West Nile virus isolates from mosquitoes in New York and New Jersey, 1999.

An outbreak of encephalitis due to West Nile (WN) virus occurred in New York City and the surrounding areas during 1999. Mosquitoes were collected as part of a comprehensive surveillance program implemented to monitor the outbreak. More than 32,000 mosquitoes representing 24 species were tested, and 15 WN virus isolates were obtained. Molecular techniques were used to identify the species represented in the WN virus-positive mosquito pools. Most isolates were from pools containing Culex pipiens mosquitoes, but several pools contained two or more Culex species.

Use of an infusion-baited gravid trap to collect adult Ochlerotatus japonicus.

Ochlerotatus japonicus adults have proven difficult to collect in significant numbers in the USA. Standard trapping and collection methods have been ineffective. Infusion-baited gravid traps, typically intended to attract Culex spp., have provided the greatest number and most consistent supply of Oc. japonicus adults collected in New Jersey.

Ochlerotatus japonicus collected from natural rockpools in New Jersey.

Ochlerotatus japonicus larvae were collected from natural rockpools in New Jersey within the Delaware Water Gap National Recreational Area. Previously reported collections of Oc. japonicus in the United States have been limited to artificial containers and an occasional treehole.

Habitat preference of host-seeking Coquillettidia perturbans (Walker) in relation to birds and eastern equine encephalomyelitis virus in New Jersey.

Coquillettidia perturbans (Walker) has been implicated as a bridge vector of eastern equine encephalomyelitis virus in North America. Eastern equine encephalomyelitis virus epizootics occur regularly in wild birds in New Jersey with little or no involvement of susceptible dead end hosts even though high populations of Cq. perturbans are present. Several factors may limit eastern equine encephalomyelitis (EEE) virus transfer from birds to mosquito bridge vectors like Cq. perturbans (Walker), including bird/mosquito density, differences in bird/mosquito habitat preference, mosquito host preference, mosquito host-seeking behavior and mosquito avoidance behavior by birds. Our investigations focused on the host-seeking activities of Cq. perturbans at Colliers Mills Wildlife Management Area in Ocean County, New Jersey. We first examined the abundance of host-seeking Cq. perturbans at a height of 1.0 m in each of three habitats: forest, open field and marsh. We collected significantly more mosquitoes in the forest as compared to the marsh. Forest and field collections did not differ significantly from one another nor did field and marsh. Second, we examined the temporal and vertical distribution of host-seeking Cq. perturbans in a sylvan habitat at three heights (1.0, 2.5, and 6.0 m) from dusk to dawn. We found that Cq. perturbans did significantly more host-seeking during the night period than either the evening or morning. Host-seeking mosquitoes also tended to stay near the ground and at no time did they ascend into the forest canopy. Host-seeking Cq. perturbans and their apparent spatial disparity to roosting/nesting birds are discussed as a possible barrier to the transmission of EEE virus in New Jersey.

Aedes (Finlaya) japonicus (Diptera: Culicidae), a newly recognized mosquito in the United States: analyses of genetic variation in the United States and putative source populations.

Introduction of potential disease vectors into a new geographic area poses health risks to local human, livestock, and wildlife populations. It is therefore important to gain understanding of the dynamics of these invasions, in particular its sources, modes of spread after the introduction, and vectorial potential. We studied the population genetics of Aedes (Finlaya) japonicus japonicus (Theobald), an Asian mosquito that was recognized for the first time in the United States in 1998. We examined patterns of genetic diversity using random amplified polymorphic DNA and sequences of ND4 of mtDNA by comparing samples from populations spanning the range of this mosquito in Japan (six samples) and the United States (nine samples) as well as specimens intercepted in New Zealand in 1999. We found geographically differentiated populations in Japan, indicating limited gene flow even on small spatial scales. In the United States, we found evidence of significant genetic differentiation between samples from New York, Connecticut, and New Jersey and those from mid-Pennsylvania and Maryland. We were unable to pinpoint the source location(s) in Japan, although some of the U.S. samples are genetically close to samples from south Honshu and western Kyushu. Further studies should include samples from Korean populations. Distinct genetic signatures in U.S. populations undergoing expansion suggest the possibility of local increases in genetic diversity if and where they meet.

Life history characteristics of Wyeomyia smithii from New Jersey.

