Machado et al. Parasites & Vectors (2015) 8:147 DOI 10.1186/s13071-015-0748-y RESEARCH Open Access Attraction of the sand fly Nyssomyia neivai (Diptera: Psychodidae) to chemical compounds in a wind tunnel Vicente Estevam Machado1, Arlene Gonçalves Corrêa2, Thais Marchi Goulart3, Flávia Benini da Rocha Silva1, Dennys Ghenry Samillan Ortiz4 and Mara Cristina Pinto1* Abstract Background: Similar to other hematophagous insects, male and female sand flies must feed on plants to obtain sugar and, subsequently, energy to complete their life cycles. A large number of compounds emitted by plants may act as volatile signals to these insects. Primary alcohols have been detected in some plants, but in small amounts. In a previous report, the attractiveness of saturated primary alcohols with 7 to 9 carbons was evaluated for Lutzomyia longipalpis, the vector of American visceral leishmaniasis, with positive results. Methods: In the present study, a wide range of primary alcohols, 3 to 10 carbons, were tested to investigate their attractiveness to another sand fly species, Nyssomyia neivai, a putative vector of American cutaneous leishmaniasis. The mixture of compounds that induced the best sand fly response was also evaluated. Results: Of the eight compounds evaluated, hexanol and octanol elicited the best attractive responses for sand fly females. Conclusion: Phytochemicals may be an interesting source of search for new sand fly attractants. Keywords: Sand flies, Wind tunnel, Attractiveness, Alcohols, Plant volatiles, Olfactometry, Nyssomyia neivai, Octanol, Hexanol Background Generally, the concept of kairomones for hematophagous insects is related to host volatiles, and the development of new lures is focused on those compounds. However, con- sidering that hematophagous insects also feed on plant tissues, phytochemical attractants are very interesting tar- gets, but less studied compared with kairomones from vertebrate animals [1]. There have been behavioural reports on the attractive- ness of floral odours or single floral compounds to mos- quitoes [2-4]. For sand flies, studies with plants [5-7] fruits [8] and honey [9] have shown their effective roles as attractants, but there has been no isolation of compounds from these different sources. * Correspondence: marap@fcfar.unesp.br 1Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Júlio de Mesquita Filho, UNESP, 14801-902 Araraquara, SP, Brazil Full list of author information is available at the end of the article © 2015 Machado et al.; licensee BioMed Centr Commons Attribution License (http://creativec reproduction in any medium, provided the or Dedication waiver (http://creativecommons.or unless otherwise stated. In a previous study conducted in a wind tunnel, the attractiveness of the host kairomone 1-octen-3-ol was compared with three saturated primary alcohols (1- heptanol, 1-octanol and 1-nonanol) for the sand fly Lutzomyia longipalpis [10]. Those saturated primary alcohols are present in small quantities in some plant volatiles. The results demonstrated a relative attractive response of female L. longipalpis to 1-heptanol and 1- nonanol. Such results indicate that other saturated pri- mary alcohols may also be attractive to sand fly species. The present study investigated the attractiveness of a broad range of saturated primary alcohols (3 to 10 car- bons) for Nyssomyia neivai, a putative vector of cutaneous leishmaniasis in South America [11]. The compounds that evoked the best responses were combined and evaluated for their attractiveness. al. This is an Open Access article distributed under the terms of the Creative ommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and iginal work is properly credited. The Creative Commons Public Domain g/publicdomain/zero/1.0/) applies to the data made available in this article, mailto:marap@fcfar.unesp.br http://creativecommons.org/licenses/by/4.0 http://creativecommons.org/publicdomain/zero/1.0/ Machado et al. Parasites & Vectors (2015) 8:147 Page 2 of 4 Methods Sand flies and laboratory maintenance Insects were collected, using manual aspiration on the wall of one house located at the edge of the Mogi-Guaçu River, northeast center of São Paulo State, Brazil (21 ° 35′ 13’S 48 ° 04′15’W). In the laboratory, the insects were maintained in netting cages with a 30% solution of sucrose fed ad libitum under controlled conditions (26 ± 1°C, 80– 90%, 12:12 (L:D) photoperiod). Experiments in the wind tunnel The experiments were performed in a wind tunnel (previously described) [12] within three to four days after the field collection of insects. Tests were performed from 9:00 to 19:00 and, at the end of the assay, the insects were identified according to the classification proposed by Galati [13]. Groups of three females were placed in a