Wooding et al. Parasites Vectors (2020) 13:80 https://doi.org/10.1186/s13071-020-3960-3 Parasites & Vectors

REVIEW Open Access Controlling mosquitoes with semiochemicals: a review Madelien Wooding1, Yvette Naudé1, Egmont Rohwer1 and Marc Bouwer2*

Abstract The use of semiochemicals in odour-based traps for surveillance and control of vector mosquitoes is deemed a new and viable component for integrated vector management programmes. Over 114 semiochemicals have been identifed, yet implementation of these for management of infectious diseases such as malaria, dengue, chikungunya and Rift Valley fever is still a major challenge. The difculties arise due to variation in how diferent mosquito spe- cies respond to not only single chemical compounds but also complex chemical blends. Additionally, mosquitoes respond to diferent volatile blends when they are looking for a mating partner, oviposition sites or a meal. Analyti- cally the challenge lies not only in correctly identifying these semiochemical signals and cues but also in develop- ing formulations that efectively mimic blend ratios that diferent mosquito species respond to. Only then can the formulations be used to enhance the selectivity and efcacy of odour-based traps. Understanding how mosquitoes use semiochemical cues and signals to survive may be key to unravelling these complex interactions. An overview of the current studies of these chemical messages and the chemical ecology involved in complex behavioural patterns is given. This includes an updated list of the semiochemicals which can be used for integrated vector control manage- ment programmes. A thorough understanding of these semiochemical cues is of importance for the development of new vector control methods that can be integrated into established control strategies. Keywords: Malaria, Vector mosquitoes, Anopheles, Aedes, Culex, Mosquito life-cycle, Semiochemicals, Chemical communication

Background some minor species are also cause for concern. For Mosquitoes transmit serious infectious diseases that example, the Asian native mosquito An. stephensi is now include dengue, chikungunya, Rift Valley fever and established in Ethiopia [4] and An. arabiensis is deemed malaria. In Africa, malaria remains as one of the most a major cause of residual malaria transmission due to the serious vector-borne diseases [1]. Malaria is spread by species’ insecticide avoidance behaviours [2]. anopheline mosquitoes that transmit malaria parasites Vector control strategies such as indoor residual spray- to humans. Of the hundreds of Anopheles species found ing (IRS) and long-lasting insecticidal net (LLIN) pro- worldwide, only a few dozen mediate the transmission of grammes have played a crucial part in the reduction of the Plasmodium parasite to humans [2]. Africa has over malaria cases between 2002 and 2017 [5–7]. Tere are 128 native Anopheles mosquito species [3], with An. gam- two main problems afecting the future use of LLINs and biae (sensu stricto), An. coluzzii and An. funestus being IRS: frst, these strategies used alone or combined will the predominant malaria vector species [4]. However, not eradicate malaria incidences in high transmission areas and secondly, insecticide resistance of the major malaria vectors in Africa is widespread and increasing *Correspondence: [email protected] 2 Forestry and Agricultural Biotechnology Institute, University of Pretoria, [6, 7]. Another major concern with these vector control Hatfeld, Pretoria, South Africa strategies is that these tools mainly target Anopheles vec- Full list of author information is available at the end of the article tors that feed and rest indoors and have a preference to

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feed at night. Changes in mosquito host preferences, time sensillum recordings (SSR) and gas chromatography cou- of feeding and an increase in outdoor feeding due to plas- pled to electroantennographic detection (GC-EAD), has ticity in mosquito behavioural responses have prompted greatly facilitated research in the feld of chemical ecol- the need for new and more environmentally friendly and ogy [14]. Chemical ecology of mosquitoes, specifcally robust vector control strategies that supplement current how mosquito behaviour is mediated by odour (Table 1), control strategies [5, 8]. Te development and incorpo- is a major feld of study that aims at improving control ration of novel vector control tools based on new scien- strategies that depend on synthetic odour lures which are tifc knowledge about mosquito behaviour and chemical popular for use in lure-and-kill strategies [9]. ecology into integrated vector management (IVM) pro- Tis literature review discusses chemical communica- grammes are needed in order to reduce the burden and tion in vector mosquitoes, with the focus on malaria vec- threat of mosquito-borne diseases [6]. tors. Key chemical cues and signals afecting diferent Studying the intricate strategies that malaria mosqui- behavioural patterns in the adult mosquito are discussed toes have evolved to survive in their environment may (Fig. 1). Te emphasis is on providing an updated list result in novel control methods for malaria. Mosquito of chemical attractants which can be used in IVM pro- survival depends not only on how successful they are at grammes. Known semiochemicals for eleven mosquito fnding suitable mating partners, oviposition sites and species are tabulated. blood or sugar meals [9], but also on how efective they are at avoiding predators by moving around undetected Semiochemicals as identifed during the diferent and fnding suitable shelter locations [10]. Mosquitoes mosquito life‑cycle stages can do this by detecting information from their environ- Chemical cues and signals mediate key behaviours during ment through a set of sensitive sensory organs [10]. Arti- the diferent stages in the life-cycle of an adult mosquito fcially interfering with these processes may result in the [15, 16]. Signals are produced for the specifc purpose ability to reduce mosquito populations and subsequently of changing the behaviour of an individual of a species, the incidence of malaria. whereas cues are not produced for any specifc pur- A range of environmental factors are detected by mos- pose, but they are detected by individuals and can lead quitoes and are used as cues that can afect their behav- to behavioural responses [17]. Host fnding by the adult iour. Changes that occur in the visual environment of the mosquito occurs through a series of behavioural steps mosquito obviously play an essential role, but other fac- that are infuenced by external chemical cues. A mos- tors such as fuctuations in the temperature or humidity quito can be primed for activity by a specifc chemical or levels also have an impact. Perhaps, the most important blend produced by a potential host. Tis is followed by cues are those that are present in the volatile chemical the initiation of fight to bring the insect within the vicin- environment that surrounds the mosquito [9]. Mosqui- ity of the source [18]. Te mosquito will then continue to toes use odours to locate their hosts and it is known that respond to external stimuli during fight for orientation diferent mosquito species depend on and detect difer- through optomotor anemotaxis (movement in response ent types of host odours [1, 11]. For example, An. arabi- to air currents and visual stimuli). Visual cues, such as ensis responds more strongly to carbon dioxide (CO­ 2) as contours against a background, that aid the mosquito a general cue to fnd a host, while An. gambiae relies on during upwind fight and physical cues, such as convec- other odours more specifc to humans together with ­CO2 tion heat and body moisture, play a role during host seek- acting as a long-range attractant [11, 12]. Understanding ing. Tese cues, together with chemical cues, infuence how these odours difer and how they are perceived by the orientation of a mosquito towards the host and can specifc mosquito species is an important feld that has induce a landing response [11, 12, 18]. Te individual been well documented but is still not understood entirely. importance of chemical, visual and physical cues that New knowledge on the chemical ecology of insects (the impact on the mosquito’s fight behaviour is well stud- study of chemical structures used by insects to mediate ied. However, the mechanism of how these cues work intra- and interspecifc interactions) has led directly to together to result in specifc types of behaviours remains the development of novel pest control strategies in agri- a topic that requires further investigation. culture and forestry [13]. Observations regarding the Another major challenge in identifying mosquito semi- interactions of insects and their chemical environments ochemicals is the degree of plasticity that is observed in together with the development of analytical techniques the behavioural responses of mosquitoes. Te internal such as gas chromatography-mass spectrometry (GC- physiological state of the mosquito will alter the behav- MS), high-performance liquid chromatography (HPLC), ioural response elicited by a chemical cue or signal [9, solid-phase microextraction (SPME) and electrophysi- 11, 19]. For example, many mosquito species have an ology, including electroantennography (EAG), single inherent preference for a specifc host (e.g. a preference Wooding et al. Parasites Vectors (2020) 13:80 Page 3 of 20 , d , d acid benzaldehyde )-β-pinene, , ( − )-β-pinene, d , 2-methylpropionic , 2-methylpropionic d acid , 3-methylbutanoic ­ , 3-methylbutanoic , ( − )-α-pinene d d acid acid , artemisia ketone, umbellulone, (Z)-3-hexenyl , artemisia (Z)-3-hexenyl umbellulone, ketone, d , hexanoic ­ d ­ acid , 2-methylbutanoic ­ , 2-methylbutanoic d ­ acetophenone )-sabinene, (E/Z)-ocimene, ­ germacrene-D, ( − )-sabinene, benzoic benzoic - phenyl-2,3-butanedi alcohol, yomogi hexyl acetate, acetate, [ 103 ] 3-hydroxy-4-phenyl-2-butanone one, ­ acid Citral [ 96 ] Citronellol [ 96 ] Citronellol Fatty acids [ 96 ] Fatty Amyl acetate [ 96 ] acetate Amyl Toluene [ 96 ] Toluene Phenylethyl alcohol [ 96 ] Phenylethyl Phenylacetaldehyde [ 96 ] Phenylacetaldehyde Lilac aldehyde [ 96 ] Lilac aldehyde )-3-Hexenyl acetate [ 96 ] acetate ( Z )-3-Hexenyl Linalool oxide [ 96 ] Linalool oxide Linalool [ 96 ] Benzaldehyde [ 96 ] Benzaldehyde Lilac alcohol [ 96 ] Acetophenone [ 96 ] Acetophenone salicylateMethyl [ 96 ] Hexanal [ 96 ] [ 96 ] 1-Hexenol ( Z )-3-Hexen-1-ol [ 96 ] [ 104 ] Benzenoids 20-Compound blend: butanoic ­ Sugar feeding Terpineol [ 96 ] Terpineol Eugenol [ 96 ] Eugenol Nonanal [ 102 ] Geraniol [ 96 ] [ 11 , 19 61 ] 2 nonanal [ 69 ] octanal, nonanal and decanal [ 112 ] [ 9 , 19 ] Hexanoic acid [ 69 ] 2-Compound blend: octanal and Geranyl acetone [ 69 , 72 ] acetone Geranyl 6-Methyl-5-hepten-2-one [ 69 ] Linalool [ 69 , 72 ] 4-Compound blend: heptanal, 4-Compound blend: heptanal, Sulcatone [ 72 ] Sulcatone Dodecanal [ 72 ] Limonene [ 72 ] 2-Ethyl hexanol [ 72 ] 2-Ethyl Butyric acid [ 72 ] Heptanoic acid [ 72 ] Octanoic acid [ 72 ] Nonanoic acid [ 72 ] Host-seeking CO ( R )-1-Octen-3-ol [ 69 , 111 ] Lactic acid [ 11 ]/( S )-lactic acid - tetradecanoic acid and methyl tetrade acid and methyl tetradecanoic canoate [ 53 ] canoate p -cresol and phenol [ 59 ] 4-Ethylphenol (stimulant) [ 40 ] 4-Ethylphenol Camphor (stimulant) [ 40 ] Camphor β-pinene (stimulant) [ 40 ] Borneol (stimulant) [ 40 ] Borneol (stimulant) [ 40 ] acetate 3-compound blend: nonanoic acid, 3-compound blend: 3-methylindole, 3-compound blend: 3-methylindole, CHs [ 24 ] 6-hexanolactone [ 55 ] Methyl dodecanoate [ 55 ] dodecanoate Methyl Dodecanoic acid [ 55 ] Methyl tetradecanoate [ 55 ] tetradecanoate Methyl Tetradecanoic acid [ 55 ] Tetradecanoic )-9-hexadecenoate [ 55 ] ( Z )-9-hexadecenoate Methyl Methyl hexadecanoate [ 55 ] hexadecanoate Methyl ( Z )-9-Hexadecenoic acid [ 55 ] Hexadecanoic acid [ 55 ] Z )-9-octadecenoateMethyl-( [ 55 ] octadecenoateMethyl [ 55 ] ( Z )-9-Octadecenoic acid [ 55 ] Oviposition Phenol [ 11 , 40 ] Phenol 3-Methylindole (skatole)3-Methylindole [ 40 ] Octadecanoic acid [ 55 ] n -Heneicosane [ 56 ] octadecanoate [ 58 ] Propyl p -Cresol [ 40 ]

