Genetic diversity of Wolbachia endosymbionts in Culex quinquefasciatus from Hawai`i, Midway Atoll, and Samoa

Abstract

Incompatible insect techniques are potential methods for controlling Culex quinquefasciatus and avian disease transmission in Hawai‘i without the use of pesticides or genetically modified organisms. The approach is based on naturally occurring sperm-egg incompatibilities within the Culex pipiens complex that are controlled by different strains of the bacterial endosymbiont Wolbachia pipientis (wPip). Incompatibilities can be unidirectional (crosses between males infected with strain A and females infected with strain B are fertile, while reciprocal crosses are not) or bidirectional (reciprocal crosses between sexes with different wPip strains are infertile). The technique depends on release of sufficient numbers of male mosquitoes infected with an incompatible wPip strain to suppress mosquito populations and reduce transmission of introduced avian malaria (Plasmodium relictum) and Avipoxvirus in native forest bird habitats. Both diseases are difficult to manage using more traditional methods based on removal and treatment of larval habitats and coordination of multiple approaches may be needed to control this vector. We characterized the diversity of Wolbachia strains in C. quinquefasciatus from Hawai‘i, Kaua‘i, Midway Atoll, and American Samoa with a variety of genetic markers to identify compatibility groups and their distribution within and between islands. We confirmed the presence of wPip with multilocus sequence typing, tested for local genetic variability using 16 WO prophage genes, and identified similarities to strains from other parts of the world with a transposable element (tr1). We also tested for genetic differences in ankyrin motifs (ank2 and pk1) which have been used to classify wPip strains into five worldwide groups (wPip1–wPip5) that vary in compatibility with each other based on experimental crosses. We found a mixture of both widely distributed and site specific genotypes based on presence or absence of WO prophage and transposable element markers on Hawai‘i Island (Volcano, Pu‘u Wa‘awa‘a, Laupāhoehoe, Kaumana, Kahuku, Nīnole, and Maulua Gulch), Kaua‘i Island (Kawaikōī, Mōhihi, Kalāheo, Lāwa‘i and Hanapepe) and Midway Atoll. Genotypes from American Samoa were unique and formed their own clade. Based on analysis of ankyrin motifs, wPip strains from Hawai‘i, Kaua‘i, and Midway Atoll were most similar to wPip5 strains of Australasian origin. By contrast, Wolbachia strains from Culex quinquefasciatus collected in American Samoa were most similar to wPip3 strains of American origin. We detected a single Culex mosquito from Pu‘u Wa‘awa‘a on Hawai‘i Island that was infected with a unique wPip3 genotype. This discovery, plus a rarefaction analysis of genotypes from Kaua‘i and Hawai‘i Islands suggests that limited sampling may have underestimated diversity of wPip in our study. Mosquitoes infected with wPip5 and wPip3 are bidirectionally compatible with each other based on prior studies, which would support their ability to coexist within the same population on Hawai‘i Island. Available evidence from prior studies suggests that genotype wPip4 from Africa, the Middle East, Europe, and Asia is bidirectionally incompatible with genotype wPip5 and varies in compatibility with genotype wPip3 depending on geographic origin. Since wPip5 appears to be the most common compatibility group in Hawai‘i based on limited sampling, logical next steps are to 1) expand the current survey to include additional islands and localities, 2) infect a laboratory colony of Hawaiian Culex with wPip4 through tetracycline treatment of Hawaiian mosquitoes and backcross with Culex from Europe, North Africa, and the Middle East that are naturally infected with wPip4, 3) conduct cage trials to confirm bidirectional incompatibilities between Hawaiian Culex infected with wPip4 and wPip5, and 4) conduct field trials to evaluate whether release of incompatible males can be applied at small scales to suppress local populations.