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Current climatic conditions limit the distribution of Aedes (Stegomyia) albopictus (Skuse, Diptera: Culicidae) in the north, but predictive climate models suggest this species could establish itself in southern Canada by 2040. A vector of chikungunya, dengue, yellow fever, Zika and West Nile viruses, the Ae. Albopictus has been detected in Windsor, Ontario since 2016. Given the potential public health implications, and knowing that Aedes spp. can easily be introduced by ground transportation, this study aimed to determine if specimens could be detected, using an adequate methodology, in southern Québec. Mosquitoes were sampled in 2016 and 2017 along the main roads connecting Canada and the U.S., using Biogent traps (Sentinel-2, Gravide Aedes traps) and ovitraps. Overall, 24 mosquito spp. were captured, excluding Ae. Albopictus, but detecting one Aedes (Stegomyia) aegypti (Skuse) specimen (laid eggs). The most frequent species among captured adults were Ochlerotatus triseriatus, Culex pipiens complex, and Ochlerotatus japonicus (31.0%, 26.0%, and 17.3%, respectively). The present study adds to the increasing number of studies reporting on the range expansions of these mosquito species, and suggests that ongoing monitoring, using multiple capture techniques targeting a wide range of species, may provide useful information to public health with respect to the growing risk of emerging mosquito-borne diseases in southern Canada.

A population of Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae), a vector of chikungunya, dengue, yellow fever, and Zika and West Nile viruses, has been detected in Windsor, Ontario, Canada from 2016 onwards. Here, we describe its seasonal distribution, as well as the various aquatic habitats from which this species was collected and its larval co-habitation. We collected immatures from tires, treeholes, extruded polystyrene foam containers, discarded plastic cups, old recycling bins and oviposition traps. Aedes albopictus larvae were collected with Aedes japonicus (Theobald), Anopheles punctipennis (Say), Culex pipiens Linnaeus, Ochlerotatus hendersoni (Cockerell), Ochlerotatus triseriatus (Say) and Orthopodomyia signifera (Coquillett). Adult female and male specimens were collected from Biogents sentinel traps (Biogents AG, Regensburg, Germany), as well as Centers for Disease Control and Prevention miniature light traps (CDC, Atlanta, GA, U.S.A.), and also as they alighted on the investigators. Peak adult collections occurred in September during epidemiological week 37. We also collected Aedes (Stegomyia) aegypti (Linnaeus), a new record for Canada, in 2016 and from two new collection sites in 2017. The 2017 collections were 3.5 km north and 19.4 km south of the index site. The present study adds to the increasing number of studies reporting range expansions of these mosquito species.

The growing awareness that microbial symbionts residing in mosquito midguts can interrupt transmission of vector-borne diseases has stimulated interest in understanding their potential role in mosquito biology. Fluorescent proteins are powerful molecular markers that provide for detailed analysis of the function and behavior of specific midgut bacterial isolates without disturbing the normal gut microbiota. The aim of this study was to label bacterial isolates from the midgut of Ochlerotatus triseriatus, the primary vector of La Crosse virus, with green, yellow, and red fluorescent proteins (GFP, YFP, RFP) via electroporation. We also assessed the stability of GFP-, YFP-, and RFP-bearing plasmids and their effect on bacterial growth. Seven of eleven bacterial species could not be labeled despite several attempts. Labeling of Escherichia coli and Enterobacter cloacae was successfully achieved with all three fluorescent proteins. In contrast, labeling of Aerococcus viridans was achieved with GFP only and labeling of Aeromonas hydrophila was achieved with GFP and YFP only. The stability of GFP plasmid varied among bacterial species with A. hydrophila followed by E. cloacae having the most stable GFP label. In contrast, YFP and RFP plasmids were very stable in all bacterial species possessing these labels. GFP plasmid reduced the growth of labeled strains relative to wild type but this effect was not evident in YFP and RFP plasmids. These findings suggest that some mosquito midgut bacterial isolates can effectively be labeled with GFP, YFP and RFP plasmids allowing non-destructive studies on their functions within the vector.

Emergence may involve invasive mosquitoes other than Asian tiger mosquitoes, climate change, and changes in wildlife densities., La Crosse encephalitis is a viral disease that has emerged in new locations across the Appalachian region of the United States. Conventional wisdom suggests that ongoing emergence of La Crosse virus (LACV) could stem from the invasive Asian tiger (Aedes albopictus) mosquito. Efforts to prove this, however, are complicated by the numerous transmission routes and species interactions involved in LACV dynamics. To analyze LACV transmission by Asian tiger mosquitoes, we constructed epidemiologic models. These models accurately predict empirical infection rates. They do not, however, support the hypothesis that Asian tiger mosquitoes are responsible for the recent emergence of LACV at new foci. Consequently, we conclude that other factors, including different invasive mosquitoes, changes in climate variables, or changes in wildlife densities, should be considered as alternative explanations for recent increases in La Crosse encephalitis.

