Introduction

The Asian tiger mosquito, Aedes albopictus (Skuse) (Culicidae: Diptera) is a day-biting mosquito, is highly anthropophilic, and resides in urban and suburban areas covered with vegetation. These mosquitoes closely interact with human beings for blood feeding. Aedes albopictus has emerged as a potential vector for both dengue and chikungunya viruses and has posed a serious threat to the human community in India and South-East Asian countries. These mosquitoes cause biting annoyance and discomfort to human beings throughout the world, and the problems become much more severe as they also act as vectors of pathogens that cause West Nile virus, yellow fever virus, St. Louis encephalitis, dengue fever, and chikungunya fever (Hochedez et al. 2006). In the recent years, the incidence of dengue and chikungunya has increased dramatically in tropical countries.

Mosquitoes can discriminate different animals due to their ability to recognize host-specific odors for blood feeding (Qiu et al. 2004; Schreck et al. 1981). An enormous attention has been paid to mosquito attractants and repellents as alternatives to pesticides by the researchers around the world in managing mosquitoes, in which many synthetic and natural volatile organic compounds possess the ability to either attract or repel adult mosquitoes (Fradin and Day 2002). The effectiveness of various attractants and other volatile organic compounds that may serve as spatial repellents has been tested extensively against Aedes aegypti (L.) mosquitoes (Bernier et al. 2003; Geier et al. 1996; Kline et al. 2003) to protect humans from mosquito biting.

It has been reported that human sweat and human skin residues are highly attractive to A. aegypti, Anopheles gambiae Giles, and Culex quinquefasciatus Say mosquitoes (Acree et al. 1968; Bosch et al. 2000; Braks and Takken 1999; Logan et al. 2008; Puri et al. 2006). A number of host skin emanation chemicals like 1-octen-3-ol, acetone, short-chain carboxylic acids, ammonia, and l-lactic acid have been found to be attractive to A. aegypti (Bosch et al. 2000; Geier and Boeckh 1999; Schreck et al. 1981). Several studies have demonstrated that ammonia, lactic acid and many carboxylic acids (Bernier et al. 2000; Bosch et al. 2000; Cork and Park 1996; Eiras and Jepson 1991; Smallegange et al. 2005, 2009), acetone, and dimethyl sulfide (Bernier et al. 2007) act as a potential attractant for Aedes and Anopheles mosquitoes.

Attractant chemicals from host skin emanations appear to provide the most immediate promise for use in traps. Nowadays, researchers are more focused on eco-friendly approaches using semiochemicals (pheromones or parapheromones) of natural and synthetic origin with multiple strategies to control hematophagous insects (Seenivasagan and Vijayaraghavan, 2010; Seenivasagan et al. 2013; 2012; 2010; 2009a; 2009b) exploiting oviposition and flight orientation behaviors. However, effective use of attractants for mosquito control requires an understanding of the mechanisms that attract mosquitoes to humans and animals. Scientific works on the use of human skin emanations to attract A. albopictus (Hao et al. 2012; Wang et al. 2006) mosquitoes are scarce. In this research paper, we report hereunder, the flight orientation response of A. albopictus females to a series of carboxylic acids (C1–C20) at various concentrations for the identification of odoriferous acids as potential attractants, repellents, or attraction inhibitors, which could be used as repellents as well as in odor-baited traps for the surveillance and control of mosquitoes.

Methods and materials

Insects

The test mosquito, A. albopictus, used in the present study was taken from the laboratory colony maintained in our insectary at 27 ± 2 °C, 70 ± 5 % RH and L10:D14 regime. Test females were kept in wooden cages (750 × 600 × 600 mm) with a sleeve opening on one side (Sharma et al. 2008), with sucrose (10 %) solution being offered ad libitum to the females. However, they did not have the access to blood meal until the beginning of experiments.

Chemicals

All the carboxylic acids used in this study viz, formic acid (C1) to arachidic acid (C20) were procured from Sigma-Aldrich/Fluka (Table 1). A stock solution (10 %) of each acid was prepared in HPLC-grade hexane (Merck) by adding the required amount (volume/weight) to the solvent. According to the purity of the chemicals, volume/weight was adjusted to prepare the stock solutions. For each chemical, five concentrations (0.0001, 0.001, 0.01, 0.1, and 1 %) were prepared by serial dilution and stored in deep freezer.

