Laboratory And Field Responses Of The

P1: KEE Journal of Chemical Ecology [joec]

pp1178-joec-485193

April 17, 2004

6:13

Style file version June 28th, 2002

C 2004) Journal of Chemical Ecology, Vol. 30, No. 5, May 2004 (°

LABORATORY AND FIELD RESPONSES OF THE MOSQUITO, Culex quinquefasciatus, TO PLANT-DERIVED Culex spp. OVIPOSITION PHEROMONE AND THE OVIPOSITION CUE SKATOLE

TIMOTHY O. OLAGBEMIRO,1 MICHAEL A. BIRKETT,2 A. JENNIFER MORDUE (LUNTZ),3 and JOHN A. PICKETT2,∗ 1 Department

of Chemistry, Abubakar Tafewa Balewa University PMB 248 Bauchi, Nigeria

2 Biological

Chemistry Division, Rothamsted Research, Harpenden Herts AL5 2JQ, United Kingdom 3 Department of Zoology, University of Aberdeen, Tillydrone Avenue Aberdeen AB24 2TZ, United Kingdom (Received March 20, 2003; accepted January 8, 2004)

Abstract—Laboratory and field studies were conducted on the oviposition behavior of the pathogen-vectoring mosquito, Culex quinquefasciatus, in response to the oviposition pheromone 6-acetoxy-5-hexadecanolide, produced from a renewable plant resource, Kochia scoparia (Chenopodiaceae) (plant-derived pheromone, PDP), and via an established synthetic route (synthetic oviposition pheromone, SOP). Responses to the oviposition cue skatole (3-methylindole), presented individually and in combination with the plant-derived and synthetic oviposition pheromone, were also studied. Both laboratory and field assays showed that PDP and SOP were equally attractive. Synergistic effects were observed with one combination of PDP and skatole combinations in laboratory assays. Synergy was also observed under field conditions. SOP and skatole combinations showed additive effects in laboratory assays, but were not tested in field bioassays. Although synergism has been previously demonstrated with combinations of SOP and polluted waters, the work presented here is the first example of synergy between a specific oviposition attractant and the oviposition pheromone. Furthermore, the efficacy of mosquito pheromone produced from a cheap, renewable botanical source has been demonstrated. Key Words—Culex quinquefasciatus, oviposition, pheromone, 6-acetoxy-5hexadecanolide, Kochia scoparia, renewable resource, skatole, synergism.

∗

To whom correspondence should be addressed. E-mail: [email protected]

965 C 2004 Plenum Publishing Corporation 0098-0331/04/0500-0965/0 °

P1: KEE Journal of Chemical Ecology [joec]

