carcasses. They are active in Europe between April and October, after which they burrow into the soil and overwinter. Using breeding and chemical analyses, we compared the fecundity and physiological state of beetles that differed in their seasonal resource use. We found seasonal variation in carrion use by wild burying beetles and correlated differences in their reproductive success and cuticular hydrocarbon profiles. Our results provide novel insight into the seasonal correlates of behaviour, physiology and life history in burying beetles.

Competition among host-specific lineages of Poecilochirus carabi mites influences the extent of co-adaptation with their Nicrophorus vespilloides burying beetle hosts.

Reciprocal selection between symbiotic organisms and their hosts can generate variations in local adaptation between them. Symbionts often form species complexes with lineages partially adapted to various hosts. However, it is unclear how interactions among these lineages influences geographic variation in the extent of host-symbiont local adaptation. We addressed this shortcoming with experiments on burying beetles Nicrophorus vespilloides and their specialist phoretic mite Poecilochirus carabi in two adjacent woodlands. Burying beetles transport these mites to vertebrate carrion upon which they both reproduce. P. carabi appears to be a species complex, with distinct lineages that specialise on breeding alongside different Nicrophorus species. We found that in one wood (Gamlingay Woods), N. vespilloides carries a mixture of mite lineages, with each lineage corresponding to one of the four Nicrophorus species that inhabits this wood. However, two burying beetle species coexist in neighbouring Waresley Woods and here N. vespilloides predominantly carries the mite lineage that favours N. vespilloides. Mite lineage mixing alters the degree of local adaptation for both N. vespilloides and the P. carabi mites, affecting reproductive success variably across different woodlands. In Gamlingay, mite lineage mixing reduced N. vespilloides reproductive success, while experimentally purifying mites lineage enhanced it. The near pure lineage of vespilloides mites negligibly affected Waresley N. vespilloides. Mite reproductive success varied with host specificity: Gamlingay mites had greatest reproductive success on Gamlingay beetles, and performed less well with Waresley beetles. By contrast, Waresley mites had consistent reproductive success, regardless of beetle's woodland of origin. We conclude that there is some evidence that N. vespilloides and its specific mite lineage have coadapted. However, neither N. vespilloides nor its mite lineage adapted to breed alongside other mite lineages. This, we suggest, causes variation between Waresley and Gaminglay Woods in the extent of local adaptation between N. vespilloides beetles and their P. carabi mites.

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.

Effect of Short-Term Desiccation, Recovery Time, and CAPA-PVK Neuropeptide on the Immune System of the Burying Beetle Nicrophorus vespilloides.

Environmental conditions, especially related to winter, are crucial for shaping activity of insect immune system. However, our previous research clearly indicates differences in the immune system functioning when the cold stress was induced in the laboratory conditions and when the beetles were collected from natural environment during winter. This is probably related to the multiplication of observed effects by simultaneous presence of different stress factors characteristic of winter, including desiccation. For these reasons, our next step was analysis of the effects of short-term desiccation and recovery time on the functioning of immune system of burying beetle Nicrophorus vespilloides. Also, the effect of Tenmo-PVK-2 (tenebrionid periviscerokinin), member of the CAPA-PVK neuropeptide family, was investigated to better understand observed changes. Short-term desiccation decreases the phagocytic activity of burying beetle haemocytes, which is correlated with a reduction in their adhesive ability. On the other hand, there was a significant increase in phenoloxidase (PO) activity and the level of proPO expression, which may suggest sealing the cuticula by melanin deposition and prevention of water loss. Additionally, the elevated level of defensin expression may be associated with the cross-talk between mechanisms, which participate in insect response to environmental stress, including pathogen infection. After 1 h of recovery time, the activity of tested cellular and humoral mechanisms was mostly back to the control level. However, inhibition of the activity of PO and down-regulation of proPO were noted. These results also indicate importance of melanin deposition during water loss. Moreover, it suggests that some changes in immune system functioning during stress conditions do not have an immune function. Interestingly, part of the effects characteristic of recovery time were also observed after the application of Tenmo-PVK-2, mainly related to haemocyte morphology. These results indicate that CAPA-PVK neuropeptides may also influence on activity of burying beetle immune system. It should be also highlighted that, because of the study of the effects of CAPA-PVK neuropeptides, homologs of vertebrate neuromedin U, the results may be interesting for search evolutionary similarities in the functioning of the neuroendocrine system of insects and vertebrates.

Antagonistic effects of intraspecific cooperation and interspecific competition on thermal performance.

