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Antibodies generated against one dengue serotype can enhance infection of another by a phenomenon called antibody-dependent enhancement (ADE). Additionally, antigenic similarities between Zika and dengue viruses can promote Zika virus infection by way of ADE in vitro using these very same anti-dengue antibodies.

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.

Dengue virus (DENV) infection is augmented by a process called antibody-dependent enhancement (ADE), in which pre-existing DENV immunity can render an individual more susceptible to a subsequent DENV exposure. Based on Zika virus (ZIKV) and DENV structural similarities, emerging evidence suggests anti-DENV antibodies can cross-react with ZIKV at non-neutralizing levels characteristic of ADE. Mast cells (MC), sentinel white blood cells integral in coordinating early immune defences, have been identified as a principle contributor to DENV vascular leakage. This clinical manifestation is attributed to significant histamine release caused by MC degranulation, a characteristic MC activation response. However, to our knowledge ZIKV-MC interactions remain unexplored. MC responses to virus can be modelled in vitro, including via use of the well-characterized KU812 cell line which express Fcγ receptors. Here, we sought to determine if the KU812 MC is susceptible to (1) direct ZIKV infection; and (2) ADE in the presence of anti-DENV antibodies that cross-react with ZIKV. A significant increase in viral titre (104 PFU/mL) was detected, by plaque assay, in MC directly infected with ZIKV compared to MC infected with UV-inactivated ZIKV (0 PFU/mL). Furthermore, a significant viral titre (106 PFU/mL) was detected in MC infected with ZIKV pre-incubated with anti-DENV antibodies when compared to MC infected with ZIKV pre-incubated with isotype control antibodies (104 PFU/mL). Furthermore, unlike DENV induced degranulation of MC, ZIKV did not activate a degranulation response in this model or in a primary murine model. Therefore, MC may be a contributor in ZIKV pathogenesis without the classical degranulation response seen in DENV infected MC.

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.

Zika virus (ZIKV), belonging to the family Flaviviridae, genus Flavivirus, is an arthropod-borne virus that was first discovered from the Zika forest in Uganda in 1947. Recent outbreaks in South America have linked ZIKV to cases of microcephaly and Guillain-Barre syndrome in humans. With the increased interest in ZIKV, protocols must be established to facilitate proper research. Here we describe the laboratory techniques required to quantify, propagate, and store ZIVK. We also review the proper safety protocol for the handling of ZIKV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention. © 2016 by John Wiley & Sons, Inc.

We tested whether the known bird-biting mosquito, Culex pipiens Linnaeus (Diptera: Culicidae), is attracted to human hosts by placing humans at ground level (∼1.5 m) or in the forest canopy (∼5 m) in a Niagara woodlot. Modified Centers for Disease Control (CDC) miniature light traps (no light, no CO2) were placed next to the human hosts to capture the attracted mosquitoes. The human-baited traps were compared with control traps (standard CDC miniature light traps with CO2, but no light). As expected from previous research, there were more C. pipiens captured at the higher elevation than at ground level. Generally, they were attracted to control traps more than to human-baited traps at 5 m, whereas at 1.5 m there was no difference between the two trap types. As a comparison, most Aedes vexans (Meigen) mosquitoes were captured at the 1.5 m elevation but there were significantly more captured in the control traps than the human-baited traps during all periods. Because C. pipiens is attracted to humans throughout the entire season at the 1.5 m height (where they might encounter humans), it is likely that C. pipiens can serve as a bridge vector of West Nile virus (WNV).

The large carpenter bees nest in reeds, stems and wood. Many other Hymenoptera, including many wasps and some bees, have strong associations between the physical elements of their nests and behavior. Nests of the North American carpenter bee, Xylocopa virginica, were collected in southern Ontario. Nest architecture elements were examined with respect to their impact on life-history elements. In particular, it was determined that the brood cells are provisioned sequentially both within and among branches. There was also no detectable pattern of sex allocation in broods. Nests with branched architecture contained more foundresses, but these foundresses did not appear to contribute in the construction of larger nests. These findings are discussed with respect to other bees, and to the social structure of Xylocopa virginica.

