Rapid induction of immune density-dependent prophylaxis in adult social insects

The innate immune system provides defence against parasites and pathogens. This defence comes at a cost, suggesting that immune function should exhibit plasticity in response to variation in environmental threats. Density-dependent prophylaxis (DDP) has been demonstrated mostly in phase-polyphenic insects, where larval group size determines levels of immune function in either adults or later larval instars. Social insects exhibit extreme sociality, but DDP has been suggested to be absent from these ecologically dominant taxa. Here we show that adult bumble-bee workers (Bombus terrestris) exhibit rapid plasticity in their immune function in response to social context. These results suggest that DDP does not depend upon larval conditions, and is likely to be a widespread and labile response to rapidly changing conditions in adult insect populations. This has obvious ramifications for experimental analysis of immune function in insects, and serious implications for our understanding of the epidemiology and impact of pathogens and parasites in spatially structured adult insect populations.     

Effects of division of labour on immunity in workers of the ant <Emphasis Type="Italic">Cataglyphis cursor</Emphasis>

Workers in social insects perform different roles, and the environment they experience differs markedly according to the tasks performed. Life history theory predicts that individuals should adapt investment in immune defences according to their cost relative to the risk of pathogen infection. Workers, which forage outside the nest, face harsher environmental conditions with increased risk of injury and infection. We then might expect higher immune defences in foragers than in intra-nidal workers which remain in the relative sanctity of the nest. However, task partitioning in social insects is often age-based, and foragers are usually the oldest individuals. Hence if immune defences degenerate with age, foragers could have lower immune defences than intra-nidal workers. In this study, we examined the difference in immune defences as assessed by the level of phenoloxidase activity (PO) between intra-nidal workers and foragers in the ant Cataglyphis cursor. In three out of four colonies tested, foragers and intra-nidal workers did not differ in their level of PO. In the final colony, the level of workers PO overall was higher than in the other three colonies, and foragers displayed a lower level of PO than intra-nidal workers. These results may suggest that, when a sustained colonial PO activity is necessary, foragers may be less able than intra-nidal workers to activate their PO. Our results are discussed in the light of previous studies that underline the diversity of PO activity patterns in social insects. The variation observed among studies and even colonies clearly emphasizes the plasticity of immune parameters.     

Immune responses of bumblebee workers as a function of individual and colony age: senescence versus plastic adjustment of the immune function

Senescence may evolve in response to externally imposed schedules of survival and reproduction. It may result from cumulative damage to several tissues and organs leading to the progressive malfunction of somatic defences including the immune system, which is a relevant and accessible trait to study the evolution and the mechanisms of senescence.
In social insects, reproduction of workers is negligible and their fitness relies on the colony reproductive success, which depends on workers' longevity. Consequently, evolution of senescence in workers will rely on age-dependent survival rate and the residual reproductive value of the colony. Using the bumblebee, Bombus terrestris , as model system, we investigated changes in constitutive and inducible immune defences. Such changes were monitored in relation to worker age and colony age and as well under a high, persistent level of parasitism. The results show that the inducible production of antibacterial peptides was not affected by the age of individual worker or the age of their colony. In contrast, constitutive defences such as haemocyte density and phenoloxidase (PO) activity decline with individual worker age and potential accumulation of physiological defects due to high and persistent levels of parasitism did not explain this pattern of immunosenescence. Interestingly, levels of worker constitutive defences were found to increase with colony age. We found antibacterial activity was strongly traded-off against PO activity. Such a relationship was not caused by resource shortage. Overall, these data suggest that patterns of variation of immune defences with worker age and colony age are likely the result of plastic temporal adjustments of immune responses in accordance with life history predictions. Hence, control of pathogenic threat in a social system may involve optimal adjustments of immune defences at both the individual and colony levels.     

Heat stress during development affects immunocompetence in workers,queens and drones of Africanized honey bees (Apis mellifera L.) (Hymenoptera: Apidae)

