West Nile Virus Vector Competency of Culex Quinquefasciatus Mosquitoes in the Galápagos Islands

West Nile Virus Vector Competency of Culex quinquefasciatus Mosquitoes in the Galápagos Islands

Gillian Eastwood ,* Laura D. Kramer , Simon J. Goodman , and Andrew A. Cunningham Institute of Zoology, Zoological Society of London, London, United Kingdom; Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom; Galápagos Genetics, Epidemiology and Pathology Laboratory, Puerto Ayora, Santa Cruz, Galápagos Islands, Ecuador; Wadsworth Center, New York State Department of Health, Slingerlands, New York

Abstract. The mosquito-transmitted pathogen West Nile virus (WNV) is not yet present in the Galápagos Archipelago of Ecuador. However, concern exists for fragile endemic island fauna after population decreases in several North American bird species and pathology in certain reptiles. We examined WNV vector competency of a Galápagos strain of mosquito ( Culex quinquefasciatus Say). Field specimens were tested for their capacity to transmit the WN02-1956 strain of WNV after incubation at 27°C or 30°C. Rates of infection, dissemination, and transmission all increased with days post-exposure to WNV, and the highest rates were observed at 28 days. Infection rates peaked at 59% and transmission rates peaked at 44% (of mosquitoes tested). Vector efficiency increased after day 14. Rates of infection but not of transmission were significantly influence by temperature. No vertical transmission was detectable. We demonstrate that Galápagos Cx. quinquefasciatus are competent WNV vectors, and therefore should be considered an animal and public health risk for the islands and controlled wherever possible.

INTRODUCTION recently, listed in the category ‘under danger’.8 Disease threats are one particular concern and several disease agents includ- West Nile virus (WNV; family Flaviviridae, genus Flavivirus ) ing avian pox virus and Salmonella spp. have already reached is a mosquito-borne virus that was originally isolated in Africa Galápagos with negative impacts on the native wildlife.9– 11 The 1 in 1937 but is now established across six continents and con- isolation of Galápagos wildlife implies they lack prior expo- sidered as causing an emerging infectious disease. This virus sure to many pathogens (i.e., they are immunologically naive) was successfully introduced to the Americas (New York City) and as such may show heightened susceptibility to WNV.12 in 1999, and although it subsequently spread west and south West Nile virus is maintained in nature in an avian host– with unprecedented intensity, and infected wildlife on several mosquito vector enzootic cycle.13 Although the highest vire- 2,3 Caribbean islands, its distribution across South America is ias generally occur in birds, the main amplification host, largely unknown. There are relatively few reports of WNV WNV is a multi-host pathogen capable also of infecting activity in South America, with several hypotheses as to why, mammals (including humans) and reptiles. Galápagos has including cross-protection of circulating viruses, lack of sur- 22 endemic reptile species, some already considered endan- 1 veillance, and dilution effect from increased biodiversity. gered. Of the 326 avian species reported with WNV infections However, WNV was isolated from horses in Argentina in in the United States, approximately 39 have been observed in 2006, and infection was serologically identified in horses in Galápagos (e.g., Pelecanus occidentalis) and additional spe- 4–6 Colombia in 2004 and birds in Venezuela in 2005, the lat- cies exist as related congenic populations (e.g., mockingbirds ter being the first confirmation of establishment on the South of the family Mimidae ).14 West Nile virus is linked to popu- American mainland. The status of WNV in mainland Ecuador, lation decreases of several North American bird species. 15, 16 and the impact on its island territory six hundred miles west, Therefore, this virus represents a substantial threat to the native the Galápagos Archipelago, has yet to be determined. This species of Galápagos should WNV reach the archipelago.17 study forms part of a more comprehensive investigation into The most likely pathway for a WNV introduction event to the extent of the threat posed by WNV to Galápagos; the dis- Galápagos is predicted to be human mediated, chiefly by the tribution, abundance, and ecology of potential mosquito vec- transport of infected mosquitoes to the islands by airplane. 