This paper reports the first tests of the suitability of the standardized mitochondrial cytochrome c oxidase subunit I (COI) barcoding system for the identification of Canadian deerflies and horseflies. Two additional mitochondrial molecular markers were used to determine whether unambiguous species recognition in tabanids can be achieved. Our 332 Canadian tabanid samples yielded 650 sequences from five genera and 42 species. Standard COI barcodes demonstrated a strong A + T bias (mean 68.1%), especially at third codon positions (mean 93.0%). Our preliminary test of this system showed that the standard COI barcode worked well for Canadian Tabanidae: the target DNA can be easily recovered from small amounts of insect tissue and aligned for all tabanid taxa. Each tabanid species possessed distinctive sets of COI haplotypes which discriminated well among species. Average conspecific Kimura two-parameter (K2P) divergence (0.49%) was 12 times lower than the average divergence within species. Both the neighbour-joining and the Bayesian methods produced trees with identical monophyletic species groups. Two species, Chrysops dawsoni Philip and Chrysops montanus Osten Sacken (Diptera: Tabanidae), showed relatively deep intraspecific sequence divergences (∼ 10 times the average) for all three mitochondrial gene regions analysed. We suggest provisional differentiation of Ch. montanus into two haplotypes, namely, Ch. montanus haplomorph 1 and Ch. montanus haplomorph 2, both defined by their molecular sequences and by newly discovered differences in structural features near their ocelli.

We studied Culex pipiens L. in the Niagara Region of Ontario, Canada, to establish whether or not these mosquitoes are attracted to hosts other than birds at different elevations or as the season progresses. Guinea-pigs and chickens were used as representative mammalian and avian hosts, respectively. Bait animals were placed next to modified CDC miniature light traps (no light and no CO2) hung 1.5 or 5 m above ground in a Niagara woodlot. The season was divided into three 6-week periods (early, middle, and late). Significantly more C. pipiens were captured at the 5 m than at the 1.5 m elevation. In general, chicken-baited traps were preferred over control and guinea-pig-baited traps, with one important exception: there was no significant difference among traps during the late period at 1.5 m elevation. The potential role of C. pipiens as a bridging vector of West Nile virus to humans is discussed.

In the wild, black flies (Diptera: Simuliidae) feed on both floral nectar and homopteran honeydew. We tested the hypothesis that sugar meal composition affects flight performance using tethered black flies that had been flown to exhaustion. Individuals were then fed distilled water (dH2O), 10% (m/v) artificial nectar (AN), or 20% (m/v) artificial honeydew (AH) sugars, and the distance, speed, and duration of the resultant flights were recorded using a computerized flight mill. Following a dH2O meal, flies were unable to regain flight. There were three different artificial honeydew diets (AHlow, AHmed, AHhigh), each differing in the proportion and composition of simple sugars (glucose, fructose, sucrose) and complex sugars (melezitose, stachyose). Flies flew the farthest and longest on AH meals that contained higher levels of complex sugars (AHmed, AHhigh) compared with flies fed on AN (simple sugars only) or AHlow. On this flight mill, the mean flight speed was 10.6 ± 3.5 m/min and was not affected by the type of sugar meal ingested.

We provide an updated checklist of 67 endemic mosquito species known from Ontario, Canada. Nine endemic species are added to the checklist found in Darsie and Ward (2005): Aedes cantator, Ae. churchillensis, Ae. nigripes, Ae. pullatus, Anopheles perplexens, An. crucians, An. smaragdinus, Culex erraticus, and Cx. salinarius. Only 4 specimens of Ae. albopictus have been recorded in Ontario since 2001 despite concerted efforts to find this species; therefore, it is considered an “accidental” species and is excluded from the checklist.

