Brood production and lineage discrimination in the red harvester ant (Pogonomyrmex barbatus)

In contrast to the system of caste determination in most social insects, reproductive caste determination in some populations of Pogonomyrmex barbatus has a genetic basis. Populations that exhibit genetic caste determination are segregated into two distinct, genetic lineages. Same-lineage matings result in female reproductives, while inter-lineage matings result in workers. To investigate whether founding P. barbatus queens lay eggs of reproductive genotype, and to determine the fate of those eggs, we genotyped eggs, larvae, and pupae produced by naturally inseminated, laboratory-raised queens. We show that founding dependent lineage queens do lay eggs of reproductive genotype, and that the proportion of reproductive genotypes decreases over the course of development from eggs to larvae to pupae. Because queens must mate with a male of each lineage to produce both workers and female reproductives, it would benefit queens to be able to distinguish males of the two lineages. Here we show that P. barbatus males from the two genetic lineages differ in their cuticular hydrocarbon profiles. Queens could use male cuticular hydrocarbons as cues to assess the lineage of males at the mating aggregation, and possibly keep mating until they have mated with males of both lineages.     

Induction of a reproductive-specific cuticular hydrocarbon profile by a juvenile hormone analog in the termite <Emphasis Type="Italic">Zootermopsis nevadensis</Emphasis>

Establishment and maintenance of the reproductive division of labor within social insect colonies relies on clear communication between nestmates. Fertile members convey their status to prevent others from becoming reproductively active. Recent findings in some basal termites indicate that cuticular hydrocarbon profiles may indicate reproductive state, but there is little evidence to show a direct link between reproductive status and hydrocarbon production—a prerequisite for an “honest” fertility signal. Here, we report that the putative signaling mechanism is influenced by juvenile hormone (JH), a primary regulator of gonadal development and activity in insects. Topical application of a JH-analog (pyriproxyfen) to reproductively inactive alates of the basal dampwood termite Zootermopsis nevadensis induced both females and males to express significantly more of a reproductive-specific hydrocarbon (6,9,17-tritriacontatriene). However, the JH-analog did not significantly enhance gonadal development or activity in treated termites beyond what is usually observed in maturing alates released from the inhibitory stimuli of their natal nest. These results suggest that a rise in JH following disinhibition drives the expression of reproductive-specific hydrocarbons, but that an individual’s hydrocarbon profile is not directly linked to its gonadal state. Rather than directly driving the expression of reproductive-specific hydrocarbons, the gonads may act indirectly through their influence on circulating JH.     

Genetic influence on caste determination underlying the asexual queen succession system in a termite

The question of how reproductives and sterile workers differentiate within eusocial groups has long been a core issue in the study of social insects. Recent studies have shown that not only environmental factors but also genetic factors affect caste differentiation. In the termite Reticulitermes speratus, queens produce their replacements (neotenics) asexually but use normal sexual reproduction to produce other colony members. Here, we demonstrate a genetic influence on caste determination underlying the asexual queen succession system in this termite species. Thelytoky in termites is accomplished by automixis with terminal fusion, yielding almost completely homozygous offspring; thus, parthenogenetically and sexually produced offspring profoundly differ in heterozygosity. An analysis of the relationship between the reproductive dominance of female neotenics obtained from experimentally orphaned colonies and their genotypes at five microsatellite loci showed that homozygosity at two loci influenced the developmental priority and/or reproductive quality of neotenics. These results suggest the existence of a multi-locus system affecting the queen fecundity and explain why parthenogens have genetic priority to become neotenics in this termite species.     

Origin of male-biased sex allocation in orphaned colonies of the termite, <Emphasis Type="Italic">Coptotermes lacteus</Emphasis>

In eusocial insects, sex allocation often constitutes a ground for intracolonial conflicts. This occurrence provides ideal opportunities to test kin-selection theory. A vast literature on this topic is available for social Hymenoptera, but the same field remains almost untouched in termites. A preeminent case is that of some species of Coptotermes, where the sex-allocation ratio in nymphs shifts from near equity to all-male when the primary reproductives are replaced by neotenics. To shed light on the developmental origin of this shift, we compared the sex ratio of the various castes and instars in primary- and neotenic-headed mature colonies of Coptotermes lacteus. The male-biased sex allocation in the latter type of colony results from two concurrent events: first, the sex ratio of the youngest instars (larvae) is male-biased by a 3:1 ratio; and second, all female larvae become workers, while a large fraction of the male larvae proceed to the nymphal and alate stages. Colony-founding experiments showed that inbreeding by itself cannot account for the male bias at hatching. We suggest that both genetic factors, due to the reproductive behaviour of neotenics, and environmental factors (colony condition and resource availability) may influence this process. Their exact nature and respective impact have not yet been clarified.     

