INTRODUCTION

The Cattail Mosquito Coquillettidia perturbans (Walker), its conspecifics and members of the related genus Mansonia have atypical immature stages within the family Culicidae. The larvae and pupae of these genera attach to roots and stems of aquatic macrophytes to obtain oxygen. Only the pupae come to the water's surface at the onset of eclosion. In New Jersey Cq. perturbans adults typically begin emerging in June and continue to do so until early September. Host-seeking adults are annoying, persistent human biters and in some areas of the country are the most common pest mosquito species (McNeel 1932; Lounibos & Escher 1983). Females oviposit throughout the summer in permanent marshes characterized by a substratum of soft flocculant organic material in a littoral zone that supports emergent aquatic vegetation. The newly hatched larvae attach to the roots of this vegetation and overwinter there, with pupation taking place the following spring. The larvae are mobile, moving vertically in the water column as well as horizontally from plant to plant as conditions change. Coquillettidia perturbans is considered to be a univoltine species in New Jersey, yet light trap records for adult host-seeking females usually show several distinct population peaks during the summer season. This phenomenon is typical of a multivoltine species. Light trap records for the southern portion of the state also suggest that equine cases of Eastern Equine Encephalitis virus (EEEV) may be associated with Cq. perturbans that emerge during late summer or early fall (Crans & Schulze 1986; Crans et al 1986). The first part of this study tested the hypothesis that the population peaks observed with this species are a result of one generation of several age cohorts overwintering and emerging temporally through the summer season.

Eastern Equine Encephalitis virus epizootics occur in New Jersey periodically and can result in numerous equine fatalities and, to a lesser extent, human fatalities. The annual EEEV cycle is not completely understood in New Jersey. Typically, the virus shows up in both Cs. melanura mosquitoes and birds in late spring and amplifies as the season progresses. How the virus overwinters is not known, and several hypotheses exist. The second part of this study tested one of those hypotheses, which suggests that the virus is transovarially transmitted from adult mosquitoes to their overwintering larvae resulting in reintroduction of virus into bird and insect populations the following spring.

MATERIAL AND METHODS

Larval collection and sorting: Field collections took place at three sites in New Jersey: Colliers Mills (Ocean County), Waterford (Camden County) and Estell Manor (Atlantic County). The Ocean County site is located in the Colliers Mills Wildlife Management Area. This site is a marsh at the mouth of a stream that creates one of several connecting lakes in the management area. The Camden County site is located in a large marsh bisected by two roads and is the result of an active beaver colony. The Atlantic County site, located within Estell Manor Park, is a large marsh crossed by several dirt access roads. Each site was sampled for larvae biweekly during quiescence as determined by water temperature. Water temperatures were recorded from the root mat of the plants sampled on each sampling date. Larvae were sampled from the roots of emergent sedges (Juncus spp.) using the modified bilge pump method (Walker & Crans 1986). Initially, a prospective plant is selected from which one suction of the bilge pump is taken. This sample is released into a floating soil sieve (.833 mm mesh size) and rinsed with clear water to assess larval presence. If larvae were present in the initial sample then five suctions of the bilge pump from each of two plants at each site were transported to the laboratory in covered containers for larval sorting.

