Detection And Identification Of Wolbachia Endosymbionts From 38 Species Of Stored Grain Insects And The Reproductive Manipulatation Of Tribolium Confusum Caused By Wolbachia

Wolbachia endosymbionts are a kind of rickettsial bacteria maternally inherited and caused various effects on the reproduction and biology of their hosts,such as cytoplasmic incompatibility,parthenogenesis,feminization and male killing.Increasingly interests in the potential use of Wolbachia to control populations of pest species were focused on many agricultural insect and vector pest species.Wolbachia was reported as a potential tool for future insect pest control strategies;however,few researches was reported in stored product insects.To advance this research in stored grain insects,we survey the prevalence and diversity of Wolbachia endosymbionts in Chinese stored grain insects.In this study,we screened 38 species(64 geographic populations from 16 provinces in China)of stored-grain insects for Wolbachia infections.Infections were detected in seven species of Coleoptera Anobiidae Lasioderma serricorne(Fabricius)and Stegobium paniceum(Linnaeus),Bruchidae Callosobruchus chinesis,Dermestidae Attagenus unicolor(Brahm),Silvanidae Oryzaephilus surinamensis(Linnaeus),Tenebrionidae Tribolium confusum(Jaquelin Du Val);and two species of Lepidoptera,Pyralidae Ephestia kuehniella Zeller and Gelechiidae Sitotroga cerealella;and one species of Psocpptera Liposcelis brostrychophilus Badonnel.In their parasitoids and predators,one species of Hymenoptera,Bracon hebtor(Say)and one species of Pseudoscorpion Cordylochernes scorpioides were detected Wolbachia infection.However,most common and epidemic pests Curculionidae Sitophilus zeamais Motschulsky and Sitophilus oryzae Linnaeus,Bostrichidae Rhyzopertha dominica(Fabricius.),Pyralidae Plodia interpunctella(Hübner)and Tenebrionidae Tribolium castaneum(Herbst)were no Wolbachia infection in all strains in these five species.Phylogenetic analyses based on wsp gene sequences identified all Wolbachia isolates as members of Supergroups A or B.This research and survey provide us with basic information for pest control strategies using Wolbachia in stored product insects in future.After the investigation of Wolbachia infection in stored-grain insects in parts of China,we chose a common stored-grain pest Tribolium confusum as the object in this study.To clarify the relationships of Wolbachia and host in T.confusum,Wolbachia-induced phenotypic alternations and their spatiotemporal infection density patterns in hosts were investigated.Results showed that:(1)Wolbachia infection was detected in two collected geographic populations with the 100% infection rate,and males and females are all fully infected.(2)Wolbachia infection density in Tribolium confusum was significantly decreased by fed with 0.03%,0.05%,0.15% and 0.3% concentrations of antibiotics(tetracycline)added in the feed.After more than 4 weeks of antibiotics treatment,Wolbachia infection in Tribolium confusum can completely removed,the removal efficiency can be reached 100%(3)During the development of T.confusum,the Wolbachia infection levels consistently increased.The levels of infection plateaued at the young adult stage(3~5 days after emergence and sexual maturity)at approximately 3.7×107 wsp copy equivalents per insect,followed by the newly emerged adult and pupal stage,the infection density also reached 1×107 level.(4)Compared with the Wolbachia infection density in different body parts and tissues of T.confusum,the infection density ranks abdomen > thorax> head.The abdominal contents of Wolbachia may account for more than 60 of the total.Moreover,Significantly density of Wolbachia in the reproductive system was significantly higher than that in the digestive system and immune system,and the content of Wolbachia in females were more than males.(5)In this study,we designed 4 different mating crosses to examined the cytoplasmic compatibility,in 4 mating crosses,only the cross of ?w+(w+,Wolbachia infected T.confusum,natural T.confusum population)×?w-(w-,Wolbachia uninfected T.confusum,Wolbachia removed by antibiotic treatment)cannot produce offspring.It indicated that Wolbachia infection results the completely cytoplasmic incompatibility.Aposymbiotic female beetles show a low fitness,crossing with Wolbachia infected males were completely incapable of producing mature embryogenesis and therefore could not reproduce even though they can produce sterile eggs.Parameters on the oviposition and developmental duration of four combinations of single females no significant differences in total egg production,However,there are significant differences in egg hatching rate,showing a maternally inherited trait.Hatching rates of eggs laied by females after the elimination of Wolbachia infection by antibiotic treatment was significantly lower than the control.(6)The experiment of Wolbachia on mating behavior and male fertility and fecundity revealed that there was no significant difference in Wolbachia infected and uninfected T.confusum mating behavior.Removing Wolbachia decreased male mating ability.(7)Through interspecific and intraspecific,heterosexual and same-sex mating experiments also revealed that neither in the intraspecific of T.confusum or in the interspecific between T.confusum and T.castaneum,no Wolbachia transmission was occurred.Compared with populations of Wolbachia eliminated by antibiotics treatment,natural Wolbachia-infected T.confusum populations have reproductive advantages.It is most benifiet to the population growth while both males and females infected with Wolbachia.This study enriches the of Wolbachia infection dynamics in stored-grain insects and regulation of Wolbachia on stored grain insect reproductive manupulated theory.It provided the basis for whether to use Wolbachia to control stored-grain insects.