Amblyospora khaliulini (Microsporidia: Amblyosporidae): Investigations on its life cycle and ecology in Aedes communis (Diptera: Culicidae) and Acanthocyclops vernalis (Copepoda: Cyclopidae) with redescription of the species.

A multi-year study was conducted to examine the natural ecology of the microsporidium Amblyospora khaliulini and more fully characterize parasite development and histopathology in all stages of its primary mosquito host, Aedes communis and intermediate copepod host, Acanthocyclops vernalis with redescription of the species. A. khaliulini exhibits polymorphic development, produces three morphologically and functionally distinct spores, and is both horizontally and vertically transmitted. Development in A. vernalis is restricted to females, occurs within the ovaries and results in death of the host. Development is haplophasic with division by binary and multiple fission producing rosette-shaped sporogonial plasmodia and conical uninucleate spores that are orally infectious to Ae. communis larvae. Both sexes are equally susceptible and infections are confined to testes in males and ovaries in females. Initial stages of development include uninucleate schizonts that undergo karyokinesis forming diplokaryotic meronts that divide repeatedly by binary fission. Sporogony occurs in both host sexes, but sporogenesis does not progress normally in adult males and elliptical, thin walled binucleate spores that function in vertical transmission of the microsporidium via infection of the ovaries and eggs are formed in adult females only. Development of vertically acquired infections in larval Ae. communis hosts occurs within fat body tissue, leads to the production of meiospores in male hosts only and results in death during the 4th larval stadium. Initial development is characterized by merogonial multiplication of diplokarya by synchronous binary division producing additional diplokarya. The cessation of merogony and the onset of sporogony are characterized by the simultaneous secretion of a sporophorous vesicle and meiotic division of diplokarya resulting in the formation of octonucleate sporonts that undergo cytokinesis and sporogenesis to form eight uninucleate, broadly ovoid meiospores enclosed within a sporophorous vesicle. The natural prevalence of patent vertically acquired fat body infections in field populations of Ae. communis ranged from 1.6% to 3.6%. Yearly infection rates in A. vernalis copepods ranged from 57.1% to 15.0%. Prevalence rates of horizontally acquired infections in emerging adult Ae. communis ranged from 69.0% to 11.9% in males and 50.0% to 16.4% in females.

From ground pools to treeholes: convergent evolution of habitat and phenotype in Aedes mosquitoes.

BACKGROUND: Invasive mosquito species are responsible for millions of vector-borne disease cases annually. The global invasive success of Aedes mosquitoes such as Aedes aegypti and Aedes albopictus has relied on the human transport of immature stages in container habitats. However, despite the importance of these mosquitoes and this ecological specialization to their widespread dispersal, evolution of habitat specialization in this group has remained largely unstudied. We use comparative methods to evaluate the evolution of habitat specialization and its potential influence on larval morphology, and evaluate whether container dwelling and invasiveness are monophyletic in Aedes. RESULTS: We show that habitat specialization has evolved repeatedly from ancestral ground pool usage to specialization in container habitats. Furthermore, we find that larval morphological scores are significantly associated with larval habitat when accounting for evolutionary relationships. We find that Ornstein-Uhleinbeck models with unique optima for each larval habitat type are preferred over several other models based predominantly on neutral processes, and that OU models can reliably simulate real morphological data. CONCLUSIONS: Our results demonstrate that multiple lineages of Aedes have convergently evolved a key trait associated with invasive success: the use of container habitats for immature stages. Moreover, our results demonstrate convergence in morphological characteristics as well, and suggest a role of adaptation to habitat specialization in driving phenotypic diversity in this mosquito lineage. Finally, our results highlight that the genus Aedes is not monophyletic.

West Nile Virus Seroprevalence, Connecticut, USA, 2000-2014.

West Nile virus (WNV) infection is mainly asymptomatic but can be severe in elderly persons. As part of studies on immunity and aging in Connecticut, USA, we detected WNV seroconversion in 8.5% of nonimmunosuppressed and 16.8% of immunosuppressed persons. Age was not a significant seroconversion factor. Our findings suggest that immune factors affect seroconversion.