Invasive mosquito species can increase the transmission risk of native mosquito-borne diseases by acting as novel vectors. In this study, we examined the susceptibility of three exotic invasive mosquito species Aedes aegypti (L.), Ae. albopictus (Skuse), and Ochlerotatus japonicus (Theobald) to La Crosse virus (LACV) relative to the native primary vector Ochlerotatus triseriatus (Say). Adult females of the four mosquito species were orally challenged with LACV; incubated for 3, 5, 7, 9, or 11 d; and their midgut infection rates, dissemination rates, and effective vector competence were determined. Overall, Oc. japonicus (2.92) had the highest effective vector competence values, followed by Ae. albopictus (1.55), Ae. aegypti (0.88), and Oc. triseriatus (0.64) . In addition, we assessed the relationship between mosquito size and LACV susceptibility for field-collected Oc. triseriatus and Oc. japonicus . We hypothesized that smaller adults would be more susceptible to LACV; however, our results did not support this hypothesis. Infected Oc. triseriatus tended to be larger than exposed but uninfected females, while infected and uninfected Oc. japonicus were similarly sized. These findings suggest that Oc. japonicus , Ae. albopictus , and Ae. aegypti have significant potential to transmit LACV and more research is needed to uncover their potential role in LACV epidemiology.

The purpose of this study was to establish geospatial and seasonal distributions of West Nile virus vectors in southern Ontario, Canada using historical surveillance data from 2002 to 2014. We set out to produce mosquito abundance prediction surfaces for each of Ontario’s thirteen West Nile virus vectors. We also set out to determine whether elevation and proximity to conservation areas and provincial parks, wetlands, and population centres could be used to improve our model. Our results indicated that the data sets for Anopheles quadrimaculatus, Anopheles punctipennis, Anopheles walkeri, Culex salinarius, Culex tarsalis, Ochlerotatus stimulans, and Ochlerotatus triseriatus were not suitable for geospatial modelling because they are randomly distributed throughout Ontario. Spatial prediction surfaces were created for Aedes japonicus and proximity to wetlands, Aedes vexans and proximity to population centres, Culex pipiens/restuans and proximity to population centres, Ochlerotatus canadensis and elevation, and Ochlerotatus trivittatus and proximity to population centres using kriging. Seasonal distributions are presented for all thirteen species. We have identified both when and where vector species are most abundant in southern Ontario. These data have the potential to contribute to a more efficient and focused larvicide program and West Nile virus awareness campaigns.

Ochlerotatus triseriatus (Say), the primary vector of La Crosse virus (LAC), develops in a variety of natural and artificial aquatic containers where it often co-occurs with larvae of other mosquito species. We conducted a field study at two woodlots (South Farms and Trelease Woods) in Urbana, IL, to examine how container type influences vector abundance, body size, and susceptibility to LAC. Mosquito pupae were collected from tree holes, plastic bins, and waste tires, and eclosing adults were identified to species morphologically. Oc. triseriatus and Ochlerotatus japonicus (Theobald) females were orally challenged with LAC and midgut infection rate, disseminated infection rate, and body titer were determined by reverse-transcriptase real-time PCR. Oc. triseriatus was the dominant species collected in tree holes while Oc. japonicus and Culex restuans (Theobald) were mostly dominant in artificial containers. Female Oc. triseriatus and Oc. japonicus collected from plastic bins were significantly larger than those collected from tree holes or waste tires. Oc. japonicus females from South Farms were also significantly larger than those from Trelease Woods. Oc. triseriatus females collected from plastic bins and waste tires were significantly more susceptible to LAC infection relative to females collected from tree holes. In addition, Oc. triseriatus females from waste tires had significantly higher LAC titer relative to Oc. triseriatus from tree holes. For each container type and study site, wing length was not correlated to infection or dissemination rates. These findings suggest that the container type in which Oc.triseriatus develop may contribute to the spatial and temporal dynamics of LAC transmission.

