The roles of kairomones, synomones and

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Acta Tropica 132S (2014) S26–S34

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The roles of kairomones, synomones and pheromones in the chemically-mediated behaviour of male mosquitoes b,c ¯ R. Jason Pitts a,∗ , Raimondas Mozuraitis , Anne Gauvin-Bialecki d , Guy Lempérière e a

Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA Laboratory of Chemical and Behavioural Ecology, Institute of Ecology, Nature Research Center, 08412 Vilnius, Lithuania Ecological Chemistry Group, Division of Organic Chemistry, Department of Chemistry, School of Chemistry and Engineering, Royal Institute of Technology, Teknikringen 36, 10044 Stockholm, Sweden d Laboratoire de Chimie des Substances Naturelles et des Sciences des Aliments, Université de La Réunion, 15 Avenue René Cassin CS 92 003, 97744 Saint-Denis cedex 9, Reunion e Institut de recherche pour le développement (IRD), Maladies infectieuses et vecteurs: écologie, génétique, évolution et contrôle (MIVEGEC), 224-CNRS 5290-UM1-UM2, Montpellier, France b c

a r t i c l e

i n f o

Article history: Received 3 April 2013 Received in revised form 7 September 2013 Accepted 7 September 2013 Available online 17 September 2013 Keywords: Male mosquito Chemical ecology Semiochemical Kairomone Synomone Pheromone Odour

a b s t r a c t Despite decades of intensive study of the chemical ecology of female mosquitoes, relatively little is known about the chemical ecology of males. This short review summarizes the current state of knowledge of the chemicals that mediate male mosquito behaviour. Various trophic interactions including insect–plant, insect–host, and insect–insect responses are emphasized. The relevance of the chemical ecology of male mosquitoes in the context of vector control programmes is discussed. Copyright © International Atomic Energy Agency 2013. Published by Elsevier B.V. All rights reserved.

1. Introduction Observations regarding the interactions of animals and their chemical environments began in earnest in the late 19th and early 20th centuries, but it was not until the middle of the 20th century that the major concepts of chemical communication and a new vocabulary were introduced by noted researchers Karlson and Lüscher (1959) and Butenandt et al. (1959). Since then, substantial progress has been made in the isolation, identification, and synthesis of chemical compounds and in the confirmation of their activities through bioassays utilizing a variety of

Abbreviations: GC–MS, gas chromatography–mass spectrometry; HPLC, highperformance liquid chromatography; SPME, solid-phase microextraction; SIT, sterile insect technique; GM, genetically-modified; GLV, green leaf volatiles; GC-EAD, gas chromatographic-electroantennographic detection; DDT, dichlorodiphenyltrichloroethane; DEET, N,N-diethyl-m-tolumide; Ors, odourant receptors; Irs, variant ionotropic receptors; Grs, gustatory receptors; Obps, odourant binding proteins. ∗ Corresponding author at: Department of Biological Sciences, Vanderbilt University, 465 21st Avenue South, Nashville, TN 37232 USA. Tel.: +1 615 343 3718. E-mail address: [email protected] (R.J. Pitts).

animal models. Over the past forty years, the development of analytical techniques like gas chromatography–mass spectrometry (GC–MS), high-performance liquid chromatography (HPLC), solid-phase microextraction (SPME) and electrophysiology have paved the way for a wide range of studies and vast publication record focused on the chemical ecology of insects. The knowledge base produced by these studies has lead directly to the production of novel control strategies for pests in agriculture and forestry (Witzgall et al., 2010). Among the earliest studies of insect chemical ecology was a report published by Willem Rudolfs that established the importance of various stimuli, including chemical compounds, in eliciting changes in mosquito behaviour (Rudolphs, 1922). Rudolfs’ use of the term “chemotropism” did not necessarily imply directed movement along a chemical gradient, which is the more modern definition of the behaviour. Instead, he used the term to describe mosquito activation and/or attraction in response to odours (Rudolphs, 1922). Studies of mosquito responses to floral odours were published in the second half of the 20th century (Sandholm and Price, 1962; Thorsteinson and Brust, 1962; Vargo and Foster, 1982; Healy and Jepson, 1988). Later, Clements’ general textbook on mosquitoes (1999) synthesized several aspects of olfaction, sensory reception and behaviour thus describing the

