External Immune Efficacy And Physiological Trade-off Regulation With Internal Immunity In The Red Palm Weevil

The red palm weevil?RPW?,Rhynchophorus ferrugineus?Olivier??Coleoptera:Curculionidae?,is a major destructive pest that has recently invaded plantations of palm species in southern China,which poses considerable losses by seriously affecting the economic and ornamental values of hosts.Because of strong migratory capability,high fecundity,limited natural enemies,concealment,and widespread resistance to insecticides,there are still no effective strategies to manage and control this prolific species.Biological control with obvious advantages is one of the most potential and sustainable measures compared with chemical control.However,when red palm weevils are infected by pathogenic microbes,it is essential for them to employ a series of immune responses primarily using external immune secretions as the core component to prevent potential threats,which will seriously impair the effectiveness of biocontrol and decrease the quantity of effective micro flora.The successful colonization of invasive species is partly due to individual strong immunity to exogenous substances,especially pathogens.External immunity and internal immunity are two kinds of immune defensive strategies for insects,but the immune trade-off and physiological regulatory mechanism of RPW are still unclear.This study was carried out based on bioassays for screening potential pathogens against RPW.The components,immunosuppressive efficiency in vitro and key genes for regulating external secretions were clarified.Meanwhile,the behaviors of individuals in response to external secretions were analyzed.To reveal the trade-off relationship and physiological regulatory mechanism between the two immune defensive strategies,the efficiency and difference of external and internal immunity and the adaptability of growth and reproduction were discussed,with the hope of providing theoretical basis for the establishment of biological management strategies against RPW.The main results are as follows:1.Screening of pathogenic microbes against red palm weevils for biocontrolA variety of pathogenic microorganisms were isolated from RPW cadavers died of natural diseases.Pathogenicity assay results of these isolates suggested that Serratia marcescens,Bacillus thuringiensis,Metarhizium anisopliae var.anisopliae and Fusarium oxysporum had the most control and application potential as microbial control agents.In addition,we found that B.thuringiensis can not only significantly prolong the incubation time of eggs,but also significantly reduce the boring activity of larvae.2.The antibacterial activity of external secretions produced by red palm weevilsData from antibacterial assay showed that external secretions produced by RPW significantly inhibited the growth of microorganisms in vitro,implying external immunizing potency against pathogens.Both the stoste and solid phase showed certain bacteriostatic activity?obvious inhibition zones?,although the liquid phase was much less evident.Besides,the extent of antibacterial activity of stoste was significantly higher than it was in the solid phase.Meanwhile,the inhibitory efficacy of these external secretions against gram-negative bacteria was significantly stronger than that against gram-positive bacteria.These results indicated that external secretions were the key factor to exert external immune function for RPW in the process of pathogen infection.3.Components and immune active substances of RPW external secretionsExternal secretions of RPW contained proteins and more than 300 chemical compounds.Metabolome analysis suggested that p-benzoquinone may be the main immune active substance responsible for external immunity.On this basis,we found that p-benzoquinone had obvious antibacterial activity in vitro.The immunoprotective efficiency of intermediate concentration of p-benzoquinone was optimum generally,which could improve the survival rate of host to the greatest extent when RPW suffered from pathogen infection.These results further confirmed p-benzoquinone to be an immunoreactive substance in RPW external secretions.4.The regulatory function of arylsulfatase B?Rf ARSBs?in the external immune response in red palm weevilsTwelve hours after injection of ds Rf ARSB-11581 and ds Rf ARSB-14322 into the body of RPW,the synthesis of p-benzoquinone in external secretions was significantly inhibited,while the concentration of p-benzoquinone was significantly increased after injection of ds Rf ARSB-0311.This indicated that Rf ARSBs could regulate the quantity of p-benzoquinone in external immune secretions produced by RPW,thus affecting the ability of external immune defense.5.Behaviors of red palm weevils in response to external immune secretionsThe results of behavioral assays indicated that external secretions produced by RPW had ability to induce the specific behavioral responses of individuals,including attractive and repellent activity.The level of response was related to the amount of secretions.Both larvae and adults exhibited the strongest attractive response to the intermediate dose of external secretions.With the increase or decrease of the amount of secretions,the attractive effect decreased gradually.It was further observed that only adults,when responding to adult abdominal secretions as high as 100 mg,showed the greatest repellency.Thus,low amounts of these external secretions function as attractants,while high amounts of these external secretions are expected to be repellents.6.The physiological regulation of immune trade-offs in red palm weevilsThe antibacterial activity of external secretions,the phenoloxidase activity of hemolymph and the antibacterial activity of hemolymph of the 7^th instar larvae were all significantly higher than those of the 3^rd instar larvae,respectively.However,the3^rd instars had a significant shorter developmental period compared with the 7^th instars.For adults,mating imposed a significant cost by reducing immunity,but with evidence for sexual dimorphism and age-associated difference.In 12-day-old individuals,mating significantly reduced the antibacterial activity of external secretions in males,while females were not affected.On the contrary,in 52-day-old individuals,mating significantly reduced the antibacterial activity of external secretions in females,while males were not affected.The phenoloxidase activity increased with age in virgin individuals,with no evidence for sexual dimorphism.However,the lifespan of mated females and mated males was significantly higher than that of virgin females and virgin males,respectively.In mated females,although the egg hatching rate of 12-day-old individuals was significantly higher than that of52-day-old individuals,the fecundity and number of offspring of the 12-day-old females were significantly lower than that of the 52-day-old females.In addition,we further found that there was no significant difference in the impact of immune challenge types on immune function.These results indicated that the changes in immune strength in vitro and in vivo were the result of the interaction of multiple factors such as age,sex,mating and immune treatment.This immune system had a direct trade-off and physiological regulation relationship with other life history components,which could affect the individual's investment in immunity,that is,the increase in immune ability,it comes at the expense of developmental duration,longevity,mating ability,and reproductive ability,and this trade-off is complicated.We can conclude from the results in the present study that external secretions of p-benzoquinone as the main immune active component play a crucial role in the external immune defense for RPW.The individual immune response?external and internal immune defense?is activated after red palm weevils are infected by pathogens,thereby weakening the effectiveness of infection,which leads to the successful invasion and colonization of the population for this invasive species.Therefore,this research will uncover invasive characteristic and adaptive variation mechanism of RPW from the perspective of external immunity through in-depth study on the immune system mechanism in the process of resisting pathogens.Also,the study will not only attribute to further analysis about the immune interaction and diversified immunization strategies between pathogen and RPW to improve the natural control effect in the field,but also provide a new idea and approach for the development of new-type pest inhibitors or behavioral interference agents targeting the insect immune system.