Hierarchical perception of fertility signals and nestmate recognition cues in two dolichoderine ants

In social insects, queens are likely to “honestly” inform their nestmates of their presence and fertility status through pheromonal communication. Cuticular hydrocarbons (CHCs) have been reported to be effective nestmate discriminators and strongly suspected to act as fertility signals, at least in some species. The use of the same chemical bouquet (i.e., the CHC profile) to convey two fundamentally different information seems puzzling. However, a recent threshold model proposes a hierarchy in the discriminating processes, i.e., fertility signals can only be perceived if nestmate recognition has been reached (Le Conte and Hefetz, Annu Rev Entomol 53:523–542, 2008). Here, we developed a simple behavioral bioassay based on chemical recruitment toward a queen placed outside the nest in two dolichoderine ants (Linepithema humile and Tapinoma erraticum), which allowed us to investigate the interplay between fertility signaling and colonial recognition. Using queen corpses of various origins (nestmates or aliens) and physiological states (fertile or infertile; mated or unmated), we demonstrated that nestmate recognition cues clearly override fertility signals under our experimental conditions. Indeed, while nestmate infertile queens were largely ignored by the workers, nestmate fertile queens (mated or not) induced worker recruitment, whereas alien fertile queens did not and were aggressed by the workers.     

Reproductive plasticity in bumblebee workers (<Emphasis Type="Italic">Bombus terrestris</Emphasis>)—reversion from fertility to sterility under queen influence

Worker sterility in the bumblebee Bombus terrestris is conditional and is linked to the social development of the colony. Workers refrain from reproducing or overtly challenging the queen until gyne production has initiated, at the so-called competition point (CP). It is not known whether this behavior is hard-wired or workers show reproductive plasticity. It also remains unclear whether worker reproductive decision is under queen and/or worker control. In this study, we tested worker reproductive plasticity in an attempt to assess whether and under which conditions worker sterility/fertility are reversible. We introduced egg-laying workers into colonies with different social structures for 1 week then monitored their reproductive status. We revealed a remarkable reproductive plasticity in the introduced workers that was social-condition-dependent. In the presence of a pre-CP queen, the introduced workers reverted to sterility, whereas in the presence of a post-CP queen, such workers remained egg-layer. Reversion to sterility does not occur when direct contact with the queen is prevented, as the introduced workers remained egg-layer in the queenright colonies with a confined queen. Egg-laying workers that were introduced into queenless colonies mostly maintained their fertility regardless of colony social phase. This shows that worker transition from cooperative to selfish behavior is reversible depending on the social context.     

Chemical ecology involved in invasion success of the cuckoo bumblebee <Emphasis Type="Italic">Psithyrus vestalis</Emphasis> and in survival of workers of its host <Emphasis Type="Italic">Bombus terrestris</Emphasis>

In bumblebees all species of the subgenus Psithyrus are social parasites in the nests of their Bombus hosts. In the bumblebee B. terrestris we investigated how colony size influences survival rates of nest entering females of the social parasite Psithyrus vestalis. Furthermore, we studied whether the host worker’s dominance status and age are reflected in its individual scent and whether Psithyrus females use volatiles to selectively kill host workers. The survival rate of Psithyrus vestalis females drops from 100%, when entering colonies with five workers, to 0% for colonies containing 50 host workers. Older host workers, born before the nest invasion, were selectively killed when Psithyrus females entered the nest. In contrast, all workers born after the nest invasion survived. The host workers’ dominance status and age are reflected by their individual odours: newly emerged workers produced a significantly lower total amount of secretions than 4-day-old workers. In chemical analyses of female groups we identified saturated and unsaturated hydrocarbons, aldehydes, and unsaturated wax-type esters of fatty acids. In a discriminant function analysis different worker groups were mainly separated by their bouquets of hydrocarbons. Killed workers release significantly more scent and of a different chemical composition, than survivors. Survivors alter scent production and increase it beyond the level of the killed workers within 1 day of the invasion. The Psithyrus female clearly maintains reproductive dominance utilizing these differences in the odour bouquets as criteria for killing workers that compete for reproduction.     

