[Identification of mosquito-parasitic microsporidia, Amblyospora rugosa and Trichoctosporea pygopellita (Microsporidia: Amblysporidae), from Acanthocyclops venustus and Acanthocyclops reductus (Copepoda: Cyclopidae), based on small subunit rDNA analysis].

Identical small subunit rDNA sequences were obtained for microsporidia Amblyospora rugosa from blood-sucking mosquitoes larvae Ochlerotatus cantans, O. cataphylla and copepods Acanthocyclops venustus, as well as for Trichoctosporea pygopellita from mosquitoes larvae Ochlerotatus cyprius, O. excrucians and copepods Acanthocyclops reductus. The data on molecular phylogeny and ecological researches show that in Siberia mosquito-parasitic microsporidia of the genera Amblyospora and Trichoctosporea have complex life cycle involving likely intermediate hosts, Acanthocyclops copepods. Life cycle of parasites is synchronized with phenology of their hosts. The phylogenetic analyses shows, that genus Trichoctosporea should be transferred from the family Thelohaniidae to the family Amblyosporidae.

Identification of bloodmeals in Anopheles quadrimaculatus and Anopheles punctipennis from eastern equine encephalitis virus foci in northeastern U.S.A.

The host-feeding patterns of Anopheles quadrimaculatus Say and Anopheles punctipennis (Say) were examined in order to evaluate their potential contributions to the transmission of eastern equine encephalitis virus (EEEv) and other arboviruses in the northeastern U.S.A. Engorged mosquitoes of the two species were collected from EEEv foci in central New York (NY) and throughout New Jersey (NJ), and their bloodmeals were identified using a polymerase chain reaction (PCR)-based assay and sequencing portions of the mitochondrial cytochrome b gene. Analysis of 131 An. quadrimaculatus and 107 An. punctipennis from NY revealed that 97.7% and 97.2%, respectively, had acquired blood solely from mammalian hosts. Similarly, examination of 288 An. quadrimaculatus and 127 An. punctipennis from NJ showed 100% and 96.0%, respectively, contained mammalian-derived bloodmeals. Mosquitoes containing mixed bloodmeals from both avian and mammalian hosts were detected in 1.6% of An. quadrimaculatus from NY, and 2.8% and 4.0% of An. punctipennis from NY and NJ, respectively. White-tailed deer (Odocoileus virginianus) constituted the most common vertebrate host for these anopheline mosquitoes, accounting for 85.8-97.7% of all bloodmeals identified. The predominance of white-tailed deer as a source of bloodmeals supports enzootic amplification of deer-associated arboviruses in this region, including Jamestown Canyon, Cache Valley and Potosi viruses. One horse- and two human-derived bloodmeals were also detected in An. quadrimaculatus collected in NJ. Limited avian-derived bloodmeals were detected from mourning dove (Zenaida macroura), sharp-shinned hawk (Accipiter striatus) and house finch (Carpodacus mexicanus), mostly in mixed bloodmeals. Occasional feeding on avian hosts suggests that these mosquitoes may participate as epizootic-epidemic bridge vectors of EEEv from viraemic birds to mammalian hosts of concern, including horses and humans. An isolate of EEEv was recovered from the head and thorax of an An. punctipennis mosquito collected in NY.

A phylogenetic approach to following West Nile virus in Connecticut.

The 1999 outbreak of West Nile (WN) virus in the northeastern United States was the first known natural occurrence of this flavivirus in the Western Hemisphere. In 1999 and 2000, 82 independent Connecticut WN virus isolates were cultured from nine species of birds, five species of mosquitoes, and one striped skunk. Nucleotide sequences obtained from these isolates identified 30 genetic changes, compared with WN-NY99, in a 921-nt region of the viral genome beginning at nucleotide position 205 and ending at 1125. This region encodes portions of the nucleocapsid and envelope proteins and includes the entire coding regions for the premembrane and membrane proteins. Amino acid changes occurred at seven loci in six isolates relative to the WN-NY99 strain. Although 34 of the isolates showed sequences identical to the WN-NY99 isolate, we were able to show geographical-based clusters of mutations. In particular, 26 isolates were characterized by mutation of C to T at position 858. This group apparently originated in Stamford, CT and disseminated to sites located as far as 54 miles from Stamford. Sequences of WN virus isolated from both brain and heart tissues from the same avian host were identical in all 14 tested individual birds, suggesting that the mutations we have documented are real and not caused by culture, RNA extraction, or PCR procedures. We conclude that this portion of the viral genome will enable us to follow the geographical and temporal movement of variant WN virus strains as they adapt to North America.

