Cryptic diversity within the Poecilochirus carabi mite species complex phoretic on Nicrophorus burying beetles: Phylogeny, biogeography, and host specificity.

Coevolution is often considered a major driver of speciation, but evidence for this claim is not always found because diversity might be cryptic. When morphological divergence is low, molecular data are needed to uncover diversity. This is often the case in mites, which are known for their extensive and often cryptic diversity. We studied mites of the genus Poecilochirus that are phoretic on burying beetles (Silphidae: Nicrophorus). Poecilochirus taxonomy is poorly understood. Most studies on this genus focus on the evolutionary ecology of Poecilochirus carabi sensu lato, a complex of at least two biological species. Based on molecular data of 230 specimens from 43 locations worldwide, we identified 24 genetic clusters that may represent species. We estimate that these mites began to diversify during the Paleogene, when the clade containing P. subterraneus branched off and the remaining mites diverged into two further clades. One clade resembles P. monospinosus. The other clade contains 17 genetic clusters resembling P. carabi s.l.. Among these are P. carabi sensu stricto, P. necrophori, and potentially many additional cryptic species. Our analyses suggest that these clades were formed in the Miocene by large-scale geographic separation; co-speciation of mites with the host beetles can be largely ruled out. Diversification also seems to have happened on a smaller scale, potentially due to adaptation to specific hosts or local abiotic conditions, causing some clusters to specialize on certain beetle species. Our results suggest that biodiversity in this genus was generated by multiple interacting forces shaping the tangled webs of life.

Appetitive olfactory learning suffers in ants when octopamine or dopamine receptors are blocked.

Associative learning relies on the detection of coincidence between a stimulus and a reward or punishment. In the insect brain, this process is carried out in the mushroom bodies under the control of octopaminergic and dopaminergic neurons. It was assumed that appetitive learning is governed by octopaminergic neurons, while dopamine is required for aversive learning. This view has recently been challenged: both neurotransmitters are involved in both types of learning in bees and flies. Here, we tested which neurotransmitters are required for appetitive learning in ants. We trained Lasius niger workers to discriminate two mixtures of linear hydrocarbons and to associate one of them with a sucrose reward. We analysed the walking paths of the ants using machine learning and found that the ants spent more time near the rewarded odour than near the other, a preference that was stable for at least 24 h. We then treated the ants before learning with either epinastine, an octopamine receptor blocker, or flupentixol, a dopamine receptor blocker. Ants with blocked octopamine receptors did not prefer the rewarded odour. Octopamine signalling is thus necessary for appetitive learning of olfactory cues, probably because it signals information about odours or reward to the mushroom body. In contrast, ants with blocked dopamine receptors initially learned the rewarded odour but failed to retrieve this memory 24 h later. Dopamine is thus probably required for long-term memory consolidation, independent of short-term memory formation. Our results show that appetitive olfactory learning depends on both octopamine and dopamine signalling in ants.

Comparative transcriptomic analysis of the mechanisms underpinning ageing and fecundity in social insects.

The exceptional longevity of social insect queens despite their lifelong high fecundity remains poorly understood in ageing biology. To gain insights into the mechanisms that might underlie ageing in social insects, we compared gene expression patterns between young and old castes (both queens and workers) across different lineages of social insects (two termite, two bee and two ant species). After global analyses, we paid particular attention to genes of the insulin/insulin-like growth factor 1 signalling (IIS)/target of rapamycin (TOR)/juvenile hormone (JH) network, which is well known to regulate lifespan and the trade-off between reproduction and somatic maintenance in solitary insects. Our results reveal a major role of the downstream components and target genes of this network (e.g. JH signalling, vitellogenins, major royal jelly proteins and immune genes) in affecting ageing and the caste-specific physiology of social insects, but an apparently lesser role of the upstream IIS/TOR signalling components. Together with a growing appreciation of the importance of such downstream targets, this leads us to propose the TI-J-LiFe (TOR/IIS-JH-Lifespan and Fecundity) network as a conceptual framework for understanding the mechanisms of ageing and fecundity in social insects and beyond. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns?'

