Abstract: Social immunity—the collective behavioural defences against pathogens—is considered a crucial evolutionary force for the maintenance of insect societies. It has been described and investigated primarily in eusocial insects, but its role in the evolutionary trajectory from parental care to eusociality is little understood. Here, we report on the existence, plasticity, effectiveness and consequences of social pathogen defence in experimental nests of cooperatively breeding ambrosia beetles. After an Aspergillus spore buffer solution or a control buffer solution had been injected in laboratory nests, totipotent adult female workers increased their activity and hygienic behaviours like allogrooming and cannibalism. Such social immune responses had not been described for a non-eusocial, cooperatively breeding insect before. Removal of beetles from Aspergillus-treated nests in a paired experimental design revealed that the hygienic behaviours of beetles significantly reduced pathogen prevalence in the nest. Furthermore, in response to pathogen injections, female helpers delayed dispersal and thus prolonged their cooperative phase within their mother's nest. Our findings of appropriate social responses to an experimental immune challenge in a cooperatively breeding beetle corroborate the view that social immunity is not an exclusive attribute of eusocial insects, but rather a concomitant and presumably important feature in the evolutionary transitions towards complex social organization.
1. Introduction
Pathogens pose a major risk to highly social animals. Insect societies, for instance, provide ideal conditions for their dissemination [1,2], because a large number of closely related individuals with potentially very similar immune defences live together in intimate contact and under homogeneous, environmentally buffered conditions. Low genetic variance has been shown to reduce the chances of successfully resisting severe fungus infections in honeybees, and in ants it reduces the effectiveness of anti-pathogen behaviours [3,4]. To counter pathogen risk, social insects evolved various physiological and behavioural strategies to inhibit the spread of diseases [5].
The innate immune system, pathogen avoidance and self-cleaning behaviours are probably the most common anti-pathogen strategies in insects. In addition to such traits that might be termed ‘non-social’, many social insects were found to express social immunity, which refers to cooperative sanitation involving the joint mechanical and chemical removal of bacterial and fungal pathogens. Originally, social immunity was regarded as a nest-wide parasite and pathogen defence mechanism that evolved in eusocial insects to counter the beforementioned inherent risks of infection caused by the social lifestyle and genetic homogeneity [5]. Importantly, this concept has highlighted the parallels between the innate non-social immune system of a single multicellular organism and a nest-wide ‘social immune system’ of a complex insect society. This idea relates to the concept of superorganismality, where a whole nest of social insects is regarded as a single reproducing entity (the ‘super organism’ [6,7]). Groups of nest members take on specialized roles, which corresponds to the differentiated cell tissues of a multicellular organism [8,9]. In the best-studied societies of ants and bees, for example, this is proposed to have led to the evolution of sophisticated group-level social immune defences by workers, including application of antimicrobial substances onto contaminated areas, removal of corpses and diseased brood, social fever and allogrooming [10–12]. Such sanitation behaviour is not restricted to eusocial insects, however, as its precursors are already present in subsocial insects with parental care (e.g. [13–15]), although empirical data from such systems are scarce. This sparked a debate about whether the concept of social immunity should be extended to include cooperative sanitation tasks performed in non-eusocial group living species, to better understand the evolutionary origins of social immunity [11,16].
This recent debate highlights that the evolution of social immunity is hitherto unclear. Either social immunity evolved as a result of increased pathogen transmission in eusocial organisms (termed the eusocial framework [5,11,16]) or sociality and social immunity co-evolved in a close feedback interaction (termed the group living framework [15–17]). In some taxa, the suppression of pathogens is a very important social task, not only exhibited by parents towards offspring, but sometimes even between all individuals in a nest or aggregation. Hence, it is conceivable that under certain circumstances, pathogens themselves may be important drivers of sociality. This might be true especially in taxa that live in permanent close contact to a decaying food source and are thus frequently in contact with various microbes (e.g. involving parental care in burying beetles, larval aggregations in Drosophila or worker specialization in attine ants [17]). Our study explored this possibility further by introducing fungus-farming bark beetles as a system for the experimental study of social immunity.
