The Effect Of Wolbachia On The Growth And Development And Endocrine Mechanism In Hylyphantes Graminicola

Wolbachia endosymbiotic bacteria are widely present in arthropods.In addition to affecting the reproduction of the host,it has been reported that Wolbachia can also affect the growth and development of the host,such as developmental duration and body weight in host.However,research on the molecular mechanism of Wolbachia affecting host development,remain largely unknown.The small spider Hylyphantes graminicola is a natural enemy of farmland pests.It can occur for multiple generations within a year and is easy to breed indoors.Our previous research results showed that the spider H.graminicola was infected with Wolbachia,and the development period of infected spiderlings was significantly lower than that of uninfected spiders,while the mature body weight of infected spiders is not significantly different from that of uninfected spiders.And ecdysone and insulin（insulin-like）play an important role in the growth and development of insects.Based on the above situation,this study examined the developmental period of H.graminicola spiderlings both in Wolbachia infected and uninfected spiders(recorded from the first day of the 2^ndinstar),and the body weight during development(recorded from the first day of the 3^rdinstar),the longevity of adult spiders were also recorded.The expression levels of 3 ecdysone-related genes and 6 insulin-related genes in each day of two infection types of the spider were measured and compared.It provides an important basis for exploring the influence of Wolbachia on host development and its molecular mechanism.The main findings are as follows:1.The effect of Wolbachia on developmental period of spiderlings,weight during development and longevity of adult spiders in H.graminicolaFemale spiders were collected from the field.After each female spider laid three egg bags,Wolbachia was detected in each mother spider,and the Wolbachia infected（W⁺）and Wolbachia uninfected（W^-）spiders from F1 generation were used to recorde the development duration,body weight and the the longevity of adult spiders.The results showed that the development duration of 2^nd,3^rd,4^th,5^th instar and the total development period in W⁺female spiders were significantly shorter than those in W^-female spiders（P<0.05）.Among male spiders,the 2^nd,5^th instar period and the total developmental period in W⁺spiders were significantly shorter than those in W^-spiders（P<0.05）.The weight of W⁺spiders in the 4^th and 5^th instars was significantly higher than that in W^-spiders（P<0.01）,but there was no difference in the weight of W⁺and W^-spiders after maturity.The longevity of W⁺spiders was significantly shorter than W^-spiders（P<0.05）.2.Screening and expression of ecdysone and insulin-related genes during the development of W⁺and W^-H.graminicolaThe spider samples of W⁺and W^-H.graminicola from the second to the fifth instar and the first day of maturity（three replicates per sample）were selected respectively.The total RNA of each sample was extracted,and the genes related to ecdysone and insulin were screened by RT q PCR based on the previous transcriptome data of H.graminicola,and the gene expression was determined.The results showed that the expression of ecdysone receptor gene Ec R,ecdysone inducible gene Ecdyin1 and Ecdyin2 reached a peak on the day before molting,and reached a highest expression peak on the day before maturation,then returned to a low level on the first day of maturation.The expression of insulin related genes（In-Like,In D,IGF2-BP1,IGF-BP1,IRS,In R）changed significantly during the whole growth stage.The expression of insulin like gene In-Like,insulin degrading enzyme gene In D,insulin like growth factor IGF2-BP1,IGF-BP1 and insulin like peptide receptor gene In R reached a peak on the day before molting in each age.However,IRS gene of insulin-like receptor substrate 2-B had a peak expression in the middle of each age,and a low expression in the day before molting.3.Differential analysis of ecdysone and insulin-related gene expression in W⁺and W^- H.graminicolaWe compared the highest expression levels in different instars of W⁺and W^-H.graminicola. The results showed that the expression level of Ecdyin1 at the 2nd,3rd,4th,and 5th instars in W⁺H.graminicola was lower than in W^-spiders,and there was no significant difference in the expression levels after maturation;The expression level of Ecdyin2 and Ec Rat the 2nd,3rd,4th,and 5th instar in W⁺H.graminicola was higher than in W^-spiders;The gene expression levels of insulin-like degrading enzyme gene（In D）,Insulin-like growth factor gene（IGF-BP1）and insulin-like receptor substrate 2-B（IRS）at the 2nd,3rd and 4th instars in W⁺H.graminicola were lower than in W^-spiders.There is no significant difference in expression between the 5th instar and mature;The expression level of IGF2-BP1 in W⁺H.graminicola was lower than W^-spiders at the 2^nd,3^rd and 5th instar.There was no significant difference in the expression levels after the 5th instar and mature;The IRS expression level in W⁺H.graminicola was higher than in W^-spiders at the 2nd,3rd and 4th instars,and there was no significant difference in the expression level at the 5th instar and after the mature spiders;The expression levels of insulin-like gene（In-Like）and insulin-like peptide receptor gene（In R）in W⁺H.graminicola were higher than in W^-spiders at the 2nd,3rd and 4th instars.