Detection And Analysis Of Wolbachia Symbiotic Bacteria And WO Phage Infection In Several Crickets

Wolbachia is a maternally inherited endosymbiotic bacteria,which is widespread in nematodes and arthropods.Because Wolbachia can regulate the reproductive mode of the host,it has attracted wide attention.Cricket are widely distributed in our country,and there are abundant species,some of which are common agricultural and forestry pests.In this study,for the first time,the infection of some cricket species Wolbachia and its WO phage was detected by PCR,and the phylogenetic analysis of the infected Wolbachia was carried out using the wsp gene and multilocus sequence types(MLST)gene sequence.Clarify the diversity of cricket Wolbachia and its WO phage infection strains,and provide a basis for studying the reproductive regulation mechanism of Wolbachia on cricket.The main results are as follows:(1)A total of 23 species of crickets in eight genera were collected from eight regions in five provinces of China.Among them,Velarifictorus micado obtained four geographic populations,Loxoblemmus doenitzi and Loxoblemmus taicoun has two different geographical populations.(2)Using Wolbachia wsp gene primers wsp-13F and wsp-13R,by PCR amplification,the collected samples of Gryllidae,23 species,eight genera,and 274 individuals were tested for Wolbachia infection.It was confirmed that 20 species of crickets from seven genera were infected with Wolbachia.The cricket Wolbachia has a high infection rate,with an infection rate of 87%.(3)Based on the wsp gene sequence and MLST typing and phylogenetic analysis,a total of 15 ST sequence types were identified,of which 12 were newly discovered ST types.Wolbachia strains infected with Loxoblemmus doenitzi(TA),Loxoblemmus spl,Loxoblemmus sp2,Loxoblemmus taicoun(LL),Mitius minor belongs to supergroup A.Wolbachia strains infected with Velarifictorus micado(LL,TA),Velarifictorus asperses?,Loxoblemmus doenitzi(LL),Loxoblemmus sp1,Loxoblemmus sp3,Loxoblemmus montanus,Loxoblemmus taicoun(JS),Dianemobius sp.,Polionemobius taprobanensis,Teleogryllus occipitalis,Comidoblemmus nipponensis belongs to the supergroup B.Wolbachia strains infected with Teleogryllus infernalis,Velarifictorus micado(ZJJ,CS),Velarifictorus khasiensis,Loxoblemmus angulatus,Loxoblemmus sp4,Teleogryllus mitratus,Loxoblemmus jacobsoni belongs to the supergroup F.Some species have dual Wolbachia infections,such as C.nipponensi infected with different individuals of Supergroup A and Supergroup B,and L.doenitzi infected with different strains of Supergroup A and Supergroup F.(4)Using RDP,the gene recombination analysis of the wsp gene was carried out,and three groups of recombination events were found,namely,wAng and wDoe1-2 as the paternal and maternal line,and the recombinant is wlnf;wDoe1-2 and wEmm as the paternal and maternal line,and the recombinant is wOcc;wDoe1-2 and wOcc are used as paternal and maternal lines,and the recombinant is wAng.(5)By using WO-F and WO-R primers of WO phage capsid protein gene orf7,20 species of crickets that have been infected with Wolbachia were detected.The results showed that T.mitratus and L.doenitzi(TA)-2 were negative,and the remaining cricket species all obtained positive gene fragments about 400 bp in length.Through sequencing and cloning,it was found that eight species of cricket Wolbachia were infected by WO phage alone,and Wolbachia of other cricket species were all infected by WO phage.After phylogenetic analysis,WO phages infected by Wolbachia crickets can be divided into eight groups.