Am. J. Trop. Med. Hyg., 81(2), 2009, pp. 264–272 Copyright © 2009 by The American Society of Tropical Medicine and Hygiene Environmental and Biological Factors Influencing Culex pipiens quinquefasciatus Say (Diptera: Culicidae) Vector Competence for Saint Louis Encephalitis Virus Stephanie L. Richards , * Cynthia C. Lord , Kendra Pesko , and Walter J. Tabachnick Florida Medical Entomology Laboratory, University of Florida, Vero Beach, Florida Abstract. Complex interactions between environmental and biological factors influence the susceptibility of Culex pipiens quinquefasciatus to St. Louis encephalitis virus and could affect the epidemiology of virus transmission. Similar interactions could have epidemiologic implications for other vector-virus systems. We conducted an experiment to exam- ine four such factors in combination: mosquito age, extrinsic incubation temperature (EIT), virus dose, and colony. The proportion of mosquitoes with body infections or disseminated infections varied between colonies, and was dependant on age, EIT, and dose. We also show that the probability of a body or leg infection interacted in complex ways between colonies, ages, EITs, and doses. The complex interactive effects of environmental and biological factors must be taken into account for studies of vector competence and epidemiology, especially when laboratory studies are used to generalize to natural transmission dynamics where the extent of variation is largely unknown. INTRODUCTION quefasciatus . 10 However, the relationship between infection and dissemination changed depending on the EIT, but not Mosquito biology and the environment can play important the dose. This inconsistency and also the limited information roles in mosquito vector competence. The effect of environ- on the impact of mosquito age on vector competence was the mental factors such as extrinsic incubation temperature (EIT) basis for the current investigation. Most laboratory studies and virus dose on vector competence for arboviruses is widely have focused on the effects of single environmental factors appreciated and has been studied and reviewed for a number with little attention to how different factors may interact with 1–6 of insect-virus systems. However, although environmental each other and how such interactions may change because of factors influence mosquito vector competence for arboviruses, biological factors such as mosquito age or mosquito strain. there has been little work to determine how various environ- St. Louis encephalitis virus (family Flaviviridae , genus mental and biological factors may interact with one another. Flavivirus ) is maintained in an enzootic cycle involving orni- For example, several studies generally show a positive rela- thophilic mosquitoes and susceptible birds. This virus threat- 1,2 tionship between EIT and vector competence in mosquitoes. ens humans whenever large numbers of infectious mosquitoes Higher virus doses overcame barriers to dissemination for exhibiting opportunistic feeding coincide with amplification Culex tarsalis infected with Western equine encephalitis virus hosts and human populations. 11–13 The range of SLEV extends 3,4 (WEEV), Cx. pipiens pipiens and Cx. p. quinquefasciatus from southern Canada to Argentina; however, most human 5 with St. Louis encephalitis virus (SLEV), and Cx. nigripalpus cases have occurred in the central and eastern portion of 6 and Cx. p. quinquefasciatus with West Nile virus (WNV). the United States. 14,15 The primary vectors of SLEV in North Biological factors such as mosquito age and mosquito strain, America have been identified as Cx. tarsalis , Cx. p. pipiens , as well as environmental factors such as EIT and dose may Cx. p. quinquefasciatus , and Cx. nigripalpus . 16–18 Culex p. quin- have indirect effects on arbovirus infection and transmission quefasciatus feeds on avian and mammalian hosts, 19–21 is a insofar as vectors must survive long enough to blood feed on competent vector of SLEV in the laboratory,22,23 and has been an infectious host, complete the extrinsic incubation period found in nature infected with SLEV. 18,24 (EIP) required for virus dissemination, oviposit, and subse- Vector competence in the present study was characterized quently feed on another host. The importance of mosquito using four epidemiologically important phenotypes: suscep- age on arbovirus transmission was shown by the observation tibility to infection, disseminated infection, virus body titer, that young Aedes aegypti have a higher survival probability and leg titer. These phenotypes are aspects of vector compe- compared with older mosquitoes, and would thereby be more tence because viruses must cause midgut infections and over- 7 likely to complete the EIP required for virus