Variation in the Microbiota of Ixodes Ticks with Regard to Geography, Species, and Sex.

Ixodes scapularis is the principal vector of Lyme disease on the East Coast and in the upper Midwest regions of the United States, yet the tick is also present in the Southeast, where Lyme disease is absent or rare. A closely related species, I. affinis, also carries the pathogen in the South but does not seem to transmit it to humans. In order to better understand the geographic diversity of the tick, we analyzed the microbiota of 104 adult I. scapularis and 13 adult I. affinis ticks captured in 19 locations in South Carolina, North Carolina, Virginia, Connecticut, and New York. Initially, ticks from 4 sites were analyzed by 454 pyrosequencing. Subsequently, ticks from these sites plus 15 others were analyzed by sequencing with an Illumina MiSeq machine. By both analyses, the microbiomes of female ticks were significantly less diverse than those of male ticks. The dissimilarity between tick microbiomes increased with distance between sites, and the state in which a tick was collected could be inferred from its microbiota. The genus Rickettsia was prominent in all locations. Borrelia was also present in most locations and was present at especially high levels in one site in western Virginia. In contrast, members of the family Enterobacteriaceae were very common in North Carolina I. scapularis ticks but uncommon in I. scapularis ticks from other sites and in North Carolina I. affinis ticks. These data suggest substantial variations in the Ixodes microbiota in association with geography, species, and sex.

Host-Feeding Behavior of Mosquitoes in the Florida Everglades.

Background: West Nile virus (WNV), Everglades virus (EVEV), and five species of Orthobunyavirus were isolated from mosquitoes collected in the Everglades in 2016-2017. Prior studies of blood meals of mosquitoes in southern Florida have related findings to acquisition and transmission of EVEV, St. Louis encephalitis virus, and WNV, but not the Orthobunyavirus viruses associated with the subgenus Melanoconion of the genus Culex. Materials and Methods: In the present study, blood-fed mosquitoes were collected in the Everglades in 2016, 2017, 2021, and 2022, and from an industrial site in Naples, FL in 2017. Blood meals were identified to host species by PCR assays using mitochondrial cytochrome b gene. Results: Blood meals were identified from Anopheles crucians complex and 11 mosquito species captured in the Florida Everglades and from 3 species collected from an industrial site. The largest numbers of blood-fed specimens were from Culex nigripalpus, Culex erraticus, Culex cedecei, and Aedes taeniorhynchus. Cx. erraticus fed on mammals, birds, and reptiles, particularly American alligator. This mosquito species could transmit WNV to American alligator in the wild. Cx. nigripalpus acquired blood meals primarily from birds and mammals and frequently fed on medium-sized mammals and white-tailed deer. Water and wading birds were the primary avian hosts for Cx. nigripalpus and Cx. erraticus in the Everglades. Wading birds are susceptible to WNV and could serve as reservoir hosts. Cx. cedecei fed on five species of rodents, particularly black and hispid cotton rats. EVEV and three different species of Orthobunyavirus have been isolated from the hispid cotton rat and Cx. cedecei in the Everglades. Cx. cedecei is likely acquiring and transmitting these viruses among hispid cotton rats and other rodents. The marsh rabbit was a frequent host for An. crucians complex. An. crucians complex, and other species could acquire Tensaw virus from rabbits. Conclusions: Our study contributes to a better understanding of the host and viral associations of mosquito species in southwestern Florida.

Prevalence of Dirofilaria immitis (Spirurida: Onchocercidae) infection in Aedes, Culex, and Culiseta mosquitoes from north San Joaquin Valley, CA.