Though social insects generally seem to have a reduced individual immunoresponse compared to solitary species, the impact of heat stress on that response has not been studied. In the honey bee, the effect of heat stress on reproductives (queens and males/drones) may also vary compared to workers, but this is currently unknown. Here, we quantified the activity of an enzyme linked to the immune response in insects and known to be affected by heat stress in solitary species: phenoloxidase (PO), in workers, queens and drones of Africanized honey bees (AHBs) experimentally subjected to elevated temperatures during the pupal stage. Additionally, we evaluated this marker in individuals experimentally infected with the entomopathogenic fungus Metarhizium anisopliae. Differences in PO activity were found between sexes and castes, with PO activity generally higher in workers and lower in reproductives. Such differences are associated with the likelihood of exposure to infection and the role of different individuals in the colony. Contrary to our expectation, heat stress did not cause an increase in PO activity equally in all classes of individual. Heat stress during the pupal stage significantly decreased the PO activity of AHB queens, but not that of workers or drones, which more frequently engage in extranidal activity. Experimental infection with Metarhizium anisopliae reduced PO activity in queens and workers, but increased it in drones. Notably, heat stressed workers lived significantly shorter after infection despite exhibiting greater PO activity than queens or drones. We suggest that this discrepancy may be related to trade-offs among immune response cascades in honey bees such as between heat shock proteins and defensin peptides used in microbial defence. Our results provide evidence for complex relationships among humoral immune responses in AHBs and suggest that heat stress could result in a reduced life expectancy of individuals.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

Generalist insects behave in a jasmonate-dependent manner on their host plants,leaving induced areas quickly and staying longer on distant parts

Plants are sessile, so have evolved sensitive ways to detect attacking herbivores and sophisticated strategies to effectively defend themselves. Insect herbivory induces synthesis of the phytohormone jasmonic acid which activates downstream metabolic pathways for various chemical defences such as toxins and digestion inhibitors. Insects are also sophisticated animals, and many have coevolved physiological adaptations that negate this induced plant defence. Insect behaviour has rarely been studied in the context of induced plant defence, although behavioural adaptation to induced plant chemistry may allow insects to bypass the host''s defence system. By visualizing jasmonate-responsive gene expression within whole plants, we uncovered spatial and temporal limits to the systemic spread of plant chemical defence following herbivory. By carefully tracking insect movement, we found induced changes in plant chemistry were detected by generalist Helicoverpa armigera insects which then modified their behaviour in response, moving away from induced parts and staying longer on uninduced parts of the same plant. This study reveals that there are plant-wide signals rapidly generated following herbivory that allow insects to detect the heterogeneity of plant chemical defences. Some insects use these signals to move around the plant, avoiding localized sites of induction and staying ahead of induced toxic metabolites.     

Adaptive social immunity in leaf-cutting ants

Social insects have evolved a suite of sophisticated defences against parasites. In addition to the individual physiological immune response, social insects also express ‘social immunity’ consisting of group-level defences and behaviours that include allogrooming. Here we investigate whether the social immune response of the leaf-cutting ant Acromyrmex echinatior reacts adaptively to the virulent fungal parasite, Metarhizium anisopliae. We ‘immunized’ mini-nests of the ants by exposing them twice to the parasite and then compared their social immune response with that of naive mini-nests that had not been experimentally exposed to the parasite. Ants allogroomed individuals exposed to the parasite, doing this both for those freshly treated with the parasite, which were infectious but not yet infected, and for those treated 2 days previously, which were already infected but no longer infectious. We found that ants exposed to the parasite received more allogrooming in immunized mini-nests than in naive mini-nests. This increased the survival of the freshly treated ants, but not those that were already infected. The results thus indicate that the social immune response of this leaf-cutting ant is adaptive, with the group exhibiting a greater and more effective response to a parasite that it has previously been exposed to.     

More than a colour change: insect melanism,disease resistance and fecundity

A ‘dark morph’ melanic strain of the greater wax moth, Galleria mellonella, was studied for its atypical, heightened resistance to infection with the entomopathogenic fungus, Beauveria bassiana. We show that these insects exhibit multiple intraspecific immunity and physiological traits that distinguish them from a non-melanic, fungus-susceptible morph. The melanic and non-melanic morphs were geographical variants that had evolved different, independent defence strategies. Melanic morphs exhibit a thickened cuticle, higher basal expression of immunity- and stress-management-related genes, higher numbers of circulating haemocytes, upregulated cuticle phenoloxidase (PO) activity concomitant with conidial invasion, and an enhanced capacity to encapsulate fungal particles. These insects prioritize specific augmentations to those frontline defences that are most likely to encounter invading pathogens or to sustain damage. Other immune responses that target late-stage infection, such as haemolymph lysozyme and PO activities, do not contribute to fungal tolerance. The net effect is increased larval survival times, retarded cuticular fungal penetration and a lower propensity to develop haemolymph infections when challenged naturally (topically) and by injection. In the absence of fungal infection, however, the heavy defence investments made by melanic insects result in a lower biomass, decreased longevity and lower fecundity in comparison with their non-melanic counterparts. Although melanism is clearly correlated with increased fungal resistance, the costly mechanisms enabling this protective trait constitute more than just a colour change.     

