Assessment of Mosquito Pollination.

Adult mosquitoes, sometimes in vast numbers, visit flowers in their search for floral nectar. However, the ability of mosquitoes to pollinate the flowers they visit is often neglected and sometimes even presumptively dismissed. Despite this, mosquito pollination has been reported in many instances, although many questions linger about its extent, importance, and the breadth of floral species or mosquito species which may be involved. In this protocol, I describe methodology to assess whether mosquitoes pollinate a flowering plant they visit, which can create a foundation for future investigations into this topic.

Mosquito Pollination and Sugar Detection Methods: An Overview.

The interactions between mosquitoes and plants and, particularly, between mosquitoes and plant sugars from flowers and other structures are often overlooked and are vastly underresearched compared to mosquito-vertebrate or mosquito-pathogen interactions. Given the importance of mosquito nectar-feeding behavior, as well as its impact on vectorial capacity and its implications for vector suppression, a better understanding of mosquito-plant interactions is needed. Direct observation of mosquitoes visiting plants to obtain sugar and other nutrients can be difficult because females may leave flowers to seek a blood meal from the observer, but this can be overcome with the right experimental procedures. This article discusses methods for the detection of sugar in mosquitoes and for assessing mosquito pollination.

Sugar Detection in Mosquitoes via Anthrone Tests.

Adult mosquitoes feed on plant sugar, frequently in the form of floral nectar. However, because of spatial and temporal variation in this behavior, as well as to the predilection of most mosquitoes to alter their behavior in the presence of a researcher, observing mosquito nectar feeding and similar behaviors directly in real time is not always feasible. In this protocol, I describe methods for hot and cold anthrone tests, which can be used to quantify the degree to which mosquito sugar feeding occurs in nature.

Mosquito (Diptera: Culicidae) Vision and Associated Electrophysiological Techniques.

Mosquitoes are considered the world's deadliest animal because of the pathogens they spread. Additionally, they are an unbearable nuisance in many areas. Visual stimuli play an important role in the mosquito life cycle, helping them find vertebrate hosts, floral nectar, and oviposition sites. Here, we review mosquito vision, including its influences on mosquito behavior, the photoreceptors involved, and mosquito spectral sensitivity, as well as provide an overview of techniques used for the analysis of mosquito vision, including electroretinograms, single-cell recordings, and the use of opsin-deficient mutants. We anticipate that this information will be useful for researchers studying mosquito physiology, evolution, ecology, and management.

Measuring Mosquito Spectral Sensitivity Using Electroretinograms.

Mosquitoes use a variety of cues to locate resources in the world around them, including olfactory, thermal, and visual stimuli. Understanding how mosquitoes perceive these stimuli is important for understanding and investigating mosquito behaviors and mosquito ecology. Specifically, mosquito vision can be studied in many ways, including by using electrophysiological recordings from their compound eyes. Electroretinograms can be used to characterize the spectral sensitivity of a mosquito species, revealing the wavelengths of light they can perceive. Here, we provide details on how to perform and analyze these recordings.

The Invasive Mosquitoes of Canada: An Entomological, Medical, and Veterinary Review.

Several invasive mosquitoes have become established in Canada, including important pathogen vectors such as Aedes albopictus, Ae. japonicus, and Culex pipiens. Some species have been present for decades, while others are recent arrivals. Several species present new health concerns and may result in autochthonous seasonal outbreaks of pathogens, particularly in southern Canada, that were previously restricted to imported cases. This review provides an overview of current knowledge of the biological, medical, and veterinary perspectives of these invasive species and highlights the need for increased monitoring efforts and information sharing.

Molecular relationships of introduced Aedes japonicus (Diptera: Culicidae) populations in British Columbia, Canada using mitochondrial DNA.

Aedes japonicus japonicus (Theobald) is a relatively recent immigrant to the Pacific Northwest, having been collected in Washington State in 2001 and in British Columbia (BC) since 2014. We applied a molecular barcoding approach to determine the phylogenetic relationship of Ae. j. japonicus populations in BC with those from around the world. We sequenced a 617 base-pair segment of the cytochrome c oxidase 1 gene and a 330 base-pair region of the NADH dehydrogenase 4 gene to find genetic variation and characterize phylogenetic and haplotypic relationships based on nucleotide divergences. Our results revealed low genetic diversity in the BC samples, suggesting that these populations arose from the same introduction event. However, our approach lacked the granularity to identify the exact country of origin of the Ae. j. japonicus collected in BC. Future efforts should focus on detecting and preventing new Ae. j. japonicus introductions, recognizing that current molecular techniques are unable to pin-point the precise source of an introduction.

Modeling the Putative Ancient Distribution of Aedes togoi (Diptera: Culicidae).

