| Both the two-spotted spider mite Tetranychus urticae Koch and the carmine spider mite T. cinnabarinus (Boisduval), which belong to Arachnida, Acari, Acariformes, Tetranychidae, Tetranychus, are very important mite pests. Wolbachia and Cardinium are maternally inherited intracellular bacteria that infect a wide range of arthropods, which could manipulate their host’s reproductive mode and thereby enhance their own spread. The effects of Wolbachia infection on reproduction include male killing, feminization, thelytokous parthenogenesis and cytoplasmic incompatibility (CI). Cardinium has been shown to cause feminization, parthenogenesis, cytoplasmic incompatibility, and be associated with a fitness benefit of infected hosts. Prior study showed that T. urticae harbors only Wolbachia, while T. cinnabarinus harbors both Wolbachia and Cardinium in China. In this study, we investigated the effect of Wolbachia on the reproduction and fitness of Chinese populations of Tetranychus urticae Koch, and identified factors that affect Wolbachia-induced CI. We also investigated the effect of both Wolbachia and Cardinium on the reproduction and fitness of T. cinnabarinus (Boisduval), and the relative contributions of Wolbachia and Cardinium, as well as the interactions occurring between the symbionts. Our results help to understand the evolutionary history of diverse host-symbiont associations and symbiont-symbiont interaction between vertically transmitted symbionts. Our results also encourage the introduction of Wolbachia into pest and vector species of economic and hygenic relevance to suppress or modify natural populations.To understand the effect of Wolbachia on the reproduction of two-spotted spider mite in China, four populations of Tetranychus urticae Koch in China, the Hunan (HN), Jiangsu (JS), Shanghai (SH) and Liaoning (LN) populations, were investigated. Our results show that Wolbachia induced strong CI in the HN and SH populations, intermediate CI in the LN population, and no CI in the JS population. CI was expressed as a reduction in egg hatchability and a male-biased sex ratio in crosses between uninfected females and infected males. This is concordant with the female mortality type of CI. We found that CI manifested not only in embryonic development but also in nymphal development in the LN and HN population. This is the first instance of a Wolbachia infection that can affect the nymphal development of hosts. Wolbachia in the LN population could induce hybrid breakdown. The Wolbachia-induced reproductive incompatibility may extend to the F2: broods of virgin F1females from the incompatibility cross suffer increased mortality rate. We found that the strength of CI enhanced after backcrossing to Wolbachia-infected males in LN population. Another interesting founding is that F1offspring produced by the cross between Wolbachia-infected females and uninfected males in the LN population showed hybrid breakdown.We found diversity of CI expression in Chinese populations of T. urticae. Several factors have been identified that influence the expression of CI phenotype, such as environmental factors including age of host and temperature, bacteria or host genes and bacterial density, mostly from drosophila data. In our experiments, we used1,3,5,7-day-old virgin males and performed experiment at25℃to investigate the influence of aging on Wolbachia-induced CI. Our results showed no effect of aging on CI, suggesting that host aging does not reduce the sperm modification induced by Wolbachia. We also studied the effect of temperature (20℃,25℃and30℃) on the CI induced by Wolbachia. Our results showed that both high and low temperatures do not influence CI. The Wolbachia-infected strains in the JS, LN, SH and HN populations of T. urticae had identical COI gene sequence, suggesting Wolbachia can severely depress levels of mtDNA diversity within an infected host species, and showing a close relationship between the four populations. The CI-Wolbachia strains in the LN, SH and HN populations of T. urticae had identical wsp gene sequence. The wsp sequence of non-CI-Wolbachia strain in the JS population had99.5%similarity (three different nucleotides out of552nucleotides) to the sequence in the LN, SH and HN populations. Therefore, we estimated the variability of CI expression among the four Chinese populations of T. urticae was due to the interaction between Wolbachia and host genotypes.In addition to the effect on reproduction, Wolbachia can also influence the fitness of the host. The Wolbachia-host interaction ranges along a continuum from mutualism to commensalism and parasitism. Fitness costs or benefits conferred by the symbiont also hinder or promote the spread of infection. To understand the effect of Wolbachia on T. urticae fitness, we measured the female ratio, host longevity, host development time and fecundity between infected and uninfected strains in the the JS, LN, SH and HN populations