Discovery of an Aedes (Stegomyia) albopictus population and first records of Aedes (Stegomyia) aegypti in 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.

Assessing natural infection with Zika virus in the southern house mosquito, Culex quinquefasciatus, during 2016 in Puerto Rico.

The epidemic of Zika in the Western hemisphere has led to intense investigations of all species important in the transmission of Zika virus (ZikV), including putative mosquito vectors. Although evidence points to Stegomyia (= Aedes) (Diptera: Culicidae) mosquitoes as the primary vectors in nature among humans, there remains the possibility that other common mosquito species may be implicated in the rapid spread of the virus. Herein, field-caught Culex quinquefasciatus (Diptera: Culicidae) collected during June 2016 in different neighbourhoods in San Juan, Puerto Rico were examined for the presence of natural infection with ZikV. Stegomyia aegypti (= Aedes aegypti) from the same locations were also analysed. None of the Cx. quinquefasciatus tested showed natural infection for ZikV, whereas S. aegypti tested positive at seven sites. The present results suggest that Cx. quinquefasciatus was not involved in the transmission of ZikV in San Juan, Puerto Rico in 2016.

First Records of Culicoides sonorensis (Diptera: Ceratopogonidae), a Known Vector of Bluetongue Virus, in Southern Ontario.

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.

Establishment of Culex (Melanoconion) erraticus (Diptera: Culicidae) in Southern Ontario, Canada.

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.

Evaluation of DNA barcoding and identification of new haplomorphs in Canadian deerflies and horseflies.

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.

Identifying Canadian mosquito species through DNA barcodes.

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%).

Honeydew sugars in wild-caught female horse flies (Diptera: Tabanidae).

Three species of horse fly, Hybomitra illota (Osten Sacken), H. affinis (Kirby), and H. zonalis (Kirby), were collected by sweep-netting around human bait at 2 sites (abandoned airfield and Davies Bog) in Algonquin Provincial Park, Ontario. There were 3 times more horse flies collected at Davies Bog than at the abandoned airfield. The crop contents of all specimens were identified by thin-layer chromatography. Using melezitose and stachyose as honeydew indicator sugars, the relative importance of homopteran honeydew and floral nectar as carbohydrate sources for these flies was determined. Of flies testing sugar-positive, 42.0% of H. illota (n = 50), 52.8% of H. affinis (n = 36), and 100% of H. zonalis (n = 4) had recently fed on honeydew. Pooled data for the 3 species showed that 38.9% of flies at the abandoned airfield (n = 18) and 51.4% of flies at Davies Bog (n = 72) were honeydew-fed. There were no significant differences among species or between sites. These findings are compared with recent black fly and deer fly studies from the same sites.

Honeydew sugars in wild-caught female deer flies (Diptera: Tabanidae).

Female deer flies (Chrysops spp.) were collected from 2 habitats in Algonquin Provincial Park, ON. Using thin-layer chromatography, 11 sugars were detected in the digestive tracts of these flies; these occurred in 41 different combinations. We argue that combinations including melezitose, stachyose, or both can be used to indicate when flies recently have fed on homopteran honeydew. Accordingly, 85.7% of Chrysops excitans (n = 49) and 61.1% of Chrysops mitis (n = 18) collected from an abandoned airfield and 69.8% of C. excitans (n = 53) and 60.0% of C. mitis (n = 15) from a bog habitat tested positive for these honeydew indicator sugars. The difference between species was significant at the airfield only. Possible reasons for this habitat-dependent difference are discussed.

Nectar versus honeydew as sources of sugar for male and female black flies (Diptera: Simuliidae).

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.

New techniques for rearing black flies from pupae (Diptera: Simuliidae).

Simple techniques are described for collecting black fly pupae from streams using plastic strips and for rearing large numbers of adult black flies in inexpensive enclosures made of chicken wire and cloth mesh netting, and 2 methods are described for rearing adult black flies individually from pupae. The first method of rearing individual black flies uses 1.5-ml microcentrifuge tubules and the second uses easy-to-construct rearing chambers that provide moisture for the developing pupa and water for the adult to imbibe. Instructions for assembly are provided. Specimens obtained using these rearing chambers are of museum quality.

Notes on sugar feeding and selected wild mammalian hosts of black flies (Diptera: Simuliidae) in Newfoundland.

This study, done on the Avalon Peninsula, Newfoundland, identified selected wild mammalian hosts and quantified the incidence of sugar feeding for several species (species complexes) of black flies. Female flies were taken from fox, snowshoe hare, lynx, caribou, and human baits. Simulium truncatum/venustum complex females took blood from fox, lynx, rabbit, and caribou and were frequently taken in sweep net collections around humans. Blood-fed females of the Simulium rostratum/verecundum complex were collected only from caribou, were rarely attracted to the other mammalian baits, and were not attracted to humans. Although most females had sugar-free before seeking a host, significant interspecific differences in the incidence of fructose-positive females were found among the four species (species complexes) examined (Prosimulium mixtum, 92.9%; S. truncatum/venustum complex, 87.4%; Stegopterna mutata (triploid cytospecies), 83.3%; Simulium vittatum complex, 67.4%). No significant differences were found in the proportion of sugar-fed flies among three habitats examined (forest, scrubland, and pasture) for either P. mixtum or the S. truncatum/venustum complex. However, the percentage of sugar-fed S. truncatum/venustum complex females was significantly higher in the evening than in the morning. Time of day did not influence the incidence of sugar-fed P. mixtum.

Blood-feeding host preferences of the isomorphic species Simulium venustum and S. truncatum.

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.

ELISA for identification of blood meal source in black flies (Diptera: Simuliidae).

A modified ELISA, in which a biotinylated second antibody and a streptavidin-biotinylated peroxidase complex are used, has been developed to identify the blood meal sources of black flies. The modified assay is sensitive enough to identify correctly 100% of blood meals at 73 h postingestion and 80% of blood meals at 116 h postingestion in black flies held in the field at ambient temperatures.