Canine heartworm is one of the most serious infections primarily affecting domestic dogs but will also infect cats and wild canids. To evaluate the potential of mosquitoes as vectors of dog heartworm, Dirofilaria immitis (Leidy) in San Joaquin County, CA, we collected mosquitoes in 2011 and analyzed for infection with heartworm by using polymerase chain reaction. Of 3,000 mosquito pools (total number of specimens = 36,554), D. immitis DNA was detected in 97 pools of seven species, and the overall minimum infection rate (MIR) for all mosquito species was 2.69: Culex pipiens L. (n = 40; MIR = 3.66), Culex tarsalis Coquillett (n = 25; MIR = 1.89), Culiseta incidens (Thomson) (n = 11; MIR = 2.81), Aedes vexans (Meigen) (n = 7; MIR = 2.18), Aedes melanimon Dyar (n = 5; MIR = 4.64), Culex erythrothorax Dyar (n = 5; MIR = 3.96), and Culiseta inornata (Williston) (n = 4; MIR = 2.65). Cx. pipiens and Cx. tarsalis had the highest number of D. immitis infections and collectively accounted for 67% of all positive pools. Ae. melanimon, Ae. vexans, and Cx. erythrothorax were found to be infected with D. immitis only in rural and agriculture areas, whereas infections in other species were identified in rural and agriculture areas, and urban and residential settings. The majority of positive pools were identified from June through November and peaked during August through October. This is the first report of D. immitis infection in Ae. melanimon, Cx. erythrothorax, Cx. tarsalis, Cs. incidens, and Cs. inornata. The frequent detection of D. immitis in field-collected Cx. pipiens and Cx. tarsalis in concert with their seasonal abundance and widespread distribution suggest a central role for these species in dog heartworm transmission. Other species, including Ae. vexans, Ae. melanimon, Cs. incidens, Cs. inornata, and Cx. erythrothorax, may play a secondary role in transmission.

Isolations of Cache Valley virus from Aedes albopictus (Diptera: Culicidae) in New Jersey and evaluation of its role as a regional arbovirus vector.

The Asian tiger mosquito, Aedes albopictus (Skuse), is an invasive species and a major pest problem in urban and suburban locales in New Jersey. To assess its potential role as an arbovirus vector, we sampled Ae. albopictus from two New Jersey counties over a 3-yr period and estimated the prevalence of virus infection by Vero cell culture and reverse transcription-polymerase chain reaction assays. Three virus isolates were obtained from 34,567 field-collected Ae. albopictus, and all were identified as Cache Valley virus by molecular methods. Ae. albopictus (N = 3,138), collected in Mercer County from late July through early September 2011, also were retested for West Nile virus (WNV) by reverse transcription-polymerase chain reaction, and all were negative. These results corroborate previous findings showing that Ae. albopictus may occasionally acquire Cache Valley virus, a deer-associated arbovirus, in nature. In contrast, we did not detect WNV infection in Ae. albopictus despite concurrent WNV amplification in this region.

Morphological and molecular characterization of a microsporidian parasite, Takaokaspora nipponicus n. gen., n. sp. from the invasive rock pool mosquito, Ochlerotatus japonicus japonicus.

A new genus and species of Microsporidia, Takaokaspora nipponicus n. gen., n. sp. is described from Ochlerotatus japonicus japonicus (Theobald) and Ochlerotatus hatorii (Yamada) based on light microscope and ultrastructural morphology, developmental features, transmission cycles and comparative sequence analyses of the small subunit ribosomal DNA (SSU rDNA). The microsporidium is both vertically and horizontally transmitted, exhibits dimorphic development alternating between diplokaryotic and monokaryotic stages and produces two morphologically distinct spores, one in larvae and another in adult females. Horizontal transmission of infection to larval mosquitoes occurs via direct oral ingestion of uninucleate spores that are produced in vertically-infected larval hosts. Development in horizontally-infected hosts is diplokaryotic following karyokinesis of uninucleate schizonts and binary fission to produce small (4.3µm × 2.0µm) membrane free, ovoid, binucleate spores that are confined to adult female reproductive tissues (ovariole sheath and oviducts). Vertical transmission of the microsporidium from adult females to larval progeny takes place via surface contamination of the egg (transovum). Microsporidian development in vertically-infected larvae is haplophasic with unpaired nuclei throughout, producing rosette-shaped sporogonial plasmodia contained within a thin non-persistent sporophorous vesicle and culminating in the formation of membrane free, uninucleate, conical spores (7.0µm×2.8µm). Development is confined to host fat body tissue which appears as swollen white masses in the thorax and selected segments of the abdomen causing larvae to appear abnormally distorted and results in death during the third and fourth instar stages. The SSU rDNA sequences obtained from the two morphologically identical microsporidia isolated from Oc. j. japonicus and Oc. hatorii were nearly identical and unique when compared with GenBank entries of all other mosquito-parasitic species. Phylogenetic trees constructed by Maximum Parsimony, Maximum Likelihood and bootstrap analyses using the Neighbor Joining search parameter yielded similar typologies. In each case, the novel microsporidium was the sister group to the clade containing Parathelohania species from Anopheles mosquitoes and the monotypic Novothelohania ovalae from Ochlerotatus caspius showing approximately 10-13% sequence divergence to those two genera providing strong support for establishment as a separate genus.

