Social transfer of pathogenic fungus promotes active immunisation in ant colonies

Due to the omnipresent risk of epidemics, insect societies have evolved sophisticated disease defences at the individual and colony level. An intriguing yet little understood phenomenon is that social contact to pathogen-exposed individuals reduces susceptibility of previously naive nestmates to this pathogen. We tested whether such social immunisation in Lasius ants against the entomopathogenic fungus Metarhizium anisopliae is based on active upregulation of the immune system of nestmates following contact to an infectious individual or passive protection via transfer of immune effectors among group members--that is, active versus passive immunisation. We found no evidence for involvement of passive immunisation via transfer of antimicrobials among colony members. Instead, intensive allogrooming behaviour between naive and pathogen-exposed ants before fungal conidia firmly attached to their cuticle suggested passage of the pathogen from the exposed individuals to their nestmates. By tracing fluorescence-labelled conidia we indeed detected frequent pathogen transfer to the nestmates, where they caused low-level infections as revealed by growth of small numbers of fungal colony forming units from their dissected body content. These infections rarely led to death, but instead promoted an enhanced ability to inhibit fungal growth and an active upregulation of immune genes involved in antifungal defences (defensin and prophenoloxidase, PPO). Contrarily, there was no upregulation of the gene cathepsin L, which is associated with antibacterial and antiviral defences, and we found no increased antibacterial activity of nestmates of fungus-exposed ants. This indicates that social immunisation after fungal exposure is specific, similar to recent findings for individual-level immune priming in invertebrates. Epidemiological modeling further suggests that active social immunisation is adaptive, as it leads to faster elimination of the disease and lower death rates than passive immunisation. Interestingly, humans have also utilised the protective effect of low-level infections to fight smallpox by intentional transfer of low pathogen doses ("variolation" or "inoculation").     

Maternal Exposure of a Beetle to Pathogens Protects Offspring against Fungal Disease

Maternal exposure to an immune challenge can convey enhanced immunity to invertebrate offspring in the next generation. We investigated whether maternal exposure of the Asian longhorned beetle, Anoplophora glabripennis, to two species of the fungus Metarhizium or the bacterium Serratia marcescens elicited transgenerational immune priming (TGIP). We tested specificity of this protection and whether occurrence of TGIP was dependent on maternal exposure to living versus dead pathogens. Our results show that TGIP occurred and protected offspring against Metarhizium brunneum. Maternal exposure to S. marcescens provided non-specific protection to offspring against a fungal pathogen, but TGIP in response to Metarhizium only occurred when offspring were exposed to the same fungal species that was used to prime mothers. Moreover, TGIP in response to M. brunneum occurred only after maternal exposure to living rather than dead fungus. Our findings suggest that occurrence of TGIP could be both specific and dependent on whether the pathogen was alive.     

Previous encapsulation response enhances within individual protection against fungal parasite in the mealworm beetle Tenebrio molitor

Immune defenses of insects show either broad reactions or specificity and durability of induced protection against attacking parasites and pathogens. In this study, we tested whether encapsulation response against nylon monofilament increases between two attempts of activation of immune system in mealworm beetles Tenebrio molitor, and whether previous exposure to nylon monofilament may also increase protection against an entomopathogenic fungus. We found that survival of beetles subjected to immune activation by nylon implant and subsequent fungal exposure a week later was significantly higher than survival of beetles which had been subjected to fungal infection only. This result suggests that previous immune activation by the nylon implant may be considered as broad spectrum “immune priming” which helps to fight not only the same intruder but also other parasites.     

Immune priming and pathogen resistance in ant queens

Growing empirical evidence indicates that invertebrates become more resistant to a pathogen following initial exposure to a nonlethal dose; yet the generality, mechanisms, and adaptive value of such immune priming are still under debate. Because life‐history theory predicts that immune priming and large investment in immunity should be more frequent in long‐lived species, we here tested for immune priming and pathogen resistance in ant queens, which have extraordinarily long life span. We exposed virgin and mated queens of Lasius niger and Formica selysi to a low dose of the entomopathogenic fungus Beauveria bassiana, before challenging them with a high dose of the same pathogen. We found evidence for immune priming in naturally mated queens of L. niger. In contrast, we found no sign of priming in virgin queens of L. niger, nor in virgin or experimentally mated queens of F. selysi, which indicates that immune priming in ant queens varies according to mating status and mating conditions or species. In both ant species, mated queens showed higher pathogen resistance than virgin queens, which suggests that mating triggers an up‐regulation of the immune system. Overall, mated ant queens combine high reproductive output, very long life span, and elevated investment in immune defense. Hence, ant queens are able to invest heavily in both reproduction and maintenance, which can be explained by the fact that mature queens will be protected and nourished by their worker offspring.     