releasing chamber, for a total of 30 different insects for each con- centration of the evaluated compounds. The releasing chambers were placed in the wind tunnel 50 cm from the stimulus. Tests lasted for 2 min, and two behaviours were evaluated: activation and attraction. Activation was defined as the number of sand flies that left the releasing chamber, and attraction was defined as the number of sand flies that reached the stimulus. The compounds evaluated were: 1-propanol (99.5%), 1-butanol (99.4%), 1-pentanol (>99.5%), 1-hexanol (98%), 1-heptanol (98%), 1-octanol (>99%), 1- nonanol (98%) and 1-decanol (>99%) (hereafter propanol, butanol, pen- tanol, hexanol, heptanol, octanol, nonanol and decanol) (Sigma-Aldrich). The compound 1-octen3-ol (hereafter octenol) has been already shown to be attractive to N. neivai [12], so it was used as a positive control. All com- pounds were diluted in hexane and tested at concentra- tions of 10%, 50% and 100% (neat). When necessary, a lower concentration (5%) was also used. Each compound was delivered (200 μL) on a filter paper (4 × 4 cm) in the entrance of the tunnel. The control was hexane presented the same way prior to testing the other compounds. After these tests, the three compounds with the best responses (heptanol, octanol, and nonanol) were com- bined (1:1:1) and subsequently evaluated in the wind tunnel. The interval between a compound and another was at least two hours, when the tunnel was cleaned with hexane and allowed to dry. Statistical analysis Chi square tests were used to evaluate the different pro- portions of sand fly females activated and attracted by each compound. Initially, the tests were conducted for all concentrations simultaneously. If a significant differ- ence was verified, each of the two groups was compared separately. The statistical analyses were performed using BioEstat (available in http://www.mamiraua.org.br/pt-br/ downloads/programas/bioestat-versao-53/). Results Considering the attraction behaviour, sand flies responded in a clear dose-dependent manner to pentanol, hexanol, octanol and octenol. Propanol and butanol were not significantly different from the control for activation (propanol X2 = 6.9, df = 3, P = 0.07; butanol X2 = 6.7 df = 3, P = 0.1) or attraction (propanol X2 = 1.1, df = 3, P = 0.8; butanol X2 = 7.6, df = 3, P = 0.1). The other evaluated com- pounds presented some degree of activation (pentanol, X2 = 32.9, df = 3, P <0.0001; hexanol X2 = 68.7, df = 3, P <0.0001; heptanol X2 = 47.7, df = 3, P <0.0001; octa- nol X2 = 60.6 df = 3, P <0 0.0001; nonanol X2 = 46.3, df = 3, P < 0.0001; decanol X2 = 21.7, df = 3, P <0.0001) or attraction (pentanol, X2 = 41.6, df = 3, P < 0.0001; hexanol X2 = 50.6, df = 3, P <0.0001; heptanol X2 = 13.8, df = 3, P = 0.008; octanol X2 = 37.9 df = 3, P < 0.0001; nonanol X2 = 22.7, df = 3, P < 0.0001; decanol X2 = 11.3, df = 3, P = 0.01) for females N. neivai sand flies (Table 1). With octenol as a positive control and 70% used as a cut off for responses, pentanol, hexanol, heptanol, octa- nol, nonanol and, to a lesser extent, decanol, elicited activation behaviours. However, according to the same criteria, hexanol and octanol were the best attractants for sand fly females. Octanol was even better than octe- nol at lower concentrations (50% and 10%). The concentration of 5% was added to trials with hep- tanol, octanol and nonanol because they elicited high responses of activation at 10%. Those compounds were mixed (1:1:1) and tested again. The results showed that 93% of females were activated and 63% were attracted by the mixture. There was an improvement in activation that was not reflected for attraction. Discussion Our study compared the attractiveness of saturated pri- mary alcohols to sand flies with 1-octen-3-ol, an unsatur- ated secondary alcohol, used as lure for hematophagous insects. The results emphasised that, similar to a previous study with the sand fly species L. longipalpis [10], such compounds also act as activators and/or attractants for N. neivai. The results are also in agreement with our prior survey [12] and showed that flies taken directly from the field can be used in laboratory olfactometer experiments, in spite of different physiological conditions, because they showed consistently higher activation and attraction re- sponses to the evaluated compounds than to air control. Octenol is a known host kairomone found in cattle [14], human breath [15] and human skin emanation [16]. For sand flies, octenol has been shown to elicit a response in L. longipalpis in electrophysiological http://www.mamiraua.org.br/pt-br/downloads/programas/bioestat-versao-53/ http://www.mamiraua.org.br/pt-br/downloads/programas/bioestat-versao-53/ Table 1 Percentages of activated and attracted N. neivai females to individual