18 tor species in Galápagos, including Culex quinquefasciatus , is Invasive insects are known to enter Galápagos by this route additionally being evaluated. (approximately one-fourth of all insect fauna in Galápagos is The wildlife species of the islands of Galápagos ( Figure 1 ) alien 19 ). Recent work detected Culex quinquefasciatus mos- embrace a distinct biodiversity that evolved in isolation over quitoes inside aircraft arriving to Galápagos (Baltra airport), thousands of years, with high levels of endemism in the flora and genetic analysis indicates there have been multiple intro- and fauna. However, the islands unique ecosystems are faced ductions of this mosquito although pathogen infection status with modern pressures from an increasing level of human was not ascertained.20 Culex spp. draw particular attention for population, tourism, invasive species, and disease introduc- those monitoring WNV outbreaks because they are the princi- 7 tion. Such pressures led to international conservation con- pal vectors; for example, accounting for more than 98% of all cern for the deteriorating quality of the island ecosystems mosquito pools positive for WNV in surveillance work in the and, as a United Nations Educational Scientific and Cultural United States. 21 Organization World Heritage Site, Galápagos was, until Three species of mosquito are currently resident in Galápagos. Aedes taeniorhynchus is ubiquitous and naturally colonized the archipelago approximately 200,000 years ago 22 (Eastwood G, * Address correspondence to Gillian Eastwood, Wildlife Epidemiol- ogy, Institute of Zoology, Zoological Society of London, Wellcome unpublished data). Aedes aegypti is a recently introduced Building, Room 29, Outer Circle Regents Park, London, NW1 4RY, anthropophilic mosquito that because of its associations United Kingdom. E-mail: [email protected] with dengue viruses has been largely controlled. Culex 426 WNV VECTOR COMPETENCY OF CX. QUINQUEFASCIATUS IN GALÁPAGOS 427

infection, dissemination beyond the midgut, and salivary transmission of WNV, even within the same state in the United States. For example, in California under comparable test conditions, one population of Cx. quinquefasciatus was refractory to infection, whilst populations from adjacent counties reached rates of 43% infection and 30% transmission. 32 Similarly, WNV vector competence of the sibling species Cx. pipiens Linnaeus, was shown to vary spatially and temporally in New York and Massachusetts. 41 If one considers vertical transmission of WNV from parent to progeny, minimum filial infection rates of approximately 3% are reported for Cx. quinquefasciatus in the United States.42 Little is known about South American Cx. quinquefasciatus , including its potential to play a significant role as a vector in WNV epidemiology in Galápagos. The available literature suggests that we cannot make assumptions about the WNV vector F igure 1. Map of the Galápagos Islands showing proximity to competency of Galápagos Cx. quinquefasciatus on the basis mainland Ecuador and South America. of studies conducted on the species elsewhere in the world. If we are to accurately model how Galápagos populations would quinquefasciatus, a wide-ranging semi-tropical/tropical species interact with WNV, it is imperative to determine the unique associated with fresh water, had been introduced to Galápagos vector characteristics specific to this strain. by 1985 and is now established around areas of human habita- 20,23 tion. Culex quinquefasciatus also has been recently found MATERIALS AND METHODS in uninhabited agricultural or tortoise reserve zones away from human habitation, in the highlands of Santa Cruz, San Mosquitoes. Field populations of Cx. quinquefasciatus were Cristobal, Isabela, and Floreana islands (Eastwood G, unpub- collected from highland and coastal areas of Santa Cruz and lished data). The invasive nature of Cx. quinquefasciatus was Isabela islands of Galápagos by using mainly gravid traps demonstrated by a study at the Miami, Florida, airport, which (CO 2 -baited light traps were also used). The resulting egg examined 102 arriving aircraft and found live specimens on rafts deposited by gravid females and first-instar larvae were 28 of these. 24 In Hawaii, an oceanic island ecosystem similar transported under U.S. Department of Agriculture (no. 47279) to Galápagos, Cx. quinquefasciatus is an introduced vector for and Centers for Disease Control and Prevention (no. 2009- the agents of avian malaria and avian pox, co-introduced dis- 06-182) permits to the Wadsworth Center Arbovirus Labora- eases associated with catastrophic decreases and extinctions tory (New York State Department of Health) during 2009. of endemic avifauna.25, 26 Mosquitoes were reared to adult stage in a walk-in chamber in Culex quinquefasciatus is of key importance to the risk of a BioSafety Level 2 quarantine insectary maintained at 28°C WNV introduction and establishment in Galápagos for sev- with a 12 hour:12 hour (light:dark) photoperiod and a relative eral reasons. First, it is a primary enzootic vector for WNV humidity of 85% to simulate natural conditions in Galápagos. in North America 1, 13 (expanded details below). Second, it Experiments to evaluate WNV vector competence took place is associated with outbreaks of related arboviruses.27 Third, under BioSafety Level 3 conditions. its feeding behavior shows preferences for bird and mam- Infection with WNV. Once maturity and mating were mal blood. 