Exposure to Bacterial Signals Does Not Alter Pea Aphids’ Survival upon a Second Challenge or Investment in Production of Winged Offspring

Pea aphids have an obligate nutritional symbiosis with the bacteria Buchnera aphidicola and frequently also harbor one or more facultative symbionts. Aphids are also susceptible to bacterial pathogen infections, and it has been suggested that aphids have a limited immune response towards such pathogen infections compared to other, more well-studied insects. However, aphids do possess at least some of the genes known to be involved in bacterial immune responses in other insects, and immune-competent hemocytes. One possibility is that immune priming with microbial elicitors could stimulate immune protection against subsequent bacterial infections, as has been observed in several other insect systems. To address this hypothesis we challenged aphids with bacterial immune elicitors twenty-four hours prior to live bacterial pathogen infections and then compared their survival rates to aphids that were not pre-exposed to bacterial signals. Using two aphid genotypes, we found no evidence for immune protection conferred by immune priming during infections with either Serratia marcescens or with Escherichia coli. Immune priming was not altered by the presence of facultative, beneficial symbionts in the aphids. In the absence of inducible immune protection, aphids may allocate energy towards other defense traits, including production of offspring with wings that could escape deteriorating conditions. To test this, we monitored the ratio of winged to unwinged offspring produced by adult mothers of a single clone that had been exposed to bacterial immune elicitors, to live E. coli infections or to no challenge. We found no correlation between immune challenge and winged offspring production, suggesting that this mechanism of defense, which functions upon exposure to fungal pathogens, is not central to aphid responses to bacterial infections.     

Ants medicate to fight disease

Parasites are ubiquitous, and the ability to defend against these is of paramount importance. One way to fight diseases is self‐medication, which occurs when an organism consumes biologically active compounds to clear, inhibit, or alleviate disease symptoms. Here, we show for the first time that ants selectively consume harmful substances (reactive oxygen species, ROS) upon exposure to a fungal pathogen, yet avoid these in the absence of infection. This increased intake of ROS, while harmful to healthy ants, leads to higher survival of exposed ants. The fact that ingestion of this substance carries a fitness cost in the absence of pathogens rules out compensatory diet choice as the mechanism, and provides evidence that social insects medicate themselves against fungal infection, using a substance that carries a fitness cost to uninfected individuals.     

Cell-cell recognition of host surfaces by pathogens. The adsorption of maize (Zea mays) root mucilage by surfaces of pathogenic fungi.

The adsorption of radioactive mucilage by pathogenic fungi was shown to be dependent upon time, the composition of mucilage, the type of fungal surface (conidia, hyphae, hyphal apices), fungal species, pH and bivalent cations. All fungal adhesins were inactivated by either proteinase or polysaccharase treatments. Adsorption was not inhibited by the numberous mono-, di- and oligo-saccharides that were tested individually, but it was inhibited absolutely by several polysaccharides. This suggested that adsorption of mucilage by pathogens involved conformational and ionic interactions between plant and fungal polymers but not fungal lectins bound to sugar residues of mucilage. Several fractionation schemes showed that pathogens bound only the most acidic of the variety of polymers that comprise mucilage. There was not any absolute distinction between ability to bind radioactive mucilage and type of pathogen or non-pathogen. However, there were notable differences in characteristics of adsorption between two types of pathogen. Differences were revealed by comparison of the adsorption capacities of conidia and germinant conidia and chromatography of radioactive mucilage on germinant conidia. An ectotrophic root-infecting fungus (a highly specialized pathogen) bound a greater proportion of mucilage than did a vascular-wilt fungus (of catholic host and tissue range) with more than one class of site for adsorption. In contrast with the vascular-wilt fungus, sites for adsorption on the specialized pathogen were present solely on surfaces formed by germination.     

