Long PCR improvesWolbachiaDNA amplification:wspsequences found in 76% of sixty‐three arthropod species

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Abstract
Bacteria belonging to the genusWolbachiaare associated with a variety of reproductive anomalies in arthropods. Allele‐specific polymerase chain reaction (= Standard PCR) routinely has been used to amplifyWolbachiaDNA from arthropods. While testing the two‐spotted spider miteTetranychus urticaeand other arthropods known to be infected withWolbachia, Standard PCR frequently produced false negatives, perhaps because the DNA from the arthropod host interfered with amplification byTaqDNA polymerase. Long PCR, which uses two enzymes (TaqandPwo), consistently amplifiedWolbachiaDNA and a sensitivity analysis indicated that Long PCR was approximately six orders of magnitude more sensitive than Standard PCR in amplifying plasmid DNA spiked into insect genomic DNA. A survey indicated that 76% of sixty‐two arthropod species and two subspecies in thirteen orders tested positive for theWolbachia wspsequence by Long PCR, which is considerably higher than the rate of 16.9% obtained previously for theftsZsequence using Standard PCR (Werren, J.H., Windsor, D. and Gao, L. (1995a)Proc R Soc Lond B262: 197–204). A subsample of Long PCR products from fourteen arthropod species and two subspecies were sequenced, both directly and after cloning. Two A‐ and eleven B‐Wolbachiastrains were detected and theirwspsequences displayed a maximum of 23.7% sequence divergence at this locus. Two new groups (named Fus and Ten) were identified in addition to nineteen reported earlier (Zhou, W., Rousset, F. and O’Neill, S.L. (1998)Proc R Soc Lond B265: 1–7;van Meer, M.M.M., Witteveldt, J. and Stouthamer, R. (1999)Insect Mol Biol8: 399–408), because they displayed more than 2.5% sequence divergence from otherWolbachia wspsequences. PCR products from seventeen of twenty‐nine (59%) arthropod species analysed could not be sequenced directly due to apparent infection by multipleWolbachiastrains. Thewspsequences cloned from two such species (Plutella xylostellaandTrichoplusia ni) indicated both A‐ and B‐Wolbachiawere present in a single individual. Hence, superinfection also may be more widespread than the 1.2% incidence previously estimated.

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