Life Cycle, Ultrastructure and Molecular Phylogeny of Hyalinocysta Chapmani (Microsporidia: Thelohaniidae), a Parasite of Culiseta Melanura (Diptera: Culicidae) and Orthocyclops Modestus (Copepoda: Cyclopidae)

The Journal of Eukaryotic Microbiology. Jul-Aug, 2002  |  Pubmed ID: 12188227

The complete life cycle of the microsporidium Hyalinocysta chapmani is described from the primary mosquito host Culiseta melanura and the intermediate copepod host Orthocyclops modestus. Infections are initiated in larval C. melanura following the oral ingestion of uninucleate spores from infected copepods. Spores germinate within the lumen of the midgut and directly invade fat body tissue where all development occurs. Uninucleated schizonts undergo binary division (schizogony) followed by karyokinesis (nuclear division) to form diplokaryotic meronts. Merogony is by synchronous binary division. The onset of sporogony is characterized by the simultaneous secretion of a sporophorous vesicle and meiotic division of the diplokaryon resulting in the formation of eight ovoid meiospores enclosed within a sporophorous vesicle. Most infected larvae die during the fourth stadium and there is no evidence of a developmental sequence leading to vertical transmission. Hyalinocysta chapmani is horizontally transmitted to O. modestus via oral ingestion of meiospores. Infections become established within ovarian tissue of females and all parasite development is haplophasic. Uninucleate schizonts divide by binary division during an initial schizogonic cycle. Newly formed uninucleate cells produce a thin sporophorous vesicle and undergo repeated nuclear division during sporogony to produce a rosette-shaped, multinucleated sporogonial plasmodium with up to 18 nuclei. This is followed by cytoplasmic cleavage, sporogenesis, and disintegration of the sporophorous vesicle to form membrane-free uninucleate spores. Infected females eventually die and there is no egg development. The small subunit rDNA sequence of H. chapmani isolated from meiospores from C. melanura was identical to the small subunit rDNA sequence obtained from spores from O. modestus, corroborating the laboratory transmission studies and confirming the intermediary role of O. modestus in the life cycle. Phylogenetic analysis was conducted with closely related microsporidia from mosquitoes. Hyalinocysta chapmani did not cluster within described Amblyospora species and can be considered a sister group, warranting separate genus status.

Epizootiology of Hyalinocysta Chapmani (Microsporidia: Thelohaniidae) Infections in Field Populations of Culiseta Melanura (Diptera: Culicidae) and Orthocyclops Modestus (Copepoda: Cyclopidae): a Three-year Investigation

Journal of Invertebrate Pathology. Oct, 2002  |  Pubmed ID: 12445795

The epizootiology, transmission dynamics and survival strategies employed by the microsporidium Hyalinocysta chapmani were examined in field populations of its primary mosquito host, Culiseta melanura and its intermediate copepod host, Orthocyclops modestus over a three-year period in an aquatic subterranean habitat. H. chapmani was enzootic and was maintained in a continuous cycle of horizontal transmission between each host. There were three distinct periods during the summer and fall when developing mosquito larvae acquired infections; each was preceded by or coincident with the detection of infected copepods. Results were corroborated in laboratory bioassays, wherein transmission was achieved in mosquito larvae that were reared in water and sediment samples taken from the site during the same time periods. The highest infection rates, ranging from 60% to 48%, were repeatedly observed during the first six weeks of larval development. These were coincident with the most sustained collections of infected copepods obtained during the year and highest levels of infection achieved in the laboratory transmission studies. The high prevalence rates of lethal infection observed in larval populations of C. melanura at this site are among the highest recorded for any mosquito-parasitic microsporidium and clearly suggest that H. chapmani is an important natural enemy of C. melanura. H. chapmani appears to overwinter in diapausing mosquito larvae but may also persist in copepods. The absence of vertical transmission in the life cycle of H. chapmani and the sole reliance on horizontal transmission via an intermediate host are unique survival strategies not seen among other mosquito-parasitic microsporidia. The epizootiological data suggest that this transmission strategy is a function of the biological attributes of the hosts and the comparatively stable environment in which they inhabit. The subterranean habitat is inundated with water throughout the year; copepods are omnipresent and C. melanura has overlapping broods. The spatial and temporal overlap of both hosts affords abundant opportunity for continuous horizontal transmission and increases the likelihood that H. chapmani will find a target host. It is hypothesized that natural selection has favored the production of meiospores in female host mosquitoes rather than congenital transfer of infection to progeny via ovarian infection as a strategy for achieving greater transmission success.

A Checklist of the Mosquitoes of Connecticut with New State Records

Journal of the American Mosquito Control Association. Mar, 2003  |  Pubmed ID: 12674540

Distribution and collection records for 3 previously unrecognized species, Anopheles barberi, Culex erraticus, and Ochlerotatus diantaeus, are included herein. This brings to 48 the number of recognized species from 11 genera in Connecticut. Also, an updated checklist of the mosquitoes of Connecticut that reflects the recent elevation of the subgenus Ochlerotatus to generic rank is presented.

Infectivity and Pathogenicity of a Novel Baculovirus, CuniNPV from Culex Nigripalpus (Diptera: Culicidae) for Thirteen Species and Four Genera of Mosquitoes

Journal of Medical Entomology. Jul, 2003  |  Pubmed ID: 14680119

The infectivity and pathogenicity of newly discovered baculovirus, CuniNPV (family Baculoviridae, genus Nucleopolyhedrovirus) originally isolated from the mosquito Culex nigripalpus Theobald, was evaluated in laboratory bioassys against thirteen species and four genera of mosquitoes native to the northeastern U.S. Purified virus at a dosage rate of 1.6 x 10(7) occlusion bodies/ml with 10 mM Mg2+ added was used in exposures with second through fourth instars at temperatures ranging from 17 to 27 degrees C. High infection rates and accompanying mortality were achieved in Cx. pipiens L. (83.0-14.4%), Cx. pipiens f. molestus (80.4% infection), and Cx. salinarius Coquillett (48.0-43.1%). Cx. restuans Theobald was also susceptible but infection rates were lower (21.3-12.5%). The gross pathology associated with infection was identical to that reported in Cx. nigripalpus. Infected larvae were lethargic and were often suspended at the water surface. Development of CuniNPV was observed in the nuclei of the midgut epitheial cells in the gastric caeca and posterior region of the stomach of host larvae. One hundred percent mortality was observed in all larvae that exhibited gross symptoms of infection within 4-d p.i. Cx. territans Walker (subgenus Neoculex Dyar) was the only Culex mosquito that was not susceptible. No infections were obtained with any species of Aedes [Ae. vexans (Meigen)], Culiseta [Culiseta morsitans (Theobald)] or Ochlerotatus [Ochlerotatus canadensis (Theobald), Oc. cantator (Coquillett), Oc. communis (De Geer), Oc. excrucians (Walker), Oc. japonicus (Theobald), Ochlerotatus stimulans (Walker), and Ochlerotatus triseriatus (Coquillett)]. The host range of CuniNPV appears to be restricted to Culex mosquitoes within the subgenus Culex. An inhibitory effect on transmission of CuniNPV was observed when a liver powder/Brewer's yeast mixture was used as a source of food reinforcing the critical role of Mg2+ and sensitivity of the infection process to the presence other divalent cations (Cu2+, Fe2+, and Zn2+) in the larval medium that interfered with the infection process. The high infectivity and pathogenicity of CuniNPV for the principal vectors of West Nile virus in North America make CuniNPV an attractive candidate for future development as a biopesticide.

Transstadial Transfer of West Nile Virus by Three Species of Ixodid Ticks (Acari: Ixodidae)

Journal of Medical Entomology. Jul, 2003  |  Pubmed ID: 14680122

Larvae and/or nymphs of four species of ixodid ticks, Ixodes scapularis Say, Amblyomma americanum (L.), Dermacentor andersoni Stiles, and Dermacentor variabilis Say, were fed to completion on laboratory hamsters or mice which had been inoculated with a West Nile (WN) virus isolate from Culex pipiens L. captured in Connecticut USA. Maximum titers in mice and hamsters were approximately 5 and two logs, respectively, lower than recorded (10 logs) in a naturally infected American crow, Corvus brachyrhynchos Brehm. WN virus was isolated in Vero cell culture from ticks and detected by TaqMan RT-polymerase chain reaction (PCR) in ticks that had completed their feeding as larvae or nymphs, and in I. scapularis, D. andersoni, and D. variabilis that had molted into the next stage of development. Naive hosts, fed upon by nymphs that as larvae had fed on viremic hosts, did not become infected. WN virus was isolated in Vero cell culture from one female I. scapularis and was detected by TaqMan RT-PCR in 24 adult I. scapularis, one D. andersoni, and two D. variabilis adults that had fed to completion as larvae on viremic hosts and as nymphs on naive mice or hamsters. Three species of ixodid ticks acquired WN virus from viremic hosts and transstadially passed the virus, but vector competency was not demonstrated.

Molecular Phylogeny and Evolution of Mosquito Parasitic Microsporidia (Microsporidia: Amblyosporidae)

The Journal of Eukaryotic Microbiology. Jan-Feb, 2004  |  Pubmed ID: 15068270

Amblyospora species and other aquatic Microsporidia were isolated from mosquitoes, black flies, and copepods and the small subunit ribosomal RNA gene was sequenced. Comparative phylogenetic analysis showed a correspondence between the mosquito host genera and their Amblyspora parasite species. There is a clade of Amblyospora species that infect the Culex host group and a clade of Amblyospora that infect the Aedes/Ochlerotatus group of mosquitoes. Parathelohania species, which infect Anopheles mosquitoes, may be the sister group to the Amblyospora in the same way that the Anopheles mosquitoes are thought to be the sister group to the Culex and Aedes mosquitoes. In addition, by sequence analysis of small subunit rDNA from spores, we identified the alternate copepod host for four species of Amblyospora. Amblyospora species are specific for their primary (mosquito) host and each of these mosquito species serves as host for only one Amblyospora species. On the other hand, a single species of copepod can serve as an intermediate host to several Amblyospora species and some Amblyospora species may be found in more than one copepod host. Intrapredatorus barri, a species within a monotypic genus with Amblyospora-like characteristics, falls well within the Amblyospora clade. The genera Edhazardia and Culicospora, which do not have functional meiospores and do not require an intermediate host, but which do have a lanceolate spore type which is ultrastructurally very similar to the Amblyospora spore type found in the copepod, cluster among the Amblyospora species. In the future, the genus Amblyospora may be redefined to include species without obligate intermediate hosts. Hazardia, Berwaldia, Larssonia, Trichotuzetia, and Gurleya are members of a sister group to the Amblyospora clades infecting mosquitoes, and may be representatives of a large group of aquatic parasites.