966

pp1178-joec-485193

April 17, 2004

6:13

Style file version June 28th, 2002

OLAGBEMIRO, BIRKETT, MORDUE (LUNTZ), AND PICKETT INTRODUCTION

Mosquitoes (Diptera, Culicidae) represent a significant threat to human health because of their ability to vector pathogens that cause diseases that afflict millions of people worldwide (WHO, 1992; Pinheiro, 1997; WHO/CTD, 1998). In addition to those major areas where mosquito-vectored diseases are currently endemic, in particular sub-Saharan Africa, problems may arise in new areas as consequences of changes in global climate patterns and increased global travel and trade (Donaldson, 2002). Thus, strategies for improved vector surveillance, as well as direct control, have been sought for the major mosquito vectors. Such strategies include the application of semiochemicals, particularly those involved in mediating oviposition site location behavior. Culex spp. mosquitoes are responsible for the transmission of a number of pathogens, notably Wucheria bancrofti, the causative agent for urban bancroftian filariasis, and arboviruses including St. Louis encephalitis virus (Riesen et al., 1992), and more recently West Nile virus in urbanized areas in the United States and Europe (Jonsson and Reid, 2000; Turell et al., 2002). Several studies have reported the roles and identities of semiochemicals mediating oviposition behavior for Culex spp. mosquitoes. The oviposition pheromone, (5R,6S)-6-acetoxy-5hexadecanolide, was originally identified over 20 years ago (Laurence and Pickett, 1982), and field trials in several countries in afflicted areas have since demonstrated the efficacy of synthetic pheromone in the field (e.g., Mboera et al., 2000a,b). Sitederived oviposition cues have been identified, with skatole (3-methylindole) being the most active component in the laboratory (Millar et al., 1992; Mordue (Luntz) et al., 1992; Blackwell et al., 1993) and in the field (Beehler et al., 1994). Recent field studies in Tanzania have shown that a potent signal for oviposition site selection comprises a synergistic combination of the pheromone and grass infusions or soakage pit water (Mboera et al., 1999, 2000b). For the development of optimized monitoring or control strategies, the use of individual components identified from organically enriched water would be advantageous, because the use of infusions and water samples with variable and undefined levels of oviposition cues could potentially lead to erratic responses (Mordue (Luntz) et al., 1992; Blackwell et al., 1993). Thus, the oviposition pheromone and skatole have been used together effectively in the field (Mbeora et al., 2000a). The attractive effect of these components suggests that they could be used in the development of control strategies, which include the use of either environmentally benign larvicides, such as the insect growth regulator pyriproxyfen, larvae-specific pathogens, such as the fungus Lagenidium giganteum Couch (Pickett and Woodcock, 1996), or trapping systems (Mboera et al., 2000b). Though many synthetic routes for production of multigram quantities of the pheromone have been published (see Olagbemiro et al., 1999, and references therein), the costs and hazardous nature of reagents have prevented large-scale pheromone production, particularly in less developed countries. It was

P1: KEE Journal of Chemical Ecology [joec]

pp1178-joec-485193

OVIPOSITION BEHAVIOR OF MOSQUITO,

April 17, 2004

Culex quinquefasciatus

6:13

Style file version June 28th, 2002

967

demonstrated recently that the pheromone could be produced rapidly, cheaply, and efficiently from a botanical source of (Z )-5-hexadecenoic acid, found as a minor component of the fixed seed oil of the summer cypress plant, Kochia scoparia (Chenopodiaceae) (Olagbemiro et al., 1999). To confirm that pheromone production from K. scoparia seed oil could be considered as a viable option for cheap pheromone production, the objective of the work reported here was to assess the biological activity of plant-derived pheromone (PDP) in inducing oviposition by gravid female Cx. quinquefasciatus mosquitoes when compared to synthetic oviposition pheromone (SOP) prepared using an established route (Dawson et al., 1990), using a previously established laboratory oviposition bioassay (Blackwell et al., 1993). To demonstrate that plantderived pheromone material was also active in the field, studies were conducted at urbanized oviposition sites in Bauchi State, Nigeria. Field bioassays were also conducted to compare the effect of skatole as an oviposition cue for Cx. quinquefasciatus in Western Africa, with that previously reported in Tanzania (Mboera et al., 2000a), and to determine its interaction with plant-derived pheromone compared to synthetic pheromone. METHODS AND MATERIALS

Chemicals Skatole (99%, 3-methylindole) was purchased from the Aldrich Chemical Co., Gillingham, UK. Solutions of skatole in hexane were prepared for laboratory bioassays, while a stock solution (100 mg) in 96% ethanol (100 ml) was used to prepare aqueous solutions for field studies. The synthetic oviposition pheromone (SOP) material was prepared using a previously established route (Dawson et al., 1990), and comprised a 1:1:1:1 mixture of the four stereoisomers of 6-acetoxy5-hexadecanolide, i.e., 25% of the material consisted of the biologically active (5R,6S)-enantiomer. The plant-derived pheromone (PDP) material was produced from the seed oil of the summer cypress plant, Kochia scoparia (Chenopodiaeae), by a route previously reported (Olagbemiro et al., 1999). The oil contained the precursor (Z )-5-hexadecenoic acid in minor amounts (approximately 7%), and was used directly in pheromone synthesis without purification of the precursor. Intermediates were used at every stage without purification, and intermediate formation was confirmed by 1 H/13 C NMR and GC analysis. Thus, the final PDP material contained the pheromone mixed with the other components in the seed oil, and included a 1:1 ratio of the active (5R,6S)-enantiomer and the inactive (5S,6R) isomer, with the pair comprising approximately 27% (w:w) of the PDP material, i.e., approximately 13.5% of each. As SOP and PDP were minor components in their respective materials, amounts of material were used in bioassays such that 5 µg and 5 mg doses of the active (5R,6S)-enantiomer were used per laboratory

