Distribution Of The Endosymbiotic Bacterium Cardinium In Chinese Populations Of Spider Mites (Acari: Tetranychidae) And Reproductive Manipulation On The Host

Cardinium, a newly discovered endosymbiont in arthropods, has been shown to causefeminization, parthenogenesis, cytoplasmic incompatibility, and be associated with a fitnessbenefit of infected hosts. Hapiodiploid host species are particularly interesting in studies ofpopulation biology and evolution of host-symbiont interactions because the development ofhaploid embryos into males widens the array of CI patterns. In arrhenotokous species,haploid embryos resulting from paternal genome elimination in fertilized eggs can developinto viable males. Consequently, in incompatible crosses, fertilized eggs can either developas haploid males or abort. The carmine spider mite Tetranychus cinnabarinus (Boisduval)and the two-spotted spider mite T. urticae Koch are both very important mite pests widelydistributed around the world. The carmine spider mite is a polyphagous herbivore attackingvegetables, cotton and many other crops, while the latter one causes damages to many fruittrees, greenhouse plants and flowers, causing great loss to agriculture. Due to their rapiddevelopments and high reproductive capacities, these mites can form new geographicalpopulations very easily. The carmine spider mite has become especially serious in Chinawith its abundant distribution as well as resistance problem in chemical control. Thediscovery of intracellular bacteria in arthropods has provided a new theoretical method forbiological control.In this study, we use molecular techniques to investigate the infection status ofCardinium among the geographical populations of T. urticae and T. cinnabarinus collectedin China. The nearly full-length 16S rDNA gene sequences were obtained and aphylogenetic tree was constructed based on the 16S rDNA gene of Cardinium from ourmite populations and other arthropods published on GenBank website. Sequencing analysisof the 16S rDNA region of the Cardiniurn in Chinese carmine spider mite indicatesevidence for horizontal transmission. Lastly, to determine the effects of Cardinium infection,we carried out crossing experiments between infected and tetracycline-treated cured mites in two populations with both high infection rates.1. The collection and rearing of experimental materialsSamples of T. cinnabarinus and T. urticae used in this study were obtained from 27and 13 geographical localities respectively, which cover an extensive geographical scalerepresenting different regions and environments in China. Mites were then rearedseparately in the laboratory on leaves of Phaseolus vulgaris L., placed on awater-saturated tissue in a glass dish. All populations were kept in an illumination culturetank at 25±1℃, 60% r.h. with a photoperiod of 16h light: 8 h dark.2. The investigation of incidence of Cardinium infection in different populations ofspider mites in ChinaFourty adult females from each population of both carmine spider mite andtwo-spotted spider mite were randomly selected and examined for the presence ofCardinium using PCR amplification with a pair of Cardiniurn-specific primers designed byWeeks et al (2003). Result showed that 6 populations of T. cinnabarinus were infected byCardinium, while no infection were detected from any of the 13 populations of T. urticae.Compared with Wolbachia infection in our mite populations, it is obvious that the infectionrate of Cardinium is much lower, suggesting a higher standard of living environment aswell as a better host for Cardinium to thrive. The infection rate in each of the sixpopulations is different from each other, probably due to the different genetic backgroundbetween hosts, climatic factors and geographical barriers.3. The clone and comparison of the 16S rDNA gene sequences of Cardinium betweengeographical populations of spider mite in China.To prevent from being mixed with the 16S rDNA of Wolbachia when using generalprimers 27 F and 1513 R, we designed a pair of primers that can specifically amplify anearly full-length of 16S rDNA of Cardinium, and generated a product of the expected sizeof about 1338 bp from the populations that were positive for the Cardinium infection.Three to five individual specimens per population were cloned and sequenced and thestandard sequences were determined by the most identical ones. When Comparing betweensix populations, the sequences of Anhui and Gansu population were identical, while theother four showed some difference in several sites, but were not significant, suggesting that they might be the same strain of Cardinium.4. The phylogenetic analyses of Cardiniurn in T. cinnabarinus in China based on 16SrDNA gene sequencesDNA sequences were submitted to the GenBank database and compared online withthe published sequences by similarity search engines such as BLAST in NCBI Web. Inorder to understand the relationships of the bacterial symbionts of T. cinnabarinus withinthe Bacteroidetes group, a phylogenetic tree was constructed by using Cardinium 16SrDNA sequences obtained in this study, as well as those from other arthropods available inGenBank. Sequences were aligned in CLUSTAL X, and subjected to distance analysis andKimura-2 neighbour-joining parameters in MEGA 2.1. The robustness of the results wasevaluated by bootstrap analysis using 1000 neighbourjoining replicates.The distance analysis-based phylogenetic tree showed that, Cardinium of T.cinnabarinus form amonophyletic group with the symbionts reported from other spidermites in Tetranychidae. This branching was highly supported by the bootstrap scores (99%).Cardinium from another phytophagous mite family Brevipalpus mites fall into a differentclade, and are more closely related to the Ixodes and Encarsia symbionts. In addition, allthe Cardinium from the Chinese populations are virtually identical.5. The crossing experiments between infected and tetracycline-treated cured mites intwo highly infected populations of Chinese carmine spider miteTo clarify the effect of Cardinium on the reproductive traits of the infected spidermites, experiments were performed in Shanxi and Gansu populations to test forCytoplasmic incompatibility (CI) phenotype previously reported to be Caused by Cardinium.Infected mites were crossed with uninfected mites for each population, and CI was inducedin both populations when infected males were crossed with uninfected females.Furthermore, the two populations tested showed different types of CI. In Shanxi population,CI resulted in a severe reduction in hatchability of eggs as well as the number of F₁ females,while in Gansu population, CI resulted in a 100% hatchability of eggs, but the F₁ generationcontained no female mite. The Cardinium strains in these two populations share very closerelationship, but the different phenomenon suggests that closely related strains can causedifferent phenotype of CI. In conclusion, by using molecular techniques, we surveyed for the presence ofCardinium in Chinese populations of spider mite, which was in addition compared with thatof Wolbachia, and determined the phylogenetic relationship of Cardinium in Chinesecarmine spider mites. Lastly, we provided evidence to show the effect of Cardinium incausing reproductive abnormalities in T. cinnabarinus. This is the first time tosystematically study Cardinium in China. Therefore, our study will establish a solidfoundation for better understanding the infection mechanism of Cardinium, and for furtherresearch on its reproductive manipulation to host species. Moreover, it will be helpful forthe use of intracellular symbionts such as Cardinium in biological control application in thefuture.