Arboviruses can have benign, deleterious, or beneficial effects on the vector. We tested the hypothesis that oral infection with La Crosse virus (LACV) will have little to no effect on mosquito longevity and fecundity, a prediction of low virulence selected in a system with frequent vertical transmission. We tested the effects of infection in native Ochlerotatus triseriatus Say and invasive Stegomyia albopicta Skuse (Diptera: Culicidae). We artificially fed adult female mosquitoes of each species with either LACV-infected or uninfected bovine blood and determined adult longevity and fecundity. For females fed LACV-infected blood, bodies and legs, respectively, were separately homogenized and assayed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to determine the LACV infection and dissemination rates. Ochlerotatus triseriatus had a higher infection and dissemination rate than St. albopicta. For both species, female size had no effect on infection status. Infection status also had no effect on longevity or fecundity for both species. We suggest that the high frequency of vertical transmission may have selected for strains of the virus with low virulence in two vectors, in spite of their different evolutionary histories with the virus.

1. It was determined if the predatory midge Corethrella appendiculata Grabham imposes a fitness cost in a native mosquito, Ochlerotatus triseriatus Say, and an invasive mosquito, Aedes albopictus Skuse. The hypothesis that decreased activity of immature prey in the presence of predator cues is associated with life history costs through all life cycle stages was tested. 2. In experiment 1, individual larvae of O. triseriatus or A. albopictus were raised in the presence or absence of predation cues at two resource levels. Prey were video recorded to detect behavioural responses and to measure development time, size at emergence, and adult longevity. In experiment 2, prey populations were reared in similar environments and the frequency of predator cue additions was varied. 3. Only O. triseriatus reduced its activity in the presence of predation cues. Predation cues were associated with longer immature development times and shorter adult life spans in O. triseriatus, whereas in A. albopictus, the cues were associated with a larger size of emerging adults. 4. In the present study, it was found that behavioural modifications during the larval stage can affect mosquitoes through multiple stages of their complex life cycle. The species-specific behavioural differences are probably attributable to the longer evolutionary history O. triseriatus has with predators, relative to the invasive A. albopictus.

Saliva of blood-sucking arthropods contains a complex mixture of peptides that affect their host's hemostasis, inflammation, and immunity. These activities can also modify the site of pathogen delivery and increase disease transmission. Saliva also induces hosts to mount an antisaliva immune response that can lead to skin allergies or even anaphylaxis. Accordingly, knowledge of the salivary repertoire, or sialome, of a mosquito is useful to provide a knowledge platform to mine for novel pharmacological activities, to develop novel vaccine targets for vector-borne diseases, and to develop epidemiological markers of vector exposure and candidate desensitization vaccines. The mosquito Ochlerotatus triseriatus is a vector of La Crosse virus and produces allergy in humans. In this work, a total of 1,575 clones randomly selected from an adult female O. triseriatus salivary gland cDNA library was sequenced and used to assemble a database that yielded 731 clusters of related sequences, 560 of which were ...

Following the discovery of the introduced mosquito, Ochlerotatus japonicus in western New Hampshire in 2000, a series of twelve used tire casings in southeastern New Hampshire was sampled, beginning in 2001, to study the occurrence of O. japonicus and its abundance over a five year period. Prior to 2003, Ochlerotatus triseriatus, a native species, was the numerically dominant mosquito species found in older water-filled tire casings. By 2004, O. japonicus had replaced O. triseriatus as the dominant mosquito species in this habitat, and by 2006 comprised 83% of the 2 species collected from the tires. Ochlerotatus japonicus has two larval population peaks annually in southeastern New Hampshire during the breeding season: mid-April to mid-May in spring, and early August to mid-September in mid-to late summer. Eggs continue to hatch and some larvae are present until freezing (ice over) occurs, usually in late October to early November. Ochlerotatus triseriatus has a single larval population peak from...

We investigated the bacterial community composition of tree holes in relation to the presence and absence of larvae of the mosquito Ochlerotatus triseriatus. Larvae were eliminated from a subset of natural tree holes with Bacillus thuringiensis serovar israelensis, and total bacterial numbers, slow- and fast-growing colony-forming units on minimal media, and 16S rRNA gene sequence data from water column and leaf material were obtained. Total bacterial counts did not change significantly with treatment; however, the number of slow-growing cultivable bacteria significantly increased in the absence of larvae. Sequence classifications and comparisons of sequence libraries using LIBSHUFF indicated that the elimination of larvae significantly altered bacterial community composition. Major groups apparently affected by larvae were Flavobacteriaceae, Rhodobacteraceae, Comamonadaceae, and Sphingomonadaceae. A clear dominance of Flavobacteriaceae in the water column after larval removal suggests members of this group are a major bacterial food source.