a [ 26 ] a [ 24 ] [ 26 ] c a ­ ethanone -heptadecane, n -penta - , n -heptadecane, 29 : Me-C b cosane, n -hexacosane cosane, one (ketoisophorone) [ 26 ] Semiochemicals involved in key behavioural strategies of mosquitoes in key strategies behavioural Semiochemicals involved Mating - 2,6,6-Trimethylcyclohex-2-ene-1,4-di 2,2,6-Trimethylcyclohexane-1,4-dione CHs 1-(4-Ethylphenyl) 1-(4-Ethylphenyl) List of identifed semiochemicals used by mosquitoes during mating, oviposition, host-seeking oviposition, during and sugar feeding mosquitoes mating, List of identifed semiochemicals used by 1 Table Mosquito species Ae. aegypti Ae. Wooding et al. Parasites Vectors (2020) 13:80 Page 4 of 20 [ 115 ] Sugar feeding – – Hexanal [ 94 ] β-Pinene [ 94 ] β-Pinene Limonene [ 94 ] β-Ocimene [ 94 ] [ 94 ] ( E )-Linalool oxide [ 94 ] ( E )-β-Farnesene β-ocimene β-pinene, 3-Compound blend: ( E )-linalool oxide, Sesquiterpenes and alkenes [ 104 ] [ 11 , 19 61 ] [ 11 , 19 61 ] [ 11 , 19 61 ] 2 2 2 [ 113 ] 2-pentadecanone 1-amine, and 1-dodecanol [ 114 ] [ 9 , 19 ] ( S )-lactic- acid and tetradeca noic acid [ 1 , 90 91 ] CO Host-seeking CO 1-Octen-3-ol [ 11 ] CO 1-Octen-3-ol [ 11 ] Octanal [ 113 ] Decanal [ 113 ] 2-Nonanone [ 113 ] [ 113 ] Benzothiazole 2-[(2-Ethylhexyl)oxy]-ethanol 5-Compound blend with butan- acid [ 84 ] ( E )-3-Methyl-2-hexenoic acid [ 84 ] ( Z )-3-Methyl-2-hexenoic 7-Octenoic acid [ 84 ] [ 11 ] Acetone Lactic acid [ 11 ]/( S )-lactic acid 1-Octen-3-ol [ 11 ] [ 11 ] 4-Methylphenol Aliphatic carboxylic acids [ 11 ] Carboxylic acids [ 9 ] Ammonia [ 9 ] 3-Compound blend: ammonia, 3-Methylindole (skatole)3-Methylindole [ 41 ] Oviposition 3-Methylindole (skatole)3-Methylindole [ 11 , 40 ] p -Cresol [ 40 , 41 ] CHs [ 24 ] p -Cresol [ 37 , 40 ] o -Cresol [ 40 ] 4-Methylcyclohexanol [ 40 ] [ 40 ] 2,4-Dimethylphenol [ 40 ] 2,3-Dimethylphenol [ 40 ] 4-Ethylphenol [ 46 ] Cedrol [ 24 ] c 31 , Me-C 30 , Me-C 29 Semiochemicals involved in key behavioural strategies of mosquitoes in key strategies behavioural Semiochemicals involved Mating – – CHs: Me-C (continued) 1 Table Mosquito species Ae. albopictus Ae. Ae. triseriatus Ae. An. gambiae Wooding et al. Parasites Vectors (2020) 13:80 Page 5 of 20 Sugar feeding Cyclic and bicyclic MonoterpenesCyclic [ 97 ] – – substitute) substitute) substitute) substitute) 2 2 2 CO CO CO [ 11 , 19 61 ] [ 11 , 19 61 ] [ 11 , 19 61 ] 2 2 2 [ 71 ] octanal, nonanal and decanal [ 115 ] nonanal and octanal [ 82 ] [ 71 ] 1-dodecanol [ 114 ] [ 71 ] tadecanone [ 114 ] blend (MB)]: butan-1-amine, blend (MB)]: butan-1-amine, ammonia, 3-methyl-1-butanol, ( S )-lactic- acid and tetradeca noic acid [ 1 , 66 ] amine, 3-methyl-1-butanol, 3-methyl-1-butanol, amine, ammonia, ( S )-lactic acid and acid [ 66 ] tetradecanoic amine, 3-methyl-1-butanol, 3-methyl-1-butanol, amine, ammonia, ( S )-lactic acid and acid [ 66 ] tetradecanoic 4-Compound blend: heptanal, 4-Compound blend: heptanal, 3-Compound blend: heptanal, 5-Compound blend with 5-Compound blend with 2-pen - CO CO CO Host-seeking 5-Compound blend [the Mbita [ 92 ] 3-Methyl-1-butanol 2-Butanone (as ­ 5-Compound blend: butan-1- 2-Butanone (as ­ [ 11 ] Acetone 1-Octen-3-ol [ 11 ] 5-Compound blend: butan-1- 2-Butanone (as ­ - -fatty acid ester, propyl octade propyl -fatty acid ester, 21 ­ C decanal, sulcatone (6-methyl-5-hepten- sulcatone decanal, 2-one), ß-pinene 3-carene, limonene, and α-pinene [ 49 ] canoate [ 58 ] canoate nonanal and ( 1R )-( + )-α-pinene [ 50 ] (1R)-( + )-α-pinene and p -cymenenal, [ 50 ] – CHs: 8-Compound blend: ß-caryophyllene, Oviposition 3-Compouond blend: benzaldehyde, 3-Compouond blend: benzaldehyde, nona - 4-Compound blend: benzaldehyde, , 29 [ 24 ] c 31 , Me-C 30 Me-C -hentriancontane, Me-CCHs: n -hentriancontane, – – Semiochemicals involved in key behavioural strategies of mosquitoes in key strategies behavioural Semiochemicals involved Mating (continued) An. arabiensis An. funestus An. stephensi 1 Table Mosquito species Wooding et al. Parasites Vectors (2020) 13:80 Page 6 of 20 Sugar feeding – – – , 2 CO [ 11 , 19 61 ] [ 11 , 19 61 ] [ 11 , 19 61 ] 2 2 2 acetone, 1-octenol-3-olacetone, and acid [ 9 ]. butonoic 3-ol [ 111 ] 1-Octen-3-ol [ 11 ]/ (R)-1-octen- CO CO Host-seeking CO ( R )-1-Octen-3-ol [ 111 ] ( E )-2-Decenal [ 85 ] Undecanal [ 85 ] Dodecanal [ 85 ] [ 85 ] Tertradecanal [ 85 ] Pentadecanal Hexadecanal [ 85 ] Heptadecanal [ 85 ] Octadecanal [ 85 ] 4-Compound blend: ­ 1-Octen-3-ol [ 11 ] oviposition pheromone) [ 42 – 44 ] pheromone) oviposition ( Culex 45 ] (repellent) [ 116 ] (repellent) indole and 4-ethylphenol, ylphenol. (skatole)3-methylindole [ 11 , 51 ] Erythro -6-acetoxy-5-hexadecanolide Erythro [ 11 , -6-acetoxy-5-hexadecanolide Oviposition Dimethyl disulfde (DMDS) [ 116 ] Dimethyl Dimethyl trisulfde (DMTS) [ 116 ] trisulfde (DMTS) Dimethyl 6-Methyl-5-hepten-2-one (sulcatone) 4-meth - 5-Compound blend: phenol, Skatole [ 40 , 41 ] [ 60 ] Acetaldehyde p -cresol [ 40 ] 2-tridecanone [ 40 ] Indole [ 40 ] [ 40 ] Trimethylamine Nonanal [ 40 ] p -cresol [ 40 ] Indole [ 40 ] Skatole [ 40 ] [ 40 ] Dimethyltrisulfde [ 40 ] Phenol Nonanal [ 40 ] Naphthalene (stimulant) [ 40 ] [ 24 ] [ 24 ] c c CHs Semiochemicals involved in key behavioural strategies of mosquitoes in key strategies behavioural Semiochemicals involved Mating – CHs - (continued) fasciatus Cx. tarsalis 1 Table Mosquito species An. coluzzii Cx. quinque Wooding et al. Parasites Vectors (2020) 13:80 Page 7 of 20 , d , d acid ­ benzaldehyde )-β-pinene, , ( − )-β-pinene, , d d , 2-methylpropionic , 2-methylpropionic d acid , 3-methylbutanoic ­ , 3-methylbutanoic , ( − )-α-pinene d d acid acid , artemisia ketone, umbellulone, (Z)-3-hexenyl , artemisia (Z)-3-hexenyl umbellulone, ketone, d , hexanoic ­ d ­ acid , 2-methylbutanoic ­ , 2-methylbutanoic d ­ acetophenone )-sabinene, (E/Z)-ocimene, germacrene-( − )-sabinene, benzoic benzoic - phenyl-2,3-butanedi alcohol, yomogi hexyl acetate, acetate, [ 103 ] 3-hydroxy-4-phenyl-2-butanone one, ­ acid Sugar feeding Terpineol [ 96 ] Terpineol Geraniol [ 96 ] [ 96 ] Eugenol Citral [ 96 ] [ 96 ] Citronellol acids [ 96 ] Fatty [ 96 ] acetate Amyl [ 96 ] Toluene alcohol [ 96 ] Phenylethyl [ 96 ] Phenylacetaldehyde [ 96 ] Lilac aldehydes [ 96 ] acetate ( Z )-3-Hexenyl [ 96 ] Linalool oxide Linalool [ 96 ] [ 96 ] Benzaldehyde Lilac alcohol [ 96 ] [ 96 ] Acetophenone salicylateMethyl [ 96 ] Hexanal [ 96 ] [ 96 ] 1-Hexenol (Z)-3-Hexen-1-ol [ 96 ] [ 104 ] Benzenoids 20-Compound blend: butanoic ­ [ 11 , 19 61 ] 2 CO Host-seeking Lactic acid [ 11 ] – Oviposition [ 24 ] c Semiochemicals involved in key behavioural strategies of mosquitoes in key strategies behavioural Semiochemicals involved Mating CHs (continued) CHs: cuticular hydrocarbons Human shared semiochemicals used during sugar feeding Human shared Aggregation pheromone Aggregation Contact pheromones Contact

