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The whitefly Bemisia tabaci (Gennadius; Hemiptera: Aleyrodidae) is a highly invasive insect pest and is considered a cryptic species complex. This study was conducted to investigate the diversity and distribution of whiteflies of the B. tabaci species complex in Pakistan, and also to investigate the diversity endosymbionts and viruses harboured. Using a well-established, PCR-based cloning and sequencing of a mitochondrial cytochrome oxidase subunit I (mtCOI) marker, six biotypes Asia II 8, Asia II 7, Asia II 5, Asia II 1, Asia 1, and MEAM 1” were identified in Pakistan. Asia II-1 was found to be the most common biotype present and thus may possibly be the most important vector of the begomovirus complex which causes cotton leaf curl disease in Pakistan. The study also highlighted changes in the prevalent biotypes in some regions in the country. Using the 16S general and specific primers, in conjunction with sequencing, six endosymbionts (Arsenophonus, Cardinium, Hamiltonella, Rickettsia, and Wolbachia) were identified. Arsenophonus found to be associated with Asia II-1 while Hamiltonella was associated with the MEAM-1. The presence and prevalence of each symbiont varied in different biotypes and also varied between regions. Endosymbionts are important in virus-vector interactions. A number of distinct begomovirus species were identified with Cotton leaf curl Burewala virus, the most prevalent virus causing CLCuD in Pakistan at this time, the frequently identified. The levels of viral and betasatellite DNA were measured at 2 weekly intervals in B. tabaci collected from cotton using a quantitative PCR assay during the cotton growing seasons of 2014, 2015 and 2016. Although there were differences between the three years, in general, it was found that the level of betasatellite rises during the cotton growing season whereas the level of virus initially rises and then decreases at the end of the season. For both virus and betasatellite the DNA levels are very low initially, when there are no CLCuD symptoms in the crop, indicating that the initial inoculum is low. This comprehensive study provides the basis for the design of vector/virus control strategies which can be tested using the methodologies devised and implemented during the study.
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