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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.

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.

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.

La Crosse virus is a leading cause of pediatric encephalitis in the United States. The mosquito Ochlerotatus triseriatus is an efficient vector for La Crosse virus, whereas the closely related O. hendersoni transmits only at very low rates. Quantitative trait loci (QTL) affecting the ability to orally transmit this virus and adult body size were identified in 164 F2 female individuals from interspecific crosses of O. hendersoni females and O. triseriatus males using a combination of composite interval mapping (CIM), interval mapping (IM) for binary traits, and single-marker mapping. For oral transmission (OT), no genome locations exceeded the 95% experimentwise threshold for declaring a QTL using IM, but single-marker analysis identified four independent regions significantly associated with OT that we considered as tentative QTL. With two QTL, an increase in OT was associated with alleles from the refractory vector, O. hendersoni, and likely reflect epistatic interactions between genes that were uncovered by our interspecific crosses. For body size, two QTL were identified using CIM and a third tentative QTL was identified using single-marker analysis. The genome regions associated with body size also contain three QTL controlling OT, suggesting that these regions contain either single genes with pleiotropic effects or multiple linked genes independently determining each trait.

Saliva was collected from 4 species of mosquitoes intrathoracically inoculated with West Nile virus (WNV). The amount of infectious virus in the saliva was quantified by plaque assay and the number of WNV genomic equivalents (GE) was measured by reverse transcriptase-polymerase chain reaction. Ochlerotatus triseriatus had the greatest mean amount of infectious virus per saliva collection, followed by Aedes albopictus, Culex pipiens, and Cx. quinquefasciatus. The mean GE/saliva collection was also greatest in Oc. triseriatus, followed by Cx. quinquefasciatus, Cx. pipiens, and Ae. albopictus. The variance of log GE/saliva collection for Ae. albopictus was significantly lower than the variance for the other 3 species. This study provides a basis for comparing this component of vector competence and for determining the amounts of virus inoculated into vertebrates in experimental host competence studies.

1. The hypothesis that selective predation on larvae of the invasive Aedes albopictus (Skuse) could account for its stable coexistence with the native mosquito species and inferior competitor Ochlerotatus triseriatus (Say) in Florida treeholes and container systems was tested experimentally.2. Functional responses of the two dipteran predators Toxorhynchites rutilus (Coquillett) and Corethrella appendiculata (Grabham) were evaluated separately for A. albopictus and O. triseriatus prey. Both predators exhibited type II functional responses and consistently consumed more of the invasive species. Handling time of T. rutilus feeding upon O. triseriatus was significantly longer than when preying upon the invasive species.3. When either predator species was offered varying ratios of the two prey species, A. albopictus was consumed preferentially. The absence of a prey ratio effect on preference indicated that switching probably does not occur.4. The higher maximum feeding rate upon, and preference for, A. albopictus suggests that differential predation may foster coexistence of the invasive and native mosquito prey species in Florida.

I surveyed treeholes in central Pennsylvania for 7 mo in 1995 to investigate relationships among insect communities and water and leaf litter resources. I used water volume, essential to growth of treehole larvae, as an indicator of habitat size. Leaf litter is the basal food resource in treehole communities, and litter volume is related to the amount of energy available. Insect species richness and larval mosquito (Ochlerotatus triseriatus) and ceratopogonid midge (Culicoides guttipennis) densities were higher in treeholes that maintained high water volumes than those with low volumes throughout the study. Treeholes with high litter volume, irrespective of water volume, had the highest densities of C. guttipennis, and higher total larval densities than those with lower litter volumes; however, litter volume did not affect insect species richness. Scirtid beetle larvae were common in all treeholes, although their densities were unaffected by both litter and water volume. Insect species richness was not related to litter volume, unlike some other studies of phytotelmata. However, my study supported earlier conclusions that both habitat size and resource availability influence structure of treehole communities and maintain species populations. The influence of each resource on partic- ular species presence and density may be related to physical aspects of treeholes, biotic interactions, and natural history of individual species.