Mosquito surveillance for West Nile virus in Connecticut, 2000: isolation from Culex pipiens, Cx. restuans, Cx. salinarius, and Culiseta melanura.

Fourteen isolations of West Nile (WN) virus were obtained from four mosquito species (Culex pipiens [5], Cx. restuans [4], Cx. salinarius [2], and Culiseta melanura [3]) in statewide surveillance conducted from June through October 2000. Most isolates were obtained from mosquitoes collected in densely populated residential locales in Fairfield and New Haven counties, where the highest rates of dead crow sightings were reported and where WN virus was detected in 1999. Minimum field infection rates per 1,000 mosquitoes ranged from 0.5 to 1.8 (county based) and from 1.3 to 76.9 (site specific). Cx. restuans appears to be important in initiating WN virus transmission among birds in early summer; Cx. pipiens appears to play a greater role in amplifying virus later in the season. Cs. melanura could be important in the circulation of WN virus among birds in sylvan environments; Cx. salinarius is a suspected vector of WN virus to humans and horses.

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.

Epizootiological studies of Amblyospora albifasciati (Microsporidiida: Amblyosporidae) in natural populations of Aedes albifasciatus (Diptera: Culicidae) and Mesocyclops annulatus (Copepoda: Cyclopidae) in a transient floodwater habitat.

The epizootiology of the microsporidium Amblyospora albifasciati was studied in natural populations of its definitive host, a multivoltine, neotropical, floodwater mosquito, Aedes albifasciatus, and its intermediate copepod host, Mesocyclops annulatus, in an ephemeral floodwater habitat during a 12-month period. A. albifasciati was enzootic in mosquitoes. Vertically (transovarially) transmitted meiospore infections occurred regularly and were detected in five of eight larval broods but the prevalence of infection was always low, ranging from 0.5 to 6.9% with an overall average of 0.7%. Horizontal transmission of A. albifasciati infection from copepods to mosquitoes was nominal and limited. It was detected at levels of 6.4 to 20% in larval Ae. albifasciatus populations on two occasions, the month of August and late September through early October. The low levels of horizontal transmission of infection to mosquito larvae appeared to be the principal limiting factor that prevented the proliferation of A. albifasciati in Ae. albifasciatus populations. Copepod populations were abundant from May through September and weekly prevalence rates of A. albifasciati averaged over 50% (range = 5.8 to 100%). The moderately high infection rates in M. annulatus copepods were inconsistent with the low prevalence of meiospore infection in Ae. albifasciatus mosquito larvae. Results suggest that either meiospores of A. albifasciati produced in the mosquito host are highly infectious to copepods or they are long-lived and remain viable within the pool as long as some standing water is present. Observations further indicate that A. albifasciati has a significant detrimental impact on M. annulatus copepod populations but minimal impact on larval populations of Ae. albifasciatus at this site.

Discovery, distribution, and abundance of the newly introduced mosquito Ochlerotatus japonicus (Diptera: Culicidae) in Connecticut, USA.

The earliest documented specimen of an exotic east Asian mosquito Ochlerotatus (Finlaya) japonicis japonicus (Theobald) in the Western Hemisphere is reported along with the results of a state wide survey to determine the distribution and abundance of this mosquito in Connecticut. Ochlerotatus japonicus was collected from 87 locations in eight counties. It is established throughout the state and occurs in a variety of natural and artificial container habitats including discarded tire casings, bird baths, wooden barrels, porcelain bath tubs (used for watering animals), plastic milk cartons, toys, vinyl tarpaulins (covering wood piles and swimming pools), exposed rock holes in stream beds, tree holes, subterranean catch basins, surface water rain pools, and spring-fed depressions. Larvae were particularly common in containers with water, decaying leaves, and algae, in shaded and sunlit areas and, in rock-pool habitats along streambeds, in association with Ochlerotatus atropalpus (Coquillett). Adult females were collected in sod grass-infused gravid and CO2- baited light traps, from early June through October, with peak collections in September. Biting females were collected by human bait method augmented with CO2, verifying its capacity to feed on humans. The ovitraps used in this study were not effective for recovering this species. Our results suggest that Oc. japonicus was introduced into Connecticut between 1992 and 1998. Because of the ability of Oc. japonicus to transmit West Nile virus, and because of the recent detection of this virus in field-collected specimens, the introduction of Oc. japonicus is considered a significant public health development.

Recovery and identification of West Nile virus from a hawk in winter.

