Studies On Evaluation Of Fertility Of Aulacocentrum Confusum And Its Regulation Of The Immune Response Of Glyphodes Pyloalis

The mulberry lesser pyralid,Glyphodes pyloalis Walker?Lepidoptera:Pyralidae?is a destructive pest of mulberry?Morus alba L.?trees,that restricts the development of sericulture industry.Aulacocentrum confusum is an effectively dominant solitary endoparasitoid of G.pyloalis,which has a high parasitic rate and a long-term effectively biocontrol.At the same time,the host of A.confusum were Ostrinia furnacalis,Clostera anachoreta and other important agricultural and forestry pests,which was a kind of biological control factors with great development porential and application value.The researches on the parasitoid wasps were still in the field and the basic biological characteristics of host selection behavior for a long time.Hope developing A.confusum as a biocontrol factor in mulberry garden,the following problems still need us to overcome:1)the parasitical fecundity potential of the stage of A.confusum;2)How to deal with the host's immune defense in the process of successfully parasitizing.To answer these scientific questions,parasitism system of A.confusum–G.pyloalis was used to explore the fecundity and progeny fitness,analyze the type and expression patterns of protein genes in the venom of A.confusum and clarify the function of calreticulin in the process of parasitism.Finally,in order to provide important support for the construction of new biological control methods of mulberry pests,the parasitism dynamics of A.confusum to G.pyloalis,and the physiological mechanism of host immune response regulated by parasitoids,this study revealed the expression pattern of immune genes and the change of hormone level after the parasitism of G.pyloalis.The main results are as follows:1.The lifetime ferility of A.confusum was preliminarily determined.We achieved the number of eggs load,immature duration of pupae,emergence date,egress date,female proportion,age of female wasps and other fitness index of 3^rdstage G.pyloalis.The results showed that the total oviposition of female wasps increased and then decreased with the increasing lifespan of female wasps and achieve the peak in 7^thday.The developmental ttime of A.confusum was 10 days and the immature duration of A.confusum was 20 days.They are barely no related to the parasitism sequence.Proportion of female offspring increased when the increase of parasitism sequence.They are barely no related to each other.When adult female wasps were 4 days,5 days and 6 days old,the quality of progeny achieve the peak that represents the maximum hind tibia,longevity and emergence rate.There was a positive correlation between the body weight and the size of the offspring,the heavier weight induce the longer hind tibia.2.In order to explore the role of venom of A.confusum in the regulation of host-parasitoid wasps,the organ morphology of the venom of the female wasp was firstly defined and the gene types of venom protein were determined by transcriptome sequencing.On this basis,the transcriptome of the venom gland was constructed for the first time,14,646 unigenes were used to analyze the transcriptome of the venom glands of A.confusum.A lot of proteases and peptidases were identified such as serine protease,trehalase,laccase,phenoloxidase and other proteases in one hand.In the other hand,9 kinds of protease inhibitors such as serine protease inhibitor,pacifastin protease inhibitor,Kunitz Trypsin Inhibitor and other protease inhibitors were also identified.The other functional proteins such as calreticulin,peptidoglycan recognition protein,tetratransmembrane protein and heat shock protein were also identified.Five venom protein genes were random Ly selected for spatiotemporal expression profiling.The results showed that these venom protein genes were highly specific expression in the gland.3.In order to reveal the role of Ac CRT in the successful parasitization of A.confusum,it was the first time to clone the calreticulin from the genome.The sequence of the calreticulin was further analyzed and found the coiled-coil domain.The prokaryotic expression and purification of the calreticulin and the calreticulin without the coiled-coil domain were achieved.The results showed that the coiled-coil domain could significantly improve the ability of Ac CRT to inhibit the expression of host immune genes and the encapsulation reaction.The results of in vitro injection showed that both Ac CRT and Mutant could significantly inhibit the expression of host immune genes,and the inhibition effect of Ac CRT was more obvious.At the same time,the encapsulation experiment showed that Ac CRT and Mutant had a significant inhibition effect on host encapsulation reaction.Finally,the fusion expression of e GFP-Ac CRT/Mutant was used,and the observation of fluorescence inversion microscope showed that Ac CRT and Mutant could accurately recognize the host immune gene expression and enter the haemolymph of G.pyloalis.4.In order to explore the different expression patterns of immune related genes in the parasitized larvae of G.pyloalis,we first identified a series of immune related genes including heat shock proteins from the transcriptome of G.pyloalis,and explored the changes of immune gene expression and hormone level in the body of G.pyloalis after being parasitized by A.confusum.At the same time,we also explored the changes of heat shock protein expression of G.pyloalis under two adversities of starvation and temperature stress,as well as under different stages.The results showed that parasitism,heat shock and starvation treatment could significantly induce the increase of expression of Gp HSPs.At the same time,the expression of Gp HSPs is low-level in the 1^st,3^rdand 5^thlarvae,but high in the prepupa,diapause and pupa,suggesting that Gp HSPs may be related to the metamorphosis and development of insects.Among the 18 immune related genes identified from the transcriptome of G.pyloalis,4 genes were significantly inhibited after parasitization,which indicated that parasitization of A.confusum could significantly inhibit the expression of immune genes,destroy the immune system of G.pyloalis,and then effectively help A.confusum to successfully parasitize.The contents of juvenile hormone and ecdysone in the parasitized larvae were significantly increased,which indicated that the parasitization of the parasitized larvae could affect the endocrine system and regulate their development and behavior.