Four synthetic mosquito repellents (Autan [10% KBR3023], IR3535 [7.5%], Off! [15% deet], Skinsations [7% deet]) and eight natural (primarily plant extracts and/or essential oils) product-based repellents (Bite Blocker [2% soybean oil], ByGone, GonE!, Natrapel [10% citronella], Neem Aura, Sunswat, MosquitoSafe [25% geraniol], and Repel [26% p-menthane-3,8-diol]) were tested in the laboratory against Aedes albopictus Skuse, Culex nigripalpus Theobald, and Ochlerotatus triseriatus (Say). When estimated mean protection time (eMPT) responses for each repellent were averaged for all three mosquito species, Autan, Bite Blocker, Off!, and Repel prevented biting foror =7.2 h; IR3535, MosquitoSafe, and Skinsations for 3.2-4.8 h; and ByGone, Natrapel, GonE, NeemAura, and SunSwat for 0.9-2.3 h. Against Ae. albopictus, the eMPT for Off! and Repel exceeded 7.0 h and ranged from 5.0 to 5.7 h for Autan, Bite Blocker, and Skinsations. Bygone, GonE, NeemAura, and SunSwat provided 0.2 h protection against Ae. albopictus and Oc. triseriatus, whereas Autan, Bite Blocker, Off., and Repel prevented bites by Oc. triseriatus foror =7.3 h. All 12 repellents provided an eMPTor =2.8 h against Cx. nigripalpus (maximum: 8.5 h for Bite Blocker). When the average eMPT for each repellent (for all species) was divided by the eMPT for 7% deet (Skinsations), the order of repellent effectiveness and the corresponding repellency index (R,) was Repel (1.7)Bite Blocker (1.5) = Autan (1.5) = Off! (1.5)Skinsations (1.0)IR3535 (0.8)MosquitoSafe (0.6)Natrapel (0.5)Neem Aura (0.3) = SunSwat (0.3) = Bygone (0.3)GonE (0.2).

From June 14 to August 30, 2001 and June 10 to August 22, 2002, the mosquitoes of Oberlin, Ohio were surveyed and tested for West Nile virus (WNV). Mosquitoes were trapped weekly using gravid traps and CO 2 -baited Centers for Disease Control light traps at seven sites in 2001 and eight sites in 2002 in woodlots within the city limits. A total of 12,151 mosquitoes, representing 14 species and one species group, were collected in gravid traps, with Culex pipiens/restuans being the most abundant, followed by Ochlerotatus triseriatus. In light traps, 12,510 mosquitoes were collected, with Aedes vexans being the most abundant, followed by Culex pipiens/restuans. All gravid trap collections were tested for WNV via reverse transcriptase-polymerase chain reaction. West Nile virus was first detected in July 2002. Of all species tested, the Cx. pipiens/restuans species group exhibited the highest minimum infection rate (MIR) and the greatest percentage of positive pools. Both the MIR and percent of positive pools increased significantly with the date, although abundance of Cx. pipiens/restuans in gravid trap collections did not.

Larvae of the mosquito Toxorhynchites rutilus (Coquillett) prey upon other container-dwelling insects, including larvae of Aedes albopictus (Skuse), which is native to Asia but was introduced into the United States, and on the native tree hole mosquito Ochlerotatus triseriatus (Say). Previous work has established that O. triseriatus adopts low-risk behaviors in the presence of predation risk from T. rutilus. It is unknown whether introduced A. albopictus show a similar response to this predator. Behavior of fourth instars of A. albopictus or O. triseriatus was recorded in water that had held either A. albopictus or O. triseriatus larvae alone (control) and in water that had held T. rutilus larvae feeding on either A. albopictus or O. triseriatus (predation). Activity and position of larvae were recorded in 30-min instantaneous scan censuses. In response to water-borne cues to predation, O. triseriatus adopted low-risk behaviors (more resting, less feeding and movement), but A. albopictus did not change its behavior. We also tested the species specificity of the cues by recording the behavior of A. albopictus in water prepared using O. triseriatus and vice versa. O. triseriatus adopted low-risk behaviors even in predation water prepared by feeding T. rutilus with A. albopictus, but A. albopictus did not alter its behavior significantly between predation and control treatments prepared using O. triseriatus. Thus, A. albopictus does not seem to respond behaviorally to cues produced by this predator and may be more vulnerable to predation than is O. triseriatus.

