WAGENINGEN UNIVERSITY LABORATORY OF ENTOMOLOGY

The effects of semiochemicals emitted by larvae of Anopheles gambiae s.s. on the oviposition behaviour of conspecific gravid adult females.

No .................................................................... 09.11 Name ............................................ Bruce Schoelitsz Period ....................... October 2008 / February 2009 Thesis/Internship ....................................ENT-80424 1st Examinator .................................... Marcel Dicke 2nd Examinator................................. Willem Takken

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The effects of semiochemicals emitted by larvae of Anopheles gambiae s.s. on the oviposition behaviour of conspecific gravid adult females.

No .................................................................... 09.11 Name ............................................. Bruce Schoelitsz Period ....................... October 2008 / February 2009 Thesis/Internship ....................................ENT-80424 1st Examinator .................................... Marcel Dicke 2nd Examinator................................. Willem Takken

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Summary

Four chemicals of larval origin were tested for their effects on oviposition behaviour of conspecific adult Anopheles gambiae females. Previous studies have shown that early larval stages had a positive effect on oviposition site selection and attracted gravid females to the oviposition cups, whereas late larval stages had a negative effect and deterred mosquitoes from oviposition cups. Two of these chemicals, dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS), were abundant only in late larval stages and were expected to be repelling/deterring. The other two chemicals, nonane and 2,4pentanedione, were found in both early and late larval stages and were expected to be attractive/stimulating. Although the effects of the chemicals on the oviposition behaviour of the females differ from effects of the larvae, they are comparable. Mosquitoes did not make a distinct choice in choice experiments, but DMDS and DMTS both had a negative effect on oviposition in terms of suppression or inhibition of oviposition, whereas nonane and 2,4pentanedione had a positive effect in which the first attracted mosquitoes to bowls in a semi-field situation and the latter seems to stimulate mosquitoes to oviposit. Although the observed oviposition behaviour differed from what was expected, it may nevertheless have important implications for mosquito monitoring and/or control. More experiments need to be performed on the effects of DMDS on development of eggs, the possible stimulating effect of 2,4-pentanedione and nonane and semi-field experiments of DMDS, 2,4-pentanedione and a blend of nonane and 2,4-pentanedione.

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Preface

This master thesis is a continuation of my previous master thesis ‘The effects of larval age structure and adult female body size on oviposition by Anopheles gambiae (Diptera: Culicidae)’, in which it was shown that oviposition site choice is affected by the larval stage of present conspecific larvae in the site and the choice of the breeding sites seem to be affected by the size of the ovipositing female. Furthermore, chemical profiles of the headspace of first and fourth instars were constructed using GC-MS and chemicals differing in the relative abundance between L1 larvae, L4 larvae and water were identified. For this thesis, I have had the opportunity to travel to Muheza, Tanzania, for which I am very grateful, to test the effects of four of those chemicals on oviposition behaviour by Anopheles gambiae, in both the laboratory as in a situation in which a field situation is imitated. This led to a successful cooperation with Victor Mwingira, a Tanzanian PhD-student active at the Ubwari field station of the National Institute of Medical Research in Muheza. I have enjoyed my stay at Muheza and have experienced the fun and exciting, but also the difficult, aspects of doing research and living in a remote place. It has been a very educational, fruitful and joyful experience!

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Table of contents

1.

Introduction.............................................................................................................. 7

2.

Research questions and hypothesis ........................................................................ 9

3.

Materials and Methods .......................................................................................... 10 3.1 Insects ................................................................................................................. 10 3.2 Chemicals............................................................................................................ 10 3.3 Dose response experiments ................................................................................ 11 3.4 Dual choice experiments ..................................................................................... 11 3.5 Sticky-screen bioassay ........................................................................................ 13 3.6 Physiological experiments ................................................................................... 14 3.7 Semi-field experiments ........................................................................................ 14

4.

Results .................................................................................................................. 16 4.1 Dose response .................................................................................................... 16 4.2 Dual choice.......................................................................................................... 17 4.3 Sticky-screen bioassay ........................................................................................ 22 4.4 Physiological experiment ..................................................................................... 22 4.5 Semi-field experiments ........................................................................................ 24

5.