Gregariousness and repellent defences in the survival of phytophagous insects

Group living has both costs and benefits for plant‐feeding insects, but defence against predators is the most widely acknowledged benefit. Gregarious folivores typically have warning coloration and elaborate anti‐predator defences. Do these defences protect these species from predation? To see if protection from predators generally results from gregariousness, I compared the shapes of published survivorship curves of externally feeding, gregarious and solitary Lepidoptera and Symphyta. Gregarious species are less likely than solitary species to die in the larval stages. However, solitary species that have anti‐predator defences do not have higher larval survival compared to gregarious species. This result, along with evidence from experimental manipulations of group size, suggests that repellent defences per se do not increase survival of gregarious larvae. Group behaviour is undoubtedly important in affecting the higher larval survival of gregarious species, but we currently cannot determine whether predator learning, dilution of risk, or rapid development contribute most to increasing survival.     

Rapid anti-pathogen response in ant societies relies on high genetic diversity

Social organisms are constantly exposed to infectious agents via physical contact with conspecifics. While previous work has shown that disease susceptibility at the individual and group level is influenced by genetic diversity within and between group members, it remains poorly understood how group-level resistance to pathogens relates directly to individual physiology, defence behaviour and social interactions. We investigated the effects of high versus low genetic diversity on both the individual and collective disease defences in the ant Cardiocondyla obscurior. We compared the antiseptic behaviours (grooming and hygienic behaviour) of workers from genetically homogeneous and diverse colonies after exposure of their brood to the entomopathogenic fungus Metarhizium anisopliae. While workers from diverse colonies performed intensive allogrooming and quickly removed larvae covered with live fungal spores from the nest, workers from homogeneous colonies only removed sick larvae late after infection. This difference was not caused by a reduced repertoire of antiseptic behaviours or a generally decreased brood care activity in ants from homogeneous colonies. Our data instead suggest that reduced genetic diversity compromises the ability of Cardiocondyla colonies to quickly detect or react to the presence of pathogenic fungal spores before an infection is established, thereby affecting the dynamics of social immunity in the colony.     

Inbred and outbred honey bees (Apis mellifera) have similar innate immune responses

Social insect colonies provide ideal conditions for the spread of pathogens. It has been proposed that the extreme polyandry and genetic diversity seen in the colonies of some eusocial insect species is central to a colony’s defence against disease. Indeed, empirically, colonies headed by polyandrous queens have lower incidence of pathogens than genetically uniform monoandrous colonies. The mechanisms of improved resistance in genetically diverse colonies could arise from the genetic diversity among worker genotypes or from increased innate immunity arising from heterozygosity at immune gene loci within individual workers. Here, we investigate the effects of heterozygosity on two components of the honey bee (Apis mellifera) innate immune system: encapsulation and phenoloxidase (PO) activity. No significant effect of heterozygosity on immune system activity was evident for either encapsulation or PO activity. Thus, we conclude that while encapsulation and PO activity are important components of the immune response, it seems that they do not underlie the positive effects of genetic diversity on parasite and pathogen resistance in honey bees.     

Behavioural ecology of defence in a risky environment: caterpillars versus ants in a Neotropical savanna

1. Predatory ants may reduce infestation by herbivorous insects, and slow‐moving Lepidopteran larvae are often vulnerable on foliage. We investigate whether caterpillars with morphological or behavioural defences have decreased risk of falling prey to ants, and if defence traits mediate host plant use in ant‐rich cerrado savanna. 2. Caterpillars were surveyed in four cerrado localities in southeast Brazil (70–460 km apart). The efficacy of caterpillar defensive traits against predation by two common ant species (Camponotus crassus, C. renggeri) was assessed through experimental trials using caterpillars of different species and captive ant colonies. 3. Although ant presence can reduce caterpillar infestation, the ants' predatory effects depend on caterpillar defence traits. Shelter construction and morphological defences can prevent ant attacks (primary defence), but once exposed or discovered by ants, caterpillars rely on their size and/or behaviour to survive (secondary defence). 4. Defence efficiency depends on ant identity: C. renggeri was more aggressive and lethal to caterpillars than C. crassus. Caterpillars without morphological defences or inside open shelters were found on plants with decreased ant numbers. No unsheltered caterpillar was found on plants with extrafloral nectaries (EFNs). Caterpillars using EFN‐bearing plants lived in closed shelters or presented morphological defences (hairs, spines), and were less frequently attacked by ants during trials. 5. The efficiency of defences against ants is thus crucial for caterpillar survival and determines host plant use by lepidopterans in cerrado. Our study highlights the effect of EFN‐mediated ant‐plant interactions on host plant use by insect herbivores, emphasizing the importance of a tritrophic viewpoint in risky environments.     