Alternative reproductive tactics and the impact of local competition on sex ratios in the ant Hypoponera opacior

The ant Hypoponera opacior exhibits alternative reproductive morphs of males and females associated with distinct sexual behaviours. Our long-term study reports strong seasonality in sexual production with a mating season in early and one in late summer. Winged (alate) reproductives emerge in June, swarm during the monsoon season and establish new colonies independently. In contrast, wingless worker-like (ergatoid) reproductives that appear in late August mate within their natal or adjacent nests and either do not disperse or establish new nests close by. These divergent dispersal patterns allowed us to analyse the impact of local factors on investment strategies by comparing sex allocation between and within the two reproductive events. The optimal sex ratio for ergatoid reproductives should be influenced both by competition for matings between brothers (local mate competition) and rivalry among young locally dispersing queens for workers, nest sites or food (local resource competition). The greater importance of local resource competition was demonstrated both by a male-biased sex ratio for wingless sexuals and a stronger increase in the number of males with total sexual production than for the number of queens. Microsatellite analysis revealed that inter-nest variation in relatedness asymmetry cannot explain split sex ratios in the August generation. Instead, nests with related ergatoid males raised a male-biased sex ratio contrary to the expectations under local mate competition. In conclusion, male bias in wingless H. opacior indicates that local mate competition is less strong than local resource competition among ergatoid queens over the help of workers during nest foundation.     

Cooperative breeding increases reproductive success in the social spider <Emphasis Type="Italic">Stegodyphus dumicola</Emphasis> (Araneae,Eresidae)

Sociality in some birds, mammals, and social insects was suggested to have evolved through the lengthening and extension of parental care behaviors to nondirect descendents. In these systems, group members care for young cooperatively and, thus, increase the reproductive success of the breeders and fitness of the young. Parental care behaviors, such as regurgitation feeding and matriphagy (consumption of the mother), occur in several subsocial and social spiders. However, it is not known whether females in a colony cooperate in caring for the young of other females and whether such cooperative care improves reproductive success. To answer this question, we created experimental colonies of the social spider Stegodyphus dumicola (Araneae, Eresidae), allowing only one female in a group to produce young, simulating reproductive skew occurring in nests in nature. In this paper, we show for the first time that females of S. dumicola cooperate in providing regurgitated food for young of other females and are even eaten by those young. Young raised by a group of females were larger and had greater survival than young raised only by their mother. Thus, fitness benefits from raising broods cooperatively may have favored the evolution of sociality in spiders.     

Dynamics of biparental care in the dampwood termite,Zootermopsis nevadensis (Hagen): response to nitrogen availability

Summary Studies were conducted on the dampwood termite, Zootermopsis nevadensis, to examine the behavioral roles of the reproductive pair during the nest-founding period and to determine the effect of nitrogen availability on their reproduction and division of labor. Nitrogen has been hypothesized to be an important limiting nutrient for founding pairs. One nitrogenous reserve, uric acid-nitrogen, was examined in reproductives and in nutrient-receiving and nutrient-gathering colony members; it was found in highest amounts in the reproductives (i.e., alates, de-alates, and primary reproductives). Young pairs may use these nitrogenous reserves to increase their chances for reproduction. In support of this hypothesis, founding pairs that were fed a diet supplemented with uric acid-nitrogen had a significantly greater probability of producing at least one offspring than did pairs fed an unsupplemented diet. Females that were fed a nitrogen-poor diet restricted their total activity while their mates sustained a high activity during colony initiation. When fed a nitrogen-rich diet, females collected pulp more often than their mates, while males collected more water, though only in the period prior to egg laying. In all pairs, males transferred proctodeal pellets (food derived from the hindgut intestine) to their mates significantly more often than females to males, and females fed on proctodeal pellets significantly more often than did their mates. The male-female asymmetries in pellet transfer and feeding were not significant in the stage after egg deposition. Once eggs and larvae were present in the nest, a male and female spend an equal percentage of time caring for eggs and feeding larvae. Proctodeal pellets examined in reproductives were found to be rich in proteins. This suggests that in the pre-egg period, the male provides nitrogen-rich substances to the female as a form of paternal investment.     