0001-706X/$ – see front matter. Copyright © International Atomic Energy Agency 2013. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.09.005

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Fig. 1. Male mosquito trophic interactions. Male mosquitoes utilize a variety of volatile and contact chemical cues to orient towards sources of sugar, vertebrate hosts, and conspecifics.

general basis of chemoreception in mosquitoes. In the 21st century, the odour-mediated host interactions of female mosquitoes have been widely investigated, but only recently have other trophic interactions like mosquito–plant interactions been studied. Several reviews have focused on specific aspects of female mosquito ecology, particularly host seeking, and are not discussed here (Takken and Verhulst, 2013; Verhulst et al., 2010; Takken and Knols, 1999; Costantini et al., 1999; Bentley and Day, 1989). Importantly, the behaviour of male mosquitoes has been largely ignored and thus there is a significant gap in our knowledge of male chemical ecology. This short review is intended to provide a summary of our current knowledge of the chemical ecology of adult male mosquitoes based upon various trophic interactions that have been described in scholarly publications: insect–plant (kairomones and synomones involved in nectar feeding), insect–insect (pheromones mediating male–female interactions), and insect–host (kairomones attracting species that mate near hosts). Studies that focus on male mosquitoes, which have paled in comparison with the numbers of studies focused on females, will not only enhance our understanding of basic mosquito biology, but are likely to directly impact vector control or surveillance. Specifically, we expect that integrated control strategies which utilize mass releases of laboratory-reared species, either in the context of the sterile insect technique (SIT) or in the use of genetically-modified (GM) mosquitoes, will benefit from a deeper understanding of the chemical ecology of male mosquitoes. 2. Sources of the semiochemicals involved in male mosquito behaviour As shown in Fig. 1, at least three chemically-mediated behaviours have been observed in male mosquitoes: the search of food sources (insect–plant), the search for hosts where conspecific females are likely to be found (insect–host), and the selection of sexual partners (insect–insect). With respect to communication level, i.e. to which species message sending and receiving individuals belong to, the relevant semiochemicals involved in each of these behaviours can thus be considered as falling into two

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categories: the allelochemicals (kairomones, and synomones), which mediate interspecific interactions, and the pheromones, which mediate intraspecific interactions (Whittaker and Feeny, 1971). Kairomones are volatile compounds emitted by one species that are beneficial to the receiver. These compounds are involved in the mosquito–plant and mosquito–host interactions. In the first case, kairomones are produced by flowers, leaves or fruits and attract both sexes towards nectar sources, or perhaps resting sites. In the second case, kairomones are produced by humans or animals as breath, sweat, or skin emanations and attract female mosquitoes towards sources of blood-feeding. These same kairomones may be utilized by males of some species for attraction to hosts for mate location. Carbon dioxide is the most common kairomone and often acts synergistically with other compounds to elicit female flight and/or attraction (Gillies, 1980; Kline et al., 1991; Cork, 1996; Takken and Knols, 1999). With respect to pheromones, there are several types of behaviourally active compounds described in insects, but in mosquitoes may include sex pheromones (Kliewer et al., 1966; Nijhout and Craig, 1971; Lang and Foster, 1976; see discussion below) and oviposition pheromones (Osgood, 1971; Starratt and Osgood, 1972; Bruno and Laurence, 1979), both classes having been recognized in various mosquito species. The fatty acid lactone, erythro-6-acetoxy-5-hexadecanolide, is produced and released by ovipositing Culex quinquefasciatus females and significantly enhances oviposition by other gravid conspecifics both in the laboratory (Bruno and Laurence, 1979; Laurence and Pickett, 1985; Otieno et al., 1988a) and in the field (Otieno et al., 1988b). Without respect to communication level (i.e. odour source), semiochemicals can be also be classified more broadly as attractants, repellents, stimulants, deterrents and arrestants. The repellent properties of many compounds against female mosquitoes have been widely documented and their use as personal protection against blood feeding has been consistently investigated and developed (Debboun and Strickman, 2013). To our knowledge there are only a few publications dealing with the repellent properties of synthetic compounds against male mosquitoes, which will be briefly discussed.