In 2004, gravid-trap infusions made from commercially available garden products were compared in field assays to determine their attractiveness to ovipositing female mosquitoes. Experiments were conducted simultaneously at a residential site and at an auto salvage yard in Lee County, AL, each site having an abundance of container-breeding mosquitoes. Several infusion types were initially screened, from which the following 4 infusions were selected to be compared using gravid traps for their attractiveness to mosquitoes under field conditions: oak leaves, pine straw, red (dyed) hardwood mulch, and composted manure. Culex quinquefasciatus was the only species collected in greater numbers with gravid traps than with light traps, and the difference was more than 5-fold at both study sites. Gravid traps collected (mean +/- SD) 3.1 +/- 4.3 and 6.9 +/- 11.5 Cx. quinquefasciatus females at the residential and auto salvage sites, respectively. The response of Cx. quinquefasciatus females to infusions tested was variable, and no single infusion was consistently more attractive throughout the experiment. Gravid traps collected 1.7 +/- 1.9 and 4.7 +/- 4.4 Aedes albopictus females, 0.3 +/- 1.4 and 0.5 +/- 1.3 Cx. nigripalpus females, and 0.3 +/- 0.9 and 0.2 +/- 0.8 Cx. restuans females at the residential and auto salvage sites, respectively. Roughly 5-, 20- and 10-fold the numbers of females of Aedes albopictus, Cx. nigripalpus, and Cx. restuans were collected by light traps than by gravid traps at both sites. Aedes albopictus females did not demonstrate a preference for any of the infusions tested. Significant difference among infusions for Cx. restuans and Cx. nigripalpus were detected on just 1 trap-night. On this occasion, traps with red (dyed) hardwood mulch collected significantly more females of Cx. restuans and Cx. nigripalpus than did traps with other infusions. This work indicates that gravid traps are effective tools for collecting Cx. quinquefasciatus females, and a wide variety of organic materials may be used to produce infusions that can be used to attract ovipositing container-breeding mosquitoes.

Insecticidal activity of eight plants collected from Burkina Faso was studied using mosquito ( Ochlerotatus triseriatus ), Helicoverpa zea and Heliothis virescens larvae and adult white fly ( Bemisia tabaci ). The n -hexane, ethyl acetate and methanol extracts of Pseudocedrela kotschyi , Strophantus hispidus , Securidaca longepedunculata , Sapium grahamii , Swartzia madagascariensis , Cassia nigricans , Jatropha curcas and Datura innoxia were used in this study. Extracts were tested at 250 μg/mL concentration. All three extracts of C. nigricans , J. curcas (skin and seeds) and D. innoxia exhibited 100% mortality on fourth instar mosquito ( O. triseriatus ) larvae. In addition, the n -hexane and ethyl acetate extracts of S. hispidus , S. longepedunculata , S. grahamii showed 100% mortality. The ethyl acetate extract of S. madagascariensis was the most active on adult white fly and exhibited 80% mortality. Extracts of all other plants exhibited 30–50% mortality on B. tabaci . In the antifeedant assays against H. zea and H. virescens , the MeOH extracts of C. nigricans , S. madagascarensis and S. hispidus were more effective against H. zea as indicated by 74% larval weight reduction as compared to the control. Since C. nigricans is commonly used in West Africa to protect grain storage from insects, we have characterized the insecticidal components present in its extract. Bioassay directed isolation of C. nigricans leaf extract yielded anthraquinones emodin, citreorosein, and emodic acid and a flavonoid, luteolin. Emodin, the most abundant and active anthraquinone in C. nigricans showed approximately 85% mortality on mosquito larvae Anopheles gambiaea and adult B. tabaci at 50 and 25 μg/mL, respectively, in 24 h. These results suggest that the extract of C. nigricans has the potential to be used as an organic approach to manage some of the agricultural pests.