Prevalence of West Nile Virus in Tree Canopy-inhabiting Culex Pipiens and Associated Mosquitoes

The American Journal of Tropical Medicine and Hygiene. Jul, 2004  |  Pubmed ID: 15238699

Culex pipiens was the dominant mosquito captured in a West Nile virus (WNV) focus in Stratford, Connecticut. More Cx. pipiens were captured in Centers for Disease Control miniature light traps baited with CO(2), quail/hamster traps, and mosquito magnet experimental (MMX) traps placed in the tree canopy than in similar traps placed near the ground. Significantly more Cx. pipiens were captured in MMX traps placed in the canopy than in the other traps tested. Ninety-two percent and 85% of the 206 and 68 WNV isolations were from Cx. pipiens in 2002 and 2003, respectively; 5% and 12% were from Cx. salinarius. Eighty-five percent and 87% of the isolates were from mosquitoes captured in the canopy in each of the two years. The significantly larger numbers of WNV isolates from Cx. pipiens captured in the canopy are attributed to the significantly larger numbers of Cx. pipiens captured in the canopy in comparison to those captured in traps near the ground.

Epidemiology of West Nile Virus in Connecticut: a Five-year Analysis of Mosquito Data 1999-2003

Vector Borne and Zoonotic Diseases (Larchmont, N.Y.). 2004  |  Pubmed ID: 15682518

Two hundred and ten isolations of West Nile virus (WNV) were obtained from 17 mosquito species in six genera in statewide surveillance conducted in Connecticut from June through October, 1999-2003. Culex pipiens (86), Culex salinarius (32), Culex restuans (26), Culiseta melanura (32), and Aedes vexans (12) were implicated as the most likely vectors of WNV in the region based on virus isolation data. Culex pipiens was abundant from July through September and is likely involved in early season enzootic transmission and late season epizootic amplification of the virus in wild bird populations. Epidemic transmission of WNV to humans in urban locales is probable. The abundance of Cx. restuans in June and July and isolations of WNV in early July suggest that this species may play an important role as an enzootic vector involved in early amplification of WNV virus among wild birds. Its involvement as a bridge vector to humans is unlikely. Culex salinarius was the most frequently captured Culex species and was abundant in August and September when virus activity was at its height. Frequent isolations of WNV from this species in September when the majority of human cases were reported in union with its abundance at this time of the year, demonstrated vector competence, and broad feeding habits, make Cx. salinarius a likely bridge vector to humans, horses and other mammals. Multiple isolations WNV from Cs. melanura collected in more rural locales in late August and September, provide supportive evidence to suggest that this predominant avian feeder may play a significant role in epizootic amplification of the virus among wild bird populations in these environs. Aedes vexans was the only species of Aedes or Ochlerotatus from which multiple isolations of WNV were made in more than one year and was among the most frequently trapped and abundant species throughout the season. Since Ae. vexans predominately feeds on mammals it is unlikely to play a significant role in epizootic amplification of WNV, however, because of its abundance and aggressive mammalian and human biting behavior it must receive strong consideration as a bridge vector to humans and horses. The occasional virus isolations obtained from Aedes cinereus (4), Uranotaenia sapphirina (3), Ochlerotatus canadensis (2), Ochlerotatus trivittatus (2), Ochlerotatus sollicitans (2), Ochlerotatus sticticus (2), Psorophora ferox (2), Anopheles punctipennis, Anopheles walkeri, Ochlerotatus cantator, Ochlerotatus taeniorhynchus, and Ochlerotatus triseriatus in conjunction with their inefficient vector competency and host feeding preferences indicate that these species likely play a very minor role in either the enzootic maintenance or epizootic transmission of WNV in this region. The principal foci of WNV activity in Connecticut were identified as densely populated (>3,000 people/mi2) residential communities in coastal Fairfield and New Haven Counties, and in the case of 2002, similar locales in proximity of the city of Hartford in central Hartford County. In almost all instances we observed a correlation both temporally and spatially between the isolation of WNV from field-collected mosquitoes and subsequent human cases in these locales. In most years the incidence of human cases closely paralleled the number of virus isolations made from mosquitoes with both peaks falling in early September. We conclude that the isolation of WNV from field-collected mosquitoes is a sensitive indicator of virus activity that is associated with the risk of human infection that habitually extends from early August through the end of October in Connecticut.

An Update on the Potential of North American Mosquitoes (Diptera: Culicidae) to Transmit West Nile Virus

Journal of Medical Entomology. Jan, 2005  |  Pubmed ID: 15691009

ABSTRACT Since first discovered in the New York City area in 1999, West Nile virus (WNV) has become established over much of the continental United States and has been responsible for >10,000 cases of severe disease and 400 human fatalities, as well as thousands of fatal infections in horses. To develop appropriate surveillance and control strategies, the identification of which mosquito species are competent vectors and how various factors influence their ability to transmit this virus must be determined. Therefore, we evaluated numerous mosquito species for their ability to transmit WNV under laboratory conditions. This report contains data for several mosquito species not reported previously, as well as a summary of transmission data compiled from previously reported studies. Mosquitoes were allowed to feed on chickens infected with WNV isolated from a crow that died during the 1999 outbreak in New York City. These mosquitoes were tested approximately 2 wk later to determine infection, dissemination, and transmission rates. All Culex species tested were competent vectors in the laboratory and varied from highly efficient vectors (e.g., Culex tarsalis Coquillett) to moderately efficient ones (e.g., Culex nigripalpus Theobald). Nearly all of the Culex species tested could serve as efficient enzootic or amplifying vectors for WNV. Several container-breeding Aedes and Ochlerotatus species were highly efficient vectors under laboratory conditions, but because of their feeding preferences, would probably not be involved in the maintenance of WNV in nature. However, they would be potential bridge vectors between the avian-Culex cycle and mammalian hosts. In contrast, most of the surface pool-breeding Aedes and Ochlerotatus species tested were relatively inefficient vectors under laboratory conditions and would probably not play a significant role in transmitting WNV in nature. In determining the potential for a mosquito species to become involved in transmitting WNV, it is necessary to consider not only its laboratory vector competence but also its abundance, host-feeding preference, involvement with other viruses with similar transmission cycles, and whether WNV has been isolated from this species under natural conditions.

Eastern Equine Encephalitis in a Flock of African Penguins Maintained at an Aquarium

Journal of the American Veterinary Medical Association. Jun, 2005  |  Pubmed ID: 15989191

Eastern equine encephalitis (EEE) was diagnosed in a flock of African penguins. Diagnosis was based on history and clinical signs and confirmed via serologic testing, virus isolation, reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and histologic examination. Clinical signs in penguins included anorexia, behavior changes, depression, regurgitation, ataxia, recumbency, and seizures, and some penguins did not have any clinical signs. Mean +/- SD number of days that affected penguins had clinical signs was 12 +/- 5 days. Abnormalities initially detected on CBC included heterophilic leukocytosis and anemia; lymphocytosis and monocytosis were detected later. Plasma biochemical abnormalities included high activities of aspartate amino-transferase and creatine kinase, hyponatremia, hypochloremia, hyperglycemia, and high concentrations of globulin, triglycerides, and cholesterol. Mean +/- SD number of days required for resolution of CBC and plasma biochemical abnormalities was 67 +/- 24 days after the onset of clinical signs. Treatment consisted of supportive therapy. All penguins survived with the exception of one that was euthanatized; histopathologic findings were consistent with encephalitis. Results of RT-PCR assays performed on tissue from the right cerebrum of the penguin that was euthanatized were positive for EEE viral RNA. An inability to isolate virus several weeks after illness suggested successful viral clearance in recovered penguins. To the authors' knowledge, EEE infection in any penguin species has not been reported.

Evolutionary Strategies and Adaptations for Survival Between Mosquito-parasitic Microsporidia and Their Intermediate Copepod Hosts: a Comparative Examination of Amblyospora Connecticus and Hyalinocysta Chapmani (Microsporidia: Amblyosporidae)

Folia Parasitologica. May, 2005  |  Pubmed ID: 16004361

The epizootiology, transmission dynamics, and survival strategies employed by two mosquito-parasitic microsporidia that utilize copepods as intermediate hosts are examined in relation to the biological attributes of their hosts and the environments in which they inhabit. Amblyospora connecticus Andreadis, 1988, a parasite of Ochlerotatus cantator (Coquillett) and Acanthocyclops vernalis (Fischer) is found in an unstable salt marsh environment that is subject to periodic flooding and drying. Both hosts have distinct non-overlapping generations. A. connecticus exhibits a well-defined seasonal transmission cycle that relies heavily on maternal-mediated transovarial transmission by female O. cantator during the summer, and horizontal transmission via the copepod host during the spring (copepod to mosquito) and fall (mosquito to copepod). Its survival strategies include: delayed virulence, low pathogenicity and high tissue specificity that allow for transstadial transmission of horizontally acquired infections and maximum spore production, reliance on living hosts throughout most of its life cycle with overwintering in the copepod, polymorphic development that is well synchronized with host physiology, and production and dissemination of infectious spores that are coincident with the seasonal occurrence of susceptible stages in each host. Hyalinocysta chapmani Hazard et Oldacre, 1975, a parasite of Culiseta melanura (Coquillett) and Orthocyclops modestus (Herrick) is found in a comparatively stable, subterranean habitat that is inundated with water throughout the year. Copepods are omnipresent and C. melanura has overlapping broods. H. chapmani is maintained in a continuous cycle of horizontal transmission between each host throughout the summer and fall but lacks a developmental sequence leading to transovarial transmission in the mosquito host. It relies on living hosts for most of its life cycle and overwinters in diapausing mosquito larvae. Transstadial transmission does not occur and there is no dimorphic development in the mosquito host. The spatial and temporal overlap of both mosquito and copepod hosts during the summer and fall affords abundant opportunity for continuous horizontal transmission and increases the likelihood that H. chapmani will find a target host, thus negating the need for a transovarial route. It is hypothesized that natural selection has favoured the production of meiospores in larval female mosquitoes rather than congenital transfer of infection to progeny via ovarian infection as a strategy for achieving greater transmission success. Analysis of the molecular phylogeny data suggest that (1) transovarial transmission and the developmental sequence leading to ovarian infection have been secondarily lost in H. chapmani, as they occur in all other closely related genera, (2) the ancestral state included complex life cycles involving transovarial transmission and an intermediate host, and (3) mosquito-parasitic microsporidia are adjusting their life cycles to accommodate host ecological conditions.

