Silencing Of PFK Gene And Infections Of Entomopathogenic Fungi Altered Locomotion And Allogrooming Behaviors In The Termite Reticulitermes Chinensis Snyder

Termites are responsible of heavy damage to agricultural crops,dams and wooden structures all over the world.Chemical pesticides are mostly used to control termites.However,harmful effects of pesticides on environment draw the public attentions towards biological control agents.Biological control agents like entomopathogenic fungi share the same habitat with termites,which makes entomopathogenic fungi effective asset for the control of termites but termites have evolved different strategies(allogrooming)to fight against entomopathogenic fungi.Locomotion is an important energy-consumed behavior in animals,which can influence almost all animal activities.However,whether and how energy metabolism and fungal infections influence locomotion and allogrooming behaviors of termites are unclear.Thus,the phosphofructokinase(pfk)gene involved in glycolytic metabolism was silenced through RNA interference and the effects of pfk silencing on physiology and behavior were evaluated in the termite Reticulitermes chinensis Snyder.Secondly,the effect of different concentrations of entomopathogenic fungus Metarhizium anisopliae(Metschnikoff)Sorokin on behavior of termites was evaluated.Thirdly,the combined effect of pfk silencing and M.anisopliae infections on physiology,immunity and behaviors of R.chinensis was analyzed.1.Silencing of pfk gene impaired glycolysis and caused abnormal locomotion and allogrooming in the termite R.chinensisPhosphofructokinase(PFK)is a rate-limiting enzyme in glycolysis,but its linkage with locomotion and allogrooming in termites is not well understood,despite the demonstrated involvement of this gene in the locomotion of different animals.Therefore,effect of the pfk gene on locomotion and allogrooming in the subterranean termite R.chinensis was studied through RNAi and Ethovision XT tracking system.The knockdown of pfk resulted in significantly decreased expression of the pfk gene in different castes(workers,soldiers and dealates)of termites.The pfk-silenced workers displayed higher levels of glucose,but lower levels of nicotinamide adenine dinucleotide(NADH)and adenosine triphosphate(ATP)production and decreased activity of the PFK enzyme.Furthermore,abnormal allogrooming(lower allogrooming frequencies)and locomotion(decreased distance travelled and velocity,but increased turn angle and angular velocity)was observed in pfk-silenced termites.We found caste-specific locomotion among workers,soldiers and dealates.Additionally,soldiers and dealates showed higher velocity in the inner zone than in the wall zone,which is considered an effective behavior to avoid predation.These findings reveal the close linkage between the pfk gene and behaviors(locomotion and allogrooming)in termites,which helps us to better understand the regulatory mechanism and caste specificity of behaviors in social insects.2.Infections of entomopathogenic fungi altered locomotion and allogrooming in the termite R.chinensisTermites,being vulnerable to entomopathogenic infections due to large number of individuals living together in colonies,have evolved various behavioral and physiological tactics to resist the infections by different entomopathogens.Locomotion can help termites to collect information on pathogens and accordingly exhibit hygienic behaviors.Termites inevitably encounter entomopathogenic fungi during nesting and foraging.However,how these fungal pathogens influence locomotion of termites and how hygienic behavior(allogrooming)benefits their survival remain unknown.Here,we examined locomotion alteration of the termite R.chinensis after infections with different concentrations of the entomopathogenic fungus M.anisopliae.When R.chinensis was isolated,the low concentration(5×10~3conidia/ml)significantly increased termite locomotion after 6,12and 24 hours compared to control.However,the high concentrations(5×10~7,5×10~9conidia/ml)significantly decreased termite locomotion after 48h,the survival rate of termites at 5×10~7 and 5×10~9conidia/ml was also significantly lower than that at low concentrations and the control.However,when fungus exposed termites were placed with their nestmates,the locomotion of fungus exposed termites significantly increased 24h after exposure to 5×10~3 and 5×10~9conidia/ml,but was normalized after 48h of exposure compared to the control.Allogrooming was significantly higher at 5×10~3and 5×10~9conidia/ml compared to the control.The fungal infection did not result in significantly higher mortality of the group termites probably owing to their allogrooming.These findings can enhance our understanding on how a termite species copes with biotic stress(i.e.,fungal infections)via adaptive behaviors.3.Alterations of locomotion and allogrooming caused by pfk silencing and fungal infections disrupted immunity and decreased survival in the termite R.chinensisTermites have evolved various behavioral and physiological adaptations to defend against pathogens,which is a great hurdle in biological control against termites.This study investigates a potential strategy against termites based on RNAi-mediated silencing of metabolic gene and infections of entomopathogenic fungus M.anisopliae,which not only is able to break the behaviors(locomotion and allogrooming)of termites but also disturb normal physiology and immunity of termites.The results showed that silencing of pfk gene resulted in the lower expression of pfk gene and lower activity of PFK enzyme in addition to declined energy production(ATP,NADH)in dspfk-injected and fungus-exposed termites.Weaker immunity(decreased immune gene expressions and anti-fungal activity)was showed by pfk-silenced termites after fungal infections.Moreover,silencing of pfk gene disrupted behaviors of infected termites placed individually and with nestmates.Lower survival and defective locomotion were exhibited by dspfk-injected termites placed individually after fungal infections.Frequencies of allogrooming towards infected individuals were declined due to pfk-silencing in nestmates,which resulted in higher mortality of termites.Lower survival was also showed by pfk-silenced nestmates of fungus exposed termites.Here,our results demonstrated that alteration of locomotion and allogrooming caused by pfk silencing and fungal infections disrupted the immunity and decreased survival in termites,which may be used as an effective strategy for the control of termites.