3. Male responses to plant volatiles Allelochemicals used by mosquitoes to locate food sources fall into two categories: (i) synomones which benefit both odour releasing and perceiving organisms, i.e. plants are pollinated and mosquitoes receive a nectar reward, and (ii) kairomones when only mosquitoes benefit from perceiving the plant signal and taking nectar from flowers without pollinating them. Despite the fact that odour-mediated sugar source seeking in mosquitoes is well documented the chemical identification of plant attractants is rather limited. Data about carbohydrate source location by mosquitoes obtained under field and laboratory conditions indicates that males and females show comparably similar responses and preferences (Jepson and Healy, 1988; Healy and Jepson, 1988; Jhumur et al., 2006, 2007a,b; Otienoburu et al., 2012); electroantennographic responses to floral odours of both sexes were also found to be similar (Jhumur et al., 2007a,b) (Fig. 2). Electrophysiological recordings revealed a high proportion of both broadly- and narrowly-tuned antennal receptor neurons of Culex pipiens pipiens L. sensitive to monoterpenes including thujone, verbenone, ␣-pinene, limonene, citral, and nerol, to sesquiterpene farnesol as well as three of each green leaf volatiles (GLVs) and fatty acid esters (Bowen, 1992). Behavioural tests showed that bicyclic terpene, thujone, at stimulus intensities within the dynamic range of the terpene-specific Ors, stimulated dose-dependent post-landing feeding responses [probing] in food-deprived, non-bloodfed female mosquitoes. In another study,

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Fig. 2. Compounds implicated in male mosquito responses. Chemical structures of plant volatile compounds that elicit electrophysiological or behavioural responses in male mosquitoes.

gas chromatographic-electroantennographic detection (GC-EAD) experiments revealed no qualitative differences and very similar quantitative antennal responses of males and females of Cx. pipiens pipiens biotype molestus to headspace flower odour samples of Silene otites (L.) consisting of 6 aromatic compounds including acetophenone, benzaldehyde, benzyl alcohol, phenyl acetaldehyde, phenylethyl alcohol, methyl salicylate four of each lilac aldehydes and lilac alcohols, three GLVs: hexanol, (Z)-3-hexen-1-ol and corresponding acetate, as well as the terpenoids linalool and binary mixes of (Z)- and (E)-isomers in both linalool oxide samples of the furanoid and pyranoid type. In bioassays, the behavioural attractiveness of the 14 single electrophysiologically active compounds were tested and ranged from 65 to 20% where acetophenone, linalool oxide (pyranoid), phenyl acetaldehyde and phenylethyl alcohol were found the most attractive in comparison to the

least attractive compound, hexanol. In a two-stimulus choice test, mosquitoes were significantly more attracted to the mixture of those four most attractive compounds compared to the mixture of all 14 compounds (Jhumur et al., 2007a,b). Furthermore, in comparison with naive mosquitoes, conditioned mosquitoes were significantly more attracted to the mixture of acetaldehyde, veratrole and 2-methoxyphenol as well as to the same volatiles tested as the single compounds (Jhumur et al., 2006). Mauer and Rowley (1999) showed that methylene chloride extracts of common milkweed flowers Asclepias syriaca L. were attractive to Cx. pipiens pipiens indicating that volatile chemicals are at least partly responsible for that attraction. GC–MS analysis revealed that 2phenylethanol and benzyl alcohol dominated the headspace profile of common milkweed flower, however, a synthetic blend of these two compounds was not attractive to mosquitoes. In another

