aen_513.fm Page 75 Tuesday, January 17, 2006 10:03 AM Blackwell Publishing AsiaMelbourne, AustraliaAENAustralian Journal of Entomology1326-67562005 Australian Entomological Society? 20054517580Original ArticleOvipositional responses of Ades aegypti to chemicals from conspecific eggs K Ganesan et al.
Australian Journal of Entomology (2006) 45, 75–80
Studies of Aedes aegypti (Diptera: Culicidae) ovipositional responses to newly identified semiochemicals from conspecific eggs Kumaran Ganesan, Murlidhar J Mendki, Malladi V S Suryanarayana, Shri Prakash and Ramesh C Malhotra* Defence Research and Development Establishment, Jhansi Road, Gwalior – 474 002, Madhya Pradesh, India.
Abstract
The chemical factors influencing the selection of oviposition site by gravid females of various mosquito species have been the subject of numerous investigations. Recent studies have revealed this behaviour to be controlled by semiochemicals. Here we report studies on semiochemicals of egg origin and their effect on the ovipositional behaviour of Aedes aegypti. The compounds present in egg extracts of Ae. aegypti mosquitoes were isolated and identified by gas chromatography-mass spectrometry. They were then evaluated for their effect on ovipositional behaviour against gravid females of Ae. aegypti mosquitoes at different concentrations. Gravid female Ae. aegypti were found to be sensitive to all the identified compounds: 6-hexanolactone, methyl dodecanoate, dodecanoic acid, methyl tetradecanoate, tetradecanoic acid, methyl (Z)-9-hexadecenoate, methyl hexadecanoate (Z)-9hexadecenoic acid, hexadecanoic acid, methyl (Z)-9-octadecenoate, methyl octadecanoate (Z)-9octadecenoic acid and octadecanoic acid. Among them, dodecanoic and (Z)-9-hexadecenoic acids showed significant positive ovipositional response at different concentrations whereas all the esters showed deterrent/repellent ovipositional effect.
Key words
Aedes aegypti, attractants, deterrents, gas chromatography-mass spectrometry, oviposition, semiochemicals.
INTRODUCTION For many mosquito species, semiochemicals play an important role in mediating the selection of oviposition sites, along with visual cues (McCall & Cameron 1995). Chemical cues may originate from natural water bodies as breakdown products of bacterial origin or from the mosquito itself as oviposition pheromone (Bentley & Day 1989). Both sources of stimuli result in the aggregation of eggs in sites suitable for the survival of its progeny. Osgood (1971) reported an oviposition pheromone like substance associated with the apical droplets of eggs rafts of Culex tarsalis Coquillett. However, the chemical structure of this substance could not be established although the active fraction separated chromatographically from the ether extract of Cx. tarsalis egg rafts showed oviposition response. Laurence and Pickett (1982) demonstrated erythro-6-acetoxy-5-hexadecanolide, the first oviposition attractant pheromone, isolated from the apical droplets of the egg rafts of Culex quinquefasciatus Say. Recently we have reported n-heneicosane of larval origin responsible for ovipositional activity in Aedes aegypti (Linnaeus) (Mendki et al., 2000). No other oviposition-stimulating semiochemicals of mosquito origin have been reported in the literature. Conversely, substantial documentation is available for the role of semiochemicals of various other origins such as soakage pits, hay and grass infusions. Ikeshoji (1975) isolated
*
[email protected] ”2006 Australian Entomological Society
trimethylphenols from extracts of wood creosote and demonstrated that several of these compounds elicited oviposition behaviour from mosquitoes in the genera Aedes, Armigeres and Culex. Later Bentley et al. (1979) isolated 4-methylphenol from decaying birch infusions and demonstrated that it was attractive to Aedes triseriatus (Say). Reiter et al. (1991) reported the attraction of Ae. aegypti to hay infusions that contain 3-methylindole. In an effort to systematically identify the environmental cues responsible for eliciting oviposition in Cx. quinquefasciatus, Millar et al. (1992) identified phenols and indoles in fermented Bermuda grass infusions, but attributed most of the biological activity to 3-methylindole alone. Thus, many of the kairomones present at natural oviposition sites have been found to contain either phenols or indoles, which are frequently associated with mammalian waste products. Of the anopheline mosquitoes, only McCrae’s (1984) work on Anopheles gambiae sensu stricto provides a published reference that semiochemicals, in conjunction with visual stimuli, may mediate oviposition behaviour. Thus, there is plethora of evidence to suggest that oviposition behaviour is mediated by semiochemicals emanating from different origins that mosquitoes recognise. Chadee et al. (1990) reported that egg-laying Ae. aegypti mosquitoes avoided sites containing eggs laid by themselves or by conspecifics. Indirect evidence presented by Apostal et al. (1994) indicated that egg density may affect the numbers of eggs deposited by gravid Ae. aegypti. Allan and Kline (1998) studied the effects of eggs from a prior oviposition on further oviposition event in mosquitoes namely Ae. aegypti doi:10.1111/j.1440-6055.2006.00513.x
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and Ae. albopictus. Their results revealed that gravid Ae. aegypti mosquitoes preferentially selected substrates where eggs already have been laid. In fact, little is known on the influence of pre-existing eggs on subsequent oviposition by female mosquitoes. It is not clear whether or not chemical factors produced by the eggs mediated oviposition behaviour. There are no reported studies aimed at identifying the components of egg origin in Ae. aegypti mosquito. Our objectives were to identify the possible semiochemicals emanated from Ae. aegypti mosquito eggs and to study whether these chemicals affect the oviposition behaviour of gravid females of the same species.
