3.1 Insect olfaction
Haematophagous insects have a highly developed olfactory system and mainly use their antennae and, in some cases, maxillary palps, to detect semiochemicals. Semiochemicals can provide information about the location, suitability or physiological state of conspecifics, hosts or breeding sites.17, 18 Semiochemicals enter through pores on the sensilla on the antennae or maxillary palps, where they are transported across the sensillum lymph by odorant binding proteins (OBPs) to the olfactory receptors on the dendrites of the olfactory receptor neurones (ORNs).18, 19 The ORNs can have extreme sensitivity and specificity, particularly with pheromones, and, when stimulated, input information directly into the central nervous system, which induces a behavioural response in the insect.19, 20 Insect olfactory systems are sensitive not only to specific molecular structures but also to ubiquitous compounds and have sophisticated mechanisms such as coincidence detection, whereby blends of volatiles in specific ratios from a host plant are detected by insects within a complex background of volatiles from non-host plants. This might be facilitated by paired or clustered ORNs that allow fine-scale resolution of such complex signals. Odours from a host are likely to occur in ‘pockets’ emanating from a host, and therefore receptor cells that respond simultaneously may indicate the presence of a host, whereas the same receptor cells being stimulated with a delay would not.21 Although this hypothesis has only been described for phytophagous (plant-feeding) insects thus far, coincidence detection might also occur in biting flies, considering the complexity of odours within their environments.
Olfactory stimuli are used by biting insects during various stages of their life cycle and thus offer more than one potential stage at which to apply control methods. During host location, mainly chemical stimuli, provided by vertebrate hosts, induce a series of behaviours that lead to the insect obtaining a blood meal.22, 23 Attaining a clear picture of the chemical basis for this process is a difficult task owing to the complexity of vertebrate odours. For example, the human body produces somewhere in the region of 300–400 volatile chemicals.23 However, a good understanding of olfactory-mediated behaviour is crucial for the development of control strategies using host-derived semiochemicals.
Olfactory cues that stimulate upwind movement by orientation to the wind (odour-mediated anemotaxis) are thought to dominate during host location and thus offer great potential for exploitation as lures in traps.24–27 As a result, many investigations have focused on the identification of such cues, also known as kairomones, from vertebrate hosts. These volatile kairomones mediate interspecific communication, where the responding insects gain a behavioural or physiological advantage, while the kairomone-emitting host does not. Many kairomones may play a major role in the ‘host-seeking’ process of haematophagous insects. Kairomones can emanate from various locations on the vertebrate, including breath, skin (which may include gland secretions and breakdown products of microorganisms), urine and faeces.
3.2 Semiochemical identification
Once an understanding is gained of whether semiochemicals are used by an insect in the relevant context, the compounds must be identified. This can be difficult, but various techniques have been developed to facilitate the process. Semiochemical isolation methods include solvent washing, solid-phase microextraction (SPME), vacuum distillation and air entrainment. Such methods have been used in the past to collect pheromones, plant volatiles and vertebrate volatiles.28, 29 Subsequent analytical chemistry techniques, such as high-resolution gas chromatography (GC) and coupled GC–mass spectrometry (GC-MS) can be applied to quantify and identify accurately compounds of interest. Combining GC with electroantennography (GC-EAG) or single-cell recordings (GC-SRC) provides a unique way of detecting individual, physiologically relevant (i.e. EAG-active) components within a complex array of chemicals in natural extracts. The detected compounds can then be tentatively identified by GC-MS and confirmed by GC coinjection with authentic samples,30 and, where necessary, by nuclear magnetic spectroscopy (NMR).31 Once identified, authentic samples of chemicals, either obtained from commercial sources or by chemical synthesis, are used to confirm both electrophysiological and behavioural activity.