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study, a significant positive upwind orientation by Cx. pipiens males and females to a pentane extract of A. syriaca was demonstrated in a dual-port flight olfactometer and the number of attractive compounds was narrowed to 3 volatiles, benzaldehyde, phenylacetaldehyde and (E)-2-nonenal whose activity was comparable to that of the extract (Otienoburu et al., 2012). In a series of laboratory bioassays, Jepson and Healy (1988) investigated the long range attraction of both male and female Aedes aegypti L. towards floral nectar sources of the ox-eye daisy, Leucothemum vulgare Lam., eliciting landings by flying mosquitoes. They showed a biphasic diel cycle of flower visits at dawn and dusk. In modified flowers with ray and disc florets removed, the landing of mosquitoes was reduced indicating their role in the location of flowers. It was also shown that male and female Ae. aegypti responded in a similar way by landing on small glass funnels through which the odour of L. vulgare, was passed in the absence of visual stimuli. Solvent extracts of flowers did not produce behavioural responses in their bioassay as compared to bioassays carried out previously by Vargo and Foster (1982). They also hypothesized that adults with low energy reserves could have a low threshold of responsiveness to plant odours. In another study using a dual port olfactometer, Kline et al. (2003) demonstrated that linalool and dehydrolinalool, which are present in floral odours of many plant species, resulted in activation and/or orientation of Ae. aegypti mosquitoes to the chemical source, linalool being the most attractive compound. GC-EAD studies conducted by Jhumur et al. (2007a,b) revealed that 15 and 18 floral volatiles of Spanish catchfly, S. otites (L.) evoked antennal responses of male and female Ae. aegypti mosquitoes, respectively. Moreover, the data showed no remarkable differences between responses of day-active Ae. aegypti and night-active Cx. pipiens pipiens mosquitoes indicating that both species use similar chemical cues for detecting S. otites as a nectar source. A wind-tunnel bioassay experiment showed that both sexes of Anopheles arabiensis Patton mosquitoes responded by flight and landing to common yarrow, Achillea millefolium L. inflorescences and to the odours released from pentane extracts of the flowers and a cyclic or bicyclic monoterpene was suspected to be the major active component of the floral odour (Healy and Jepson, 1988). Dual choice olfactometer assays revealed that female An. gambiae Giles discriminated between odours released by three suspected host plants Santa Maria feverfew, Parthenium hysterophorus L., castor oil plant, Ricinus communis L. and Cobbler’s pegs Bidens pilosa L. where the first and the last species were found the most and the least attractive, respectively. Of the six EAD-active components tested at the doses 10, 20, 40, 80, and 160 ng/dispenser, hexanal, ␣-pinene, limonene and (E)-linalool oxide, were highly attractive at 40 ng/dispenser, while ␣-ocimene and (E)-␣-farnesene were optimally attractive at 20 ng/dispenser. It was found that hexanal remained attractive at all five doses, while females demonstrated avoidance behaviour to the other five compounds presented at a dose of 160 ng/dispenser (Nyasembe et al., 2012). Strong fragrances of flowers or volatiles originating from fermenting fruits which mosquitoes use for long-range sugar source location (Foster, 1995; Takken and Knols, 1999; Mauer and Rowley, 1999; Jhumur et al., 2006, 2007a,b, 2008; Otienoburu et al., 2012) got use in mosquito control programmes in particular attract and kill strategy. Attractive toxic sugar bait (ATSB) method consists of fruit or flower scent as an attractant, sugar solution for feeding, and low-risk oral toxins such as boric acid mixed with sugar to kill the mosquitoes. Attractive toxic sugar formulations are either sprayed on plants or provided in bait stations. Successful ATSB field trials have been reported to decrease local populations of Anopheline and Culicine mosquito species in Israel (Beier et al., 2012; Müller and Schlein, 2006, 2008; Müller et al., 2008, 2010a; Schlein and Müller, 2008), to control Cx. quinquefasciatus Say in Florida, USA

