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.

Jamestown Canyon virus comes into view: understanding the threat from an underrecognized arbovirus.

This review examines the epidemiology, ecology, and evolution of Jamestown Canyon virus (JCV) and highlights new findings from the literature to better understand the virus, the vectors driving its transmission, and its emergence as an agent of arboviral disease. We also reanalyze data from the Connecticut Arbovirus Surveillance Program which represents the largest dataset on JCV infection in mosquitoes. JCV is a member of the California serogroup of the genus Orthobunyavirus, family Peribunyaviridae, and is found throughout much of temperate North America. This segmented, negative-sense RNA virus evolves predominately by genetic drift punctuated by infrequent episodes of genetic reassortment among novel strains. It frequently infects humans within affected communities and occasionally causes febrile illness and neuroinvasive disease in people. Reported human cases are relatively rare but are on the rise during the last 20 yr, particularly within the northcentral and northeastern United States. JCV appears to overwinter and reemerge each season by transovarial or vertical transmission involving univoltine Aedes (Diptera: Culicidae) species, specifically members of the Aedes communis (de Geer) and Ae. stimulans (Walker) Groups. The virus is further amplified in a mosquito-deer transmission cycle involving a diversity of mammalophilic mosquito species. Despite progress in our understanding of this virus, many aspects of the vector biology, virology, and human disease remain poorly understood. Remaining questions and future directions of research are discussed.

Establishing Colonies from Field-Collected Mosquitoes: Special Accommodations for Wild Strains.

A researcher may have many reasons for wanting to establish new laboratory colonies from field-collected mosquitoes. In particular, the ability to study the diversity found within and among natural populations in a controlled laboratory environment opens up a wide range of possibilities for understanding how and why burdens of vector-borne disease vary over space and time. However, field-collected mosquitoes are often more difficult to work with than established laboratory strains, and considerable logistical challenges are involved in safely transporting field-collected mosquitoes into the laboratory. Here, we provide advice for researchers working with Aedes aegypti, Anopheles gambiae, and Culex pipiens, as well as notes on other closely related species. We provide guidance on each stage of the life cycle and highlight the life stages for which it is easiest to initiate new laboratory colonies for each species. In accompanying protocols, we provide methods detailing Ae. aegypti egg collection and hatching as well as how to transport larvae and pupae from the field.

Collecting, Storing, and Hatching Aedes aegypti Eggs.

Laboratory study of natural populations of mosquitoes can play a key role in determining the underlying causes of variation in burdens of mosquito-borne disease. Aedes aegypti is the main vector of the viruses that cause dengue, chikungunya, Zika, and yellow fever, making it a high priority for laboratory study. Ae. aegypti eggs provide an ideal starting point for new laboratory colonies. Eggs can be collected using ovicups, which are small plastic cups lined with seed-germination paper and partially filled with leaf-infused H2O. Once collected, dry eggs will remain viable for months and can be safely transported long distances back to the laboratory as long as they are properly stored. This protocol provides step-by-step instructions for preparing for collecting, storing, and hatching Ae. aegypti eggs and has successfully yielded laboratory colonies from locations across both the native and invasive range of this species.

Mosquito Larvae and Pupae Transport from the Field.

Laboratory study of field-collected mosquitoes can allow researchers to better understand the ways variation within and among mosquito populations shapes burdens of mosquito-borne disease. The Anopheles gambiae complex comprises the most important vectors of malaria, but it can be challenging to keep in the laboratory. For some species of mosquitoes, especially An. gambiae, it is very difficult to bring viable eggs into the laboratory. Instead, it is preferable to collect larvae or pupae and then transport them as carefully as possible back to the laboratory. This simple protocol allows a researcher to start new laboratory colonies from larvae or pupae collected from natural breeding sites or proceed directly to their planned experiments. The use of natural breeding sites provides additional reassurance that the resulting colonies are representative of natural populations.

Evidence of Uranotaenia sapphirina (Diptera: Culicidae) feeding on annelid worms in the Northeastern United States.