A short fragment of mt DNA from the cytochrome c oxidase 1 (CO1) region was used to provide the first CO1 barcodes for 37 species of Canadian mosquitoes (Diptera: Culicidae) from the provinces Ontario and New Brunswick. Sequence variation was analysed in a 617-bp fragment from the 5' end of the CO1 region. Sequences of each mosquito species formed barcode clusters with tight cohesion that were usually clearly distinct from those of allied species. CO1 sequence divergences were, on average, nearly 20 times higher for congeneric species than for members of a species; divergences between congeneric species averaged 10.4% (range 0.2-17.2%), whereas those for conspecific individuals averaged 0.5% (range 0.0-3.9%).

The black fly subgenus Inseliellum is present on a series of archipelagos in the South Pacific. In this study, larval polytene chromosome maps of six Inseliellum species are presented. Chromosomal relationships among taxa were determined through shared fixed inversions or chromosomal landmark positioning. Three fixed inversions (IL-2, IIS-1, and IIIS-1) were shared among species, as was the position of the nucleolar organizer (NO) (IL or IIL). Comparisons to two previously studied species of Inseliellum are included to produce a cytological transformation series among eight taxa. The NO position defines two clades in the phylogeny of Inseliellum, herein named the NO-IL and NO-IIL clades. The utility of this cytological data set is discussed.

The polytene chromosome banding pattern of Simulium exasperans (Craig, 1987) is described for the first time. Three populations of S. exasperans from Moorea and Tahiti were examined cytologically. Interspecific comparisons revealed that S. exasperans is homosequential in chromosomal banding pattern to Simulium cataractarum (Craig, 1987), but contains three species-characteristic floating inversions: IL-1ex.2ex, IIL-1ex,2ex, and IIIL-1ex. No sex-linked inversions were identified; males and females had undifferentiated sex chromosomes. The IIL-1ex,2ex inversion was in Hardy-Weinberg disequilibrium in two of the three populations. From this preliminary data, two cytotypes may exist: cytotype A (Belvedre Cascade population) and cytotype B (1st Afareaitu Cascade and Jardin Public Vaipahi populations). Contrary to phylogenetic and biogeographic data, it is hypothesized that the Belvedre Cascade population (cytotype A) is ancestral to the populations containing cytotype B. Furthermore, a heterozygote advantage may exist in cytotype B.

Polytene chromosomes from the salivary glands of Simulium cataractarum larvae from the islands of Moorea and Tahiti were examined. Intraspecific comparisons revealed a polymorphism in the short arm of chromosome I, termed the IS-1cainversion. This inversion sequence was significantly heterozygous in the Tahitian populations (s/i), while the Moorean populations were all homozygous for the standard sequence (s/s). These two island populations seem to be reproductively isolated from one another. Thus, two cytotypes are recognized. Based on biogeographical relationships between the islands, we argue that the Moorean cytotype is likely to be ancestral to the Tahitian cytotype. Furthermore, a heterozygote advantage may exist in the Tahitian cytotype. Chromosomal maps for S. cataractarum are presented.

Simulium craigi Adler and Currie is a polymorphic species based on polytene chromosome banding patterns in the long arm of chromosome III (IIIL). Three cytotypes are described based on the predominant IIIL sequences. These correspond to three broad geographic areas: cytotype ‘CC’ from Pennsylvania; cytotype ‘AF’ from Ontario and Manitoba; and cytotype ‘ACF/BCF’ from New Hampshire. In the absence of sympatric populations, these cytotype differences are best explained by clinal variation within a single species. The relationship of S. craigi to other described members of the S. vernum group is discussed.