Table 1 Carboxylic acids used in the flight orientation experiments against Aedes albopictus females
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Y-tube olfactometer

Flight orientation of A. albopictus females was studied using Y-tube olfactometer as previously described by Seenivasagan et al. (2009a), with a modification that the mosquitoes used in these experiments were 5–7-day-old nonblood-fed and hungry host-seeking females. In each replicate, a set of 20 hungry female mosquitoes, which readily oriented to human hand, were aspirated out from the cage and used in the experiments. Seven replicates were performed for each concentration of the carboxylic acids. Before stimulation, the mosquitoes were given 5-min time to acclimatize in the release chamber. Between the replicates, a constant stream of fresh air was purged into the olfactometer. Orientation experiments in dual-choice conditions were performed, in which precisely 200 μl of the test chemicals at various concentrations was applied onto a piece of filter paper and then loaded onto an odor cartridge after evaporation of the solvent. The air flow was maintained at 50 l/min during the experiment. Between replicates the positions of test stimuli were alternated. The bioassay was conducted for 3 min until all the mosquitoes fled from the releasing chamber and entered into the upwind end of the olfactometer. The olfactometer was cleaned thoroughly after testing each of the chemicals. The number of mosquitoes that entered into treatment and control chambers in a replicate was considered for analysis.

Statistical analysis

Data of flight orientation response in a Y-tube olfactometer were converted to orientation index, by the formula Orientation Index = (Nt − Nc)/(Nt + Nc), where Nt is the number of mosquitoes caught in the treatment chamber and Nc is the number of mosquitoes caught in the control chamber, and subjected to two-way analysis of variance (Table 2) using SigmaStat v2.03 (SPSS Inc, Chicago, IL).The differences between the treatment means were separated by least significant difference (LSD), and p value <0.05 was considered statistically significant among the test chemicals.

Table 2 Results of the two-way analysis of variance on the orientation indices exhibited by Aedes albopictus females to carboxylic acid odors in the Y-tube olfactometer
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Results

Orientation index values of different carboxylic acids apparently demonstrated the flight choice of A. albopictus females to the test odor plumes over control stimulus in terms of both attractant and repellent effects. Carboxylic acids with carbon atom atom C1, C2, C3, C5, C6, C8, C9, C10, C12, C14, C16, C18, and C20 were attractive to A. albopictus females. The odor plumes of C4, C7, C11, C15, C17, and C19 acids repelled the host-seeking females. Dose-dependent increase in orientation response was observed for formic, pentanoic, nonanoic, decanoic, dodecanoic, and stearic acids. At the highest dose (10−2 g), A. albopictus females exhibited a reversal in the orientation from attractance to repellence for pentanoic, nonanoic, decanoic, tridecanoic, heptadecanoic, and eicosanoic acids. Similarly, hexanoic acid which did not elicit any significant attractance at lower doses (10−6g to 10−4g); attracted 20 % and 30 % of A. albopictus females respectively at 10−3g and 10−2g odor plume.

Females of A. albopictus displayed a characteristic orientation behavior demonstrating the behavioral threshold by dose-dependent reversal of their orientation to tridecanoic acid (C13) in which the odor plume (10−6 and 10−5 g) at lower doses did not elicit any significant response, while 10−4 g plume attracted the females. In contrast, 10−3 and 10−2 g plumes repelled the females. Similarly, heptadecanoic acid (C17) at 10−6 g attracted the females while all other doses turned repulsive to A. albopictus females (Fig. 1). Eicosanoic acid showed attractance at 10−5 to 10−3 g plumes, however, repelled the mosquitoes at the highest dose. Repellent effect of few carboxylic acids viz butanoic, heptanoic, undecanoic, and pentadecanoic acids increased in dose-dependent manner as the negative orientation index increased with increasing doses. Undecanoic and nonadecanoic acids at higher doses exerted a consistent repellency to test mosquitoes, as evidenced by a slight increase in mosquito orientation to control chamber.

Fig. 1
figure 1

Flight orientation response of Aedes albopictus females to various concentrations of carboxylic acids (C1–C20, Table 1). Carboxylic acid legends with different alphabet superscripts are significantly different (two-way ANOVA, P < 0.001, Table 2)

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Discussion

Laboratory studies aimed at elucidating the volatiles from human skin emanations that mosquitoes utilize for host location have yielded several active mixtures and individual substances, having carboxylic acids as an important component of human sweat (Cork and Park 1996). In subsequent studies, volatile organic compounds (VOCs) emerging from sweat, breath, and other emanations of hosts constituting a complex mixture of volatiles including short- and long-chain hydrocarbons, alcohols, carboxylic acids, ketones, and aldehydes have been identified (Bernier et al. 2000; Braks et al. 2001; Curran et al. 2005; Gallagher et al. 2008; Penn et al. 2007). In earlier studies, mostly C3–C18 acids have been evaluated, and they were found to be attractive to A. aegypti (Bosch et al. 2000), A. gambiae s.s. (Smallegange et al. 2005), and C. quinquefasciatus (Puri et al. 2006).