1 Table Mosquito species a b c d Cx. pipiens Wooding et al. Parasites Vectors (2020) 13:80 Page 8 of 20

Fig. 1 An overview of the key behavioural patterns, infuenced by semiochemicals, discussed in this review. The four main behaviours that are targeted include mating, oviposition, host-seeking and sugar feeding for birds or mammals). Tis preference is easily coun- related species that co-occur in the same environment termanded by internal physiological factors, such as the sometimes have very similar odour cues and signals and, level of starvation, and by external factors such as host in such cases, isolation occurs by having mating period availability [11, 19]. If there is an urgent need to feed, at diferent times of the day [21, 22]. Tis mechanism then the mosquito may settle for hosts that are normally serves to prevent interspecies hybridisation [11]. Vola- not preferred. Te physiological state of the mosquito tile chemicals that form part of the signals and cues used will thus infuence its response to certain behavioural during these behaviours are thus not the same for the dif- cues. Tis state varies depending on the age, size, nutri- ferent species and need to be studied separately if specifc tional status and gonotrophic stage of the mosquito. control applications are to be developed. In this section Once the optimal internal state for a specifc reaction a concise review of current insights into mate fnding, is met then the mosquito will respond in the appropri- swarm formation and mating cues with the emphasis on ate manner to the correct stimulus [11]. Tis plasticity semiochemicals will be discussed. in mosquito behaviour can impede the identifcation of important mosquito semiochemicals because the species Mate‑fnding being studied needs to be in the correct state to respond. Chemical cues and signals are used by both male and Tis adds to the analytical challenge of correctly identify- female mosquitoes to fnd a conspecifc mating partner. ing semiochemicals that mediate certain behaviours. Tis behaviour is crucial for mosquito reproductive suc- cess rates [11]. Diferences exist between species regard- Mating behaviour ing the type of chemical compounds that they use. In Semiochemicals are known to be involved in the mating some species, females need to locate male swarms and behaviour of mosquitoes. Tere are diferent behavioural they do this by using olfactory cues and perhaps the patterns that occur at diferent mating stages and these same visual cues that guide male mosquitoes to form also vary for diferent species [11, 20]. Mating behav- swarms [20]. While in other species males fnd foraging iour includes maturation feeding (feeding required for females by following kairomones, chemicals involved an insect’s gonads to mature for gamete production) in mosquito-host interactions, that are indicative of the followed by swarm formation, in some species, and a presence of a female during her blood-feeding behaviour sequence of mate-fnding behaviours that are guided by a [11, 20]. During this process, female mosquitoes are frst set of chemical mating cues and signals [11]. Mating cues attracted to a host, be it animal or human, through the and signals are important to ensure the survival of a spe- kairomones that the host produces in breath, sweat or cies and these cues and signals may help isolate a species, skin emanations [14]. Male mosquitoes, of certain spe- especially if they are unique to that species. Diferent, but cies, then locate blood-feeding females by following the Wooding et al. Parasites Vectors (2020) 13:80 Page 9 of 20

