Decapitation improves detection of Wolbachia pipientis (Rickettsiales: Anaplasmataceae) in Culex pipiens (Diptera: Culicidae) mosquitoes by the polymerase chain reaction

Abstract

Polymerase chain reaction (PCR) is often used to detect microorganisms, pathogens, or both, including the reproductive parasite Wolbachia pipientis (Rickettsiales: Anaplasmataceae), in mosquitoes. Natural populations of Culex pipiens L. (Diptera: Culicidae) mosquitoes are infected with one or more strains of W. pipientis, and crosses between mosquitoes harboring different Wolbachia strains provide one of the best-known examples of cytoplasmic incompatibililty (CI). When we used PCR to monitor Wolbachia in the Buckeye strain of Culex pipiens, and in a Wolbachia-cured sister colony obtained by tetracycline treatment, we noted false negative PCR reactions with DNA samples from infected mosquitoes; these results were inconsistent with direct microscopic observation of Wolbachia-like particles in gonads dissected from mosquitoes in the same population. Assays with diluted template often improved detection of positive samples, suggesting that DNA prepared from whole mosquitoes contained an inhibitor of the PCR reaction. We reconciled discrepancies between PCR and microscopy by systematic measurement of the PCR reaction in the presence of an internal standard. Mosquito decapitation before DNA extraction restored the reliability of the PCR reaction, allowing accurate determination of Wolbachia infection status in infected and tetracycline-cured mosquito populations, consistent with microscopic examination. Using PCR primers based on the Tr1 gene, we confirmed that the Wolbachia infection in the Buckeye strain of Culex pipiens belongs to the genotype designated wPip1. Finally, to explore more widely the distribution of PCR inhibitors, we demonstrated that DNA isolated from the cricket, Acheta domesticus (L.); the beetle, Tenebrio molitor L.; the honey bee, Apis mellifera L.; and the mosquito, Anopheles punctipennis Say also contained PCR inhibitors. These results underscore the importance of measuring the presence of inhibitors in PCR templates by using a known positive standard, and provide an approach that will facilitate use of PCR to monitor environmental samples of mosquitoes that harbor endosymbionts or pathogenic organisms.

Fig. 1

Variable PCR-based detection of Wolbachia in adult Cx. pipiens. Lanes 1 and 19: positive control; lanes 2 and 20: negative control; lanes 3–10 are females, and lanes 11–18 are males. DNA was extracted from individual mosquitoes as described in the Materials and Methods. Lanes 21–23 show positive PCR identification of Wolbachia in egg rafts: lane 21, one egg mass; lane 22, pool of five egg masses; lane 23, pool of 10 egg masses.

Fig. 2

Effect of template volume on the PCR reaction. Lanes 1 and 12 show positive controls; lanes 2 and 13 are negative controls; lanes 3–11: 1 μl to 9 μl of DNA template, respectively. Lanes 14–18 all contain positive control DNA as in lane 12, with no additional mosquito DNA (lane 14) and 1 μl to 4 μl of mosquito template DNA (lanes 15–18, respectively).

Fig. 3

Preparation of template DNA from decapitated mosquitoes removes the inhibitor. Lanes 1, 2, and 3 show DNA ladder, positive control and negative control, respectively. Lanes 4–9 show PCR template DNA prepared from decapitated mosquitoes. For lanes 10–15, mosquitoes were homogenized intact. Even lanes used 1 μl of template DNA; odd lanes had 9 μl of template DNA.

Fig. 4

Survey for PCR inhibitors from various insects. Positive control DNA (1 μl; lanes 1 and 27) was combined with 8-μl template DNA from various extractions (lanes 3–34). Lanes with reduced PCR product, relative to the positive control show evidence for an inhibitor. Panels A and B show lanes from the same gel, whereas Panel C is from a separate gel. Lanes 2 and 28 are negative controls without DNA. Lanes 3–5 show M. autumnalis head DNA and 6–8 show M. autumnalis decapitated, whole body DNA. Lanes 9–11 show A. domesticus head DNA and 12–14 show A. domesticus decapitated, whole body DNA. Lanes 15–17 show T. molitor head DNA and 18–20 contain T. molitor decapitated, whole body DNA. Lanes 21–23 show A. mellifera head DNA and 24–26 show A. mellifera decapitated, whole body DNA. Lanes 29–30 are An. punctipennis whole mosquitoes and 32–34 are decapitated An. punctipennis mosquitoes.