Preliminary Research On Reproductive-related Genes Function In Phytoseiulus Persimilis

Phytoseiid mites(Chelicerata: Arachnida: Acari)play important roles in agroecosystems,as highly effective biocontrol agents of many small pests,including spider mites,whiteflies,and thrips.Studies on phytoseiids have mainly focused on their biology,and many interesting phenomena were observed and reported.For example,they have a reproductive pattern called “paternal genome elimination”,referring to males that develop from fertilized eggs but become haploid with their paternal genomes lost at early embryonic stages.They also have a relatively stable offspring sex ratio and the first egg always develop into males.However,the mechanisms behind such a special pattern are still unclear.One major reason is that their highly ossified small bodies became barriers,thus blocking successful applications of many molecular biological techniques.Another reason is that the embryogenesis had started before egg was laid.These two factors limited the studies of mechanisms on reproduction.Phytoseiulus persimilis is a specialist predator and has a huge predation on spider mites.It is one of the earliest phytoseiids commercially used in agriculture.Studies on its reproductive mechanism are helpful for reasonably regulating sex ratio in mass rearing.Therefore,we improve the efficiency of RNA interference(RNAi)in Phytoseiulus persimilis.Based on the application of RNAi,we explore molecular characterization,expression,and function of Vitellogenin and doublesex genes in P.persimilis,which may be related to embryogenesis and sex determination and differentiation.The results will provide some clues for future research in embryogenesis of P.persimilis.Star Polycation is a new nanomaterial commonly used as a carrier of ds RNA in RNAi.It can be used as carriers to transfer exogenous ds RNA into cells efficiently and rapidly.In the present study,five genes of P.persimilis(Pp ATPb,Pp ATPd,Pp Rp L11,Pp Rp S2,and Pptra-2)were selected to verify whether SPc promotes the delivery of ds RNA into P.persimilis through soaking.Correspondingly,when the target gene was interfered,P.persimilis expression of Pp Rp L11 reduced by 64 %,while that of the other four genes reduced by at least 80 %.When each of the five genes were interfered using SPc-mediated ds RNA,the total number of success offspring produced per female in six days decreased by ca.92,92,91,96,and 64 %.Compared with oral feeding,the efficiency of this delivery system is improved by 6 % to 70 % and the effective time can be as long as one week.Two orthologs of Vg gene were obtained from the genome of P.persimilis and termed as Pp Vg1 and Pp Vg2.Both orthologs belong to the large lipid transfer protein(LLTP)superfamily.The expression of Pp Vg1 and Pp Vg2 was low in immatures and male adults,and increased rapidly in female adults after mating,and reached a peak before the first egg was laid(168× and 21× the level in virgin females,respectively).When Pp Vg1 and Pp Vg2 were interfered with ds RNA,the relative expression decreased by 81 and 31%,respectively,and 8 and 31 % interfered individuals died within 24 h.Among surviving individuals,ca.51 and 45 % are infertile.Two orthologs of Vg gene were obtained from the genome of P.persimilis and termed as Ppdsx1 and Ppdsx2.Both expressions had a higher level in eggs and females 10 h after mating.In 1st and 2nd eggs laid by each female,which generally develop into males and females,expressions of Ppdsx1 were both higher than Ppdsx2.While in 10 h after mating,both genes are similar.When interfered,expression of Ppdsx1 and Ppdsx2 decreased by 67 and 34 %,respectively.After interfered,about 60 and 75 %individuals died.In offspring,ca.4 and 27 % of the 1st eggs developed into females,respectively.In addition,when Ppdsx1 was interfered,the sex ratio(female)decreased by about 20 %.But when Ppdsx2 was interfered,ca.13 % of the 2nd eggs developed into males.To further explore the mechanism of the above phenomenon,Yeast c DNA library of P.persimilis was constructed and sequences of Ppdsx1 and Ppdsx2 were further cloned to the vector p GBKT7,which were used as baits to screen interacting genes.There were 34 and 70 positive clones that were screened,respectively.After sequencing and analysis,we selected Pp Vg1,Pp Vg2,facr1 and zfnanos1 related to embryonic development for point-to-point verification,and the results showed that they all had interaction.The results of the study contribute to our preliminary understanding of the mechanisms regulating embryonic development and sex differentiation in P.persimilis,enriching the content of sex differentiation in arthropods,and providing us with ideas to regulate the sex ratio of the population during mass rearing.