Mosquito host-feeding behavior is an important parameter for determining the vector potential of mosquito species in a given locale. Despite the recent discovery of Uranotaenia sapphirina Osten Sacken feeding on annelid hosts in Florida, host association studies for this mosquito species in the United States remain limited. To investigate the blood-feeding pattern of Ur. sapphirina in the northeastern United States, mosquitoes were collected from Massachusetts, Connecticut, and New Jersey using CDC miniature light traps, peat fiber resting boxes, gravid traps, and backpack aspirators. Vertebrate and invertebrate hosts of this mosquito species were identified through PCR amplification and nucleotide sequencing of portions of the mitochondrial cytochrome b gene and the 28S ribosomal RNA gene, respectively. Of 21 (24.7%) specimens successfully identified to host species, 47.6% contained solely annelid blood, 14.3% mammalian blood, 14.3% avian blood, and 23.8% with mixed blood of annelid and avian origin. The mud earthworm, Sparganophilus tennesseensis Reynolds (Haplotaxida: Sparganophilidae), was identified as the most common host (n = 14, including mixed bloods), followed by American robin, Turdus migratorius (n = 7, including mixed bloods). Testing of these blood engorged mosquitoes for West Nile virus and eastern equine encephalitis virus did not result in any positive specimens. This is the first report of Ur. sapphirina feeding on annelids and on both vertebrate and invertebrate hosts in mixed bloodmeals in the northeastern United States. Our findings support the recent report of Ur. sapphirina feeding on invertebrates and further emphasizes the inclination of some mosquito species to feed on a wider range of hosts spanning nontraditional taxonomic groups.

Dynamics of eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, similar to previous years, cases were driven by multiple independent but short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019, although the ecology of EEEV is complex and further data is required to explore these in more detail. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Dynamics of Eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans, and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, like previous years, cases were driven by frequent short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

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.

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.

Impacts of Lysinibacillus sphaericus on mosquito larval community composition and larval competition between Culex pipiens and Aedes albopictus.

Effectiveness of mosquito larvicide active ingredients (AI), such as Lysinibacillus sphaericus, varies between species, yet little is known regarding how differential effectiveness manifests in larval communities in applied settings. To examine how differential effectiveness of L. sphaericus influences larval community dynamics, we performed two experiments. We performed a field experiment in which containers were seeded with a standardized nutrient treatment, mosquitoes colonized the containers, and then containers received one of three L. sphaericus applications. We then performed competition assays between Culex pipiens and Aedes albopictus in low nutrient environments using multiple interspecific ratios and the presence/absence of a low dose of L. sphaericus. Field results demonstrated elimination of Culex spp. from treated containers while container breeding Aedes spp. proliferated across all treatments. Lysinibacillus sphaericus did not influence competition between Cx. pipiens and Ae. albopictus, and the L. sphaericus application eliminated Cx. pipiens in all treatment replicates while survival of Ae. albopictus was similar between treated and untreated containers across interspecific ratios. Lysinibacillus sphaericus is an effective AI for control of Culex spp. However, different AIs should be utilized in habitats containing non-Culex genera while a mix of AIs should be utilized where coexistence of multiple genera is expected or confirmed.

Spatiotemporal distribution, abundance, and host interactions of two invasive vectors of arboviruses, Aedes albopictus and Aedes japonicus, in Pennsylvania, USA.

BACKGROUND: Aedes albopictus and Aedes japonicus, two invasive mosquito species in the United States, are implicated in the transmission of arboviruses. Studies have shown interactions of these two mosquito species with a variety of vertebrate hosts; however, regional differences exist and may influence their contribution to arbovirus transmission. METHODS: We investigated the distribution, abundance, host interactions, and West Nile virus infection prevalence of Ae. albopictus and Ae. japonicus by examining Pennsylvania mosquito and arbovirus surveillance data for the period between 2010 and 2018. Mosquitoes were primarily collected using gravid traps and BG-Sentinel traps, and sources of blood meals were determined by analyzing mitochondrial cytochrome b gene sequences amplified in PCR assays. RESULTS: A total of 10,878,727 female mosquitoes representing 51 species were collected in Pennsylvania over the 9-year study period, with Ae. albopictus and Ae. japonicus representing 4.06% and 3.02% of all collected mosquitoes, respectively. Aedes albopictus was distributed in 39 counties and Ae. japonicus in all 67 counties, and the abundance of these species increased between 2010 and 2018. Models suggested an increase in the spatial extent of Ae. albopictus during the study period, while that of Ae. japonicus remained unchanged. We found a differential association between the abundance of the two mosquito species and environmental conditions, percent development, and median household income. Of 110 Ae. albopictus and 97 Ae. japonicus blood meals successfully identified to species level, 98% and 100% were derived from mammalian hosts, respectively. Among 12 mammalian species, domestic cats, humans, and white-tailed deer served as the most frequent hosts for the two mosquito species. A limited number of Ae. albopictus acquired blood meals from avian hosts solely or in mixed blood meals. West Nile virus was detected in 31 pools (n = 3582 total number of pools) of Ae. albopictus and 12 pools (n = 977 total pools) of Ae. japonicus. CONCLUSIONS: Extensive distribution, high abundance, and frequent interactions with mammalian hosts suggest potential involvement of Ae. albopictus and Ae. japonicus in the transmission of human arboviruses including Cache Valley, Jamestown Canyon, La Crosse, dengue, chikungunya, and Zika should any of these viruses become prevalent in Pennsylvania. Limited interaction with avian hosts suggests that Ae. albopictus might occasionally be involved in transmission of arboviruses such as West Nile in the region.

