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Insect pests such as termites cause damages to crops and man-made structures estimated at over $30 billion per year, imposing a global challenge for the human economy. Here, we report a strategy for compromising insect immunity that might lead to the development of nontoxic, sustainable pest control methods. Gram-negative bacteria binding proteins (GNBPs) are critical for sensing pathogenic infection and triggering effector responses. We report that termite GNBP-2 (tGNBP-2) shows β(1,3)-glucanase effector activity previously unknown in animal immunity and is a pleiotropic pattern recognition receptor and an antimicrobial effector protein. Termites incorporate this protein into the nest building material, where it functions as a nest-embedded sensor that cleaves and releases pathogenic components, priming termites for improved antimicrobial defense. By means of rational design, we present an inexpensive, nontoxic small molecule glycomimetic that blocks tGNBP-2, thus exposing termites in vivo to accelerated infection and death from specific and opportunistic pathogens. Such a molecule, introduced into building materials and agricultural methods, could protect valuable assets from insect pests.

In social insects, alerting nestmates to the presence of a pathogen should be critical for limiting its spread and initiating social mechanisms of defense. Here we show that subterranean termites use elevated vibratory alarm behavior to help prevent fatal fungal infections. The elevated alarm leads to elevated social hygiene. This requires that termites coalesce so that they can groom each other’s cuticular surfaces of contaminating conidial spores. Groups of 12 Reticulitermes flavipes workers varied in their response when immersed in conidia solutions of nine different strains of Metarhizium. Pathogen alarm displays of short 2–7-second bursts of rapid longitudinal oscillatory movement (LOM), observed over 12 min following a fungal challenge, were positively correlated with the time that workers spent aggregated together grooming each other. The frequency of these LOMs was inversely correlated with fatal fungal infections. The variation in fatalities appeared to be largely attributable to a differential response to Metarhizium brunneum and Metarhizium robertsii in the time spent in aggregations and the frequency of allogrooming. Isolated workers challenged with conidia did not display LOMs, which suggests that the alarm is a conditional social response. LOMs appear to help signal the presence of fungal pathogens whose virulence depends on the level of this emergency alert.

Allogrooming appears to be essential in many social animals for protection from routine exposure to parasites. In social insects, it appears to be critical for the removal of pathogenic propagules from the cuticle before they can start an infectious cycle. For subterranean termites, this includes fungal spores commonly encountered in the soil, such as Metarhizium conidia, that can quickly germinate and penetrate the cuticle. We investigated whether there is a difference in reliance on social and innate immunity in two closely related subterranean termites for protection from fatal infections by two locally encountered Metarhizium species. Our results indicate that relatively weak innate immunity in one termite species is compensated by more sustained allogrooming. This includes enhanced allogrooming in response to concentrations of conidia that reflect more routine contamination of the cuticle as well as to heavy cuticular contamination that elicits a networked emergency response.

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. Reduced genetic diversity through inbreeding can negatively affect pathogen resistance. We investigated this hypothesis in termites by testing whether the level of inbreeding within a colony affected the survival of workers challenged with two fungal pathogens. We found a bimodal distribution of colony survival, whereby colonies exhibit either high or low susceptibility. However, this difference was not explained by the level of inbreeding within the colony, suggesting that colony immunity may rely more on a specific genetic background, rather than its overall genetic diversity.

The evolution of termite eusociality has been influenced by their nesting and foraging ecology. This includes the evolution of a separate developmental line for specialized workers that forego direct reproduction (true workers), which coincides with the transition from inhabiting a dead-wood nest to foraging for food outside the nest. Foraging for extranidal food requires that termites move through an entomopathogen-rich rhizosphere. This suggests that improved defenses against these pathogens were required for the successful transition to foraging outside the nest. Soil is especially rich in insect pathogens such as Metarhizium, and termites use secreted salivary β-1,3-glucanases for protection from this fungus. These enzymes are likely to be dependent on hygiene behaviors, such as allogrooming of external surfaces after contact with fungal conidial spores or ingestion or burying of infected nestmates prior to sporulation of the cadaver. These social mechanisms of defense could compensate for internal innate mechanisms of defense and even relax selective pressure on these innate mechanisms. Here, we investigated whether the selective pressure was intensified or relaxed on secreted β-1,3-glucanases as well as internal innate immune proteins, especially those putatively involved in antifungal defense. An analysis of the molecular evolution of two termite β-1,3-glucanases (GNBP1,2) indicates that the intensity of selection on them significantly increased with the transition to foraging. The shift to foraging for extranidal food apparently required adaptive modification of secreted GNBPs to help cope with increased exposure to pathogenic conidia in the soil. This included modification of a conserved binding site in GNBP2. In contrast, there was either significant relaxation or no change of selection pressure on Toll and phenoloxidase pathway immune genes with the transition to foraging. Relaxation was also observed with the evolution of drywood termites, but this likely reflects a transition to a microhabitat with fewer pathogens. Selection intensified on a subset of immune genes that regulate intestinal microbes with the more recent radiation of the Termitidae and the diversification of their feeding strategies.Significance statementThe evolution of termite worker eusociality coincides with a relaxation of selective pressure on innate components of the immune system, especially those involved in antifungal defense. In contrast, the selective pressure intensified on two β-1,3-glucanases that contribute to the elimination of fungal pathogens by social behaviors such as allogrooming, cannibalism, and undertaking. This change in selective pressure appears to reflect an increased reliance on social immunity by dedicated altruistic workers in response to novel or enhanced threat by fungal pathogens. This supports other studies in social insects that indicate that the evolution of social immunity not only compensates for an increased vulnerability to infectious disease associated with living in crowded conditions but relaxes selective pressure on components of the internal innate immune system. Our results also indicate that a shift to microbially depauperate habitats relaxed selective pressure on termite immune defenses whereas a more recent diversification of diet intensified selection on mechanisms for regulating intestinal microbes.