West Nile Virus (WNV) first arrived in Ontario, Canada in 2001 and has since spread throughout most of the province, causing disease in humans. The provincial government established a province-wide surveillance program to monitor WNV transmission throughout the 36 regional health units. Here we have acquired records of WNV human and mosquito surveillance from 2002 to 2013 to describe seasonal and geographic trends in WNV activity in southern Ontario. Additionally, we obtained climate data from seven municipalities to investigate how temperature and precipitation affect WNV transmission dynamics. We identified a strong quadratic relationship between the number of confirmed human cases and positive Culex mosquito pools recorded at the end of each year (R2 = 0.9783, p < 0.001). Using Spearman rank correlation tests, we identified that the minimum infection rate of Culex pipiens/restuans pools are the strongest predictor of human cases at a 1 week lag period. We also identified positive correlations between minimum infection rates, temperature, vector abundance, and cumulative precipitation. Global Moran's I index indicates strong positive autocorrelation and clustering of positive Culex pool counts in southern Ontario. Local indicators of spatial association tests revealed a total of 44 high-high and 1 high-low trap locations (n = 680). In the current work we have identified when and where hot spots of WNV activity have occurred in southern Ontario. The municipalities surrounding the western shore of the Lake Ontario and Windsor-Essex County have the largest records of positive mosquitoes and human cases. We identified that positive mosquitoes are a strong indicator of human cases to follow in the coming weeks. An epidemic action threshold of cumulative positive Culex pools was established, allowing Ontario public health officials to predict an epidemic at epidemiological week 34 (rho = 0.90, p < 0.001). These data have the potential to contribute to more efficient larvicide programs and awareness campaigns for the public.

Culex (Melanoconion) erraticus (Dyar and Knab) is now established in southern Ontario, Canada. This species was first discovered in 2002 during a province-wide adult mosquito surveillance program for West Nile virus. Using CO2-baited CDC miniature light traps, a few Cx. erraticus were collected from 2002 to 2011, but the total number increased during the 2012 and 2013 seasons. The number of Ontario Public Health Units with records for Cx. erraticus has also increased since 2002, demonstrating that the geographic distribution of this species is expanding northward. Cx. erraticus is a potential arboviral bridge vector for a number of pathogens and its establishment in Ontario should be considered a potential public health concern.

It is often said that on the Canadian Shield, black flies pollinate the sweet lowbush blueberry, because years with high black fly populations also tend to be those with large blueberry crops. This folklore has never been tested experimentally. Here we report on research designed to test whether or not black flies can act as pollinators for two species of ericaceous plants, sweet lowbush blueberry (Vaccinium angustifolium) and leatherleaf (Chamaedaphne calyculata). In enclosures, black flies may assist in leatherleaf pollination but there is no evidence that they increase fruit set in sweet lowbush blueberry. However, we do not exclude the possibility that in the wild, they act as opportunistic nectar thieves of sweet lowbush blueberry.

We present information on an aniline dye marking method for black flies. In the laboratory, adults were sprayed with 2% aqueous solutions of four colors of aniline dyes; brilliant blue and methyl orange gave the best results in longevity trials. In field trials we were able to recapture 1.3% of newly emerged marked and released flies at oviposition sites. Mark–release–recapture experiments were designed to distinguish among three competing models concerning oviposition site selection by gravid female black flies: (1) larval site fidelity (“Do flies return to the site that they experienced as larvae?”), (2) adult site fidelity (“Do flies return to the site that they experienced as adults?”), and (3) no site fidelity (“Do flies oviposit at random, i.e., without regard to adult or larval experience?”). Models 1 and 2 were rejected. There is, however, no reason to reject Model 3, the no site fidelity model. Thus, we conclude that for members of the S. venustum/verecundum complex (i.e., S. rostratum, S. venustum, and S. truncatum) females find an “apparently suitable” waterway in which to oviposit; this may or may not be their natal site.

The number of primary rays in the labral fan of filter-feeding black fly (Diptera: Simuliidae) larvae has been used by some authors to aid in species identification. We examined the number of primary labral fan rays in field-collected last-instar larvae of Simulium rostratum and Simulium decorum. Significant inter- and intra-specific differences were found both spatially and temporally. Simulium rostratum and S. decorum larvae were also reared to last instar (from eggs or first- or second-instar larvae) under three feeding regimes to test the effect of seston load on the number of primary labral fan rays. In both species the fan-ray number decreased when the food supply was increased. We conclude that the primary fan ray number should be used cautiously in species diagnosis because it is a phenotypically plastic trait that varies with seston load. A potential feedback mechanism to explain the observed differences in fan-ray number is hypothesised.