Semi-field and surveillance data define the natural diapause timeline for Culex pipiens across the United States.

Reproductive diapause serves as biological mechanism for many insects, including the mosquito Culex pipiens, to overwinter in temperate climates. While Cx. pipiens diapause has been well-studied in the laboratory, the timing and environmental signals that promote diapause under natural conditions are less understood. In this study, we examine laboratory, semi-field, and mosquito surveillance data to define the approximate timeline and seasonal conditions that contribute to Cx. pipiens diapause across the United States. While confirming integral roles of temperature and photoperiod in diapause induction, we also demonstrate the influence of latitude, elevation, and mosquito population genetics in shaping Cx. pipiens diapause incidence across the country. Coinciding with the cessation of WNV activity, these data can have important implications for mosquito control, where targeted efforts prior to diapause induction can decrease mosquito populations and WNV overwintering to reduce mosquito-borne disease incidence the following season.

Evaluation of a Methoprene Aerial Application for the Control of Culiseta melanura (Diptera: Culicidae) in Wetland Larval Habitats.

Eastern equine encephalitis virus (EEEV) is an arbovirus endemic to the eastern United States. Human cases are rare but can be serious. The primary enzootic vector is Culiseta melanura (Coquillett) (Diptera: Culicidae), an ornithophagic mosquito. We conducted an aerial application of a granular methoprene formulation in Hockomock Swamp (Massachusetts), which represents a focus of EEEV transmission. Water collected from inside and outside Cs. melanura crypts was evaluated in bioassays of early fourth instar Cs. melanura larvae using treated and untreated water. Adult eclosion rates were 36% significantly lower in treated compared with untreated water (P < 0.05). Eclosion rates for water collected from inside crypts were significantly higher (62%) than rates from outside crypts (30%) (P < 0.05), indicating higher efficacy outside crypts. We tested whether reduced methoprene efficacy inside the crypts was due to reduced chemical penetration into this habitat. Chemical water analyses confirmed that methoprene concentrations were lower inside the crypts (0.1 ± 0.05 ppb) compared to water from outside crypts (1.79 ± 0.41 ppb). The susceptibility of Cs. melanura to methoprene was also determined to allow for comparison against concentrations observed in water collected from the field (LC-95: 1.95 ± 0.5 ppb). Overall, methoprene-treated water prevented mosquito development for up to 4 wk, but with a reduction in efficacy between 4- and 6-wk post-application. Our results suggest that aerial methoprene applications can effectively treat open water in wetlands but may not provide efficacious control of Cs. melanura due to an inability to penetrate larval habitats.

Host interactions of Aedes albopictus, an invasive vector of arboviruses, in Virginia, USA.