The coastal rock pool mosquito, Aedes (Tanakius) togoi (Theobald) (Diptera: Culicidae), is found in coastal east Asia in climates ranging from subtropical to subarctic. However, a disjunct population in the Pacific Northwest of North America has an ambiguous heritage. Two potential models explain the presence of Ae. togoi in North America: ancient Beringian dispersal or modern anthropogenic introduction. Genetic studies have thus far proved inconclusive. Here we described the putative ancient distribution of Ae. togoi habitat in east Asia and examined the climatic feasibility of a Beringian introduction into North America using modern distribution records and ecological niche modeling of bioclimatic data from the last interglacial period (~120,000 BP), the last glacial maximum (~21,000 BP), and the mid-Holocene (~6000 BP). Our results suggest that suitable climatic conditions existed for Ae. togoi to arrive in North America through natural dispersal as well as to persist there until present times. Furthermore, we find that ancient distributions of suitable Ae. togoi habitat in east Asia may explain the genetic relationships between Ae. togoi populations identified in other studies. These findings indicate the utility of ecological niche modeling as a complementary tool for studying insect phylogeography.

Ultraviolet inflorescence cues enhance attractiveness of inflorescence odour to Culex pipiens mosquitoes.

Inflorescence patterns of ultraviolet (UV) absorption and UV-reflection are attractive to many insect pollinators. To understand whether UV inflorescence cues affect the attraction of nectar-foraging mosquitoes, we worked with the common house mosquito, Culex pipiens and with two plant species exhibiting floral UV cues: the tansy, Tanacetum vulgare, and the common hawkweed Hieraciumm lachenalii. Electroretinograms revealed that Cx. pipiens eyes can sense UV wavelengths, with peak sensitivity at 335 nm. Behavioural bioassays divulged that UV inflorescence cues enhance the attractiveness of inflorescence odour. In the presence of natural floral odour, female Cx. pipiens were attracted to floral patterns of UV-absorption and UV-reflection but preferred uniformly UV-dark inflorescences. Moreover, Cx. pipiens females preferred UV-dark and black inflorescence models to UV-dark and yellow inflorescence models. With feathers and pelts of many avian and mammalian hosts also being UV-dark and dark-coloured, foraging Cx. pipiens females may respond to analogous visual cues when they seek nectar and vertebrate blood resources.

Author Correction: Multimodal floral cues guide mosquitoes to tansy inflorescences.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Modeled distributions of Aedes japonicus japonicus and Aedes togoi (Diptera: Culicidae) in the United States, Canada, and northern Latin America.

The Asian bush mosquito, Aedes japonicus japonicus, and the coastal rock pool mosquito, Aedes togoi, are potential disease vectors present in both East Asia and North America. While their ranges are fairly well-documented in Asia, this is not the case for North America. We used maximum entropy modeling to estimate the potential distributions of Ae. togoi and Ae. j. japonicus in the United States, Canada, and northern Latin America under contemporary and future climatic conditions. Our results suggest suitable habitat that is not known to be occupied for Ae. j. japonicus in Atlantic and western Canada, Alaska, the western, midwestern, southern, and northeastern United States, and Latin America, and for Ae. togoi along the Pacific coast of North America and the Hawaiian Islands. Such areas are at risk of future invasion or may already contain undetected populations of these species. Our findings further predict that the limits of suitable habitat for each species will expand northward under future climatic conditions.

Lemongrass and Cinnamon Bark: Plant Essential Oil Blend as a Spatial Repellent for Mosquitoes in a Field Setting.

Plant essential oils (EOs) have been considered as spatial repellents to help disrupt the pathogen transmission cycle of mosquitoes. Our objective was to assess spatial repellency effects of EOs on the tropical yellow fever mosquito, Aedes aegypti (L.) (Diptera: Culicidae) and on local mosquito populations in coastal British Columbia (Canada). In laboratory experiments using protocols of the World Health Organization, three of the solitary EOs tested proved repellent to Ae. aegypti: cinnamon bark, lemongrass, and rosemary. Binary combinations of select EOs enhanced the repellent effect of single EOs through synergistic interactions. The EO blend of geranium and peppermint lowered the RD50 (the dose required to obtain 50% repellency) of each solitary EO by >1,000-fold. Compared with binary EO blends, ternary EO blends were typically less repellent to mosquitoes, possibly due to a dilution effect of the most effective EO constituent(s) in the blend. In field experiments, the EO blend of lemongrass and cinnamon bark expressed spatial repellency towards the cool weather mosquito, Culiseta incidens (Thomson) (Diptera: Culicidae), even when this blend was disseminated from devices as much as 1 m away from a sentinel trap releasing attractive vertebrate host odorants and CO2. Deployment of EOs as spatial repellents in small outdoor gatherings could help protect humans from mosquito-borne diseases, particularly when this tactic is coupled with other tools of mosquito management.