Seasonality of Mosquitoes in North Dakota, 2003-2006, with Four New State Records.

Thirty-seven species and subspecies of mosquitoes were identified from 3,580,610 specimens collected in eastern (Cass, Nelson, and Richland counties) and western (Williams County) North Dakota in 2003-2006. Four species were new state records (Aedes schizopinax, Psorophora ciliata, Ps. ferox, and Ps. horrida). Aedes vexans was dominant (82.9%). Other relatively abundant species were Ae. trivittatus (7.7%), Ae. melanimon (2.7%), Culex tarsalis (2.6%), Ae. dorsalis (1.6%), Ae. sticticus (1.0), and Culiseta inornata (0.9%). The seasonality of the species is presented.

Vector host-feeding preferences drive transmission of multi-host pathogens: West Nile virus as a model system.

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.

Weak larval competition between the invasive mosquito Aedes japonicus japonicus (Diptera: Culicidae) and three resident container-inhabiting mosquitoes in the laboratory.

The spread of exotic mosquito species into new environments can introduce shifts in mosquito populations and potentially alter public health risks to mosquito-borne diseases. The successful establishment of exotic species may occur due to their competitive advantage over other cohabitating species. We hypothesized that the recently introduced exotic mosquito Aedes japonicus japonicus (Theobald) would be a more effective competitor than Aedes atropalpus (Coquillett) and Aedes triseriatus (Say), and an equal competitor to Culex pipiens (L.) based on larval abundance data within tire habitats. Impacts of competition were measured using the larval developmental rate and survival of larvae, adult mortality, wing length, and sex ratio. We found that intraspecific competition acted strongest against Ae. japonicus versus the other three resident mosquito species by delaying larval development and increasing adult mortality. Interspecific competition was generally weak and significant main effects were only detected for species and density. Overall, our results show that larval competition between Ae. japonicus and the three resident species was weak when present, indicating that other ecological or behavioral factors may be influencing the invasion success for Ae. japonicus in North America.

Diversity of Wolbachia pipientis strain wPip in a genetically admixtured, above-ground Culex pipiens (Diptera: Culicidae) population: association with form molestus ancestry and host selection patterns.

Analysis of molecular genetic diversity in nine marker regions of five genes within the bacteriophage WO genomic region revealed high diversity of the Wolbachia pipentis strain wPip in a population of Culex pipiens L. sampled in metropolitan Chicago, IL. From 166 blood fed females, 50 distinct genetic profiles of wPip were identified. Rarefaction analysis suggested a maximum of 110 profiles out of a possible 512 predicted by combinations of the nine markers. A rank-abundance curve showed that few strains were common and most were rare. Multiple regression showed that markers associated with gene Gp2d, encoding a partial putative capsid protein, were significantly associated with ancestry of individuals either to form molestus or form pipiens, as determined by prior microsatellite allele frequency analysis. None of the other eight markers was associated with ancestry to either form, nor to ancestry to Cx. quinquefasciatus Say. Logistic regression of host choice (mammal vs. avian) as determined by bloodmeal analysis revealed that significantly fewer individuals that had fed on mammals had the Gp9a genetic marker (58.5%) compared with avian-fed individuals (88.1%). These data suggest that certain wPip molecular genetic types are associated with genetic admixturing in the Cx. pipiens complex of metropolitan Chicago, IL, and that the association extends to phenotypic variation related to host preference.

