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An Initial Survey for Wolbachia (Rickettsiales: Rickettsiaceae) Infections in Selected California Mosquitoes (Diptera: Culicidae) JASON L. RASGON1

AND

THOMAS W. SCOTT

Department of Entomology, University of California Davis, Davis, CA 95616

J. Med. Entomol. 41(2): 255Ð257 (2004)

ABSTRACT Knowledge of biogeographic variation in Wolbachia infection rates and inferred susceptibility to infection among different mosquito taxa has fundamental implications for the design and successful application of Wolbachia-based vector-borne disease control strategies. Using a WolbachiaspeciÞc polymerase chain reaction assay, we tested 14 North American mosquito species in Þve genera (Aedes, Anopheles, Culiseta, Culex, and Ochlerotatus) for Wolbachia infection. Wolbachia infections were only detected in members of the Culex pipiens (L.) species complex. KEY WORDS Wolbachia, wsp, mosquito, classiÞcation, transgenic

THE MATERNALLY INHERITED BACTERIAL endosymbiont Wolbachia is widespread among arthropods. Estimated frequencies of infected species range from ⬇20 (Werren et al. 1995) to 76% (Jeyaprakash and Hoy 2000). Wolbachia infection is associated with numerous host reproductive alterations, including cytoplasmic incompatibility (CI) (Stouthamer et al. 1999). With CI, infected males are reproductively incompatible with uninfected females (matings are partially or completely sterile). Infected females are reproductively compatible with both infected and uninfected males (Stouthamer et al. 1999). The phenomenon of CI gives infected females a reproductive advantage and allows Wolbachia to drive rapidly through host populations (Turelli and Hoffmann 1999). The ability of Wolbachia to spread through populations has generated an applied interest in using the symbiont as a mechanism to drive introduced transgenic traits into vector populations to control mosquito-borne diseases (Pettigrew and OÕNeill 1997, Turelli and Hoffmann 1999, Rasgon and Scott 2003). It is crucial to know which Wolbachia strains are already present in populations before releasing infected individuals because preexisting natural infections can interact with and alter the dynamics of introduced strains. For example, the dynamics of Wolbachia spread can become more complicated as the number of incompatible Wolbachia strains present in the population increase (Hoffmann and Turelli 1997). In addition, understanding the similarities and differences between Wolbachia strains infecting different mosquito species is fundamental for estimating how often (in evolutionary time) mosquitoes encounter Wolbachia-horizontal transmission events in na1 Current address: Department of Entomology, North Carolina State University, Raleigh, NC 27695.

ture. Similarities between strains infecting different mosquito species are one indication of how easily mosquitoes encounter and acquire new infections. This kind of information has important implications for the design and success of artiÞcial introductions of Wolbachia into natural mosquito populations to control disease because it may provide insight into whether introduced infections are likely to be maintained. The diversity of Wolbachia infection in mosquito taxa was investigated in Southeast Asia (Kittayapong et al. 2000), and Europe and Africa (Ricci et al. 2002), but little is known about variation in Wolbachia infection in mosquitoes in North America. In California mosquitoes, Wolbachia infection has only been characterized in the Culex pipiens (L.) species complex (Barr 1980, 1982; Rasgon and Scott 2003), which consists of the subspecies Cx. pipiens pipiens and Culex pipens quinquefasciatus (Say) (Cornel et al. 2003). There is no information on the infection status of other mosquito taxa. To address this lack of knowledge, we surveyed selected North American mosquito taxa, primarily from California, for Wolbachia infection. We focused our effort on those taxa of primary importance as vector or pest species. Materials and Methods Mosquito Collections. Except where noted, mosquitoes were collected from wild populations (Table 1). Larvae were collected by dip sampling appropriate habitats. Adults were collected by CO2 traps, CDC light traps, or by aspiration from resting boxes (Service 1993). Larvae were transported live to the University of California, Davis (UCD), reared to adults, sorted to species, and stored at ⫺80⬚C until processed for DNA extraction. Adults were frozen, transported to UCD on

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Table 1. Wolbachia prevalence as determined by PCR amplification of 16S rDNA sequences in mosquitoes collected in North America Genus

Species

n

Collection location

Aedes Ochlerotatus

vexans dorsalisa melanimon nigromaculis sierrensis triseriatusb triseriatus washinoi incidens melanurac erythrothorax p. pipiensa,d p. quinquefasciatusa,d stigmatosoma tarsalis tarsalisc freeborni

20 20 20 1 2 25 10 4 20 20 20 45 30 10 20 10 19

Kern Co., CA San Luis Obisbo Co., CA Contra Costa Co., CA Contra Costa Co., CA Contra Costa Co., CA Lacrosse Co., WI Houston Co., MN Contra Costa Co., CA Contra Costa Co., CA Middlesex Co., NJ Contra Costa Co., CA Shasta Co., CA Riverside Co., CA Kern Co., CA Kern Co., CA Kern Co., CA Kern Co., CA