Discussion ............................................................................................................. 25 5.1

DMDS and DMTS: Negative effects on oviposition ........................................ 25

5.2

Nonane and 2,4-pentanedione: Positive effects on oviposition....................... 28

5.3

Comparison between chemicals and larvae ................................................... 30

5.4

Application and further research .................................................................... 31

5.5

Acknowledgement.......................................................................................... 31

6.

Literature references.............................................................................................. 32

7.

Appendix ............................................................................................................... 37 7.1

Dose response............................................................................................... 37

7.2

Dual choice .................................................................................................... 42

7.3

Sticky-screen bioassay .................................................................................. 48

7.4

Physiological experiments .............................................................................. 50

7.5

Semi-field experiments................................................................................... 52

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1.

Introduction

Many mosquito species use chemical cues to locate oviposition sites. Culex quinquefasciatus Say has been shown to be attracted to a variety of volatiles from breeding sites, including oviposition pheromones (Otieno et al., 1988) and organic material such as grass infusions and skatole (Mboera et al., 2000a). It has since been shown that odour blends can be used to manipulate egg-laying females of Cx. quinquefasciatus and can therefore be used for mosquito control (Mboera et al., 2000b). Oviposition traps are suitable for surveillance programs for they attract a higher proportion of gravid, blood fed females that may have received pathogenic organisms from a blood meal, than CO2, light or vertebrate baited traps, which predominantly attract host-seeking females, many of which have not taken a blood meal (Millar et al., 1994). The identification of oviposition attractants can help to improve existing methods of monitoring mosquito populations (Allan et al., 2005), and baited oviposition traps may be a useful addition to current mosquito control tools (Perich et al., 2003). Larvae of An. gambiae play a role in oviposition behaviour of conspecific females (McCrae, 1984; Sumba et al., 2008). Recently, we have found that the African malaria vector An. gambiae Giles s.s uses olfactory cues in its oviposition behaviour. It was found that young instars produce attracting chemical substances while older instars produce repelling compounds. Thus the larval stages can manipulate their parents’ oviposition behaviour and enhance the fitness of their generation. The chemical compounds produced by late instars are believed to serve as semiochemicals signalling gravid females the presence of an unsuitable breeding site, while the ones produced by early instars are believed to inform the presence of a suitable breeding site (Mwingira et al, in prep.; Schoelitsz, 2008). We have demonstrated that cues mediating oviposition behaviour were released by larvae and oviposition behaviour in An. gambiae is mediated by olfactory cues (Mwingira et al, in prep). These findings have important implications for the manipulation of population development of this important vector through the use of commercially produced pheromones. In the field settings, potential breeding sites differ in a range of characteristics both biotic and abiotic (Gimning et al, 2001), which either singly or synergistically (Beehler et al, 1993) influence the choice of oviposition site by gravid An. gambiae. The reported ‘olfactory mediated’ oviposition behaviour of An. gambiae in the laboratory has yet to be demonstrated in the field with wild type mosquitoes. Demonstrating that this

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behaviour occurs in the wild would be complicated and would be subject to influences from many external factors such as multiple breeding sites, competitors and predators. However, the Mosquito Spheres at the Amani Medical Research Centre, Tanzania, provide the ideal site for investigating oviposition behaviour of An. gambiae under semifield conditions, where many of the external variables in the field can be controlled. Using coupled gas chromatography-mass spectrometry (GC-MS) to study the volatiles produced by larvae, four chemical compounds were identified recently, that might affect the oviposition behaviour of An. gambiae (Schoelitsz, 2008). Nonane and 2,4-pentanedione were present in both L1 and L4 larvae, whereas dimethyl disulfide and dimethyl trisulfide was present only in the fourth instars. The roles of these chemical compounds on oviposition behaviour of An. gambiae sensu stricto have been investigated, for both the single compounds as for blends in different concentrations. Adult females have been exposed to the four candidate compounds under controlled circumstances and in a semi-field situation in Muheza and the oviposition behaviour has been examined. This report covers the exploration of the effects of the semiochemicals of larval origin in mediating oviposition behaviour of Anopheles gambiae in laboratory and semi-field situations.

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2.

Research questions and hypothesis

What are the effects of the identified chemicals of larval origin, dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), nonane and 2,4-pentanedione, on the oviposition behaviour of conspecific adult females of Anopheles gambiae, under laboratory and semi-field situations?