The techniques described above, along with laboratory and field-based behavioural assays, can identify semiochemicals that can be exploited to control and monitor vector insects, as well as enhance the knowledge and understanding of their behaviour and evolution. The majority of semiochemicals with potential applications can be obtained from commercial sources and via chemical synthesis from fine chemical precursors. In the latter instance, however, cheap and efficient synthesis is sometimes difficult to achieve, and higher plants offer alternative cheap and renewable resources for semiochemical production, especially in resource-poor afflicted countries. For example, (Z)-5-hexadecenoic acid, a precursor to the Cx. quinquefasciatus oviposition pheromone (5R,6S)-6-acetoxy-5-hexadecanolide, can be obtained from seed oil of a renewable plant resource, Kochia scoparia (L.) Roth (Chenopodiaceae).32–34 The pheromone can be obtained efficiently from the precursor at a cost of $3 g−1, which compares favourably with conventional synthetic materials, an example of whose production35 has been costed at $15 g−1.33 Similarly, the sandfly pheromone, (S)-9-methylgermacrene-B, can be synthesised from germacrone, a major component of Geranium macrorrhizum L. (Geraniaceae) essential oil.36 Although not directly related to biting fly chemical ecology, the most advanced example of plant-based semiochemical production is the aphid sex pheromone, (4aS, 7S, 7aR)-nepetalactone, which can be obtained by steam distillation from Nepeta cataria L. (Lamiaceae) plants at a cost 1000 times cheaper than its production from fine chemicals.37 Although availability of semiochemicals will facilitate the development of control strategies, registration is also an important issue in the implementation of semiochemicals in control technologies, and can prove to be an expensive process. As a result, it can be difficult to exploit semiochemicals, especially those that are relevant to small markets (such as species-specific semiochemicals).
3.3 Using semiochemicals in traps
Traps can be used for monitoring populations of vector insects and are important for several reasons. Firstly, they provide early warning of insect activity to local authorities, which can facilitate rapid control measures and warnings to be issued. Secondly, they provide a better understanding of the relationship between arboviruses, vectors and their environment, which can aid and maximise control attempts. The development of trapping systems that utilise semiochemicals as lures depends upon the identification of behaviourally active compounds.38 Such traps are relatively new for management of adult mosquito populations.39 Currently, trapping systems baited with carbon dioxide are most widely used for monitoring and control. However, trapping systems that incorporate other volatile chemicals have been developed to include host-derived volatile compounds, pheromones and oviposition site cues. These traps have been mainly used to catch mosquitoes and Culicoides spp. biting midges and are described below.
The Centre for Disease Control (CDC) light trap attracts mosquitoes by emitting light (and sometimes carbon dioxide) and can be used in conjunction with human-baited bed nets.40 Counterflow geometry (CFG) traps, originally developed by American Biophysics Corporation (ABC), are used to sample mosquitoes, and are efficient at trapping many species using various baits41 such as (5R,6S)-6-acetoxy-5-hexadecanolide, the Cx. quinquefasciatus oviposition pheromone,42, 43 grass infusions that are associated with breeding sites44 and host odours such as carbon dioxide and 1-octen-3-ol.45 The host odour compounds are used with great success to trap both mosquitoes and midges, and ABC now markets various models of the CFG trap (Mosquito Magnet®) globally. The suction motor on the trap is powered by burning butane, which conveniently also generates carbon dioxide to form part of an attractive lure alongside a racemic mixture of 1-octen-3-ol. Human landing catches (HBCs) have also been used successfully, and HBCs can be a reliable method for monitoring mosquito populations.46, 47 However, this method is now ethically unacceptable owing to the pathogen transmission potential. Alternatively, odour-baited entry traps47–50 or light traps used in conjunction with bed nets can be used to evaluate the extent of contact between mosquitoes and human beings.51