Sorting Cq. perturbans larvae is more time consuming than for most species of mosquitoes for several reasons. First, the larvae must be separated from the flocculant detritus in the sample; second, in winter the larvae are cold, less mobile and at times feign death; and third, Cq. perturbans larvae have a tendency not to come to the surface of the sample to respire as most mosquitoes must. Several methods for sorting larvae have been proposed (Clark et al. 1985; Morris et al. 1985; Batzer 1993). Although less time consuming, these methods do miss numerous larvae for reasons stated previously. Typically, white porcelain pans are used to sort and count mosquito larvae. However, because Cq. perturbans larvae are nearly white with a greenish thorax, the porcelain pans used in sorting for this study were painted black to show the larvae more clearly. A small portion of sample material was poured into a black porcelain pan, to which clean tap water was added to dilute the sample. With this sorting method, the detritus and flocculant material in the sample is spread out making the larvae more visible. This sampling method, albeit time consuming, is the most accurate in determining the make up of the larval cohort in the sample. Larval instars were sorted according to head capsule width using the method described by Nemjo and Slaff (1984). Each larval instar was counted, recorded, and divided into pools of 50 individuals. These pools were first rinsed with deionized water then with both ultra pure and DEPC treated water. All pools were frozen to -70o C in micro-centrifuge tubes for EEEV isolation using the reverse transcriptase polymerase chain reaction (RT-PCR) technique (Monroy et al. 1996).

Adult collection and sorting: A variety of techniques are used to assess adult mosquito populations. Historically, New Jersey light traps and the Centers for Disease Control (CDC) light traps have been two of the most effective tools used to monitor host-seeking female mosquitoes. The population dynamics of host-seeking Cq. perturbans in this study were monitored at weekly intervals using ABC (American Biophysics Corp., Jamestown RI) traps supplemented with CO2. The host-seeking Cq. perturbans caught in the ABC traps were placed on dry ice on site and returned to the laboratory where they were dissected for parity by the methods of Detinova (1962). Adult collections were compared to the densities of the sampled larval instars that overwintered to determine if a correlation existed between densities of overwintering larvae and the population dynamics of adult Cq. perturbans during summer emergence.

RESULTS AND DISCUSSION

Colliers Mills Site (Ocean Co. NJ). Winter larval sampling showed the overwintering population to be comprised of 11% 4th instars, 86% 3rd instars and 3% 2nd instars. Trap collections for host-seeking adult females began in mid-June with the initial collection showing a parity rate of <30%. This is consistent with a newly emerging cohort. Trap collections increased steadily and peaked on July 26th followed by a steady decline to zero by mid-Sept. Following the first collection in mid-June, the remaining collections showed a parity rate of >60%. This may indicate that there was no new discreet emergence of adults from the site or females were long-lived which diluted any indication of a new discreet emergence. The single prolonged emergence peak of adults at this site in mid-summer correlates with the >80% overwintering 3rd instar larval cohort and is consistent with the prediction of the hypothesis.

Estelle Manor (Atlantic Co. NJ). The winter larval sampling at this site was very different than that of Collier's Mills and comprised 42% 4th instars and 58% 3rd instars. Trap collections for host-seeking adult females also began in mid-June with the initial collection showing a parity rate of >50%. The start date of trapping at this site may have missed an earlier emergence of nulliparous host-seeking females and could account for the high percentage of parous individuals on the first trap date. Trap collections rose steadily and peaked on July 1st with a subsequent steady decline to nearly zero on Aug. 5th. Trap collections rose steadily again to another albeit much smaller peak on Aug. 26th and declined to zero on Sept. 23rd. The initial peak on July 1st showed a parity rate of <40% and increased until July 29th. The Aug. 5th trap catch consisted of only nulliparous females, but parity did increase to >50% until Sept. 16th. The two separate emergence peaks of adults at this site correlates with the 42% overwintering 4th instar larval cohort and the 58% overwintering 3rd instar larval cohort and is consistent with the prediction of the hypothesis. However, prior to the second peak, the beaver dam at this site was destroyed resulting in a drop in water levels. Larvae are mobile and are able to move vertically if water levels decrease, however, pupae are firmly attached to roots and are unable to move once their breathing trumpets have been inserted into plant tissues. The drop in water levels at this site is likely to have had an impact on the pupae thereby reducing the number of ecloding adults and may explain the much smaller peak on Aug 26th.