Isolations of Potosi Virus from Mosquitoes (Diptera: Culicidae) Collected in Connecticut

Journal of Medical Entomology. Sep, 2005  |  Pubmed ID: 16363172

Potosi virus (POTV) (Bunyaviridae: Orthobunyavirus) was first isolated from Aedes albopictus (Skuse) collected in Potosi, MO, in 1989, and subsequent isolations were reported from Illinois, Michigan, Ohio, and the Carolinas. To determine whether the distribution of this virus extends into the northeastern United States, we analyzed arboviruses acquired from mosquitoes collected in Connecticut from 1998 to 2004. In 2001, a bunyavirus was isolated from Aedes vexans (Meigen) that was different from other arboviruses known to occur in Connecticut by cross-neutralization and reverse transcription-polymerase chain reaction (RT-PCR) assays. Nucleotide and encoded amino acid sequences of a portion of the G2 envelope gene were 99 and 100% similar to POTV, respectively, yet distinct from indigenous strains of Jamestown Canyon (JCV), Cache Valley (CVV), and Trivittatus virus (TVTV). Viral isolates obtained from the statewide surveillance program were retested by RT-PCR coupled with restriction enzyme analysis to distinguish POTV from other bunyaviruses. POTV isolates, previously typed by neutralization, were correctly identified by RT-PCR; however, many isolates classified as JCV or CVV by enzyme-linked immunosorbent assay proved to be POTV by molecular assays. In total, 92 strains of POTV were isolated from 12 mosquito species in 2000, 2001, and 2003, whereas POTV was not detected in mosquitoes sampled during 1998, 1999, 2002, and 2004. Viral isolation rates were highest for Anopheles punctipennis (Say) (3.2-11.3 infection rate per 1,000 mosquitoes), whereas the greatest number of isolates came from Ochlerotatus trivittatus (Coquillett) (8-16 isolates). This finding represents the first detection of POTV in the northeastern United States where it infects a diverse array of mosquito species.

Detection of West Nile Virus RNA from the Louse Fly Icosta Americana (Diptera: Hippoboscidae)

Journal of the American Mosquito Control Association. Dec, 2005  |  Pubmed ID: 16506578

West Nile virus (WNV) was detected by Taqman reverse transcription-polymerase chain reaction in 4 of 85 (4.7%) blood-engorged (n = 2) and unengorged (n = 2) Icosta americana (Leach) hippoboscid flies that were collected from wild raptors submitted to a wildlife rehabilitation center in Mercer County, NJ, in 2003. This report represents an additional detection of WNV in a nonculicine arthropod in North America and the first documented detection of the virus in unengorged hippoboscid flies, further suggesting a possible role that this species may play in the transmission of WNV in North America.

Host Feeding Patterns of Culex Mosquitoes and West Nile Virus Transmission, Northeastern United States

Emerging Infectious Diseases. Mar, 2006  |  Pubmed ID: 16704786

To evaluate the role of Culex mosquitoes as enzootic and epidemic vectors for WNV, we identified the source of vertebrate blood by polymerase chain reaction amplification and sequencing portions of the cytochrome b gene of mitochondrial DNA. All Cx. restuans and 93% of Cx. pipiens acquired blood from avian hosts; Cx. salinarius fed frequently on both mammals (53%) and birds (36%). Mixed-blood meals were detected in 11% and 4% of Cx. salinarius and Cx. pipiens, respectively. American robin was the most common source of vertebrate blood for Cx. pipiens (38%) and Cx. restuans (37%). American crow represented <1% of the blood meals in Cx. pipiens and none in Cx. restuans. Human-derived blood meals were identified from 2 Cx. salinarius and 1 Cx. pipiens. Results suggest that Cx. salinarius is an important bridge vector to humans, while Cx. pipiens and Cx. restuans are more efficient enzootic vectors in the northeastern United States.

Molecular Phylogeny and Evolutionary Relationships Among Mosquitoes (Diptera: Culicidae) from the Northeastern United States Based on Small Subunit Ribosomal DNA (18S RDNA) Sequences

Journal of Medical Entomology. May, 2006  |  Pubmed ID: 16739399

The phylogenetic relationships of Culicidae native to the northeastern United States were investigated by analyzing small subunit ribosomal DNA (18S rDNA) sequences obtained from 39 species representing nine genera. Molecular phylogenies were consistent with traditional classifications based on morphological characters except for the placements of Psorophora Robineau-Desvoidy and Uranotaenia Lynch Arribalzaga. In our analyses, 1) Anopheles Meigen was strongly supported as the sister taxon to the remaining Culicidae; 2) Toxorhynchites Theobald was represented as a distinct monophyletic sister group to the Culicinae; 3) Psorophora formed a basal clade to Culiseta Felt, Coquillettidia Dyar, and Culex L. but also was shown as a sister taxon to Aedes Meigen and Ochlerotatus Lynch Arribalzaga; 4) Coquillettidia perturbans (Walker) seems to be a sister group to Culiseta; 5) placement of Uranotaenia was inconclusive and seemed to be either a sister group to the Aedes and Ochlerotatus or a basal taxon to all other culicines; and 6) Aedes and Ochlerotatus formed two separate and distinct clades, providing phylogenetic data consistent with the recent elevation of Ochlerotatus to the generic level as proposed by Reinert (2000). The utility of 18S rDNA for evaluating phylogenetic and evolutionary relationships among mosquito taxa was demonstrated at the genus and species levels. To our knowledge, this study represents the first molecular-based phylogenetic study of mosquito species occurring within this geographic region of North America and contains the largest number of species that have been examined among the genera Aedes and Ochlerotatus.

A New Genetic Variant of La Crosse Virus (bunyaviridae) Isolated from New England

The American Journal of Tropical Medicine and Hygiene. Sep, 2006  |  Pubmed ID: 16968927

La Crosse virus (LACV) is found primarily in the Midwestern and Appalachian regions of the United States where it is a leading cause of mosquito-borne encephalitis in children. To determine whether the distribution of this virus extends further east into New England, we analyzed a bunyavirus that was isolated from a pool of eastern tree-hole mosquitoes, Ochlerotatus triseriatus (= Aedes triseriatus), collected from Fairfield, Connecticut (CT) in 2005. Nucleotide and encoded amino acid sequences from portions of the S, M, and L segments were more similar to the prototype strain of La Crosse virus than that of closely related snowshoe hare virus. Phylogenetic analysis of sequences from the M segment indicated that the CT isolate represents a distinct lineage of La Crosse virus, diverging earliest from other strains found in southeastern, central, and northeastern United States. Despite low sequence homology with other viral strains, the CT isolate was antigenically similar to the prototype strain of LACV by plaque-reduction neutralization tests with polyclonal and monoclonal antibodies. This represents the first isolation of LACV in New England to our knowledge and suggests long-term independent evolution of the CT isolate.

Modeling the Spatial Distribution of Mosquito Vectors for West Nile Virus in Connecticut, USA

Vector Borne and Zoonotic Diseases (Larchmont, N.Y.). 2006  |  Pubmed ID: 16989568

The risk of transmission of West Nile virus (WNV) to humans is associated with the density of infected vector mosquitoes in a given area. Current technology for estimating vector distribution and abundance is primarily based on Centers for Disease Control and Prevention (CDC) light trap collections, which provide only point data. In order to estimate mosquito abundance in areas not sampled by traps, we developed logistic regression models for five mosquito species implicated as the most likely vectors of WNV in Connecticut. Using data from 32 traps in Fairfield County from 2001 to 2003, the models were developed to predict high and low abundance for every 30 x 30 m pixel in the County. They were then tested with an independent dataset from 16 traps in adjacent New Haven County. Environmental predictors of abundance were extracted from remotely sensed data. The best predictive models included non-forested areas for Culex pipiens, surface water and distance to estuaries for Cx. salinarius, surface water and grasslands/agriculture for Aedes vexans and seasonal difference in the normalized difference vegetation index and distance to palustrine habitats for Culiseta melanura. No significant predictors were found for Cx. restuans. The sensitivity of the models ranged from 75% to 87.5% and the specificity from 75% to 93.8%. In New Haven County, the models correctly classified 81.3% of the traps for Cx. pipiens, 75.0% for Cx. salinarius, 62.5% for Ae. vexans, and 75.0% for Cs. melanura. Continuous surface maps of habitat suitability were generated for each species for both counties, which could contribute to future surveillance and intervention activities.