Modeling social insect populations II: Optimal reproductive strategies in annual eusocial insect colonies

Summary We examined the optimal reproductive strategies for eusocial insects with an annual life cycle. A model is constructed for the growth of workers and reproductives as a function of the resources allocated to each. We then compute the allocation schedule which yields the maximum number of reproductives by season's end. Field observations appear to confirm the prediction of our model: the optimal strategy is to invest in colony growth until approximately one generation before the season's end, whereupon worker production ceases and reproductive effort is switched entirely to producing queens and males. Similar all-or-none strategies are observed in other biological contexts, and their optimality is proven here. Finally, we calculate the fraction of the total season's resources allocated to reproductives from which we can estimate the ergonomic efficiency of the colony with respect to reproductive success.     

Conserved male-specific cuticular hydrocarbon patterns in the trap-jaw ant Odontomachus brunneus

Cuticular hydrocarbons have been identified as the source of sex-recognition signals for many insects, but for social insects, specifically ants, cuticular hydrocarbon profiles of males are often ignored. This study reports male-specific cuticular hydrocarbon patterns for the trap-jaw ant Odontomachus brunneus. Analysis of samples from four Florida populations demonstrated that male-specific overabundance of four hydrocarbons is conserved across populations despite population-level divergence of the remainder of the profile. In addition, hydrocarbon patterns unique to adult males were not present on the cuticle of final instar male larvae, indicating that male-specific profiles arise late in development. The pattern of an abundant subset of conserved cuticular hydrocarbons characteristic of males across divergent populations was compared to earlier findings of the conservation of fertility signals of females across these same populations.     

Experimental evidence that workers recognize reproductives through cuticular hydrocarbons in the ant Odontomachus brunneus

Eusociality is characterized by a reproductive division of labor, wherein workers respond to the presence of reproductive individuals by refraining from reproduction themselves and restricting the reproductive efforts of others. Our understanding of how eusociality is maintained therefore depends on characterizing the mechanism by which workers detect the presence of a reproductive. Variations in cuticular hydrocarbons correspond to changes in reproductive ability in ants, and experimental studies are beginning to reveal the function of hydrocarbons as signals. In this study, we compare the cuticular hydrocarbon profiles of dominant and reproductive workers and queens of the ant Odontomachus brunneus with profiles of non-reproductive workers. Using split/reunification tests we document the existence of worker policing in both queenless and queenright colonies; supernumerary reproductives were treated aggressively by nestmates. Finally, we induce aggression and replicate queen-like submissive nestmate responses by supplementing the hydrocarbon profile of workers with (Z)-9-nonacosene, a compound that was significantly more abundant on the cuticles of reproductives. In three bioassays, we compare this manipulation to various control manipulations of the hydrocarbon profile and demonstrate that workers gauge the reproductive activity of nestmates through changes in their cuticular hydrocarbon profiles.     

Profiles of cuticular hydrocarbons mediate male mate choice and sexual isolation between hybridising <Emphasis Type="Italic">Chrysochus</Emphasis> (Coleoptera: Chrysomelidae)

Summary. Chemical signals frequently underlie sexual isolation between insect species. Our understanding of the evolutionary forces influencing these signaling systems is known for very few systems, challenging both our efforts to understand insect speciation, and our ability to predict long-term changes in the chemical communication systems of insects. Thus, we are in need of more systems in which both the chemical signals causing sexual isolation and the evolutionary forces driving sexual isolation are understood. Sexual isolation in the hybrid zone between Chrysochus cobaltinus and C. auratus has apparently increased in response to natural selection against hybridisation (i.e. reinforcement). Previous experiments suggested that this isolation was due, at least in part, to male preferences for conspecific females. Here, we confirm this role of male choice, and document that male mate choice in this system is influenced by cuticular hydrocarbon (CHC) profiles. Specifically, male C. cobaltinus responses to control cadavers and conspecific female cadavers painted with different cuticular hexane extracts, together with analyses of the composition of those extracts, revealed that male mate choice is governed by CHC profiles. Multivariate analyses of GC profiles demonstrated that those profiles are indeed both sex- and species-specific. Although GC-MS enabled identification and quantification of the specific cuticular hydrocarbons, we have not yet determined which individual compounds govern mate choice. Having established that CHCs influence sexual isolation in this system, we can now assess the evolutionary lability of these cues, which will inform both our understanding of speciation, and of the conditions under which the chemical signaling systems that influence mate choice in insects can evolve.     