Waterford (Camden Co. NJ). The larval sampling at this site was similar to that found at Estelle Manor and comprised 51% 4th instars, 48% 3rd instars and 1% 2nd instars. Trap collections for host-seeking adult females also began in mid-June with the initial collection showing a parity rate of >50%. Trap collections increased steadily and peaked in mid-July. As the collections increased the parity rate decreased until the parity rate was <15% on July 22nd. Following this peak, trap catches began to decrease until Aug. 5th. There was a second dramatic increase in the trap collection on Aug. 12th that showed a parity rate of <30%. The two separate emergence peaks of adults at this site correlates with the 51% overwintering 4th instar larval cohort and the 48% overwintering 3rd instar larval cohort and is consistent with the prediction of the hypothesis. This site as with the Estelle Manor site experienced a drop in water levels due to the destruction of a beaver dam. This alteration in the habitat may explain the low adult trap catches experienced at this site.

The PCR technique for isolating and amplifying EEEV was performed on 60 pools total of 50 larvae each from all three sites. There were no positive virus isolations of EEEV from any of the three sites.

CONCLUSIONS

The preliminary results suggest a correlation between overwintering larval populations of Cq. perturbans and discreet adult population peaks through the summer season. However, low trap catches and the disturbance of larval habitat during the initial season indicate that further work is needed for this portion of the study.

The lack of a single positive virus isolation for EEEV via the PCR technique indicates that EEEV is not transmitted transovarially in Cq. perturbans. This work is a result of one season's collection and may not reflect the aforementioned finding in years of high virus activity.

REFERENCES CITED

Batzer, P.B. 1993. Technique for surveying larval populations of Coquillettidia perturbans. J. Am. Mosq. Control Assoc. 9(3):349-351.

Clark, G.G., W.J. Crans and C.L. Crabbs. 1985. Absence of eastern equine encephalitis (EEE) virus in immature Coquillettidia perturbans associated with equine cases of EEE. J. Am. Mosq. Control Assoc. 1(4):540-542.

Crans, W.J. and T.L. Schulze. 1986. Evidence incriminating Coquillettidia perturbans as an epizootic vector of eastern equine encephalitis. I. Isolation of EEE virus from Cq. perturbans during an epizootic among horses in New Jersey. Bull. Soc. Vector Ecol. 11:178-184.

Crans, W.J., L.J. McCuiston and T.L. Schulze. 1986. Evidence incriminating Coquillettidia perturbans as an epizootic vector of eastern equine encephalitis. II. Ecological investigations following an inland epizootic in New Jersey. Bull. Soc. Vector Ecol. 11:185-190.

Detinova, T.S. 1962. Age-grouping methods in Diptera of medical importance with special reference to some vectors of malaria. Monogr. Ser. W.H.O. 47:1-216.

Lounibos, L.P. and R.L. Escher 1983. Seasonality and sampling of Coquillettidia perturbans (Diptera: Culicidae) in south Florida. Environ. Entomol. 12(4):1087-1093.

McNeel, T.E. 1932. Observations on the biology of Mansonia perturbans (Walk.) Diptera, Culicidae. Proc. N.J. Mosq. Control Assoc. 19th Annu. Meet. pp.125-128.

Monroy, A.M., T.W. Scott and B.A. Webb. 1996. Evaluation of reverse transcriptase polymerase chain reaction for the detection of eastern equine encephalomyelitis virus during vector surveillance. J. Med. Entomol. 33:449-457.

Morris, C.D., J.L. Callahan and R.H. Lewis. 1985. Devices for sampling and sorting immature Coquillettidia perturbans. J. Am. Mosq. Control Assoc. 1:247-250.

Nemjo, J. and M. Slaff. 1984. Head capsule width as a tool for instar and species identification of Mansonia dyari, Mansonia titillans, and Coquillettidia perturbans (Diptera: Culicidae). Ann. Entomol. Soc. Am. 77:633-635.

Walker, E.D. and W.J. Crans. 1986. A simple method for sampling Coquillettidia perturbans larvae. J. Am. Mosq. Control Assoc. 2(2): 239-240.

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