28, 29 Although local feeding patterns in Galápagos achieved (allowing 96 hours after emergence of adult females), require further investigation, the broad diet of Cx. quinque- mosquitoes were fed a viral preparation by using the WNV fasciatus known from elsewhere indicates that this mosquito strain WNV02-1956 isolated from an American crow. The virus would act as a bridge vector for WNV between bird and mam- had been passaged once in African green monkey kidney (Vero) mals in Galápagos.23, 30, 31 cells and amplified once in Ae. albopictus (C6/36) cells to yield

North American Cx. quinquefasciatus populations are a stock concentration of 9 log 10 PFU/mL, which was stored reported as competent vectors of WNV, and in southern at –80°C in multiple aliquots. To encourage feeding, blood United States this is the principal mosquito species associ- meals consisting of diluted (1:20) virus suspended in 9 mL of ated with WNV. 28, 30 Infection rates of up to 100% have been defibrinated goose blood containing 50% sucrose and 100 μL observed (Florida and Texas), and high WNV transmission (0.2 mM) ATP were presented to mosquitoes overnight in a rates of up to 52% (southern California), calculated as a pro- sausage skin membrane and also soaked onto gauze pledgets. portion of the number of mosquitoes tested.30, 32– 35 There are Prior experience showed this combined technique to yield noticeable influences on mosquito susceptibility to WNV higher feeding success in field mosquitoes. The virus level was infection including extrinsic (e.g., initial viral dose, temper- determined both pre-feeding and post-feeding to ensure titers ature, and length of incubation) and intrinsic (e.g., genetic had remained constant during overnight feeding. Infected 32,36–38 strain, midgut physiology) factors. Generally, longer incu- blood meals had a median WNV dosage of 7.7 log 10 PFU/mL. bation periods, higher viral doses, and higher temperatures The titer used was relatively high; comparative studies showed 30,32,39 40 > 32 are expected to yield higher rates of infection. However, a titer range from 4.1 log10 PFU/mL to 8.0 log10 PFU/mL. Cx. quinquefasciatus in Australia was shown to be a highly However, such virus levels realistically occur in nature in competent vectors when exposed to a dose of only 10 4.0±0.3 passerine birds.43 40 plaque-forming units (PFU)/mL of WNV (NY99 strain). After feeding, mosquitoes were anaesthetized with CO2 , Extensive geographic variation can arise in the rates of basic and engorged female mosquitoes were separated from unfed 428 EASTWOOD AND OTHERS

or male mosquitoes and sorted randomly into groups incu- Vertical transmission. The F 1 offspring obtained from field- bated at either 27°C or 30°C. Mosquitoes were held at the collected mosquitoes that had been infected with WNV in the relevant temperature in mesh-topped 0.47-liter cartons with above experiments were reared to adulthood and tested in access to 10% sucrose solution ad libitum until transmission 8 pools of up to 30 mosquitoes in each pool for WNV by using testing. Light cycling was 12 hour:12 hour (light:dark) at 85% the plaque assay described. relative humidity for the duration of each incubation period. Statistical analysis. Infection rates were calculated as the

An ovicup was included in each carton to harvest F 1 mosqui- percentage of mosquitoes tested in each group that were toes for vertical transmission testing. found to contain WNV in their body. An infection limited Because viral development changes over time and is lia- to the midgut was determined to have taken place when the ble to interact non-linearly with temperature, 36, 38, 44 we chose mosquito body sample showed a positive plaque assay result to examine the status of mosquito infection and transmis- but the corresponding leg sample showed a negative result. sion ability and consider the effect of (i)incubation time Dissemination rates were calculated as the percentage of all post-exposure and (ii)temperature variation. The Galápagos mosquitoes tested with virus-positive leg samples. Recovery ecosystem experiences seasonal variation with a hot, humid, of virus in the salivary secretion of mosquitoes indicated that rainy season with temperatures exceeding 30°C in January– the virus could be transmitted by bite. Thus, transmission rates April. In contrast, June–September are cooler months with were calculated as this proportion, of all mosquitoes, that temperatures