Quantitative trait loci (QTL) affecting the ability of the Eastern treehole mosquito, Ochlerotatus triseriatus, to transovarially transmit (TOT) La Crosse virus (LAC) were mapped in an F1 intercross. The Holmen strain of O. triseriatus, previously selected for TOT refractoriness, was crossed to the AIDL strain that had been selected for TOT permissiveness. In P1 and F1 parents and 49 F2 individuals, regions of 10 cDNA loci were analysed with single strand conformation polymorphism (SSCP) analysis to identify and orientate linkage groups. Genotypes were also scored at fifty-six random amplified polymorphic DNA (RAPD)-SSCP loci. Twenty-eight F2 offspring were individually analysed for TOT. Three QTL for TOT were detected with standard interval mapping on chromosomes II and III. Alleles at the three loci contributed additively towards determining the overall TOT rate and cumulatively accounted for approximately 53% of the phenotypic variance in TOT.

In tree holes, leaf scraping scirtid beetles increase the rate at which leaf litter is converted to fine particles, which may benefit fine particle feeding mosquitoes if these fine particles are valuable to mosquitoes. We tested whether the products of scirtid feeding are a valuable food resource for mosquito larvae [Ochlerotatus triseriatus (Say)] by introducing different amounts of scirtid feces to mosquito larvae and measuring mosquito performance. Mosquito larvae survived longer and developed to later instars in treatments with many scirtids (and, therefore, a lot of feces) compared to treatments with few or no scirtids. This result suggests that scirtid feces (and attached microorganisms) constitute a valuable food resource for O. triseriatus. Thus, other members of tree hole communities may have complex effects on the population growth of O. triseriatus.

Larvae of 12 mosquito species were collected from abandoned tire piles at peridomestic and forested sites in Nicholas County, WV, from March through November of 2001. No larvae were found in March, but the numbers of species increased to 10 by July and remained relatively constant, at 9-11 in any given month, throughout November. Larvae of Ochlerotatus triseriatus (Say), the most commonly encountered species in every month of collection, were significantly more likely to be found in forested tire pile sites. Conversely, Culex restuans Theobald, Anopheles punctipennis (Say), Cx. territans Walker, and Aedes albopictus (Skuse) larvae were significantly more likely to be found in peridomestic tire piles. Larvae of the remaining seven species were either found in equal proportions at peridomestic and woodland sites, or there were too few collections to make statistical inferences. Opportunities for competitive interactions between Ae. albopictus and Oc. triseriatus in Nicholas County would be minimized because the peak occurrence of the two species differ temporally and spatially.

1. Scirtid beetles may benefit mosquitoes Ochlerotatus triseriatus (Say) by consuming whole leaves and leaving behind fine particles required by mosquito larvae. Such interactions based on the sequential use of a resource that occurs in multiple forms are known as processing chains. 2. Models of processing chains predict that interactions can vary from commensal (0, +) to amensal (0, –), depending on how quickly resource is processed in the absence of consumers. 3. The scirtid–O. triseriatus system was used to test the prediction derived from processing chain models that, as consumer-independent processing increases, scirtids benefit mosquitoes less. Consumer-independent processing rate was manipulated by using different leaf species that vary in decay rate, or by physically crushing a single leaf type to different degrees. 4. Although scirtids increased the production of fine particles, the effects of scirtids on mosquitoes were weak and were not dependent on consumer-independent processing rate. 5. In the leaf manipulation experiment, a correlation between scirtid feeding and consumer-independent processing was detected. Numerical simulations suggest that such a correlation may eliminate shifts from commensal to amensal at equilibrium; because mosquito populations are typically not at equilibrium, however, this correlation may not be important. 6. There was evidence that mosquitoes affected scirtids negatively, which is inconsistent with the structure of processing chain interactions in models. Processing chain models need to incorporate more detail on the biology of scirtids and O. triseriatus, especially alternative mechanisms of interaction, if they are to describe scirtid–O. triseriatus dynamics accurately.