Queen loss increases worker survival in leaf-cutting ants under paraquat-induced oxidative stress.

Longevity is traded off with fecundity in most solitary species, but the two traits are positively linked in social insects. In ants, the most fecund individuals (queens and kings) live longer than the non-reproductive individuals, the workers. In many species, workers may become fertile following queen loss, and recent evidence suggests that worker fecundity extends worker lifespan. We postulated that this effect is in part owing to improved resilience to oxidative stress, and tested this hypothesis in three Myrmicine ants: Temnothorax rugatulus, and the leaf-cutting ants Atta colombica and Acromyrmex echinatior. We removed the queen from colonies to induce worker reproduction and subjected workers to oxidative stress. Oxidative stress drastically reduced survival, but this effect was less pronounced in leaf-cutting ant workers from queenless nests. We also found that, irrespective of oxidative stress, outside workers died earlier than inside workers did, likely because they were older. Since At. colombica workers cannot produce fertile offspring, our results indicate that direct reproduction is not necessary to extend the lives of queenless workers. Our findings suggest that workers are less resilient to oxidative stress in the presence of the queen, and raise questions on the proximate and ultimate mechanisms underlying socially mediated variation in worker lifespan. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns?'

Oxidative stress and senescence in social insects: a significant but inconsistent link?

The life-prolonging effects of antioxidants have long entered popular culture, but the scientific community still debates whether free radicals and the resulting oxidative stress negatively affect longevity. Social insects are intriguing models for analysing the relationship between oxidative stress and senescence because life histories differ vastly between long-lived reproductives and the genetically similar but short-lived workers. Here, we present the results of an experiment on the accumulation of oxidative damage to proteins, and a comparative analysis of the expression of 20 selected genes commonly involved in managing oxidative damage, across four species of social insects: a termite, two bees and an ant. Although the source of analysed tissue varied across the four species, our results suggest that oxidative stress is a significant factor in senescence and that its manifestation and antioxidant defenses differ among species, making it difficult to find general patterns. More detailed and controlled investigations on why responses to oxidative stress may differ across social species may lead to a better understanding of the relations between oxidative stress, antioxidants, social life history and senescence. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns?'

From the host's point of view: Effects of variation in burying beetle brood care and brood size on the interaction with parasitic mites.

The fitness and virulence of parasites is often determined by how many resources they can wrangle out of their hosts. Host defenses that help to keep resources from the parasites will then reduce virulence and parasite fitness. Here, we study whether host brood care and brood size regulation can protect host fitness and harm a parasite. We use the biparental brood-caring burying beetle Nicrophorus vespilloides and its phoretic Poecilochirus carabi mites as a model. Since paternal brood care does not seem to benefit the offspring in a clean laboratory setting, the male presence has been suggested to strengthen the defense against parasites. We manipulated male presence and found no effect on the fitness of the parasitic mites or the beetle offspring. We further manipulated beetle brood size and found larger broods to reduce parasite fitness. The specific pattern we observed suggests that beetle larvae are strong competitors and consume the carrion resource before all parasites develop. They thus starve the parasites. These results shed new light on the observation that the parasites appear to reduce host brood size early on-potentially to avert later competition their offspring might have to face.

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.

Sociality and communicative complexity: insights from the other insect societies.

Recognition and communication are essential processes when it comes to interaction of organisms with their biotic environment. As especially social interactions are coordinated by communication, it has been predicted that social evolution drives communicative complexity. However, studies comparing olfactory signals or receptor repertoires of solitary and eusocial insects found only mixed evidence for the social complexity hypothesis. We present some possible explanations and especially argue that our current knowledge of intermediate levels of sociality is insufficient to fully test the hypothesis, for which a more balanced comparative dataset would be required. We illustrate with chosen examples how complex communication within the other insect societies can be: Many messages are not unique to eusocial insects. Studying the other insect societies will provide us with a more detailed picture of the link between social and communicative complexity.