kairomone trail [11, 14]. Te behaviour of a male mos- conclusively show evidence for the existence of an aggre- quito intercepting a female near a host is known to occur gation pheromone. Tese studies should also consider for Aedes spp. and Mansonia spp. of the Culicidae [11]. the combined infuence of visual and chemical cues on Hapairai et al. [23] reported collecting a larger number of swarm formation. Such studies will be essential for the male than female Ae. aegypti when using human bait col- elucidation of the cause of the aggregation behaviour and lections. Tis fnding shows that the males of some spe- for fnding evidence of the existence of aggregation pher- cies are attracted to and orientate towards host cues in omones for other mosquito species [24]. the feld [23]. Correctly identifying kairomones for the control of Mating cues mosquitoes is possible but it may be that efective blends Te use of contact sex pheromones in dipterans for spe- are diferent for each of the sexes. Furthermore, the chal- cies recognition during mating is well documented, lenge lies not only in identifying these chemicals but leading to the assumption that this mode of species rec- also to realise that diferences exist for diferent species. ognition is also utilised by mosquitoes [14, 24]. Obser- Close-range species recognition cues that ultimately vations in support of sex pheromones for mosquitoes allow male mosquitoes to correctly recognise their con- include studies done on adult male crabhole mosquitoes specifc females could potentially be useful as a variety of (Deinocerites cancer) [27] and winter mosquitoes (Culi- mosquito species will likely be present near a host [11]. seta inornata) [28] that revealed the male’s ability to Te identifcation of a species-specifc sex pheromone locate female pupae to mate with newly emerged virgin that is useful for selective control of vector mosquito spe- female mosquitoes [27]. Mating has also been perceived cies remains elusive. Te existence of such pheromone between males and empty pupal cases or recently dead can pave the way towards selective control of vector mos- females for winter and crabhole mosquito species [27, quitoes: the ultimate goal. 28]. Culiseta inornata males have been observed touch- ing conspecifc mates with their front leg tarsi [28]. Swarm forming Furthermore, a decrease in mating attempts in Ae. albop- It is proposed that both male and female mosquitoes pro- ictus males have been recorded when their terminal tar- duce aggregation pheromones that attract both sexes to someres of the prothoracic and mesothoracic tarsi were the mating swarm [24]. How females locate male swarms treated with a solvent mixture [14, 24, 29]. Tese stud- is still unclear. Females may use similar visual cues (i.e. ies found that short-range contact pheromones are most swarm markers) as males and olfactory cues, such as probably used by male and female mosquitoes in a swarm aggregation pheromones, to locate swarms [11, 20]. to recognise their own species. Te volatility of aggrega- Laboratory studies have shown that when swarming Ae. tion pheromones makes these unlikely candidates for aegypti males or females are placed upwind in an olfac- close range species recognition inside the swarm. Tis is tometer a fight response is elicited in female mosqui- due to the dynamic nature of swarms, specifcally mixed toes, suggesting that both female and male Ae. aegypti species swarms [30]. produce a volatile attractant as one of many signals to Although cuticular hydrocarbons (CHs) normally act initiate swarm formation [14, 24, 25]. Fawaz et al. [26] as a desiccation barrier for insects they are potential con- have identifed 2,6,6-trimethylcyclohex-2-ene-1,4-dione tact pheromones for some mosquito species. It is possible (ketoisophorone), 2,2,6-trimethylcyclohexane-1,4-dione that low volatility contact pheromones play an important (the saturated analogue of ketoisophorone) and 1-(4-eth- part in species recognition, especially when a range of ylphenyl) ethanone as aggregation pheromones for Ae. other mosquito species are present near a host [11, 14]. aegypti. Tese compounds may be the same or structur- Female Cx. quinquefasciatus Say, Cx. tarsalis Coquillett ally related to aggregation pheromones that related mos- and Cx. pipiens L. are attracted to benzene extracts of quito species use and should be evaluated further. conspecifc males indicating that the extracted CHs may Host odours also play a role in aggregation and mat- act as pheromones during mating [24]. Cuticular hydro- ing as many mosquito species mate near their host [20]. carbon profles appear to be diferent between mated and For example, Ae. albopictus form small swarms of 3 to 40 unmated An. gambiae (s.s.) and Ae. aegypti females. In individuals around the ankles and feet of human hosts Ae. aegypti, the ratio of n-heptadecane, n-pentacosane and Ae. aegypti has been observed to do the same [14, and n-hexacosane is altered drastically after mating 24]. A study by Cabrera and Jafe [25] revealed that Ae. [24]. Such changes could potentially serve to fngerprint aegypti males can be induced to form swarms in response females that are already mated. to host volatiles. Although the above-mentioned stud- Qualitative diferences in CH profles between the two ies have provided insights into how swarms are formed sexes of An. gambiae and age-dependent changes of the through host odours, more studies are needed to CH profles of the diferent sexes suggest that CHs are Wooding et al. Parasites Vectors (2020) 13:80 Page 10 of 20

possible cues for mate choice and sex recognition [24, 31]. Oviposition cues Hydrocarbon analysis has also been used to discriminate Reviews by Navarro-Silva et al. [39] and Affy and Gali- between closely related mosquito species or populations zia [40] highlight the importance of semiochemicals that and this tool is useful for discriminating morphologically mediate oviposition in mosquitoes. Odour attractants similar species such as An. gambiae and An. arabiensis involved in oviposition site selection have been identifed [24]. Qualitative diferences in CH profles, specifcally from plant material, faeces from mosquito larvae as well n-hentriacontane (Me-C31), n-nonacosane (Me-C29) and as secretions from mosquito larvae, pupae and eggs [41, n-triacontane (Me-C30), are used to discriminate between 42]. Te frst breakthrough on the use of chemical cues these sympatric species and Me-C29 has been identifed for oviposition site selection was with the identifcation as a pheromone candidate for Ae. aegypti [24, 32–34]. of the oviposition pheromone, erythro-6-acetoxy-5-hex- Mosquito hydrocarbons might consequently not only be adecanolide, which was extracted from the apical droplet important for species diferentiation in order to obtain at the tip of the eggs of Cx. quinquefasciatus by Laurence vital epidemiological data for vector control, but they and Pickett [43, 44]. Studies reviewed by Takken and are also potential sex pheromones which can be used Knols [11] have shown that gravid conspecifcs, as well in future lure-and-kill control strategies [24]. Te chal- as Cx. tarsalis, are highly attracted to the erythro-6-ace- lenge lies in how exactly these short-range chemical cues toxy-5-hexadecanolide, and electrophysiological activity may be used in vector control strategies. Te most likely has also been demonstrated by Cx. quinquefasciatus in strategy is with the interference of the mosquito’s landing response to this pheromone [45]. Another major devel- response. opment on oviposition odour attractants was achieved Te importance of acoustic cues and wingbeat fre- by Lindh et al. [46] during a study conducted in Kenya quency in mosquito mating behaviour has been doc- which identifed the frst oviposition attractant for gravid umented by various researchers and should not be female An. gambiae, namely cedrol. A follow-up study, overlooked when implementing vector control strategies conducted by Eneh et al. [47], on the natural source of the during mating [14] (reviewed by Gibson, Warren & Rus- chemical, found that cedrol is produced by fungal spe- sell [35]). Te unique properties of the mosquito anten- cies found on grass rhizomes near the natural mosquito nae and Johnston’s organ, which is located at the base of breeding site. Tis brought to light the infuence of both each antenna, allow mosquitoes to be more sensitive to microbes and vegetation for oviposition site selection in sound than any other insect [35]. mosquitoes. Te infuence of grass volatiles on oviposition site selec- Oviposition tion was further demonstrated by Asmare et al. [48], as Te selection of an oviposition site for many mosquito blood-fed An. arabiensis females showed an attraction to species is determined by visual and chemical cues [11]. these volatiles; the volatiles in question are yet to be iden- Chemical cues, produced by conspecifcs, used as ovi- tifed. Te importance of vegetation infuencing oviposi- position attractants include egg raft, larval and pupal tion site selection was highlighted by Wondwosen et al. pheromones [11, 36]. Te presence of conspecifc larvae [49] in a study which revealed that rice volatiles attract and their associated semiochemicals at oviposition sites gravid An. arabiensis females. Eight compounds, namely indicates suitable conditions for larval development [36]. ß-caryophyllene, decanal, sulcatone (6-methyl-5-hepten- Breakdown products, in natural water bodies, of bacte- 2-one), limonene, 3-carene, ß-pinene, and α-pinene, elic- rial origin are also used as chemical cues for oviposition ited antenna responses during GC-EAD experiments. [11]. Te chemical ecology and oviposition behaviour Furthermore, the study revealed that a complete blend of of gravid mosquitoes, including attractants and deter- the compounds, released at the lowest efective dose (10 rents such as water salinity, phytochemicals and insecti- ng), was needed to induce the full behavioural response cides, was comprehensively reviewed by Bentley and Day of attraction and oviposition [49]. Sugarcane pollen-asso- [37]. An updated list and further research on oviposi- ciated volatiles attracted and induced egg laying in An. tion attractants for mosquito larvae control is needed as arabiensis females during a still air two-port olfactom- targeting mosquito larvae is deemed a more successful eter bioassay. Two blends were identifed as being attrac- long-term approach to vector control. Targeting larvae tive for gravid An. arabiensis females, namely a three removes vectors before they can reproduce and trans- compound synthetic blend of benzaldehyde, nonanal mit diseases [38]. Tis section highlights the infuence and (1R)-(+)-α-pinene and a four compound synthetic of semiochemicals, singular or blends of chemical com- blend of benzaldehyde, nonanal, (1R)-(+)-α-pinene and pounds, on oviposition site selection as well as egg-laying p-cymene [50]. Blend composition appears to be critical behaviour in mosquitoes. for eliciting the oviposition behaviour. Wooding et al. Parasites Vectors (2020) 13:80 Page 11 of 20

