**6. Attraction of phlebotomine to volatile organic compounds**

It is well known that insects use olfactory and visual stimuli during different stages of its life cycle like for the search for a blood source or suitable place for oviposition [123]. In the case of olfactory stimuli, the orientation of these insects towards the sources of attractive odors depends on the time and space distribution of odorant molecules spread on the environment. The molecules associated with olfactory responses are volatile organic compounds that disperse following the gas laws, forming clouds of odors and are responsible for chemical communication between the vector insect and host [124].

#### **6.1. Chemical ecology of insects**

The chemical communication occurs when an individual (transmitter) sends chemicals which are transmitted through a medium (air, water, substrate) and are captured by another individual (receiver). These chemicals are called semiochemicals and can be toxins, nutrients, or the substances responsible for the proper transmission of information, infochemicals [125].

Infochemicals are chemical substances that provide information during the interaction between two individuals, resulting in a behavior or physiological response of the receptor. Communication is considered intraspecific when infochemicals called pheromones are secreted by an individual and released to the environment, resulting on a specific behavior reaction of another individual of the same specie [6].

The substances involved in the case of interspecific communication are called allelochemicals and are classified based on harm and benefits to the organisms. The kairomone is a type of allelochemicals which evokes an adaptive response that is unfavorable to the transmitter, but favorable to the receiver [6]. Typical example of kairomone are the volatile organic compounds produced by hosts and are detected by receivers and antennae of insects during the search process and location of food source [126]. The ability to detect these volatile compounds makes easier the specific recognition of the host and increases the range foraging by the vector [127].

Several animals produce a huge number of volatile compounds derived from their own skin, breathing, urine and feces. The detectability of these substances by receivers depends upon the amount that are produced and dispersed over the environment, distance between the insect and the host; wind speed, turbulence and specificity of these molecules to receptor cell of insects which are primarily present in the antennas of insects [128].

The importance of some chemical compounds to the guidance of hematophagous insects has been demonstrated. Carbon dioxide is a by-product of cellular respiration that is released in large amounts by potential hosts and it is a wildly studied semiochemical such as its attractive effect has already been reported for *Aedes aegipty* [129], *Culex quinquefasciatus* [130], *Lutzo‐ myia* sandflies [131] and some species of tsetse fly (*Glossina* spp.) [132]. Another extensively studied compound is 1-octen-3ol which was initially identified from volatile compound of expiration of bovine animals and its attractive effect was evaluated in several species of hematophagous insects [123], including sandflies [124]. Lactic acid is the main component of human sweat and effect was evaluated, being considered attractive when used together with CO2 [133].

These chemical compounds are produced in large scale by several species of animals, therefore they can be regarded as universal kairomones. They attract insects that have opportunistic feeding habits, since they indicate the presence of live vertebrate at a nonspecifically way [134]. Additionally, these orientation behaviours are complicated and potentially plastic, may be modulated by environmental factors [135].

#### **6.2. Chemical ecology of phlebotomines**

**6. Attraction of phlebotomine to volatile organic compounds**

communication between the vector insect and host [124].

330 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

reaction of another individual of the same specie [6].

**6.1. Chemical ecology of insects**

non-infected dogs (B) [41].

It is well known that insects use olfactory and visual stimuli during different stages of its life cycle like for the search for a blood source or suitable place for oviposition [123]. In the case of olfactory stimuli, the orientation of these insects towards the sources of attractive odors depends on the time and space distribution of odorant molecules spread on the environment. The molecules associated with olfactory responses are volatile organic compounds that disperse following the gas laws, forming clouds of odors and are responsible for chemical

**Figure 5.** PCA scores graph based on chromatographic peak areas of VOC of the hair of leishmaniasis infected (A) and

The chemical communication occurs when an individual (transmitter) sends chemicals which are transmitted through a medium (air, water, substrate) and are captured by another individual (receiver). These chemicals are called semiochemicals and can be toxins, nutrients, or the substances responsible for the proper transmission of information, infochemicals [125].