It is expected that DMDS and DMTS have a repelling/deterring effect on oviposition, whereas nonane and 2,4-pentanedione have an attracting/stimulating effect. DMDS and DMTS were found in headspace of deterring L4 larvae significantly more than in headspace of attracting L1 larvae. There was no difference between L1 and L4 larvae, were nonane and 2,4-pentanedione were concerned, suggesting that they are attractive and that their effect is masked by DMDS and DMTS in L4 larvae. Furthermore, it is expected that the effects found under semi-field situations are comparable to the effects found in the laboratory.

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3.

Materials and Methods

3.1 Insects Larvae of the species Anopheles gambiae were reared in round aluminium pans, with a diameter of 27 cm, filled with water to a depth of 2 cm. Larvae were fed on Tetramin (Tetra, Germany) and were kept in a 12 L : 12 D light regime. The temperature in the insectarium was 29 ±1 oC. Pupae were removed from the trays and were placed in mosquito cages of 30 * 30 * 30 cm. For the experiments, 5-7 day old mosquitoes were moved to another cage, in which they were fed blood at dusk, provided by a human arm. Together with females, some males were moved as well, to increase the chance of insemination. For the experiments, 7-9 day old mosquitoes were used. 3.2 Chemicals The chemicals of which their effects on oviposition were tested during this study were dimethyl disulfide (DMDS ≥ 99.0%, Sigma Aldrich Chemie BV, Zwijndrecht, The Netherlands), dimethyl trisulfide (DMTS ≥ 98.0%, Sigma Aldrich Chemie BV, Zwijndrecht, The Netherlands), nonane (Lot and fillingcode: 1329952 35107188, ≥ 99.0%, contains naphtene and isoparaffin, Sigma Aldrich Chemie BV, Zwijndrecht, The Netherlands) and 2,4-pentanedione (Acetylacetone ReagentPlus® Lot code S47392138, ≥ 99.0%, Sigma Aldrich Chemie BV, Zwijndrecht, The Netherlands). None of these chemicals were soluble in water. They were dissolved in methanol and Tween20, according to the following formula: 55% DMDS

+ 40% Methanol

+ 5% Tween20

55% DMTS

+ 40% Methanol

+ 5% Tween20

55% nonane

+ 40% Methanol

+ 5% Tween20

55% 2,4-pentanedione

+ 40% Methanol

+ 5% Tween20

Hereafter, the chemicals could be dissolved in distilled water. Dilution series were performed to produce the concentrations needed for the experiments, diluting the chemical with one magnitude with each step. Dilutions were performed with distilled water, and were conducted in plastic cups of 50 ml with a plastic cap.

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3.3 Dose response experiments To determine the most effective concentration of the chemicals on the behaviour of gravid mosquitoes, 10 gravid mosquitoes (Anopheles gambiae s.s., Ifakara-strain) were placed in a cage containing a control cup (distilled water), and cups containing concentrations of 5.5 * 10-7 ml / ml, 5.5 * 10-8 ml / ml and 5.5 * 10-9 ml / ml of one of the chemicals to be tested. This was repeated with a control cup (distilled water) and cups containing concentrations of 5.5 * 10-10 ml / ml, 5.5 * 10-11 ml / ml and 5.5 * 10-12 ml / ml of one of those chemicals. Cups contained 30 ml of the solutions. The cages measured 30 * 30 * 30 cm and each cup was placed in one of the four corners, at a distance from each other of approximately 20 cm. On each cup, a filter paper cone was placed, as an oviposition paper. The tip of the cone was submerged in the liquid at all times, making sure that the oviposition paper remained wet. The mosquitoes were provided a glass bottle with a filter paper and a 6% glucose solution as an additional food source. Mosquitoes were placed 48 hours after taking a blood meal. After 12 and 36 hours (1 and 2 nights) the number of eggs was counted. The determination of the most effective concentration was based on the total number and percentage of eggs found in both control and treatment cups. Each treatment was repeated three times. Based on the results from these experiments, dual choice experiments were designed and performed. Data of number of eggs in the cups was analyzed with the Friedman test. Data between the treatments was analyzed with Mann Whitney U test. 3.4 Dual choice experiments Dual choice experiments were done with single compounds and with blends of two compounds. The following concentrations of single compounds were tested: DMDS: 5.5 * 10-7 ml / ml and 5.5 * 10-9 ml / ml DMTS: 5.5 * 10-9 ml / ml and 5.5 * 10-11 ml / ml Nonane: 5.5 * 10-11 ml / ml 2,4-pentanedione: 5.5 * 10-10 ml / ml