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(Müller et al., 2010b) and An. gambiae s.l. in Mali (Müller et al., 2010c). The experiments in Israel were carried out in arid areas where typically there is scarcity of attractive floral sources for mosquitoes. Further experiments are required for testing the function of the ATSB in a less advantageous environment. On the other hand, it is well known that floral volatiles are responsible for attraction of pollinators and this aspect was not evaluated or even discussed, thus more extensive use of toxic sugar formulations could have undesirable impact on pollinators. One final point of consideration would be the use of plant volatiles in mosquito surveillance programmes, perhaps for both males and females. Odour-bated trapping systems are commonly used to monitor mosquito population dynamics and infection and often rely on attracting host-seeking or gravid males (Silva et al., 2005; Allan et al., 2005; Kline et al., 2006; Farajollahi et al., 2009; Mukabana et al., 2012; Tchouassi et al., 2013). In contrast, a recent study reported significantly higher than expected collections of male Ae. aegypti in French Polynesia using odour-bated Biogents (BG) Sentinel Traps (Hapairai et al., 2013). Perhaps plant volatile-based trapping systems would offer a complementary or alternative method of surveillance. Such systems may even be more sensitive than current strategies as they would potentially attract both males and females in all gonotrophic states and in reproductive diapause. Moreover synthetic host odour, or plant infusion odour-bated trapping systems are often very biased in the species collected, such that plant volatile odour-bated traps may attract a more diverse profile of mosquito species.

4. Host odours and candidate pheromones in male swarming Males of numerous Dipterans, including mosquitoes, form swarms as a prerequisite to mating (Downes, 1969). Swarming behaviour in both the Anophelines and Culicines is well documented. Species like An. gambiae form large swarms in the absence of host animals, presumably using visual cues (Charlwood and Jones, 1980). The univoltine species Ae. communis and Ae. stimulans have been observed forming swarms in large walk-in cages and mating pairs form in flight (McDaniel, 1986). In other species, like Mansonia uniformis and Ma. africana, swarming is rarely observed and males are known to orient towards host animals where they will presumably find females for mating (Gillies and Wilkes, 1975; McIver et al., 1980). Still, males of other species that are not normally associated with swarming have been observed to aggregate near hosts in nature. For example, small swarms of 3–40 Ae. albopictus males congregated around the feet and ankles of human observers (Gubler and Bhattacharya, 1972). Females entering these swarms were engaged by males and copulation ensued (Gubler and Bhattacharya, 1972). Similar observations have been made for Ae. aegypti in nature (Lumsden, 1957; McClelland, 1959; Hartberg, 1971). Moreover, a group of researchers working in French Polynesia reported collecting a higher proportion of males than females in human bait collections, which supports the concept that males of some species orient towards hosts in the field (Hapairai et al., 2013). In the laboratory, Ae. aegypti males can be induced to form leks by stimulation with a host odour at the onset of darkness (Cabrera and Jaffe, 2007). Swarming males, when placed upwind in an olfactometer elicited female flight activity, leading the authors to suggest that males produce an aggregation pheromone (Cabrera and Jaffe, 2007). While this is formally possible, other explanations can be offered. Indeed females placed upwind elicited female flight equally well, while air alone also induced flight activity, albeit at statistically reduced levels (Cabrera and Jaffe, 2007). The authors do not mention any attempt to remove human odours during

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the olfactometer experiments, potentially confounding the results. Similarly, glass cylinders containing Cx. pipiens pipiens, Cx. pipiens quinquefasciatus, and Cx. tarsalis males, alive or dead, attracted significantly more conspecific females than empty cylinders in a dual choice olfactometer (Gjullin et al., 1967). In some cases this attractiveness was significant even between heterospecifics (Gjullin et al., 1967). While the aforementioned studies provide insight into lekking behaviours, follow-up studies are needed and direct evidence for aggregation pheromones in mosquitoes remains elusive.