Infochemicals are chemical substances that provide information during the interaction between two individuals, resulting in a behavior or physiological response of the receptor. Communication is considered intraspecific when infochemicals called pheromones are secreted by an individual and released to the environment, resulting on a specific behavior Among the studies performed to identify VOC involved in chemical communication of phlebotomines most of them use *L. longipalpis* as target species, which is why there are few knowledge about other species of phlebotomines.

In the late 80s the sex pheromone of *L. longipalpis* was discovered and since then many studies have been carried out. The pheromone is produced in glandular tissue located in the abdomen of males [136, 137] and is primarily composed of terpenes, a class of natural products that contains in their structure three or four isoprene units with five carbon atoms. More recent works have shown that there are differences in the chemical composition of the sexual pheromone according to the geographical location of *L. longipalpis* the male that produces it, suggesting that this variation is a result of reproductive isolation [138]. This result supports the hypothesis that the *L. longipalpis* is formed by a complex of cryptic species with different morphological and behavioral characteristics [139, 140].

Despite the proven efficacy of sexual pheromone for attraction of *L. longipalpis* by both laboratory [141] as well as field studies [142], this variation in chemical composition restricts the practical use of pheromone at different locations, increasing the importance of the study of kairomones for chemical ecology of phlebotomines. Furthermore, Bray e Hamilton [143] when conducting behavioral studies in the laboratory, they found that the attractiveness of females of *L. longipalpis* nearly doubled when the odor of hosts was added to sex pheromone.

Even so, there are few studies in the literature that evaluate the attractiveness of the phlebot‐ omine to host odors. For example, Hamilton and Ramsoondar [144] found that females and males of *L. longipalpis* were attracted by emitted volatile organic compounds from human skin and there was a difference in attractiveness among the volunteers.

Likewise Rebollar-Tellez *et al.* [145] found different levels of attractiveness to human volun‐ teers, suggesting that this variation may be due to different VOCs emitted by humans. It was also observed that females of *L. longipalpis* preferred to bite human's ear over other parts of the body, considering the number of bites over the exposed area. It has been shown that insects were attracted by odors extracted from human ear, showing that this preference is related to the VOC emitted this region of body [145]. Also in this work, females of *L. longipalpis* coming from the city of Jacobina (Bahia, Brazil) were more anthropophilic than insects from other geographical locations (Marajó, Pará, Brazil and Curiágua, Venezuela). This difference in response of female of *L. longipalpis* from different sites was also found by Rebollar-Tellez *et al.* [146] suggesting that there is a local adaptation of the complex of *L. longipalpis* species to particular hosts or to a specific set of odors and this behavior is inborn and genetically controlled.

For other host species it had been shown that sixteen chemical compounds from anal and caudal glands of European foxes (*Vulpes vulpes*) perform electrophysiological and behavioral action on *L. longipalpis* [147], suggesting that there is a complexity of components that can act on chemical communication between hosts and phlebotomines.

It has been observed that in the case of canine VL phlebotomines are more attracted to feed from infected dogs than from healthy dogs and this preference is probably related to the different odors emitted by infected animals [128]. As demonstred by Oliveira *et al.* [41], in fact volatile organic compounds emitted by the dog clearly differ between animals infected by *Leishmania sp.* and healthy ones, previously identified for ELISA and PCR. O´Shea *et al*. [148] when studying the attractiveness of *L. longipalpis* to hamsters it was observed that the vector is more attracted by individuals infected by *Leishmania infantum* in comparison with healthy ones. After analyzing the volatile organic compounds emitted by rodents using gas chroma‐ tography O´Shea *et al*. [148] described the presence of 10 chromatographic peaks of substances in infected animals which were not detected in those healthy.