Summary 1. Researchers often use the spatial distribution of insect offspring as a measure of adult oviposition preferences, and then make conclusions about the consequences of these preferences for population growth and the relationship between life-history traits (e.g. oviposition preference and offspring performance). However, several processes other than oviposition preference can generate spatial patterns of offspring density (e.g. dispersal limitations, spatially heterogeneous mortality rates). Incorrectly assuming that offspring distributions reflect oviposition preferences may therefore compromise our ability to understand the mechanisms determining population distributions and the relationship between life-history traits. 2. The purpose of this study was to perform an empirical study at the whole-system scale to examine the movement and oviposition behaviours of the eastern tree hole mosquito Ochlerotatus triseriatus (Say) and test the importance of these behaviours in determining population distribution relative to other mechanisms. 3. A mark‐release‐recapture experiment was performed to distinguish among the following alternative hypotheses that may explain a previously observed aggregated distribution of tree hole mosquito offspring: (H 1 ) mosquitoes prefer habitats with particular vegetation characteristics and these preferences determine the distribution of their offspring; (H 2 ) mosquitoes distribute their eggs randomly or evenly throughout their environment, but spatial differences in developmental success generate an aggregated pattern of larval density; (H 3 ) mosquitoes randomly colonize habitats, but have limited dispersal capability causing them to distribute offspring where founder populations were established; (H 4 ) wind or other environmental factors may lead to passive aggregation, or spatial heterogeneity in adult mortality (H 5 ), rather than dispersal, generates clumped offspring distributions. 4. Results indicate that the distribution of tree hole mosquito larvae is determined in part by adult habitat selection (H 1 ), but do not exclude additional effects from passive aggregation (H 4 ), or spatial patterns in adult mortality (H 5 ). 5. This research illustrates the importance of studying oviposition behaviour at the population scale to better evaluate its relative importance in determining population distribution and dynamics. Moreover, this study demonstrates the importance of linking behavioural and population dynamics for understanding evolutionary relationships among life-history traits (e.g. preference and offspring performance) and predicting when behaviour will be important in determining population phenomena.

Predator-mediated coexistence of competitors occurs when a species that is superior in competition is also more vulnerable to a shared predator compared to a poorer competitor. The invasive mosquito Aedes albopictus is usually competitively superior to Ochlerotatus triseriatus. Among second instar larvae, A. albopictus show a lesser degree of behavioral modification in response to water-borne cues from predation by the larval midge Corethrella appendiculata than do O. triseriatus, rendering A. albopictus more vulnerable to predation by C. appendiculata than O. triseriatus. The hypothesis that C. appendiculata predation favors coexistence of these competitors predicts that C. appendiculata abundances will be negatively and positively correlated with A. albopictus and O. triseriatus abundances, respectively, and that coexistence will occur where C. appendiculata are common. Actual abundances of O. triseriatus, A. albopictus, and C. appendiculata in three habitats fit this prediction. In natural container habitats like tree holes, C. appendiculata were abundant and competitors co-existed at similar densities. In cemeteries and tires, which occur primarily in non-forested, human-dominated habitats, A. albopictus dominated, with abundances twice those found in tree holes, but C. appendiculata and O. triseriatus were rare or absent. We also tested for whether antipredatory behavioral responses of A. albopictus differed among habitats or populations, or were correlated with local C. appendiculata abundances. We could detect no differences in A. albopictus antipredatory behavioral responses to water-borne cues from predation. Tree hole habitats appear to promote co-existence of O. triseriatus and A. albopictus through interactions with predatory C. appendiculata, and this predator effect appears to limit invasion success of A. albopictus in tree holes. There are many studies on predator-mediated coexistence in natural habitats but to our knowledge this is the first study to suggest differential predator-mediated coexistence between natural and man-made habitats.

Invasive arthropods that vector pathogens have the potential to influence pathogen transmission both directly, by becoming a novel pathogen vector, or indirectly, by interacting with native vectors. Adult mosquito size is influenced by food availability in the larval stage, and smaller, nutrient-deprived mosquitoes are, in some studies, more efficient viral vectors in the laboratory. This is the first study to examine the indirect impacts that larval competition between Aedes albopictus, an introduced mosquito species, and Ochlerotatus triseriatus, a native mosquito species and the primary vector for La Crosse virus (LACV) in the US, has on native mosquito larval survival, adult size, and vector competence. A. albopictus presence decreased Oc. triseriatus larval survival, but surviving Oc. triseriatus females were larger, potentially owing to a release from intraspecific competition. These larger, native females were more likely to develop both midgut and disseminated LACV infections than females emerging from monospecific treatments. Collectively, these results suggest a need to better understand the ecology of both native and invasive vector species, their interactions, and the potential for those interactions to alter vector-borne disease transmission.