5. Auditory and chemosensory cues in mosquito mating Whatever the ultimate causes of mosquito swarming, including aggregation above or near visible swarm markers (Marchand, 1984; Yuval and Bouskila, 1993; Charlwood et al., 2002, 2003; Manoukis et al., 2009; Tuten et al., 2013), considerable evidence for the importance of audition in the act of mating pair formation has been documented by many researchers (reviewed in Gibson and Warren, 2010). Numerous studies have shown that conspecific pairs modulate their wingbeat vibrations when brought into close proximity, such that one or more of the harmonic frequencies converge (Gibson and Russell, 2006; Cator et al., 2009; Gibson and Warren, 2010; Pennetier et al., 2010). The ability of mating pairs to successfully converge also correlates with mating success in the next generation of males (Cator and Harrington, 2011). Incredibly, M and S forms of An. gambiae perform harmonic wingbeat convergence much more readily with like-form partners (Pennetier et al., 2010). The authors conclude that such convergence contributes to assortative mating and incipient speciation, although they also speculate that this mechanism alone is insufficient to explain the behaviour. We propose that in these and similar experiments where mosquitoes come into very close proximity, although not necessarily touching, a form of chemical communication takes place where males (and perhaps females) recognize a low volatility, most likely contact, sex pheromone produced by conspecifics. Contact sex pheromone production in Dipterans has long been recognized and mediates mating responses in houseflies, tsetse flies and Drosophila melanogaster (Antony and Jallon, 1982; Tillman et al., 1999; Ferveur, 2005). Their existence in the fly lineages implies that mosquitoes may also utilize a similar mode of species recognition. Observations collected from various mosquito species offers insight into this question, where in some species the sound stimulus seems to be less important than a chemical one. For example, adult males of the crabhole mosquito, Deinocerites cancer, and the winter mosquito, Culiseta inornata, locate female pupae, await their emergence, and mate with newly eclosed virgins (Haeger and Phinizee, 1959; Kliewer et al., 1966). The sensory basis of this behaviour is presumed to be chemical as males also attempt copulation with empty pupal cases shortly after females have emerged (Haeger and Phinizee, 1959) or with recently dead females (Kliewer et al., 1966; Lang and Foster, 1976). Moreover, males search for potential mates by touching conspecifics of either sex with their front leg tarsi. Ultimately, males that encounter a receptive female will initiate copulation attempts (Haeger and Phinizee, 1959; Kliewer et al., 1966). These observations alone suggest the presence of a female-produced chemical stimulus that is sensed by contact and modifies male behaviour. In the case of Cs. inornata, neither male antennae nor female wings are strictly required conspecific recognition as antennalectomized males and wingless females exhibit high frequencies of mating (Kliewer et al., 1966). Based on simple olfactometery attraction assays, Kliewer et al. (1966) also described evidence for a volatile sex pheromone in Cs. inornata, a result that was not repeatable by later experimenters (Lang and Foster, 1976). In hindsight, the results of the former

experiments lacked statistical significance in almost all trials (8 of 10), but were considered significant in the aggregate (Kliewer et al., 1966). Despite this, the suggestion of a possible contact sex pheromone in Cs. inornata by Kliewer et al. was supported by Lang and Foster (1976) who found that the legs of females were necessary for full stimulation of male copulation attempts and sufficient for males to discriminate between conspecifics and heterospecifics (Lang and Foster, 1976). Furthermore, upon closer examination the copulation-releasing substance found in female Cs. inornata legs remained active for several days after legs were removed from female bodies, was relatively heat stabile, and could be removed by washing the legs in certain solvents (Lang, 1977). These experiments offer strong support for the existence of a contact pheromone in this mosquito. To our knowledge, follow-up studies have been lacking, despite the potential importance of identifying mosquito sex pheromones. Nijhout and Craig demonstrated that several species of the subgenus Stegomyia were able to correctly choose between conspecific and heterospecific females while free flying in cages (Nijhout and Craig, 1971). Ae. albopictus males were especially adept at correctly choosing conspecific females. Males would approach heterospecific females that were suspended in cages, but would not attempt copulation with them once contact was initiated (Nijhout and Craig, 1971). Similar to Cs. inornata, Ae. albopictus males would attempt copulation with living or recently dead conspecific females, when first being attracted by an electronic sound source mimicking the female wingbeat. Perhaps most telling, the percentage of copulation attempts by Ae. albopictus males was dramatically reduced when the terminal tarsomeres of the prothoracic and mesothoracic tarsi were treated with a solvent mixture (Nijhout and Craig, 1971). These results provided evidence for a contact sex pheromone in several Aedes species and implicated the terminal tarsomeres as the sites of pheromone perception by male Ae. albopictus (Nijhout and Craig, 1971). As with Cs. inornata, the evidence for the existence of contact sex pheromones in Aedes species has been available for several decades. We suggest that future studies into this critical aspect of mosquito biology are highly warranted. Such studies would offer important new insights into mosquito chemical ecology and reproduction and may impact future surveillance or control strategies. 6. Male responses to synthetic repellents Recent publications by Said et al. (2009) and Kongmee et al. (2010) showed that males of Ae. aegypti test populations were significantly repelled when exposed to DDT at the test dose of 25 nmol/cm2 , whereas ␣-cypermethrin and deltamethrin failed to elicit directional movements. Bioassay tests using control odourant-free and DEET-treated arenas revealed that Cx. quinquefasciatus mosquitoes of both sexes significantly avoided landing on DEET-treated filter paper, indicating that DEET repellency is not sex specific, but exerts its effect through common mechanisms in adults (Syed and Leal, 2008). Nonetheless, these results suggest that further investigation into the potential spatial repellency of insecticides or personal protective compounds is needed, as some may differentially affect males and females, or adults in various physiological states. If such differences exist, they could help reveal modes of action as well as inform control and surveillance programmes. 7. Molecular biology of male chemosensation Recent work has begun to elucidate the molecular mechanisms that underlie chemosensation in mosquitoes (Leal, 2013; Rützler and Zwiebel, 2005). The genomes of three medically important