#### **6.3. Main techniques used for chemical ecology of phlebotomines**

The insects have a well developed sense of smell which odors are mainly picked up by the antennae for the detection and discrimination of chemical compounds present in the environ‐ ment. This signal reception in most insects is performed by sensilla, small hairs that are located in the antennae, and is processed as follows: when the molecules responsible for the emission of odors reach the antennae sensilla they cross through the cuticle pores until they attain sensillum lymph where they bind to odors ligand protein and they are carried to receiving neuron, generating a cation flow through the membrane of the axon which is then converted to electrical stimulation [149]. This electrical stimulation can be measured by a technique known as electroantennography, wherein the insect antenna is used as a detector of chemical substances. In this technique the amplitude of the voltage produced by the stimulus increases proportionally with concentration of the substance until saturation threshold is reached.

Despite the proven efficacy of sexual pheromone for attraction of *L. longipalpis* by both laboratory [141] as well as field studies [142], this variation in chemical composition restricts the practical use of pheromone at different locations, increasing the importance of the study of kairomones for chemical ecology of phlebotomines. Furthermore, Bray e Hamilton [143] when conducting behavioral studies in the laboratory, they found that the attractiveness of females of *L. longipalpis* nearly doubled when the odor of hosts was added to sex pheromone.

Even so, there are few studies in the literature that evaluate the attractiveness of the phlebot‐ omine to host odors. For example, Hamilton and Ramsoondar [144] found that females and males of *L. longipalpis* were attracted by emitted volatile organic compounds from human skin

Likewise Rebollar-Tellez *et al.* [145] found different levels of attractiveness to human volun‐ teers, suggesting that this variation may be due to different VOCs emitted by humans. It was also observed that females of *L. longipalpis* preferred to bite human's ear over other parts of the body, considering the number of bites over the exposed area. It has been shown that insects were attracted by odors extracted from human ear, showing that this preference is related to the VOC emitted this region of body [145]. Also in this work, females of *L. longipalpis* coming from the city of Jacobina (Bahia, Brazil) were more anthropophilic than insects from other geographical locations (Marajó, Pará, Brazil and Curiágua, Venezuela). This difference in response of female of *L. longipalpis* from different sites was also found by Rebollar-Tellez *et al.* [146] suggesting that there is a local adaptation of the complex of *L. longipalpis* species to particular hosts or to a specific set of odors and this behavior is inborn and genetically

For other host species it had been shown that sixteen chemical compounds from anal and caudal glands of European foxes (*Vulpes vulpes*) perform electrophysiological and behavioral action on *L. longipalpis* [147], suggesting that there is a complexity of components that can act

It has been observed that in the case of canine VL phlebotomines are more attracted to feed from infected dogs than from healthy dogs and this preference is probably related to the different odors emitted by infected animals [128]. As demonstred by Oliveira *et al.* [41], in fact volatile organic compounds emitted by the dog clearly differ between animals infected by *Leishmania sp.* and healthy ones, previously identified for ELISA and PCR. O´Shea *et al*. [148] when studying the attractiveness of *L. longipalpis* to hamsters it was observed that the vector is more attracted by individuals infected by *Leishmania infantum* in comparison with healthy ones. After analyzing the volatile organic compounds emitted by rodents using gas chroma‐ tography O´Shea *et al*. [148] described the presence of 10 chromatographic peaks of substances

The insects have a well developed sense of smell which odors are mainly picked up by the antennae for the detection and discrimination of chemical compounds present in the environ‐ ment. This signal reception in most insects is performed by sensilla, small hairs that are located

and there was a difference in attractiveness among the volunteers.

332 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

on chemical communication between hosts and phlebotomines.

in infected animals which were not detected in those healthy.

**6.3. Main techniques used for chemical ecology of phlebotomines**

controlled.

The electroantennography is a sensitive and specific technique since the insect antenna is much more sensitive than equipments usually used for the detection of chemical substances, therefore it is able to detect minimum amounts of a particular substance.