West Nile Virus from Female and Male Mosquitoes (Diptera: Culicidae) in Subterranean, Ground, and Canopy Habitats in Connecticut

Journal of Medical Entomology. Sep, 2006  |  Pubmed ID: 17017241

In total, 93,532 female mosquitoes (Diptera: Culicidae) were captured in traps placed in subterranean (catch basin), ground (approximately 1.5 m above ground), and canopy (approximately 7.0 m above ground) habitats in Stamford and Stratford, CT, during 2003-2005. Culex pipiens L. was the most abundant (64.8%) of the 31 species identified. Significantly greater numbers of Cx. pipiens were captured in canopy-placed mosquito magnet experimental traps, and significantly greater numbers were collected in catch basin-placed (Centers for Disease Control) CDC traps than in CDC traps placed elsewhere. Culex restuans Theobald was captured in significantly greater numbers in traps placed in catch basins. Aedes vexans (Meigen), Aedes cinereus Meigen, and Aedes cantator (Coquillett) were significantly more abundant in ground traps. In total, 429 isolations of West Nile virus (WNV) were made from seven species of mosquitoes from late June through the end of October during 2003 through 2005. Three hundred ninety-eight (92.8%) isolates were from Cx. pipiens. Others were from Cx. restuans (n = 16), Culex salinarius Coquillett (n = 5), Ae. vexans (n = 4), Ae. cantator (n = 3), Aedes triseriatus (Say) (n = 2), and Ae. cinereus (n = 1). Multiple isolates from Cx. pipiens were made each week, primarily during the later part of July through the end of September. Weekly minimum infection rates (MIRs) were lower in 2004 (highest weekly MIR = 7.1) when no human cases were reported in Connecticut in comparison with 2003 and 2005 (highest weekly MIR = 83.9) when human cases were documented. Frequencies of infected pools were significantly higher in Cx. pipiens captured in traps in the canopy and significantly higher in catch basin placed traps than in traps at ground level. The physiological age structure of Cx. pipiens captured in the canopy was significantly different from that of Cx. pipiens collected in catch basins. Invariably, Cx. pipiens captured in the canopy were nulliparous or parous with ovaries in Christophers' stage 2, whereas 58.7% of the females captured in catch basins possessed ovaries filled with mature oocytes in Christophers' stage 5. Our results suggest that females in the canopy are seeking hosts, and after digestion of the bloodmeal and development of mature oocytes, they descend to catch basins for shelter and deposition of eggs. WNV was isolated from three, one, and two pools of male Cx. pipiens captured in catch basin-, ground-, and canopy-placed traps, respectively, and from six nulliparous Cx. pipiens females collected in the canopy. Weekly MIR ranged from 1.2 to 31.1 per 1,000 male specimens. These data show that mosquitoes become infected by means other than by blood feeding, possibly by transovarial transmission. The placement of traps in tree canopies and in catch basins can be used to augment current practices of placement of traps near the ground for surveillance of mosquitoes infected with WNV and for studies of the ecology of WNV.

Identification of Avian- and Mammalian-derived Bloodmeals in Aedes Vexans and Culiseta Melanura (Diptera: Culicidae) and Its Implication for West Nile Virus Transmission in Connecticut, U.S.A

Journal of Medical Entomology. Sep, 2006  |  Pubmed ID: 17017250

To evaluate the host-feeding patterns of Aedes vexans (Meigen) and Culiseta melanura (Coquillett) as secondary vectors of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in Northeastern United States, we identified the source of vertebrate bloodmeals by sequencing portions of the cytochrome b gene of mitochondrial DNA. Analysis of polymerase chain reaction products from a total of 119 Ae. vexans revealed that 92.4% of individuals acquired blood solely from mammalian and 2.5% from avian hosts. Mixed bloodmeals from both avian and mammalian hosts were detected in 5% of individuals of this species. Ae. vexans obtained vertebrate bloodmeals most frequently from white-tailed deer (80%) followed by domestic horse, American robin, eastern cottontail, and domestic cat. In contrast, Cs. melanura fed predominantly on avian species (89.6%) but exhibited some inclination for mammalian blood (4.2%). Individual mosquitoes containing mixed bloodmeals were also identified in 6% of Cs. melanura. American robin was the most common source of vertebrate blood for Cs. melanura (23%), followed by wood thrush and gray catbird. American crow represented only 2% of the bloodmeals identified in Cs. melanura, as was similarly found with other recognized Culex vectors of WNV in the northeast. These findings support the view that Ae. vexans is likely to be a relatively important "bridge vector" to large mammals, including deer and horse, whereas Cs. melanura likely plays a secondary role in enzootic transmission of WNV among free-ranging birds in more rural environs.

Molecular Identification of Blood-meal Sources in Culiseta Melanura and Culiseta Morsitans from an Endemic Focus of Eastern Equine Encephalitis Virus in New York

The American Journal of Tropical Medicine and Hygiene. Dec, 2006  |  Pubmed ID: 17172382

Eastern equine encephalitis (EEE) virus perpetuates in an enzootic cycle involving ornithophilic mosquito vectors, principally Culiseta melanura (Coquillett) and avian amplification hosts. To better understand the role of Cs. melanura and Culiseta morsitans (Theobald) in the epizootiology of EEE virus, we collected blood-fed mosquitoes between 31 May and 15 October 2004 at two sites associated with an EEE virus focus in central New York and identified the source of vertebrate blood by nucleotide sequencing of polymerase chain reaction (PCR) products of the cytochrome b gene. Analysis of 484 Cs. melanura and 122 Cs. morsitans revealed that 94.2% and 86.9%, respectively, acquired blood solely from avian hosts. Blood meals derived exclusively from mammals were detected in 0.8% of Cs. melanura and 1.6% of Cs. morsitans. Individual mosquitoes containing mixed-blood meals from both avian and mammalian hosts were also detected in 5.0% of Cs. melanura and 11.5% of Cs. morsitans. Wood thrush constituted the most common vertebrate host for Cs. melanura (23.6%) and Cs. morsitans (30.9%), followed by American robin, song sparrow, ovenbird, red-eyed vireo, and common yellowthroat. Mammalian-derived blood meals were identified as white-tailed deer, horse, domestic cat, and eastern pipistrelle bat. There were three isolations of EEE virus from Cs. melanura and one from Cs. morsitans. These results suggest that wood thrush and a few other passerine birds may play key roles in supporting EEE virus transmission in the northeast and possibly throughout the geographic range of EEE in North America. The frequency of mammalian feedings also suggests that Cs. melanura and Cs. morsitans may play a role in the transmission of EEE virus to equines, in addition to maintaining enzootic transmission among avian hosts. We report the first isolation of arboviruses from mosquito vectors concomitant with the identifications of their blood meal sources.

The Phylogenetic Position of Ovavesicula Popilliae (Microsporidia) and Its Relationship to Antonospora and Paranosema Based on Small Subunit RDNA Analysis

Journal of Invertebrate Pathology. Nov, 2007  |  Pubmed ID: 17599347

Comparative small subunit rDNA sequence analyses, indicate that Ovavesicula popilliae, a microsporidian parasite of the Japanese beetle, Popillia japonica, represents a distant sister group to Paranosema and Antonospora. These three genera represent a second major group (the Nosema/Vairimorpha clade representing the first) of Microsopridia which infect terrestrial insects, suggesting independent origins for both groups. Phylogenetic analyses of Ovavesicula and other Microsporidia having a multi-sporous sporogony reveal that this condition is found in several unrelated taxa implying either that multi-sporous sporogony is the ancestral condition for Microsporidia or that it has multiple origins.

Host Feeding Pattern of Culex Quinquefasciatus (Diptera: Culicidae) and Its Role in Transmission of West Nile Virus in Harris County, Texas

The American Journal of Tropical Medicine and Hygiene. Jul, 2007  |  Pubmed ID: 17620633

The vertebrate hosts of 672 blood-engorged Culex quinquefasciatus Say, collected in Harris County, Texas, during 2005, were identified by nucleotide sequencing PCR products of the cytochrome b gene. Analysis revealed that 39.1% had acquired blood from birds, 52.5% from mammals, and 8.3% were mixed avian and mammalian blood meals. Most frequent vertebrate hosts were dog (41.0%), mourning dove (18.3%), domestic cat (8.8%), white-winged dove (4.3%), house sparrow (3.2%), house finch (3.0%), gray catbird (3.0%), and American robin (2.5%). Results are interpreted in conjunction with concurrent avian and mosquito West Nile virus (WNV) surveillance activities in Harris County. We conclude that Cx. quinquefasciatus is an opportunistic feeder and principal mosquito vector of WNV in this metropolitan area; however, transmission by other mosquito species or by other modes of infection, such as ingestion, must account for the high WNV infection rates among local blue jays and American crows.

A Two-year Evaluation of Elevated Canopy Trapping for Culex Mosquitoes and West Nile Virus in an Operational Surveillance Program in the Northeastern United States

Journal of the American Mosquito Control Association. Jun, 2007  |  Pubmed ID: 17847845

The effectiveness of CO2-baited Centers for Disease Control and Prevention miniature light traps elevated in the tree canopy (approximately 7.6 m) was compared with light traps placed at ground level (approximately1.5 m) and grass-sod infused gravid traps for collecting Culex pipiens, Culex restuans, and Culex salinarius and detecting West Nile virus (WNV) activity in an operational surveillance program that encompassed 12 ecologically diverse sites in Connecticut in 2004 and 2005. More than twice as many Cx. pipiens were collected on average in light traps suspended in the tree canopy than in either light or gravid traps placed at ground level. This difference was generally restricted to those collection sites where markedly greater numbers of Cx. pipiens were collected with all trapping methods but was not associated with site-specific urbanization indices. Culex restuans was not preferentially attracted to light traps suspended in the tree canopy. No differences in the overall abundance of this species were recorded with either of the 2 trapping procedures, but both light traps were more effective than the gravid traps. Culex salinarius was significantly more attracted to ground-based light traps than traps suspended in the tree canopy, while gravid traps were ineffective at all sites regardless of the level of urbanization or any other specific land-use characteristic. CO2-baited light traps placed in the tree canopy were generally superior to ground-based light traps for detecting WNV in Cx. pipiens. West Nile virus-infected females were collected more regularly, and the frequency of infected pools was significantly greater. Twofold higher minimum field infection rates (maximum likelihood estimation [MLE] = 6.7 vs. 3.0 per 1,000 mosquitoes) were also recorded from canopy collections of this species, and virus was detected in canopy-collected females several weeks before it was detected in collections from light traps at ground level. We conclude that the use of CO2-baited light traps placed in the tree canopy for targeted trapping of Cx. pipiens and subsequent detection of WNV are likely to yield better overall results than light traps placed at ground level in this region of the northeastern United States. The virus isolation data obtained from Cx. pipiens collected in gravid traps compared favorably both temporally and spatially with results from canopy trap collections. There were no significant differences in the overall frequency of WNV-infected pools or MLEs for Cx. pipiens, but fewer total WNV isolations were made from Cx. pipiens collected in the gravid traps and virus was detected more infrequently. Results reaffirmed the utility of gravid traps as effective surveillance tools for detection of WNV in Cx. pipiens in the northeastern United States. However, findings also demonstrated that CO2-baited light traps placed in the tree canopy provided more consistent results where weekly detection of virus amplification is a critical objective. The comparative effectiveness of ground- and canopy-based light traps for detection of WNV-infected Cx. restuans and Cx. salinarius was inconclusive owing to the limited number of virus isolations that were made from these species during the 2 years of study. However, WNV virus isolations were made several weeks earlier and more frequently from Cx. restuans collected in traps placed in the canopy rather than at ground level in 2004. Results support the view that ground-based light traps are more effective for detection of WNV in Cx. salinarius.