Mixed messages: fertility signaling interferes with nestmate recognition in the monogynous ant Camponotus floridanus

Chemical communication is crucial for the organization of social insect colonies. However, with the heavy use of one communication modality, problems may arise such as the interference of different types of information. This study investigated how information about fertility and colony membership is integrated in the ant Camponotus floridanus. We introduced into mature, queenright colonies (a) the nestmate queen, (b) a nestmate worker, (c) a foreign, high-fertility queen, (d) a foreign, low-fertility queen, and (e) a foreign worker. As expected, workers did not attack their nestmate queen or a nestmate worker but responded aggressively to foreign workers and foreign, low-fertility queens. Surprisingly, workers did not attack foreign, high-fertility queens. Chemical analysis demonstrated that the cuticular hydrocarbon profile of C. floridanus encodes information about fertility status in queens and workers and colony membership in workers. We suggest that ants respond to this information in the cuticular hydrocarbon profile: individuals with strong fertility signals are accepted regardless of their colony membership, but individuals without strong fertility signals are tolerated only if their cuticular hydrocarbon profile matches that of colony members. Learning how social insects respond to multiple types of information presented together is critical to our understanding of the recognition systems that permit the complex organization of social insect colonies.     

Recognition of caste and mating status maintains monogyny in the ant Aphaenogaster senilis

In ants dispersing through colony fission, queens mate near their natal nest and found a new society with the help of workers. This allows potential future queens to challenge the mother queen’s reproductive monopoly. Conflicts might be resolved if the mated queen signals her presence and the workers control the developmental fate of the diploid larvae (whether they develop to worker or queen). In this study we sought to determine whether, in the fission-performing ant Aphaenogaster senilis, conflicts between queens for control of the colony are resolved by the resident queen signalling her mating status. Virgin queens were less effective than newly mated queens in inhibiting queen rearing. Moreover, potential challenger queens were recognized and heavily aggressed independent of mating status. Chemical analyses showed that mating status was associated with changes in cuticular hydrocarbon and poison gland composition, but not in Dufour’s gland composition. Cuticular dimethylalkanes were identified as potential constituents that signal both caste (present in queens only) and mating status (mated queens have higher amounts). We hypothesised that pheromone emission by virgin queens did not reach the threshold needed to fully inhibit larval development into queens but was sufficiently high to stimulate overt aggression by mated queens. These findings provide evidence for the complexity of chemical communication in social insects, in which a small number of signals may have a variety of effects, depending on the context.     

The role of the queen mandibular gland pheromone in honeybees (<Emphasis Type="Italic">Apis mellifera</Emphasis>): honest signal or suppressive agent?

Queen pheromones interfere with worker reproduction in social insects. However, there is still an unresolved question as to whether this pheromone acts as an “honest” signal for workers, giving a reliable indication of the queen’s reproductive value, or as a suppressive agent, inhibiting worker reproduction independent of the queen’s reproductive capacity. In honeybees (Apis mellifera), the queen’s mandibular gland secretion, a mix of fatty acids and some aromatic compounds, is crucial for regulating the reproductive division of labor in the colony inhibiting ovary development in workers. We quantified the mandibular gland secretions of virgin, drone-laying, and naturally mated queens using gas chromatography to test whether the queens’ mating, ovary activation, or the reproductive value for workers correlated with the composition of the secretion. Although the absolute amounts of the “queen substance” 9-oxo-2(E)-decenoic acid (9-ODA) were similar among the three groups, the proportions of 9-ODA decreased with increasing reproductive quality. Furthermore, the ratios of queen to worker compounds were similar in all three treatment groups, irrespective of the reproductive capacity. A multivariate analysis including all six compounds could not separate drone-laying queens from naturally mated ones, both with active ovaries but only the latter ensuring colony survival. We suggest that the mandibular gland pheromones are unlikely to function as reliable indicators of queen reproductive value and rather operate as an agent to suppress worker reproduction. This does not exclude the possibility that other “honest” pheromone signals exist in the honeybee colony, but these would have to arise from other semiochemicals, which could be produced by both the queen and the brood.     

