jmbReview Wolbachia Sequence Typing in Butterflies Using Pyrosequencing

Wolbachia is an obligate intracellular symbiotic bacterium. It belongs to the order Rickettsiales and is known to infect numerous species of arthropods and nematodes. The bacterium is known to manipulate the reproduction of its insect hosts via cytoplasmic incompatibility, parthenogenesis, male-killing, or feminization [8, 18, 30, 31]. Wolbachia species are classified into 11 (A–K) supergroups [22] based on multilocus sequence typing (MLST) and Wolbachia surface protein (WSP) typing [2]. The standard MLST system for Wolbachia determines a sequence type (ST) based on the combination of alleles for five conserved housekeeping genes (ftsZ, gatB, coxA, hcpA, and fbpA). The WSP typing system determines STs using four hypervariable regions (HVRs) of WSP: HVR1 (aa 52 to 84), HVR2 (85 to 134), HVR3 (135 to 185), and HVR4 (186 to 222) [1]. A host can be infected with a single strain of Wolbachia, or multiple strains, as has been observed in a wide range of insects [13, 20, 21]. Classical MLST can be used for the universal characterization of Wolbachia, but its application is currently limited to hosts infected with a single strain of Wolbachia [2]. To assess the diversity of Wolbachia in insects infected with multiple strains, sequencing of the wsp gene is generally conducted [32]. Amplification of ST-specific regions is also used, but this method has limited application because the design of specific primers is only possible after a strain’s sequence is known. Improved methods for determining STs in insects infected with multiple strains would contribute to understanding Wolbachia diversity. Received: March 26, 2015 Revised: June 29, 2015 Accepted: July 1, 2015

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