Multimodal floral cues guide mosquitoes to tansy inflorescences.

Female mosquitoes exploit olfactory, CO2, visual, and thermal cues to locate vertebrate hosts. Male and female mosquitoes also consume floral nectar that provides essential energy for flight and survival. Heretofore, nectar-foraging mosquitoes were thought to be guided solely by floral odorants. Using common tansies, Tanacetum vulgare L., northern house mosquitoes, Culex pipiens L., and yellow fever mosquitoes, Aedes aegpyti (L.), we tested the hypothesis that the entire inflorescence Gestalt of olfactory, CO2 and visual cues is more attractive to mosquitoes than floral odorants alone. In laboratory experiments, we demonstrated that visual and olfactory inflorescence cues in combination attract more mosquitoes than olfactory cues alone. We established that tansies become net producers of CO2 after sunset, and that CO2 enhances the attractiveness of a floral blend comprising 20 synthetic odorants of tansy inflorescences. This blend included nine odorants found in human headspace. The "human-odorant-blend" attracted mosquitoes but was less effective than the entire 20-odorant floral blend. Our data support the hypothesis that the entire inflorescence Gestalt of olfactory, CO2 and visual cues is more attractive to mosquitoes than floral odorants alone. Overlapping cues between plants and vertebrates support the previously postulated concept that haematophagy of mosquitoes may have arisen from phytophagy.

Attraction of Female Aedes aegypti (L.) to Aphid Honeydew.

Plant sugar is an essential dietary constituent for mosquitoes, and hemipteran honeydew is one of the many forms of plant sugar that is important to mosquitoes. Many insects rely on volatile honeydew semiochemicals to locate aphids or honeydew itself. Mosquitoes exploit volatile semiochemicals to locate sources of plant sugar but their attraction to honeydew has not previously been investigated. Here, we report the attraction of female yellow fever mosquitoes, Aedes aegypti, to honeydew odorants from the green peach aphid, Myzus persicae, and the pea aphid, Acyrthosiphon pisum, feeding on fava bean, Vicia faba. We used solid phase micro-extraction and gas chromatography-mass spectrometry to collect and analyze headspace odorants from the honeydew of A. pisum feeding on V. faba. An eight-component synthetic blend of these odorants and synthetic odorant blends of crude and sterile honeydew that we prepared according to literature data all attracted female A. aegypti. The synthetic blend containing microbial odor constituents proved more effective than the blend without these constituents. Our study provides the first evidence for anemotactic attraction of mosquitoes to honeydew and demonstrates a role for microbe-derived odorants in the attraction of mosquitoes to essential plant sugar resources.

Populations of the gall midge Dasineura oxycoccana on cranberry and blueberry produce and respond to different sex pheromones.

We identified and field-tested the sex pheromones of Dasineura oxycoccana (Johnson) (Diptera: Cecidomyiidae) midges collected from cranberry, Vaccinium macrocarpon Aiton, and from highbush blueberry, Vaccinium corymbosum L., commonly named cranberry tipworm (CTW) and blueberry gall midge (BGM), respectively. Coupled gas chromatographic-electroantennographic detection (GC-EAD) analyses of pheromone gland extract from the ovipositor of calling CTW females revealed one component (<10 pg per ovipositor/pheromone gland) that elicited antennal responses from CTW males. Stepwise identification was based on its mass spectrum in a concentrated sample with 300 pheromone gland equivalents, retention indices (RI) on three GC columns (DB-5, DB-23, and DB 210), RI inter-column differentials, and RIs and double bond positions of other midge pheromones. These analyses indicated that (8Z)-2,14-diacetoxy-8-heptadecene (2,14-8Z-17) was the candidate pheromone of the CTW. GC-EAD analysis of pheromone gland extract from calling BGM females revealed two components that elicited antennal responses from BGM males. Retention times on the three GC columns were consistent with 2,14-8Z-17 and 2,14-17, indicating that these were candidate pheromone components of the BGM. The four stereoisomers of 2,14-8Z-17 were stereoselectively synthesized and field-tested in cranberry. Delta-type traps baited with SS-2,14-8Z-17 captured significantly more CTW males than did traps baited with any other single stereoisomer or with all four stereoisomers combined. In blueberry, delta-type traps baited with RR-2,14-8Z-17 captured significantly more BGM males than did traps baited with any other single stereoisomer or with all four stereoisomers combined. Subsequent field experiments demonstrated that RR-2,14-17 is the major pheromone component of BGM, and that RR-2,14-8Z-17 is a pheromone component that does not enhance attractiveness of RR-2,14-17. The BGM pheromone RR-2,14-17 has no antagonistic effect on the CTW pheromone SS-2,14-8Z-17 and vice versa. Our results substantiate the conclusion that populations of D. oxycoccana on cranberry and blueberry represent two cryptic species.