P1: KEE Journal of Chemical Ecology [joec]

968

pp1178-joec-485193

April 17, 2004

6:13

Style file version June 28th, 2002

OLAGBEMIRO, BIRKETT, MORDUE (LUNTZ), AND PICKETT

and field experiment, respectively. Solutions of PDP and SOP in hexane, with the concentration of the active (5R,6S) enantiomer at 1 mg/ml, were used for laboratory studies. For field studies, blank effervescent tablets (see Otieno et al., 1988, for formulation) were laced with pheromone material (PDP) prior to use. Tablets were spread individually on a clean piece of paper, and pheromone (5 mg active enantiomer per tablet) was added as a hexane solution (0.1 ml) to each tablet. The tablets were left to dry for a few minutes at room temperature before use. All solvents were freshly distilled prior to use. Insect Behavior Laboratory Bioassays. Cx. quinquefasciatus Say (Lagos strain) mosquitoes were maintained on a LD 12:12 hr photoperiod at 27 ± 2◦ C, 55–60% humidity. Larvae were fed daily on desiccated liver powder. Adults were fed on a sucrose solution, and females were transferred after 8 d to goose blood (Alsevers), fed through a membrane feeder (Hemotek 5W1 system, Discovery Workshops, Accrington, UK). For behavior experiments, gravid females were taken from the colony 4 d after feeding (7- to 10-d-old). Trials were carried out in muslin-covered wooden framed cages (31 × 31 × 31 cm) with Perspex fronts and muslin sleeves. Each trial involved 20 gravid female Cx. quinquefasciatus and was replicated over several nights in randomized blocks. For two-choice experiments, two glass bowls containing 100 ml distilled water were placed at diagonally opposite corners of the cages. For four-choice experiments, glass bowls were placed in each of the four corners. The cages were left overnight (for 17 hr) under the same conditions as described for colony maintenance. The number of egg rafts in the bowls was recorded the following morning and converted to percentages of the total number of rafts in both bowls for each cage. Control bowls contained distilled water (100 ml) plus solvent where appropriate. Skatole and pheromone doses used were based on those shown previously to be the most effective in laboratory tests (Mordue (Luntz) et al., 1992; Blackwell et al., 1993). Skatole was applied directly to the water, and pheromone was applied to glass coverslips floated on plastic caps to simulate release from egg rafts. Thus, test bowls contained distilled water (100 ml) plus pheromone material (5 µg, PDP or SOP) and/or skatole at one of two concentrations (10−4 or 10−5 µg/l). The following experiments were conducted: (a) two-choice experiments, PDP vs. control and SOP vs. control; (b) four-choice experiments, control vs. skatole vs. PDP vs. skatole/PDP and control vs. skatole vs. SOP vs skatole/SOP. Each trial was repeated 10–20 times depending on mosquito availability. Field Studies. All experiments were conducted in Bauchi (10◦ 170 N, 9◦ 490 W), Nigeria. The mean annual temperature is 38◦ C, and the average rainfall of 1000 mm falls mainly in a short rainy season (June–September). Twelve pit latrines were selected for the experiments, located at four sites. Each of the pit latrines consisted