Host-derived volatile semiochemicals, which have the potential to improve the efficacy of targets and traps for tsetse flies, have been identified from excretory products such as urine, dung or breath, or glandular secretions and exudates from hosts. Carbon dioxide, acetone and 1-octen-3-ol, which are present in exhaled ruminant breath, are known to be attractive,52–55 and multicomponent baits including these compounds, along with butanone, 4-methylphenol and propylphenol, have been shown to be effective in trapping populations in the field.56–58 Reports suggested that these baits are less than 50% as attractive as natural cattle odour when released at naturally occurring concentrations, implying that other unidentified attractive components of the host odour need to be identified.56, 59 However, by increasing their release to 10–100 times their natural dose, tsetse trap catches can be increased 20-fold.56, 59
Tsetse fly attractants have also been evaluated for their ability to trap other Dipterous pests in the Muscidae, Tabanidae and Ceratopogonidae. For the stable fly Stomoxys calcitrans L., olfactory and behavioural responses to 1-octen-3-ol, acetone and cattle odour were observed.60–63 1-Octen-3-ol has also been shown to increase catches of Stomoxys spp.64 Female Haematococcus pluvialis Flotow, Tabanus bromius L., T. maculicornis Zetterstedt, Chrysops relictus Meigen and Hybomitra bimaculata Macquart were also collected in significantly higher numbers in traps baited with 1-octen-3-ol than in traps baited with acetone or ammonium hydroxide, and some species were also attracted to a combination of acetone and aged horse urine.65, 66
The traps described here are valuable tools for monitoring populations of insect vectors and can provide localised control of vector populations.41, 67 However, a trap that matches or exceeds the ability to lure as many insects as a natural vertebrate host does not exist, and most traps up until now have used carbon dioxide as part of the lure, which can be problematic in remote field locations. However, a new trap called the BioGents Sentinel trap, which does not use carbon dioxide, was recently developed and has been demonstrated to be extremely effective in catching mosquitoes (particularly Aedes sp.) many times. This trap releases lactic acid, ammonia and a fatty acid over a larger surface area and mimics convection currents from a human body.67–72
3.4 Semiochemicals and differential attraction
Biting insects may use several semiochemicals during the location of a suitable host. The numerous compounds that are known to play a role are not discussed here, but are summarised in Table 1. Instead, this section will focus on a particular aspect of host location that has received much attention recently. Many semiochemicals have recently been identified that are thought to be involved in the differential attraction of biting insects to individual vertebrate hosts. Research indicates that the host location process also involves the detection of repellents or ‘non-host’ compounds as well as attractants, especially during the discrimination between different hosts.23, 28, 73–75 Many biting insects show preferences for certain host species and even individuals within a host species. By understanding this complex interaction, researchers have uncovered new semiochemicals with potential control applications.
Table 1. Reported semiochemicals for various biting insects
|Chemical stimulus||Insects that bite humans||Insects that bite animals|
|Carbon dioxide||Mosquitoes: Ae. aegypti; Cx. quinquefasciatus; An. quadrimaculatus||Tsetse flies: Glossina spp.|
| ||Midges: C. impunctatus; C. furens; C. stellifer; C. mississippiensis||Stable fly: Stomoxys calcitrans|