Understanding how climate-mediated biotic interactions shape thermal niche width is critical in an era of global change. Yet, most previous work on thermal niches has ignored detailed mechanistic information about the relationship between temperature and organismal performance, which can be described by a thermal performance curve. Here, we develop a model that predicts the width of thermal performance curves will be narrower in the presence of interspecific competitors, causing a species' optimal breeding temperature to diverge from that of its competitor. We test this prediction in the Asian burying beetle Nicrophorus nepalensis, confirming that the divergence in actual and optimal breeding temperatures is the result of competition with their primary competitor, blowflies. However, we further show that intraspecific cooperation enables beetles to outcompete blowflies by recovering their optimal breeding temperature. Ultimately, linking abiotic factors and biotic interactions on niche width will be critical for understanding species-specific responses to climate change.

Insects, reptiles and many other animals are often referred to as being 'cold-blooded' because, unlike mammals and birds, their body temperature fluctuates with the temperature of their surrounding environment. As a result, many cold-blooded animals are very sensitive to changes in local climate. Environmental factors, such as temperature and precipitation, as well biotic factors, such as two species competing for food or the presence of a predator, may influence how well an animal performs at different temperatures. However, few studies have examined how both environmental and biotic factors affect the range of temperatures in which a cold-blooded animal is able to survive and reproduce. When Asian burying beetles reproduce, they lay their eggs around buried animal carcasses that can provide food for their offspring. Previous studies have found that individual burying beetles can cooperate with each other to defend themselves against their main competitor, blowflies, which also lay their eggs on animal carcasses. Here, Tsai et al. used mathematical and experimental approaches to study how blowflies affect the range of temperatures in which burying beetles are able to live under different environmental conditions. The experiments showed that when blowflies were present, the range of temperatures that burying beetles were able to survive and reproduce in was smaller. Furthermore, the optimal temperature for the burying beetles to live in shifted back, away from that of their competitor. Larger groups of burying beetles were able to survive and reproduce in a greater range of temperatures than smaller groups, even when blowflies were present. This suggests that increasing the amount bury beetles cooperate with each other may make them more resilient to changes in temperature. The Earth is currently experiencing a period of climate change and therefore it is important to understand how different species of animals may respond to to changing temperatures. These findings reinforce the idea that even a small change in temperature may lead to changes in how different species interact with each other, which in turn influences the ecosystem in which they live.

Temperature stress induces mites to help their carrion beetle hosts by eliminating rival blowflies.

Ecological conditions are known to change the expression of mutualisms though the causal agents driving such changes remain poorly understood. Here we show that temperature stress modulates the harm threatened by a common enemy, and thereby induces a phoretic mite to become a protective mutualist. Our experiments focus on the interactions between the burying beetle Nicrophorus vespilloides, an associated mite species Poecilochirus carabi and their common enemy, blowflies, when all three species reproduce on the same small vertebrate carrion. We show that mites compete with beetle larvae for food in the absence of blowflies, and reduce beetle reproductive success. However, when blowflies breed on the carrion too, mites enhance beetle reproductive success by eating blowfly eggs. High densities of mites are especially effective at promoting beetle reproductive success at higher and lower natural ranges in temperature, when blowfly larvae are more potent rivals for the limited resources on the carcass.

Larval morphology of Nicrophorus (Nicrophorus) nepalensis Hope (Coleoptera: Silphidae: Nicrophorinae).

Larval morphology of all three instars of Nicrophorus (Nicrophorus) nepalensis Hope, 1831 (Coleoptera: Silphidae: Nicrophorinae) is described and illustrated, based on reared larvae. The eastern Palaearctic and Oriental N. (N.) nepalensis is similar to the western Palaearctic N. (N.) humator (Gleditsch, 1767) in a number of larval characters. This is congruent with recent classification of the genus Nicrophorus Fabricius, 1775 by Sikes, who suggested a close phylogenetic affinity of the N. nepalensis species group (with 16 species) with the N. humator species group (2 species). The generic description of larvae of Nicrophorus Fabricius, 1775 is expanded, based on detailed SEM observation.

Rapid local adaptation linked with phenotypic plasticity.

Models of "plasticity-first" evolution are attractive because they explain the rapid evolution of new complex adaptations. Nevertheless, it is unclear whether plasticity can facilitate rapid microevolutionary change between diverging populations. Here, we show how plasticity may have generated adaptive differences in fecundity between neighboring wild populations of burying beetles Nicrophorus vespilloides. These populations occupy distinct Cambridgeshire woodlands that are just 2.5 km apart and that probably originated from a common ancestral population about 1000-4000 years ago. We find that populations are divergently adapted to breed on differently sized carrion. Adaptive differences in clutch size and egg size are associated with divergence at loci connected with oogenesis. The populations differ specifically in the elevation of the reaction norm linking clutch size to carrion size (i.e., genetic accommodation), and in the likelihood that surplus offspring will be lost after hatching. We suggest that these two processes may have facilitated rapid local adaptation on a fine-grained spatial scale.