These so-called ambrosia beetles offer a unique opportunity for studying the evolution of social traits because closely related species express various social structures ranging from uniparental care to eusociality [18]. Cooperative breeders are of particular interest for experimentally studying social evolution, as here adult females delay dispersal and act as helpers or temporary workers. The length of dispersal delay is affected by the presence and quantity of dependent offspring in the colony and the level of nutrition [19,20], but it might also be affected by the presence and load of pathogens.
All ambrosia beetles live in close mutualistic relationships with different fungi and bacteria, which they farm as their sole source of nutrition within tunnel systems in the heartwood of trees. The main mutualists are so-called ambrosia fungi, primarily from the ascomycete orders Microascales and Ophiostomatales [21–23]. These fungi are taken up from the natal nest by dispersing adult females in special spore carrying organs called mycetangia and subsequently spread on the walls of newly excavated tunnel systems. Finally, they are cultivated and possibly protected from other (fungal) pathogens or competitors [18,23,24]. In addition to these fungal mutualists, several other fungi have been isolated from beetle nests, many of which are pathogens for the beetles or at least competitors of the beetles' fungal mutualists [21,23]. The genera Aspergillus and Beauveria, for example, can directly infect and kill adults and brood of ambrosia beetles [23,25–28]. Other fungi compete with the ambrosia fungi and thus deplete the food source of the beetles (e.g. Penicillium sp., Chaetomium sp., Nectria sp. [29]). Such pathogens and competitors are probably the primary threat for the beetles because within the wood, they are well protected from most other natural enemies.
Morphological castes such as those in eusocial insects do not exist in ambrosia beetles. Instead, many species show division of labour among totipotent adult and larval offspring, with adults overtaking nest protection and sanitation, and larvae engaging in nest enlargement and packing of frass (i.e. sawdust, faeces and possibly pathogens) [19]. Larvae and adults join forces to pack and expel pellets of waste through the nest entrance. One of the most common behaviours in both adults and larvae is allogrooming of each other and the brood, probably against pathogens. Diseased individuals are either cannibalized or removed from the nest [19]. Currently, it is unknown, however, if ambrosia beetle larvae and/or adults can detect pathogens and actively suppress their load within nests. Some bark beetles have been shown to exude secretions from their mouth to kill pathogenic fungi [30]. Others, like the species Dendroctonus frontalis, are associated with bacteria that produce antibiotics which selectively kill antagonistic microorganisms threatening their fungal associates [31]. Indications for such a bacterial defence mechanism that specifically targets fungal pathogens and not the fungal cultivars have been recently also found in our model species Xyleborinus saxesenii [32].
Recent advancements in laboratory rearing, observation and in situ manipulation techniques [24,33,34] allow studies of social pathogen defence in ambrosia beetles. Previous studies revealed vigorous cleaning behaviours by adult offspring and even larvae. Since all ambrosia beetles live in close contact to a rich microbial environment, similar to some of the best-described models for social immunity in eusocial insects, we expect to find convergent behavioural adaptations to increased pathogen exposure. In addition, the naturally very high inbreeding rate found in cooperatively breeding ambrosia beetles is assumed to create a condition similar to eusocial insects, where the genetic homogeneity of nestmates renders group members highly vulnerable to microbial attack.
To test this idea, we used the cooperatively breeding and naturally highly inbred species X. saxesenii Ratzeburg to determine the effect of Aspergillus fungal pathogens on beetle social behaviours and potential social immunity. This pathogen was chosen because it has been repeatedly isolated from diseased individuals from X. saxesenii nests (see electronic supplementary material, figure S1) and it is well known for its pathogenicity for many insects (including other bark beetles [26,27]), which is a result of produced aflatoxins [35,36]. Aspergillus spores were experimentally injected in laboratory nests, and effects were determined on (i) the social behaviours displayed by larvae and adults and (ii) the timing of dispersal of adult offspring from the natal nest. In addition, (iii) we assessed the effectiveness of the beetles' hygienic behaviours on pathogen spore loads, by comparing pathogen spore loads of nest parts with beetles present against parts where beetles had been experimentally removed after injection of the pathogen. We predict that the group members increase nest sanitation in response to the introduced pathogen and that this behaviour reduces pathogen spore loads. Furthermore, daughters will either delay their dispersal to help with nest hygiene and thus increase their indirect fitness benefits or disperse earlier to protect their individual health and direct fitness gains.
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