alcohols and mixture of three alcohols (n = 30 for each compound) Compound Activated (%) Attracted (%) Control 13 a 7 a Propanol 10% 7 a 7 a Propanol 50% 7 a 3 a Propanol 100% 27 a 10 a Control 13 a 7 a Butanol 10% 27 a 10 a Butanol 50% 30 a 13 a Butanol 100% 43 a 30 a Control 13 a 7 a Pentanol 10% 40 ab 10 a Pentanol 50% 70 bc 63 b Pentanol 100% 80 c 67 b Control 13 a 7 a Hexanol 10% 30 a 17 a Hexanol 50% 90 b 57 b Hexanol 100% 100 b 87 c Control 13 a 7 a Heptanol 5% 53 b 47 b Heptanol 10% 83 b 43 b Heptanol 50% 87 bc 40 b Heptanol 100% 77 b 33 a Control 13 a 7 a Octanol 5% 77 b 57 b Octanol 10% 80 b 70 b Octanol 50% 83 b 70 b Octanol 100% 97 b 73 b Control 13 a 7 a Nonanol 5% 70 b 53 b Nonanol 10% 87 b 43 b Nonanol 50% 83 b 63 b Nonanol 100% 70 b 47 b Control 13 a 7 a Decanol 10% 40 a 20 a Decanol 50% 70 b 37 b Decanol 100% 57 b 40 b Control 13 a 7 a Octenol 10% 13 a 10 a Octenol 50% 73 b 47 b Octenol 100% 80 b 80 bc Mixture* 97 63 For each compound, different letters within a column indicate significant differences (P < 0.05). Bold signs indicates values ≥ 70%; a cut off for responses based on octenol. *Mixture = heptanol, octanol and nonanol (1:1:1). Machado et al. Parasites & Vectors (2015) 8:147 Page 3 of 4 recordings [17] and attractive behaviours in a wind tunnel [10]. For Nyssomyia intermedia and N. neivai, octenol also elicited attractive responses in field studies [18,19]. In addition to its occurrence in mammals, octenol is also identified in many studies with volatile organic compounds (VOC) in different species of plants such as watermelon [20], flowers of lucerne [21] and mango [22]. The alcohols used in the present study are not host kairomones, but they are present in plant volatiles. For example, octanol and hexanol are present in watermelon [20] and strawberry [23]; and butanol, pentanol, hexanol, and octanol are present in guava [24]. Considering that sand flies require sugar meals from plants to survive, those compounds could be considered to be phytochem- ical attractants. Previous reports have demonstrated the attractiveness of plants to sand flies [7,8,25]. A laboratory study in a wind tunnel evaluated unifloral honey odours as attractants for different populations of three sand flies species, and the responses were species and population specific [9]. Despite the evidence of plant attractiveness to sand flies, there is a gap in the identification of phytochemical attractants isolated from the plants. The main advantage of phytochemical attractants for hematophagous insects is that they lure males and females of all ages and females in all gonotrophic states [1]. Our results show that, in addition to host kairomones, other compounds, such as phytochemical, should also be investigated as attractants for sand flies. Conclusions The compounds hexanol and octanol elicited the best attractive responses for N. neivai females in bioassays. The next step is to evaluate if those compounds will be attractive for other sand fly species in field. Competing interests The authors declare that they have no competing interests. Authors’ contributions VM, MP and AC conceived and executed the study and drafted the manuscript. VM, TG and FS conducted field captures of sandflies and bioassay experiments. DO contributed to field captures and the manuscript. All authors read and approved the final manuscript. Acknowledgements MP received a grant (F-4587-1) from the International Foundation for Science (IFS) to build the wind tunnel and financial support for publication from Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas at UNESP (PADC/FCFAr). Author details 1Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Júlio de Mesquita Filho, UNESP, 14801-902 Araraquara, SP, Brazil. 2Departamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos, SP, Brazil. 3Departamento de Zoologia Animal, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil. 4Universidade de Franca, 14404-611 Franca, SP, Brazil. Machado et al. Parasites & Vectors (2015) 8:147 Page 4 of 4 Received: 8 November 2014 Accepted: 17 February 2015 References 1. Foster WA. Phytochemicals as population sampling lures. J Am Mosq Control Assoc. 2008;24(1):138–46. 2. Healy TP, Jepson PC. The location of floral nectar sources by mosquitoes: the long-range responses of Anopheles arabiensis Patton (Diptera: Culicidae) to Achillea millefolium flowers and isolated floral odour. Bull Entomol Res. 1988;78(04):651–7. 3. Jhumur US, Dotterl S, Jurgens A. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Abstract Background Methods Results Conclusion Background Methods Sand flies and laboratory maintenance Experiments in the wind tunnel Statistical analysis Results Discussion Conclusions Competing interests Authors’ contributions Acknowledgements Author details References