less than 26°C. Variation in climate influences would be competent vectors for WNV. Confidence intervals host–pathogen interactions and, plausibly, disease dynamics, at the α = 0.05 level of significance were calculated for rates varying host-contact rates or the duration in which infections of vector competency or disseminated infection in accordance 45 develop. It is therefore reasonable to hypothesize that the with the Wald interval ( pˆ ± za / 2 pˆ (1- pˆ )/ n ). WNV vector competency of Galápagos Cx. quinquefasciatus Generalized linear regression (GLM) models based on esti- may vary over the course of a year. mation of binomial parameters were constructed to compare Transmission testing. Mosquito groups at each temperature infection, dissemination, and transmission rates across EIP (27°C or 30°C) were held for an extrinsic incubation period and temperature to determine if these parameters resulted (EIP) of 7, 11, 14 or 21 days post-feeding to determine the in any significant differences. The fitted GLM models were influence of time on the extent of infection and/or transmission used to fit predicted future rates according to varying temper- of WNV. A single group held at 27°C only was included 28 days ature and EIP. post-exposure; survival rates of Cx. quinquefasciatus at 30°C restricted testing beyond 21 days for the higher temperature. RESULTS At each time point, approximately 30 mosquitoes from each temperature group were immobilized by using triethylamine Groups of Galápagos Cx. quinquefasciatus F0 mosquitoes (Sigma, St. Louis, MO). To investigate WNV infection status, collected in Galápagos were tested for WNV vector compe- bodies of mosquitoes were examined for midgut infections. tency at two temperatures (27°C or 30°C) and various time Legs were used to check for disseminated infections spread- periods (7, 11, 14, 21, or 28 days). Each group showed some ing beyond the midgut as described. 46 Briefly, mosquito legs degree of midgut infection and further dissemination of WNV were first removed from bodies and separately triturated ( Table 1 ). Overall WNV infection rates of exposed mosqui- in a microfuge tube containing 1 mL of mosquito diluent toes averaged 35.5% across all time points from 7 to 28 days (phosphate-buffered saline, 20% fetal bovine serum [FBS], post-feeding. and antibiotics) and a ball-bearing. To check for transmission To test the hypothesis that the rates of infection, dissemina- ability, salivary secretions were obtained by using a modi- tion, and transmission increased as a function of temperature fied in vitro capillary transmission assay36, 47 in which the mos- and days post-feeding, a regression model was constructed. quito proboscis is inserted into a capillary tube containing a This model also generated predictive rates for Galápagos Cx. 1:1 mixture of FBS and 50% sucrose. This procedure invokes quinquefasciatus WNV vector competency on the basis of a reflex action by the live mosquito to secrete its saliva into influences found to be significant. Binomial data, i.e., a posi- the solution and after 30 minutes the contents of the capillary tive or negative result for the viral screening of each tissue are dispensed into 300 μL of mosquito diluent. All samples suspension, were weighted to eliminate any effects of size were stored at –80°C. After homogenization by using a grinder variation. Overdispersion of generalized linear models was mill at 24 cycles/seconds for 30 seconds and centrifugation at avoided by fitting a quasi-binomial model (logit function) 12,000 rpm, the resultant suspensions were screened for virus. where necessary. Virus assay. A plaque assay was used to screen mosquito Effect of incubation period. An increase in the rates of body, legs, and salivary sample for infectious virus by using six- infection, virus dissemination, and transmission were evident well Vero cell culture plates (Costar, Corning, NY) basically with increasing period post-feeding (Table 1). Longer EIPs, as described by Payne and others. 48 Briefly, cell monolayers which enabled WNV to replicate further within mosquitoes, were inoculated with 100 μL of each sample, then incubated at significantly increased detection for percentage infected (F = < 37°C in an atmosphere of 5% CO 2 for 1 hour to enable virus 8.01, degrees of freedom [df] = 7, P 0.05), percentage with to enter cells. An overlay of 10% FBS Eagles-Agar containing disseminated infections (F = 20.4, df = 7, P < 0.01), and the fungizone (amphotericin B) to limit contamination, was then percentage transmitting (F = 20.3, df = 7, P < 0.01). added. After an additional incubation for 48 hours at 37°C, Effect of temperature. Although there appeared to be a second overlay of 2% FBS containing 2% neutral red was increased WNV development at the higher of the two applied. Following a further 12 hours of incubation plates temperatures tested ( Figure 2 ), this effect was only found were examined for development of plaques to determine the to be significant for basic infection rates. As confirmed by presence of virus in each sample. the associated models, rates of disseminated infection and WNV VECTOR COMPETENCY OF CX. QUINQUEFASCIATUS IN GALÁPAGOS 429