Ultrastructural characterization and comparative phylogenetic analysis of new microsporidia from Siberian mosquitoes: evidence for coevolution and host switching.

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.

The contribution of Culex pipiens complex mosquitoes to transmission and persistence of West Nile virus in North America.

Mosquitoes within the Culex pipiens complex have been implicated as major vectors of West Nile virus (WNV) in North America due to their seasonal abundance, vector competence and high field infection rates. However, the role of Cx. p. pipiens complex mosquitoes in enzootic amplification of WNV among avian hosts and epidemic transmission to humans varies throughout its geographical distribution. In the northeastern United States, Cx. p. pipiens is recognized as the primary enzootic vector responsible for amplification of virus among wild bird populations. However, because this mosquito is strongly ornithophilic, its role in transmission to humans appears to be more limited in this region. In the north central and Mid-Atlantic States by contrast, Cx. p. pipiens shows an increased affinity for human hosts and has been incriminated as a key bridge vector. In southern regions of the United States, Culex p. quinquefasciatus are more opportunistic feeders, and are thought to be principal enzootic and epidemic vectors. In western regions of the United States where Culex tarsalis predominates, especially in rural areas, Cx. p. pipiens and Cx. p. quinquefasciatus play roles that are more limited and are recognized as secondary vectors. In the southwestern United States Cx. p. quinquefasciatus also appears to be the predominant vector in urban habitats, but only a secondary vector in more rural environs. The direct involvement of Cx. p. pipiens form molestus in WNV transmission is largely unknown, but human-biting Cx. p. pipiens are more likely to have a probability of genetic ancestry with Cx. p. pipiens form molestus. The detection of WNV from overwintering populations of diapausing Cx. p. pipiens and non-diapausing Cx. p. quinquefaciatus and their role in local overwintering of WNV are addressed.

Field observations on the overwintering ecology of Culiseta melanura in the northeastern USA.

The overwintering ecology of Culiseta melanura was studied in a seasonally flooded evergreen forest swamp in south central Connecticut in an effort to clarify which larval stages successfully overwinter in the northeastern USA, and to determine the degree to which larval development and/or mortality occur during the winter months. A total of 8,626 immature Cs. melanura were collected weekly for analysis from subterranean crypts and cavities located under the roots of trees from December 13, 2011 to May 31, 2012. Despite the formation of ice on the surface water at the entrance holes to the crypts, water temperatures within the cavities remained above freezing (average = 1.8 degrees C) throughout the coldest winter months of January and February. A heterogeneous population of 2nd, 3rd, and 4th instars were recovered throughout the winter and early spring in the same relative proportions (30%, 30%, 40%, respectively), with no significant change in their comparative abundance during this period, providing unequivocal evidence that all 3 instars successfully overwinter in the region. Findings further demonstrate that larvae undergo no development during the winter and do not appear to be impacted by any measurable mortality. The cessation of larval diapause and a resumption of development was observed in mid-April and was coincident with a gradual increase in water temperature within the crypts to 9 degrees C, in agreement with a previously calculated developmental thermal minimum of 8.5 degrees C for Cs. melanura. This resulted in a protracted period of pupation that encompassed a minimum of 5 wk, followed by a staggered emergence of adults and an overlap of the residual overwintering population with larvae of the 1st summer generation.

Vector-host interactions of Culex pipiens complex in northeastern and southwestern USA.