West Nile virus was recovered from the brain of a red-tailed hawk that died in Westchester County, N.Y., in February 2000. Multiple foci of glial cells, lymphocytes, and a few pyknotic nuclei were observed in the brain. Three to 4 days after inoculation of Vero cells with brain homogenates, cytopathic changes were detected. The presence of West Nile virus antigen in fixed cells or cell lysates was revealed by fluorescent antibody testing or enzyme-linked immunosorbent assay, respectively. Furthermore, Reverse transcriptase-PCR with primers specific for the NS3 gene of West Nile virus resulted in an amplicon of the expected size (470 bp). Electron microscopy of thin sections of infected Vero cells revealed the presence of viral particles approximately 40 nm in diameter, within cytoplasmic vesicles. The demonstration of infection with the West Nile virus in the dead of the winter, long after mosquitoes ceased to be active, is significant in that it testifies to the survival of the virus in the region beyond mosquito season and suggests another route of transmission: in this case, prey to predator.

Isolation of West Nile virus from mosquitoes, crows, and a Cooper's hawk in Connecticut.

West Nile (WN) virus, a mosquito-transmitted virus native to Africa, Asia, and Europe, was isolated from two species of mosquitoes, Culex pipiens and Aedes vexans, and from brain tissues of 28 American crows, Corvus brachyrhynchos, and one Cooper's hawk, Accipiter cooperii, in Connecticut. A portion of the genome of virus isolates from four different hosts was sequenced and analyzed by comparative phylogenetic analysis. Our isolates from Connecticut were similar to one another and most closely related to two WN isolates from Romania (2.8 and 3.6 percent difference). If established in North America, WN virus will likely have severe effects on human health and on the health of populations of birds.

Epizootiology of Amblyospora stimuli (Microsporidiida: Amblyosporidae) infections in field populations of a univoltine mosquito, Aedes stimulans (Diptera: Culicidae), inhabiting a temporary vernal pool.

The epizootiology of the microsporidium Amblyospora stimuli was studied in natural populations of a univoltine mosquito, Aedes stimulans, inhabiting a temporary vernal pool over an 18-year period. The yearly prevalence of benign oenocytic infections in adult females was variable, ranging from 1.0 to 9.6% (mean = 5.1%). The yearly prevalence of transovarially transmitted meiospore infections in larval populations was consistently lower but less variable, ranging from 1.3 to 5.9% (mean = 3.5%). Meiospore infections in F(1)-generation larvae were significantly correlated with infections in parental-generation females, thus suggesting that larval infection rates could be substantially increased if methods were available to facilitate transmission of A. stimuli to a larger portion of the female population via inundative or inoculative release of infected copepods. No correlation was found when infections in filial-generation adult females were measured against meiospore infections in larvae from the preceding year. Analysis of yearly prevalence data using Fine's Fundamental Vertical Transmission Equation revealed low rates of horizontal transmission from the intermediate copepod host to female larvae in most years, ranging from 0.1 to 8.7% (mean = 3.1%). A. stimuli is enzootic, persists at a very low level, and has minimal impact on Ae. stimulans populations at this site. The low incidence rate of horizontal transmission to larvae appears to be due largely to a paucity of copepods and is a major factor that limits the abundance and subsequent proliferation of A. stimuli in Ae. stimulans populations at this locale. Results support the view that host-parasite cospeciation is an important mechanism of evolution in this group of mosquito/copepod microsporidia.

Multiple isolations of eastern equine encephalitis and highlands J viruses from mosquitoes (Diptera: Culicidae) during a 1996 epizootic in southeastern Connecticut.

Thirty-six isolations of eastern equine encephalitis virus were obtained from 8 species of mosquitoes collected from 5 September through 18 October 1996 during an epizootic in southeastern Connecticut. These included Culiseta melanura (Coquillett) (19 isolates), Culex pipiens L. (8), Culiseta morsitans (Theobald) (3), Aedes sollicitans (Walker) (2), Aedes cantator (Coquillett) (1), Aedes trivittatus (Coquillett) (1), Aedes vexans (Meigen) (1), and Coquillettidia perturbans (Walker) (1). Isolations from Ae. cantator and Ae. trivittaus are new to North American records, and those from Ae. cantator and Ae. sollicitans represent the first infections of human-biting, salt-marsh mosquitoes with eastern equine encephalitis virus in Connecticut. With one exception, eastern equine encephalitis-infected Cs. melanura were found at all sites where eastern equine encephalitis virus was isolated. The large number of eastern equine encephalitis isolations from Cs. melanura and the collection of infected mosquitoes in residential woodlots and coastal salt marshes away from traditional red maple or white cedar swamp habitats, reaffirm the importance of local populations of this mosquito for viral amplification and dispersal from swamp foci. Highlands J virus was more widespread geographically, but fewer isolations of this virus were made from fewer species of mosquitoes. These included Cs. melanura (8 isolates), Cx. pipiens (5), Ae. vexans (3), Aedes canadensis (Theobald) (1), Ae. cantator (1) and Cs. morsitans (1). No human or horse cases of eastern equine encephalitis were reported, although this represents the largest number of isolations for eastern equine encephalitis ever recovered from field-collected mosquitoes in Connecticut.

