Wolbachia wsp gene hypervariable region specific PCR primers detect multiple strain infections in northern corn rootworm (Diabrotica barberi)

Wolbachia wsp gene hypervariable region specific PCR primers detect multiple strain infections in northern corn rootworm (Diabrotica barberi) Richard L. Roehrdanz* and Sheila G. Sears,Insect Genetics & Biochemistry Research Unit, USDA-ARS-RRVARC, Fargo, ND 58102-2765

The northern corn rootworm (Diabrotica barberi)(Coleoptera: Chrysomelidae) in eastern and central North America exhibits at least three distinct populations with respect to Wolbachia infection: uninfected; singly-infected; multi-infected. The infected states are associated with different mtDNA haplotypes and reduced mitochondrial DNA variability. Both wsp and MLST sequencing indicated that the multiple infections include at least five distinct strains. The recovery of the different wsp hypervariable regions among 55 clones obtained with universal primers from several individuals was unequal. The most common was obtained 33 times, the least common only once. We designed a group of primers specific for the different HV regions. Various primer combinations can amplify each of the five strains to determine their presence or absence in single beetles. The primers were tested on beetles that had previously shown to be harboring certain wsp variants. Some of these amplicons were sequenced to verify that they contained the anticipated HV regions. In the process we have discovered a fourth variant of hypervariable region 1 (HV1 Bar4) that was not observed via the cloning experiments. Four of the five identified Wolbachia strains appear to be widely distributed with one of the four strains also found in a singly infected population. The fifth Wolbachia strain seems to be rare.