Responsive Expression And Reproductive Regulation Of Vg Genes In Ophraella Communa Feeding On Ambrosia Artemisiifolia Under CO₂ And Heat Wave Stress

Vitellogenin（Vg）is a female multifunctional reproductive protein,which provides functional materials for developing oocytes.Ophraella communa is an important natural enemy of invasive weed Ambrosia artemisiifolia,and its strong reproductive ability is the key key of effective control against A.artemisiifolia.Previous studies have shown that the fecundity and fertility of the O.communa decreased under the background of climate warming.However,it is still unclear whether it is related to a decreased expression level of the Vg gene.Based on the early phenomenon that the O.communa feeding on ragweed caused by CO₂and heat wave stress led to a decrease in its fertility.Applying insect physiology,molecular cloning,RT-q PCR,RNAi,and enzyme linked immunosorbent techniques,we further explored the functional analysis of the Vg genes on the reproductive function of the O.communa,and analyzed the response expression of the Vg genes in the O.communa that fed on ragweed under CO₂and heat wave stress.This study will provide a basis for better understanding of the reproductive molecular mechanism and the internal mechanism of fitness generation of the O.communa,and it is of great significance for protecting and better utilizing the resources of the O.communa in China.The main research results are as follows:1.Cloning and spatiotemporal expression pattern of the Vg genes in O.communaAccording to the genome and transcriptome database of the O.communa,the CDS region coding sequences of three Vg genes were cloned using RT-PCR technology.The results showed that the open reading frame（ORF）lengths of the OcVg1,OcVg2,and OcVg3 genes were 5310 bp,5322 bp,and 5298 bp,respectively,corresponding to 1769,1773,and 1765 amino acids,and the protein relative molecular weights were 202.64 k D,203.21 k D,and 202.48 k D,respectively.The isoelectric point（PI）of amino acidswas5.50,6.37,and 5.96,respectively.The three Vg proteins of female insects have typical conserved domains,including the N-terminal domain（LPD＿N）,the domain of unknown function（DUF1943）,and the von Willebrand factor type D domain（VWD）.Simultaneously containing N-terminal polyserine regions,conserved GL/ICG,R/KXXR/K,DGXR motifs,and C-terminal cysteine residues.The results of phylogenetic analysis showed that OcVgs neatly clustered on a branch of Coleoptera insects and were closely related to the Diabrotica virgifera virgifera.The spatiotemporal expression changes of Vg1,Vg2,and Vg3 genes in the O.communa were detected by real-time fluorescence quantitative PCR（RT-q PCR）.The results showed that the three Vg genes showed a consistent expression pattern in different tissues,which were specifically overexpressed in the fat bodies of female adults.The three Vg genes also exhibited a similar trend of high specific expression at different developmental stages,with no expression at the larval stage.They began to be highly expressed in newly emerged females,especially after the females mature sexually.2.The role of Vg genes in the reproductive regulation of the O.communaThe RNAi technique was used to silence the Vg genes expression in the female of the O.communa,in order to reveal the potential function of the Vg genes in the female reproductive physiology.After injecting dsOcVg1,dsOcVg2,and dsOcVg3 in the newly emerged females,m RNA transcription levels significantly decreased by more than 98%when compared with the control females injected with dsGFP.After the injection of dsOcVg1,dsOcVg2,and dsOcVg3,there was no significant difference in the amounts of egg laid by the females in comparison with.After injection of dsOcVg1,and dsOcVg3,there was no significant difference in egg hatchability compared to the control group.After the injection of dsOcVg2,egg hatching rate was almost zero,the egg particles blackened,and the larvae lost their vitality and failed to hatch properly.It indicated that the Vg genes may play a joint regulatory role in the reproduction of the female species of the O.communa.Subsequently,a combined interference experiment was conducted on three Vg genes of female insects,and it was found that after any two Vg genes were silenced,the m RNA transcription level of a single Vg gene was significantly reduced,while the expression level of the other Vg gene had a significant compensatory effect,resulting in no statistically significant difference in the egg production of female insects after interference.In the combined interference group injected with dsVg1+dsVg2,dsVg1+dsVg3,and dsVg2+dsVg3,the hatching rate of their eggs was significantly reduced compared to the control group.After simultaneously silencing three Vg genes,their expression levels were reduced by 85.6%,87.9%,and 74.9%compared to the control group injected with dsGFP,respectively.The amounts of egg laid by females was significantly reduced compared to the control,and the eggs hardly hatch.3.The expression effect of Vg genes in the body of the Ophraella communa fed on ragweed under CO₂and heat wave stressVitellogenin（Vg）provided essential nutrients for developing oocytes.After feeding on ragweed under CO₂and heat wave stress,the expression level of Vg gene in the body of the Ophraella communa significantly changed.The results indicate that,in the context of global climate change,the reproductive ability of the widespread firefly beetle may decrease,which may affect the control effect on ragweed.The expression level of Vg gene in female Ophraella communa fed on ragweed under CO₂and heat wave stress was significantly lower than that in the control group,especially in the early sexual maturity,the key to the success of female reproduction,the expression level of Vg gene was significantly inhibited.4.Changes in lipid and hormone content in the body of the Ophraella communa fed on ragweed under CO₂and heat wave stressLipidsbeing as the main energy substance in organisms,directly affected the normal physiological activities of insects.The lipid and hormone content in the body of the female Ophraella communa feeding on ragweed under different treatments also showed significant changes.At the early stage of sexual maturity（0～48 h）,the content of lipid,juvenile hormone and ecdysone in the body of the Ophraella communa fed on ragweed under high CO₂concentration or heat wave treatment significantly decreased.In the late stage of sexual maturity（3～15 d）,lipid and hormone content tended to increase,and this response change may affect the synthesis and expression of Vg.