Table 1 West Nile virus Vector competence of Culex quinquefasciatus mosquitoes of Galápagos * Dissemination, % of Transmission, % of Efficiency of dissemination, Efficiency of transmission, Temperature (°C) No. tested Days post-feeding Infection, % (95% CI) no. tested (95% CI) no. tested (95% CI) % of no. infected % of no. infected 27 57 7 21.1 (10–32) 5.3 (0–11) 0 25 0 27 62 11 21.0 (11–31) 8.1 (1–15) 3.2 (0–8) 38.5 15.4 27 31 14 38.7 (22–56) 32.3 (16–49) 22.6 (8–37) 83.3 58.3 27 32 21 50.0 (33–67) 50.0 (33–67) 34.4 (18–47) 100 68.8 27 27 28 59.3 (41–78) 55.6 (37–74) 44.4 (26–63) 93.8 75 30 54 7 38.9 (26–52) 9.3 (2–17) 0 23.8 0 30 55 11 25.5 (14–37) 12.7 (4–22) 5.5 (0–11) 50 21.4 30 31 14 54.8 (37–72) 45.2 (28–63) 22.6 (8–37) 82.4 41.2 30 20 21 50.0 (28–72) 40.0 (19–61) 20.0 (2–38) 80 40 * Values are percentages of mosquitoes in each group with tissues positive for West Nile virus or with virus in salivary secretion. CI = confidence interval. All groups were exposed to the WNV02-

1956 strain of West Nile virus at a median titer of 7.65 log10 plaque-forming units/mL of blood. subsequent transmission by Galápagos Cx. quinquefasciatus quitoes tested could transmit WNV, and significantly higher showed no significant relationship to temperature even when rates of transmission occurred over time (F = 20.3, df = 7, P < taking possible interaction effects with time into consideration. 0.01), 31.3% by day 21 (27°C), and 44% by day 28 (27°C). No Infection rates showed a significant relationship with both significant difference between temperatures was discernable. temperature (c2 = 9.6, P < 0.05) and time post-feeding (c2 = Predicted WNV transmission rates obtained by our model 14.1, P < 0.01), although no interaction between these two for Galápagos Cx. quinquefasciatus were 3.3% (day 7), 6.3% factors occurred. The fitted model (Proportion Infected = (day 11), 10.0% (day 14), 26.9% (day 21), and 54.9% (day 28). 0.08 * EIP + 0.16 * Temperature, AIC = 51.4, P < 0.05) based Lack of vertical transmission. No virus was detected in on incubation period and temperature produced the following any of the 8 pools (comprising a total of 200 mosquitoes) of likelihoods for midguts of Galápagos Cx. quinquefasciat to F 1 adult progeny tested to investigate vertical passage of WNV contain detectable WNV post-feeding: 21.1%/30.4% infected in Galápagos Cx. quinquefasciatus . (by day 7) at 27˚C/30˚C respectively, 26.9%/37.6% (day 11), Increased efficiency with time. If one considers those 31.9%/43.4% (day 14), 45.2%/57.4% (day 21), and 59.1% mosquitoes already showing detectable infections, the (day 28). corresponding rate of WNV dissemination beyond the midgut Dissemination of virus showed no relationship with tem- also increased significantly with incubation period (c2 = 9.6, perature. However, incubation time post-feeding significantly df = 7, P < 0.001) and showed up to 100% efficiency, Also, influenced the extent that WNV moves beyond the midgut consequent rates of transmission increased (c2 = 15.9, df = 7, epithelial cells of the mosquito (F = 20.4, df = 7 P < 0.01). The P < 0. 0001) and an efficiency up to 75% was observed ( Table 1 ). fitted model produced the following likelihoods for WNV Once the virus disseminated beyond the midgut of Galápagos dissemination rates in Galápagos Cx. quinquefasciatus : 9.4% Cx. quinquefasciatus, transmission was highly likely, i.e., up to (day 7), 15.5% (day 11) and 22.0% (day 14), 43.4% (day 21), 80% of mosquitoes with disseminated-infections could then and 67.6% (day 28). transmit WNV. No viral transmission was observed until day 11 at either temperature, and even then the rate of transmission was low: DISCUSSION 3.2% at 27°C, 5.5% at 30°C. However, by day 14, 22.6% of mos- We demonstrated WNV vector competency for a unique population of Cx. quinquefasciatus mosquitoes within South America. West Nile virus has yet to reach Galápagos, but with a presence in South America, this pathogen has the potential to reach these islands in the near future. 