ARTICLE. Department of Entomology, University of Massachusetts, Amherst, MA 01003

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1 ARTICLE Effect of Eastern Equine Encephalomyelitis Virus on the Survival of Aedes albopictus, Anopheles quadrimaculatus, and Coquillettidia perturbans (Diptera: Culicidae) ABELARDO C. MONCAYO, 1 JOHN D. EDMAN, 2 AND MICHAEL J. TURELL 3 Department of Entomology, University of Massachusetts, Amherst, MA J. Med. Entomol. 37(5): 701Ð706 (2000) ABSTRACT The effect of eastern equine encephalomyelitis (EEE) virus on the survivorship of Aedes albopictus (Skuse), Anopheles quadrimaculatus Say, and Coquillettidia perturbans (Walker) was determined experimentally. Female mosquitoes were allowed to feed on EEE viremic chicks, and survival rates were compared for infected and uninfected mosquitoes. Additionally, the survival of female Cq. perturbans and An. quadrimaculatus intrathoracically (IT) inoculated with EEE was compared with controls receiving diluent inoculations. Infection with EEE signiþcantly reduced survival in Cq. perturbans compared with uninfected individuals in per os infection experiments. IT infections of Cq. perturbans did not reduce survival when compared with diluent inoculated groups. In contrast, infection with EEE did not affect the survival of Ae. albopictus after per os infection or An. quadrimaculatus after either IT or per os infections. KEY WORDS Aedes albopictus, Anopheles quadrimaculatus, Coquillettidia perturbans, eastern equine encephalomyelitis virus, survivorship ALTHOUGH IT WAS believed for many years that infection with an arbovirus had no deleterious effects upon its arthropod host (LaMotte 1960, DeFoliart et al. 1987, Hardy 1988, Morris 1988), recent studies indicated that such viral infections may not be benign. Viral infections in mosquitoes can reduce fecundity (Turell et al. 1985), the ability to obtain a blood meal (Grimstad et al. 1980, Turell et al. 1985), and even survival (Faran et al. 1987, Turell 1992, Scott and Lorenz 1998) when compared with uninfected individuals. In North America, eastern equine encephalomyelitis (EEE) virus is maintained in nature in an enzootic cycle involving the transmission of virus among passarine birds by the ornithophagic mosquito, Culiseta melanura (Coquillett). Occasionally EEE transmission spills over to humans and horses by generalistfeeding mosquitoes that serve as bridge or epizootic vectors between avian and mammalian hosts. In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. The facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. 1 Current address: Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555Ð Current address: Center for Vector-Borne Disease Research, School of Veterinary Medicine, University of California, Davis, CA Virology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702Ð It has been shown that EEE causes pathology and affects the survival of Culiseta melanura (Weaver et al. 1988, Scott and Lorenz 1998). The effect that an arbovirus has on the survival of its mosquito host is important in determining vectorial capacity. The inßuence of EEE on the mortality of bridge or epizootic vectors of EEE has not been examined previously. In eastern Massachusetts, suspected epizootic vectors of EEE include: Culex salinarius (Coquillett), Aedes canadensis (Theobald), Coquillettidia perturbans (Walker), Anopheles quadrimaculatus Say, Aedes vexans (Meigen), and Anopheles punctipennis (Say) (Vaidyanathan et al. 1997, Moncayo and Edman 1999). We evaluated the effect that infection with EEE has on the survivorship of Cq. perturbans, An. quadrimaculatus, and Aedes albopictus (Skuse), the Asian tiger mosquito. Ae. albopictus was included because it is well established in the southeastern United States and it is a competent laboratory vector of EEE (Turell et al. 1994). Materials and Methods Mosquitoes, Virus, and Viral Assays. Adult female Cq. perturbans were captured with light traps (American Biophysics Corporation, East Greenwich, RI) supplemented with CO 2 operated at a cattail swamp in the Quabbin Reservoir in Shutesberry, MA, in early July Emerging populations of Cq. perturbans were monitored via the light traps mentioned above with CO 2 at this site beginning in early June. Mosquitoes were captured 1 wk before these experiments. Because Cq. perturbans populations began to emerge