Te presence of bacteria at oviposition sites has also investigated by Seenivasagan et al. [57] using electroan- been shown to impact the selection of suitable ovipo- tennogram (EAG) techniques. Tese authors showed sition sites. Culex quinquefasciatus was found to be that EAG response increased with an increasing stimulus attracted to a fve-compound blend, composed of phenol, concentration and behavioural repellence was reported 4-methylphenol, 4-ethylphenol, indole, and 3-methylin- for concentrations above 50 ppm [57]. Dose-dependent dole (skatole), produced by bacteria found in hay infused EAG responses from the antenna of gravid female Ae. waterbodies that are used as oviposition sites [11, 51]. In aegypti and An. stephensi mosquitoes were recorded for addition, olfactory responses with egg-laying behaviour the ­C21-fatty acid ester, propyl octadecanoate [58]. in Culex mosquitoes have been found to be induced by Te signifcance of compound blends, rather than sin- the oviposition site volatile 3-methylindole [52]. Elec- gle impact compounds, responsible for attracting gravid trophysiological studies done revealed that Ae. albopic- mosquitoes has gained more attention in recent litera- tus responds to 3-methylindole and Ae. aegypti respond ture as is evident from the aforementioned studies. Te to phenol [11]. Field studies done in Tanzania revealed use of compound blends in vector control strategies tar- a synergistic oviposition response of Cx. quinquefascia- geting oviposition was demonstrated in a study by Baak- tus to 6-acetoxy-5-hexadecanolide blended with either Baak et al. [59] where a blend of synthetic chemicals, viz 3-methylindole or volatiles produced by bacteria found 3-methylindole, p-cresol and phenol, induced a response in soakage pit water [11]. Bacterial kairomones have in gravid Ae. aegypti female mosquitoes. Te authors been shown to afect oviposition in Ae. aegypti in a dose demonstrated that an ovitrap baited with a blend of cer- dependent manner. A nonanoic acid (16%), tetradecanoic tain attractants can be used to monitor and potentially acid (83%) and methyl tetradecanoate (1%) blend was control mosquito populations [59]. Te synergistic and/ found to stimulate oviposition, whereas hexadecenoic or antagonistic oviposition response of gravid mosqui- acid methyl ester deterred oviposition [53]. Te impor- toes to complex chemical blends adds to the already chal- tance of bacterial profles in combination with physi- lenging analytical task of not only identifying individual ochemical properties and semiochemicals for oviposition oviposition chemical cues but also the ratio in which they site selections was demonstrated by Eneh et al. [54] in a occur. study showing that on average twice as many An. arabi- A recent study by Choo et al. [60] used a reversed ensis instar larvae were found in freshwater ponds when chemical ecology approach to identify potential semio- compared to aged ponds (4-days-old). Tis indicates that chemicals for vector control strategies. Te reverse volatiles of bacterial origin play a crucial role for oviposi- chemical ecology approach is described as an alternative tion site selection. process to identify active semiochemicals by screening Odour cues originating from mosquito eggs or larvae for the ligand specifcity of olfactory receptor proteins. can potentially be as used as attractants or repellents Olfactory proteins, specifcally odour receptor proteins in vector control strategies. Cuticular hydrocarbons of expressed in the antennae of Cx. quinquefasciatus, were mosquito eggs play a vital role in preventing egg desic- screened with a panel of 230 odourants for activity. Tis cation and have been investigated as a potential source of approach bypassed the often time-consuming traditional oviposition pheromones. Cuticular hydrocarbons were bioassay-guided approach to identify semiochemicals listed as oviposition pheromones for Ae. aegypti and are from natural sources. Electroantennogram recordings known behavioural modifers at breeding sites for Ae. from the antennae of the female mosquito and cage albopictus [24]. Ganesan et al. [55] identifed compounds oviposition as well as dual-choice assays demonstrated that induce an ovipositional response in gravid female Ae. the potential of acetaldehyde as a potential oviposition aegypti. Tese compounds included 6-hexanolactone, attractant over a wide range of concentrations. Tis methyl dodecanoate, dodecanoic acid, methyl tetrade- was verifed by positive results from the newly identifed canoate, tetradecanoic acid, methyl (Z)-9-hexadecenoate, attractant [60]. A better understanding of receptor pro- methyl hexadecanoate, (Z)-9-hexadecenoic acid, hexade- teins and the genes that encode them will greatly facili- canoic acid, methyl (Z)-9-octadecenoate, methyl octade- tate the analytical challenge of identifying chemical cues canoate, (Z)-9-octadecenoic acid, and octadecanoic acid. used by mosquito vectors. A signifcant positive ovipositional response was obtained Volatile oviposition stimulants could potentially facili- in gravid female Ae. aegypti with dodecanoic and (Z)- tate the selective trapping of female mosquitoes. Te 9-hexadecenoic acids whereas all the esters investigated challenge lies in the fact that diferent mosquito species induced a deterrent/repellent ovipositional efect [55]. detect and respond to diferent oviposition stimulant Mendki et al. [56] identifed n-heneicosane as an oviposi- blends. Tese blends will be unique for each species that tion attractant pheromone from the larval cuticle of Ae. needs to be controlled. aegypti. Te dose-dependent efect of n-heneicosane was Wooding et al. Parasites Vectors (2020) 13:80 Page 12 of 20