The genetic organization of colonies of the subterranean termite Reticulitermes flavipes in two subpopulations in Massachusetts was explored using five polymorphic allozymes and double-strand conformation polymorphism (DSCP) analysis of the mitochondrial control region. Empirically obtained estimates of worker relatedness and F-statistics were compared with values generated by computer simulations of breeding schemes to make inferences about colony organization. In one study site (G), worker genotypes indicated the presence of a mixture of colonies headed by monogamous outbred primary reproductives and colonies headed by inbreeding neotenic reproductives, both colony types having limited spatial ranges. A second site (S) was dominated by several large colonies with low relatedness among nestmates. Mixed DSCP haplotypes in three colonies indicated that nestmates had descended from two or three unrelated female reproductives. Computer simulations of breeding schemes suggested that positive colony inbreeding coefficients at site S resulted from either commingling of workers from different nests or different colonies. Such an exchange of workers between nests corresponds to the multiple-site nesting lifetype of many subterranean termites and resembles colony structure in polycalic Formica ants. Our study demonstrates considerable variation in R. flavipes colony structure over a small spatial scale, including colonies headed by monogamous outbred primary reproductives, colonies containing multiple inbred neotenic reproductives and large polydomous colonies containing the progeny of two or more unrelated queens, and suggests that the number of reproductives and nestmate relatedness change with colony age and size.

We identified the antifungal gene termicin in three species of Cryptocercus woodroaches. Cryptocercus represents the closest living cockroach lineage of termites, which suggests that the antifungal role of termicin evolved prior to the divergence of termites from other cockroaches. An analysis of Cryptocercus termicin and two β-1,3-glucanase genes ( GNBP1 and GNBP2 ), which appear to work synergistically with termicin in termites, revealed evidence of selection in these proteins. We identified the signature of past selective sweeps within GNBP2 from Cryptocercus punctulatus and Cryptocercus wrighti . The signature of past selective sweeps was also found within termicin from Cryptocercus punctulatus and Cryptocercus darwini . Our analysis further suggests a phenotypically identical variant of GNBP2 was maintained within Cryptocercus punctulatus , Cryptocercus wrighti , and Cryptocercus darwini while synonymous sites diverged. Cryptocercus termicin and GNBP2 appear to have experienced similar selective pressure to that of their termite orthologues in Reticulitermes . This selective pressure may be a result of ubiquitous entomopathogenic fungal pathogens such as Metarhizium . This study further reveals the similarities between Cryptocercus woodroaches and termites.

Termicin is an anti-fungal defensin that is disseminated from termite salivary glands. The peptide appears to be critical for the elimination with mutual grooming (allogrooming) of pathogenic spores (conidia) that have attached to the insect cuticle. There has been a recent selective sweep for an advantageous variant of this peptide in the subterranean termite Reticulitermes flavipes . We tested the anti-mycotic activity of a recombinant termicin corresponding with this variant against the conidia of different Metarhizium fungal isolates from soil close to foraging R. flavipes workers. Termicin was most effective against isolates that had previously been shown to elicit a relatively weak alarm response, as indicated by brief bouts of rapid longitudinal oscillatory movement (LOM). These isolates that elicited weak alarm were also the deadliest apparently because the survival of termites exposed to the fungus depends on a strong social immune response (LOMs and allogrooming). The selective pressure for a single termicin variant may have been driven by the most dangerous isolates that elicit a weak behavioral response. The correlation between termicin anti-fungal activity and LOM suggests that pathogen-associated molecular patterns that affect termite recognition of conidial contamination and the onset of elevated allogrooming also affect the vulnerability of conidia to the disruption of their cell membranes by termicin.

Loss of aggression between social groups can have far-reaching effects on the structure of societies and populations. We tested whether variation in the genetic structure of colonies of the termite Nasutitermes corniger affects the probability of aggression toward non-nestmates and the ability of unrelated colonies to fuse. We determined the genotypes of workers and soldiers from 120 colonies at seven polymorphic microsatellite loci. Twenty-seven colonies contained offspring of multiple founding queens or kings, yielding an average within-colony relatedness of 0.33. Genotypes in the remaining 93 colonies were consistent with reproduction by a single queen and king or their progeny, with an average within-colony relatedness of 0.51. In standardized assays, the probability of aggression between workers and soldiers from different colonies was an increasing function of within-colony relatedness. The probability of aggression was not affected significantly by the degree of relatedness between colonies, which was near zero in all cases, or by whether the colonies were neighbors. To test whether these assays of aggression predict the potential for colony fusion in the field, we transplanted selected nests to new locations. Workers and soldiers from colonies that were mutually tolerant in laboratory assays joined their nests without fighting, but workers and soldiers that were mutually aggressive in the assays initiated massive battles. These results suggest that the presence of multiple unrelated queens or kings promotes recognition errors, which can lead to the formation of more complex colony structures.