2 702 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 37, no. 5 Table 1. Dissemination and infection rates for per os-exposed Cq. perturbans, An. quadrimaculatus, and Ae. albopictus Species Host viremia (n) a Day 7 b % diss. (n) Final day c % diss. (n) Final day % infected (n) Cq. perturbans 8.3 (27) 19 (5) 30 (8) 44 (12) 9.3 (24) 42 (10) 50 (12) 71 (17) Combined 29 (15) 39 (20) 57 (29) An. quadrimaculatus 6.0 (39) 5 (2) 13 (5) 44 (17) 8.6 (37) 27 (10) 35 (13) 81 (30) 9.2 (28) 28 (8) 43 (12) 89 (25) Combined d 28 (18) 38 (25) 85 (55) Ae. albopictus 8.9 (43) 58 (25) 93 (40) 93 (40) 9.1 (15) 87 (13) 100 (15) 100 (15) Combined 66 (38) 95 (55) 95 (55) a Log 10 PFU/ml of blood during mosquito feeding (number on day 7). b Day 0 is 7 d after infectious blood meal. c Final day is day of death or end of experiment. d Combined rates for experiments with viremias of 8.0 PFU/ml of blood during mosquito feeding. 2 wk before capture, we estimated our experimental population to be 1Ð3 wk of age. Eggs of An. quadrimaculatus were provided by the USDAÐARS Laboratories in Gainesville, FL. This An. quadrimaculatus colony originated from Orlando, FL, in the 1950s and most likely belongs to sibling species A. These eggs were hatched at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) insectary and raised to adults. Inseminated adult females (4Ð8 d old) were used for survivorship experiments. A long-standing colony Ae. albopictus (OAHU strain) adults were provided by the Virology Division at USAMRIID. Inseminated 4- to 8-d-old adult females were used for survivorship experiments. Immature and adult mosquitoes were maintained at 26 C with a photoperiod of 16:8 (L:D) h. Larvae were fed ground Þsh food. Adults were provided apple slices and water. Humidity levels were increased by placing moist gauze pads on top of adult mosquito cages. An EEE isolate (90Ð122) made from a pool of Cs. melanura collected in Halifax, MA, in 1990 was used after one passage in chick embryo tissue culture. Mosquito bodies and legs were triturated separately in 1 ml mosquito diluent (10% heat-inactivated fetal bovine serum in Medium 199 with EarleÕs salts, antibiotics, and sodium bicarbonate) and stored at 70 C until assayed on Vero cell monolayers as described by Gargan et al. (1983), except that cells were stained at 2 d rather 4 d after the original overlay. Survival Experiments with Mosquitoes Intrathoracically Inoculated with EEE. EEE was inoculated intrathoracically (IT) into mosquitoes to observe the effect of bypassing midgut infection and escape barriers on survivorship and to increase the sample size of mosquitoes with a disseminated infection. In two experiments with Cq. perturbans and one with An. quadrimaculatus, females were inoculated with 0.3 l of either diluent or a suspension containing 10 3 PFU ( PFU/ml) of EEE in diluent. All three experiments were conducted blind so that the investigator performing the inoculations was unaware of whether the inoculum contained virus or diluent. Each cohort was placed in a 3.8-liter cardboard container with netting on top. If mortality occurred within 24 h, it was considered to be caused by inoculation trauma. Cages were observed for mortality twice daily. Survival Experiments with Mosquitoes Orally Exposed to EEE. Adult female mosquitoes were allowed to blood-feed on anesthetized chicks (2 or 3 d old) 24 h after they had been inoculated subcutaneously with 10 5 plaque-forming units (PFU) of EEE in 0.2 ml of mosquito diluent. Serial 10-fold dilutions of chick blood at the time of mosquito feedings were assayed for virus on Vero cell monolayers to determine feeding dose. A control groupñmosquitoes fed on an uninfected chick 2 or 3 d oldñwas included in each experiment. Engorged mosquitoes were placed in a 3.8- liter cardboard container maintained at 26 C and observed daily for mortality. Mosquitoes were coldanesthetized 7 d after the infectious blood meal and one mesothoracic leg was removed from each female, triturated in 1 ml of mosquito diluent and stored at 70 C for viral assay. Each mosquito then was placed in a 0.5-liter cardboard container, fed 5% corn syrup, and observed twice daily for mortality until the experiment was terminated (14 d following the infectious blood meal for Cq. perturbans and An. quadrimaculatus and 20 d following the infectious blood meal for Ae. albopictus). Dead mosquitoes were removed from the containers and processed for virus assay as described above. Mosquito bodies and the remaining legs were triturated separately at the end of each experiment and stored at 70 C. We considered a mosquito that had virus recovered from its body, but not its legs, to have a nondisseminated infection limited to its midgut. In contrast, if virus was recovered from both body and leg suspensions, we considered that the mosquito had a disseminated infection (Turell et al. 1984). We deþned the dissemination rate as the percentage of all mosquitoes tested that had virus detected in their legs (Table 1). Because we were interested in the effect of a disseminated infection on survivorship, we compared infected mosquitoes exhibiting disseminated infections by day 7 after the