Persistence and diuretic shedding of hepatitis B virus (HBV) by mosquitoes (Diptera: Culicidae) was studied by using infectious blood feedings, intrathoracic inoculations, and detection of virus by polymerase chain reaction (PCR) and Southern hybridization. Results showed that both Anopheles stephensi Liston and Ochlerotatus triseriatus (Say) shed HBV during diuresis for up to 72 h after feeding on an HBV-positive serum drawn from a human donor. HBV did not persist in the bodies of either An. stephensi or Oc. triseriatus past 72 h by infectious feeding or intrathoracic inoculation of HBV suspension. Viral dissemination did not occur by infectious feeding in An. stephensi or Oc. triseriatus, or by intrathoracic inoculation in An. stephensi, Oc. triseriatus, or Culex quinquefasciatus Say. These results suggest that HBV could be transmitted to humans by a stercorarian route, especially if mosquitoes that fed on an HBV-positive human are interrupted during feeding and move to another person to resume feeding.

When a species is introduced into a new location, it may escape, at least temporarily, from its natural enemies. In field surveys, we found that when the exotic, invasive mosquito, Aedes albopictus, invades new sites, it initially experiences reduced infection by its gut parasite, Ascogregarina taiwanensis. To determine the effect of this escape from parasitism on the competitive ability of A. albopictus, we performed a laboratory competition experiment in which infected and uninfected A. albopictus larvae were reared in microcosms alone and in competition with larvae of the native mosquito, Ochlerotatus triseriatus. We analyzed the effect of parasitism by A. taiwanensis on A. albopictus performance when subjected to intra- and interspecific competition across a range of larval densities, as well as the effect of A. albopictus parasitism by A. taiwanensis on the competitive impact of A. albopictus on O. triseriatus. At a density of 30 O. triseriatus larvae, O. triseriatus survivorship was significantly reduced by the addition of 30 unifected A. albopictus, but not by addition of 30 infected A. albopictus, and not by addition of 15 A. albopictus whether infected or uninfected. Although estimated finite rate of population increase (λ') showed similar trends, and was significantly affected by treatments, no pairwise differences in rate of increase were significant. Infection by A. taiwanensis also significantly prolonged A. albopictus female development time and reduced the intraspecific competitive effect of increased density of A. albopictus, but did not affect A. albopictus survivorship, mass, or estimated finite rate of population increase. Thus, when A. albopictus escapes from this parasite as it colonizes new sites, this escape may give it a small, but significant, added competitive advantage over O. triseriatus, which may facilitate range expansion of A. albopictus and enhance A. albopictus's initial impact on resident species.