Host‑seeking studied [11, 61]. Te gas is responsible for priming the Takken [18] described the host-seeking behaviour of fight response and guiding mosquitos to their hosts [11, blood-feeding arthropods as “the orientation to a host 19, 61, 66]. Mosquitoes have been shown to respond to from a distance”. Host-seeking generally involves a series very small changes in atmospheric CO­ 2 concentrations of behavioural steps that starts when the insect is acti- (~ 0.035%) [12, 67]. Host recognition through changes vated by a host stimulus and ends when the mosquito in ­CO2 levels usually occurs over distances of approxi- lands on a suitable feeding area on the host [18]. Dif- mately 10 m when the ambient ­CO2 level changes by the ferent behavioural cues are used during host-seeking addition of a breath plume containing ~ 4% ­CO2 [12]. A and includes visual cues, long distance chemical stimu- source of ­CO2 is often added to traps as an additional lants (such as ­CO2, that activates and induces upwind attractant. Tis can be done by adding dry ice, releasing fight), heat and moisture cues that play an important CO­ 2 from gas canisters, or fermenting sugar and molas- role in behavioural responses in close vicinity of the ses in a trap to produce CO­ 2. Carbon dioxide substi- host, and skin odour cues that infuence landing and bit- tutes such as 2-butanone can also be added to traps [66, ing site selection [18, 61–63]. Semiochemical cues used 68–71]. in the host-seeking process are discussed in this section. Carbon dioxide is a general cue indicative of the pres- Emphasis is placed on the importance of carbon dioxide ence of a vertebrate host but mosquitoes do not only in host fnding, the use of host skin volatiles in host selec- rely on ­CO2 because it does not convey information on tion and/or preference, and lastly the efect of compound the potential suitability of the host [11, 19, 61]. Tere is blends on host preference. also variation in how diferent mosquito species use and Te use of odour cues during host-seeking behaviour respond to CO­ 2. Some opportunistic mosquito species, was described by Rudolfs [64] as early on as 1922. Olfac- such as An. arabiensis, feed on both humans and animals tion is now deemed to be the most important stimu- and use CO­ 2 as a general host cue whereas other species, lus used by mosquitoes during host-seeking [11, 19, 63]. such as An. gambiae, prefer humans and rely on ­CO2 and Mosquitoes have very complex olfactory systems contain- other odours that are specifc to humans to guide them ing hundreds of receptor proteins from three diferent during fight [19, 66]. Te generality of using CO­ 2 in host- families. As many as 131 and 79 putative odour receptors seeking is thought to increase with the degree of zooph- have been identifed in the Ae. aegypti and An. gambiae ily in mosquito feeding behaviour [61]. Carbon dioxide, genomes, respectively [41]. Te receptor families include by itself, may not be suitable as a chemical lure for selec- the olfactory receptors (ORs), ionotropic receptors (IRs) tively trapping vector mosquitoes and in particular not and gustatory receptors (GRs) [41, 65]. Te olfactory for selectively trapping anthropophilic vector mosqui- sensilla that express these proteins are located on the toes. However, ­CO2 remains an important synergist of antenna, maxillary palps and proboscis [41]. host specifc chemical cues. Further feld work is needed Sexual dimorphism is present in all the olfactory organs to improve the efectiveness and specifcity of ­CO2 as a of mosquitoes [9]. Te dimorphism correlates with the lure. importance of olfactory cues for female mosquitoes dur- Behavioural responses of mosquitoes do not only ing host location. For example, the females of An. gam- depend on the presence of CO­ 2. Carbon dioxide, human biae have three to four times more antennal sensilla than odour and heat was needed to elicit a robust feeding males [41]. Te neurons housed in these sensilla are response in wild-type Ae. aegypti mosquitoes. Te study, thought to express odourant-binding proteins that func- by Raji et al. [72] highlighted the importance of consid- tion specifcally for female host location [41]. Further ering synergistic chemical compounds, as well as the studies on the role of sexual dimorphism in the mosquito interaction between chemical and physical cues when olfactory apparatus are needed to clarify how this diver- studying vector-host interactions. Removing either ­CO2, sity infuences the detection of odour cues during the heat or odour resulted in a reduced feeding response [72]. mosquito life-cycle [9, 41]. Such studies can potentially Te intricate combination of both chemical and physical lead to new discoveries that can enhance our understand- cues that mosquitoes rely on during vector-host inter- ing of how odour cues are detected by the diferent sexes. actions complicates the development of artifcial lures. Tis could then enable the development of gender spe- Tis is because chemical lures by themselves do not rep- cifc vector control strategies. licate the physical cues that may be required to elicit the desired behavioural response from the mosquito. The importance of CO­ 2 Carbon dioxide is the best-known mosquito kairomone and its role in mosquito host location has been intensively Wooding et al. Parasites Vectors (2020) 13:80 Page 13 of 20

Host skin volatiles Certain mosquito species prefer human hosts that are Skin volatiles play an important part in host preference already infected with the malaria parasite [19, 80–82]. for those mosquito species that are specialists on selected Plasmodium falciparum produces an isoprenoid precur- hosts [19]. Human skin volatiles have been studied inten- sor, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate sively and over 500 compounds have been identifed from during certain stages of the parasite’s life-cycle, which human skin secretions [73, 74]. Variation in attractive- stimulates red blood cells to increase ­CO2, aldehydes and ness between diferent individuals has been attributed monoterpene release rates that, when combined, enhance to diferences in skin-odour profles as well as difer- vector attraction and stimulate vector feeding in An. ences in human skin microbial fora [9, 11, 75]. Te Knols gambiae (s.l.) [83]. Te aldehydes heptanal, nonanal and group provided the frst evidence of attraction of highly octanal were detected in larger amounts on individuals anthropophilic mosquitoes to a non-human odour source infected with the Plasmodium parasite. Te study, con- with experiments conducted on Limburger cheese. Te ducted by Robinson et al. [82], revealed an increase in smell of Limburger cheese is similar to sweaty human attraction by An. gambiae (s.s.) towards a synthetic blend feet. Tis unique odour was traced back to the coryne- containing the above-mentioned aldehydes. form cheese bacteria, Staphylococcus epidermidis, that Tere is evidence that human skin volatiles interfere belong to the same genus as the coryneform bacteria with ­CO2 detection in mosquitoes. Costantini et al. [84] found between the toes of human feet. Traps baited with investigated electrophysiological responses of An. gam- Limburger cheese caught signifcantly more An. gambiae biae to human-specifc sweat components using EAG. (s.s.) than the control traps [11, 76, 77]. A large amount Te carboxylic acids, (E)- and (Z)-3-methyl-2-hexenoic of research has been done since the Limburger cheese acid and 7-octenoic acid were found to reduce the mos- experiments confrming the efect of chemical signals quitoes’ response to ­CO2 indicating a possible antago- derived from skin microorganisms on the host-seeking nistic efect. Wind tunnel bioassays conducted by Lacey behaviour in mosquitoes [78]. et al. [62] found that skin volatiles collected from feet Te intensity and composition of human skin odours onto glass beads override the CO­ 2 behavioural responses is directly related to the type and amount of certain skin in Ae. aegypti at close proximity to the host. Tis result bacteria [78]. Skin bacteria metabolise the components confrmed that ­CO2 is involved in long-range activation of sweat giving sweat its characteristic odour. Te vola- and is a less important cue for anthropophilic species tile chemicals released by skin microorganisms have when they are near the host [62]. therefore become a major focal point for studying how Odour cues are also of importance in zoophilic mos- mosquitoes distinguish between hosts [78]. Tese micro- quito species. Cooperband et al. [85] reported an activa- bially produced volatiles vary between individuals and are tion response in female Cx. quinquefasciatus mosquitoes thought to aid mosquitos when they need to discriminate to avian faecal odours. Culex quinquefasciatus blood- between diferent hosts [78, 79]. A recent study of Busula feeds on birds and is an important vector of the West et al. [79] showed that mosquito species that feed oppor- Nile virus. Headspace volatiles from chicken faeces were tunistically also respond behaviourally to a wider array collected using SPME whereafter eight volatile alde- of volatiles. Tese authors showed that the more oppor- hydes, namely (E)-2-decenal, undecanal, dodecanal, ter- tunistic An. arabiensis responds behaviourally to the tradecanal, pentadecanal, hexadecanal, heptadecanal skin microbiota of all the vertebrate species that it nor- and octadecanal, showed EAG responses when using the mally feeds on. It was also shown that traps containing antennae (four antennae were used in parallel to elicit an bacteria of human origin caught higher proportions of improved response) from Cx. quinquefasciatus [85]. Te An. gambiae than An. arabiensis while traps containing behavioural response of Cx. quinquefasciatus to these bacterial volatiles from chickens caught more An. arabi- volatiles still needs to be confrmed. ensis than An. gambiae. Anopheles arabiensis responded equally to all species of bacteria tested whilst An. gam- Host skin volatiles and analytical approaches biae responded only to four specifc bacteria [79]. Tere Te vast amount of skin-associated volatiles makes bio- is currently a need for further studies on more oppor- assays aimed at determining the behavioural responses of tunistic mosquito species as the majority of research mosquitoes almost impossible. Fortunately, sophisticated regarding skin microorganisms and mosquito behaviour analytical techniques are now being applied for eluci- have been done on the more anthropophilic species such dating the identity of both specifc semiochemicals and as An. gambiae [78]. Te chemical compounds eliciting potential semiochemical blends. Electroantennogram the behavioural response from these sources must be (EAG) recordings can be used to investigate the anten- identifed. nal responses of mosquitoes to blends of chemicals and single sensillum recording (SSR) studies can be used to Wooding et al. Parasites Vectors (2020) 13:80 Page 14 of 20