Rapid adaptation in phoretic mite development time.

Strong ecological selection can erode genetic variation and render populations unable to deal with changes in ecological conditions. In the adaptation of the phoretic mite Poecilochirus carabi to its host, the burying beetle Nicrophorus vespilloides, the timing of reproduction is crucial. Safe mite development is only possible during the beetles' brood care; mites that develop too slowly will have virtually zero fitness. If the strong specialisation in development time leaves no room for standing genetic variation to remain, changes in beetle brood care are disastrous. Beetle brood care depends on temperature and is thus vulnerable to changing climate. Accidental host switches to another beetle species with shorter brood care would also have negative effects on the mites. Only sufficient standing genetic variation could allow mismatched mite lines to survive and adapt. To test whether such rapid adaptation is possible in principle, we artificially selected on mite generation time. We were able to speed up, but not to slow down, mite development. We conclude that there is enough standing genetic variation in development time to allow P. carabi to quickly adapt to new host species or climate conditions, which could potentially lead to the evolution of new mite species.

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.

A critical look at proximate causes of social insect senescence: damage accumulation or hyperfunction?

Social insects have received attention for their extreme lifespan variation and reversal of the fecundity/longevity trade-off. However, proximate causes of senescence in general are disputed, and social insects often fail to meet the predictions of prevailing models. We present evidence for and against the long-held free radical theory of aging in social insects, and consider the application of the competing hyperfunction theory. Current results present problems for both theories, and a more complex picture of the biological processes involved emerges. The eusocial life style might allow colonies to allocate damage in ways that create seemingly senescence-free life histories. Only experimental approaches characterizing multiple senescence factors simultaneously will shed light on how social insects defy the conventions of senescence.

Chemical disguise of myrmecophilous cockroaches and its implications for understanding nestmate recognition mechanisms in leaf-cutting ants.

BACKGROUND: Cockroaches of the genus Attaphila regularly occur in leaf-cutting ant colonies. The ants farm a fungus that the cockroaches also appear to feed on. Cockroaches disperse between colonies horizontally (via foraging trails) and vertically (attached to queens on their mating flights). We analysed the chemical strategies used by the cockroaches to integrate into colonies of Atta colombica and Acromyrmex octospinosus. Analysing cockroaches from nests of two host species further allowed us to test the hypothesis that nestmate recognition is based on an asymmetric mechanism. Specifically, we test the U-present nestmate recognition model, which assumes that detection of undesirable cues (non-nestmate specific substances) leads to strong rejection of the cue-bearers, while absence of desirable cues (nestmate-specific substances) does not necessarily trigger aggression. RESULTS: We found that nests of Atta and Acromyrmex contained cockroaches of two different and not yet described Attaphila species. The cockroaches share the cuticular chemical substances of their specific host species and copy their host nest's colony-specific cuticular profile. Indeed, the cockroaches are accepted by nestmate but attacked by non-nestmate ant workers. Cockroaches from Acromyrmex colonies bear a lower concentration of cuticular substances and are less likely to be attacked by non-nestmate ants than cockroaches from Atta colonies. CONCLUSIONS: Nest-specific recognition of Attaphila cockroaches by host workers in combination with nest-specific cuticular chemical profiles suggest that the cockroaches mimic their host's recognition labels, either by synthesizing nest-specific substances or by substance transfer from ants. Our finding that the cockroach species with lower concentration of cuticular substances receives less aggression by both host species fully supports the U-present nestmate recognition model. Leaf-cutting ant nestmate recognition is thus asymmetric, responding more strongly to differences than to similarities.

Ecology and Evolution of Communication in Social Insects.

