| Lipocellis entomophila(Enderlein)(Psocoptera: Liposcelididae)that with small body size,have led to frequent outbreaks that seriously threaten agricultural products and increase environmental pollution owing to strong adaptability,mixed eating habits,and high fecundity.As a common pest in granaries,L.entomophila produces resistance due to its unique biological characteristics and exceptionally strong environmental resistance,especially resistance against phosphine.In addition,they have also produced significant reproductive adaptability to the current ‘double-low’ green storage technology.It has become one of the most difficult controlled pests to completely eradicate from stored grains.Therefore,it is particularly urgent to explore new control methods and develop new specific insecticidal technologies for L.entomophila.Insulin is a multifunctional peptide hormone that has evolved conservatively in insects.The insulin signaling pathway has attracted more and more research attention due to its involvement in various insect physiological processes such as growth,development,reproduction,metabolism,aging,and lifespan.By targeting the insulin signaling pathway that regulates insect vitellogenesis,the molecular mechanisms of key genes in the insulin signaling and JH signaling pathways in the collective regulation of vitellogenesis in L.entomophila were explored.The exogenous bovine insulin was applied to determine the impact of insulin on the reproduction of L.entomophila.The ILPs that specifically regulated vitellogenesis were cloned and verified of L.entomophila.The downstream genes of the insulin signaling pathway of L.entomophila,Le ERK and Le Akt in mitogen-activated protein kinase(MAPK)and phosphotidylinositol 3-kinase(PI3K)were cloned and functional analysized.The roles of these genes on expresss of vitellogenesis gene of L.entomophila were explored.The functional of Le Akt on the L.entomophila reproduction,Vg synthesis and juvenile hormone production was researched.The detailed research results are as follows:(1)Exogenous insulin induction promotes oviposition in L.entomophilaExogenous bovine insulin was used to investigate the effect of insulin on oviposition in L.entomophila.The results showed that a slight increase in mortality of psocids was observed at one week therapy of bovine insulin(0.1,1.0 and 10 μg/μL)compared with that in the solvent control(HEPES buffer).However,a high concentration of bovine insulin(100μg/μL)treatment resulted in significant mortality(reached 51.88%).In addition,the results showed that bovine insulin(1.0 μg/μL)exhibited a continuous effect on egg production in L.entomophila for the next two weeks.This study provided the first evidence that the insulin signaling pathway was involved in the regulation of vitellogenesis in L.entomophila.(2)The Le ILP genes in the insulin signaling pathway regulate vitellogenesis of L.entomophilaBased on the transcriptome database established by our research group,three insulin-like peptides(ILP)were cloned from L.entomophila for the first time,and identified as Le ILP1,Le ILP2,and Le ILP7.The CDS lengths of Le ILP1,Le ILP2,and Le ILP7 were 471,381,and 420 bp,encoding 157,127,and 140 amino acids,respectively.The molecular weights of the encoded proteins were 37.96,30.79,and 34.13,and their isoelectric points were 8.383,9.562,and 8.56,respectively.Le ILP1,Le ILP2,and Le ILP7 belong to the insulin genes superfamily and have highly conserved structures,including signal peptide,B chain,C peptide,and A chain in their precursor.The C-terminal extension of Le ILP7 is a characteristic of insulin-like growth factors(IGFs).Le ILP1,Le ILP2,and Le ILP7 were specifically expressed at different developmental stages of L.entomophila,but the relative expression levels varied significantly among different stages(P < 0.05).The relative expression levels of the Le ILP1 and Le ILP2 genes in the seventh instar female adults were higher than in the nymphal stage,while the expression level of Le ILP7 was higher in the nymphal stage.Le ILP1,Le ILP2,and Le ILP7 are all highly expressed in the head.Compared with that of the control group,the transcription levels of Le ILP1,Le ILP2,and Le ILP7 after treatment with exogenous insulin for 6 h significantly upregulated by 19.4,20.1,and 35.6times,respectively.By continuously feeding ds Le ILP1,ds Le ILP2,and ds Le ILP7,the transcription levels of these genes are effectively suppressed in L.entomophila.The egg production and hatchability declined,Vg expression was significantly decreased and ovaries atrophied with less yolk protein deposition after RNAi-mediated knockdown of Le ILP2.(3)The regulatory role of MAPK-Le ERK gene in vitellogenesis of L.entomophilaThe extracellular signal-regulated kinase gene(Le ERK)was cloned from L.entomophila.The CDS lengths of Le ERK was 1107 bp,encoding 368 amino acids.The molecular weight and isoelectric point of L.entomophila ERK were predicted 42.4 k Da and6.14,respectively.Le ERK belongs to the phosphotransferase,serine or threonine specific kinase subfamily.Phylogenetic analysis revealed that Le ERK of L.entomophila is more closely related to the Hemipterans.Le ERK was specifically expressed in all developmental stages of L.entomophila,with significant differences in its relative expression levels among different stages(P < 0.05).Le ERK had the highest relative expression level in the seventh instar female adults,with high expression in the head and ovary.Compared with that of the control group,the transcription levels of Le ERK after treatment with exogenous insulin for12 h significantly upregulated by 4.75 times.Continuous feeding of ds Le ERK effectively inhibited the transcription level of Le ERK gene in L.entomophila.The egg production and hatchability declined,Vg synthesis inhibition and ovaries atrophied with less yolk protein deposition after RNAi-mediated knockdown of Le ERK.These results suggest that Le ERK plays an important role on vitellogenesis of L.entomophila.(4)The regulatory effect of Le Akt gene in the PI3 K pathway downstream of insulin signaling on JH synthesis and vitellogenesis in L.entomophilaThe protein kinase B gene(Le Akt)was cloned from L.entomophila.The CDS lengths of Le Akt was 1587 bp,encoding 529 amino acids.The molecular weight and isoelectric point of L.entomophila Akt were predicted 59.43 k Da and 5.81,respectively.Le Akt belongs to the serine or threonine specific kinase subfamily and exhibits high conservation among its structural domains.The amino acid sequences of Le Akt are highly consistent with those of Hemiptera insects.Le Akt gene was specifically expressed in all developmental stages of female L.entomophila,with the highest relative expression levels in the seventh and thirteenth instar stages.Le Akt had higher relative expression levels in the head and ovary of female adults,suggest that Le Akt is related to ovarian development.Compared with that of the control group,the transcription levels of Le Akt after treatment with exogenous insulin for12 h significantly upregulated by 3.49 times.Continuous feeding of ds Le Akt effectively inhibited the transcription level of Le Akt gene of L.entomophila,leading to inhibition of Vg synthesis and incomplete ovarian tube development,this indicates that Le Akt plays an important regulatory role in the vitellogenesis and ovarian development of L.entomophila.Silencing of Le Akt decreased JH-III titer and suppressed JH synthesis,resulting in reduced normal reproductive ability of L.entomophila.The interference process also caused a certain degree of mortality due to hormone transmission blockage.This research explored the interaction between the Le Akt gene and key genes in the JH signaling pathway.Silencing of Le Akt resulted the expression level of JH biosynthesis gene(Le JHAMT)was significant reduction,and the JH receptor gene(Le Met)and downstream transcription factor(Le Kr-h1)were significantly inhibited.The expression levels of two JH degradation genes(Le JHE and Le JHEH)were significantly increased.This demonstrates that activation of the insulin pathway is necessary for JH synthesis,the insulin and JH signaling pathway jointly regulate vitellogenesis and reproductive processes.However,further clarification is needed on how the interaction between insulin pathway and JH signal pathway regulates the reproduction of L.entomophila. |