transmission. come barriers to dissemination. Female mosquitoes that have However, there may also be a direct effect of vector age on no body infection show a midgut infection barrier (MIB), and viral infection and dissemination rates. Decreased abdomen females having an infected body with no dissemination to the infection rates were found in Cx. tritaeniorhynchus fed WNV legs show a midgut escape barrier (MEB).25 Basic knowledge at 12 days post-emergence, compared with mosquitoes fed at of individual properties of the MIB and MEB under different 8 four and eight days post-emergence. However, there were no conditions, as well as the relationship between the MIB and differences in transmission of Japanese encephalitis virus by MEB is needed to develop mechanistic hypotheses and design Cx. tritaeniorhynchus that were infected at 10 or 24 days post- further studies of vector competence. We use virus titers in 9 emergence. the body and leg as a quantitative measure of the ability of A positive relationship was observed between EIT and dose the viruses to replicate in mosquito tissues. Although other with WNV infection and dissemination rates in Cx. p. quin- investigators have shown a positive relationship between dis- seminated infection and transmission for Cx. p. pipiens and WNV, 26,27 further studies are needed to directly address the * Address correspondence to Stephanie L. Richards, Institute of Food relationship between dissemination out of the midgut and sal- and Agricultural Sciences, Florida Medical Entomology Laboratory, University of Florida, 200 9th Street Southeast, Vero Beach, FL 32962. ivary gland infection with regard to actual vector transmission E-mail: [email protected] under varying environmental and biological conditions. 264 CULEX MOSQUITO VECTOR COMPETENCE 265 The intra-population and inter-population variability in medium effect sizes in infection rates. Sample sizes (and interactions between environmental and biological factors therefore power to detect small differences between groups) is virtually unknown. These interactions must be character- for dissemination rates were expected to be lower because we ized and the responsible mechanisms ultimately elucidated to did not expect 100% infection rates. understand the impact of vector competence on the epidemi- Blood meal preparation. Previous results showed that ology of arbovirus cycles. Results from the current study are fresh preparation of low-titered SLEV is an effective method expected to inform mechanistic studies of vector competence for infecting mosquitoes by artificial feeding systems.31 by elucidating some of the factors and complexity between Consequently, a T-75 cm 2 flask of confluent African green factors and the effect on vector competence. We explore the monkey (Vero) cells was inoculated with 0.15 mL of SLEV complexity of environmental and biological effects on vec- stock previously determined by plaque assay to have 6.0 logs tor competence for arboviruses using Cx. p. quinquefasciatus plaque-forming units (PFU)/mL. Then, 12 mL of Medium and SLEV. 199 (with Earle’s salts, 10% fetal bovine serum, penicillin/ streptomycin, and mycostatin) was added to the flask and incubated at 35°C in an atmosphere of 5% CO . At 72 hours MATERIALS AND METHODS 2 post-inoculation, the supernatant was mixed with citrated Mosquitoes. Culex p. quinquefasciatus from two colonies bovine blood to create blood meals of low and high dose. were used for experiments. The first colony was established in Because of a lower feeding success rate of the 2007 colony, a 1995 from a collection from Alachua County in north central second set of flasks was inoculated with SLEV 24 hours after > Florida (generation F40 ) and will be referred to as the 1995 the first flask so that another feeding could be attempted the colony. The second colony was established in 2007 from a day after the initial feeding. Two 0.1-mL samples of the blood collection of 32 egg rafts from Indian River County in east meal were each added to separate tubes containing 0.9 mL 32 central Florida (generation F3 ) and will be referred to as the BA-1 diluent and stored at −80°C until tested to determine 2007 colony. Mosquitoes were reared at 28°C and maintained blood meal titer. under a 14:10 (light:dark) cycle. Rearing conditions were Mosquito infection. The TBH28 strain of SLEV (passaged stan dardized to generate similar sized individuals. For each three times in Vero cells) was isolated from a human in colony, three egg rafts were placed in each of approximately Florida in 1962. More than 200 female mosquitoes were 15 enameled pans (24 cm × 36 cm × 5 cm) containing approx- offered a blood meal for each colony, age group, dose, and EIT imately 700 mL water. Larvae were fed daily with a slurry to achieve