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Evaluation of an oviposition-stimulating kairomone for the yellow fever mosquito, Aedes aegypti, in Recife, Brazil

2010; Wiley; Volume: 35; Issue: 1 Linguagem: Inglês

10.1111/j.1948-7134.2010.00078.x

1948-7134

Rosângela Maria Rodrigues Barbosa, André Freire Furtado, Lêda Regis, Walter Leal Filho,

Malaria Research and Control

A synthetic mixture of an oviposition-stimulating kairomone for the yellow fever mosquito, Aedes aegypti, comprising of 83% tetradecanoic acid, 16% nonanoic acid and 1% tetradecanoic acid methyl ester (NTT, in short) was tested in a dengue endemic area in Recife, Brazil. Gravid female mosquitoes confined to a cage under semi-field conditions deposited significantly higher numbers of eggs in traps baited with NTT at doses ranging from 0.6 to 600 ng/μl than in control (water) traps. When tested in homes, egg-laying in traps baited with 60 ng NTT/μl (final concentration in trap, ≈3.33 ng/ml) and in control traps was not significantly different, but egg deposited in traps with lower dosage (6 ng NTT/μl; final concentration in trap, ≈0.33 ng/ml) was significantly higher than in control traps. In subsequent trials, the numbers of eggs laid in traps baited with 0.6 ng NTT/μl (final concentration in trap, ≈0.033 ng/ml) were not significantly different from the numbers deposited in trap loaded with 6 ng NTT/μl. Egg-laying was significantly higher in these treatments than in control traps. The yellow fever mosquito, Aedes aegypti (Diptera; Culicidae) is the primary vector of dengue throughout the tropical and subtropical world, thus accounting every year for several million cases globally (Gubler 1997). Prevention and control of dengue depend on controlling the mosquito vector, Ae. aegpti, in and around the home where most transmission occurs, with the most effective way being larval source reduction (Gubler 1998). Ovitraps were initially developed as a surveillance tool for Ae. aegypti (Fay and Perry 1965, Fay and Eliason 1966), but their use has been extended to control strategies, including lethal ovitraps (for a review, see Morrison et al. 2008). The success of these "attract-and-kill" strategies in Aedes depend heavily on lures that stimulate oviposition in target containers thus reducing "skip oviposition." Recently, a promising kairomone blend was isolated from natural sources using a bioassay-guided approach (Ponnusamy et al. 2008). A synthetic mixture of the identified semiochemicals, comprised of 83% tetradecanoic acid, 16% nonanoic acid and 1% tetradecanoic acid methyl ester (hereafter named NTT), was demonstrated in laboratory bioassays to induce egg-laying but at an extremely narrow dosage range, with the optimal dose being 10 ng/30 ml (Ponnusamy et al. 2008). These findings prompted us to evaluate this new lure under field conditions to determine its suitability and the appropriate dosage for practical applications. Here, we report that when tested under semi-field conditions in a dengue-endemic area in Recife, Brazil, gravid Ae. aegypti deposited significantly more eggs in traps baited with NTT than in control traps at all doses tested. However, when tested in residential areas the synthetic lures performed better than control traps only at a specific dosages, thus suggesting its potential application as long as lures at the right dosages are deployed. Tetradecanoic acid (=myristic acid) (95%, Aldrich Chemical. Co.), nonanoic acid and methyl myristate (=tetradecanoic acid methyl ester) (Acros Organics) were dissolved in ethanol (HPLC grade, Fisher Scientific) to prepare a stock solution, which was sealed in glass ampoule at UC Davis, shipped to Brazil, and kept at -20° C until use. Aliquots were kept at room temperature in the laboratory to monitor for possible chemical changes by gas chromatography (GC)-mass spectrometry (MS). Analyses were performed on a 6890 series GC with a 5973 Network Mass Selective Detector (Agilent Technologies, Palo Alto, CA) equipped with a HP-5MS column (25 m × 0.25 mm; 0.25 μm; Agilent Technologies) operated at 70° C for 1 min, increased to 250° C at a rate of 10° C/min, and held at the final temperature for 5 min. We based our calculations for the lures on the optimal dosage of 10 ng/30 ml in laboratory bioassays (Ponnusamy et al. 2008) and a trap volume of 1.8 l. Thus, an ovitrap loaded with an equivalent concentration of the kairomone would require 600 ng of NTT. Considering that 100 μl aliquots of lures were to be