Control of Neglected Disease Insect Vectors : Future Prospects for the Use of Tools Based on Behavior Manipulation-Interference

A expressão “doenças tropicais negligenciadas” (NTDs) representa uma série de doenças que afetam a população de países de baixa renda, sendo insetos vetores os responsáveis pela transmissão de grande parte dessas doenças. Ferramentas de controle são necessárias para impedir o contato entre humanos e insetos vetores destas doenças. Este controle é comumente realizado pelo uso intensivo de inseticidas, porém, resistência a estes xenobióticos ocorre nos casos mais relevantes de espécies de vetores de doenças. Portanto, metodologias alternativas para o controle de vetores são urgentemente necessárias para evitar a transmissão da maioria das NTDs. Nesta revisão, uma série de compostos químicos que podem auxiliar no desenvolvimento de ferramentas de controle desses vetores, as quais se baseiam na manipulação do comportamento dos insetos associado à estas moléculas, são descritas. Tais relações incluem os odores de hospedeiros usados por artrópodes na busca de fontes de sangue, assim como feromônios utilizados por estes em diversos contextos como, por exemplo, na reprodução. Adicionalmente, são apresentados caminhos recentemente explorados na busca de compostos capazes de modificar comportamentos através de metodologias de alto rendimento. Especificamente, são mostrados exemplos de como estas metodologias mediam a busca por novos repelentes e atrativos.


Neglected Tropical Diseases and Control Tools
5][6] Many of these illnesses are transmitted by vector organisms, mostly blood-feeding arthropods.Even though several of these diseases can be prevented or treated, it is clear that most vector-borne neglected maladies cannot be currently controlled by means other than extensive use of insecticides to eliminate their vector insects. 7Diseases like dengue fever, leishmaniasis, Chagas disease, lymphatic filariasis, African sleeping sickness and onchocerciasis are considered NTDs and represent the main vector-borne maladies affecting humans around the globe together with malaria.
The virus causing dengue fever, for example, is transmitted by the mosquito Aedes aegypti (Figure 1a).Brazil officially reported more than 200,000 cases to the Pan-American Health Organization in 2013. 8The estimated annual cost with dengue fever in that country amounts to approximately US$ 132 million, including activities of vector control; surveillance; information, education and communication; direct and indirect medical costs. 9From this global cost, the average investment in control represents 25%, while surveillance requires 4%. 9 The yearly global cost due to dengue fever infections is estimated in 390 million (including symptomatic and asymptomatic cases). 10e century after the discovery of Chagas disease, a curative agent is still not available.It is one of the leading causes of mortality/morbidity in Latin America, with approximately 8 million people infected worldwide, more than 25 million people at risk of infection, and approximately 15,000 deaths each year. 11This debilitating zoonosis is caused by infection with the flagellate protozoan Trypanosoma cruzi.The primary vectors in South America are Triatoma infestans (Figure 1b) and Rhodnius prolixus, however, species such as Panstrongylus megistus and Triatoma brasiliensis, play an important role in transmission in some regions of Brazil. 12nother example of neglected disease caused by a protozoan parasite and transmitted by a vector insect is leishmaniasis, which is caused by parasites of the genus Leishmania.Its transmission to humans happens via the bite of phlebotomine sandflies, e.g., Lutzomyia longipalpis (Figure 1c) and 70-90% of reported cases come from a list of 12 countries that includes Brazil, Colombia and Peru. 13rrently, the intense use of insecticides needed for controlling some of these and other diseases represents an enormous chronic expenditure for the public agencies that combat their transmission, both in terms of cost of active principles and logistics necessary to apply them. 7,14evertheless, in most of the cited cases these xenobiotics currently represent our best tools to avoid disease spread. 15

The Use of Insecticides: Future Limitations
Historic records show that the use of insecticides has allowed effective disease control in many cases. 15This sustained use and its eventual failures have promoted the appearance of insecticide resistance in many of the vector species transmitting NTDs. 16,17Resistance to insecticides is widespread for important disease vectors such as mosquitoes, 18,19 triatomines [20][21][22] and ticks. 23Populations of other vector arthropods, e.g., sandflies, which have been subjected to intensive selective pressure by insecticide use, will probably develop similar tolerance to current xenobiotics in the future.5][26][27] Due to this, the search for new tools that could act as control alternatives has been an active trend in the field of insect vector biology in the last decade.][36][37] What follows is a series of topics in which we summarize current knowledge on diverse aspects of the chemistry, biology and chemical ecology of vector arthropods, which could lead to the development of tools to assist their control.In connection to this, we suggest several perspectives that could be pursued in the field in order to offer alternatives for control agencies.