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species, An. gambiae, Ae. aegypti, and Cx. quinquefasciatus, have led to the identification of major chemosensory gene families including odourant receptors (Ors), gustatory receptors (Grs), variant ionotropic receptors (Irs), and odourant binding proteins (Obps), among others (Hill et al., 2002; Biessmann et al., 2002; Xu et al., 2003, 2010; Li et al., 2005; Bohbot et al., 2007; Kent et al., 2008; Pelletier et al., 2010; Liu et al., 2010; Croset et al., 2010). Although comparisons between males and females are generally lacking, some studies have indicated that males and females express similar arrays of chemosensory genes, albeit at different relative levels (Biessmann et al., 2005; Bohbot et al., 2007; Schymura et al., 2010; Pitts et al., 2011; Pelletier and Leal, 2011). These results suggest that males and females possess qualitatively similar chemical discriminatory abilities, but that females by virtue of significantly higher expression of these genes may exceed males in their thresholds of chemical sensitivity. Direct examination of this hypothesis, both electrophysiologically and behaviourally would be of great interest. Moreover, the global examination of transcript and/or protein expression in male legs, which are the sites of contact pheromone perception in Dipterans like Glossina morsitans, Musca domestica, and D. melanogaster (Langley et al., 1975; Schlein et al., 1980, 1981; Bray and Amrein, 2003), could be used to identify candidate sex pheromone receptors. In the fruitfly, D. melanogaster, gustatory receptors DmGr39a and DmGr68a, and a novel chemosensory protein, CheB42a, are expressed in the forelegs and are involved in male courtship behaviour (Bray and Amrein, 2003; Watanabe et al., 2011; Park et al., 2006). DmGr68a is also important for detection of moving females by males and courtship initiation (Ejima and Griffith, 2008). Alternative splicing of DmGr39a implies the possibility of diverse function of isoforms expressed in different tissues. Interestingly, homologues of these 2 genes are found in the An. gambiae and Ae. aegypti genomes, each with predicted alternative splice forms (Hill et al., 2002; Kent et al., 2008) and may be the most likely candidates to encode sex pheromone receptors in those species. In addition, an An. gambiae gustatory receptor, AgGr33, is highly enhanced in male antennae (Pitts et al., 2011). Perhaps this receptor is involved in one or more male-specialized functions in An. gambiae, which may include either hearing reception of an unrecognized volatile sex pheromone. Although direct evidence for volatile pheromones in mosquitoes is lacking, it is worth noting that D. melanogaster utilizes both contact pheromones like the female-produced cuticular hydrocarbon, heptacosadiene, and volatile pheromones like the male-produced compound, 11-cis vaccenyl acetate, as well as auditory cues in its complex courtship behaviour (reviewed in Smith, 2012).