P1: KEE Journal of Chemical Ecology [joec]

pp1178-joec-485193

OVIPOSITION BEHAVIOR OF MOSQUITO,

April 17, 2004

Culex quinquefasciatus

6:13

Style file version June 28th, 2002

969

of 1 m2 slabs of concrete provided with a drop hole (12 × 22 cm) in the center, suspended over a soakage pit, and mounted on a total floor area of 4 m2 . Experiments were performed during the rainy seasons of 2001 and 2002. In each of the trials, treatments comprised black plastic bowls containing 1000 ml of non-chlorinated water plus either skatole and/or an effervescent pheromone tablet, placed 1 m apart on the floor of a pit latrine building. Treatments alternated between the four sites for 4 nights and were rerandomized within each latrine between experiments. Experiments started at 18:00 hr local time and were stopped at 08:00 hr the following morning. In all experiments, collected egg rafts were taken to the laboratory, sorted by shape (Edwards, 1942; Gillett, 1972), and counted. The number of Cx. quinquefasciatus egg rafts in the bowls was converted to percentages of the total number of rafts in both bowls. Eggs from other Culex spp. mosquitoes were not counted in this study, which was directed exclusively to Cx. quinquefasciatus. The following experiments were conducted and replicated 12 times (a) response of Cx. quinquefasciatus to SOP, PDP, or a control. One of the bowls was treated with a control tablet (0.1 ml hexane only). The two remaining bowls were treated with SOP or PDP tablets, respectively; (b) oviposition response of Cx. quinquefasciatus to skatole. To determine the optimum dose of skatole at the field sites, the stock solution of skatole in ethanol was subjected to serial dilution using non-chlorinated water to make the following concentrations: 10−4 , 10−5 , 10−6 , and 10−7 µg/l; (c) oviposition responses of Cx. quinquefasciatus to control (hexane only), PDP, skatole (10−5 gµg/l), and PDP + skatole (10−5 gµg/l). Data Analysis Laboratory Bioassays. In laboratory bioassays, data were analyzed by nonparametric tests because data were not normally distributed in some cases. Factorial experiments were subjected to Kruskal–Wallis analysis, followed by a Wilcoxon paired test to compare differences between treatment means. Synergism between treatments was tested using Student’s t-test to compare the sum of effects of skatole and oviposition pheromone presented separately to the effects of skatole and pheromone presented together. Field Studies. All field data were log(x + 1) transformed, and means of treatments were compared by Student’s t-tests. Means of factorial experiments were subjected to ANOVA, and an F test significant at P < 0.05 was followed by a Least Significant Difference test to compare treatment means.

RESULTS

Laboratory Bioassays. Laboratory oviposition bioassays showed that the number of egg rafts laid by gravid Cx. quinquefasciatus in response to the mixture

P1: KEE Journal of Chemical Ecology [joec]

970

pp1178-joec-485193

April 17, 2004

6:13

Style file version June 28th, 2002

OLAGBEMIRO, BIRKETT, MORDUE (LUNTZ), AND PICKETT

FIG. 1. Oviposition behavior of gravid females of Culex quinquefasciatus in the laboratory in the presence of 5 µg plant-derived (PDP) and 5 µg synthetic oviposition pheromone (SOP) (N = 20 and 10, respectively). Vertical bars represent standard errors. Statistical analysis was carried out using Kruskall–Wallis and Wilcoxon paired tests. ∗ P < 0.05 and ∗∗∗ P < 0.001.

of (5R,6S) and (5S,6R) stereoisomers of the oviposition pheromone 6-acetoxy-5hexadecanolide (5 µg dose), prepared from the seed oil of K. scoparia (PDP) and the mixture of all four stereoisomers prepared via an established synthetic route (SOP), was significantly different from the control (Figure 1; PDP vs. control, mean ± SE 81.6 ± 3.7% vs. 18.4 ± 3.7%, P < 0.001, N = 20; SOP vs. control, 68 ± 3.8% vs. 32 ± 3.8%, P < 0.05, N = 10). In multiple choice laboratory bioassays using skatole and pheromone, in the experiment using the lower skatole concentration (10−5 µg/l), skatole and pheromone individual treatments received more egg rafts than the control (Figure 2, control 4.3 ± 2.8%, skatole 19.6 ± 7%; P < 0.05, PDP 19.6 ± 6.4%; P < 0.05; Figure 3, control 9.7 ± 4.9%, skatole 22.2 ± 6.9%; P < 0.05, SOP 27.4 ± 7.3%; P < 0.05). Using the higher skatole concentration (10−4 µg/l), a similar pattern was observed (Figure 2, control 4.1 ± 2.1%, skatole 18 ± 4.1%, P < 0.01, PDP 14.1 ± 3.5%; P < 0.05; Figure 3, control 4.2 ± 2.1%, skatole 25.6 ± 3.7%; P < 0.01, SOP 17.2 ± 4.5%; P < 0.05). When skatole and pheromone treatments were combined, a difference in response was observed between PDP and SOP. For the skatole + SOP combination, additive effects were observed at both concentrations of skatole when compared to individual treatments (Figure 3, 40.7