Host Kairomones and their Potential for Vector Detection/Capture
9][40][41][42][43][44][45][46][47][48][49] This is true for diverse hematophagous arthropod orders and evidence gathered so far indicates that CO 2 is an almost universal cue used by these arthropods to detect and find their hosts. 50The detection of other substances emitted by hosts is also frequently exploited in order to find emission sources.Examples of female mosquitoes orienting to host odors are diverse 42,[51][52][53][54][55] and highlight how much these insects rely on a widespread use of chemical signals to recognize their sources of blood.
L-Lactic acid was identified as the major constituent of acetone extracts from human skin and showed to be attractive to female Ae.aegypti when used jointly with CO 2 . 42Later, by employing ammonia in combination with CO 2 and L-lactic acid, a strong synergistic effect between those compounds was observed using Y-tube olfactometry. 44 blend of short carboxylic acids (e.g., propionic, butyric, isovaleric, hexanoic) combined with L-lactic acid was shown to be more attractive to Ae. aegypti females than L-lactic acid alone.45 The effect of kairomones in host location by the malaria transmitting mosquito Anopheles gambiae was tested by using traps in controlled cages.A synergistic effect was found when ammonia, lactic acid and fatty acids were presented as a blend into trap devices.However, the tripartite synergistic effect observed for An.gambiae differed from Ae. aegypti, since lactic acid alone attracted Ae. aegypti but not An.gambiae.51 An. gambiae females preferred human odor over clean air in a Y-tube and avoided cow odor over clean air, 54 a feature that might reinforce the effectiveness of their orientation to human hosts.Analysis of skin rubbings from various vertebrates indicated that human skin levels of L-lactic acid are the highest found between studied hosts, suggesting that the lower levels of L-lactic acid found on other animals contribute to their lower attractiveness to An. gambiae.55 The genus Culex comprises the vectors of pathogens that cause diverse human diseases, including St. Louis encephalitis, Japanese encephalitis, Venezuelan equine encephalitis, Western equine encephalitis, lymphatic filariasis and West Nile Virus. 56ulex quinquefasciatus are strongly attracted by rabbit chow-baited traps and the bioactive compounds responsible for this were studied.By using solid phase micro-extraction (SPME), gas chromatography-electroantennographic detection (GC-EAD) and gas chromatography-mass spectrometry (GC-MS) techniques three compounds were identified as trimethylamine, nonanal and 3-methylindole.56 These compounds were tested in the field and were shown to be attractive in binary and ternary combinations.56 Odorants from humans from diverse ethnic backgrounds were collected by SPME and the major components identified were 6-methyl-5-hepten-2-one, nonanal, decanal, and geranyl acetone.57 Volatiles emitted by birds, the principal hosts of Cx. quinquefasciatus, were also analysed and nonanal was shown to be their main component.57 Electroantennography recordings with female Cx.quinquefasciatus antennae tested against volatiles from chicken, pigeons and humans showed a consistent response to nonanal.57 Behavioral experiments showed strong attraction of Cx. quinquefasciatus to traps baited with nonanal alone or in combination with CO 2 .57 Triatomine bugs, another group of relevant vector arthropods that transmit Chagas disease to humans, have also been shown to exploit an array of chemical substances emitted by their hosts.[58][59][60][61][62][63][64][65] For Rhodnius prolixus, CO 2 and human odor proved to be attractive, as well as the odor of a hamster, while on the other hand, L-lactic acid alone did not seem to play an important role in orientation.58 A mixture of lactic, propionic, butyric and valeric acid showed a synergistic effect with CO 2 (300 ppm above the ambient level) on the attraction of Triatoma infestans, evoking a behavioral response comparable in intensity to that induced by a live mouse.59 By using electrophysiological single sensillum recordings coupled to gas chromatography, nonanal (from sheep wool and chicken feathers) and isobutyric acid (from rabbit odor) were identified as chemostimulants to receptor neurons from Triatoma infestans.Behavioral bioassays showed that nonanal causes the activation of the bugs, while isobutyric acid induces their attraction.62 1-Octen-3-ol, which was first isolated from cattle odors and is also present in human sweat, was shown to be attractive to Triatoma infestans, even in the absence of CO 2 .][67][68][69][70][71] Male and female Lu.longipalpis sandflies showed attraction to human skin extracts 67,69,70 and also to CO 2 emitted at human-equivalent rates.70 Single sensillum recordings coupled to gas chromatography showed that sixteen compounds from the odor-producing glands of the fox Vulpes vulpes stimulate the olfactory organs of sandflies.Lu. longipalpis were as attracted to fox extracts as to a synthetic mixture containing all the synthetic compounds (4-methyl-2-pentanone, 2-hexanone, 4-hydroxy-4-methyl-2-pentanone, 3-hydroxy-2-butanone, 4-methylheptanone, 2-butanol, 3-methylbutan-1-ol, 3-methyl-3-buten-1-ol, 1-pentanol, propanoic acid, 2-methyl-propanoic acid, butanoic acid, 3-methyl butanoic acid, pentanoic acid, benzaldehyde and hexanal).68 Simulids are a group of small flies that can transmit serious diseases, as onchocerciasis, to humans and seem to rely on host odors in order to find a bloodmeal.][77][78][79] Relevant vector flies such as African tripanosomiasis-transmitting tsetse flies also orient to their hosts by means of olfactory cues.80,81 This array of examples highlights the potential of chemical substances emitted by hosts and, their associated skin microorganisms, as attractants to manipulate vector behavior, as summarized in Table 1.The search for appropriate formulations of behaviorally active blends is a field that needs attention to allow the development of efficient baits.Particularly, long and sustained emission of a stable blend formulation seems critical for proper bait function under field conditions.