We colonized Wyeomyia smithii (Coquillett) from southern New Jersey and studied life history characteristics in the laboratory. Males and females showed no significant difference in time spent from first to third instar, but female larvae remained in fourth instar 2.1 days longer than males. At 22 +/- 2 degrees C females emerged 22.6 +/- 3 days after egg hatch; males emerged approximately two days earlier. Male emergence peaked five hours after dawn; females showed a trend to emerge late in the day. Rotation of male terminalia was completed 9 to 11 hours after emergence. Females were capable of mating immediately after emergence. Wyeomyia smithii females laid their first egg batch four to six days after emergence. Females were capable of laying up to seven batches of eggs, however the mean number of eggs per oviposition decreased significantly as the number of oviposition cycles increased.

An updated checklist of the mosquitoes of New Jersey.

The last checklist of New Jersey mosquitoes was published in 1983 and contained 59 species from 10 genera. Since that time 4 additional species have been collected in New Jersey: Aedes thibaulti, Aedes infirmatus, Aedes aegypti, and Aedes albopictus. Aedes aegypti was not able to overwinter and is not part of New Jersey's mosquito fauna. As a result, the addition of 3 species brings the updated checklist of New Jersey mosquitoes to 62.

Aedes (Finlaya) japonicus japonicus (Theobald), a new introduction into the United States.

Aedes (Finlaya) japonicus japonicus is recorded for the 1st time in the United States. Four adult females were collected in light traps at 2 sites in New York and one site in New Jersey during the months of August and September 1998. Notes on bionomics are provided. Illustrations of the adult female, male, and larva are included.

Ovarian development and parity determination in Culiseta melanura (Diptera: Culicidae).

A laboratory colony of Culiseta melanura (Coquillett) was used to follow ovarian development from emergence to oviposition and to validate the accuracy of using follicular dilatations to age grade females. We observed no change in the size of the primary follicles in unfed females from emergence to 3 d of age. Sugar feeding stimulated follicular growth and produced the following 3 types of ovarioles: (1) large primary follicles that eventually developed into functional ovarioles; (2) lesser numbers of small primary follicles that developed small amounts of yolk after blood feeding but degenerated in the latter stages of development; and (3) small primary follicles that did not develop yolk after blood feeding, exhibited accelerated growth, and became rogue ovarioles with multiple false dilatations. The yolk of the small primary follicles that degenerated after blood feeding was resorbed during stage IV of egg development, and the degenerating follicles resembled gonotrophic dilatations in the latter stages of the cycle. This process produced gravid females with some secondary follicles that appeared to possess a gonotrophic dilatation. Other authors have termed these artifacts false or agonotrophic dilatations. Degenerating ovarioles bearing these artifacts were used to determine physiological age in blood fed and gravid Cs. melanura. Nulliparous females in the latter stages of gonotrophic development have single false dilatations on degenerating ovarioles, 1-parous females have 2 false dilatations on degenerating ovarioles. In unfed, nulliparous females, false dilatations can be distinguished from true dilatations because they are attached to secondary follicles that are much smaller than the primary functional follicles that fill most of the ovary. In blood fed and gravid females, follicles that support false dilatations always lack yolk. Rogue ovarioles are unreliable indicators of physiological age and should not be used for diagnostic purposes.

Effect of temperature on the development of Culiseta melanura (Diptera: Culicidae) and its impact on the amplification of eastern equine encephalomyelitis virus in birds.

Eastern equine encephalomyelitis virus (EEE) is perpetuated in a maintenance cycle that involves Culiseta melanura (Coquillett) as the enzootic vector and passerine birds as the amplifying hosts. Amplification of virus in any given year requires an influx of nulliparous Cs. melanura in the presence of susceptible avian hosts. We conducted laboratory experiments at constant temperatures from 10 to 34 degrees C to develop thermal heat summation models to predict emergence in nature. Embryonic development progresses slowly at 10 degrees C, and the time to eclosion decreased significantly as temperatures increased to 28 degrees C. High temperature were lethal and eggs failed to hatch at 32 degrees C. The thermal minimum (t0) for embryonic development was 9.38 degrees C, and 38.46 degrees-days (DD) were required for egg hatch. The time for larval development decreased with increasing temperatures. Cs. melanura larvae develop in subterranean habitats (crypts) where water temperatures remain below 20 degrees C throughout the summer. Under controlled conditions, egg hatch to emergence took 8 mo at 10 degrees C, 3 mo at 16 degrees C, and 1 mo at 22 degrees C. The thermal minimum for larval development (t0) was 8.5 degrees C, and 467.29 DD were required from eclosion to adult emergence. Our findings indicate that Cs. melanura is well suited to develop in cold water crypts where the larvae are collected most frequently. The mosquito appears to be bivoltine in the northeast with an overwintering generation of larvae that emerges as a spring brood of adults and a summer generation of larvae that emerges in fall. Higher than normal water temperatures hasten development of the summer generation and increase the probability for amplification of EEE by bringing large numbers of nulliparous mosquitoes into contact with recrudescing virus for subsequent transfer to the growing population of susceptible juveniles. Water temperatures in the crypts also may regulate the northern limit for virus amplification each year.