To identify which VOC present in particular extract exhibit biological activity on *L. longipal‐ pis* gas chromatography coupled to an electroantennographic detector (GC-EAD) can be used, which is an instrument capable of separating the organic compounds present in the sample and measuring electrical signals emitted by antennae of insects used as biological detectors (Figure 6).

**Figure 6.** Gas chromatograph coupled with flame ionization detector and electroantennogram recorder, adapted of [150].

Thus, by inserting the sample into the GC injector it is vaporized and the compounds are transported by a carrier gas traveling across the chromatographic column where separation of substances is performed. When leaving the chromatographic column the flow is splitted into two and one part goes to the detector that identifies the chemical compound and the other part goes to the electroantennographic detector. In this way, the first detector provides a chroma‐ togram containing all compounds detected while the second detector produces an electroan‐ tennogram exhibiting only the compounds that are biologically active to that insect (Figure 6).

This technique has been used successfully in several studies involving chemical ecology of insects, especially to search for compounds which are used for agricultural pest management [151, 152]. In research involving phlebotomines this procedure has also been used [147, 153], but it is underexplored and still needs further study due to the great potential for discoveries [154]. The electroantennography serves to specify which compound the insect actually reacts [155]. However, to specify the biological role of this compound laboratory bioassays and field tests must be carried out.

The laboratory bioassays are conducted in closed systems attempting to obtain data about the behavior of insects when they are induced to respond to a chemical stimulus. Bioassay in the detector is a living organism or part of your sensory system. In bioassay the detector is a living being or part of its sensory system. For the realization of bioassays can be used olfactometers, wind tunnels, testing arena, among others. It is important to highlight that the design and size oftheseapparatusvarygreatlydependingonthesizeandbiologicalcharacteristicsofeachinsect.

There are several advantages of performing bioassay under laboratory conditions compared to field tests: (I) control of environmental conditions, (II) control of the physiological conditions of the organism, (III) elimination of external stimuli, (IV) better interpretation of complex responses.

The bioassay most widely used for phlebotomines is the wind tunnel. In this methodology the flight of insects is allowed while they follow a track of chemical compounds established by an air current which is released on the opposite side where the insects are arranged (Figure 7). The behavior of the insect due to this stimulus is recorded by direct observation or by video cameras and special software. The first wind tunnel study using *L. longipalpis* phlebotomine species was performed by Morton and Ward [136] in which the authors found that virgin females aged 3-6 days came faster and in greater numbers to the other side of the tunnel when they were used hamster and pheromone extracted from attractive males as compared to the control. For these tests it was used a wind tunnel of 240 cm, but since then, relatively few studies have been conducted using this methodology [147, 148, 153, 156].

**Figure 7.** Schematic drawing of wind tunnel [156]

Despite several advantages, bioassays have significant drawbacks: (I) organisms up under laboratory conditions may have different behaviors compared with wild organisms, (II) environment standardization in the laboratory is scarcely repeated at field conditions. Thus, field tests are considered as essential for the validation of a semiochemical and they are the final phase for determining the attraction of phlebotomines to a compound or mixture of compounds. In these studies chemical compounds are placed as bait in traps and their own attractiveness is tested in a proven endemic area for the particular studied insect [124].

#### **6.4. Application of chemical ecology for vector control**

but it is underexplored and still needs further study due to the great potential for discoveries [154]. The electroantennography serves to specify which compound the insect actually reacts [155]. However, to specify the biological role of this compound laboratory bioassays and field

The laboratory bioassays are conducted in closed systems attempting to obtain data about the behavior of insects when they are induced to respond to a chemical stimulus. Bioassay in the detector is a living organism or part of your sensory system. In bioassay the detector is a living being or part of its sensory system. For the realization of bioassays can be used olfactometers, wind tunnels, testing arena, among others. It is important to highlight that the design and size oftheseapparatusvarygreatlydependingonthesizeandbiologicalcharacteristicsofeachinsect. There are several advantages of performing bioassay under laboratory conditions compared to field tests: (I) control of environmental conditions, (II) control of the physiological conditions of the organism, (III) elimination of external stimuli, (IV) better interpretation of complex