Pheromones and their Potential for Vector Detection/Capture
Pheromones, the chemical signals used by animals to communicate with their conspecifics, represent a powerful alternative for the development of behavior-modifying tools.Pheromones have been described for triatomine bugs, flebotomine sandflies, mosquitoes, ticks and other vector arthropods.The appealing potential of pheromones relies in their specificity, powerful behavioral effect and low tendency of target species for developing resistance, if used in control tools.A diverse range of functions can be described for arthropod pheromones and these include the recognition of sexual partners, the promotion of species-specific aggregations, the marking of oviposition sites and shelters, and the rapid communication of the presence of menaces.

Next Generation Sequencing Techniques and Functional Characterization of Molecular Targets to Interfere in Insect Olfaction
The last two decades represent a historical turning point in terms of multidisciplinarity in chemical and biological research.One of the key events driving this phenomenon was the advent of sequencing techniques and, specially, the so-called next generation sequencing techniques (NGS).This allowed accessing the sequence identity of significant genes and even promoting the mass identification of genes actively expressed in target tissues.Likely, insect science benefited from this and the genes and genomes of model insects, e.g., Drosophila, 116 have been exhaustively studied through these methodologies.Among diverse outcomes that deserve mentioning, the discovery of several families of sensory receptors was made possible, representing a breakthrough in our understanding of the molecular basis of insect sensory perception.8][119] The identity of other insect sensory receptors, like thermoreceptors (TRPs), 120,121 water and contact chemoreceptors (ppks) 122,123 and opsins (visual pigments), 124 has also been clarified and their roles are under intense functional evaluation.As receptor proteins are exposed in the membrane of sensory neurons normally housed in insect sensilla, the detection of stimuli depends on their efficiency to bind specific ligands or to react when confronted to specific patterns of energy, e.g., thermal.These functions are considered fundamental for the proper detection of relevant resources and therefore, become natural targets for interfering with the life of pest  12), (S)-2-butanol (13), 2-methyl-3-buten-2-ol ( 14), 3-methyl-2-butanol (15), 3-pentanol ( 16), (S)-2-pentanol (17), (E)-2methyl-3-penten-2-ol ( 18), (S)-4-methyl-2-pentanol ( 19), (S)-3-hexanol (20), 2-methyl-1-butanol ( 21) and (±)-4-methyl-3-penten-2-ol (22).(23) and hexacosanoic acid (24).Vol. 25, No. 10, 2014   insects.The development of specific blocking agents or antagonist molecules that impede key sensory detection would allow affecting the recognition of hosts or sexual partners, interfering with fundamental activities such as feeding and reproduction.Recent advances 125,126 suggest that this approach to the development of a new generation of xenobiotics is feasible 127 and deserves attention due to its alternative and specific way of action.Interestingly, the first evidence of the effectiveness of blocking olfactory receptors to interfere with the detection of food sources came from the discovery of a set of substances capable of blocking the CO 2 receptors of An. gambiae, Ae. aegypti and C. quinquefasciatus, the three main mosquitoes vectoring human diseases. 125,126It is therefore clear that this new perspective should be explored to determine whether other functions such as the detection of pheromones emitted by sexual partners could also be impeded to interfere with the normal development of insect vectors.