identify specifc neuron function and type [11]. Gas chro- Volatile chemicals emitted by the host can be subdi- matography coupled to electroantennogram (GC-EAG) vided into diferent categories. Tese include primary recordings can then be used to separate components odours that do not change when the host diet changes, of blends and investigate their individual electrophysi- secondary odours that are dependent on the host diet ological activity [11]. Electroantennogram responses and environmental factors, and lastly, tertiary odours that are generally measured from the excised head of a mos- come from the application of, for example, lotions and quito mounted between two glass capillaries containing make-up [88]. Verhulst et al. [89] found that the attrac- a saline solution and gold or silver electrode wires. Te tiveness of studied volunteers is dependent on the appli- grounded indiferent electrode is inserted into the back cation of skincare products. When volunteers stopped of the decapitated head whilst the recording electrode is applying a specifc product, An. coluzzi mosquitoes lost connected to the clipped antenna [57, 58, 84, 85]. their ability to distinguish between body parts. Te skin- Comparing profles of human skin volatiles is not an care product reduced the attractiveness of the skin region easy task especially when diferent sampling methods where it was applied [89]. Tis led to the conclusion that have been used between studies. Te sampling method skincare products may afect a person’s attractiveness to directly afects how the true chemical profle is repre- mosquitoes counter to the expectation that mosquitoes sented in the sample. Tis factor should not be under- have a preference to bite specifc skin regions. estimated (see review by Dormont et al. [73]). Sampling Various studies focussed on developing an attractive methods that have been used include solvent back extrac- synthetic odour blend (i.e. “man-in-a-bottle”) in the past tion from a cotton swabs, dynamic headspace adsorp- [11]. Van Loon et al. [1] formulated a fve-component tion onto various polymers, solid phase microextraction odour blend for the purpose of mosquito control. Te (SPME) [73], and recently (2018), sorptive extraction blend was developed by adding two compounds, butan- directly from the skin using polydimethylsiloxane 1-amine and 3-methyl-1-butanol, to the synergistic (PDMS) loops that can be worn as bracelets or anklets three component blend of ammonia, (S)-lactic acid and [86]. Collection of skin volatiles onto glass beads and iso- tetradecanoic acid which was previously developed by lating volatiles from worn nylon stockings and T-shirts Smallegange et al. [90, 91]. Te new blend was found to have been reported [9, 11, 62, 66, 69, 87]. Dynamic sam- be even more efective when combined with CO­ 2 which pling techniques have been developed where the sam- increased the attractiveness of the blend synergistically pled body part is wrapped in a polyvinyl-acetate bag, [1]. Te slow release of the standard three component clean air is then passed over the skin before exiting the synthetic blend with the addition of 3-methyl-1-butanol bag through a polymer containing flter. Te flter is then from low-density polyethylene material attracted more either thermally desorbed or extracted with a solvent An. gambiae than when using the standard blend alone [82]. Some studies attempt to standardise volatile pro- [92]. Te challenge in fnding the right blend was demon- fles by asking participants to follow particular diets and strated by Smallegange et al. [91]. Te synergism between use specifc soaps and shampoos for a time before sam- ammonia, lactic acid and carboxylic acids was investi- pling commences [73]. Such procedures could potentially gated. Lactic acid alone attracted Ae. aegypti but not An. reduce variation in volatile profles caused by factors that gambiae. In contrast, ammonia on its own attracted more are unknown. An. gambiae (the addition of lactic acid did not increase its attractiveness); however, ammonia was only attractive Compound blends to Ae. aegypti in combination with lactic acid. Further- It is not very likely that all skin volatile compounds, with more, combining ammonia to either lactic or carboxylic over 500 described in the literature, are simultaneously acids in a binary blend did not increase the attractiveness involved in host-seeking, landing and probing by mosqui- of ammonia on its own to An. gambiae. Te authors also toes. Semiochemical cues are rather thought to be spe- found that the repellent efect of a 12-compound car- cifc combinations of a subset of these volatiles. Specifc boxylic acid blend was suppressed with the addition of chemical combinations can arise out of combining sub- ammonia [91]. sets of only the semiochemicals that occur in the profles Te aforementioned studies demonstrate the vast of hosts. For example, it is known that specifc compound range of information on mosquito-host volatile chemical blends of human skin volatiles are more likely to afect cues that have emerged in recent years. Analytical tech- anthropophilic mosquito species such as Ae. aegypti and niques such as GC-EAD and GC–MS have greatly facili- An. gambiae [9]. Tis fnding can be rationalised because tated this process. New analytical methods and novel mosquitoes with diferent host preferences are more sampling techniques have made it possible to identify likely to respond to specifc blends of volatile compounds a vast range of compounds potentially useful for vector that are associated with their preferred hosts [78]. control strategies. Te studies highlight the complexity Wooding et al. Parasites Vectors (2020) 13:80 Page 15 of 20

and analytical challenge in determining the chemical identifed as the potential active component from these composition of host cues that can change depending on a fowers [97]. variety of factors. Chemical cues can originate from bac- A study conducted by Gouagna et al. [99] revealed teria which difer between and within human and animal that An. arabiensis males are able to discern between hosts, or can be infuenced by illness or skincare prod- potential sugar sources in their environment and that ucts. Synergistic and antagonistic efects of certain com- they preferentially feed on plant species that provide the pounds need to be identifed. Diferent mosquito species highest metabolic pay of. Field work [100] and labora- respond to diferent chemical compounds and/or blends tory studies, using Y-tube olfactometer assays [100] and from hosts diferently and this also makes it more dif- two-choice wind tunnel olfactometer bioassays [101], cult to sample and identify efective blends for specifc showed that An. gambiae (s.s.) had a preference for spe- species correctly. cifc plants (namely Mangifera indica, Delonix regia, Te efectiveness of using odour-baited traps for future Tevetia neriifolia, Senna didymobotrya, Senna siamea, vector control strategies was recently demonstrated by Cassia sieberiana and Parthenium hysterophorus) [100, Homan et al. [70] in a stepped-wedge cluster-randomised 101]. Lahondère et al. [102] demonstrated how orchid trial. Solar-powered odour-baited mosquito trapping sys- odours mediate mutualistic relationships between Ae. tems (SMoTS), baited with the fve-component blend, aegypti and the orchid Platanthera obtusata. Monitoring were installed in households on the Rusinga Island in the mosquito’s antennal lobe revealed that both lilac alde- Lake Victoria, western Kenya. Tis study showed that hyde and nonanal are detected by mosquitoes. However, malaria prevalence can be reduced by 29.8% in areas the level of these two attractants infuence the attrac- where the traps are deployed [70]. Te human-biting rate tiveness of the orchid to the mosquito. Higher levels of of Ae. albopictus in France was reduced to nearly zero nonanal are released by the orchid species visited by the with ­CO2 based barrier trap systems [93]. mosquito, whereas higher levels of lilac aldehyde were released by an orchid not visited by mosquitoes [102]. Sugar‑feeding Te identifcation of the semiochemicals responsible Kairomones play a key role in locating vital energy for the behavioural response (i.e. attraction to the sugar sources for fight and other metabolic activities in mos- source) could potentially lead to new efective synthetic quitoes [11, 94]. Male mosquitoes feed exclusively on attractants that target both male and female mosquitoes. carbohydrate sources. In certain mosquito species, the Treating natural or artifcial sugar sources, i.e. attrac- female mosquitoes will take a sugar meal prior to blood- tive toxic sugar baits (ATSB), with insecticides is another feeding, whilst in other species the female will take a approach employed in vector population reduction pro- sugar meal after the blood meal [11, 94]. Here we review grammes [2, 14]. Te ATSB technique is limited by the recent literature on how mosquitoes fnd these sugar availability of suitable plant products as baits and the meals with the focus on olfactory cues. non-selectivity towards trapping mosquitoes. Tis tech- nique relies on the use of ripening fruits, fruit juices and Olfactory cues fowers to attract vectors [8]. Te limitations associated Sandholm and Price [95] identifed the potential of plant with the ATSB technique have resulted in the emer- volatiles for luring mosquitoes as early on as 1962. Tese gence of research aimed at identifying semiochemicals authors observed that certain mosquito species were that mediate plant-mosquito interactions and ultimately attracted to light-coloured fowers with distinct odours using these semiochemicals as lures in plant-based vector in the feld [95]. Various studies have since been done control strategies [94, 96]. Tese lures have the potential on the odour mediated sugar source-seeking behaviour to be used in surveillance programmes and mass trapping in mosquitoes (reviewed by Nyasembe and Torto [96]). operations, or for contaminating mosquitoes with selec- Tese studies revealed that foral odours play an impor- tive insecticides and entomophathogenic agents such tant role for both male and female mosquitoes during as fungi and viruses [96]. A recent study by Peach et al. sugar feeding [14, 94]. In 1988, Healy and Jepson [97] [103] found that ­CO2, produced by plants as a metabolite demonstrated an upwind fight and landing response for from cellular respiration, works in a multimodal way with An. arabiensis and Ae. aegypti to the inforescences of olfactory and visual cues to attract mosquitoes to plants. Achillea millefolium and Leucanthemum vulgare, respec- Lures based on plant volatiles need to compete with sur- tively, during wind tunnel experiments [96–98]. Male rounding vegetation when used in the feld and they are and female An. arabiensis mosquitoes responded to the often not suitable for long distance attraction of mos- inforescence as well as pentane extracts of A. millefolium quitoes under these conditions. However, by employing fowers. A cyclic or bicyclic monoterpene was tentatively plant-based lures the cost concerns of using CO­ 2, such as in the form of gas canisters, dry ice, sugar fermentation Wooding et al. Parasites Vectors (2020) 13:80 Page 16 of 20