BACKGROUND: As an invasive mosquito species in the United States, Aedes albopictus is a potential vector of arboviruses including dengue, chikungunya, and Zika, and may also be involved in occasional transmission of other arboviruses such as West Nile, Saint Louis encephalitis, eastern equine encephalitis, and La Crosse viruses. Aedes albopictus feeds on a wide variety of vertebrate hosts, wild and domestic, as well as humans. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate blood feeding patterns of Ae. albopictus, engorged specimens were collected from a variety of habitat types using the Centers for Disease Control and Prevention light traps, Biogents Sentinel 2 traps, and modified Reiter gravid traps in southeast Virginia. Sources of blood meals were determined by the analysis of mitochondrial cytochrome b gene sequences amplified in PCR assays. Our aims were to quantify degrees of Ae. albopictus interactions with vertebrate hosts as sources of blood meals, investigate arboviral infection status, assess the influence of key socioecological conditions on spatial variability in blood feeding, and investigate temporal differences in blood feeding by season. Analysis of 961 engorged specimens of Ae. albopictus sampled between 2017-2019 indicated that 96%, 4%, and less than 1% obtained blood meals from mammalian, reptilian, and avian hosts, respectively. Domestic cats were the most frequently identified (50.5%) hosts followed by Virginia opossums (17.1%), white-tailed deer (12.2%), and humans (7.3%), together representing 87.1% of all identified blood hosts. We found spatial patterns in blood feeding linked to socioecological conditions and seasonal shifts in Ae. albopictus blood feeding with implications for understanding human biting and disease risk. In Suffolk Virginia in areas of lower human development, the likelihood of human blood feeding increased as median household income increased and human blood feeding was more likely early in the season (May-June) compared to later (July-October). Screening of the head and thorax of engorged Ae. albopictus mosquitoes by cell culture and RT-PCR resulted in a single isolate of Potosi virus. CONCLUSION AND SIGNIFICANCE: Understanding mosquito-host interactions in nature is vital for evaluating vectorial capacity of mosquitoes. These interactions with competent reservoir hosts support transmission, maintenance, and amplification of zoonotic agents of human diseases. Results of our study in conjunction with abundance in urban/suburban settings, virus isolation from field-collected mosquitoes, and vector competence of Ae. albopictus, highlight the potential involvement of this species in the transmission of a number of arboviruses such as dengue, chikungunya, and Zika to humans. Limited interaction with avian hosts suggests that Ae. albopictus is unlikely to serve as a bridge vector of arboviruses such as West Nile and eastern equine encephalitis in the study region, but that possibility cannot be entirely ruled out.

The community-wide effectiveness of municipal larval control programs for West Nile virus risk reduction in Connecticut, USA.

BACKGROUND: Mosquito larval control through the use of insecticides is the most common strategy for suppressing West Nile virus (WNV) vector populations in Connecticut (CT), USA. To evaluate the ability of larval control to reduce entomological risk metrics associated with WNV, we performed WNV surveillance and assessments of municipal larvicide application programs in Milford and Stratford, CT in 2019 and 2020. Each town treated catch basins and nonbasin habitats (Milford only) with biopesticide products during both WNV transmission seasons. Adult mosquitoes were collected weekly with gravid and CO2 -baited light traps and tested for WNV; larvae and pupae were sampled weekly from basins within 500 m of trapping sites, and Culex pipiens larval mortality was determined with laboratory bioassays of catch basin water samples. RESULTS: Declines in 4th instar larvae and pupae were observed in catch basins up to 2-week post-treatment, and we detected a positive relationship between adult female C. pipiens collections in gravid traps and pupal abundance in basins. We also detected a significant difference in total light trap collections between the two towns. Despite these findings, C. pipiens adult collections and WNV mosquito infection prevalence in gravid traps were similar between towns. CONCLUSION: Larvicide applications reduced pupal abundance and the prevalence of host-seeking adults with no detectable impact on entomological risk metrics for WNV. Further research is needed to better determine the level of mosquito larval control required to reduce WNV transmission risk.

Local persistence of novel regional variants of La Crosse virus in the Northeast USA.

BACKGROUND: La Crosse virus (LACV) (genus Orthobunyavirus, family Peribunyaviridae) is a mosquito-borne virus that causes pediatric encephalitis and accounts for 50-150 human cases annually in the USA. Human cases occur primarily in the Midwest and Appalachian regions whereas documented human cases occur very rarely in the northeastern USA. METHODS: Following detection of a LACV isolate from a field-collected mosquito in Connecticut during 2005, we evaluated the prevalence of LACV infection in local mosquito populations and genetically characterized virus isolates to determine whether the virus is maintained focally in this region. RESULTS: During 2018, we detected LACV in multiple species of mosquitoes, including those not previously associated with the virus. We also evaluated the phylogenetic relationship of LACV strains isolated from 2005-2018 in Connecticut and found that they formed a genetically homogeneous clade that was most similar to strains from New York State. CONCLUSION: Our analysis argues for local isolation and long-term persistence of a genetically distinct lineage of LACV within this region. We highlight the need to determine more about the phenotypic behavior of these isolates, and whether this virus lineage poses a threat to public health.

Increased mosquito abundance and species richness in Connecticut, United States 2001-2019.

