| Endosymbionts are widely spread in arthropods and nematodes,which play an important role in developmental processes of hosts.They could not only influence the host's fitness,but also they have many reproductive manipulations on hosts.Wolbachia is the most well-studied endosymbiont,it can infect about 40%of all arthropod species.Wolbachia could manipulate host's reproduction in many ways,such as inducing feminization,parthenogenesis inducing,cytoplasmic incompatibility and male killing.Meanwhile,Wolbachia is regarded to participate in some nutrients synthesis processes of host arthropods.Wolbachia transmission modes are diverse,including vertical(heritable)transmission and horizontal transmission(by medium such as plant).Spiroplasma is another heritable transmitted endosymbiont,it could be found in many arthropods and a few plants.In insect hosts,Spiroplasma is regarded as a male-killer;however,in plant hosts,Spiroplasma is often considered as a pathogen.Previous study had revealed that in many arthropod species,Wolbachia and Spiroplasma could co-infect the same host and have distinct influence on host arthropods compared to symbiont singly-infected strains.In a survey of natural Tetranychus tuncatus,we found most mites were co-infected by Wolbachia and Spiroplasma,which symbionts co-infected proportion was significantly higher than singly-infected and uninfected proportions.Due to this phenomenon,we intended to explore the interactions between Wolbachia,Spiroplasma and host T.truncatus in many ways:1)the influence of ensosymbiont infection on fitness and fecundity of host spider mites;2)host mites' genetic backgrouds' influence on symbionts' effects;3)competetive interations between Wolbachia and Spiroplasma;4)influence of different symbionts' infections on host mits' gene expressions;5)the symbiont-infection influence on the bacterial communities in T.truncatus.Based on all these studies.we clarified the interactions between symbionts and host mites.revealed the patterns and mechanisms of reproductive manipulations by symbionts on host mites preliminarily;further more.we also investigated the interactions between symbionts and other bacteria in spider mites.In total,our experiments gave a favourable basics for further study on machanisms of symbionts'reproductive manpulations on host mites,in addition,we also provided a definite theoretical basis for managing spider mites and other pests by symbionts means.1.The influence of endosymbionts Wolbachia or/and Spiroplasma on natural host T.truncatus' fitness and fecundityIn the year 2014,we collected 132 Tetranychus truncatus individuals from six sites of north Chia.By specific primers of PCR detection,we found 16.7%of all mites were infected with Wolbachia,only 3.8%mites were infected with Spiroplasma,the rest 79.5%mites were infected with both Wolbachia and Spiroplasma.Interestingly,we didn't find any symbiont uninfected mites in fields.Based on the infected traits in natural fields,we isolated three isofemale lines in laboratory:Wolbachia singly-infected strain W+S-,Spiroplasma singly-infected strain W-S+and symbiont co-infected strain W+S+.After that,we treated W-S+strain for consistent 3 generations by 1 g/L tetracycline concentrations to get a symbiont uninfected strain W-S-.We designed crossing experiments between each two of all these 4 different symbiont-infected T.truncatus strains,including a total of 16 combinations.Based on crossing experiment,we found that in natural spider mite strains,symbiont doubly-infected strain had the highest fecundity and fastest developmental time(from egg stage to adult).At the same time,Wolbachia and Spiroplasma co-infection could induce CI in host mites,resulting in lowing hatchability of co-infected males mated with other symbiont-infected females' offspring.However,Wolbachia singly-infection could not induce CI phenomenon.Although symbiont-infection could not influence host mites'longevity and sex ratio,our results still revealed that in fields' populations,symbiont coinfection could confer competetive advantages for host mites compared to symbiont singlyor un-infected mites,however.this advantage was due to spider mites' discongruent genetic backgrouds or symbionts' influence was still needed to be explored.2.Effects of symbionts were influenced by host mites' genetic backgroudsTo eliminate the influence of inconsistent genetic backgrounds on results.based on introgression,we used males of W-S-strain to mate with females of other 3 strains(W-S+.W+S-and W+S+strains)and female offspring for continuous 7 generations.Finally,we