1. The hypothesis that size-selective predation and species-specific prey behaviours facilitate the coexistence between larvae of invasive Aedes albopictus (Skuse) and U.S.A.-native Ochlerotatus triseriatus (Say) was tested experimentally with the predator Corethrella appendiculata (Grabham). 2. Larval behaviours associated with a higher risk of predation were identified, and prey behavioural responses were tested in either the physical presence of predators or in water containing predation cues. Larvae that thrashed on container bottoms had a higher risk of being captured by fourth instar C. appendiculata than did larvae resting on the water surface. Ochlerotatus triseriatus, but not A. albopictus, adopted low-risk behaviours in response to water-borne cues to predation. Both prey species reduced risky behaviours in the physical presence of the predator, but O. triseriatus showed a stronger response. 3. The vulnerability of 2nd and 3rd instar prey to predation was compared, and behavioural responses were correlated with prey vulnerability. Second instars of both species were more vulnerable to predation by C. appendiculata than were 3rd instars, and the 3rd instar A. albopictus was more vulnerable than O. triseriatus of the same stage. All instars of O. triseriatus showed a similar reduction of risky behaviours in response to the presence of C. appendiculata despite 4th instar prey being relatively invulnerable to size-selective predation. 4. Weaker predator avoidance, coupled with superior competitive ability, of invasive A. albopictus is likely to contribute to its coexistence with O. triseriatus in containers of the south-eastern U.S.A., where C. appendiculata can be abundant.

A total of 36,495 larvae consisting of 45 species from 11 genera were collected from 7,189 sites from southern New Jersey, Burlington County between the months of March and October, 2001-2014. Density and seasonal distribution were determined among natural and artificial habitat. The most dominant species collected from natural habitat was Aedes vexans (Meigen) followed by Ochlerotatus canadensis canadensis (Theobald), Culex restuans Theobald, Culex pipiens L., and Culex territans Walker. The most dominant species collected from artificial habitat was Aedes albopictus (Skuse) followed by Ochlerotatus japonicus japonicus (Theobald), Cx. restuans, Cx. pipiens, and Ochlerotatus triseriatus (Say). Cx. restuans and Cx. pipiens were the only species categorized as dominant among both natural and artificial habitat and comprised greater than half the total density. Sympatry was common among dominant species from artificial habitat where a significant percentage of the total collection contained multiple species. The most common types of natural habitats were forested depressions and stream flood plains whereas rimless vehicle tires and various plastic containers were the most common artificial habitats. The pitcher plant Sarracenia purpurea L. was the only habitat exclusive to one species.

A state-wide survey of tire-breeding mosquitoes in Alabama was conducted in 2004 and 2005. Tire sites in all 67 counties in the state of Alabama were sampled for mosquito larvae. A total of 13,022 mosquito larvae, representing 12 species in 7 genera, was collected. The most frequently collected species were Aedes albopictus (70.4%), Culex territans (8.0%), and Ochlerotatus triseriatus (7.1%). The following species were also collected: Cx. restuans (6.0%), Cx. salinarius (2.7%), Orthopodomyia signifera (2.4%), and Cx. quinquefasciatus (1.4%). Ochlerotatus atropalpus, Toxorhynchites rutilus, Anopheles punctipennis, An. quadrimaculatus, and Psorophora columbiae each represented

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.

We conducted experiments in laboratory microcosms to simulate input of stemflow nutrients and flushing of metabolites in the tree hole habitats of larval Ochlerotatus triseriatus (Say). In the first experiment, we simultaneously examined the effects of nutrient additions (nitrogen, phosphorus, glucose, or combination) and flushing (removal of one-half of water volume replaced by deionized water) on mosquito production. The combination of nutrients had the greatest positive effects on mosquito production, with nitrogen (N) likely accounting for most of the increase in adult emergence and adult mass. Dilution of the nutrient pool via simulated flushing reduced mosquito growth, suggesting that the primary effect of stemflow input was nutrient addition as opposed to dilution of any latent toxic metabolites. In a second experiment, N additions were crossed with larval presence or absence to examine effects on key microbial processes. N increased leaf decay rates, soluble carbohydrate concentrations, fungal biomass and leaf-associated carbohydrase activity, but it did not stimulate bacterial productivity. Leaf decay was enhanced and bacterial production on leaves and in the water column was depressed in the presence of larvae. We conclude that the inputs of soluble N stimulated fungal growth, which made more fungal biomass available because of both its absolute increase and via the softening of the leaf particulate matter that could allow direct ingestion by larvae.