Verification of intermediate hosts in the life cycles of microsporidia by small subunit rDNA sequencing.

Small subunit rDNA sequences were obtained from field-collected Amblyospora connecticus (Microsporida: Amblyosporidae) spores isolated from the mosquito, Aedes cantator (Diptera: Culicidae), and from field collected spores isolated from the putative intermediate host, Acanthocyclops vernalis (Copepoda: Cyclopidae). The ribosomal DNA sequences of the spores isolated from the two hosts were identical. These findings corroborate previous laboratory transmission studies and validate the intermediary role of A. vernalis in the life cycle of this microsporidium. These data represent the first comparative sequence analysis of a microsporidium isolated from its definitive and intermediate hosts. The results demonstrate the effectiveness of using rDNA sequence data for screening potential intermediate hosts. Unlike laboratory transmission tests, which can take months or years to complete, this technique can be completed in days and can be performed on a single infected organism.

Phylogeny of amblyospora (Microsporida: amblyosporidae) and related genera based on small subunit ribosomal DNA data: A possible example of host parasite cospeciation.

Small subunit ribosomal RNA (SSU rRNA) gene sequences were analyzed for six species and four genera of microsporidia from mosquito hosts; Amblyospora stimuli (Aedes stimulans), Amblyospora californica (Culex tarsalis), Amblyospora sp. (Culex salinarius), Edhazardia aedis (Aedes aegypti), Culicosporella lunata (Culex pilosus), and Parathelohania anophelis (Anopheles quadrimaculatus). Comparison of these sequences to those of other microsporidia show that these sequences are longer with the SSU rRNA gene of E. aedis being the longest microsporidia sequenced to date (1447 base pairs). Parsimony, maximum likelihood, and distance methods produced identical trees, suggesting that the above microsporidian taxa, contrary to current classification schemes, form a monophyletic group. Relationships within this group are further supported by high bootstrap and decay analysis values. Based on the molecular analysis, P. anophelis is the most divergent species in this group of mosquito parasites. Amblyospora is paraphyletic with A. californica and Amblyospora sp., forming a sister taxon to a clade composed of E. aedis and A. stimuli. Culicosporella lunata comprises a sister taxon to the Amblyospora/Edhazardia clade. The pattern of host relationships on the tree provides preliminary evidence that the branching pattern seen here may indicate that host-parasite cospeciation is an important mechanism of evolution in this group.

Amblyospora salinaria n. sp. (Microsporidia: amblyosporidae), parasite of Culex salinarius (Diptera: culicidae): its life cycle stages in an intermediate host.

Horizontal transmission testing with an Amblyospora species from the mosquito Culex salinarius has documented the involvement of a copepod intermediate host. Meiospores (one type of uninucleate spore) of the Amblyospora sp. were infectious per os to female Macrocyclops albidus adults. All developmental stages in the copepod had unpaired nuclei (were haplophasic), starting with the sporoplasms from the meiospore, continuing as a succession of schizonts undergoing binary division and ending with sporulation, producing a second type of uninucleate spore. These spores, formed in the ovaries of M. albidus, were lanceolate, slightly curved and measured 13.23 x 3.85 microm. They infected C. salinarius larvae, both male and female, when ingested. In addition, cross-infectivity testing was conducted and demonstrated that while A. californica from C. tarsalis will infect C. salinarius, it does not complete its life cycle in this host. Based on these findings, we conclude that Amblyospora sp. from Culex salinarius is a distinct species and assign it the name Amblyospora salinaria n. sp.

Ultrastructural differentiation of the genogroups in the genus Ehrlichia.