transferred by glass capillaries (Fisher Scientific) to traps filled with 1.8 l of water, this would require a 6 ng/μl solution of NTT. The stock solution was prepared at 100X this concentration, i.e., 600 ng/μl, and decadic dilutions were made with ethanol. Thus, the following dosages were tested: 1 (undiluted, 600 ng/μl, 100 μl; final concentration in trap, ≈33.33 ng/ml), 1:10 (60 ng/μl; final concentration in trap, ≈3.33 ng/ml), 1:100 (6 ng/μl; final concentration in trap, ≈0.33 ng/ml), and 1:1000 (0.6 ng/μl; final concentration in trap, ≈0.033 ng/ml). Therefore, our NTT sample of 1:100 is equivalent to 10 ng/30 ml water in the indoor tests (Ponnusamy et al. 2008). For convenience, we will refer hereafter to the dilutions or the concentrations of the lure (before they were diluted from 100 μl to 1,800 ml with water in the traps). Ovitraps were made of a black conical plastic cup (height, 15 cm; diameter at the base, 14 cm; diameter at top, 18 cm). One wooden paddle (5 × 15 cm, Eucatex, S/A Ind.) was attached with a paper clip to the inner wall of each cap to serve as oviposition substrate. Traps were filled with 1.8 l of tap water to which the test formulation was transferred with a 100 μl capillary tube. The numbers of eggs laid on paddles for the duration of the experiments (see below) were counted under a stereomicroscope. Evaluations under semi-field conditions were conducted from August to November, 2008. A field cage (2 × 1.5 m; height, 1.65 m) was made with a wood frame, covered with nylon mosquito net with an entrance door (height 1.4 m; width, 60 cm). The cage was placed in the surrounding of the Centro de Pesquisas Aggeu Magalhães, Recife, Brazil, and five traps (one close to each corner and one in the center) were deployed. Forty gravid females were released in the cage and left for five days. Then, all mosquitoes were removed with a back aspirator, the traps were collected, inspected, and the numbers of eggs laid were recorded. Treatment and control traps were randomly placed for the first trial, and rotated for subsequent experiments. Field evaluations were performed in the metropolitan area of Recife, PE, Brazil (Rua Jacome de Araújo, Mustardinha) from January 2009 to February 2010. Due to space limitations in homes and to avoid compromising intertrap distances, we deployed only three traps per home, with two treatments (different doses of the same kairomone) and one control per residence. Traps were replaced every seven days and the number of eggs counted and recorded. Preliminary tests were also conducted with the oviposition-stimulating kairomone plus nonanal, which has been demonstrated to be an attractant for Culex quinquefasciatus (Leal et al. 2008, Syed and Leal 2009). In these experiments, three traps were deployed per home, one as control (water) trap, one loaded with NTT (6 ng/μl) and a third trap with NTT (6 ng/μl) plus nonanal. A pellet containing 0.1 mg of nonanal as a slow-release device (Syed and Leal 2009) was placed on the top of a small piece of polystyrene sheet which was allowed to float on the trap water surface. Data representing the number of egg on paddles were transformed to log (x+1). All statistical analyses were based on transformed data, but untransformed means are presented in figures. Data were analyzed by ANOVA and compared by Tukey's honestly significant differences (HSD) at 5% probability using KaleidaGraph (Synergy Software, Reading, PA). In an attempt to identify the best doses of a previously identified oviposition-stimulating kairomone for gravid Ae. aegypti for in-depth field evaluation, we tested under semi-field conditions (N=10) four doses of NTT, including a dose (1:100 dilution; 6 ng NTT/μl; final concentration in trap, ≈0.33 ng/ml) equivalent to the optimal dose in indoor two-choice bioassays (10 ng/30 ml) (Ponnusamy et al. 2008). The numbers of eggs deposited in traps baited with NTT were significantly higher (≈3x) than the number of eggs deposited in control traps (Figure 1), but there was no significant difference among treatments. Therefore, we could not eliminate some treatments on the basis of these preliminary experiments. Considering the space limitation of the test homes, we limited the number of traps per residence to three so as to allow an intertrap distance of at least 5 m. Ae. aegypti eggs deposited in traps baited with a kairomone (NTT) and water (control) traps by gravid females confined in cages under semi-field conditions. Four doses of the kairomone were