Activation of prophenoloxidase and inhibition of melanization in the haemolymph of immune Galleria mellonella larvae

After immunization of Galleria larvae with bacterial lipopolysaccharide, inhibition of haemolymph melanization developed parallel with antibacterial immunity. Failure of melanization was correlated with significantly decreased amounts of active cell-associated phenoloxidase (PO). In normal, nonimmune haemolymph, cellular PO originated from plasma proPO activated by, and largely attached to, the haemocytes; activation was maximal by 150 min. With immune haemolymph, such cellular activation of plasma proPO did not occur. Immune plasma proPO was activated by homogenization, but not by freeze-thawing. Normal plasma proPO was activated by freeze-thawing, but only to a very slight extent by homogenization. Mixing immune plasma with the freeze-thaw activated PO in normal plasma (1:2, v:v) caused a 37%, average reduction in PO activity. The results suggest that inhibition of melanization in immune Galleria haemolymph is caused by plasma factor(s) inhibiting the activation of plasma proPO by haemocytes.     

Not one odour but two: A new model for nestmate recognition

Recognition systems play a key role in a range of biological processes, including mate choice, immune defence and altruistic behaviour. Social insects provide an excellent model for studying recognition systems because workers need to discriminate between nestmates and non-nestmates, enabling them to direct altruistic behaviour towards closer kin and to repel potential invaders. However, the level of aggression directed towards conspecific intruders can vary enormously, even among workers within the same colony. This is usually attributed to differences in the aggression thresholds of individuals or to workers having different roles within the colony. Recent evidence from the weaver ant Oecophylla smaragdina suggests that this does not tell the whole story. Here I propose a new model for nestmate recognition based on a vector template derived from both the individual’s innate odour and the shared colony odour. This model accounts for the recent findings concerning weaver ants, and also provides an alternative explanation for why the level of aggression expressed by a colony decreases as the diversity within the colony increases, even when odour is well-mixed. The model makes additional predictions that are easily tested, and represents a significant advance in our conceptualisation of recognition systems.     

Juvenile immune system activation induces a costly upregulation of adult immunity in field crickets Gryllus campestris

Inducible immune defence may allow organisms a state-dependent upregulation of costly immunity in order to minimize the risk of anticipated future parasitism. The basic costs of elevated immune activity might involve a reduction in other fitness-related traits as well as an increased risk of immunopathology. In male field crickets Gryllus campestris we experimentally investigated the condition-dependent effects of immune system activation in nymphs on immunity and physiological condition during adulthood. Following a nymphal injection of bacterial lipopolysaccharides, adult males showed significantly elevated levels of two major immune parameters, i.e. haemolymph antibacterial activity and the concentration of prophenoloxidase (proPO). By contrast, the active enzyme, phenoloxidase (PO), did not increase, suggesting a strategic long-term upregulation of the inactive proenzyme proPO only. This may help avoid the cytotoxic effects associated with high standing levels of the active enzyme. The nymphal immune insult further caused a reduction in adult haemolymph protein load, suggesting a long-term decline in overall metabolic condition. Nymphal food availability positively affected adult lysozyme activity, while PO and proPO concentrations were not affected. Our data thus suggest the long-term upregulation of immunity in response to antigenic cues as an adaptive, yet costly, invertebrate strategy to improve resistance to future parasitism.     

The strepsipteran endoparasite Xenos vesparum alters the immunocompetence of its host, the paper wasp Polistes dominulus

It is unexplained how strepsipteran insects manipulate the physiology of their hosts in order to undergo endoparasitic development without being entrapped by the innate immune defences of the host. Here we present pioneering work that aimed to explore for the first time several components of the cellular and humoral immune response among immature stages of the paper wasp Polistes dominulus, in both unparasitized insects and after infection by the strepsipteran endoparasite Xenos vesparum. We carried out hemocyte counts, phagocytosis assays in vitro and antibacterial response in vivo. On the whole, hemocyte load does not seem to be drastically affected by parasitization: a non-significant increase in hemocyte numbers was observed in parasitized wasps as respect to control, while the two dominant hemocyte types were present with similar proportions in both groups. On the other hand, phagocytosis was significantly reduced in hemocytes from parasitized wasps while the antibacterial response seemed to be less effective in control. These somewhat unexpected results are discussed, along with the implications of a multiple approach in immune response studies.