Insect life strategies comprise all levels of sociality from solitary to eusocial, in which individuals form persistent groups and divide labor. With increasing social complexity, the need to communicate a greater diversity of messages arose to coordinate division of labor, group cohesion, and concerted actions. Here we summarize the knowledge on prominent messages in social insects that inform about reproduction, group membership, resource locations, and threats and discuss potential evolutionary trajectories of each message in the context of social complexity.

Variation in nestmate recognition ability among polymorphic leaf-cutting ant workers.

A key feature for the success of social insects is division of labour, allowing colony members to specialize on different tasks. Nest defence is a defining task for social insects since it is crucial for colony integrity. A particularly impressive and well-known case of worker specialization in complex hymenopteran societies is found in leaf-cutting ants of the genera Atta and Acromyrmex. We hypothesized that three morphological worker castes of Acromyrmex echinatior differ in their likelihood to attack intruders, and show that major workers are more aggressive towards non-nestmate workers than medium and minor workers. Moreover, minors do not discriminate between nestmate and non-nestmate brood, while larger workers do. We further show that A. echinatior ants use cuticular chemical compounds for nestmate recognition. We took advantage of the natural variation in the cuticular compounds between colonies to investigate the proximate factors that may have led to the observed caste differences in aggression. We infer that major workers differ from medium workers in their general propensity to attack intruders (the "action component" of the nestmate recognition system), while minors seem to be less sensitive to foreign odours ("perception component"). Our results highlight the importance of proximate mechanisms underlying social insect behaviour, and encourage an appreciation of intra-colony variation when analysing colony-level traits such as nest defence.

Kin-informative recognition cues in ants.

Although social groups are characterized by cooperation, they are also often the scene of conflict. In non-clonal systems, the reproductive interests of group members will differ and individuals may benefit by exploiting the cooperative efforts of other group members. However, such selfish behaviour is thought to be rare in one of the classic examples of cooperation--social insect colonies--because the colony-level costs of individual selfishness select against cues that would allow workers to recognize their closest relatives. In accord with this, previous studies of wasps and ants have found little or no kin information in recognition cues. Here, we test the hypothesis that social insects do not have kin-informative recognition cues by investigating the recognition cues and relatedness of workers from four colonies of the ant Acromyrmex octospinosus. Contrary to the theoretical prediction, we show that the cuticular hydrocarbons of ant workers in all four colonies are informative enough to allow full-sisters to be distinguished from half-sisters with a high accuracy. These results contradict the hypothesis of non-heritable recognition cues and suggest that there is more potential for within-colony conflicts in genetically diverse societies than previously thought.

Ants recognize foes and not friends.

Discriminating among individuals and rejecting non-group members is essential for the evolution and stability of animal societies. Ants are good models for studying recognition mechanisms, because they are typically very efficient in discriminating 'friends' (nest-mates) from 'foes' (non-nest-mates). Recognition in ants involves multicomponent cues encoded in cuticular hydrocarbon profiles. Here, we tested whether workers of the carpenter ant Camponotus herculeanus use the presence and/or absence of cuticular hydrocarbons to discriminate between nest-mates and non-nest-mates. We supplemented the cuticular profile with synthetic hydrocarbons mixed to liquid food and then assessed behavioural responses using two different bioassays. Our results show that (i) the presence, but not the absence, of an additional hydrocarbon elicited aggression and that (ii) among the three classes of hydrocarbons tested (unbranched, mono-methylated and dimethylated alkanes; for mono-methylated alkanes, we present a new synthetic pathway), only the dimethylated alkane was effective in eliciting aggression. Our results suggest that carpenter ants use a fundamentally different mechanism for nest-mate recognition than previously thought. They do not specifically recognize nest-mates, but rather recognize and reject non-nest-mates bearing odour cues that are novel to their own colony cuticular hydrocarbon profile. This begs for a reappraisal of the mechanisms underlying recognition systems in social insects.