We examined how leaf litter of alien invasive honeysuckle (Lonicera maackii Rupr.) either alone or in combination with leaf litter of one of two native tree species, sugar maple (Acer saccharum Marshall) and northern red oak (Quercus rubra L.), affects the ecology of Culex restuans Theobald, Ochlerotatus triseriatus Say, and Ochlerotatus japonicus Theobald. Experimental mesocosms containing single species litter or a mixture of honeysuckle and one of two native tree species litter were established at South Farms and Trelease Woods study sites in Urbana, IL, and examined for their effect on 1) oviposition site selection by the three mosquito species, and 2) adult production and body size of Oc. triseriatus and Oc. japonicus. There were no significant effects of study site and leaf treatment on Oc. japonicus and Oc. triseriatus oviposition preference and adult production. In contrast, significantly more Cx. restuans eggs rafts were collected at South Farms relative to Trelease Woods and in honeysuckle litter relative to native tree species litter. Significantly larger adult females of Oc. japonicus and Oc. triseriatus were collected at South Farms relative to Trelease Woods and in honeysuckle litter relative to native tree species litter. Combining honeysuckle litter with native tree species litter had additive effects on Cx. restuans oviposition preference and Oc. japonicus and Oc. triseriatus body size, with the exception of honeysuckle and northern red oak litter combination, which had antagonistic effects on Oc. triseriatus body size. We conclude that input of honeysuckle litter into container aquatic habitats may alter the life history traits of vector mosquito species.

Aedes triseriatus is the principal vector of La Crosse virus (LACv), which is the most common cause of pediatric arboviral encephalitis in North America. Here we report a novel species-specific polymerase chain reaction (PCR) assay that differentially identifies Ae. triseriatus and Ae. hendersoni. Because these 2 sibling species differ in their abilities to transmit LACv, accurate identification is critical for surveillance, research, and control programs. This duplex assay can detect the presence of both species in a single PCR reaction and is therefore simpler and faster than previously reported methods.

The Asian Tiger Mosquito Aedes albopictus arrived in the USA in 1985 in used automobile tires from Japan and became established in Texas. This species has since spread to become the most abundant container-inhabiting mosquito in the southeastern USA, including Florida, where it has reduced the range of another non-indigenous mosquito, Aedes aegypti. To assess the accuracy of predictions that A. albopictus would competitively exclude the native Eastern Treehole Mosquito Aedes triseriatus from tires but not from treeholes (Livdahl and Willey (1991) Science 253: 189–191), we extensively monitored the abundances of mosquito immatures before and after the Asian Tiger invaded these habitats in south Florida. These field data failed to demonstrate exclusion of A. triseriatus from treeholes following the establishment of A. albopictus in this microhabitat in 1991. However, A. albopictus had significantly higher metamorphic success and showed a significant increase in mean crowding on A. triseriatus in treeholes monitored from 1991 to 1999. In urban and suburban sites, A. triseriatus was uncommon in abandoned tires even before the arrival of A. albopictus. In some wooded sites, there is evidence for a decline in numbers of A. triseriatus in used tires and cemetery vases, but the native species has not been excluded from these habitats. Overall, the negative effect of A. albopictus on A. triseriatus has been less severe than that on A. aegypti. Experiments outdoors in surrogate treeholes showed that A. albopictus was more successful than A. triseriatus in survival to emergence in the presence of predatory larvae of the native mosquito Toxorhynchites rutilus when first instar predators encountered both prey species shortly after their hatch. Eggs of A. albopictus also hatched more rapidly than those of A. triseriatus, giving larvae of the invasive species an initial developmental advantage to escape predation. Biological traits that may favor A. albopictus are offset partly by greater treehole occupancy by A. triseriatus and the infrequency of the invasive mosquito species in undisturbed woodlands, which mitigates against displacement of the native mosquito in these habitats.

Aedes triseriatus (Say) population density patterns and La Crosse encephalitis virus infection rates were evaluated in relation to a variety of habitat parameters over a 14-wk period. Ovitraps and landing collections were used in a La Crosse virus-enzootic area in Nicholas County, WV. Study sites were divided into categories by habitat type and by proximity to the residences of known La Crosse encephalitis cases. Results demonstrated that Ae. triseriatus population densities were higher in sugar maple/red maple habitats than in hemlock/mixed hardwood habitats or in a site characterized by a large number of small red maple trees. Sites containing artificial containers had higher population densities than those without. La Crosse virus minimum infection rates in mosquitoes collected as eggs ranged from 0.4/1,000 to 7.5/1,000 in the 12 study sites, but did not differ significantly among sites regardless of habitat type or proximity to human case residences. La Crosse virus infection rates in landing Ae. triseriatus mosquitoes ranged from 0.0/1,000 to 27.0/1,000. La Crosse virus was also isolated from host-seeking Ae. canadensis (Theobald) in two study sites, at rates similar to those found in the Ae. triseriatus populations. The Ae. triseriatus oviposition patterns and La Crosse virus infection rates suggest that this mosquito species disperses readily in the large woodlands of central West Virginia. The La Crosse enzootic habitats in Nicholas County, WV, are contrasted with those studied in other geographic regions where La Crosse virus is found.