P1: KEE Journal of Chemical Ecology [joec]

pp1178-joec-485193

OVIPOSITION BEHAVIOR OF MOSQUITO,

April 17, 2004

Culex quinquefasciatus

6:13

Style file version June 28th, 2002

971

FIG. 2. Oviposition behavior of gravid females of Culex quinquefasciatus in the laboratory in the presence of skatole at two concentrations (10−4 and 10−5 µg/l) and 5 µg plantderived pheromone (PDP) (N = 12 and 16, respectively). Vertical bars represent standard errors. Statistical analysis was carried out using Kruskall–Wallis and Wilcoxon paired tests, comparing test with control data, ∗• P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

± 6.6%; P < 0.01 and 53 ± 6.2%; P < 0.01, respectively). For skatole + PDP combinations, an additive effect was observed for the lower skatole concentration (Figure 2, 56.5 ± 10.2% vs. 39.2% for summed individual treatments; P < 0.001), whereas for the higher concentration of skatole, a synergistic effect was observed (Figure 2, 63.8 ± 5.9% vs. 32% for summed individual treatments, P < 0.01). Field Studies. In field oviposition bioassays, no significant differences were observed in the responses of Cx. quinquefasciatus to PDP (46.7 ± 1.4%) and SOP (49.3 ± 1.4%) (Table 1; P > 0.05). Significantly fewer egg rafts were laid in control bowls containing tap water (4.8 ± 0.7%, P < 0.05). In the four-choice field experiment to determine the effect of skatole concentration on oviposition, responses differed across the range of concentrations used. Concentrations of 10−4 µg/l (31.2 ± 2.6%) and 10−5 µg/l (37.8 ± 3.1%) were equivalent (Figure 4; P > 0.05) and concentrations of 10−6 µg/l (15.2 ± 2.1%) and 10−7 µg/l (16.1 ± 2.8%) also were not significantly different (P > 0.05). However, responses to the lower two concentrations were lower than the upper two concentrations (P < 0.05). A four-choice field experiment using skatole, PDP, skatole + PDP combination, and a control was conducted using the 10−5 µg/l skatole concentration. The skatole treatment was different from the control (Table 2, 15.5 ± 2% vs. 5.4 ± 1.4%, P < 0.05) and the PDP treatment, which was no different

P1: KEE Journal of Chemical Ecology [joec]

972

pp1178-joec-485193

April 17, 2004

6:13

Style file version June 28th, 2002

OLAGBEMIRO, BIRKETT, MORDUE (LUNTZ), AND PICKETT

FIG. 3. Oviposition behaviour of gravid females of Culex quinquefasciatus in the laboratory in the presence of skatole at two concentrations (10−4 and 10−5 µg/l) and 5 µg synthetic pheromone (SOP) (N = 12 and 16, respectively). Vertical bars represent standard errors. Statistical analysis was carried out using Kruskall–Wallis and Wilcoxon paired tests, comparing test with control data, ∗• P < 0.05 and ∗∗•• P < 0.01.

from the control (8.3 ± 1.7%, P > 0.05). The skatole + PDP combination received most of the egg rafts (73.6 ± 1.8%). The substantially larger response to the skatole + PDP combination compared to the summed responses to skatole and PDP presented individually indicated synergism between the two components (P < 0.05). TABLE 1. FIELD EXPERIMENT SHOWING NUMBER OF EGG RAFTS OF Culex quinquefasciatus DEPOSITED IN WATER TREATED WITH PLANT-DERIVED OVIPOSITION PHEROMONE (PDP) AND SYNTHETIC OVIPOSITION PHEROMONE (SOP) VERSUS TAP WATER (CONTROL)a Egg rafts Treatment Control PDP SOP aN

Total no.