Locusts increase carbohydrate consumption to protect against a fungal biopesticide

There is growing evidence to suggest that hosts can alter their dietary intake to recoup the specific resources involved in mounting effective resistance against parasites and pathogens. We examined macronutrient ingestion and disease-resistance in the Australian plague locust (Chortoicetes terminifera), challenged with a fungal pathogen (Metarhizium acridum) under dietary regimes varying in their relative amounts of protein and digestible carbohydrate. Dietary protein influenced constitutive immune function to a greater extent than did carbohydrate, indicating higher protein costs of mounting an immune defence than carbohydrate or overall energy costs. However, it appears that increased immune function, as a result of greater protein ingestion, was not sufficient to protect locusts from fungal disease. We found that locusts restricted to diets high in protein (P) and low in carbohydrate (C) were more likely to die of a fungal infection than those restricted to diets with a low P:C ratio. We hypothesise that the fungus is more efficient at exploiting protein in the insect’s haemolymph than the host is at producing immune effectors, tipping the balance in favour of the pathogen on high-protein diets. When allowed free-choice, survivors of a fungus-challenge chose a less-protein-rich diet than those succumbing to infection and those not challenged with fungus locusts. These results are contrary to previous studies on caterpillars in the genus Spodoptera challenged with bacterial and baculoviral pathogens, indicating that nutrient ingestion and pathogen resistance may be a complex interaction specific to different host species and disease agents.     

Insect immune priming: ecology and experimental evidences

1. Immune priming refers to improved protection of the host after a second encounter with the same parasite or pathogen. This phenomenon is similar to that of adaptive immunity in vertebrates. 2. There is evidence to suggest that this improved protection can be species/strain‐specific and can protect organisms for a lifetime. These two attributes, along with a biphasic immune response, are essential characteristics of immune priming and form the basis for the effectiveness of resistance to parasites and pathogens. 3. This paper considers the effect of immune priming within and across generations, the influence of a heterologous challenge during immune priming and the importance of testing the immune response with natural pathogens. 4. The analysis presented takes into account the multifaceted nature of the invertebrate immune response. The lack of evidence suggesting that the bacterial microbiome plays a complementary role in the immune priming outcome is discussed. 5. Finally, the cost of immune priming is explored. This is a poorly investigated issue, which could help to explain why there is a paucity of evidence in support of immune priming.     

Weeding and grooming of pathogens in agriculture by ants

The ancient mutualism between fungus-growing ants and the fungi they cultivate for food is a textbook example of symbiosis. Fungus-growing ants' ability to cultivate fungi depends on protection of the garden from the aggressive microbes associated with the substrate added to the garden as well as from the specialized virulent garden parasite Escovopsis. We examined ants' ability to remove alien microbes physically by infecting Atta colombica gardens with the generalist pathogen Trichoderma viride and the specialist pathogen Escovopsis. The ants sanitized the garden using two main behaviours: grooming of alien spores from the garden (fungus grooming) and removal of infected garden substrate (weeding). Unlike previously described hygienic behaviours (e.g. licking and self-grooming), fungus-grooming and garden-removal behaviours are specific responses to the presence of fungal pathogens. In the presence of pathogens, they are the primary activities performed by workers, but they are uncommon in uninfected gardens. In fact, workers rapidly eliminate Trichoderma from their gardens by fungus grooming and weeding, suggesting that these behaviours are the primary method of garden defence against generalist pathogens. The same sanitary behaviours were performed in response to the presence of the specialist pathogen Escovopsis. However, the intensity and duration of these behaviours were much greater in this treatment. Despite the increased effort, the ants were unable to eliminate Escovopsis from their gardens, suggesting that this specialized pathogen has evolved counter-adaptations in order to overcome the sanitary defences of the ants.     

Insect immunity shows specificity in protection upon secondary pathogen exposure

Immunological memory in vertebrates, conferring lasting specific protection after an initial pathogen exposure, has implications for a broad spectrum of evolutionary, epidemiological, and medical phenomena . However, the existence of specificity in protection upon secondary pathogen exposure in invertebrates remains controversial . To separate this functional phenomenon from a particular mechanism, we refer to it as specific immune priming. We investigate the presence of specific immune priming in workers of the social insect Bombus terrestris. Using three bacterial pathogens, we test whether a prior homologous pathogen exposure gives a benefit in terms of long-term protection against a later challenge, over and above a heterologous combination. With a reciprocally designed initial and second-exposure protocol (i.e., all combinations of bacteria were tested), we demonstrate, even several weeks after the clearance of a first exposure, increased protection and narrow specificity upon secondary exposure. This demonstrates that the invertebrate immune system is functionally capable of unexpectedly specific and durable induced protection. Ultimately, despite general broad differences between vertebrates and invertebrates, the ability of both immune systems to show specificity in protection suggests that their immune defenses have found comparable solutions to similar selective pressures over evolutionary time.     