obtained 4 spider mite strains shared with similar genetic backgrouds and performed another crossing experiment using these 4 newly obtained spider mite strains.The results of crossing experiment by spider mites shared with concurrent genetic backgrouds indicated that symbiont-infection made host mites suffer fitness costs,mainly showed in lower fecundity than uninfected strains.Wolbachia could induce female-killing CI in T.truncatus which resulted in declining female proportion of incompatible combinations.Symbiontinfection could not influence longevity and developmental time of host mites.Male ages could not influence the results of crossing experiment.Symbiont titer would increase with the development of host mites and Wolbachia titer was significantly higher than Spiroplasma titer in co-infected strain.Our results suggested that influence of genetic backgrouds was much greater than symbiont-infection on T.truncatus and Wolbachia dominate Spiroplasma in host mites.3.Competetive interations between Wolbachia and SpiroplasmaIn host T.truncatus with different and consistent genetic backgrouds,we performed QPCR experiments for three symbiont-infected strains(co-infected strain W+S+,Wolbachia singly-infected strain W+S-and Spiroplasma singly-infected strain W-S+),the results indicated that:in female mites,symbiont titer was higher in singly-infected strains;however,in male mites,Wolbachia titer was higher in co-infected strain while Spiroplasma titer was still higher in singly-infected strain.In symbiont co-infected strain,Wolbachia titer was much higher than Spiroplasma titer.These results proved that Wolbachia dominate Spiroplasma in host T.truncatus.4.Influence of different symbionts' infections on host mits' gene expressionsVia high-throughput sequencing for 7-day-old female adults'(the age which symbiont titer was highest)transcriptome,we investigated the influence of Wolbachia and Spiroplasma on host T.truncatus' gene expression.Sequencing results indicated that there were many genes related to molecular function and biological process catogeries differently expressed by different symbiont-infected status.Wolbachia infection could induce many fecundity-realted genes(such as histone gene and vitellogenin genes)differently expressed,this difference could be related to CI induced by Wolbachia.What's more,co-infection of Wolbachia and Spiroplasma could induce many immune-related genes upregulation,which potentially showed a protective effect by symbiont co-infection.Finally,we found gene expression pattern of Wolbachia singly-infected strain W+S-was highly consistent with coinfected strain W+S+,this results also revealed that Wolbachia could offer much more influence on host mites' gene expression than Spiroplasma.At the same time,the possible protective effect by co-infection could partly explain why symbiont co-infected strain was so widely distributed in fields.5.The symbiont-infection influence on the bacterial communities in T.truncatusBased on results of 16s rRNA gene sequencing for 1-day-old female spider mite adults,we found the bacterial communities in T.truncatus were very abundant,Wolbachia and Spiroplasma were the two most abundant bacteria species in symbiont-infected strains.At the same time,symbiont-infection could not influence Alpha-diversity index(including Shannon,Simpson,ACE and Chao 1 diversity index)of bacterial communities in T.truncatus,while we found different symbiont-infected T.truncatus strains were varied each other by PCoA(Principal Coordinates Analysis).After removing OTUs of Wolbachia and Spiroplasma,we repeated PCoA analysis of bacterial communities in T.truncatus and found no significant difference between all tested 4 spider mite strains.These results proved that symbiont had no significant influence on bacterial communities in T.truncatus.Above all,we verified the dominated status of Wolbachia in T.truncatus by biological experiment(Wolbachia could induce CI in host mites),16s rRNA gene sequencing experiment(Wolbachia was most abundant in Wolbachia-infected strains)and highthroughput sequencing experiment(gene expression pattern of Wolbachia singly-infected strain W+S-was highly consistent with gene expression pattern of co-infected strain W+S+).What's more,we also explored the effects of symbiont-infections on host mites'fecundity,fitness,bacterial communities and gene expression.At last,the molecular machanisms of reproductive manpulations by symbionts(such as Wolbachia-induced CI)in T.truncatus still needed to be explored. |
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