Reproductive isolation between intertidal and subtidal<Emphasis Type="Italic"> Scoloplos armiger</Emphasis> (Polychaeta,Orbiniidae) indicates sibling species in the North Sea

Two distinct modes of development in the common polychaete Scoloplos armiger (O. F. Müller, 1776) occur in the North Sea region: holobenthic development in egg cocoons and pelagic larvae hatching from suspended eggs. In the northern Wadden Sea near the island of Sylt, we observed that egg cocoons are produced intertidally while pelagic larvae originate from the adjacent subtidal zone. A previous genetic comparison between these subtidal and intertidal populations revealed distinct gene pools, suggesting that reproductive differences are not phenotypic but heritable. In this study, crossbreeding experiments show that intertidal and subtidal populations are reproductively isolated. Couples with males and females from different habitats had no offspring. Production of egg cocoons is determined by female origin from the intertidal zone. Pelagic larvae occurred only in couples with subtidal females and subtidal males. Intertidal males have spermatozoa with heads twice as long as those from subtidal males and a significantly shorter flagellum. We suspect that deviating sperm morphology may cause the reproductive breakdown at the fertilization stage. Juveniles hatching from cocoons have shorter anal cirri compared to juveniles that metamorphosed from pelagic larvae. We conclude there to be two sympatric sibling species in S. armiger: 'type I' in intertidal areas, which have egg cocoons, no pelagic larvae, elongated sperm heads, shortened sperm flagella and anal cirri; and a subtidal 'type S', lacking egg cocoons but with pelagic larvae, short sperm heads, long sperm flagella and anal cirri.Communicated by O. Kinne, Oldendorf/Luhe     

Changes in the cuticular hydrocarbons of incipient reproductives correlate with triggering of worker policing in the bulldog ant Myrmecia gulosa

In social insects, conflicts over male parentage can be resolved by worker policing. However, the evolution of policing behavior is constrained by the ability of individuals to identify reproductive nestmates, or their eggs. We investigated the occurrence of worker policing and its underlying chemical communication in the bulldog ant Myrmecia gulosa. Although workers have functional ovaries and can lay male-destined eggs, they do not reproduce in queenright colonies. To determine if their sterility is a consequence of worker policing, we experimentally induced worker reproduction in the presence of a queen. Some individuals were seized and immobilized by nestmates, and sometimes killed as a consequence. Although the ovarian development of immobilized individuals was variable, their cuticular hydrocarbon profiles were intermediate between reproductive and nonreproductive workers, indicating they were in the process of starting to reproduce. Approximately 29% of these incipient reproductive workers were successfully policed. To test for policing on eggs, we transferred viable worker eggs to queenright colonies and monitored their acceptance. Furthermore, we compared the surface hydrocarbons of the different types of eggs to determine whether these chemicals could be involved in egg recognition. We found that although there were differences in hydrocarbon profiles and discrimination between queen and worker-laid eggs, viable eggs were not destroyed. Our results strongly support the idea that cuticular hydrocarbons are involved in the policing of reproductive workers. A low level of worker policing appears sufficient to select for self-restraint in workers when few fitness benefits are gained by selfish reproduction. Policing of eggs may thus be unnecessary.     

Social context-dependent immune gene expression in bumblebees (<Emphasis Type="Italic">Bombus terrestris</Emphasis>)

Social insects are prone to attack by parasites as they provide numerous resources of food and brood, homeostatic nest conditions and a high density of individuals, enhancing the transmission of parasites. The defence of social insects might occur at different levels, the individual and the group. Individual defence occurs in part via the innate immune system resulting in the expression of antimicrobial substances. Group level defences, summarised as ‘social immunity’, represent a suite of behavioural and organisational features. Here, all effects contributing to social immunity except for the social context were removed from bumblebee (Bombus terrestris) workers, kept either in groups or solitarily. The gene expression of six effector molecules of the immune system was monitored in both groups and in controls from the same source colonies. The social treatment has a highly significant effect on immune gene expression, with groups exhibiting higher levels of two antimicrobial peptides (AMPs) and two lysozymes. Phenoloxidase is affected at the regulatory level, with a strong upregulation of its suppressor Spn27A in groups suggesting a trade-off with antimicrobial activity. AMPs are strongly upregulated in groups, whereas lysozymes are strongly downregulated in solitary treatments suggesting another trade-off. Clearly, social immunity impacts elements of individual immunity.