Culiseta Culex

Anopheles

Specimens were collected as wild adults except where noted. Collected as larvae. Arthropod-borne Infectious Diseases Laboratory colony. c UCD colony. d Wolbachia-positive, 100% infection frequency. a

b

dry ice, and stored at ⫺80⬚C until processed for DNA extraction. DNA Extraction. Mosquito genomic DNA was extracted using DNEasy spin columns (QIAGEN, Valencia, CA). Individual insects were homogenized in 200 ␮l of phosphate-buffered saline and DNA extracted according to the manufacturerÕs suggested protocol for cultured cells. DNA was stored at ⫺20⬚C until used for polymerase chain reaction (PCR). Wolbachia-Specific PCR Assay. PCR was conducted using primers 99F and 994R, which amplify an ⬇0.9kilobase (kb) fragment from Wolbachia 16S rDNA and are designed to be speciÞc to all strains of Wolbachia (OÕNeill et al. 1992). DNA template quality was assessed by successful ampliÞcation of an ⬇0.4-kb fragment of insect mitochondrial DNA by using primers 12SA1 and 12SB1 (Simon et al. 1991). PCR primer sequences and conditions were as stated in the respective cited references. Ready-To-Go PCR beads (Amersham Biosciences Inc., Piscataway, NJ) were used for all reactions. AmpliÞed fragments were separated by agarose gel electrophoresis, stained with ethidium bromide (1 ␮g/ml) and visualized with UV light. Known colony positive and tetracycline-cured negative specimens of Cx. pipiens were included in every reaction as positive and negative controls, respectively, and always gave the expected result. Wolbachia Strain Sequencing. A 558-base pair fragment of the Wolbachia wsp gene was ampliÞed from Þve specimens from all positive species by using primers 81F and 691R. Primer sequences and conditions were as stated by Zhou et al. (1998). The resulting PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide (1 ␮g/ml) for visualization under UV light, puriÞed from the gel by Qiaquick PCR puriÞcation columns (QIAGEN), and directly sequenced in both directions using an ABI

Vol. 41, no. 2

Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA). Sequence data were aligned using Sequencher DNA Sequence Analysis Software version 4.0.5 (Gene Codes Corporation, Ann Arbor, MI).

Results and Discussion In total, we tested 296 individuals from 14 species across Þve genera for Wolbachia infection (Table 1). Wolbachia infections have previously been identiÞed in members of the Cx. pipiens species complex (Barr 1980, 1982; Rasgon and Scott 2003). We included these mosquitoes in our survey as a detection control. All Cx. pipiens complex specimens tested were positive for infection. All obtained wsp sequences were identical within and between subspecies, with no ambiguous bases to indicate the presence of multiple Wolbachia strains. Homology searches of the GenBank database conÞrmed strain identiÞcation as wPip, the type strain associated with Cx. pipiens (Zhou et al. 1998). The obtained sequence was identical to existing GenBank wsp sequences from California Cx. pipiens complex mosquitoes. The fact that both taxa are infected with the same Wolbachia strain is not surprising, because there is extensive documented hybridization and gene ßow between California Cx. pipiens complex members (Urbanelli et al. 1997, Cornel et al. 2003). In contrast, all mosquitoes tested in species from the genera Aedes, Anopheles, Culiseta, and Ochlerotatus did not contain detectable infections. Similarly, except for the Cx. pipiens complex, we did not detect Wolbachia in the genus Culex. Counting the Cx. pipiens complex as one species, we found that 7% (1/14) of tested species were infected. In contrast, in Europe 19% (5/26, discounting Þlarial Wolbachia infections) were infected (Ricci et al. 2002) and 28% (25/89) of mosquito species tested in Southeast Asia contained detectable infections (Kittayapong et al. 2000). In addition, 48% (12/25) of infected Southeast Asian species harbored multiple Wolbachia strains (Kittayapong et al. 2000). However, infection frequencies in California were not signiÞcantly different from those observed in Asia (Fisher exact test, P ⫽ 0.08) or Europe (Fisher exact test, P ⫽ 0.3). Studies investigating similarities and differences in Wolbachia infection patterns are critical to understand processes that govern Wolbachia-mosquito interactions. Consistent patterns, such as the complete absence of infection in Anopheles (Kittayapong et al. 2000, Ricci et al. 2002; Table 1), may reßect a different history of horizontal Wolbachia transfer events over evolutionary time, possible physiological differences in the ability of different mosquitoes to harbor infection or may be a biogeographic relic of the evolutionary history between Wolbachia and mosquitoes. Knowledge of geographic variation in the extent of natural mosquito Wolbachia infections will be essential for designing CI-based strategies for disease control. Surveys for Wolbachia infection in different mosquito taxa over a variety of geographic locations and

March 2004

RASGON AND SCOTT: Wolbachia IN CALIFORNIA MOSQUITOES

scales will provide basic, descriptive information necessary to help resolve these issues. Acknowledgments We thank William K. Reisen, Ann Donohue, Steve Shutz, Laura Goddard, Barry J. Beaty, and the Mosquito and Vector Control Association of California for providing mosquitoes and assisting with mosquito collections. This research was supported by the University of California Mosquito Research Program.

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