Terrestrial invertebrate carcasses are an important resource for insects developing in pitcher plants. However, little is known of the role of these carcasses in other containers, which also receive leaf fall and stemflow inputs. This experiment investigated effects of accumulated invertebrate carcasses as a resource for two competing mosquitoes, Aedes albopictus (Skuse) and Aedes aegypti (L.), whether either species differentially benefited from accumulated carcasses, and if such a benefit affected interspecific competition. First, we measured accumulation of invertebrate carcasses in standard containers at a field site. We then used a replacement series with five different species ratios at the same total density, and varied the input of invertebrate carcasses [dead Drosophila melanogaster (Meigen)] in three levels: none, the average input from our field site, or the maximum input recorded at our field site. Survivorship, development time, and mass were measured for each mosquito species as correlates of population growth, and were used to calculate a population performance index, λ′. There were strong positive effects of invertebrate carcass additions on all growth correlates and λ′. Differences in performance between species were pronounced in small or no carcass additions and absent in large inputs of invertebrate carcasses, but there was little evidence that inputs of invertebrate carcasses altered the competitive advantage in this system. These results suggest that terrestrial invertebrate carcasses may be an important resource for many types of container communities, and large accumulations of dead invertebrates may reduce resource competition between these mosquitoes, thus favoring coexistence. We propose that the total amount of resource, including accumulated invertebrate carcasses, may explain observed patterns of replacement involving these mosquitoes.

Mechanisms by which an introduced container-dwelling mosquito, Aedes albopictus, may cause declines in a resident container-dwelling mosquito, Aedes aegypti, in South Florida were tested using a combination of field experiments and field observations. Field experiment 1 tested which species has a competitive advantage as larvae developing in water-filled tires. Densities and availability of resources (leaf litter, which is a substrate for microorganisms fed upon by larvae) were manipulated in a factorial design. Contrary to previous laboratory experiments, A. albopictus was clearly the superior competitor in this tire environment, maintaining positive population growth at higher combined density and lower per capita resource availability than did A. aegypti. The primary determinant of success in this experiment was survivorship to adulthood, and A. aegypti only survived well in this environment when raised alone at low density, with high resource availability. Field experiment 2 tested whether this advantage for A. albopictus resulted from apparent competition mediated by shared protozoan parasites in the genus Ascogregarina. In field experiment 2, A. albopictus larvae had moderate to high levels of parasitism, but A. aegypti larvae were virtually free from Ascogregarina in all experimental tires, implying that Ascogregarina played little or no role in producing the advantage for A. albopictus in field experiment 1. Thus, apparent competition does not appear to be necessary to account for the replacement of A. aegypti by A. albopictus. As a first step toward understanding variation in the outcome of this invasion, numbers of Aedes immatures and masses of adults from field collected pupae (indicators of the intensity of competition) were compared for three sites with known histories of invasion by A. albopictus and decline of A. aegypti. Differences among sites in both number of Aedes per container and masses of adults of both species were consistent with the hypothesis that intensity of competition varies among sites, and suggest that A. aegypti persists only at sites where interspecific competition is less intense. Resource competition among larvae appears to be sufficient to account for replacement of A. aegypti by A. albopictus in suburban and rural areas of South Florida, which may have been marginal habitats for A. aegypti.