Multiple predator species can interact as well as strongly affect lower trophic levels, resulting in complex, nonadditive effects on prey populations and community structure. Studies of aquatic systems have shown that interactive effects of predators on prey are not necessarily predictable from the direct effects of each species alone. To test for complex interactions, the individual and combined effects of a top and intermediate predator on larvae of native and invasive mosquito prey were examined in artificial analogues of water-filled treeholes. The combined effects of the two predators were accurately predicted from single predator treatments by a multiplicative risk model, indicating additivity. Overall survivorship of both prey species decreased greatly in the presence of the top predator Toxorhynchites rutilus. By itself, the intermediate predator Corethrella appendiculata increased survivorship of the native prey species Ochlerotatus triseriatus and decreased survivorship of the invasive prey species Aedes albopictus relative to treatments without predators. Intraguild predation did not occur until alternative prey numbers had been reduced by approximately one-half. Owing to changes in size structure accompanying its growth, T. rutilus consumed more prey as time progressed, whereas C. appendiculata consumed less. The intermediate predator, C. appendiculata, changed species composition by preferentially consuming A. albopictus, while the top predator, T. rutilus, reduced prey density, regardless of species. Although species interactions were in most cases predicted from pairwise interactions, risk reduction from predator interference occurred when C. appendiculata densities were increased and when the predators were similarly sized.

SUMMARY 1. Variation in detritus quality and quantity can have significant effects on aquatic invertebrate food webs. Allochthonous inputs of detritus are the principal energy source for organisms in aquatic tree hole microsystems. We compared the effects of two major detritus types found in tree holes, senescent leaves (Sugar Maple and White Oak) and invertebrate carcasses (dead adult fruit flies and crickets), on several water quality characteristics of laboratory microcosms as well as on mass, survival and population performance of the dominant tree hole consumer, Ochlerotatus triseriatus (Diptera: Culicidae). To date, no study has documented the effects of animal detritus in tree hole microsystems or on resident consumers. 2. Aquatic environments receiving invertebrate carcasses had significantly greater total nitrogen, total reactive phosphorus and higher pH, than leaf-based environments. Decay rate of invertebrate carcasses was greater compared to leaf material. Consumption of O2 by micro-organisms increased with increasing detritus amounts, but we detected no difference between detritus types. 3. Ochlerotatus triseriatus larvae grew faster in animal-based treatments, and mean mass of larvae was significantly greater when more animal detritus was used. The effect of animal-based treatments on larvae translated into higher performance for adults, which were three times heavier than counterparts from plant-based containers. Survivorship and estimated population growth rates were significantly greater for O. triseriatus reared on animal-based versus plant-based detritus. 4. We hypothesise two mechanisms for the pronounced effect of invertebrate carcasses on mosquito performance relative to that associated with leaf detritus: (i) invertebrate carcasses decompose more quickly and release nutrients more effectively into the aquatic environment; or (ii) O. triseriatus larvae may directly ingest nutrient-rich components of invertebrate carcasses. Because even relatively small animal detritus additions can have strong effects on O. triseriatus populations, studies need to be conducted to explore the overall role of animal detritus in tree holes in nature.

Eleven species of mosquito larvae were collected in 568 collection visits to 209 abandoned tire pile sites in 33 West Virginia counties from April through October of 2003. Ochlerotatus j. japonicus supplanted the native Oc. triseriatus as the most common tire-colonizing species in the state. In general, the exotic Oc. j. japonicus was a better colonizer of tires than Oc. triseriatus in peridomestic, sunlit conditions throughout the state, whereas both species were equally likely to colonize tires in nonperidomestic, shaded settings. Aedes albopictus competed effectively with Oc. triseriatus from mid-summer through October in low elevation western counties but was not well represented in central counties of the state. Aedes albopictus was seldom collected at higher elevations in eastern counties. Culex pipiens, like Ae. albopictus, was more likely to be in collections from the western portion of West Virginia throughout the study period. Frequency of occurrence of Cx. restuans peaked in early season collections and declined as the season progressed, but Anopheles punctipennis and the medically unimportant Cx. territans gradually increased in frequency each month through September. Typically, species in the genus Culex and An. punctipennis were predisposed to peridomestic, sunlit habitats. Larvae of the predatory Toxorhynchites rutilus septentrionalis were found mostly in nonperidomestic, shaded settings. This species initially appeared in mid-summer, and by August and September it was found in as many as 30% of the collection sites in western and eastern counties. Larvae of An. barberi, Oc. atropalpus, and Orthopodomyia signifera were poorly represented in collections.