source and MS quadrupole were kept at 250°C, 280°C, 230°C and 150°C, respectively. Helium was used as the carrier gas at a flow rate of 1.2 mL min−1. The mass spectrometer was operated in the electron impact ionisation mode at 70 eV. Samples were introduced by splitless injection and 1 µL of concentrated solution was injected into the GC-MS system each time. NIST MS library search program was used for the identification (matching >95%) and then confirmed them using authentic chemicals of >98% purity obtained from M/s Aldrich/Sigma by comparing their retention times and mass spectral data under same GC-MS operating conditions.
Test chemicals MATERIALS AND METHODS Test insects Aedes aegypti mosquitoes were obtained from a stock colony being maintained in the insectary at 27 ± 1°C and 75 ± 5% relative humidity. Larval stages were maintained in enamel bowels filled with dechlorinated tap water. Brewer’s yeast powder was provided as food and water was changed twice per week. Pupae were collected and kept in a cage and the adults emerged were provided with 10% sucrose. Female mosquitoes were fed on rabbit blood 4–5 days after emergence. Five days after blood feeding, the gravid female mosquitoes were used for all bioassay experiments.
Extraction of semiochemicals from Ae. aegypti eggs Approximately 10 000 Ae. aegypti eggs were obtained from the stock colony being maintained in the insectary. The eggs were rinsed with distilled water, filtered through Whatman No. 1 paper and transferred into a test tube and 10 mL of methanol (2 × 5 mL, HPLC Grade purchased from Merck, Germany) was added. The organic layer was decanted and concentrated to 500 µL by nitrogen purging.
Gas chromatography-mass spectrometry analysis of eggs extract Gas chromatography coupled with mass spectrometry (GCMS) is a powerful tool used for the separation, analysis and identification of compounds in a mixture. The methanol extract obtained from the eggs of Ae. aegypti mosquitoes was analysed by GC-MS using a capillary column with a polar stationary phase. In the present studies, a HP 6890 gas chromatograph equipped with a split/splitless injector and coupled to a HP 5973-quadrupole mass spectral detector was used throughout the analysis. The analytical column used was a 30 m × 0.32 mm ID, 0.25 µm film thickness, BP-5 stationary phase (SGE, Australia). The chromatograph was programmed from an initial temperature of 50°C, held at isothermal for 2 min and then increased at a rate of 10°C min−1 to a final temperature 280°C, kept isothermal for 5 min (total run time 30 min). The temperature of the injector, MS interface, MS ”2006 Australian Entomological Society
All the identified chemicals were procured from Aldrich/ Sigma, USA and hexane (HPLC Grade, Merch, Germany) was used as solvent to prepare the stock solutions. The stock solutions were diluted further to get the required concentrations for the bioassay.