Growth of larvae of the eastern tree-hole mosquito, Aedes triseriatus (Say), measured as survival to adulthood, development time, and body mass at adult emergence, was significantly better when larvae were provided with fresh rather than senescent beech leaves as food substrate. Leaf type affected larval growth performance independently of ration of leaf available per larva when the ration level was high and larvae were not densely crowded, but leaf type and ration level had interactive effects on larval growth performance when ration was more limiting and larvae denser. Fresh leaves contained about twice as much nitrogen, had a lower carbon:nitrogen ratio, leached more mass into water, and contained significantly more soluble protein and carbohydrate than did senescent leaves. Thus, the observed growth responses could be explained on the basis of variation in nutrient content of, and greater leaching from, the fresh leaves. Larval growth was also significantly better on intact senescent beech leaves than on leaves that had been leached to remove soluble protein and carbohydrate; it was also significantly better on leaves whose surfaces had flourishing bacterial growth than on leaves with disinfected surfaces. Growth of female larvae fit well a pupation window model where larvae attained a minimum mass to emergence past a minimum development time across a range of leaf substrates varying in quality and quantity.

1. Hypotheses about declining populations of container-inhabiting Aedes mosquitoes following the invasion by additional species were tested. 2. The larval competition hypothesis was studied experimentally in pure and mixed cultures of Aedes aegypti (L.), A.albopictus (Skuse) and A.triseriatus (Say). The experiments used decomposing leaf litter in the laboratory, as opposed to most previous research which used non-natural food. 3. Resistance to starvation is introduced as a new measure of larval performance and competitiveness. The hypothesis is that more successful larvae store larger energy reserves and resist the lack of food longer. 4. Contrary to previous research showing better performance of A.aegypti in mixed cultures, A.albopictus developed faster and had greater survival when natural food was used. 5. Resistance to starvation was greater in the better performing species (i.e. A.aegypti with non-natural food and A.albopictus with leaf litter). Oxygen consumption by starved larvae was similar in the three container species, and in the ground-water mosquito, A.taeniorhynchus (Wied.), whose resistance to starvation was comparatively very low.

1 We tested the effects of food, in the form of leaf litter, and density on population growth and fitness correlates of the tree hole mosquito Aedes triseriatus. Our field experiment used a 2 x 2 randomized block design, with three holes as blocks. In cages within three holes, we manipulated densities of Aedes triseriatus larvae, and presence of leaf litter. Our laboratory experiment used a 2 x 3 factorial design in which we also manipulated densities of larvae and availability of leaf litter within similar cages (inside, outside, or absent). For both experiments we determined effects on survival, days to and mass at eclosion of both sexes, and a composite index of population performance λ′, that estimates finite rate of increase. 2 In the field experiment, we found significant and large effects of leaf litter and of density on the performance of A. triseriatus. The effect of density did not depend on leaf litter availability. We documented significant variation among tree holes for most correlates of fitness. Often, the effects of treatments varied significantly depending on the tree hole tested. 3 In the laboratory experiment we found significant effects of density and litter, and that direct browsing on leaf litter is necessary for the effect of litter on performance of A. triseriatus. In the laboratory, the negative effect of increased density was dependent on leaf litter availability. 4 Our results demonstrate that leaf litter has important effects on population performance of tree hole mosquitoes in natural tree holes. Direct browsing on the surface of leaf litter is the most likely mechanism by which litter enhances population growth. The impact of litter on populations varies among tree holes.