New Repellents Based on Olfactory Receptor Characterization
Chemical substances considered as repellents have the common feature of avoiding contacts between their users, normally humans, and organisms that could otherwise feed upon them.These can be used to impregnate skin or clothes or, even to fumigate in open areas by means of candles and other emission devices.Technically accepted repellents are very few and vary in their protective power, both in terms of effectiveness to avoid vector-host contacts and duration of protection. 128he first synthetic repellent of massive commercial use was N,N-diethyl-3-methylbenzamide (DEET). 1280][131] Nevertheless, it is still considered an adequate personal protection method against dengue and other vector-borne diseases. 128][138][139][140] The recent use of cheminformatic pipelines to predict receptor-odorant interactions and subsequent molecular modeling strategies to uncover shared structural features allowed screening in silico for new candidate ligands from libraries including thousands of potential volatiles (i.e., a high throughput search).This approach allowed identifying new compounds potentially tuned to chemosensory receptors that have potential application in avoiding vector-human contacts. 126,127,141This seems to be a promising area in which molecules could be selected or designed in order to find new effective ligands with high affinity to receptors expressed in neurons known to mediate repellency-related behaviors.

Final Remarks
As shown in this revision of our current knowledge on the potential of chemical manipulation of insect behavior vectoring human diseases, there is an arsenal available that needs further evaluation for the purpose of developing control tools.The relevance of this collection of compounds relies in the fact that most of them represent critical signals that have key roles in controlling adaptive vector behaviors.As such, the substances listed, and others still to be uncovered, represent more sustainable alternatives in terms of resistance development probabilities, as insects rely on their use for properly triggering behaviors that are critical for their biology.Apart from chemical approaches, strategies involving genetically modified organisms (GMO) have also been studied.Vector transgenesis and paratransgenesis are new control methods intended to diminish the capacity of vectors to transmit pathogens.Transgenesis is the direct manipulation of a vector to render it incompetent for pathogen transmission. 142ikewise, paratransgenesis induces genetic alterations on vector symbionts or comensals so that they produce toxic compounds that impede pathogen development, and therefore, transmission. 143Diverse practical limitations exist in terms of their current application, e.g., governmental agencies, as well as public opinion, are concerned with potential side effects of GMO release.These difficulties must be addressed before any of these promising techniques can allow decreasing vector-borne pathogen transmission. 144Future years should allow translational research to transform these propositions into tangible realities that help controlling NTDs.

Marcelo Gustavo Lorenzo graduated in Biological Sciences at the Buenos Aires University (1991).
He holds a PhD in Biological Sciences from the Buenos Aires University (1997).He had posdoctoral trainings at the Buenos Aires University (1998-1999), CPqRR-Fiocruz  (1999-2002) and the Swedish Agricultural University (2009-2011).Since 1999, he is a researcher at Oswaldo Cruz Foundation at Belo Horizonte, Brazil.He has experience in physiology with emphasis in behavioral physiology, mainly on the following themes: behavior, pheromones, kairomones, electrophysiology, olfaction functional genomics, triatomines, development of traps and baits for vector control.

Figure 1 .
Figure 1.Examples of vectors of neglected tropical diseases: (a) Aedes aegypti, the main vector of dengue virus; (b) Triatoma infestans, an important vector of the flagellate protozoan Trypanosoma cruzi, etiological agent of Chagas disease; and (c) Lutzomyia longipalpis, a phlebotome sandfly that can be parasitized by Leishmania flagellates, etiological agents of leishmaniasis.
Chemistry at the Federal University of Paraná (UFPR).He holds a Master's degree in Organic Chemistry from UFPR.Since 2012, he is a PhD student at UFPR (CNPq fellowship holder).He has experience in chemistry with emphasis in organic synthesis, pheromone chemistry and chemical ecology.Paulo Henrique G. Zarbin is associate professor of the Department of Chemistry at UFPR, where he served as chair of the graduate program in Chemistry.He obtained his PhD in 1998 at UFSCar, with part of his thesis developed at the National Institute of Sericultural and Entomological Science, in Tsukuba, Japan.He is former president of the International Society of Chemical Ecology (ISCE), member of the editorial board of the Journal of Chemical Ecology and editor of Chemoecology.His main research interest is focused on the identification, synthesis, biosynthesis and field evaluation of insect pheromones and other semiochemicals.

Table 1 .
Kairomones emitted by hosts used by arthropods to search for blood sources