| Neoseiulus californicus is an important natural enemy for the control of the red spider mites and small sucking pests.For the predatory mites,biology,ecology,largescale breeding and application has been well documented worldwide.However,little has been known about the sex determination and reproduction mechanisms at the gene level to date.It is important and necessary to clarify its sex determination and reproduction regulation in theory and application.A significant negative effect was found in offspring sex ratio when preys are insufficient.How the negative effect comes about? In order to understanding the mechanism of the prey-mediated negative sex ratio,transcriptomes of the reproductive stage of adult female mites under prey stress and sufficient prey were constructed to explore the genes related to reproduction and sex determination,and to clarify their functions by using q PCR and RNAi methods in N.californicus.The study may lay the foundation for fully interpreting molecular mechanism of reproduction and sex determination in the N.californicus and even phytoseiid mites.The main results are as follows:1 Effects of prey density stress on reproduction and expression differences in transcriptome of N.californicusIt is found that under the stress of prey,the N.californicus adapts by reducing the number and size of eggs and adjusting the offspring sex ratio(male/female)through statistical analysis.The fecundity was 40.80 per female,the egg size was 0.0153 μl,the female size was 357.12 μm,and the female offspring ratio was 0.72(female biased)in N.californicus when 75 eggs of Tetranychus urticae were provided per day,which was significantly higher than the fecundity(21.63 eggs/female),the egg size(0.0136 μl),the female size(345.23 μm)and the female offspring ratio(0.53,male biased)when only 5 prey eggs were provided.Through comparative transcriptome analysis,a total of 4201 differentially expressed genes(DEGs)were identified,including 1875 upregulated genes and 2326 down-regulated genes,under the conditions of sufficient and insufficient prey.The signaling pathways and genes that affect reproduction and sex determination were identified and analyzed.It was found that three vitellogenin and one receptor gene,and four sex determination genes(tra2,grou,da and YTDC1)were significantly down regulated under prey stress.In addition,KEGG enrichment analysis of the DEGs revealed that signal pathways regulating reproductive and ovarian development,such as insulin signaling pathway,TOR signaling pathways,fat digestion and absorption,JAK-STAT signaling pathway and notch signaling pathway,were significantly enriched.2 Identification,homologies and expression patterns of sex determination genes and genes involved in reproduction regulationA total of 20 candidate homologous protein sequences were obtained by identifying the transcriptome database of N.californicus,including the genes of doublesex-like(dsx1-like and dsx2-like),transformer-2(tra2),intersex(ix)and fruitless-like(BTB1 and BTB2),but not containing the upstream genes for transformer(tra)and sex lethal(sxl).Compared with the homologous protein sequences of the identified sex determination genes,except for the functional conserved structure domain,the sex determination genes of N.californicus have relative low homology with those of insects.The phylogenetic tree showed that the sex determination genes of N.californicus had the closest genetic relationship with acarine species,indicating that the sex determination genes of acarina were evolutionarily conserved.The genes highly expressed in different stages were as follows: nine genes(BTB1,BTB2,ix,snf,hop,mls3,ime4,YTDC1 and MAGO)were highly expressed in fertilized adult female mites(45%),six genes(tra2、da、grou、run、fl(2)d and vir)in eggs(30%),two genes(stat92E and dsx2-like)in unfertilized female mites(10%),one gene(dsx1-like)in male adults(5%),and two genes(dsf and ovo)in the protonymph(10%).Three Vgs and one Vg R gene were identified in the N.californicus.All Vgs gene belong to the lipid transporter family and have specific Vg gene conserved domain.By q PCR,it was found that Vg1,Vg2 and Vg R were highly expressed in fertilized adult female mites,and Vg3 gene was also highly expressed in the developmental stage.Seven insulin signaling pathway genes,including ILP1,ILP2,INSR,PDK1,Akt,FOXO and PI3 K were identified.Phylogenetic tree showed that the seven insulin pathway genes of N.californicus were genetic relationship to acarina.Their expression patterns varied greatly at different developmental stages.The INSR and Akt genes were highly expressed in eggs,PDK1 and PI3 K genes highly expressed in fertilized female adult,implying these two genes involved in reproduction regulation.3 Functions of sex determination genes and genes involved in reproduction regulationThe functions of tra2,dsx1-like,dsx2-like,BTB1,BTB2,ix,snf and da genes in the sex determination and reproduction of N.californicus were investigated by RNAi technology.After delivered the target genes of ds RNA or si RNA into the body,the transcription level of target gene m RNA was significantly inhibited in N.californicus.When the tra2 gene silenced,the oviposition of the female was significantly reduced and a small number of eggs failed to hatch normally.After silencing BTB2 gene,the oviposition was also significantly reduced,the oviposition period was shortened,and a small number of eggs could not hatch normally,but the offspring female ratio was not affected.It was observed that most female adults suspended spawning on the third to fourth days,during the oviposition period,and then resumed spawning.Silencing snf gene mites caused serious reproductive disorder of female adult,of them the most female did not lay eggs or produced seriously teratoid eggs until death.Inhibiting the expression of ix gene resulted in the decrease of egg hatching rate and offspring female ratio.Silencing dsx1-like,dsx2-like,BTB1 and da genes had no significant effect on reproduction or sex ratio.After silencing Vg1 and Vg2 genes,the fecundity of female adults decreased by14.66% and 11.59% respectively compared with the control group.At the same time,silencing Vg1 gene caused about 5% of eggs failing to hatch normally and reduced the offspring female ratio.However,feeding ds Vg3 and ds Vg R had no significant affected on their oviposition and egg hatching rate.Silencing ILP1,PDK1,Akt,FOXO and PI3 K genes resulted in a significant decrease in the oviposition of female mites.The egg hatching rate was decreased by2.49% and 2.46% respectively when feed ds RNA of ILP2 and PDK1 genes compared with the control.At the same time,the offspring female ratio was decreased when silencing PDK1,INSR,FOXO and PI3 K genes.After silencing of BTB2,snf and ix genes,the expression level of Vg and Vg R genes was detected by q PCR technology,on the first and fifth days of oviposition period,and it was found that the expression decreased in varying degrees,indicating that BTB2,snf and ix genes could regulate reproduction by regulating Vgs and Vg R.After silencing seven insulin signaling pathway genes,the expression levels of Vgs and Vg R genes were detected on the first and fifth days of oviposition period,and it was found that the expression of the two Vg genes were also decreased in varying degrees.In addition,it was found that there was no obvious regulatory relationship between the sex determination genes of BTB2,snf and ix and insulin signaling pathway genes.In summary,this dissertation constructed a transcriptome library based on the biological phenotypes that adult female mite reproduction and progeny sex ratio were affected under prey density stress.To identified and annotated genes related to sex determination and reproduction showed that the genes of snf,BTB2 and ix genes were involved in the regulation of reproduction or offspring sex ratio.The functions of Vg and Vg R in the regulation of reproduction or offspring sex ratio are clarified.It is revealed that insulin signaling pathway is involved in the regulation of reproduction or sex ratio by regulating Vg and Vg R.This paper provides a new idea for in-depth study of the reproduction and sex determination mechanism of predatory mites. |
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