Mean % ± SE

75 937 988

4.8 ± 0.7 b 46.7 ± 1.4 c 49.3 ± 1.4 c

= 12. Means followed by a different letter are significantly different at P < 0.05.

P1: KEE Journal of Chemical Ecology [joec]

pp1178-joec-485193

OVIPOSITION BEHAVIOR OF MOSQUITO,

April 17, 2004

6:13

Style file version June 28th, 2002

Culex quinquefasciatus

973

FIG. 4. The dose-response of Culex quinquefasciatus to skatole in four-choice bioassays in the field (N = 12). Vertical bars represent standard errors. Means followed by a different letter are significantly different at P < 0.05. DISCUSSION

In laboratory oviposition bioassays, the pheromone prepared from a botanical precursor (PDP) was as effective in stimulating egg-laying by gravid Cx. quinquefasciatus mosquitoes as synthetic oviposition pheromone (SOP) prepared from fine chemicals, confirming the viability of pheromone production using the botanical route. Production of the pheromone from a precursor from renewable plant TABLE 2. FIELD EXPERIMENT SHOWING NUMBER OF EGG RAFTS OF Culex quinquefasciatus DEPOSITED IN WATER TREATED WITH SKATOLE (10−5 µg/l), PLANT-DERIVED OVIPOSITION PHEROMONE (PDP), AND SKATOLE + PDP, VERSUS TAP WATER (CONTROL)a Egg rafts Treatment

Total no.

Mean % ± SE

Control Skatole PDP Skatole + PDP

95 373 153 1681

5.4 ± 1.4 b 15.5 ± 2 c 8.3 ± 1.7 b 73.6 ± 1.8 d

= 12. Means followed by a different letter are significantly different at P < 0.05.

aN

P1: KEE Journal of Chemical Ecology [joec]

974

pp1178-joec-485193

April 17, 2004

6:13

Style file version June 28th, 2002

OLAGBEMIRO, BIRKETT, MORDUE (LUNTZ), AND PICKETT

resource represents an important step forward in the development of sustainable, cheap, and efficient pheromone production. Laboratory bioassays involving skatole and pheromone (PDP and SOP) treatments used concentrations of skatole (10−4 and 10−5 µg/l) shown previously to be the most effective in increasing oviposition (Blackwell et al., 1993). An additive effect was observed for skatole + SOP combinations, in agreement with previous studies (Blackwell et al., 1993). Similar additive effects in laboratory bioassays have been observed with the pheromone and polluted water (Mordue (Luntz) et al., 1992; Blackwell et al., 1993), and the pheromone with a synthetic mixture of oviposition attractants (Millar et al., 1994). However, in this study, additive effects were obtained with PDP + skatole at one concentration (10−5 µg/l), whereas PDP + skatole at a higher concentration (10−4 µg/l) appeared to act synergistically. Field bioassays using PDP and SOP showed no difference in the two materials, similar to the laboratory bioassays. Field assays using skatole were then conducted using a range of concentrations, to determine the optimum concentration for later use with PDP. The results indicate that skatole is used by West African populations of Cx. quinquefasciatus as an oviposition cue, similar to populations in Eastern Africa (Mboera et al., 2000a) and the United States (Millar et al., 1992). The dose-response study showed that the highest proportion of egg rafts of Cx. quinquefasciatus were laid in skatole solutions of 10−4 and 10−5 µg/l, similar to the most active concentrations (10−5 and 10−6 µg/l) observed in Tanzania (Mboera et al., 2000a). In contrast to field studies in Tanzania (Mboera et al., 2000a), where additive effects were observed between skatole and SOP, skatole and PDP appeared to act synergistically. Although synergy between either grass infusions or soakage pit water and SOP has been observed in the field (Mboera et al., 1999, 2000b), the work presented here is the first example of synergy between a specific mosquito oviposition attractant and the oviposition pheromone in the laboratory and in the field. Because PDP was synthesized from an unpurified precursor obtained from the seed oil of K. scoparia (Olagbemiro et al., 1999), it contains numerous additional components some of which may be attractive to Cx. quinquefasciatus. Work is underway to chemically characterize these possible additional components. It is also possible that the difference in activity of skatole and pheromone combinations in the field for West and East African populations of Cx. quinquefasciatus may be due to variation in the populations between the two areas (Tanzania and Nigeria). Previous studies have shown that gravid Cx. quinquefasciatus are able to distinguish between sites with and without the oviposition pheromone at distances of up to 10 m in the field (Otieno et al., 1988). In pit latrines used in the field studies here, treatments were separated by a distance of only 1 m. The data in this study, which showed a much higher preference for the skatole + PDP combination compared to individual treatments, confirm that the blend of site-derived and pheromonal oviposition cues stimulate optimum oviposition activity by gravid