| ||Tsetse flies: Glossina spp.|| |
|Lactic acid||Mosquitoes: Ae. aegypti; Cx. pipiens; Ae. atropalpus An. gambiae|| |
| ||Midges: C. impunctatus|| |
| ||Triatoma bugs: Triatoma infestans|| |
|Amino acids||Mosquitoes: Ae. aegypti|| |
|Acetone||Midges: C. impunctatus||Tsetse flies: Glossina spp.|
| ||Mosquitoes: An. gambiae; An. stephensi||Stable fly: Stomoxys calcitrans|
| || ||Midges: C. nubeculosus|
|Ammonia||Triatoma bugs: Triatoma infestans||Clegs: Haematopota pluvialis; Hybomitra expollicata|
|Carboxylic acids||Mosquitoes: Ae. aegypti; An. gambiae|| |
| ||Triatoma bugs: Triatoma infestans|| |
|1-Octen-3-ol||Mosquitoes: An. gambiae; C. impunctatus, 15 Aedes spp. including Ae. aegypti||Tsetse flies: Glossina spp.|
| ||5 Anopheles spp.; 10 Culex spp.; 2 Culiseta spp.; 2 Mansonia spp.; 5 Psorophora spp.; 2 Wyeomyia spp.||Clegs: Haematopota pluvialis; Hybomitra expollicata|
| ||Midge: C. impunctatus||Midges: C. nubeculosus|
| ||Sandfly: L. longipalpis|| |
| ||Triatoma bugs: Triatoma infestans|| |
|Phenols||Mosquitoes: An. gambiae||Tsetse flies: Glossina spp.|
| ||C. impunctatus||Stable fly: Stomoxys calcitrans|
| || ||Midges: Culicoides spp.|
|Other|| || |
|2-Oxopentanoic acid||Mosquitoes: An. gambiae|| |
|Lysine, cadaverine, estradiol||Mosquitoes: An. stephensi|| |
|Butanone||Midges: C. impunctatus|| |
|7-Octenoic acid, (E) & (Z)-3-methyl-2-hexanoic acids||Mosquitoes: An. gambiae|| |
|Methyl salicylate; isothiocyanates: allyl, butyl, phenyl, 2-phenyl||Midges: C. impunctatus|| |
|6-Methyl-5-hepten-2-one; 2-pentanone; 3-pentanone; butanone;||Mosquitoes: Ae. aegypti|| |
| 4-hydroxy-4-methyl-2-pentanone,|| || |
| 4-methyl-2-pentanone.||Sandfly: L. longipalpis|| |
Many studies have demonstrated that haemato- phagous insects show feeding preferences for certain host species, which has given rise to specialist and generalist species.75, 76 For example, recent studies are starting to elucidate the semiochemical basis of the natural differential attractiveness of certain vertebrate species by tsetse flies. The preference of Glossina mortisans mortisans Westwood and G. pallipides Austen for buffalo, Syncerus caffer (Sparrmann), and ox hosts, compared with the non-host waterbuck, Kobus defassa Rüppel, is caused by fewer aldehydes and more phenolic components, octalactone and a series of methyl ketones (C8C13) in the non-hosts, which were not detected or only present in trace amounts in the two preferred hosts.75 It was proposed that the blend of waterbuck-specific components may function as long- or medium-range allomones against tsetse flies, and that the blend of aldehydes may be an unidentified part of the attraction system for these insects. In behavioural tests, G. m. mortisans were shown to be attracted to the host-specific aldehyde blend, but they preferred control stimuli when presented with the waterbuck-specific blend.77 Lactic acid, 2-methoxyphenol and acetophenone could also play a role, as they have been shown to reduce trap catches of G. pallipides and G. m. mortisans.78 Furthermore, the addition of pentanoic acid, hexanoic acid, acetophenone or 2-methoxyphenol to traps baited with an attractive odour blend substantially reduces trap catches.79
The differential attractiveness of vertebrate hosts within a single species, as opposed to that observed for tsetse flies, has been observed for cattle flies (Diptera: Muscidae).80 The role of cattle-derived semiochemicals in differential attractiveness was established through isolation of volatiles from Holstein-Friesian heifers. Twenty-three compounds were identified by coupled GC-EAG.28 Of these, 1-octen-3-ol, 6-methyl-5-hepten-2-one and 3-octanol were identified as attractants, whereas naphthalene, propyl butanoate and linalool were identified as repellents. When applied as slow-release formulations in the field, however, 6-methyl-5-hepten-2-one reduced fly populations on individual animals. The identification of attractants and repellents demonstrated the potential that these compounds have for monitoring and controlling cattle fly populations.