Bioassay Laboratory bioassay on oviposition attractancy was carried out in fine wire mesh fitted cages 750 × 600 × 600 mm with a sleeve opening on one side. Glass Petri dishes (55 mm diameter) containing 18 mL of control or chemical-treated deionised water were provided as oviposition sites. In each bioassay, females were able to choose a control or treated water Petri dish. Bioassays were conducted using 100 gravid females added to the cage. For the bioassay, oviposition Petri dishes were removed every day, numbers of eggs were counted and fresh solutions in fresh Petri dishes were placed in cages. One millilitre of the hexane solution containing the desired quantity of the compound (to make 1, 10 and 100 ppm concentrations) was added to the Petri dish containing deionised water. In controls, 1 mL of hexane was added to control for the presence of this solvent. Hexane was allowed to evaporate at room temperature. The treated dishes along with controls were subjected to bioassay for 22 h. The four Petri dishes, namely control, 1, 10 and 100 ppm treatments, were placed at equidistant from each other. The Petri dishes were rotated clockwise everyday to avoid any bias for site preference. There were three replicates (in three cages) of each assay per day. Each chemical was evaluated separately. Experiments were conducted for 5 continuous days. The basis for measuring the oviposition response was the number of eggs in both control and treatment dishes. The oviposition activity was expressed as oviposition activity index (OAI) and calculated as follows (Kramer & Mulla 1979): OAI =
N T - NS N T + NS
where NT denotes the mean number of eggs in the treated water and NS denotes the mean number of eggs laid in the control water. All the index values fall within the range of +1 to −1. A positive value indicates that more eggs occur in the treated
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Ovipositional responses of Aedes aegypti to chemicals from conspecific eggs than in the control, thus signalling the treated chemical to be oviposition attractant. Conversely, more eggs laid in the control than in the treated water would result in the negative OAI value, indicating the chemical to be oviposition deterrent. As suggested by Kramer and Mulla (1979), compounds with OAI of +0.30 and above are considered as attractants, while those with −0.30 and below are considered as repellents. Data were subjected to t-test for statistical analysis.
RESULTS Semiochemicals identification The reconstructed total ion chromatogram of the methanol extract obtained from Ae. aegypti eggs was found to contain about 25 peaks of varying intensities (Fig. 1). Thirteen compounds were identified using the NIST MS library search program having >95% matching and further they are confirmed by comparing their retention times and fragmentation patterns with the authentic samples analysed under the same gas chromatographic operating conditions. The chemical con-
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stituents of the egg cuticle were found to contain higher fatty acids of carbon chain-length C12 to C18 and their methyl esters along with a lactone (6-hexanolactone). All the identified chemicals are listed in Table 1.
Oviposition behaviour of mosquitoes in response to identified chemicals In the laboratory bioassay tests, dodecanoic acid (C12) and (Z)9-hexadecenoic acid (C16, with double bond at ninth position) showed significant positive ovipositional response at concentrations of 10 and 100 ppm (OAI > +0.30, P < 0.01–0.05) (Table 2). In dodecanoic acid, the attractant activity increased as the concentration increased from 1 to 100 ppm, whereas in (Z)-9-hexadecenoic acid, the attractant activity increased as the concentration increases from 1 to 10 ppm but remained the same after a further increase to 100 ppm. Tetradecanoic acid (C14) showed significant positive oviposition response at lower concentrations of 1 and 10 ppm (OAI > +0.30, P < 0.001) whereas negative response at higher concentration of 100 ppm which was not significant; the attractant activity was found to decrease as the concentration increases. Hexadecanoic acid
Fig. 1. Reconstructed total ion chromatogram obtained from the methanol extract of the Aedes aegypti mosquito eggs. ”2006 Australian Entomological Society
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(C16) showed positive oviposition response at lower concentrations of 1 and 10 ppm, whereas negative response at higher concentration of 100 ppm and both were insignificant; the attractant activity was found to decrease as the concentration increases. Again octadecanoic acid (C18) showed concentration-dependent ovipositional activity, namely positive at 1 ppm and negative at higher concentrations and OAI were insignificant. However, (Z)-9-octadecenoic acid (C18, with double bond at ninth position) showed poor ovipositional activity, varying with concentration and were not significant at all. In contrast to the acids, the esters showed a negative ovipositional response and were also significant (OAI < −0.30, P < 0.001–0.05) in all concentrations except methyl dodecanoate (1 ppm) and methyl octadecanoate (10 and 100 ppm)
Table 1 Compounds in the methanol extract of Aedes aegypti mosquito eggs identified by gas chromatography-mass spectrometry (GC-MS) analysis No.