8. Concluding remarks Compared with past studies of female mosquitoes, the chemical ecology of males has been much less intensively investigated and is therefore poorly understood. Certainly this is expected given that females are responsible for disease transmission in mosquitoes. Nonetheless, the biology of male mosquitoes should be of interest from a basic research perspective, especially as comparisons between sexes for any given species may themselves provide valuable insights into the behaviour of females. Moreover, past successes in controlling insects of agricultural and veterinary importance have generated a renewed interest in the use of SIT or GM mosquitoes as components of vector control strategies (Coluzzi and Costantini, 2002; Tabachnick, 2003; Riehle et al., 2003; Malcolm et al., 2009; Nolan et al., 2011). Such programmes necessitate a deeper understanding of male mosquito behaviour in general, and male chemical ecology specifically. Despite the legitimate ethical concerns and the many technical and regulatory difficulties of implementing SIT or

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GM on a large scale, sterile male releases have occurred in the past with some early successes in El Salvador and the Florida Keys (reviewed by Dame et al., 2009; Benedict and Robinson, 2003). Conceptually similar methodologies for mosquito population suppression based on the RIDL technology are again moving forward very rapidly either in practice or in concept (Harris et al., 2011; Lacroix et al., 2012; Marinotti et al., 2013). Moreover, paratransgenic releases (McGraw and O’Neill, 2013) or gene drive systems will ultimately depend on successful mating between modified, laboratory-reared individuals and their wild type counterparts, which again necessitates a deeper understanding of male-female interactions and a well-conceived, balanced research agenda (Tabachnick, 2003). Even when laboratory experiments confirm the mating competitiveness of modified males, moving from the laboratory to the field can be confounding. For example, male Cx. tritaeniorhynchus that carried a complex chromosomal inversion performed well in laboratory mating experiments, but demonstrated strong assortative mating upon release, generally failing to make with wild females (Baker et al., 1979; Reisen et al., 1980). This effect was observed in consecutive years of release, despite a concerted effort to outcross the inversion into the local genetic background in the second year of release (Reisen et al., 1980). Given these arguments, we have identified some relevant questions that should be addressed: (1) Are laboratory-modified males equally able to locate and utilize sugar sources compared to wild males? (2) Do modified males correctly recognize conspecifics? (3) Is the host seeking and/or swarming behaviour of modified males compromised? and (4) Do males respond to, and mate efficiently with, modified females? The answers to these questions are likely to be complex and context dependent; their context perhaps strongly influenced by the chemical ecology of males. Therefore, urgent attention is needed to ensure the fitness and competitiveness of modified males and/or the potential weaknesses of population replacement strategies and gene drive systems that rely on releasing either females or both sexes. Without this knowledge, the efficacies of release programmes are likely to fall short of expectations. Moreover, chemical modifications of males prior to release may actually improve their competitiveness. For example, males that are “preconditioned” by being placed in proximity to volatile odour compounds for an empirically determined amount of time prior to release could show increase survivorship or mating competitiveness and therefore enhance vector control outcomes. The pre-exposure odours could either represent naturally occurring compounds that would otherwise attract males to local sugar sources or they could be odours that are provided in artificial sugar sources at release sites. Precedent for this concept has been demonstrated in the Mediterranean fruit fly, Ceratitis capitata, whereby irradiated males that had been exposed to ginger root oil displayed dramatically improved mating success compared to unexposed males when placed in competition with unexposed wild males in laboratory settings (Shelly et al., 2003; Shelly, 2001; Shelly and McInnis, 2001) and in field trials (Shelly et al., 2004, 2007). The presumed effect of this treatment is the presence of ␣-copaene in ginger oil, which is a known male attractant that is important for medfly lekking and stimulates male release of calling pheromone (Nishida et al., 2000; Shelly et al., 1996). Although the mating behaviours of medflies and mosquitoes may be quite distinct, very little is known about the potential chemical compounds involved in mosquito mating. The availability of a wide range of investigative approaches, including chemical separation/identification, electrophysiology, behaviour, and molecular biology, should help produce a wealth of new knowledge relating to the chemical ecology of mosquitoes. A renewed appreciation for the importance of male mosquitoes in vector research programmes is needed as these studies may significantly contribute

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