Studies on the vector-host interactions of Culex pipiens complex mosquitoes by sequencing portions of mitochondrial cytochrome b gene indicate that Cx. p. pipiens f. pipiens predominantly feed on avian hosts (93.1%), and focus feeding activity on several key bird species, in particular the American robin, the gray catbird, and the house sparrow in Connecticut. However, Cx. p. quinquefasciatus indiscriminately feed on both birds and mammals. Culex p. quinquefasciatus in Harris County - Texas and southern California acquired 39.1% and 88.2% of bloodmeals from birds, respectively. Mammalian-derived bloodmeals constituted 52.5% and 9.6% in the two regions, respectively. The most frequent avian hosts for this mosquito species in the southwestern U.S. were the mourning dove, the white-winged dove, the house sparrow and the house finch. Humans infrequently served as the source of bloodmeals for Cx. p. pipiens and Cx. p. quinquefasciatus. Microsatellite analysis of mosquitoes from Chicago, Illinois showed that Cx. p. pipiens f. pipiens with mammalian- derived bloodmeals had significantly higher ancestry and proportion of hybrids from Cx. p. pipiens f. molestus than did those with avian-derived bloodmeals.

Ecology and Epidemiology of Eastern Equine Encephalitis Virus in the Northeastern United States: An Historical Perspective.

In the current review, we examine the regional history, ecology, and epidemiology of eastern equine encephalitis virus (EEEV) to investigate the major drivers of disease outbreaks in the northeastern United States. EEEV was first recognized as a public health threat during an outbreak in eastern Massachusetts in 1938, but historical evidence for equine epizootics date back to the 1800s. Since then, sporadic disease outbreaks have reoccurred in the Northeast with increasing frequency and northward expansion of human cases during the last 20 yr. Culiseta melanura (Coquillett) (Diptera: Culicidae) serves as the main enzootic vector that drives EEEV transmission among wild birds, but this mosquito species will occasionally feed on mammals. Several species have been implicated as bridge vectors to horses and humans, with Coquilletstidia perturbans (Walker) as a leading suspect based on its opportunistic feeding behavior, vector competence, and high infection rates during recent disease outbreaks. A diversity of bird species are reservoir competent, exposed to EEEV, and serve as hosts for Cs. melanura, with a few species, including the wood thrush (Hlocichia mustelina) and the American robin (Turdus migratorius), contributing disproportionately to virus transmission based on available evidence. The major factors responsible for the sustained resurgence of EEEV are considered and may be linked to regional landscape and climate changes that support higher mosquito densities and more intense virus transmission.

Seasonal Dynamics of Mosquito-Borne Viruses in the Southwestern Florida Everglades, 2016, 2017.

Mosquitoes were collected for 12 consecutive months beginning June 2016, from 11 locations in the Florida Everglades, Collier County, and tested for viruses by isolation in Vero cells and subsequent identification. One species complex and 31 species of mosquitoes were identified from 668,809 specimens. Ochlerotatus taeniorhynchus comprised 72.2% of the collection. Other notable species were Anopheles crucians complex, Culex nigripalpus, Cx. erraticus, and Cx. cedecei. Seven species of virus were identified from 110 isolations: Everglades, Gumbo Limbo, Mahogany Hammock, Pahayokee, Shark River, Tensaw, and West Nile viruses. Everglades, West Nile, Tensaw, and Mahogany Hammock viruses were most frequently isolated. Largest numbers of viruses were identified from Cx. cedecei, Cx. nigripalpus, and An. crucians complex. Five species of virus were isolated from Cx. cedecei. Viruses were isolated from mangrove, cypress swamp, hardwood hammock, and sawgrass habitats. West Nile virus was isolated August through October when Cx. nigripalpus was most abundant. Everglades virus was the most frequently isolated virus from nine species of mosquitoes collected from June through August. Tensaw virus was isolated primarily from Anopheles species. Isolations were made in July, August, January, February, and April, suggesting that this virus may be present in host-seeking mosquitoes throughout the year. Mahogany Hammock, Shark River, Gumbo Limbo, and Pahayokee viruses were isolated primarily from Cx. cedecei from June through December. Shotgun metagenomic sequencing was used to document that seven pools of Cx. cedecei were infected with two arboviruses. As communities expand into the Everglades, more humans will become exposed to arboviruses.

Control of mosquitoes in catch basins in Connecticut with Bacillus thuringiensis israelensis, Bacillus sphaericus, [corrected] and spinosad.

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.