Microsporidian Parasites of Mosquitoes

Journal of the American Mosquito Control Association. 2007  |  Pubmed ID: 17853594

Nocturnal Activity of Mosquitoes (Diptera: Culicidae) in a West Nile Virus Focus in Connecticut

Journal of Medical Entomology. Nov, 2007  |  Pubmed ID: 18047212

Six species of mosquitoes (Diptera: Culicidae) were collected in sufficient numbers for analysis in segregating traps set at 2-h intervals by using CO2 and light as attractants in a West Nile virus (family Flaviviridae, genus Flavivirus, WNV) focus in Stratford, CT. The Kolmogorov-Smirnov one-sided test for two samples was used to analyze the data. Mosquito activity began shortly before sunset and continued until shortly after sunrise the next morning. All species had geometric means that were significantly higher during the 2-h period shortly after sunset compared with the 2-h collection before sunset. Species, known to be naturally infected with WNV, were often attracted to these traps in about equal numbers at 2-h intervals during an 8- to 10-h period commencing shortly after sunset. Differences of geometric means were not significant among the four or five 2-h collection periods commencing at sunset for Aedes vexans (Meigen), Culex salinarius Coquillett, and Aedes cinereus Meigen. Aedes cantator (Coquillett) had a significantly higher geometric mean for the 2-h period commencing at sunset, and Coquillettidia perturbans (Walker) was captured in significantly greater numbers during the 2-h period starting at sunset compared with periods commencing 6 h after sunset. Culex pipiens L. tended to have an activity pattern that was primarily nocturnal. Time of night, not meteorological conditions, was the most important factor in determining the nightly variation in the number of trapped mosquitoes. Parity rates of Cx. pipiens collected during specific periods of the night were not significant. In total, 39 isolations of WNV were made from seven species collected primarily during periods of total darkness. Humans are at risk of being bitten by infected mosquitoes throughout the night.

Genetic Relationships of Jamestown Canyon Virus Strains Infecting Mosquitoes Collected in Connecticut

The American Journal of Tropical Medicine and Hygiene. Dec, 2007  |  Pubmed ID: 18165540

Jamestown Canyon virus (JCV) (family Bunyaviridae, genus Orthobunyavirus) is maintained in a mosquito-deer cycle and has been implicated in the etiology of meningitis and encephalitis with human cases reported from Ontario, Canada, Michigan, Connecticut, and New York. Despite the recognition of symptomatic cases in the northeastern United States, little is known about the genetic relationships of JCV variants circulating in this region. Accordingly, we compared the phylogenetic relationships of 56 JCV isolates from mosquitoes collected in Connecticut over a 40-year period to evaluate their evolutionary history and characterize patterns of genetic diversity in the state. We distinguished at least two major lineages in Connecticut on the basis of phylogenetic reconstruction of small (S), medium (M), and large (L) segment nucleotide sequences. Viruses representing each lineage infected a diverse group of mosquito species over multiple years of sampling and appeared to be geographically structured along an east-west axis. One of these lineages was detected in Connecticut from 1966 through 2006 with few mutational changes accumulating over time. Phylogenetic trees generated from portions of the M and L segments yielded different topologies from S segment sequences as three clades became consolidated into two. Although direct evidence for genetic exchange by reassortment was lacking among cocirculating strains in Connecticut, molecular trees from S, M, and L segments were incongruent, which suggests a distinct evolutionary history or process for each genomic segment. These results suggest that JCV variants are stably maintained in Connecticut where they infect a wide diversity of mosquito species.

A Rift Valley Fever Risk Surveillance System for Africa Using Remotely Sensed Data: Potential for Use on Other Continents

Veterinaria Italiana. Jul-Sep, 2007  |  Pubmed ID: 20422546

The authors developed a monitoring and risk mapping system using normalized difference vegetation index (NDVI) times series data derived from the advanced very high resolution radiometer (AVHRR) instrument on polar orbiting national oceanographic and atmospheric administration (NOAA) satellites to map areas with a potential for a Rift Valley fever (RVF) outbreaks in sub-Saharan Africa. This system is potentially an important tool for local, national and international organisations involved in the prevention and control of animal and human disease, permitting focused and timely implementation of disease control strategies several months before an outbreak. We are currently developing a geographic information system (GIS)-based remotely sensed early warning system for potential RVF vectors in the United States. Forecasts of the potential emergence of mosquito vectors will be disseminated throughout the United States, providing several months' warning in advance of potentially elevated mosquito populations. This would allow timely, targeted implementation of mosquito control, animal quarantine and vaccine strategies to reduce or prevent animal and human disease.

Isolations of Jamestown Canyon Virus (Bunyaviridae: Orthobunyavirus) from Field-collected Mosquitoes (Diptera: Culicidae) in Connecticut, USA: a Ten-year Analysis, 1997-2006

Vector Borne and Zoonotic Diseases (Larchmont, N.Y.). Apr, 2008  |  Pubmed ID: 18386967

Jamestown Canyon virus (JCV) (Bunyaviridae: Orthobunyavirus) is a mosquito-borne zoonosis belonging to the California serogroup. It has a wide geographic distribution, occurring throughout much of temperate North America. White-tailed deer, Odocoileus virginianus are the principal amplification hosts, and boreal Aedes and Ochlerotatus mosquitoes are the primary vectors. A 10-year study was undertaken to identify potential mosquito vectors in Connecticut, quantify seasonal prevalence rates of infection, and define the geographic distribution of JCV in the state as a function of land use and white-tailed deer populations, which have increased substantially over this period. Jamestown Canyon virus was isolated from 22 mosquito species. Five of them, Ochlerotatus canadensis, Oc. cantator, Anopheles punctipennis, Coquillettidia perturbans, and Oc. abserratus were incriminated as the most likely vectors, based on yearly isolation frequencies and the spatial geographic distribution of infected mosquitoes. Jamestown Canyon virus was isolated from Oc. canadensis more consistently and from a greater range of collection sites than any other species. Frequent virus isolations were also made from Aedes cinereus, Aedes vexans, and Oc. sticticus, and new North American isolation records were established for Anopheles walkeri, Culex restuans, Culiseta morsitans, Oc. sticticus, Oc. taeniorhynchus, and Psorophora ferox. Other species from which JCV was isolated included C. melanura, Oc. aurifer, Oc. communis, Oc. excrucians, Oc. provocans, Oc. sollicitans, Oc. stimulans, Oc. triseriatus, and Oc. trivittatus. Jamestown Canyon virus was widely distributed throughout Connecticut and found to consistently circulate in a diverse array of mosquito vectors. Infected mosquitoes were collected from June through September, and peak infection rates paralleled mosquito abundance from mid-June through mid-July. Infection rates in mosquitoes were consistent from year to year, and overall virus activity was directly related to local mosquito abundance. Infected mosquitoes were equally distributed throughout the state, irrespective of land use, and infection rates were not directly associated with the abundance of white-tailed deer, possibly because of their saturation throughout the region.

Tracking Eastern Equine Encephalitis Virus Perpetuation in the Northeastern United States by Phylogenetic Analysis

The American Journal of Tropical Medicine and Hygiene. Aug, 2008  |  Pubmed ID: 18689638

Epidemics and epizootics of eastern equine encephalitis virus (EEEV) occur sporadically in temperate regions where transmission is seasonal from late summer to early fall. These outbreaks may derive from virus that overwinters locally or perhaps results from reintroduction of virus from other sites. To evaluate these possibilities, we compared the phylogenetic relationships of EEEV isolates obtained from mosquitoes collected during statewide arbovirus surveillance in Connecticut, in addition to isolates from concurrent outbreaks in southern New Hampshire and upstate New York. In Connecticut, viral isolates grouped into temporally discrete clades by year of isolation or over 2 years of sampling. Two or more clades arose in 2000, 2001, 2003, 2004, and 2006, possibly the result of separate introduction events into the state, whereas viruses from upstate New York and New Hampshire segregated into single clades that persisted for 2 or more years. New Hampshire viruses shared recent common ancestry to those isolated in Connecticut suggesting viral dispersal among these regions. These results provide additional evidence for independent episodes of EEEV overwintering in northern foci.

Molecular and Ultrastructural Characterization of Andreanna Caspii N. Gen., N. Sp. (Microsporida: Amblyosporidae), a Parasite of Ochlerotatus Caspius (Diptera: Culicidae)

Journal of Invertebrate Pathology. Nov, 2008  |  Pubmed ID: 18700147

A new genus and species of microsporidia, Andreanna caspii n. gen., n. sp. is described from the mosquito, Ochlerotatus caspius (Pallas) based on ultrastructural morphology, developmental characteristics, and comparative sequence analyses of the small subunit (SSU) ribosomal DNA (rDNA). Parasite development is confined to fat body tissue and infected larvae appear swollen with dull white masses within the thorax and abdomen. Meronts have diplokaryotic nuclei and are delineated by a simple plasmalemma contiguous with the host cell cytoplasm. Merogony occurs by synchronous binary division followed by cytokinesis. Diplokaryotic sporonts undergo meiosis and synchronous nuclear division forming sporogonial plasmodia with two, four and eight nuclei enclosed within a persistent sporophorous vesicle. Cytokinesis of sporogonial plasmodia results in the formation of eight uninucleate spores. The episporontal space of early sporonts is filled with a homogeneous accumulation of electron dense granular inclusions and ovoid vesicles of various dimensions, transforming into an interwoven matrix during the initial phase of sporogenesis. Spores are oval, uninucleate and measure 4.8+/-0.3 x 3.1+/-0.4 microm (fixed). The spore wall is 260 microm thick with an irregular exospore consisting of two layers (150-170 microm) and a thinner endospore (90-100 microm). The anchoring disk is well developed and is contiguous with a lamellar polaroplast that occupies the anterior third of the spore and possess more narrow lamellae on the posterior end. The polar filament is gradually tapered and arranged in a single row consisting of six coils ranging from 180 to 150 microm in diameter. The posterior vacuole (posterosome) is moderately sized and filled with a matrix of moderate electron density. Phylogenetic analysis of SSU rDNA from A. caspii and 30 other species of microsporidia including 11 genera parasitic in mosquitoes using maximum parsimony, neighbor joining and maximum likelihood methods showed A. caspii to be a sister group to the clade containing all of the Amblyospora species, including Culicospora, Edhazardia and Intrapredatorus, as well as Culicosporella and Hyalinocysta thus providing strong support for establishment of Andreanna as a separate genus.