The host-seeking behaviour of the Culicidae and Ceratopogonidae has received much attention because of the close association between these insect families and human beings. In recent years, many scientists have focused on providing systematic evidence for the differential attraction of mosquitoes to human beings, which is well described anecdotally. Although there is now scientific evidence that this phenomenon may be mediated by chemical stimuli in mosquitoes,49, 81–87 so far investigations have merely scratched the surface in their attempts to explain the chemical basis for this complicated event. Differential attraction has also been demonstrated for sandflies88 and for blackflies.89
Until recently, evidence for the involvement of specific olfactory cues in differential attraction of mosquitoes to human hosts was relatively scarce or contradictory. Some authors believe that a lack of ‘attractive’ stimuli or host kairomones is the main cause. For example, adding lactic acid to the skin of formerly non-attractive individuals increases mosquito responses towards them.23, 90–92 Lower levels of lactic acid contribute to the lesser attractiveness of non-human vertebrates to An. gambiae mosquitoes.93 Lactic acid has also been shown to repel Aedes mosquitoes when applied to normally attractive human and mouse skin.94Anopheles mosquitoes, which normally respond behaviourally to sweat,95, 96 show no response to lactic acid when it is presented at the same concentration on its own.97 Therefore, lactic acid could be involved in making a host more or less attractive to anthropophilic mosquitoes. Carbon dioxide has also been suggested to play a role in differential attraction. For example, the removal of exhaled air and standardising carbon dioxide has been shown to eliminate differential attraction of humans to blackflies and mosquitoes.49, 89 However, Costantini et al.48 demonstrated that standardising carbon dioxide does not equalise responses of An. gambiae to individual human beings. Additionally, no study has accurately quantified carbon dioxide in odour profiles of individuals and related it directly to biting insect behaviour. Therefore, the true role of carbon dioxide in differential attraction is unknown. However, this type of compound is not expected to be associated with differential attraction, as it is a product of primary metabolism. Secondary metabolites comprising other volatile organic compounds are more likely to be involved, and are commonly found in animals and plants as defences against parasites, diseases and predators.
One study that attempted to quantify compounds in association with differential attraction indicated that several chemicals may have contributed to differential responses of Ae. aegypti to two volunteers or the same volunteer over time.23 Some components were described as weak attractants or repellents, but the results were inconclusive owing to small sample sizes and the inability to distinguish between the chemicals that were physiologically relevant (i.e. EAG-active) to mosquitoes and the chemicals that simply comprised human odour, since electrophysiological techniques were not used. Additionally, an accurate quantification of the chemicals from the volunteers was not performed. However, the authors reported that lactic acid, butanone, 2-pentanone, 3-pentanone and 6-methyl-5-hepten-2-one were ‘weak attractants’ for Ae. aegypti mosquitoes and implied that these might be responsible for making people more attractive to this species. The remaining components tested did not elicit a response. However, the authors suggested that some of the chemicals could also be repellents.23
It is unknown whether the production of semiochemicals that negatively affect insect behaviour (i.e. repellents) has evolved from selective pressures or is a byproduct of metabolic processes.98 If differential attraction has evolved as a natural defence trait in some human beings, the existence of genetic evidence for this might be expected. Although there is no evidence at present, in one study, 80% of malaria cases were found to be prevalent in only 20% of the population.99 However, the extent to which differential attraction was involved in creating this statistic is unknown. There is evidence that the production of repellents in cattle is genetically determined. For livestock, this could lead to breeding programmes to select for unattractive individuals.100 If this is also true for human beings, a screening process could identify the most vulnerable individuals, allowing for targeted control efforts.
Most chemicals that are described as causing positive behavioural responses in haematophagous insects, such as carbon dioxide, 1-octen-3-ol, lactic acid, ammonia, acetone and fatty acids, are found ubiquitously in all humans and in many other vertebrates. Therefore, chemicals such as these might comprise a basic ‘core’ suite of olfactory signals that, when present, convey information to an insect that a vertebrate is nearby. The addition or increase of certain other chemicals that either repel or ‘mask’ the activity of these core attractants could be a way by which inappropriate or unsuitable vertebrate hosts are avoided by host-seeking insects.