The epidemiological consequences of immune priming

Exposure to low doses of pathogens that do not result in the host becoming infectious may ‘prime’ the immune response and increase protection to subsequent challenge. There is increasing evidence that such immune priming is a widespread and important feature of invertebrate host–pathogen interactions. Immune priming clearly has implications for individual hosts but will also have population-level implications. We present a susceptible–primed–infectious model—in contrast to the classic susceptible–infectious–recovered framework—to investigate the impacts of immune priming on pathogen persistence and population stability. We describe impacts of immune priming on the epidemiology of the disease in both constant and seasonal environments. A key result is that immune priming may act to destabilize population dynamics. In particular, when the proportion of individuals becoming primed rather than infected is high, but this priming does not confer full immunity, the population may be strongly destabilized through the generation of limit cycles. We discuss the implications of our model both in the context of invertebrate immunity and more widely.     

Natural variation in colony inbreeding does not influence susceptibility to a fungal pathogen in a termite

Reduced genetic diversity through inbreeding can negatively affect pathogen resistance. This relationship becomes more complicated in social species, such as social insects, since the chance of disease transmission increases with the frequency of interactions among individuals. However, social insects may benefit from social immunity, whereby individual physiological defenses may be bolstered by collective‐level immune responses, such as grooming or sharing of antimicrobial substance through trophallaxis. We set out to determine whether differences in genetic diversity between colonies of the subterranean termite, Reticulitermes flavipes, accounts for colony survival against pathogens. We sampled colonies throughout the United States (Texas, North Carolina, Maryland, and Massachusetts) and determined the level of inbreeding of each colony. To assess whether genetically diverse colonies were better able to survive exposure to diverse pathogens, we challenged groups of termite workers with two strains of a pathogenic fungus, one local strain present in the soil surrounding sampled colonies and another naïve strain, collected outside the range of this species. We found natural variation in the level of inbreeding between colonies, but this variation did not explain differences in susceptibility to either pathogen. Although the naïve strain was found to be more hazardous than the local strain, colony resistance was correlated between two strains, meaning that colonies had either relatively high or low susceptibility to both strains regardless of their inbreeding coefficient. Overall, our findings may reflect differential virulence between the strains, immune priming of the colonies via prior exposure to the local strain, or a coevolved resistance toward this strain. They also suggest that colony survival may rely more upon additional factors, such as different behavioral response thresholds or the influence of a specific genetic background, rather than the overall genetic diversity of the colony.     

When subterranean termites challenge the rules of fungal epizootics

Over the past 50 years, repeated attempts have been made to develop biological control technologies for use against economically important species of subterranean termites, focusing primarily on the use of the entomopathogenic fungus Metarhizium anisopliae. However, no successful field implementation of biological control has been reported. Most previous work has been conducted under the assumption that environmental conditions within termite nests would favor the growth and dispersion of entomopathogenic agents, resulting in an epizootic. Epizootics rely on the ability of the pathogenic microorganism to self-replicate and disperse among the host population. However, our study shows that due to multilevel disease resistance mechanisms, the incidence of an epizootic within a group of termites is unlikely. By exposing groups of 50 termites in planar arenas containing sand particles treated with a range of densities of an entomopathogenic fungus, we were able to quantify behavioral patterns as a function of the death ratios resulting from the fungal exposure. The inability of the fungal pathogen M. anisopliae to complete its life cycle within a Coptotermes formosanus (Isoptera: Rhinotermitidae) group was mainly the result of cannibalism and the burial behavior of the nest mates, even when termite mortality reached up to 75%. Because a subterranean termite colony, as a superorganism, can prevent epizootics of M. anisopliae, the traditional concepts of epizootiology may not apply to this social insect when exposed to fungal pathogens, or other pathogen for which termites have evolved behavioral and physiological means of disrupting their life cycle.     