Genetic Insights into the Population Structure of Culex Pipiens (Diptera: Culicidae) in the Northeastern United States by Using Microsatellite Analysis

The American Journal of Tropical Medicine and Hygiene. Oct, 2008  |  Pubmed ID: 18840738

Members of the Culex pipiens complex are considered as biting nuisance and vectors of important arbo-viruses including West Nile virus (WNV). To analyze the genetic structure of urban and rural populations of Cx. pipiens form pipiens and gain insights into behavioral implications, mosquitoes were collected from established WNV transmission foci in Connecticut from October 2006 through October 2007, examined by using microsatellite markers, and compared with other populations from neighboring states in the northeastern United States. The mean numbers of alleles per locus for the aboveground Cx. pipiens form pipiens populations ranged from 11.5 +/- 2.3 to 13.2 +/- 2.4 and were not significantly different. In contrast, Cx. pipiens form molestus had greatly reduced allelic diversities with an average of 4.4 +/- 1.2 alleles per locus, which was significantly lower than that of any of the Cx. pipiens form pipiens populations analyzed. We did not detect significant genetic differences between urban and rural populations of Cx. pipiens form pipiens from Connecticut nor did we observe temporal genetic changes. However, in a comparative analysis with populations of neighboring states, New Jersey, New York, and Massachusetts, genetic variations associated with geographic distance were identified. In the analyses of Bayesian clustering and principal component analysis, we identified two clusters separating Cx. pipiens form molestus from Cx. pipiens form pipiens populations, indicating that Cx. pipiens form molestus was genetically distinct from any of the Cx. pipiens form pipiens populations examined during this study.

Host-feeding Patterns of Potential Mosquito Vectors in Connecticut, U.S.A.: Molecular Analysis of Bloodmeals from 23 Species of Aedes, Anopheles, Culex, Coquillettidia, Psorophora, and Uranotaenia

Journal of Medical Entomology. Nov, 2008  |  Pubmed ID: 19058640

We evaluated the blood-feeding patterns in several mosquito species that may serve as vectors of disease agents in the northeastern United States. Blood-fed mosquitoes were collected from 91 different sites throughout Connecticut over a 6-yr period (June-October 2002-2007), and the host-feeding patterns of 23 mosquito species representing six genera were examined by using a polymerase chain reaction-based assay and sequencing portions of the cytochrome b gene of mitochondrial DNA. This study was part of a statewide surveillance program and for some of the mosquito species a limited number of specimens were examined [e.g., Aedes communis (De Geer) (1), Anopheles barberi Coquillett (1), Uranotaenia sapphirina (Osten Sacken) (5)]. With the exception of Culex territans Walker that acquired bloodmeals from all four classes of vertebrates--birds, reptiles, amphibians, and mammals--all species of Aedes, Anopheles, Coquillettidia, Psorophora, and to a lesser degree, Uranotaenia, were found to feed predominately upon mammalian hosts. Fourteen mammalian species were identified as sources of blood, but the majority of feedings were taken from the white-tailed deer, Odocoileus virginianus. Human-derived bloodmeals were identified from 13 of the 23 mosquito species. Limited avian-derived bloodmeals were detected in Aedes canadensis (Theobald), Aedes cantator (Coquillett), Aedes cinereus Meigen, Aedes triseriatus (Coquillett), Aedes trivittatus (Coquillett), Coquillettidia perturbans (Walker) Cx. territans, Psorophoraferox (von Humboldt), and Ur. sapphirina. American robin, Turdus migratorius, was the most common source of avian blood, followed by a few other mostly Passeriformes birds. We conclude that the white-tailed deer serve as the main vertebrate host for these mammalophilic mosquitoes in this region of the United States. This feeding pattern supports enzootic amplification of arboviruses, including Jamestown Canyon, Cache Valley, and Potosi viruses that perpetuate in cervid hosts. Occasional feeding on avian hosts suggests that some of these mosquito species, such as Cq. perturbans, also could facilitate transmission of West Nile and eastern equine encephalitis viruses from viremic birds to mammalian hosts.

Genetic Variation Associated with Mammalian Feeding in Culex Pipiens from a West Nile Virus Epidemic Region in Chicago, Illinois

Vector Borne and Zoonotic Diseases (Larchmont, N.Y.). Dec, 2009  |  Pubmed ID: 19281434

Mosquitoes of the Culex pipiens complex are important vectors of West Nile virus in the United States. We examined the genetic variations of Cx. pipiens mosquitoes from Chicago, Illinois that were determined to be principally ornithophilic but exhibited a relatively higher inclination for mammalian hosts including humans. Microsatellite analysis of 10 polymorphic markers was performed on 346 engorged Cx. pipiens specimens with identified avian or mammalian blood meals. Our results indicated that there were no significant differences in allelic richness, the pattern of conformity to Hardy-Weinberg equilibrium, and linkage disequilibrium, nor was there overall genetic differentiation between specimens with avian- and mammalian-derived blood meals. However, Cx. pipiens form pipiens with mammalian- (including human-) derived blood meals had significantly higher ancestry (p < 0.001) and proportion of hybrids (p < 0.01) from the Cx. pipiens form molestus (population from New York City) than did those with avian-derived blood meals. By contrast, there were no significant differences in the ancestry (p > 0.05) and the proportion of hybrids (p > 0.05) from Cx. quinquefasciatus (population from Harris Country, Texas). No temporal genetic variation was detected in accordance with the observation that there was no shift in blood feeding from birds to mammals. The results of this study in conjunction with regional host-feeding behavior suggest that the probability of genetic ancestry from Cx. pipiens f. molestus may predispose mosquitoes to feed more readily on mammals; however, the genetic mechanisms are unknown.

Failure of Aedes Albopictus to Overwinter Following Introduction and Seasonal Establishment at a Tire Recycling Plant in the Northeastern USA

Journal of the American Mosquito Control Association. Mar, 2009  |  Pubmed ID: 19432065

In July 2006, an introduction of the Asian tiger mosquito, Aedes albopictus, was documented for the first time at a commercial tire recycling plant in northeastern Connecticut, USA. The introduction likely occurred via transport of infested tires originating from northern New Jersey or metropolitan New York City. Efforts were made to determine seasonal establishment and overwintering success by assessing adult biting and oviposition activity in the surrounding woodlands. The first adult female was collected in a CO2-baited Mosquito Magnet Liberty trap within the confines of the tire plant during the week of July 28. Additional females were collected intermittently thereafter through October 16. Host-seeking female Ae. albopictus attempting to alight on human subjects and larvae hatching from eggs collected in ovitraps placed in the woodlands surrounding the tire plant were detected weekly from August 21 through October 2, denoting seasonal establishment in the adjoining woodlands. However, no larvae of Ae. albopictus were recovered from eggs collected in ovitraps that were placed in the surrounding woodlands or in traps placed 1.0-1.6 km away, nor were any host-seeking females detected by human subjects the following season (July to October 2007), indicating that the species did not survive winter conditions to enable successful colonization. The failure of Ae. albopictus to overwinter and establish itself in the forested woodlands following several weeks of seasonal breeding and oviposition during the summer and early fall were most likely due to winter egg mortality, interspecific competition from Aedes triseriatus and Aedes japonicus, and/or other ecological barriers. Permanent establishment of Ae. albopictus in New England is unlikely despite the recurring importation of infested used tires into recycling facilities. However, continued monitoring of such facilities for potential reinvasion is warranted especially in urban/suburban environs where global warming and milder winter temperatures may provide more suitable conditions in the future for colonization.

Factors Affecting Horizontal Transmission of the Microsporidium Amblyospora Albifasciati to Its Intermediate Copepod Host Mesocyclops Annulatus

Journal of Invertebrate Pathology. Jul, 2009  |  Pubmed ID: 19465025

Factors that directly impact horizontal transmission of the microsporidium Amblyospora albifasciati to its intermediate copepod host, Mesocyclops annulatus were examined in laboratory bioassays. Results were evaluated in relation to life history strategies that facilitate persistence of the parasite in natural populations of its definitive mosquito host, Ochlerotatus albifasciatus. A moderately high quantity of meiospores from mosquito larvae was required to infect adult female copepods; the IC50 was estimated at 3.6 x 10(4) meiospores/ml. Meiospore infectivity following storage at 25 degrees C was detected up to 30 days, while meiospores stored at 4 degrees C remained infectious to copepods for 17 months with virtually no decline in infectivity. Uninfected female M. annulatus are long-lived; no appreciable mortality was observed in field-collected individuals for 26 days, with a few individuals surviving up to 70 days. The pathological impact of A. albifasciati infection on M. annulatus resulted in a 30% reduction in survivorship after 7 days followed by gradual progressive mortality with no infected individuals surviving more than 40 days. This moderate level of pathogenicity allows for a steady continual release of spores into the environment where they may be ingested by mosquito larvae. Infected female copepods survived in sediment under conditions of desiccation up to 30 days, thus demonstrating their capacity to function as a link for maintaining A. albifasciati between mosquito generations following periods of desiccation. The susceptibility of late stage copepodid M. annulatus to meiospores of A. albifasciati and subsequent transstadial transmission of infection to adult females was established.