Ultrastructural characteristics of 15 strains and isolates of ehrlichiae belonging to three genogroups, or clades of genetically related organisms united in the genera Ehrlichia, Cowdria, Anaplasma, Neorickettsia and a strain of Wolbachia pipientis which represents a fourth genogroup in this cluster of species, were studied in continuous cell culture or in vivo: E. canis (Oklahoma strain and VHE isolate), E. muris (AS 145), E. chaffeensis (Arkansas, 91HE17 and Sapulpa), human granulocytic ehrlichiae (HGE)(BDS, 96HE27, 96HE37, #54, #55 and #72), E. equi (MRK), E. sennetsu (Miyayama), E. risticii (HRC-IL). Wolbachia pipientis was studied in the naturally infected Aedes albopictus mosquito cell line Aa23. All organisms were similar in the normal ultrastructure of individual cells and in the ability to form abnormal, pathological ehrlichial cells of the same type irrespective of the species. Normally all ehrlichiae studied in cell culture existed in two morphological forms - reticulate and dense-cored cells, both of which could divide by binary fission. Most alterations were related to their membranes, especially the cell wall. Differences in the structure of intravacuolar microcolonies (morulae) of ehrlichiae and their inter-relations with the host cells allowed differentiation of the genogroups: the E. canis-E. chaffeensis-E. muris genogroup formed large morulae, with many ehrlichiae, often suspended in a fibrillar matrix, and the host cell mitochondria and endoplasmic reticulum usually aggregated near the morulae and were in contact with the morula membrane; the E. phagocytophila-E. equi-HGE group morulae had no fibrillar matrix, no contacts with host cell mitochodria, and they did not aggregate around the morulae; E. sennetsu-E. risticii group usually developed in small individual vacuoles that did not fuse with each other and divided along with the ehrlichiae.

Serologic evidence of Jamestown Canyon virus infection in white-tailed deer populations from Connecticut.

We determined the prevalence and distribution of Jamestown Canyon (JC) virus antibody in white-tailed deer (Odocoileus virginianus) populations in Connecticut, USA. Sera were collected from hunter-killed deer during 1993. Antibody to JC virus was detected by enzyme-linked immunosorbent assay (ELISA) in 92 (21%) of 446 deer sera, and was uniformly distributed among geographic sites. Twenty-one ELISA-positive sera were tested and confirmed positive by plaque reduction neutralization testing. This represents the first serologic evidence of JC virus in a reservoir host population from the northeastern United States. No cross-reactivity was seen with California encephalitis, Keystone, or snowshoe hare viruses, but a varying degree of cross-reactivity was obtained with Guaroa, Jerry Slough, La-Crosse, San Angelo, and trivittatus viruses. We conclude from this investigation and previous isolations of JC virus from mosquitoes in the state that JC virus occurs enzootically in Connecticut.

Intraspecific variation in key morphological characters of Culiseta melanura (Diptera:Culicidae).

Culiseta melanura (Coq.), the enzootic vector of eastern equine encephalitis in North America, is polymorphic for a trait used as a key diagnostic character. The absence of white abdominal bands distinguishes this species in several prominent keys to North American mosquitoes. However, this is an environmentally induced, nongenetic trait that cannot be used as a key character for diagnosing Cs. melanura. In light trap collections, banded specimens occur in early spring and summer, and nonbanded adults appear in late summer-autumn. Larvae reared in laboratory conditions produce nonbanded adults. Progeny reared from banded mothers are uniformly nonbanded. Biochemical genetic results indicate that banding is not correlated with a distinctive genotype or presence of cryptic species. In 18 enzyme loci screened, neither diagnostic alleles nor large differences in allele frequencies were detected between field-collected representatives of the two forms. Genetic variability was relatively low in the 28-year-old laboratory colony (average heterozygosity = 7%; average number of alleles per locus = 1.4), whereas in field samples, the variability was typical of field populations (average heterozygosity = 12-19%; average number of alleles per locus = 1.6-1.8), with the presence of both polymorphic and private alleles. The population genetic profile and comparisons among geographically distinct populations represent the first such presentation for any species in the genus Culiseta.

In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line.

A continuous cell line, Aa23, was established from eggs of a strain of the Asian tiger mosquito, Aedes albopictus, naturally infected with the intracellular symbiont Wolbachia pipientis. The resulting cell line was shown to be persistently infected with the bacterial endosymbiont. Treatment with antibiotics cured the cells of the infection. In the course of establishing this cell line it was noticed that RFLPs in the PCR products of two Wolbachia genes from the parental mosquitoes were fixed in the infected cell line. This indicates that the mosquito host was naturally superinfected with different Wolbachia strains, whereas the infected cell line derived from these mosquitoes only contained one of the original Wolbachia strains. The development of an in vitro culture system for this fastidious microorganism should facilitate molecular analysis of the reproduction distorting phenotypes it induces in natural arthropod hosts.