18 The research we report forms part of a larger risk framework evaluating the threat posed to the islands from this flavivirus. Knowledge of how Galápagos strains of mosquito would interact with WNV is an essential component of this framework. The concern underly- ing the study was that Cx. quinquefasciatus would be a highly competent and efficient vector for WNV, able to circulate the virus rapidly among rare fauna, and perpetuate harmful conse- quences of an introduction event to Galápagos. The results of our study do not alleviate concerns because they suggest that the strain of Cx. quinquefasciatus in Galápagos could have a role in WNV maintenance should the pathogen be introduced into the islands. This mosquito does show susceptibility to viral infection from feeding on an infected blood meal, and subsequently approximately 50% would be expected to able to transmit WNV to future hosts with an optimal timeframe of 28 days. F igure 2. Influence of temperature upon rates of West Nile virus Prior to 14 days post-exposure to WNV, Galápagos popula- infection, dissemination (of those tested), and transmission (of those tested) in Galápagos Culex quinquefasciatus . Dashed lines indicate tions of Cx. quinquefasciatus demonstrated low rates of WNV upper and lower 25% quartiles. infection and dissemination in comparison to North American 430 EASTWOOD AND OTHERS strains. Studies in Florida showed much higher infection and seasonal temperature fluctuations in Galápagos would alter dissemination rates at one-week post-exposure at compa- the epidemiology of an outbreak on the islands. One excep- rable or lower doses, even at lower temperatures (transmis- tion to that conclusion is the possible transfer of WNV via the sion rates were not mentioned).30 Similarly, although studies consumption of infected mosquitoes by vertebrates, a pathway in California (which did cite transmission rates) showed which has been shown to produce host infection. 43 Although variability in results on the basis of comparable viral titers, this mechanism for WNV maintenance is not well docu- mosquitoes there tended to have higher vector competency mented, regular vector ingestion during hotter periods when than those of Galápagos. 32, 34 In our own study, transmission we predict more vectors to be a) infected, and b) increased in ability in the first two weeks of infection was less than 10%, abundance (Eastwood, unpublished data), could have impli- which suggests a lower WNV risk for Galápagos fauna if other cations for virus epidemiology. It is hence important to under- more-competent mosquito vectors are not present. However, stand vector-pathogen-host-environment interactions.36 increasing rates of WNV infection, dissemination, and trans- Although we report infection and virus dissemination rates, mission in Galápagos Cx. quinquefasciatus were observed it is the transmission rate that is key to determining the extent from 14 days onwards (until the end of the study at day 28). to which a mosquito species is a competent vector. For exam- Given that mosquitoes have a limited lifespan, survival issues ple, Cx. quinquefasciatus from Florida is considered to be a become more relevant with longer incubation periods, i.e., if competent but only a moderately efficient vector: despite high a longer period of time is required for virus to develop in the infection rates, the virus often does not disseminate beyond mosquito body, there is reduced opportunity for vector-host the midgut (22% being the greatest rate). 33 However, when contact while the mosquito is in an infectious state. It becomes exposed to intrathoracic inoculation of WNV (mimicking increasingly likely that the mosquito will die before it reaches virus dissemination), 94% of the Florida population could the period of optimal transmission. Nevertheless, even at 30°C, transmit WNV.33 In the case of Galápagos Cx. quinquefascia- Cx. quinquefasciatus generally survive for longer than 28 days tus, we were more interested in what could occur in nature on (Eastwood G, unpublished data).49 Thus, virus transmission the islands, and although only a proportion of infected mosqui- would be expected to occur and maintain pathogen cycling in toes showed dissemination beyond the midgut with a smaller Galápagos, assuming susceptible vertebrate hosts are present proportion further able to transmit virus, the vector efficiency that support sufficiently high virus titers to infect mosquitoes. greatly increases over time ( Table 1 ). Our results indicate that The dosage of virus used must be taken into account for vec- the Galápagos strain of Cx. quinquefasciatus, although less tor competency studies and should represent the range of competent than strains from the United States, is a competent titers realistically found in nature. A WNV titer of 7.3–8.3 log10 and efficient vector for WNV that is capable of transmission PFU/mL of blood was used in blood meal preparations and once viral infection extends beyond the midgut. produced infection in 39–55% of Galápagos Cx. quinquefas- It should be noted that even when a mosquito is capable ciatus at day 14. This titer is consistent with those found in of transmitting virus, an infection may still not develop in the nature and those used in other studies. However, studies else- vertebrate host on which the mosquito feeds, depending on where have used lower doses to yield higher rates of WNV the viral dose transmitted and the susceptibility of that host. infection in populations of Cx. quinquefasciatus . 