Response of A. albopictus females to a series of carboxylic acids (C1–C20) has not been previously reported. In the Y-tube olfactometers, A. albopictus females were exposed to concentrated kairomone plumes in confined spaces, less than 2 m from the odor source, facilitating odor-mediated upwind flight that confirms the attractive, repulsive, or inhibitory nature of test odor plumes. In this study, carboxylic acids viz C1–C3, C5, C6, C8–C10, C12, C14, C16, C18, and C20 were attractive, while C4, C7, C11, C15, and C19 repelled A. albopictus females. Puri (2003) has found that C3, C6–C13, and C17 plumes were attractive to A. aegypti at 500 μl of 10 ppm of respective acids loaded in an olfactometer, while C14–C16 and C18 repelled the females at the same dose. In case of C. quinquefasciatus, C3, C6–C11, C13–C14 elicited increased orientation, while C15–C18 reduced flight orientation response at 10 ppm (Puri 2003). In contrast, Anopheles stephensi Liston females were repelled by C10, C11, C16, and C17 plumes, while other acids showed attractance (Puri 2003). Till date, nonadecanoic and eicosanoic acids have not been reported as components of human sweat. Yet, eicosanoic acid presented a clear case of insect behavioral threshold, wherein at 10−6 g, no attraction was observed; at 10−5 to 10−3 g, attraction was observed; and at 10−2 g, repulsion of mosquitoes was observed, indicating that optimum concentration of a volatile elicits significant behavioral response. In contrast, C19 showed varying levels of repellency to A. albopictus at all concentrations tested.

Bosch et al. (2000) have reported that the addition of C1–C3, C5–C8, and C18 to lactic acid attracted the females of A. aegypti, while the addition of C9, C11, and C14 reduced their attraction. In contrast, C3, C4, 3mC4 (3-methyl-butanoic), C5, C7, C8, and C14 acids when added to ammonia + lactic acid blend resulted in an increase in the number of A. gambiae females flying into the olfactometer at one or more of the tested concentrations (Smallegange et al. 2009). In our experiments, butanoic, heptanoic, and pentadecanoic acids repelled A. albopictus females at all tested concentrations. Undecanoic acid (C11) repelled 63 % of the female Aedes albopictus at 10−4 g, and further increase in concentration resulted in reduced orientation, possibly due to the saturation of olfactory receptors present in the antennal sensilla (Seenivasagan et al. 2009b) of A. albopictus mosquitoes. Ali et al. (2012) have reported that octanoic, decanoic, undecanoic, dodecanoic, and tridecanoic acids repelled A. aegypti mosquitoes at 25 nmol/cm2 concentration, displaying >0.80 biting deterrence index (BDI). Tetradecanoic acid was attractive over a range of concentrations to A. aegypti and A. stephensi (Puri 2003), A. gambiae (Smallegange et al. 2009), and C. quinquefasciatus (Puri et al. 2006). Although C14 was attractive to A. albopictus females in our experiments, a slight decline in the orientation was observed at 10−3 and 10−2 g plumes; however, a significant repellence at 10−5g plume was observed against A. aegypti females (Puri 2003).

Our results suggest that only a few of these carboxylic acids, depending on their chain length and concentration, contributed to the orientation of A. albopictus to human skin emanations. This study also demonstrated that aliphatic acids can act in both ways (attractant/repellent) depending on their concentration and molecular structure. Further pieces of evidence have emerged based on the dose-dependent reversal in the orientation behavior of female mosquitoes to C5, C9, and C10 at 10−2 g plume, tridecanoic acid (C13) at 10−3 and 10−2 g, and heptadecanoic acid (C17) at 10−5 to 10−2 g wherein the lower doses of these acids were slightly attractive to the test insects. Different mosquito species may not possess a similar host odor preference, and there cannot be a single lure for all mosquitoes, which can be exploited for use in traps. Our results combined with the previous findings of various research groups suggest that further research is required to develop an optimum lure for diurnally active A. albopictus mosquitoes and its evaluation under laboratory, semi field, and field conditions for successful integrated vector management programs.