Myotropic activity and immunolocalization of selected neuropeptides of the burying beetle Nicrophorus vespilloides (Coleoptera: Silphidae).

Burying beetles (Nicrophorus sp.) are necrophagous insects with developed parental care. Genome of Nicrophorus vespilloides has been recently sequenced, which makes them interesting model organism in behavioral ecology. However, we know very little about their physiology, including the functioning of their neuroendocrine system. In this study, one of the physiological activities of proctolin, myosuppressin (Nicve-MS), myoinhibitory peptide (Trica-MIP-5) and the short neuropeptide F (Nicve-sNPF) in N. vespilloides have been investigated. The tested neuropeptides were myoactive on N. vespilloides hindgut. After application of the proctolin increased hindgut contraction frequency was observed (EC50 value was 5.47 × 10-8 mol/L). The other tested neuropeptides led to inhibition of N. vespilloides hindgut contractions (Nicve-MS: IC50 = 5.20 × 10-5 mol/L; Trica-MIP-5: IC50 = 5.95 × 10-6 mol/L; Nicve-sNPF: IC50 = 4.08 × 10-5 mol/L). Moreover, the tested neuropeptides were immunolocalized in the nervous system of N. vespilloides. Neurons containing sNPF and MIP in brain and ventral nerve cord (VNC) were identified. Proctolin-immunolabeled neurons only in VNC were observed. Moreover, MIP-immunolabeled varicosities and fibers in retrocerebral complex were observed. In addition, our results have been supplemented with alignments of amino acid sequences of these neuropeptides in beetle species. This alignment analysis clearly showed amino acid sequence similarities between neuropeptides. Moreover, this allowed to deduce amino acid sequence of N. vespilloides proctolin (RYLPTa), Nicve-MS (QDVDHVFLRFa) and six isoforms of Nicve-MIP (Nicve-MIP-1-DWNRNLHSWa; Nicve-MIP-2-AWQNLQGGWa; Nicve-MIP-3-AWQNLQGGWa; Nicve-MIP-4-AWKNLNNAGWa; Nicve-MIP-5-SEWGNFRGSWa; Nicve-MIP-6- DPAWTNLKGIWa; and Nicve-sNPF-SGRSPSLRLRFa).

Dose-independent virulence in phoretic mites that parasitize burying beetles.

Virulence, the negative impact of parasites on their hosts, typically increases with parasite dose. Parasites and hosts often compete for host resources and more parasites will consume more resources. Depending on the mechanism of competition, increasing host resources can benefit the host. Additional resources can also be harmful when the parasites are the main beneficiaries. Then, the parasites will thrive and virulence increases. While parasite dose is often easy to manipulate, it is less trivial to experimentally scale host resources. Here, we study a system with external host resources that can be easily manipulated: Nicrophorus burying beetles reproduce on vertebrate carcasses, with larger carcasses yielding more beetle offspring. Phoretic Poecilochirus mites reproduce alongside the beetles and reduce beetle fitness. The negative effect of mites could be due to competition for the carrion between beetle and mite offspring. We manipulated mite dose and carcass size to better understand the competition between the symbionts. We found that mite dose itself was not a strong predictor of virulence. Instead, the number of mite offspring determined beetle fitness. At larger doses, there was strong competition among adult parental mites as well as mite offspring. While increasing the carcass size increased both host and parasite fitness, it did surprisingly little to alleviate the negative effect that mites had on beetles. Instead, relative virulence was stronger on large carcasses, indicating that the parasites appropriate more of the additional resources. Our results demonstrate an ecological influence on the selection of parasites on their hosts and suggest that virulence can be dose-independent in principle.

The early-life environment and individual plasticity in life-history traits.