Cocoon spinning was analysed, using video recording and playback, in eighteen Nearctic black fly species, comparing nine Simulium species, six Eusimulium species, Stegopterna mutata, Cnephia dacotensis and Prosimulium mixtum. Fourteen behavioural characters were revealed that produced twenty-two equally parsimonious trees (CI = 0.93, RI = 0.96). Another tree was constructed on the basis of five characters relating to the cocoon structure (end-product characters). The goal of the study was to determine whether characters relating to behavioural components of black fly cocoon spinning or those based on end-products of the behaviour are superior for revealing phylogenetic relationships. This was accomplished by comparing both data sets to a phylogeny constructed with the use of cytological and morphological characters. If taxa are grouped according to end-products (the cocoons) there are some spurious groupings. The behavioural analysis only required one extra step to duplicate the morphological and cytological tree. In the case of black flies, it is more informative to use characters resulting from the analysis of the cocoon spinning behaviour than cocoon morphology.

Black flies were collected twice daily using a vehicle-mounted insect net on 8 sample days between 20 May and 9 July 1993. The contents of the crops and midguts of 160 male and 160 female Simulium venustum Say were analyzed individually by thin-layer chromatography. Fourteen different combinations were identified of the following sugars: fructose, glucose, sucrose/turanose, melezitose, raffinose, and stachyose. Presence of melezitose and stachyose indicated that black flies had fed on homopteran honeydew. Significantly more female (40.0%) than male flies (27.5%) fed on honeydew, and this difference was caused by a skewedness in the morning samples.

Black flies (Diptera: Simuliidae) were collected from a tamarack stand,Larix laricina(Du Roi) Koch, heavily infested withAdelges lariciatus(Patch) (Homoptera: Adelgidae). Insect nets were used to sweep the tamarack branches to capture black flies associated with the trees. Six black fly species were sweep-netted, with 85.5% of all flies belonging toSimulium venustumSay complex. Samples of honeydew and the crops and midguts of individual black flies were tested by thin layer chromatography using fructose, glucose, sucrose, turanose, melezitose, raffinose, and stachyose as standards. The sugars fructose, glucose, sucrose, raffinose, and stachyose were found in the adelgid honeydew samples. Of the 201 black flies tested, 194 contained sugars, which occurred in 16 combinations. It is argued that stachyose can be used to indicate when black flies have fed on the adelgid honeydew. We conclude that 49.7% of theS.venustumcollected from the tamarack had fed recently on this honeydew source. In addition, it was observed that black flies reared in the laboratory readily ingested freshly excreted and older (dry) honeydew when presented with branches from the tamarack stand.

Adult black flies were sampled by sweep-netting vegetation in four habitats within Algonquin Provincial Park, Ontario: Davies Bog, the airfield, deciduous habitat, and coniferous habitat. Sugars in the crops and midguts of female flies (n = 773) were tested by thin-layer chromatography to determine whether the flies had fed on nectar or homopteran honeydew. Melezitose and stachyose were used as honeydew-indicator sugars. For Simulium venustum, it was found that significantly fewer black flies (19%) from the airfield contained honeydew sugars than black flies from the other three sites (34% from Davies Bog; 36% from deciduous habitat; 25% from coniferous habitat). We argue that black flies will feed on nectar or honeydew according to availability.

Predator:prey experiments were conducted in the laboratory using snipe fly (Atherix lantha Webb) larvae as predators and hydropsychid caddisflies (Cheumatopsyche spp.) as prey. The effects on feeding rate of two different temperature regimes (20 and 10 °C) and five different predator:prey ratios (3:1, 2:1, 1:1, 1:2, 1:3) were tested. The number of caddisflies consumed per A. lantha larva per day was significantly greater at 20 °C than at 10 °C for the 2:1, 1:1, and 1:2 ratios (2×, 7×, and 2× greater, respectively). There were no significant differences between the 20 and 10 °C treatments at either the 3:1 or 1:3 ratio.