or propane combustion, and as well as the logistical chal- review by Nyasembe and Torto [96]. Tese unknowns lenges of transporting CO­ 2 into remote areas are elimi- provide potential study areas and opportunities to imple- nated [96]. ment novel mosquito control and surveillance pro- Te use of semiochemicals in vector control strategies grammes that are based on plant attractants [94]. Adding was demonstrated by Nyasembe et al. [94] for An. gam- to the complexity is the multimodal approach used by biae. Six EAD-active plant compounds were identifed, mosquitoes to locate sugar sources. Peach et al. [103] viz hexanal, β-pinene, limonene, β-ocimene, (E)-linalool concluded that odour, ­CO2 and visual cues are used by oxide and (E)-β-farnesene [94]. In a follow-up study, the mosquitoes to locate inforescences. Visual cues were authors used these six compounds as a blend in a plant- deemed more important as an attractant than odour cues based lure. Te study revealed that the plant-based lure alone. Furthermore, the authors showed an overlap of competed well with the synthetic human odours for semiochemicals used by mosquitoes for blood-host fnd- trapping malaria vectoring mosquitoes. Furthermore, ing and sugar-feeding [103]. Omondi et al. [106] demon- linalool oxide used on its own or in combination with strated the difculties of using both plant- and human CO­ 2 showed signifcant potential for use in plant-based odour cues together. Te authors used a blend of linalool odour traps [8]. Te challenge in using plant specifc oxide and hexanoic acid that were individually identi- kairomones in mosquito lures is substantial. Not only fed as attractive plant- and human cues for Ae. aegypti, should the preferred sugar source be identifed for each respectively, but when they were combined a decrease in mosquito species, but each source should be investigated the amount of Ae. aegypti occurred [106]. Te frequency analytically to identify potential attractants. Tis presents of these shared semiochemicals between the two meal a huge analytical undertaking due to the chemical com- types needs further investigation to specifcally eluci- plexity present in plant-based samples. date diferences that mosquitoes use for discrimination. Te use of DNA barcoding to profle plant species An even greater challenge exists when it becomes neces- fed upon by vectors is now possible due to advances in sary to measure variation of semiochemical profles and genetic and molecular science. Tis method utilises spe- to show how these patterns change between preferred cifc plant markers to identify plants the mosquitoes pre- plant species and how they are detected by the two sexes viously fed on. Tese new molecular approaches were of the mosquito. Diferences in their responses need to used by Nyasembe et al. [104] to identify compounds be quantifed and the multimodal efect of chemical and involved in specifc mosquito-plant interactions. Gas physical cues needs to be elucidated. Te combination of chromatography coupled to electroantennogram detec- heat and odour cues appears to function synergistically in tor (GC-EAD) recordings were employed to identify the absence of ­CO2, and it is known that a complicated odour cues. Unique classes of volatile compounds were multimodal interaction occurs between ­CO2, human detected by vectors from their respective preferred odour and heat when mosquitoes locate their blood host plants. Tese compounds were benzenoids eliciting a [107]. An improved understanding on multimodal cues response from Ae. aegypti, aldehydes and a benzenoid can provide a valuable approach to selectively target vec- eliciting a response from Ae. mcintoshi and sesquiterpe- tor species. nes and alkenes eliciting a response from An. gambiae [104]. Conclusions Te list of attractive volatile chemicals from preferred Tere is an urgent need to develop new odour-based plants varies considerably between mosquito species traps to be used in IVM programmes in order to achieve and even the compounds that were found to be common the ambitious goals set out by the World Health Organi- across plant species vary in attraction based on difer- sation (WHO) and the complete eradication of malaria ences in the released ratios. Te variation is potentially goal set by the Bill and Melinda Gates Foundation [5]. To due to an adaptive or innate evolution in mosquitoes that ensure the success of odour-based traps, the focus of lure help them to distinguish their plant food source by using development strategies using semiochemicals needs to specifc and general cues in certain ratios [96]. Terpe- shift to include other types of important vector species, noids, such as α-pinene, D-limonene and β-mycrene are such as An. arabiensis and An. funestus, as the major- often described as mosquito repellents and are present in ity of research to date, has been done on An. gambiae. several plant families known to be attractive for An. gam- Traps need to be able to target mosquito vectors during biae thereby adding to the complexity [105]. diferent life stages and key behaviours as well as target Te list of known plant attractants and moreover the diferent sexes while being species-specifc. Even though chemical composition of the plant volatiles remain lim- semiochemicals alone might not be adequate as a control ited [14, 94, 105]. Plant volatile compounds identifed as tool against mosquitoes, using them in IVM programmes mosquito semiochemicals can be found in an extensive can provide a powerful tool that can help reduce and Wooding et al. Parasites Vectors (2020) 13:80 Page 17 of 20

even eliminate vector populations. Odorants can be Authors’ contributions MW wrote the manuscript. MW, MB, YN and ERR sourced publications. MB and used to control mosquitoes by repelling, or by masking YN contributed to drafting the manuscript. All authors read and approved the human odours, or by attracting mosquitoes with lures fnal manuscript. into traps. Tere are, however, substantial shortcomings Funding with current attractants and repellents available to the Not applicable. public. N,N-Diethyl-meta-toluamide (DEET) for exam- ple requires high doses, is costly and has an unpleasant Availability of data and materials Not applicable. smell. Current commercial traps are expensive and bulky as they require a ­CO2 source and usually contain foul Ethics approval and consent to participate smelling synergists. Alternative traps made from recy- Not applicable. cled materials [108], such as buckets (for example the Consent for publication Gravid Aedes Trap) [109] and sticky traps, using prod- Not applicable. ucts available in the home, such as castor oil [110], need Competing interests further investigation as these can provide a low-cost and The authors declare that they have no competing interests. practical solution for vector control in developing coun- tries. Te focus of most of the research has primarily Author details 1 Department of Chemistry, University of Pretoria, Hatfeld, Pretoria, South been on single olfactory compounds or blends contain- Africa. 2 Forestry and Agricultural Biotechnology Institute, University of Preto- ing less than fve compounds. Te efect of odour blends, ria, Hatfeld, Pretoria, South Africa. synergistic or antagonistic, on mosquito host selection Received: 7 September 2019 Accepted: 11 February 2020 requires exploring, especially considering that more than 500 diferent chemicals are associated with human skin of which only a handful will be physiologically active in mosquitoes [65, 73, 74]. Mosquitoes rely on semiochemi- References cals when searching for mates, oviposition sites, sugar 1. Van Loon JJ, Smallegange RC, Bukovinszkiné-Kiss G, Jacobs F, De Rijk M, meals and blood-hosts. Tese semiochemicals are poten- Mukabana WR, et al. Mosquito attraction: crucial role of carbon dioxide in formulation of a fve-component blend of human-derived volatiles. J tially valuable for enhancing the selectivity and efcacy of Chem Ecol. 2015;41:567–73. odour-based traps and such traps can form an important 2. Killeen GF. Characterizing, controlling and eliminating residual malaria part of the IVM strategy. 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