39, 40 Ability to induce or develop infection may be restricted until Contrary to evidence of vertical transmission in North viral titers reach a certain threshold. To clarify the WNV titer American Cx. quinquefasciatus , 42, 50 Galápagos populations that could be inoculated by Galápagos Cx. quinquefasciatus , a showed no transmission to progeny, which suggests that ver- subset of our samples were tested beyond the basic assay for tical transmission may have a limited role in maintenance presence of virus, and the range deduced was comparable with 53 of WNV in Galápagos. However, the number of progeny we those of other studies (1–5 log10 PFU/mL). Therefore, a vari- tested was limited and only progeny from the first oviposition able amount of WNV could be inoculated by the vector while cycle, 14 days after feeding on the infectious blood meal, could they feed on live hosts. be tested. Because the mosquitoes in our study did not live In addition to the presence of susceptible hosts, establish- long enough to complete the second and third ovarian cycles, ment of WNV in Galápagos would require hosts that also it is difficult to draw firm conclusions. amplify WNV. There may be reduced risk posed to systems such South America has a unique climate, geology, avifauna, and as Galápagos when WNV amplification cycles are restricted genetically distinct mosquitoes.21, 51 We simulated seasonal cli- to involving only species circulating a high dose of virus. For mate variation by testing WNV vector competency at two example, West Nile virus titers less than 4 log10 PFU/mL rarely temperatures, and hypothesized that temperature would influ- appear to induce infection in Cx. quinquefasciatus (although ence viral transmission in Galápagos Cx. quinquefasciatus . further experimentation with Galápagos Cx. quinquefascia- The impact that changes in global climate would have on dis- tus to establish a 50% infectious dose is recommended). Bird ease dynamics is a hotly debated topic, with some predicting species such as chickens or psittacines rarely produce viremias increased disease emergence and serious implications for pub- exceeding 3 log 10 PFU/mL. Thus, some species encountered in 38,52 lic health control. We demonstrated an environment with Galápagos, for example Tyto alba (peak viremia 3.6 log10 PFU/ higher temperatures to influence mosquito infection rates. mL when tested in the United States), would have a limited This suggests that if WNV emerges in Galapagos during the role in WNV amplification by being unlikely to develop titers hotter months of rainy season, more mosquitoes are likely to high enough to infect mosquitoes.54 However, passerine birds become infected. However, there was no significant indication have viremic profiles with the highest magnitude of titers; cer- 43 in our study for transmission being influenced by temperature tain species peak at 11 log10 PFU/mL. (despite adequately sized samples). Therefore, despite studies A study to determine the host susceptibility and likely mor- using other Culex species demonstrate even small temperature bidity of Galápagos native vertebrates is required. Galápagos gradients to influence vector competency,36 it is unlikely that has an abundance of passerines that could be important for WNV VECTOR COMPETENCY OF CX. QUINQUEFASCIATUS IN GALÁPAGOS 431 the epidemiology of WNV, including 13 unique species of finch penultimately from continental South America (currently all famed by Charles Darwin. Finch species in United States show flights pass through Guayaquil or Quito, Ecuador), and the high susceptibility to WNV infection, high mortality rates, and risk assessment of WNV reaching Galápagos performed by 18 reservoir competence with viremias greater than 5 log10 PFU/ Kilpatrick and others was based on an assumption of WNV mL sustained for 6 days.43, 55 The susceptibility of Galápagos being established in mosquitoes at the ports of origin for air- finches and other key wildlife species, and the likelihood they craft and vessels traveling to the islands. Therefore, determin- would support WNV viremias high enough to infect a feeding the status of WNV activity on mainland Ecuador is a key ing mosquito, needs to be determined. Although such testing requirement for determining the risk of an introduction event is notably politically sensitive, it would ascertain the vulner- into Galápagos itself. ability and potential role of Galápagos fauna to understand We also recommend that current measures to minimize the the threat