Symbiont-mediated protection against fungal infection in the dampwood termite, Zootermopsis angusticollis

Termites have a long co-evolutionary history with prokaryotic and eukaryotic gut microbes. Historically, the role of these obligate symbionts has been attributed to the nutritional welfare of the host. We hypothesize, however, that the nature of this mutualistic interaction extends beyond the nutritional benefits to the host and propose that termite gut symbionts enhance the host's defenses against pathogens. To test this hypothesis, a series of experiments were devised using the primitive dampwood termite Zootermopsis angusticollis and the entomopathogenic fungus Metarhizium anisopliae. In order to examine the role of symbionts in host protection, termites were experimentally defaunated (obligate anaerobic hindgut symbionts removed) with oxygen and compared with control animals in in vivo and in vitro experiments.;First, both defaunated and faunated control termites were exposed to the conidia of M. anisopliae or to a control Tween 80 solution and their survival was tracked for 21 days. In vivo data indicate that normally faunated termites are significantly less susceptible to M. anisopliae infection than their defaunated counterparts. In addition, defaunated termites are more susceptible to opportunistic pathogens, such as Serratia sp., than their faunated nestmates. The hindgut community of Z. angusticollis appears to play an important role in pathogen defense. To further characterize this role, two additional experiments were conducted.;Faunated and defaunated Z. angusticollis individuals were tested for beta-1,3 glucanase (betaGlu) activity. The gut of Z. angusticollis has several active betaGlu's. We have determined that this betaGlu activity is of symbiont-origin and that their presence in the gut may help explain the higher survival of faunated fungal-infected termites compared to defaunated fungal-infected nestmates. We have concluded that these betaGlu's likely play an important role in termite pathogen defense. In addition to the symbiont derived betaGlu's, we have also identified two betaGlu's of potential termite origin. Furthermore, the eastern subterranean termite, Reticulitermes flavipes, also exhibits symbiont-produced betaGlu's, indicating that this could be a widespread phenomenon in the phylogenetically basal ("lower") Isoptera.;Lastly, via in vitro experiments, we investigated the ability of termite tissue to inhibit the germination of M. anisopliae by incubating conidia with termite extracts. The fungistatic effect of faunated guts, measured by colony forming units (CFUs), tended to be stronger than that of the faunated gutted body, demonstrating that factors in intact guts make it particularly inhospitable to fungi. To test if the fungistatic nature of the gut was due to the presence of symbionts, extracts of faunated and defaunated insects were also incubated with fungal conidia. There were no significant differences in median number of CFUs between conidia incubated with faunated and defaunated termite extracts. One possible explanation for this is that bacterial symbionts not eliminated during oxygenation could be "compensating" by producing more or different antifungal compounds. Blocking the betaGlu activity with D-Glucon-1,5 lactone (GDL) in an attempt to "rescue" conidia viability following incubation with termite extracts did not have a consistent effect on M. anisopliae germination. This could potentially be connected to incomplete betaGlu inhibition by the GDL. The main contribution of this research lies in establishing a novel disease defense role for the mutualistic association between termites and their hindgut symbionts, which in turn contributes to a better understanding of the evolution of this mutualism, as well as the evolution of termite sociality and disease resistance.