Fungus Exposed Solenopsis invicta Ants Benefit from Grooming

We investigated aspects of resistance to entomopathogenic fungi in the social insect Solenopsis invicta, the red imported fire ant (RIFA). RIFA reared individually were significantly more susceptible to the entomopathogenic fungi Metarhizium anisopliae var. anisopliae M09 than reared in groups. Fungus exposed ants performed more self-grooming behavior when isolated as individuals and received more allo-grooming when accompanied with four healthy nestmates. Using fluorescence microscopy, we counted the number of fluorescein isothiocyanate (FITC)-labeled conidia on the cuticle of fungus exposed ants reared individually or as groups. The number of conidia on the surface of grouped ants decreased more rapidly than on isolated individuals. Allo-grooming behavior appears to be important in removing the conidia on the surface of RIFA. Individuals help fungus exposed ants by performing intensive grooming behaviors, which either risk infecting themselves or get them immunized as social immunity. We show evidence that contacting with fungus exposed ants would decrease susceptibility of nestmates to the fungus. All these results indicate that RIFA benefit from grooming behavior to fight against the fungal pathogens. Future advances in biological control of RIFA with entomopathogenic fungi are also discussed.     

Host–pathogen interaction after infection of Galleria mellonella with the filamentous fungus Beauveria bassiana

The filamentous fungus Beauveria bassiana is a natural pathogen of the greater wax moth Galleria mellonella. Infection with this fungus triggered systemic immune response in G. mellonella; nevertheless, the infection was lethal if spores entered the insect hemocel. We observed melanin deposition in the insect cuticle and walls of air bags, while the invading fungus interrupted tissue continuity. We have shown colonization of muscles, air bags, and finally colonization and complete destruction of the fat body—the main organ responsible for the synthesis of defense molecules in response to infection. This destruction was probably not caused by simple fungal growth, because the fat body was not destroyed during colonization with a human opportunistic pathogen Candida albicans. This may mean that the infecting fungus is able to destroy actively the insect's fat body as part of its virulence mechanism. Finally, we were unable to reduce the extremely high virulence of B. bassiana against G. mellonella by priming of larvae with thermally inactivated fungal spores.     

The presence of conifer resin decreases the use of the immune system in wood ants

Abstract.  1. Wood ants ( Formica paralugubris ) incorporate large amounts of solidified conifer resin into their nest, which reduces the density of many bacteria and fungi and protects the ants against some detrimental micro-organisms. By inducing an environment unfavourable to pathogens, the presence of resin may allow workers to reduce the use of their immune system.
2. The present study tested the hypothesis that the presence of resin decreases the immune activity of wood ants. Specifically, three components of the humoral immune defences of workers kept in resin-rich and resin-free experimental nests (antibacterial, lytic, and prophenoloxidase activities) were compared.
3. The presence of resin was associated with reduced bacterial and fungal densities in nest material and with a small decrease in worker antibacterial and lytic activities. The prophenoloxidase activity was very low in all workers and was not affected by the presence of resin.
4. These results suggest that collective medication with resin reduces pathogen pressure, which in turn decreases the use of the inducible part of the immune system. More generally, the use of plant secondary compounds might be an efficient and economical way to fight pathogens.     

Divergent immune priming responses across flour beetle life stages and populations

Growing evidence shows that low doses of pathogens may prime the immune response in many insects, conferring subsequent protection against infection in the same developmental stage (within‐life stage priming), across life stages (ontogenic priming), or to offspring (transgenerational priming). Recent work also suggests that immune priming is a costly response. Thus, depending on host and pathogen ecology and evolutionary history, tradeoffs with other fitness components may constrain the evolution of priming. However, the relative impacts of priming at different life stages and across natural populations remain unknown. We quantified immune priming responses of 10 natural populations of the red flour beetle Tribolium castaneum, primed and infected with the natural insect pathogen Bacillus thuringiensis. We found that priming responses were highly variable both across life stages and populations, ranging from no detectable response to a 13‐fold survival benefit. Comparing across stages, we found that ontogenic immune priming at the larval stage conferred maximum protection against infection. Finally, we found that various forms of priming showed sex‐specific associations that may represent tradeoffs or shared mechanisms. These results indicate the importance of sex‐, life stage‐, and population‐specific selective pressures that can cause substantial divergence in priming responses even within a species. Our work highlights the necessity of further work to understand the mechanistic basis of this variability.