Historical declines in multiple insect taxa have been documented across the globe in relation to landscape-level changes in land use and climate. However, declines have either not been universally observed in all regions or examined for all species. Because mosquitoes are insects of public health importance, we analyzed a longitudinal mosquito surveillance data set from Connecticut (CT), United States (U.S.) from 2001 to 2019 to identify changes in mosquito community composition over time. We first analyzed annual site-level collections and metrics of mosquito community composition with generalized linear/additive mixed effects models; we also examined annual species-level collections using the same tools. We then examined correlations between statewide collections and weather variables as well as site-level collections and land cover classifications. We found evidence that the average trap night collection of mosquitoes has increased by ~ 60% and statewide species richness has increased by ~ 10% since 2001. Total species richness was highest in the southern portion of CT, likely due to the northward range expansion of multiple species within the Aedes, Anopheles, Culex, and Psorophora genera. How the expansion of mosquito populations in the northeast U.S. will alter mosquito-borne pathogen transmission in the region will require further investigation.

Evaluation of Novel Trapping Lures for Monitoring Exotic and Native Container-Inhabiting Aedes spp. (Diptera: Culicidae) Mosquitoes.

Surveillance for diurnal container-inhabiting mosquitoes such as Aedes albopictus (Skuse), Aedes japonicus japonicus (Theobald), and Aedes triseriatus (Say) have routinely relied on the deployment of multiple trap types, including CO2-baited light traps, gravid traps, oviposition traps, and BG-Sentinel. These trap configurations have met with varying degrees of effectiveness and in many instances likely under-sample these key mosquito vectors. Most recently, the BG-Sentinel trap used in conjunction with the human-scent lure has been largely accepted as the gold-standard for monitoring Ae. albopictus. However, its ability to attract other container-inhabiting Aedes species has not been fully evaluated. During 2018, we tested new scent lures, TrapTech Lure-A and Lure-H (Bedoukian Research, Inc.), using BG-Sentinel traps with CO2 in two regions of Connecticut, Stamford and Hamden, against the BG-Lure. Pooled mosquitoes were additionally screened for arbovirus infection. A total of 47,734 mosquitoes representing 8 genera and 32 species were captured during the study, with the Stamford site deriving on average three times as many mosquitoes per trap, adjusting for sampling effort. Lure-A and Lure-H outperformed the BG-Lure in terms of total numbers, diversity evenness, and the proportion of both Ae. j. japonicus and Ae. triseriatus. There were no significant differences among lures in capturing Ae. albopictus, and in terms of species richness. Fifty-seven isolates of virus (West Nile, Jamestown Canyon, and La Crosse viruses) were obtained during the study, with no significant difference between trap-lure. We highlight both novel lures as effective attractants for use in mosquito surveillance=, which either outperform, or equal, BG-Lure.

Successive blood meals enhance virus dissemination within mosquitoes and increase transmission potential.

The recent Zika virus (ZIKV) and chikungunya virus epidemics highlight the explosive nature of arthropod-borne viruses (arboviruses) transmitted by Aedes spp. mosquitoes1,2. Vector competence and the extrinsic incubation period (EIP) are two key entomological parameters used to assess the public health risk posed by arboviruses3. These are typically measured empirically by offering mosquitoes an infectious blood meal and temporally sampling mosquitoes to determine the infection and transmission status. This approach has been used for the better part of a century; however, it does not accurately capture the biology and behaviour of many mosquito vectors that refeed frequently (every 2-3 d)4. Here, we demonstrate that acquisition of a second non-infectious blood meal significantly shortens the EIP of ZIKV-infected Aedes aegypti by enhancing virus dissemination from the mosquito midgut. Similarly, a second blood meal increases the competence of this species for dengue virus and chikungunya virus as well as Aedes albopictus for ZIKV, suggesting that this phenomenon may be common among other virus-vector pairings and that A. albopictus might be a more important vector than once thought. Blood-meal-induced microperforations in the virus-impenetrable basal lamina that surrounds the midgut provide a mechanism for enhanced virus escape. Modelling of these findings reveals that a shortened EIP would result in a significant increase in the basic reproductive number, R0, estimated from experimental data. This helps to explain how A. aegypti can sustain explosive epidemics such as ZIKV despite relatively poor vector competence in single-feed laboratory trials. Together, these data demonstrate a direct and unrecognized link between mosquito feeding behaviour, EIP and vector competence.