P1: KEE Journal of Chemical Ecology [joec]

pp1178-joec-485193

OVIPOSITION BEHAVIOR OF MOSQUITO,

April 17, 2004

Culex quinquefasciatus

6:13

Style file version June 28th, 2002

975

Cx. quinquefasciatus. Further studies to assess the distance over which skatole + PDP combinations may act will determine the potential for the use of traps at nonbreeding sites. Oviposition attractants/stimulants have shown promise in increasing the sensitivity of gravid Culex mosquito traps for monitoring and control of populations in the United States (Reiter, 1983, 1986; Millar et al., 1994) and in Tanzania (Mboera et al., 1999, 2000b), respectively. The use of specific attractants such as skatole, rather than grass infusions or soakage pit water (cf. Mboera et al., 2000b), is desirable in order to standardize attractants. Accurate and reliable information regarding the breeding habits and distribution of Cx. quinquefasciatus mosquitoes, and Culex spp. mosquitoes in general, is an essential requisite for improved management strategies for these important disease vectors. Acknowledgments—The authors thank various staff for assistance with laboratory and field bioassays (Craig Rogers, James Logan, Momoh Shaibu, Emilia Ugwu, and Gbemga Oke) and Rev. B. A. Adewusi for approving the use of pit latrines and facilities at First Baptist Church, Bauchi. This work was supported by a Wellcome Trust Collaborative Research Initiative Grant (Ref. No. 060796/Z/00/Z). Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom.

REFERENCES BEEHLER, J. W., MILLAR, J. G., and MULLA, M. S. 1994. Field evaluation of synthetic compounds mediating oviposition in Culex mosquitoes (Diptera, Culicidae). J. Chem. Ecol. 20:281–291. BLACKWELL, A., MORDUE (LUNTZ), A. J., HANSSON, B. S., WADHAMS, L. J., and PICKETT, J. A. 1993. A behavioural and electrophysiological study of oviposition cues for Culex quinquefasciatus. Physiol. Entomol. 18:343–348. DAWSON, G. W., MUDD, A. L., PICKETT, J. A., PILE, M. M., and WADHAMS, L. J. 1990. Convenient synthesis of mosquito oviposition pheromone and a highly fluorinated analog retaining biological activity. J. Chem. Ecol. 16:1779–1789. DONALDSON, L. J. 2002. Getting Ahead of the Curve: A Strategy for Combating Infectious Diseases. UK Department of Health report, London. EDWARDS, F. W. 1942. Mosquitoes of the Ethiopian Region III—Culicine Adults and Pupae. Adlard, London. GILLETT, J. D. 1972. Common African Mosquitoes and Their Medical Importance. William Heinemann Medical Books, London. JONSSON, N. N. and REID, S. W. J. 2000. Global climate change and vector-borne diseases. Vet. J. 160:87–89. LAURENCE, B. R. and PICKETT, J. A. 1982. erythro-6-Acetoxy-5-hexadecanolide, the major component of an oviposition attractant pheromone. Chem. Commun. 1:59–60. MBOERA, L. E. G., MDIRA, K. Y., SALUM, F. M., TAKKEN, W., and PICKETT, J. A. 1999. The influence of synthetic oviposition pheromone and volatiles from soakage pits and grass infusions upon oviposition site-selection of Culex mosquitoes in Tanzania. J. Chem. Ecol. 25:1855–1865. MBOERA, L. E. G., TAKKEN, W., MDIRA, K. Y., CHUWA, G. J., and PICKETT, J. A. 2000a. Oviposition and behavioural responses of Culex quinquefasciatus to skatole and synthetic oviposition pheromone in Tanzania. J. Chem. Ecol. 26:1193–1203.