3 September 2000 MONCAYO ET AL.: SURVIVAL OF MOSQUITOES INFECTED WITH EEE 703 Fig. 1. Survival curve for Cq. perturbans IT-inoculated with EEE or mosquito diluent. blood meal with those without a disseminated infection and with uninfected controls. Survival rates for Cq. perturbans and An. quadrimaculatus were calculated based on the number of individuals surviving 14 d after the blood meal. We chose to end our experiments at 14 d for Cq. perturbans and An. quadrimaculatus because most females had died by this time and the probability of refeeding has been found to become signiþcantly reduced at about this time in another vector species (Scott and Weaver 1989). Survival rates for Ae. albopictus were calculated up to 20 d following the infectious blood meal. The later time point was extended in Ae. albopictus studies, because no mortality was observed at 14 d following the blood meal. Statistical Analysis. Comparison between survival rates was performed using Pearson chi-square and the Fisher exact test at the 0.05 signiþcance level. Kaplan- Meier survival curves were compared by a log-rank test (Matthews and Farewell 1996). Results Infection and Dissemination Rates in Mosquitoes Orally Exposed to EEE. Viremias of chicks during the infectious blood meals were and PFU/ml of blood for Cq. perturbans; ,10 8.6, for An. quadrimaculatus; and and for Ae. albopictus. All three species were susceptible to infection with EEE, with infection rates of 57, 85, and 95% for Cq. perturbans, An. quadrimaculatus and Ae. albopictus, respectively, that ingested 10 8 PFU/ml of EEE (Table 1). Dissemination rates in these species were 39, 38, and 95%, respectively. Survival of Mosquitoes Inoculated with EEE. Survivorship curves up to day 14 were similar (P 0.25) for virus- (n 94) and diluent- (n 93) inoculated Cq. perturbans when replicates were combined (Fig. 1). Likewise survivorship was similar (P 0.25) for An. quadrimaculatus inoculated with virus (n 53) or diluent (n 46) (Fig 2.). Fig. 2. Survival curve for An. quadrimaculatus IT-inoculated with EEE or mosquito diluent.

4 704 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 37, no. 5 Fig. 3. Survival curve of infected (disseminated versus nondisseminated) and uninfected (exposed to viremic chick versus control naõ ve chick) Cq. perturbans mosquitoes in per os experiments. Comparison of Survival Rates of Per Os Experiments. Coquillettidia perturbans (Fig. 3) with disseminated infections had a higher mortality rate than uninfected cohorts feeding on the same viremic chick and control females feeding on a naõ ve chick (P 0.05 and P 0.025, respectively). There were no signiþcant differences between overall survival curves of mosquitoes having disseminated infections versus nondisseminated infections (P 0.25), although those mosquitoes with a disseminated infection had signiþcantly poorer survival between 12 and 14 d postinfectious blood meal than did those without a disseminated infection (P 0.007). In contrast, no signiþcant effect of EEE infection was observed on the survival of An. quadrimaculatus (Fig. 4), with the exception of the survivorship of cohorts with disseminated infections versus the control group that fed on a naõ ve chick (P 0.025). All Ae. albopictus in both the infected and uninfected cohorts survived throughout the entire 20-d experimental period. Discussion Mosquito daily survival relative to the extrinsic incubation period is one of the most important factors in determining vectorial capacity (Smith 1987, Freier 1989). This difference determines the length of time that a vector would be able to transmit a virus and the Fig. 4. Survival curve of infected (disseminated versus nondisseminated) and uninfected (exposed to viremic chick versus control naõ ve chick) An. quadrimaculatus mosquitoes in per os experiments.