Human Bloodfeeding by the Recently Introduced Mosquito, Aedes Japonicus Japonicus, and Public Health Implications

Journal of the American Mosquito Control Association. Jun, 2009  |  Pubmed ID: 19653507

Knowledge of the host-feeding behavior and extent of interactions with human hosts are important in evaluating the role and vector potential of invasive mosquitoes in transmission of native arboviruses. We collected blood-engorged females of the recently established exotic species Aedes japonicus japonicus from sites in New Jersey during 2000 to 2007 and identified the sources of vertebrate blood meals by sequencing portions of the cytochrome b gene of mitochondrial DNA. Over 1/3 (36%, n = 36) of the engorged mosquitoes acquired blood meals from humans. Other mammalian hosts included white-tailed deer (53%), fallow deer (5%), horse (3%), and Virginia opossum (3%). No avian, amphibian, reptilian, or mixed blood meals were identified. Our detection of a comparatively high prevalence of human bloodfeeding in Ae. j. japonicus in association with its local abundance, vector competence, and repeated detection of West Nile virus from field-collected specimens illustrates the potential for this invasive mosquito to serve as a "bridge" vector in transmission of West Nile and other mosquito-borne viruses in North America.

Avian Host-selection by Culex Pipiens in Experimental Trials

PloS One. 2009  |  Pubmed ID: 19924251

Evidence from field studies suggests that Culex pipiens, the primary mosquito vector of West Nile virus (WNV) in the northeastern and north central United States, feeds preferentially on American robins (Turdus migratorius). To determine the contribution of innate preferences to observed preference patterns in the field, we conducted host preference trials with a known number of adult female C. pipiens in outdoor cages comparing the relative attractiveness of American robins with two common sympatric bird species, European starling, Sternus vulgaris and house sparrow, Passer domesticus. Host seeking C. pipiens were three times more likely to enter robin-baited traps when with the alternate host was a European starling (n = 4 trials; OR = 3.06; CI [1.42-6.46]) and almost twice more likely when the alternative was a house sparrow (n = 8 trials; OR = 1.80; CI = [1.22-2.90]). There was no difference in the probability of trap entry when two robins were offered (n = 8 trials). Logistic regression analysis determined that the age, sex and weight of the birds, the date of the trial, starting-time, temperature, humidity, wind-speed and age of the mosquitoes had no effect on the probability of a choosing a robin over an alternate bird. Findings indicate that preferential feeding by C. pipiens mosquitoes on certain avian hosts is likely to be inherent, and we discuss the implications innate host preferences may have on enzootic WNV transmission.

Avian Communal Roosts As Amplification Foci for West Nile Virus in Urban Areas in Northeastern United States

The American Journal of Tropical Medicine and Hygiene. Feb, 2010  |  Pubmed ID: 20134014

West Nile virus (WNV) perpetuates in an enzootic transmission cycle involving Culex mosquitoes and virus-competent avian hosts. In the northeastern United States, the enzootic vectors, Cx. pipiens and Cx. restuans, feed preferentially on American robins (Turdus migratorius), suggesting a key role for this bird species in the WNV transmission cycle. We examined the role of American robin communal roosts as virus amplification foci in greater New Haven, Connecticut. Robin communal roosts were located by radio tracking. After mid-August, when most robins were using the roosts, Cx. pipiens and Cx. restuans fed often on robins and were significantly more infected with WNV at communal roosts than at non-roosting sites. We also identified 6.4% human-derived blood meals in Aedes vexans in communal roosts. Our results indicate that communal roosts act as late-season amplification foci facilitating transmission to humans because of high infection rates, high abundance, and feeding patterns of enzootic and bridge vectors.

Evidence for Reduction of Native Mosquitoes with Increased Expansion of Invasive Ochlerotatus Japonicus Japonicus (Diptera: Culicidae) in the Northeastern United States

Journal of Medical Entomology. Jan, 2010  |  Pubmed ID: 20180307

Ochlerotatus japonicus japonicus (Theobald) is an invasive mosquito native to Japan, Korea, and eastern China. The species was first detected in the northeastern United States in 1998 and has rapidly spread throughout much of eastern North America. In addition to used tire casings, Oc. j. japonicus develops in a wide variety of artificial and natural container habitats, especially rock pools along stream beds. In an effort to evaluate the invasion success and impact of Oc. j. japonicus on populations of native container dwelling species, waste tire disposal sites and natural rock pool habitats were sampled for mosquito larvae throughout Connecticut in 2005, and data were compared with results from prior surveys made in 1987 and 1999. Oc. j. japonicus was the predominant species collected at the waste tire disposal sites regardless of surrounding landscape features, accounting for 55.9% of all larvae. A comparison with collections from prior surveys revealed a 90% reduction in the relative abundance of larval populations of Oc. triseriatus (Say) and significant reductions among larval populations of Oc. atropalpus (Coquillett) and Culex restuans Theobald. Oc. j. japonicus was also the most abundant mosquito collected in rock pool habitats, accounting for nearly 80% of all collected larvae, except where water temperatures exceeded 30 degrees C. This was concomitant with significant declines in the relative abundance of Oc. atropalpus and Cx. restuans. We conclude that Oc. j. japonicus is a potentially effective competitor in rock pool and tire environments and may be responsible for reducing populations of several native species occupying these habitats through interspecific competition for limited resources. The exclusion of Oc. j. japonicus from warm water pools further suggests that a temperature barrier may exist for Oc. j. japonicus and that populations may not be able to colonize southern regions of the United States with relatively high summer temperatures.

Phylogenetic Position of Octosporea Muscaedomesticae (Microsporidia) and Its Relationship to Octosporea Bayeri Based on Small Subunit RDNA Analysis

Journal of Invertebrate Pathology. Nov, 2010  |  Pubmed ID: 20688077

Comparative phylogenetic analysis of the small subunit rDNA sequence of Octosporea muscaedomesticae (Flu, 1911) (type species) (Microsporidia) isolated from the blowfly Phormia regina (Diptera:Calliphoridae) is presented. Neighbor Joining bootstrap, Maximum Parsimony and Maximum Likelihood analyses with 38 microsporidian taxa representing five major clades of Microsporidia placed O. muscaedomesticae on a separate branch within a clade containing parasites of freshwater hosts. O. muscaedomesticae differed from Octosporea bayeri, a parasite of the microcrustacean, Daphnia magna (Cladocera:Daphniidae) by 29% demonstrating that the latter microsporidium is not closely related to the type species at the generic level, and should not be placed within the genus Octosporea, a conclusion that is further supported by morphological and developmental differences. Considering the number of disparately related hosts from which Octosporea species have been previously described based mostly on developmental and morphological characters it is likely that many will not fit the current definition of the genus, and it is possible that molecular analysis of these species will show that this genus as defined represents a polyphyletic grouping of unrelated taxa.

Studies on Hibernating Populations of Culex Pipiens from a West Nile Virus Endemic Focus in New York City: Parity Rates and Isolation of West Nile Virus

Journal of the American Mosquito Control Association. Sep, 2010  |  Pubmed ID: 21033052

A 3-year study was undertaken to examine the parity status, survival, and prevalence of West Nile virus (WNV) in overwintering populations of Culex pipiens pipiens collected from a hibernaculum located in a WNV endemic region in New York City. Nearly 6,000 females were collected from December through April. Parity rates were highest among females collected in December and January, ranging from 12.3% to 21.9%, depending on the year. In each year of the study, the proportion of parous females declined significantly during the course of the winter; the percentage of parous females found in April ranged from 0.9% to 10%. Results provide unequivocal evidence that parous Cx. p. pipiens females from this region of the northeastern US enter hibernacula in the fall in comparatively high proportions not previously recognized for this species, and while these females experience significant mortality during the winter, some survived to April to emerge in the spring. The absence of any detectible blood remnants in overwintering females reaffirms that blood feeding does not occur among diapausing females during the winter. The possibility that a portion of the diapausing population may be autogenous as a result of hybridization with sympatric belowground populations of Cx. p. pipiens "form molestus" is discussed. A single isolation of WNV was obtained in Vero cell culture from a pool of 50 females collected on January 11, 2007, representing an infection prevalence of 0.07% in the overwintering population in 2007 (n = 1,370 mosquitoes, 33 pools). No isolations of WNV were made from mosquitoes collected in 2008 (n = 1,870 mosquitoes, 190 pools) or 2009 (n = 1,767 mosquitoes, 184 pools). Findings provide further evidence for local overwintering of WNV in diapausing Cx. p. pipiens, albeit at very low rates, consistent with the paucity of WNV-positive mosquitoes detected in June and early July despite the emergence of females from hibernacula in early May in this region.

Vector-host Interactions Governing Epidemiology of West Nile Virus in Southern California

The American Journal of Tropical Medicine and Hygiene. Dec, 2010  |  Pubmed ID: 21118934

Southern California remains an important focus of West Nile virus (WNV) activity, with persistently elevated incidence after invasion by the virus in 2003 and subsequent amplification to epidemic levels in 2004. Eco-epidemiological studies of vectors-hosts-pathogen interactions are of paramount importance for better understanding of the transmission dynamics of WNV and other emerging mosquito-borne arboviruses. We investigated vector-host interactions and host-feeding patterns of 531 blood-engorged mosquitoes in four competent mosquito vectors by using a polymerase chain reaction (PCR) method targeting mitochondrial DNA to identify vertebrate hosts of blood-fed mosquitoes. Diagnostic testing by cell culture, real-time reverse transcriptase-PCR, and immunoassays were used to examine WNV infection in blood-fed mosquitoes, mosquito pools, dead birds, and mammals. Prevalence of WNV antibodies among wild birds was estimated by using a blocking enzyme-linked immunosorbent assay. Analyses of engorged Culex quinquefasciatus revealed that this mosquito species acquired 88.4% of the blood meals from avian and 11.6% from mammalian hosts, including humans. Similarly, Culex tarsalis fed 82% on birds and 18% on mammals. Culex erythrothorax fed on both birds (59%) and mammals (41%). In contrast, Culex stigmatosoma acquired all blood meals from avian hosts. House finches and a few other mostly passeriform birds served as the main hosts for the blood-seeking mosquitoes. Evidence of WNV infection was detected in mosquito pools, wild birds, dead birds, and mammals, including human fatalities during the study period. Our results emphasize the important role of house finches and several other passeriform birds in the maintenance and amplification of WNV in southern California, with Cx. quinquefasciatus acting as both the principal enzootic and "bridge vector" responsible for the spillover of WNV to humans. Other mosquito species, such as Cx. tarsalis and Cx. stigmatosoma, are important but less widely distributed, and also contribute to spatial and temporal transmission of WNV in southern California.