Aedes thibaulti in northern New Jersey.

Aedes thibaulti Dyar and Knab occurs from southern Louisiana, USA, to Ontario, Canada, but has an exceptionally patchy distribution over much of its range. Typical breeding habitat for this univoltine species includes cavities at the bases of trees growing in low-lying swampy areas or dark recesses within the root balls of upturned trees. Larvae have never been collected in the northeastern portion of the range, but adult records suggest that breeding populations are present. The lack of low-lying swampland in northern areas where adults have been collected suggests that the breeding habitat for this species may not be as specific as previously believed. On April 20, 1997, we collected Ae. thibaulti larvae from the flooded cavity of a red maple tree (Acer rubrum) growing next to a temporary snow pool in northern New Jersey. Larvae persisted in this habitat until mid-May when the cavity dried completely. The collection suggests that Ae. thibaulti is a cavity breeder in the northern portion of its range, but is able to utilize cavity habitats associated with temporary pools in dry forested areas.

Larval competition in Aedes triseriatus (Diptera: Culicidae): effects of density on size, growth, sex ratio, and survival.

We assessed the effect of increased larval density on selected life table attributes of larval Aedes triseriatus. Larvae were reared at densities of 0.125, 0.250, and 0.375 larvae/cm3 of water. The parameters we evaluated included duration of each developmental instar, percent survival to adult emergence, sex ratio, and size of early and late emerging adults. The amount of time spent in the first and second instar was not affected by density, but crowding lengthened duration of later instars significantly. Males emerged earlier than females at all densities, and there was no difference in the ratio of males to females due to crowding. Crowding did increase the overall developmental time of both sexes and produced significantly smaller adults of both sexes. Densities of 0.375 larvae/cm3 resulted in a significant increase in mortality. Results suggest that the effects of crowding in container habitats should be allowed for in the construction of time-specific life tables where duration of larval instars is used in the calculations. The importance of smaller body size as a result of crowding in container habitats is discussed in terms of vector competence of Ae. triseriatus in the transmission of LaCrosse encephalitis virus.

Observations on multiple bloodfeeding in field-collected Culiseta melanura.

This study examined field populations of Culiseta melanura for evidence of partial blood meals and multiple feeding behavior. Engorged specimens that had 1/3 or less abdominal distention together with eggs in stage I-II of ovarian development were recorded as mosquitoes that had taken partial blood meals. Multiple blood meals were credited to engorged specimens with fresh blood as well as partially digested blood surrounded with separate peritrophic membranes and eggs in stage III or IV of ovarian development. Results indicated that only 17 of 532 Cs. melanura were captured with a partial meal, indicating that natural populations feed to repletion more than 95% of the time. Less than 1% of the specimens showed physical evidence of feeding on multiple hosts. Culiseta melanura appears to be highly efficient at obtaining a full blood meal and seems to be extremely reluctant to refeed once ovarian development has been initiated. As a result, multiple feeding does not appear to be important in the amplification of virus by this species.

A thermal heat summation model to predict the duration of the gonotrophic cycle of Culiseta melanura in nature.

This study determined the effect of temperature on the gonotrophic cycle of Culiseta melanura and developed a thermal heat summation model to calculate its duration under field conditions. A colony of Cs. melanura was used from New Jersey (F13-F17 generation) and the length of the gonotrophic cycle was observed at 2, 10, 16, 22, 28, 32 and 34 degrees C. None of the mosquitos survived at 2 degrees C or 34 degrees C and none laid fertile eggs at 32 degrees C. A linear regression analysis on the data showed that the thermal minimum for ovarian development was 6.4 degrees C and 95.87 degree days were required above 6.4 degrees C to complete one gonotrophic cycle. A thermal heat summation model is presented to allow calculation of the duration of the gonotrophic cycle under field conditions when average temperatures are known.

First record of Aedes albopictus from New Jersey.

In early August 1995, Aedes albopictus was detected in a light trap collection at a coastal location of Monmouth County, NJ. Larval surveillance indicated the species was breeding in a variety of containers over an area of at least 1.0 km2. This New Jersey record currently represents the northernmost breeding population of Ae. albopictus on the eastern seaboard. The collection site is very close to the 0 degree C daily mean January isotherm that has been used as a conservative estimate for the northern limit of this mosquito's overwintering range.