of T. urticae. Our results indicate that Wolbachia bacteria can have variable affects on T. urticae fitness. Although Wolbachia strains in the LN, SH and HN populations shared identical wsp sequences, they apparently affected fitness in different ways. Wolbachia infection had a positive effect on fecundity in the HN population. By contrast, no fecundity change was observed in the other strains studied. Wolbachia enhanced female ratio of infected females in the JS population, while showed no effect on the female ratio of other populations. We found evidence for both positive and negative fitness effects associated with Wolbachia bacteria. Wolbachia shortened the longevity of infected females in the LN population and prolong the lifespan of infected females in the JS population. This is the first report of a Wolbachia infection affecting longevity of infected females in T. urticae. We also found Wolbachia infection prolonged the development time of the JS and HN populations and shortened the development time of the LN and SH populations. The variety of fitness effects may be attributed to the interaction of Wolbachia and host genotype.Prior study on spider mites showed that incompatibility among populations was caused by Wolbachia infection, nuclear-cytoplasmic interaction or nuclear-nuclear interactions. Most commonly, Wolbachia cause sperm-egg incompatibility know as cytoplasmic incompatibility. In concert with geographic or genetic barriers to gene flow, such cytoplasmic incompatibility could promote the evolution of reproductive isolation, a crucial component in speciation. We conducted cross experiments between the JS and LN populations of T. urticae to determine weather reproductive incompatibility exist and how Wolbachia are related to the incompatibility in this species. No uni-or bidirectional incompatibility caused by nuclear-cytoplasmic interaction and nuclear-nuclear interaction were observed. Wolbachia infection in the males of the LN population induced unidirectional incompatibility and hybrid breakdown between the two populations, and Wolbachia infection in the males of the JS population could not induce CI and hybrid breakdown. An interesting founding is that the F1offspring produced by the cross between Wolbachia-infected females in the LN population and uninfected males in the JS population and the cross between Wolbachia-infected females in the JS population and uninfected males in the LN population showed hybrid breakdown. We suggested that Wolbachia infection in females could affect the reproduction of hosts. Wolbachia and Cardinium have been found coinfecting in the same host apecies. Although both Wolbachia and Cardinium bacteria have been documented to cause cytoplasmic incompatibility, little is known about their interaction. In this study, we investigated the effects of both Wolbachia and Cardinium, and their interactions, in the Jiangsu (JS), Shanxi (SN) and Yunnan (YN) populations of T. cinnabarinus (Boisduval). We successfully separated Wolbachia and Cardinium from double infected lines, by treating doubly infected lined with tetracycline, penicillin G and high temperature. Our results showed that the Wolbachia strains in the JS and SN populations of T. cinnabarinus (Boisduval) had identical wsp gene sequence, belonging to Con subgroup, and the Wolbachia strain in the YN population belongs to Ori subgroup. Nucleotide sequences of the16SrDNA genes of Cardinium amplified from the three populations showed little variation, with97-99%similarity. All the doubly infected lines in the three populations could induce CI, and the strength of CI in the YN population was the strongest. Wolbachia did not induce CI in the YN population, and induced CI in the JS and SN populations. Cardinium in the JS and SN populations induced strong CI, and strength of CI was not different from that in the doubly infected line. Cardinium in the YN population induced intermediate CI, which was much weaker than the strength of CI in doubly infected line. We suggested Wolbachia in the YN population may provide benefit to Cardinium. We did not find evidence that Cardinium was able to modify or rescue Wolbachia-induced CI, nor did we found Wolbachia was able to modify or rescue Cardinium-induced CI in the JS population. Q-PCR results showed that the interaction between Wolbachia and Cardinium was cooperative among the four days after adult emergence. Four days later, they act as competitive interactions. We also studied the influence of aging on CI induced by Wolbachia and Cardinium. Our results showed no effect of aging on Wolbachia-CI, but Cardinium-CI levels declined rapidly with male age. We studied the effect of Wolbachia and Cardinium on the fitness of T. cinnabarinus (Boisduval). Our results indicated that both Wolbachia and Cardinium could provide benefit to the fitness of T. cinnabarinus (Boisduval) in the JS population. |
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