5 September 2000 MONCAYO ET AL.: SURVIVAL OF MOSQUITOES INFECTED WITH EEE 705 relative size of the infective vector population. This, in turn, determines the rate of virus replication in nature. Dissemination of the Massachusetts EEE strain (90Ð 122) from the midgut signiþcantly reduced the number of Cq. perturbans that survived 7Ð14 d after an infective blood meal compared with uninfected controls. This demonstrated major differences in survival during the time when transmission may occur after an infectious blood meal. Of the Cq. perturbans that took an infectious blood meal, 29% developed disseminated infections by 7 d. Only 4% of Cq. perturbans exposed to infectious blood meals transmitted in recent studies (Vaidyanathan et al. 1997). Although able to transmit EEE, our survivorship data indicated that the transmission potential may be reduced in Cq. perturbans developing disseminated infections 7 d after an infectious blood meal. Disseminated infection did not reduce the survivorship of An. quadrimaculatus in our per os experiments. The intrathoracic inoculation experiments agreed with this lack of signiþcant difference between disseminated and nondisseminated groups. EEE-induced mortality could not be demonstrated in this species by our methods. Infection by EEE had no observable effect on the survivorship of Ae. albopictus with disseminated versus nondisseminated infections. Although 95% of Ae. albopictus developed a disseminated infection after a viremic(blood meal, neither they nor the negative controls died within 20 d. Based on this Þnding, along with the opportunistic feeding behavior of Ae. albopictus (Savage et al. 1993), EEE isolation from this mosquito in the Þeld (Mitchell et al. 1992), and vector competence for EEE (Scott et al. 1990, Turell et al. 1994), it would appear that this newly introduced species could be important as an epidemic vector of EEE at any endemic focus in the eastern United States, where it becomes well established. Infection and dissemination rates observed in this study for Cq. perturbans, An. quadrimaculatus, and Ae. albopictus were consistent with those reported in the literature (Scott et al. 1990, Vaidyanathan et al. 1997). Weaver et al. (1988) described the following pathological changes in Cs. melanura after EEE infection: sloughing of infected gut epithelial cells, degeneration of cells within the gut epithelium, loss of brush border, and disruption of the basal lamina. This pathology can serve to modulate virus infection in the mosquito by the sloughing of heavily infected luminal cells, which could result in the reduction of viral load. Midgut pathology also may facilitate the dissemination of virus to the salivary glands as a result of basal lamina disruption. EEE infection reduces survival in Cs. melanura (Scott and Lorenz 1998). Similarly, we found that EEE reduced the survival of Cq. perturbans with disseminated infections compared with uninfected controls in per os experiments. Reduction in survivorship following EEE infection may be the result of pathological events occurring when EEE exits the midgut epithelial cells. Intrathoracic inoculation experiments supported our per os experiment results. The similarity in the survival proþle of IT infected and uninfected groups indicate the importance of gut pathology on survivorship. Romoser et al. (1992) found that the midgut of Culex pipiens L. was much more likely to become infected with Rift Valley fever virus after per os rather than IT infection. If a similar phenomenon exists with Cq. perturbans and EEE, the reduced survivorship we observed after per os rather than IT infections suggests that infection of the midgut and the subsequent pathological changes in midgut cells may inßuence survival. Midgut pathology could be linked to the ability to digest sugar and blood meals and hence the ability to acquire energy for survival. We found signiþcant differences between the An. quadrimaculatus control group that fed on an uninfected host and females with a disseminated infection. However, because we were not able to duplicate this Þnding between the uninfected group that had been exposed to a viremic host and the disseminated infection group (P 0.10), the difference between uninfected and disseminated groups was not proven. Increased mortality of mosquito species that are susceptible to arbovirus infections occurs with some, but not all, virusðvector systems. We found that Cq. perturbans, a likely epizootic or bridge vector in the transmission of EEE, may experience a reduction in survivorship between 7Ð14 d postinfection if virus disseminates after ingesting an infectious blood meal. Cq. perturbans has been known to engage in multiple feedings (Magnarelli 1977), and it is important to determine how soon after an infectious blood meal that an infective Cq. perturbans is likely to refeed. If there is a high probability of refeeding, and therefore transmitting EEE before the onset of virus-induced mortality, then the overall vectorial capacity may not be signiþcantly affected. However, the survivorship, and therefore vectorial capacity, of the older infectious population component may be affected by EEE. Studies to answer this question are needed to fully understand the epidemiological signiþcance of reduced survivorship during these time periods. Observations of reduced survivorship by a vector caused by arbovirus exposure should be incorporated in any model describing the vectorial capacity of a given arthropod for its viral pathogen. Acknowledgments We thank Daniel L. Kline and John Jackson (USDAÐARS, Gainesville, FL) for providing the An. quadrimaculatus Orlando used in this study. Special thanks go to Dave Dohm (USAMRIID) for his excellent technical assistance. This research was funded by the Massachusetts Department of Public Health and Hatch support from the Massachusetts Agricultural Experiment Station. References Cited Defoliart, G. R., P. R. Grimstad, and D. M. Watts Advances in mosquito-borne arbovirus-vector research. Annu. Rev. Entomol. 32: 479Ð505. Faran, M. E., M. J. Turell, W. S. Romoser, R. G. Routier, P. H. Gibbs, T. L. Cannon, and C. L. Bailey Reduced

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