Experimental infection of wading birds with eastern equine encephalitis virus.

To study the susceptibility of wading birds to eastern equine encephalitis (EEE) virus and to determine their potential as reservoir or amplifying hosts, fledgling glossy ibises (Plegadis falcinellus) and snowy egrets (Egretta thula) were captured in New Jersey (USA) and shipped to Colorado (USA) where they were experimentally inoculated with EEE virus. All 16 snowy egrets and 14 (93%) of 15 of the glossy ibises inoculated became viremic with moderate titers, and all survivors developed neutralizing antibody. Six ibises and two egrets died during the first week after inoculation, and EEE virus was isolated from the tissues of three birds. Our experimental results support field evidence about the relative involvement of glossy ibises and snowy egrets in the epizootiology of EEE virus in New Jersey.

Eastern equine encephalomyelitis virus in relation to the avian community of a coastal cedar swamp.

Eastern equine encephalomyelitis virus (EEEV) is perpetuated in eastern North America in a mosquito-wild bird maintenance cycle that involves Culiseta melanura (Coquillett) as the principal enzootic vector and passerine birds as the primary amplifying hosts. We examined the role of birds in the EEEV cycle at a site in southern New Jersey where EEEV cycles annually at high levels. Birds and mosquitoes were sampled during three epiornitics and one season of limited virus activity. We examined antibody prevalence in birds in relation to eight physical and natural history characteristics. Our goal was to compare EEEV cycling in C. melanura and the primary avian hosts better to understand the mechanisms that initiate annual epiornitics. Antibody prevalence was highest in the Blue Jay (62%), Wood Thrush (60%), and Tufted Titmouse (44%). Resident status of birds was the natural history characteristic most closely linked to participation in the EEEV cycle. Species spending the greatest amount of time at our study site (permanent residents, summer residents) had the highest antibody rates. We captured viremic birds as early as 25 May, 51 d before we first detected virus in C. melanura. We recaptured 10 after hatching year adults and one hatching year (HY) bird that seroconverted before we detected virus in C. melanura. We also found EEEV antibody in 15 HY birds up to 31 d before we isolated EEEV from C. melanura. We provide evidence that a cryptic cycle develops weeks before epiornitic cycling is detected in C. melanura by traditional laboratory techniques, indicating that the early season cycle is initiated by the recrudescence of latent virus in previously infected birds.

Susceptibility parameters of Aedes albopictus to per oral infection with eastern equine encephalitis virus.

Aedes albopictus (Skuse) mosquitoes were fed on snowy egrets, Egretta thula (Thayer and Bangs), that had been infected by subcutaneous inoculation of eastern equine encephalitis (EEE) virus. Freshly fed mosquitoes were frozen and tested to determine how much virus they had ingested. Other fed mosquitoes from the same lots were incubated for 7 d at 27 degrees C before testing. Seven lots of Ae. albopictus fed on viremic birds. Based on average amounts of virus ingested and day 7 virus infection rates in mosquitoes from the same lots, the amount of virus required to infect 50% of the mosquitoes was calculated to be 10(2.8) Vero cell plaque-forming units (PFU). The infection threshold (i.e., the amount of virus required to infect from 1 to 5% of mosquitoes) was determined to be < or = 10 PFU per blood meal. These parameters indicate that Ae. albopictus is sufficiently susceptible to infection with EEE virus to enable it to acquire infectious doses from a wide variety of viremic birds and possibly from equines.

The relationship between size and parity status of field collected Culiseta melanura.

The relationship between body size and parity status of adult female Culiseta melanura collected from 3 locations in northeastern United States was studied by measuring wing lengths and examining ovaries of individual mosquitoes. Virus isolation was attempted from Cs. melanura collected in Maryland and in New Jersey. At all 3 locations, the size of Cs. melanura collected varied from large in the spring, to small in the summer. In New Jersey and Maryland mosquitoes collected in the fall were again large. The size of Massachusetts mosquitoes collected in the summer versus the fall was not different. In general, parous mosquitoes were larger than nulliparous mosquitoes in the spring but smaller than nulliparous ones in the fall. Eastern equine encephalomyelitis (EEE) and Highlands J (HJ) viruses were recovered from Cs. melanura only during the late summer when mosquitoes were small or during the fall months when larger mosquitoes were collected. We conclude that there is no detectable association between Cs. melanura size and parity status and that there is no obvious effect of mosquito size on EEE or HJ virus transmission.