P1: KEE Journal of Chemical Ecology [joec]

976

pp1178-joec-485193

April 17, 2004

6:13

Style file version June 28th, 2002

OLAGBEMIRO, BIRKETT, MORDUE (LUNTZ), AND PICKETT

MBOERA, L. E. G., TAKKEN, W., MDIRA, K. Y., and PICKETT, J. A. 2000b. Sampling gravid Culex quinquefasciatus (Diptera, Culicidae) using traps baited with synthetic oviposition pheromone and grass infusions in Tanzania. J. Med. Entomol. 33:172–176. MILLAR, J. G., CHANEY, J. D., BEEHLER, J. W., and MULLA, M. S. 1994. Interaction of the Culex quinquefasciatus egg raft pheromone with a natural chemical associated with oviposition sites. J. Am. Mosq. Control Assoc. 10:374–379. MILLAR, J. G., CHANEY, J. D., and MULLA, M. S. 1992. Identification of oviposition attractants for Culex quinquefasciatus from fermented Bermuda grass infusion. J. Am. Mosq. Control Assoc. 8:11–17. MORDUE (LUNTZ), A. J., BLACKWELL, A., HANSSON, B., WADHAMS, L. J., and PICKETT, J. A. 1992. Behavioural and physiological evaluation of oviposition attractants for Culex quinquefasciatus Say (Diptera: Culicidae). Experientia 48:1109–1111. OLAGBEMIRO, T. O., BIRKETT, M. A., MORDUE (LUNTZ), A. J., and PICKETT, J. A. 1999. Production of (5R,6S)-6-acetoxy-5-hexadecanolide, the mosquito oviposition pheromone, from the seed oil of the Summer Cypress plant, Kochia scoparia (Chenopodiaceae). J. Agric. Food Chem. 47:3411– 3415. OTIENO, W. A., ONYANGO, T. O., PILE, M. M., LAURENCE, B. R., DAWSON, G. W., WADHAMS, L. J., and PICKETT, J. A. 1988. A field trial of the synthetic oviposition pheromone with Culex quinquefasciatus Say (Diptera, Culicidae) in Kenya. Bull. Ent. Res. 78:463–470. PICKETT, J. A. and WOODCOCK, C. M. 1996. The role of mosquito olfaction in oviposition site location and in the avoidance of unsuitable hosts, pp. 109–123, in R. G. Bock and G. Cardew (eds.). Olfaction in Mosquito–Host Interactions. Wiley, Chichester, UK. PINHEIRO, F. 1997. Global situation of dengue and dengue haemorrhagic fever, and its emergence in the Americas. World Health Stat. Q. 3–4:161–169. REITER, P. 1983. A portable battery-powered trap for collecting gravid Culex mosquitoes. Mosq. News 43:496–498. REITER, P. 1986. A standardized procedure for the quantitative surveillance of certain Culex mosquitoes by egg raft collection. J. Am. Mosq. Control Assoc. 2:219–221. RIESEN, W. K., MILBY, M. M., PRESSER, S. B., and HARDY, J. L. 1992. Ecology of mosquito and St. Louis encephalitis virus in Los Angeles basin of California. J. Med. Entomol. 29:582–598. TURELL, M. J., SARDELIS, M. R., O’GUINN, M. L., and DOHM, D. J. 2002. Potential vectors of West Nile virus in North America, pp. 241–252, in J. S. McKenzie, A. D. T. Barrett, and V. Deubel (eds.). Current Topics in Microbiology and Immunology. Japanese Encephalitis and West Nile Viruses. Springer, Berlin. WHO. 1992. Lymphatic Filariasis: The Disease and Its Control. WHO report, Geneva. WHO/CTD. 1998. Malaria Prevention and Control. WHO report, Geneva.