The antennae of Hypoderma bovis (Diptera: Oestridae) females were examined using scanning electron microscopy. Each antenna is composed of 3 parts: the scape, the pedicel, and the funiculus, with a large, protruding arista. Mechanoreceptors are found on the proximal and lateral margins of the scape and pedicel, respectively. Microtrichia, which are presumably non-innervated, are located evenly over both the outer surface of the scape and the inner surface of the pedicel. A narrow band of microtrichia is present proximally on the outer surface of the pedicel. The entire funicular surface is densely covered with microtrichia. Small patches lacking these microtrichia appear as depressions or “pits” (8–20 μm in diameter) on the surface of the funiculus. Olfactory sensilla found on the funicular surface include basiconica type 1, basiconica type 2, and trichoid sensilla. The sensilla basiconica commonly occur in pits on the anterodorsal surface of the funiculus. Trichoid sensilla are abundant on the posteroventral surface of the funiculus and do not appear to occur in pits. In addition, clavate and peg sensilla, whose functions are unknown, are found in low numbers on the funicular surface. There may be as many as 300 olfactory pits on the anterodorsal surface of each funiculus. These are single-chambered and contain 6 or fewer sensilla basiconica. We propose that a relatively high number of pits may be characteristic of flies in Superfamily Oestroidea (as compared with those of Superfamily Muscoidea), but that pit morphology within the Calyptratae is not Superfamily-specific.

Ceratopogonidae (Diptera) were collected on sheep farms in southern Ontario to establish whether Culicoides spp. pose a threat to the livestock industry. Specimens were collected in modified CO2-baited Centers for Disease Control and Prevention light traps, returned to the laboratory, freeze-killed, and identified to species under a microscope. In addition to Culicoides variipennis (Coquillet), we found that Culicoides sonorensis Wirth & Jones occurred on a number of farms over a 2-yr period. These records represent a significant departure from C. sonorensis' previously known geographical distribution. We present spatial and temporal distribution data for both species, with an emphasis on C. sonorensis. DNA sequence information is presented so that researchers lacking the necessary taxonomic skills can determine whether C. sonorensis is present in their collections. To differentiate C. sonorensis from C. variipennis, taxonomically reliable and informative traits were found in EF1α and, to a lesser extent, in ITS1, whereas the universal barcode region of cytochrome oxidase subunit 1 (CO1) was unsuitable.

Recent collection data indicate that at least four potential malaria vectors occupy more widespread distributions within the Andean highlands than in the past. Since habitat elimination is an important aspect of malaria control, it is vital to characterize larval habitats for Anopheles species within both lowland and highland sites. To that end, 276 sites within Ecuador were surveyed between 2008 and 2010. Characteristics of Anopheles-present sites for four species were compared to Anopheles-absent sites within the same geographical range and also to Anopheles-absent sites within a highland range representing potential future habitats. Thermochron iButtons(©) were used to describe the daily temperature variation within a subset of potential habitats. Anopheles albimanus (W.) was positively associated with permanent habitats, sand substrates, floating algae (cyanobacterial mats), and warmer temperatures in both comparisons. Anopheles pseudopunctipennis (T.) was associated with floating algae (cyanobacterial mats), warmer temperatures, and higher water clarity in both comparisons. Anopheles punctimacula (D.K.) was negatively associated with floating algae and positively associated with dissolved oxygen in both comparisons. Anopheles oswaldoi s.l. (P.) was not significantly associated with any parameters more often than expected given larval-absent sites. The results indicate that minimum water temperatures might limit the upper altitudinal distribution of An. albimanus (18.7° C) and An. pseudopunctipennis (16.0° C).