tested, namely, 1 (600), 1:10 (60), 1:100 (6), and 1:1000 (0.6 ng/μl). Numbers of eggs deposited in traps baited with NTT were significantly higher than the number of eggs deposited in control traps, regardless of the dose. First, we compared the dosage (1:100 dilution; 6 ng NTT/μl), which is equivalent to the optimal dosage in indoor two-choice bioassays (Ponnusamy et al. 2008), to a higher dosage (1:10). In these experiments (N=24; three homes, eight trials), the number of eggs deposited in the 1:100 traps were significantly higher than the number of eggs laid in control traps (Figure 2A). As opposed to what has been observed in indoor bioassays (Ponnusamy et al. 2008), egg-laying in traps baited with a 10x more concentrated lure (1:10) and in control traps was not significantly different (Figure 2A). Therefore, we did not test the 100x higher dose of the synthetic kairomone. Next, we compared the performance of traps baited with 6 ng NTT/μl (1:100 dilution) with those loaded with a 10x lower dose of the kairomone. In these experiments (N=27; three homes, nine trials), the numbers of eggs deposited in traps baited with oviposition-stimulating kairomone were significantly higher than those laid in control traps (Figure 2B). Although there was no significant difference in the numbers of egg deposited in the 1:100 dilution (6 ng NTT/μl) and 10x lower dose (1:1000), the number of eggs laid in the former (357±56) was ≈2.6x higher than in control traps (139±25), whereas ≈2x more eggs were deposited in 1:1000 traps (286±49) than in control traps (Figure 2B). Interestingly, the optimal dose observed under field conditions is equivalent to that obtained by indoor two-choice studies (Ponnusamy et al. 2008). Likewise, there was no significant difference in egg-laying in traps loaded with 6 ng NTT/μl (1:100) and those with 0.6 ng NTT/μl (1:1000). However, a higher dose (60 ng NTT/μl; 1:10) did not differ from the control, as opposed to what has been observed in indoor tests (Ponnusamy et al. 2008). We monitored the quality of the samples by GC-MS. Even after 18 months at room temperature, samples of the two acids did not undergo esterification, whereas no transesterification of the methyl ester was observed. Egg-laying by Ae. aegypti in traps deployed at homes in a dengue endemic area in Recife, Brazil. (A) Significantly more eggs were laid in trap loaded with 6 ng/μl (1:100) than in traps with 10x more concentrated lure (1:10) and in control traps. (B) Egg-laying was not significantly different in traps baited with 6 ng/μl (1:100; final concentration in trap, ≈0.33 ng/ml) and a 10x dilution (1:1000, 0.6 ng/μl; final concentration in trap, ≈0.033 ng/ml), but significantly higher than in control (water) traps. We reasoned that attractants added to this oviposition-stimulating kairomone would enhance trap performance. Considering that nonanal is an attractant for both host-seeking (Syed and Leal 2009) as well as gravid female Culex mosquitoes (Leal et al. 2008), we tested next whether nonanal would enhance the performance of NTT-baited traps. Preliminary experiments (N=12; three homes, four trials) showed no significant difference in the number of eggs laid in traps baited only with NTT and those baited with NTT and nonanal at the dose tested (0.1 mg per pellet). On the basis of our preliminary experiments we cannot address the question whether nonanal is a female attractant as the traps were not designed to capture adult mosquitoes. However, nonanal did not enhance egg-laying. The discovery of attractants that enhance egg-laying in gravid female Aedes mosquitoes when used in combination with the oviposition-stimulating kairomone NTT remains an exciting area for future research. In summary, field evaluations of a previously identified kairomone (NTT) suggest that traps loaded with 6 ng NTT/μl (final concentration in trap, ≈0.33 ng/ml) enhance the performance of the Aedes ovitraps. These traps may be deployed in mosquito-abatement programs for monitoring and/or surveillance. In addition, this kairomone may be used as a bait in lethal ovitraps in combination with insecticides (Perich et al. 2003, Ritchie et al. 2008) or biological agents toxic to larvae (Barbosa et al. 2010). The authors thank Zainulabeuddin Syed and Julien Pelletier (UC Davis) for their critique of an earlier version of the manuscript. This work was supported in part by a cooperative agreement with Bedoukian Research Inc. (Danbury, CT) and gifts from Fuji Flavor Co. (Tokyo, Japan).