Eastern Equine Encephalitis Virus in Mosquitoes and Their Role As Bridge Vectors

Emerging Infectious Diseases. Dec, 2010  |  Pubmed ID: 21122215

Eastern equine encephalitis virus (EEEV) is maintained in an enzootic cycle involving Culiseta melanura mosquitoes and avian hosts. Other mosquito species that feed opportunistically on mammals have been incriminated as bridge vectors to humans and horses. To evaluate the capacity of these mosquitoes to acquire, replicate, and potentially transmit EEEV, we estimated the infection prevalence and virus titers in mosquitoes collected in Connecticut, USA, by cell culture, plaque titration, and quantitative reverse transcription-PCR. Cs. melanura mosquitoes were the predominant source of EEEV (83 [68%] of 122 virus isolations) and the only species to support consistently high virus titers required for efficient transmission. Our findings suggest that Cs. melanura mosquitoes are primary enzootic and epidemic vectors of EEEV in this region, which may explain the relative paucity of human cases. This study emphasizes the need for evaluating virus titers from field-collected mosquitoes to help assess their role as vectors.

Control of Mosquitoes in Catch Basins in Connecticut with Bacillus Thuringiensis Israelensis, Bacillus Sphaericus, [corrected] and Spinosad

Journal of the American Mosquito Control Association. Mar, 2011  |  Pubmed ID: 21476447

Catch basins are a major source of Culex pipiens pipiens, Cx. restuans, and Aedes japonicus in northeastern USA. VectoBac CG (Bacillus thuringiensis israelensis [Bti]), VectoLex CG (Bacillus sphaericus [Bs]), and VectoBac 12AS (Bti), each applied at maximum label rate of 1.8 g, 1.8 g, and 0.193 ml per catch basin, respectively, significantly reduced the numbers of larvae for 1 wk. The dosages on the labels for treatment of mosquito larvae in catch basins, where mosquito breeding is continuous, are not adequate for providing long-term control in the northeastern USA without the need for frequent retreatment. When applied at 3 times the maximum label rate, VectoLex CG, VectoBac 12AS, and VectoBac CG significantly reduced the numbers of larvae for 5, 4, and 2 wk, respectively. A single application of VectoMax WSP (Bti + Bs) (1 pouch containing 10 g) per catch basin significantly reduced the numbers of 3rd and 4th instars and healthy pupae in catch basins in 2008, but numbers of 3rd and 4th instars in treated catch basins at 21 days after treatment had increased to 40% of the numbers in untreated catch basins. A 2nd treatment of 1 pouch per catch basin reduced the numbers of 3rd and 4th instars and healthy pupae to near zero for the next 4 wk, into the middle of September 2008. In 2009, VectoMax applied as 1 pouch per catch basin on July 1 and again on August 18 significantly reduced the numbers of healthy pupae throughout the summer until the end of September. A 2nd application of VectoMax to catch basins is likely needed during summer, when rainfall averages 13.7 in. (approximately 34.25 cm) during June through September, to keep the numbers of Culex and Ae. japonicus significantly reduced to lower risk of human exposure to West Nile virus. The application of 1 Natular XRT tablet, each weighing approximately 40.5 g (6.25% spinosad), to individual catch basins in 2009 significantly reduced the total numbers of larvae for 5 wk.

Molecular Evolution of West Nile Virus in a Northern Temperate Region: Connecticut, USA 1999-2008

Virology. Aug, 2011  |  Pubmed ID: 21723580

West Nile virus (WNV) has become firmly established in northeastern US, reemerging every summer since its introduction into North America in 1999. To determine whether WNV overwinters locally or is reseeded annually, we examined the patterns of viral lineage persistence and replacement in Connecticut over 10 consecutive transmission seasons by phylogenetic analysis. In addition, we compared the full protein coding sequence among WNV isolates to search for evidence of convergent and adaptive evolution. Viruses sampled from Connecticut segregated into a number of well-supported subclades by year of isolation with few clades persisting ≥2 years. Similar viral strains were dispersed in different locations across the state and divergent strains appeared within a single location during a single transmission season, implying widespread movement and rapid colonization of virus. Numerous amino acid substitutions arose in the population but only one change, V→A at position 159 of the envelope protein, became permanently fixed. Several instances of parallel evolution were identified in independent lineages, including one amino acid change in the NS4A protein that appears to be positively selected. Our results suggest that annual reemergence of WNV is driven by both reintroduction and local-overwintering of virus. Despite ongoing evolution of WNV, most amino acid variants occurred at low frequencies and were transient in the virus population.

Reexamination of Culex Pipiens Hybridization Zone in the Eastern United States by Ribosomal DNA-based Single Nucleotide Polymorphism Markers

The American Journal of Tropical Medicine and Hygiene. Sep, 2011  |  Pubmed ID: 21896800

Mosquitoes in the Culex pipiens complex are important vectors of several disease-causing pathogens, including West Nile virus. In North America, the complex consists of Cx. pipiens pipiens form pipiens, Cx. pipiens pipiens form molestus, Cx. pipiens quinquefasciatus, and their hybrids that exhibit substantial diversity in physiology, behavior, and geographic range. Hybridization among these mosquitoes is of concern because of potential implications for disease transmission. Currently, several morphological and molecular markers exist for differentiating members of the Cx. pipiens complex; however, these markers have specific limitations. We report here two highly reliable ribosomal DNA-based single nucleotide polymorphism (SNP) markers, CxpG2T and CxpA2d, for detecting Cx. pipiens complex mosquitoes containing Cx. p. quinquefasciatus alleles. Both CxpG2T and CxpA2d contain one allele that is present in all members of the Cx. pipiens complex, and the other allele is specific to Cx. p. quinquefasciatus. Testing of field populations from the eastern United States showed that these two SNP markers are capable of identifying a south to north gradient of Cx. p. quinquefasciatus and hybrids. The northern limit of detection of Cx. p. quinquefasciatus alleles in this study was in Fort Totten, NY (40.79°N), whereas the southern boundary was determined between Atlanta, GA (33.81°N) and Gainesville, FL (29.64°N). CxpG2T and CxpA2d were more accurate than the ACE-2 marker, and they may conceivably provide comparable resolution with microsatellite markers for detecting Cx. p. quinquefasciatus alleles.

Ultrastructural Characterization and Comparative Phylogenetic Analysis of New Microsporidia from Siberian Mosquitoes: Evidence for Coevolution and Host Switching

Journal of Invertebrate Pathology. Jan, 2012  |  Pubmed ID: 22001630

A survey of mosquito larvae infected with microsporidia was conducted from 2005 to 2008 in the Tomsk, Kemerovo and Novosibirsk regions of western Siberia, Russia. Twenty-one morphologically and genetically unique species of microsporidia were isolated from nine species of Anopheles, Aedes, Culex and Ochlerotatus mosquitoes including: (1) 14 proposed new species of Amblyospora (A. bakcharia, A. baritia, A. bogashovia, A. chulymia, A. hristinia, A. jurginia, A. kazankia, A. mavlukevia, A. mocrushinia, A. modestium, A. salairia, A. severinia, A. shegaria, and A. timirasia); (2) a newly proposed genus and species, Novothelohaniaovalae and; (3) six species of Amblyospora (A. flavescens, A. kolarovi, A. rugosa), Parathelohania (P. divulgata and P. tomski) and Trichoctosporea (T. pygopellita) from which gene sequences had not been previously obtained. Detailed ultrastructure of meiospores revealed unique cytological features associated with the length, arrangement and ratio of broad to narrow coils of the polar filament, comparative thickness of the exospore and endospore, and overall size of each species reaffirming their value in distinguishing taxonomic relationships. SSU rDNA sequences obtained from each species of microsporidia were unique when compared with GenBank entries. Phylogenetic trees constructed by Maximum Parsimony, Maximum Likelihood and Neighbor Joining analyses yielded similar topologies with a high degree of congruence between parasite and host at the generic level. Species that parasitize Aedes/Ochlerotatus and Culex mosquitoes segregate into distinct monophyletic groupings mirroring their host phylogeny, while species from Anopheles mosquitoes group as a sister clade basal to the entire group of mosquito-parasitic microsporidia as their Anopheles hosts cluster as a sister clade to the entire group of culicine mosquitoes. This provides strong evidence for host-parasite coevolution by descent at the generic level and limited host lineage switching between unrelated taxa. Among parasites of Aedes/Ochlerotatus and Anopheles mosquitoes, we found several instances where a single mosquito species serves as a host for two or more related species of microsporidia, an observation consistent with host switching and independent parasite speciation. Among the microsporidian parasites of Culex mosquitoes, we found only one parasite per host indicating a higher degree of host specificity and less host switching among parasites of this genus. Findings suggest a degree of host-parasite co-speciation with host switching occurring occasionally when the "normal" host is unavailable in the aquatic ecosystem. Frequency of host switching seems to be occurring in proportion to host relatedness and does not cross generic boundaries in this system.

Vector Host-feeding Preferences Drive Transmission of Multi-host Pathogens: West Nile Virus As a Model System

Proceedings. Biological Sciences / The Royal Society. Mar, 2012  |  Pubmed ID: 21849315

Seasonal epizootics of vector-borne pathogens infecting multiple species are ecologically complex and difficult to forecast. Pathogen transmission potential within the host community is determined by the relative abilities of host species to maintain and transmit the pathogen and by ecological factors influencing contact rates between hosts and vectors. Increasing evidence of strong feeding preferences by a number of vectors suggests that the host community experienced by the pathogen may be very different from the local host community. We developed an empirically informed transmission model for West Nile virus (WNV) in four sites using one vector species (Culex pipiens) and preferred and non-preferred avian hosts. We measured strong feeding preferences for American robins (Turdus migratorius) by Cx. pipiens, quantified as the proportion of Cx. pipiens blood meals from robins in relation to their abundance (feeding index). The model accurately predicted WNV prevalence in Cx. pipiens at three of four sites. Sensitivity analysis revealed feeding preference was the most influential parameter on intensity and timing of peak WNV infection in Cx. pipiens and a threshold feeding index for transmission was identified. Our findings indicate host preference-induced contact heterogeneity is a key mediator of vector-borne pathogen epizootics in multi-species host communities, and should be incorporated into multi-host transmission models.