| Termite societies are characterized by high density,frequent contact and close relative,contributing to pathogen transmission.To defend against pathogenic infections,termite societies have evolved a series of behavioral,physiological and organisitional defences,which is called social immunity.At present,studies on termite social immunity mainly focus on the behavioral and physiological changes against diseases at the colony level,including grooming,trophallaxis,isolation,aggression,cellular immunity,humoral immunity and so on.However,its molecular regulatory mechanisms are still few.Thus,we address this problem by using the termite R.chinensis and the entomopaghenic fungus M.anisopliae as experimental materials.The main conclusions are as follows:1 The termite R.chinensis employs active immunization to defend against the pathogenic fungus M.anisopliaeTermites exposed to the pathogenic fungus M.anisopliae exhibited significantly increased allogrooming frequency(p<0.05).By fluorescence microscope,we found that na?ve termite nestmates obtained a low dose of conidia from fungus-exposed termites via the social contact.Hence,the colony forming units(CFUs) in the nestmates of fungus-exposed termites were significantly lower than in the fungus-exposed termites(p<0.01),causing lower mortality of the nestmates(13%) when compared to the exposed termites(50%).However,the nestmates of fungus-exposed termites exhibited singnificantly higher antifungal activity,the activites of antioxidases(catalase and superoxide dismutase) and expressions of immune genes(Phenoloxidase,transferrin and termicin) compared with the nestmates of unexposed termites(p<0.05).These results indicated that active social transfer of the low-dose conidia via allogrooming behavior could activate the immune system of the nestmates of the fungus-exposed termites,resulting in increased number of immune individuals and hence decreased mortality of individuals.This immune strategy in termite societies is similar to the utilization of the variolation in humans to fight against smallpox,namely active immunization in this study.2 Screening of potential proteins associated with active immunization in the termite R.chinensisWe found 40 upregulated and 22 downregulated proteins in nestmates of fungus-exposed termites by using iTRAQ techonology,in which 20 differently expressed proteins were associated with immunity including 12 stress response proteins,six immune siginalling proteins and two immune effectors.Subsequently,14 differently expressed proteins from iTRAQ techonology were validated by multiple reaction monitoring(MRM) technology.Among them,there were one metabolic poritein(isocitrate dehydrogenase [NAD~+] subunit alpha,IDH-α),three stress response proteins(glutathione S-transferase D1,60S ribosomal protein L23 and cuticle protein 19) and one immune signaling protein(ubiquitin conjugating enzyme).IDH-αis the subunit of the isocitrate dehydrogenase[NAD~+]that is the rate-limiting enzyme in tricarboxylic acid(TCA) cycle.Considering that IDH-αwas significantly upregulated during active immunization of the termite R.chinensis,we predict that IDH-αmay play an important role in active immunization of R.chinensis.Certainly,this hypothesis needs to be further vadilated in the later experiments.3 The influence of IDH-αpotentially associated with active immunization on metabolism of the termite R.chinensisRNAi-mediated IDH-αsilencing significantly decreased mRNA level(p<0.05) and protein level of IDH-α,reduced the isocitrate content(p<0.05),but increased the NADH level(p<0.05),indicating an impaired NAD~+-IDH reaction in the termite R.chinensis.Meanwhile,IDH-αsilencing also caused significantly increased glycogen,glucose,lactate and triglycerides(p<0.05),but significantly reduced pyruvate(p<0.05),implying a disrupted carbohydrate metabolism in which pyruvate is outside of the TCA cycle and converted to lactate and triglycerides.In addition,IDH-αsilenced termites also exhibited significantly decreased chromium and manganese(p<0.05),and marginally significantly decreased ferrum(p=0.078) and selenium(p=0.057),but significantly increased zinc(p<0.05),suggesting a disturbed homeostasis of trace elements.Moreove,IDH-αsilenced termites exhibited reduced amino acids including arginine,proline,glycine,alanine,valine,leucine and isoleucine(p<0.05),significantly altered the expressions of five genes involved in amino acid metabolism(p<0.05),and significantly increased uric acid and urea(p<0.05).These results suggested that an aberrantly enhanced amino acid metabolism was activated to produceα-ketoglutarate which enters into the TCA cycle for energy compensation.Thus,the knockdown of IDH-αcan result in metabolic disorders of R.chinensis.4 The regulatory effect of IDH-αon active immunization of the termite R.chinensisAlthough IDH-αsilencing had no significant influence on either allogrooming frequency in fungus-exposed termites or immune gene expressions in nestmates of fungus-exposed termites,it significantly diminished the antifungal activity(p<0.01) and increased mortality(p<0.05) in the nestmates of fungus-exposed termites,indicating that significant downregulation of IDH-αcould disrupted active immunization.Furthermore,the IDH-αsilenced nestmates of fungus-exposed termites exhibited significantly increased caspase 3 activity(p<0.05),upregulated expressions of apoptotic genes(p<0.01),and reduced ATP content(p<0.01).They also exhibited more apoptotic cells in different body parts,including heads,thorax,abdmen and guts and had significantly higher apoptosis rate(p<0.05) when compared to the control.Also,their guts showed significantly more colony forming units(CFUs) (p<0.01).Therefore,IDH-αsilencing can cause metabolic disorders and excessive apoptosis of the termite R.chinensis,which leads to the increased risk of invastion and penetration by M.anisopliae conidia in the termite guts and makes low-dose fungal conidia cause the death of termites.In conclusion,the knockdown of IDH-αcan result in the disruption of active immunization against the pathogenic fungus in the termites R.chinensis.In conclusion,this study found that termite colonies can employ active mmunization to defend against diseases,which is similar to the variolation in human beings.Meanwhile,the key metabolic gene IDH-αis shown to play an important role in regulation of active immunization in termites.Theoretically,these findings enrich the theory of social immunity in insects.Practically,our results provide a novel way to enhance the efficiency of the entomopaghenic fungus M.anisopliae against termites in the field. |
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