Pheromones which mediate interactions between members of the same species can be divided into different categories, depending upon the type of behaviour that is mediated, e.g. mating, aggregation, oviposition (egg laying) and invitation behaviour, and each class of pheromone has the potential to be utilised in traps. The function of sex pheromones, which are released by one sex, is to initiate either attraction or behaviour associated with mating in the opposite sex. Aggregation pheromones usually promote aggregation of both sexes, while oviposition pheromones promote egg-laying behaviour by gravid (egg-bearing) females.
Pheromones, although not always directly related to the vectoring component of the life cycle, represent a potentially potent means of vector detection through the deployment of pheromone baits in trapping systems. Such monitoring systems are typically highly species specific. Culex quinquefasciatus females utilise an oviposition pheromone, (5R,6S)-6-acetoxy-5-hexadecanolide, which is released from egg rafts laid on the water surface.33 Field trials in several countries and in areas where the pathogen is prevalent have demonstrated the efficacy of the synthetic pheromone in the field,32, 42, 44 particularly when used in conjunction with site-derived oviposition cues found in grass infusions or soakage pit water, such as 3-methylindole (skatole).32, 42, 101, 102 This could be deployed effectively to monitor Culex spp. mosquito populations or even to control them if used in conjunction with environmentally benign larvicides, such as the insect growth regulator pyriproxyfen or larvae-specific pathogens, such as the fungus Lagenidium gigantuem Couch.103 In spite of the demonstrated value of this pheromone in the trapping systems described above, it has not yet been exploited to a great extent in monitoring or control systems. However, the recent spread of West Nile virus by Culex species mosquitoes in the developed world means that there is renewed interest in this pheromone, which is now commercially available for trapping systems.
Sex pheromones produced by male sandflies of the Lutzomyia longipalpis (Lutz & Neiva) species complex are novel homosesquiterpenes characterised as 3-methyl-α-himachalene (L. longipalpis from State of Bahia, Brazil) and (S)-9-methylgermacrene-B (L. longipalpis from State of Minas Gerais, Brazil),88, 104–106 or a diterpene (L. longipalpis from State of Ceará, Brazil). The compounds act as sex pheromones to attract females and maintain species isolation.88 More recently, (1S,3S,7R)-3-methyl-α-himachalene was shown also to cause male aggregation.107 An oviposition pheromone has also been identified for L. longipalpis as dodecanoic acid.108 These pheromones are not currently used in the control of sandfly populations but show potential. A female-produced sex pheromone has also been identified for the farmyard midge, Culicoides nubeculosus (Meigen), as n-heptadecane.25 For the face fly, Musca autumnalis Deg., and the stable fly, S. calcitrans, sex pheromone components are straight-chain monoalkenes and mono- and dimethyl branched alkanes.109 Contact sex pheromones have been reported for the tsetse fly Glossina tachinoides Westwood as comprising long-chain dimethylalkanes,110 and for G. austeni Newstead 13,17-dimethylpentatriacont-1-ene is highly stimulatory to males. For the screwworm, Cochliomyia hominivorax Coq., a contact sex pheromone has been postulated but not identified.111
Some biting flies, including mosquitoes, blackflies and Culicoides midges, are believed to produce a pheromone while feeding on a host, although the semiochemicals involved have not yet been identified. These compounds are believed to be aggregation or ‘invitation’ pheromones that may attract conspecifics during feeding.112–114
Pheromones are an excellent resource as baits in trapping systems that target specific species. However, with the exception of the sandfly pheromone, sex pheromones often trap only males, the sex that usually does not bite. While this can give an indication of species populations as a whole, oviposition pheromones and host-derived kairomones have greater potential for monitoring and controlling the female vectors.