Genetic variability, community structure, and horizontal transfer of endosymbionts among three Asia II‐Bemisia tabaci mitotypes in Pakistan

Endosymbionts associated with the whitefly Bemisia tabaci cryptic species are known to contribute to host fitness and environmental adaptation. The genetic diversity and population complexity were investigated for endosymbiont communities of B. tabaci occupying different micro-environments in Pakistan. Mitotypes of B. tabaci were identified by comparative sequence analysis of the mitochondria cytochrome oxidase I (mtCOI) gene sequence. Whitefly mitotypes belonged to the Asia II- 1, -5, and -7 mitotypes of the Asia II major clade. The whitefly-endosymbiont communities were characterized based on 16S ribosomal RNA Operational Taxonomic Unit (OTU) assignments, resulting in 43 OTUs. Most of the OTUs occurred in the Asia II-1 and II-7 mitotypes (r2=0.9, p<0.005) while the Asia II-5 microbiome was less complex. The microbiome OTU groups were mitotype-specific, clustering with a basis in phylogeographical distribution and the corresponding ecological niche of their whitefly host, suggesting mitotype-microbiome co-adaptation. The primary endosymbiont Portiera was represented by a single, highly homologous OTU (0-0.67% divergence). Two of six Arsenophonus OTUs were uniquely associated with Asia II-5 and -7, one occurred exclusively in Asia II-1, two only in Asia II-5, and one in both Asia II-1 and -7. Four other secondary endosymbionts, Cardinium, Hemipteriphilus, Rickettsia, and Wolbachia OTUs were found at ≤29% frequencies. The most prevalent Arsenophonus OTU was found in all three Asia II mitotypes (55% frequency), whereas the same strain of Cardinium and Wolbachia were found in both Asia II-1 and -5, and a single Hemipteriphilus OTU occurred in Asia II-1 and -7. This pattern is indicative of horizontal transfer, suggestive of a proximity between mitotypes sufficient for gene flow at overlapping mitotype ecological niches.
Methods
The 16S rDNA (~1500 bp) sequence was PCR-amplified using ‘universal’ primers 27F (5'-AGAGTTTGATCMTGGCTCAG) and 1513R (5'-ACGGYTACCTTGTTACGACTT) (0.4 μM) [64, 65]. The PCRs were set as explained above using 20ng of whitefly DNA. Cycling parameters were an initial denaturation at 94°C for 2 min, and 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min, with a final extension at 72°C for 10 min. Amplicon sizes were verified by agarose gel electrophoresis and cloned. Twelve clones per whitefly were selected by colony PCR amplification, and sequenced.