Larval habitat for three highland Anopheles species: Anopheles albimanus Wiedemann, Anopheles pseudopunctipennis Theobald, and Anopheles punctimacula Dyar & Knab was related to human land uses, rivers, roads, and remotely sensed land cover classifications in the western Ecuadorian Andes. Of the five commonly-observed human land uses, cattle pasture (n=30) provided potentially suitable habitat for An. punctimacula and An. albimanus in less than 14% of sites, and was related in a Principal Components Analysis (PCA) to the presence of macrophyte vegetation, greater surface area, clarity and algae cover. Empty lots (n=30) were related in the PCA to incident sunlight and provided potential habitat for An. pseudopunctipennis and An. albimanus in less than 14% of sites. The other land uses surveyed (banana, sugarcane and mixed tree plantations; n=28, 21, 25, respectively) provided very little standing water that could potentially be used for larval habitat. River edges and eddies (n=41) were associated with greater clarity, depth, temperature and algae cover, which provide potentially suitable habitat for An. albimanus in 58% of sites and An. pseudopunctipennis in 29% of sites. Road-associated water bodies (n=38) provided potential habitat for An. punctimacula in 44% of sites and An. albimanus in 26% of sites surveyed. Species collection localities were compared to land cover classifications using Geographic Information Systems software. All three mosquito species were associated more often with the category "closed/open broadleaved evergreen and/or semi-deciduous forests" than expected (P ≤ 0.01 in all cases), given such a habitat's abundance. This study provides evidence that specific human land uses create habitat for potential malaria vectors in highland regions of the Andes.

Larvae of Atherix lantha Webb (Diptera: Athericidae) are piercing – sucking predators. Predator–prey experiments were conducted in beakers containing gravel substrates and airstone-oxygenated water. The predator:prey ratio used in all experiments was 5:5. One prey type was tested at a time. Prey tested included large and small heptageniid mayflies, hyalellid amphipods, and hydropsychid caddisflies. The effect of predator size was examined using two size classes of A. lantha larvae. Large A. lantha consumed more mayflies than did small A. lantha. However, amphipod mortality was the same with large as with small A. lantha. The effect of prey size on predation success was tested using two size classes of mayflies. Statistically, small A. lantha fed on equal numbers of small and large mayflies, whereas large A. lantha consumed more large than small mayflies. Data for species-wise comparisons are only available for small A. lantha; according to our results, hydropsychid caddisflies (average mortality (m) = 0.5346) are more vulnerable to predation than are hyalellid amphipods (m = 0.2041) and heptageniid mayflies (m = 0.1135–0.1813). However, the mortality of large mayflies caused by large A. lantha larvae (m = 0.5375) is the same as that of caddisflies caused by small A. lantha larvae. Thus, the vulnerability of prey species depends, in part, on predator size.

To control malaria effectively, it is essential to understand the current knowledge, beliefs, concerns, land management practices, and mosquito bite protection methods in use by citizens. This study presents a comparative, quantitative, interview-based study of land owners and/or managers ( 𝑛 = 2 6 2 ) in the Ecuadorian lowlands (presently considered malarious) ( 𝑛 = 1 3 1 ) and highlands (potentially malarious in the future) ( 𝑛 = 1 3 1 ). Although respondents had a strong understanding of where the disease occurs in their own country and of the basic relationship among standing water, mosquitoes, and malaria, about half of respondents in potential risk areas denied the current possibility of malaria infection on their own property. As well, about half of respondents with potential anopheline larval habitat did not report its presence, likely due to a highly specific definition of suitable mosquito habitat. Most respondents who are considered at risk of malaria currently use at least one type of mosquito bite prevention, most commonly bed nets.

Animal-baited traps, using fox, mink, rabbits and ducks, were used in host preference experiments in two habitats (Beach and Woods). A generalized linear model of blood-feeding host preference is presented to test for significant differences between the isomorphic species Simulium venustum and Simulium truncatum. The S. truncatum population peaked before that of S. venustum. On any given day the two species divided their blood-feeding efforts among the different hosts in the same proportions. On the Beach, fox was the preferred host at the beginning of the season, but by the end of the season flies fed with equal frequency on the fox and the mink. In the Woods fox remained the preferred host throughout the season (late May to early July). Possible reasons for these feeding trends are discussed.

The original Centers for Disease Control and Prevention (CDC) gravid trap, with its mesh collection bag, can cause considerable damage to the captured mosquitoes. In an effort to reduce this damage and keep costs at a minimum, we modified our existing CDC gravid traps to accept a collection cup from a CDC light trap. This modification allowed for easier transport, freezing, storage, and removal of the mosquitoes. Modifying the CDC gravid traps takes little expertise or resources and can be completed quickly.