GC retention time (in minutes)
Compounds identified
1 2 3 4 5 6 7 8 9 10 11 12 13
09.33 14.24 14.83 16.56 17.10 18.48 18.67 19.08 19.27 20.37 20.58 20.88 21.06
6-Hexanolactone Methyl dodecanoate Dodecanoic acid Methyl tetradecanoate Tetradecanoic acid Methyl (Z)-9-hexadecenoate Methyl hexadecanoate (Z)-9-Hexadecenoic acid Hexadecanoic acid Methyl (Z)-9-octadecenoate Methyl octadecanoate (Z)-9-Octadecenoic acid Octadecanoic acid
(Table 3). Further, it is interesting to note that the response was found to be concentration-dependent. As the concentration of the esters increased from 1 to 100 ppm, the ovipositional deterrent effect also increased in all cases except the ester of saturated C18 acid. Methyl octadecanoate showed deterrent effect at the intermediate concentration of 10 ppm. The only lactone identified, 6-hexanolactone, showed positive response at all concentrations although it was not significant (OAI < +0.30) (Table 3).
DISCUSSION Chemicals of egg origin from Ae. aegypti mosquito have been identified and evaluated for oviposition activity and are reported here for the first time. The fatty acids of chain-length C16 to C18 and their methyl esters were the predominant compounds in the egg extract. Earlier, various fatty acids and their esters were subjected to several investigations for their effect on oviposition activity in mosquitoes (Clements 1999). Hwang et al. (1980) studied the ovipositional behaviour of Cx. quinquefasciatus to lower aliphatic acids such as acetic, propionic, isobutyric, butyric, isovaleric and caproic acids which were found to be oviposition deterrents. Further, the oviposition activity of homologues C5 to C13 fatty acids were evaluated in laboratory bioassay against Ae. aegypti, Cx. quinquefasciatus and Culex tarsalis (Hwang et al. 1982). In general, it was observed that all species were sensitive to these compounds and activity was greatest in the higher acids from C8 to C10, with C9 eliciting significant negative responses in Ae. aegypti at the lower concentration. Interestingly, our present studies with C12 and monounsaturated C16 fatty acids showed that they are oviposition attractants at the concentra-
Table 2 Ovipositional response of Aedes aegypti to acids of eggs origin Semiochemical used in the test solution
Concentration (in ppm)
Dodecanoic acid
Tetradecanoic acid
(Z)-9-Hexadecenoic acid
Hexadecanoic acid
(Z)-9-Octadecenoic acid
Octadecanoic acid
1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100
*P < 0.05; **P < 0.01; ***P < 0.001; NS, non-significant. Values are mean ± SE, n = 15. OAI, oviposition activity index. ”2006 Australian Entomological Society
Mean number of eggs Control 67.4 ± 11.51 114.20 ± 25.24 21.65 ± 4.65 78.27 ± 18.59 116.73 ± 15.94 116.98 ± 17.94
OAI Treated
122.53 ± 20.74* 145.13 ± 31.94* 227.73 ± 52.34** 352.73 ± 36.39*** 323.67 ± 37.16*** 81.67 ± 15.75NS 39.75 ± 11.99NS 73.97 ± 14.15** 74.23 ± 12.54** 134.60 ± 37.57NS 120.73 ± 16.95NS 31.07 ± 8.13* 72.53 ± 13.49* 135.53 ± 16.22NS 106.67 ± 22.53NS 237.33 ± 50.17* 44.33 ± 11.38** 14.20 ± 5.34***
+0.29 +0.37 +0.54 +0.51 +0.48 −0.17 +0.29 +0.55 +0.55 +0.26 +0.21 −0.43 −0.23 +0.07 −0.04 +0.34 −0.45 −0.78
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Table 3 Ovipositional responses of Aedes aegypti to esters and a lactone of eggs origin Semiochemical used in the test solution Esters Methyl dodecanoate
Methyl tetradecanoate
Methyl (Z)-9-hexadecenoate
Methyl hexadecanoate
Methyl (Z)-9-octadecenoate
Methyl octadecanoate
Lactone 6-Hexanolactone
Concentration (in ppm)
Mean number of eggs Control
1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100
144.20 ± 28.17 141.60 ± 31.00 113.93 ± 16.59 203.80 ± 33.97 82.60 ± 15.02 157.20 ± 31.68
95.27 ± 25.84
OAI
Treated 83.07 ± 14.52NS 56.13 ± 11.14** 5.73 ± 3.78*** 67.0 ± 10.49* 50.6 ± 12.81* 18.0 ± 3.51** 43.47 ± 10.94** 21.67 ± 3.64*** 8.87 ± 2.41*** 114.67 ± 18.71* 50.80 ± 11.57*** 13.00 ± 6.31*** 45.67 ± 9.42* 16.87 ± 6.02*** 4.13 ± 3.05*** 48.40 ± 8.76** 129.47 ± 26.08NS 90.20 ± 17.25NS
−0.27 −0.44 −0.92 −0.36 −0.47 −0.77 −0.45 −0.68 −0.86 −0.28 −0.60 −0.88 −0.29 −0.66 −0.90 −0.53 −0.10 −0.27
118.13 ± 39.03NS 110.73 ± 29.61NS 144.13 ± 28.99NS
+0.10 +0.08 +0.20
*P < 0.05; **P < 0.01; ***P < 0.001; NS, non-significant. Values are mean ± SE, n = 15. OAI, oviposition activity index.