We tested whether the early-life environment can influence the extent of individual plasticity in a life-history trait. We asked: can the early-life environment explain why, in response to the same adult environmental cue, some individuals invest more than others in current reproduction? Moreover, can it additionally explain why investment in current reproduction trades off against survival in some individuals, but is positively correlated with survival in others? We addressed these questions using the burying beetle, which breeds on small carcasses and sometimes carries phoretic mites. These mites breed alongside the beetle, on the same resource, and are a key component of the beetle's early-life environment. We exposed female beetles to mites twice during their lives: during their development as larvae and again as adults during their first reproductive event. We measured investment in current reproduction by quantifying average larval mass and recorded the female's life span after breeding to quantify survival. We found no effect of either developing or breeding alongside mites on female reproductive investment, nor on her life span, nor did developing alongside mites influence her size. In post hoc analyses, where we considered the effect of mite number (rather than their mere presence/absence) during the female's adult breeding event, we found that females invested more in current reproduction when exposed to greater mite densities during reproduction, but only if they had been exposed to mites during development as well. Otherwise, they invested less in larvae at greater mite densities. Furthermore, females that had developed with mites exhibited a trade-off between investment in current reproduction and future survival, whereas these traits were positively correlated in females that had developed without mites. The early-life environment thus generates individual variation in life-history plasticity. We discuss whether this is because mites influence the resources available to developing young or serve as important environmental cues.

Divergent coevolutionary trajectories in parent-offspring interactions and discrimination against brood parasites revealed by interspecific cross-fostering.

In animal families, parents are expected to adapt to their offspring's traits, and offspring, in turn, are expected to adapt to the environment circumscribed by their parents. However, whether such coevolutionary trajectories differ between closely related species is poorly understood. Here, we employ interspecific cross-fostering in three species of burying beetles, Nicrophorus orbicollis, Nicrophorus pustulatus and Nicrophorus vespilloides, to test for divergent co-adaptation among species with different degrees of offspring dependency on parental care, and to test whether they are able to discriminate against interspecific parasites. We found that offspring survival was always higher when offspring were reared by conspecific rather than heterospecific parents. In the case of N. orbicollis raising N. pustulatus, none of the larvae survived. Overall, these results indicate that parent and offspring traits have diverged between species, and that the differential survival of conspecific and heterospecific larvae is because of improper matching of co-adapted traits, or, in the case of N. orbicollis with larval N. pustulatus, because of selection on parents to recognize and destroy interspecific brood parasites. We suggest that burying beetles experiencing a high risk of brood parasitism have evolved direct recognition mechanisms that enable them to selectively kill larvae of potential brood parasites.

Impact of cold on the immune system of burying beetle, Nicrophorus vespilloides (Coleoptera: Silphidae).

Insect overwintering is one of the most astonishing phases of the insect life cycle. Despite vast amounts of knowledge available about the physiological mechanisms of this phenomenon, the impact of stress factors on insect immune system functioning during the winter is still unknown. The aim of this study is to analyze how low temperatures influence the immune system of the beetle Nicrophorus vespilloides. The results show that the beetle's immune system is differently modulated by cold induced in laboratory settings than that which occurs in natural conditions. Among beetles cultured in conditions similar to summer, low temperatures, did not influence the number of circulating haemocytes, phenoloxidase activity, haemocytes morphology, and percentage ratio of haemocyte types. In these beetles, differences were noted only in the ability of haemocytes to perform phagocytosis. Individuals acclimated in natural conditions in autumn had a higher level of humoral response and a different percentage ratio of haemocyte types. During the winter period, the number of haemocytes in the beetles decreased, but the percentage ratio of phagocytic haemocytes increased. Furthermore, we noted an increase of phenoloxidase activity. Our study also showed mitotic divisions of haemocytes in haemolymph collected from burying beetles after cold exposure and from burying beetles collected from natural conditions during autumn and winter. Differences in response to low temperatures in laboratory conditions and the natural environment suggest that the simultaneous presence of other stress factors during winter such as desiccation and starvation have a significant influence on the activity of burying beetle's immune system.

Phoretic Poecilochirus mites specialize on their burying beetle hosts.

Recurring species interactions can cause species to adapt to each other. Specialization will increase the fitness of symbionts in the coevolved association but may reduce the flexibility of symbiont choice as it will often decrease fitness in interactions with other than the main symbiont species. We analyzed the fitness interactions between a complex of two cryptic mite species and their sympatric burying beetle hosts in a European population. Poecilochirus mites (Mesostigmata, Parasitidae) are phoretic on burying beetles and reproduce alongside beetles, while these care for their offspring at vertebrate carcasses. While Poecilochirus carabi is typically found on Nicrophorus vespilloides beetles, P. necrophori is associated with N. vespillo. It has long been known that the mites discriminate between the two beetle species, but the fitness consequences of this choice remained unknown. We experimentally associated both mite species with both beetle species and found that mite fitness suffered when mites reproduced alongside a nonpreferred host. In turn, there is evidence that one of the beetle species is better able to cope with the mite species they are typically associated with. The overall fitness effect of mites on beetles was negative in our laboratory experiments. The Poecilochirus mites studied here are thus specialized competitors or parasites of burying beetles.