Potential larval competition between Aedes albopictus (Skuse) and Aedes triseriatus (Say) was examined with replacement series experimental design, an analysis previously used to evaluate competition between plant species. Experimental design was a factorial treatment arrangement with four replications of larval diet (high and low) and species ratio (proportions of A. triseriatus/A. albopictus larvae 1:0, 3:1, 1:1, 1:3, and 0:1). Adult emergence, adult biomass, and sex ratio were evaluated. No competitive effects were observed in the high-diet treatments; however, significant competitive relationships were observed for all parameters in the low-diet treatments. Competition was evaluated by replacement diagrams, relative crowding coefficients, and analysis of variance. All techniques indicated that A. albopictus was a superior competitor to A. triseriatus under low diet. The use of the replacement series design was a powerful method for examining competition through a single experiment. The results indicate that replacement series experimental design can be used to characterize competition between animal species, providing that individual animals can be placed in an experimental habitat in appropriate proportions.

Comparative sequence analysis of mosquito vitellogenin (Vg) genes was carried out to gain a better understanding of their evolution. The genomic clones of vitellogenin genes were isolated and sequenced from all three subfamilies of the family Culicidae including Culicinae (Aedes aegypti, Ochlerotatus atropalpus, Ae. polynesiensis, Ae. albopictus, Ochlerotatus triseriatus and Culex quinquefasciatus), Toxorhynchitinae (Toxorhynchites amboinensis), and Anophelinae (Anopheles albimanus). Genomic clones of vitellogenin genes Vg-B and Vg-C were isolated from Ae. aegypti and sequenced. A comparison of Vg-B and Vg-C, with the previously characterized vitellogenin gene, Vg-A1, suggests that Vg-A1 and Vg-B probably arose by a recent gene duplication, and Vg-C apparently diverged from the two other members of the gene family in an earlier gene duplication event. Two vitellogenin genes orthologous to Vg-C were cloned from a Cx. quinquefasciatus DNA library, one of which is truncated at the N-terminal end. Single vitellogenin genes, orthologous to Vg-C, were cloned from the An. albimanus and Tx. amboinensis libraries. Incomplete sequences orthologous to Vg-B and Vg-C were isolated from the Oc. atropalpus library. Only partial sequences were isolated from Ae. polynesiensis, Ae. albopictus and Oc. triseriatus. Inferred phylogenetic relationships based on analysis of these sequences suggest that Vg-C was the ancestral gene and that a recent gene duplication gave rise to Vg-A1 and Vg-B after the separation of the genus Aedes. The deduced amino acid composition of mosquito vitellogenin proteins exhibits higher tyrosine and phenylalanine composition than other mosquito proteins except for the hexamerin storage proteins. Analysis of vitellogenin coding sequences showed that a majority of amino acid substitutions were due to conserved and moderately conserved changes suggesting that the vitellogenins are under moderately selective constrains to maintain tertiary structure. The vitellogenin genes of the three anautogenous mosquitoes, that require a blood meal to develop eggs, had very high synonymous codon usage biases similar to highly expressed genes of other organisms. On the other hand, the vitellogenin genes of autogenous mosquitoes, that develop at least one batch of eggs without a blood meal, exhibited low synonymous codon usage bias. An unusual pattern of synonymous codon usage was observed in the first 15 amino acid residues encoding the signal peptide in the vitellogenin genes, where a high number of rarely used synonymous codons are present. It is hypothesized that rare synonymous codons have selectively accumulated in the signal peptide region to down-regulate the rate of translation initiation in the absence of a blood meal. Real-time PCR gene expression experiments showed that all three Ae. aegypti vitellogenin genes were highly expressed after a blood meal, and expressed in non-blood-fed females, males, larvae and pupae at trace levels. Sequences were deposited in GenBank (accession numbers: Ae. aegypti Vg-B, AY380797, Vg-C, AY373377; Oc. atropalpus Vg-B, AY691321, Vg-C, AY691322; Ae. polynesiensis Vg-A1, AY691318, Vg-B, AY691319, Vg-C, AY691320; Ae. albopictus Vg-A1, AY691316, Vg-C, AY691317; Oc. triseriatus Vg-C, AY691323; Cx. quinquefasciatus Vg-C1, AY691324, Vg-C2, AY691325; Tx. amboinensis Vg-C, AY691326; An. albimanus Vg-C, AY691327).