tions of 10 and 100 ppm (OAI > +0.30). Moreover, C14 and C16 fatty acids also showed significant oviposition attractant activity at lower concentrations, but as the concentration increases to 100 ppm, they showed deterrent activity. These observations indicate that these acids of egg origin may be playing some role in the signalling of oviposition sites to gravid females. Unsaturated fatty acids with carbon chain lengths between 16 and 22 carbon atoms elicited negative oviposition activity indices when bioassayed against Cx. quinquefasciatus at 10−4 M concentrations (Hwang et al. 1984). In a similar manner, the oviposition deterrent activity was noted presently with (Z)-9-octadecenoic acid when bioassayed with Ae. aegypti. However (Z)-9-hexadecenoic acid differed in its response between Cx. quinquefasciatus and Ae. aegypti. Positive ovipositional responses were observed with Ae. aegypti at 1, 10 and 100 ppm concentrations with the same oviposition activity indices at 10 and 100 ppm concentrations. The major component of oviposition attractant pheromone of Cx. quinquefasciatus, erythro-6-acetoxy-5-hexadecanolide, is also a C16 compound derived from (Z)-5-hexadecenoic acid (Olagbemiro et al. 1999). Few studies have been carried out on the effect of esters of fatty acids on the ovipositional behaviour of mosquitoes (Perry & Fay 1967) and the esters of lower fatty acids (C < 6) induced negative or weak positive responses. In the current study, the methyl esters of all the fatty acids of eggs origin showed an oviposition deterrent effect. This may be helpful in controlling the density of eggs at a particular site and thus helps to prevent overcrowding in the development of the species in better con-
dition. Such oviposition deterrent chemicals were known to occur in various lepidopteran and dipteran species. Behan and Schoonhoven (1978) reported that female cabbage white butterflies (Pieris brassicae and P. rapae) add a host-marking chemical to eggs during egg-laying which deters other females of the same species from ovipositing on the same place. Similar marking chemicals deterring repeated ovipositions at one place were reported in apple maggot flies (Prokopy 1972, 1975). Further, it is evident from our oviposition activity data that deterrent effect of esters increases as the concentration increases, and this deterrent effect will be reached when the number of eggs at a site is very high. As the density of eggs increases, the concentration of fatty acids will also increase, leading to a stronger deterrent effect. Thus, the equilibrium between the concentration of these acids and the esters present in the cuticle of the eggs may be playing some role in the control of overcrowding of conspecific eggs at a particular site. Chadee (1993) and Apostal et al. (1994) reported that gravid mosquitoes disperse their eggs over several sites with approximately 11–30 eggs per oviposition container – a pattern of ‘skip oviposition’ reported by Corbet and Chadee (1993) and others. Thus, the present study is merited on several grounds: the effect of chemicals of Ae. aegypti eggs origin on the oviposition patterns and chemical treatment of all potential breeding sites of Ae. aegypti with the esters identified from its conspecific eggs leading to forced egg-retention benefits (Chadee 1997). The OAI of 6-hexanolactone is similar to the results obtained with various ketones studied earlier in different mos”2006 Australian Entomological Society
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quitoes (Ikeshoji & Mulla 1974; Knight & Corbet 1991). The ketones generally show positive ovipositional response although it is not significant. Given that these chemicals are so cheap and easy to obtain, the ovipositional deterrent properties of the esters originating from Ae. aegypti eggs can potentially be exploited for the control of this species. Further work is in progress to study the highly active chemicals as oviposition repellents in semifield and field conditions.
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Accepted for publication 11 August 2005.