The bioassay most widely used for phlebotomines is the wind tunnel. In this methodology the flight of insects is allowed while they follow a track of chemical compounds established by an air current which is released on the opposite side where the insects are arranged (Figure 7). The behavior of the insect due to this stimulus is recorded by direct observation or by video cameras and special software. The first wind tunnel study using *L. longipalpis* phlebotomine species was performed by Morton and Ward [136] in which the authors found that virgin females aged 3-6 days came faster and in greater numbers to the other side of the tunnel when they were used hamster and pheromone extracted from attractive males as compared to the control. For these tests it was used a wind tunnel of 240 cm, but since then, relatively few

Despite several advantages, bioassays have significant drawbacks: (I) organisms up under laboratory conditions may have different behaviors compared with wild organisms, (II) environment standardization in the laboratory is scarcely repeated at field conditions. Thus, field tests are considered as essential for the validation of a semiochemical and they are the

studies have been conducted using this methodology [147, 148, 153, 156].

**Figure 7.** Schematic drawing of wind tunnel [156]

tests must be carried out.

334 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

responses.

Despite recurrent attempts to control and prevent visceral *Leishmania*sis, it is booming worldwide, particularly in Brazil [7, 8].

The situation becomes more alarming when it evaluates the current trend towards urbaniza‐ tion of the disease [10], associated with increasing environmental degradation that favors its spread [157], as well as exposure of susceptible populations and the ineffectiveness of current measures to combat disease [158].

Currently, vector control is based only on the use of environmentally-destructive chemical insecticides [159], however this measure is highly debatable due the risks of environmental contamination and development of resistant insects [160]. Thus, the vector control based on chemical manipulation of vectors is gaining prominence.

In this direction, the identification of semiochemical that can be used as bait in traps to capture phlebotomine can provide a significant improvement for disease control. Light traps are commonly used to monitor populations of phlebotomines and are important for several reasons: (I) enable early identification of the presence of disease vectors in areas previously considered as non-transmission, making it easier to decision making authorities, (II) allow the knowledge about distribution of the vector in a given region as well as monitoring the spread of vector in these locations, (III) and finally, they allow a better understanding of the relation‐ ship between parasite, phlebotomine and environment, thus maximizing the attempts to control the disease.

The trap advocated for capture of phlebotomines is the CDC light [11] and these traps have some operational limitations such as the high cost, little specific and contestable accuracy for areas with low phlebotomines occurrence [161]. The use of chemical compounds along with traps aims to make them more specific and efficient, which has also been demonstrated in studies of phlebotomine [124, 162, 163], as well as other insect vectors of diseases [164, 165].

Another option arising from the identification of semiochemicals attractive to phlebotomine is their use integrated with insecticides. The use of semiochemicals with insecticides to control sand fly populations would be an example of integrated vector control. Similar techniques are used to control agricultural pests [152, 166]. Configures as an ecological approach for the control of insect vectors of disease and it is a rational and integrated use of various techniques available and necessary to a unified program.

The use of semiochemicals in integrated vector management reduces the amount of insecticide used, reducing accidents and environmental risks to public health, as well as decreasing risks of development of resistance in insects. The use of semiochemicals has other advantages, such as focus control on single specie, since most compounds is specie-specific, nontoxic, need to be used in small amounts and are biodegradable

In the case of phlebotomines control, Bray *et al.* [167] used sex pheromone of *L. longipalpis* as attractive on walls treated with insecticides and sticky traps, obtaining satisfactory results. However more studies are needed to increase the efficiency of the system, including the use of host odors to maximize the attraction of insects.