presented by WNV. Should Galápagos wildlife incursion of live mosquitoes from the mainland (e.g., aircraft prove susceptible or reach viremias high enough to enable the disinfection) are rigorously enforced and additional measures virus to become established, the island fauna face a serious (e.g., disinfection of sea vessels traveling to Galápagos) are threat. There is particular concern for endemic Galápagos spe- introduced. With increasing human populations and visitor cies, which given their evolution in isolation from flavivirus numbers, management authorities overseeing the Galápagos are likely to have limited resistance to WNV. The generally Islands are already faced with the challenge of conserv- small population sizes exacerbate vulnerability from stochas- ing the unique range of wildlife that in addition to invalu- tic events such as disease outbreaks. able natural history value, provide considerable economic Our finding that Galápagos Cx. quinquefasciatus has the benefit, through ecotourism, to Ecuador. West Nile virus has ability to infect its hosts with WNV prompts the research shown unprecedented activity on arriving in the Americas question: what exactly is the feeding behavior of this species in and placed substantial pressure on resources in the United Galápagos? Although it has been suggested that Cx. quinque- States, also identifying flaws in the existing public health sys- fasciatus can feed on mammals and birds, the relative propor- tem (e.g., recording issues).60 In addressing the WNV threat tion of preferred host order varies greatly among studies and to Galápagos ecosystems, it is advised that identification of vertebrate availability.28, 31, 56, 57 Knowledge of local diet prefer- the principal risks from this pathogen is fully explored. This ences specific to Galápagos is critical to determine which ver- includes greater knowledge of the ecological characteristics tebrate species would most likely be affected. of Galápagos mosquitoes, how they interact with both native There are other potential vectors of WNV in addition to Cx. species, and the increasing influx of invasive species. quinquefasciatus in Galápagos. In particular, given the prolific presence of a Galápagos strain of Ae. taeniorhynchus , determin- Received December 31, 2010. Accepted for publication May 9, 2011. ing the WNV vector competency of this mosquito is necessary Acknowledgments: We thank Marilyn Cruz, Pamela Martinez, to understand the potential dynamics of WNV in Galápagos. Alberto Velez, and Macarena Piaz for support at the Galápagos field Aedes taeniorhynchus is far more widely distributed through- site; Yongjing Jia, Pam Chin, Joe Maffei, Mary Franke, and Alex Ciota out the archipelago than Cx. quinquefasciatus (Eastwood G, for laboratory support in the United States; and Virna Cedeño for sci- unpublished data), has a broad host preference, and is another entific and logistical support in Ecuador. We also thank the Galápagos 21 National Park for cooperation and support of the overall project potential WNV vector. Aedes taeniorhynchus has been found (PNG09-21) and for granting access for sample collection. in WNV-positive surveillance pools in the United States since Financial support: This study was supported by a Natural Environ- 2002, which reflects the southerly spread of WNV to the hot- mental Research Council Doctoral Training Grant to the Faculty of ter areas where this and other species including Cx. quinque- Biological Sciences, University of Leeds, with additional support from fasciatus are found.58 A study using Ae. taeniorhynchus from Darwin Initiative grant EIDPO15. the United States found that although partially refractory to Authors’ addresses: Gillian Eastwood, Institute of Zoology, Zoological WNV infection (infection rates of only 12% occurred even Society of London, London, United Kingdom and University of Leeds, Leeds, United Kingdom, E-mail: [email protected] . Laura when exposed to titers exceeding 7 log10 PFU/mL), mosquitoes when inoculated showed transmission rates of 93%.59 D. Kramer, New York State Department of Health, Slingerlands, NY, E-mail: [email protected] . Simon J. Goodman, Institute Because there is a potential for WNV to arrive in Galápagos of Integrative and Comparative Biology, University of Leeds, Leeds, 18 in the near future without appropriate mitigation measures, United Kingdom, E-mail: [email protected] . Andrew A. we recommend that determination of the WNV vector compe- Cunningham, Wildlife Epidemiology, Institute of Zoology, Zoological tency of Galápagos Ae. taeniorhynchus is a priority to inform Society of London, London, United Kingdom, E-mail: a.cunningham@ control measures to alleviate the future impact of WNV in the ioz.ac.uk . Galápagos Islands. 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