We report 2 polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar gregarine species based on amplification of variable regions of the internal transcribed spacer region of ribosomal DNA. The gregarines we investigated were Ascogregarina barretti (Vavra), A. culicis (Ross), and A. taiwanensis (Lien and Levine), parasites of the mosquitoes Ochlerotatus triseriatus (Say), Aedes aegypti (Linnaeus), and Ae. albopictus (Skuse), respectively. These 3 important vector mosquitoes often utilize the same container habitats, where larval development and infection by the parasite occurs, leaving ample opportunity for cross-species gregarine infection. Because previous studies have shown that the parasites A. culicis and A. taiwanensis variably affect fitness in both normal and abnormal mosquito hosts, distinguishing parasite infection and species is important. The task is complicated by the fact that these 2 parasite species are virtually identical in morphology, whereas A. barretti is morphologically distinct. Of the 2 PCR-based assays reported here, the first provides a rapid, sensitive, and straight- forward means of general ascogregarine detection based on a single PCR amplification. The second method provides a means of differentiation between A. culicis and A. taiwanensis based on a species-specific PCR assay. Together, these assays allow whole mosquitoes to be tested for the presence of Ascogregarina species as well as identification of both A. culicis and A. taiwanensis singly or in dual infections. Ascogregarina (Apicomplexa: Lecudinidae) contains a group of protozoan parasites that naturally infect certain insects. In mosquitoes (Diptera: Culicidae), infections have only been de- scribed from some container-breeding species (Beier and Craig, 1985). Three such species, Aedes aegypti (Linnaeus), Ae. al- bopictus (Skuse), and Ochlerotatus triseriatus(Say), are known or potential vectors of a number of arboviruses (Calisher, 1994). Each of the 3 mosquito species can naturally harbor a specific gregarine parasite that allows the host mosquito to carry out life functions relatively unaffected by its presence. The parasite is transmitted to other mosquitoes in the aquatic environment when feeding larvae ingest oocysts released by defecating or dead adult mosquitoes. Gregarine trophozoites feed intracellu- larly in the midgut epithelium and increase in size during mos- quito larval development; gametogony and sporogony in the Malphigian tubules during the pupal and early adult stages re- sult in new oocysts that are released back into the larval envi

The saliva of a blood-feeding insect can facilitate the intake of blood and effect the transmission of a pathogen. Apyrase is a salivary enzyme that inhibits the aggregation of platelets by hydrolyzing the activating molecule ADP. Apyrase also hydrolyzes ATP, which is a signal for neutrophil activation. Investigators have reported that malaria vector species in the Anopheles gambiae species complex and the genus Simulium had more apyrase activity than sibling species that were non-vectors. In this study, salivary gland extracta from sibling species Ochlerotatus triseriatus (Say), vector of LaCrosse virus, and the non-vector Oc. hendersoni Cockerell were examined. Apyrase activity was characterized from both species, but no difference in activities was observed. Differences in days to maximal apyrase activity after eclosion and apyrase levels after a blood meal were detected between Oc. triseriatus and Aedes aegypti L. (Rockefeller strain). These differences indicate that Ae. aegypti may be able to feed sooner and more often than Oc. triseriatus.

A population genetics study of the mosquito Ochlerotatus triseriatus was performed on 36 collections from adjoining regions of Iowa, Minnesota, and Wisconsin covering approximately 120 km 2 . Single nucleotide polymorphism analysis was used to estimate variation in the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene. The heated oligonucleotide ligation assay was used to identify the ND4 haplotype of each mosquito. No evidence of genetic isolation by distance was found, nor did Interstate 90 or the Mississippi River serve as barriers to gene flow. The effective migration rate varied from 18 to 45 reproductive migrants/generation, which is similar to estimates from an earlier study. The collections belong to a single, large, panmictic population. However, within this panmictic population, local genetic drift arises, possibly due to one or a few females ovipositing in larval breeding containers. From generation to generation, there is sufficient gene flow to mix families arising from individual breeding sites and eliminate founder effects due to drift.

Studies in aquatic systems have shown that habitat complexity may provide refuge or reduce the number of encounters prey have with actively searching predators. For ambush predators, habitat complexity may enhance or have no effect on predation rates because it conceals predators, reduces prey detection by predators, or visually impairs both predators and prey. We investigated the effects of habitat complexity and predation by the ambush predators Toxorhynchites rutilus and Corethrella appendiculata on their mosquito prey Aedes albopictus and Ochlerotatus triseriatus in container analogs of treeholes. As in other ambush predator-prey systems, habitat complexity did not alter the effects of T. rutilus or C. appendiculata whose presence decreased prey survivorship, shortened development time, and increased adult size compared to treatments where predators were absent. Faster growth and larger size were due to predator-mediated release from competition among surviving prey. Male and female prey survivorship were similar in the absence of predators, however when predators were present, survivorship of both prey species was skewed in favor of males. We conclude that habitat complexity is relatively unimportant in shaping predator-prey interactions in this treehole community, where predation risk differs between prey sexes.