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Based on the results from the dose response experiments, DMDS and DMTS were combined (Blend R), and nonane and 2,4-pentanedione (Blend A) were combined. Three different concentrations of the blends were tested. For Blend R, these were 5.5 * 10-7 ml / ml DMDS with 5.5 * 10-11 ml / ml DMTS, 5.5 * 10-9 ml / ml DMDS with 5.5 * 10-13 ml / ml DMTS, and 5.5 * 10-10 ml / ml DMDS with 5.5 * 10-14 ml / ml DMTS.

For Blend A these different concentrations were: 5.5 * 10-11 ml/ ml nonane with 5.5 *10-10 ml / ml 2,4-pentanedione, 5.5 * 10-13 ml / ml nonane with 5.5 * 10-12 ml / ml 2,4-pentanedione, and 5.5 * 10-14 ml / ml nonane with 5.5 * 10-13 ml / ml 2,4-pentanedione. Gravid mosquitoes were given a choice between cups containing one of these concentrations and a control cup, containing the solvent in the same concentration as the treatment. One female was placed in a cage measuring 30 * 30 * 30 cm, 48 hours after blood feeding, and was provided a 6% glucose solution. The two cups contained 30 ml of the solution and were placed in opposing corners with a distance of approximately 25 cm from each other. On top of the cups, white filter papers were placed as oviposition paper. After approximately 12 and 36 hours the number of eggs was counted. The oviposition activity was expressed as an ovipositional activity index (OAI) and was calculated as follows (Kramer and Mulla, 1979):

OAI=

Nt - Nc Nt + Nc

Where Nt denotes the mean number of eggs laid in the treatment cup and Nc denotes the mean number of eggs laid in the control cups. The index values range from -1 to +1. A positive value indicates that more eggs were found in the treatment than in the control, thus signalling the chemical of treatment to be an oviposition attractant. On the other hand more eggs laid in the control than in the treatment would result in the negative OAI

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value indicating the chemical to be oviposition repellent. Kramer and Mulla (1979), suggested that compounds with OAI of +30 and above are considered to be attractants, while those with -0.30 and below are considered to be repellents. After 36 hours, the mosquitoes were stored in a 75% methanol solution for dissection. Mosquitoes were dissected and retained eggs were counted using a microscope. Data concerning number of eggs between cups was analyzed with the Wilcoxon signed rank test. The number of eggs retained with exposure to different treatments was analyzed with the Mann Whitney U test. Each treatment was repeated for at least six times. 3.5 Sticky-screen bioassay To determine the actual effect on the ovipositional behaviour of mosquitoes, a stickyscreen bioassay was performed with DMDS (5.5 * 10-7 ml / ml), DMTS (5.5 * 10-11 ml / ml), nonane (5.5 * 10-11 ml / ml) and 2,4-pentanedione (5.5 * 10-10 ml / ml), against a control, containing the solvent in the same concentration as in the treatment. 30 mL of the solutions were kept in plastic cups. Instead of an oviposition paper, as in the experiments described above, a piece of net, mesh size measuring 1.2 mm, was placed on top of the ovipostition cup, and fastened with a rubber band. After the nets were attached to the cups containing the solutions, a thin layer of Tanglefoot (PRI Pherobank, Wageningen) was evenly spread on the net. The small holes that automatically formed in the glue made sure that the glue did not block the odours coming from within the cups. Together with the cups, 10 female mosquitoes were placed in a cage of 30 * 30 * 30 cm, 48 hours after blood feeding. When a mosquito came in contact with the Tanglefoot, it was trapped (Figures 1a and 1b). After approximately 12 and 36 hours, the number of mosquitoes trapped on each cup was counted. Mosquitoes caught after one night were removed from the glue, but were not replaced.

a

b

Figure 1: Side view of an oviposition cup covered with a net, on which Tanglefoot is applied. Mosquitoes are trapped on the Tanglefoot (1a). Figure 1b shows a mosquito incapable of escaping from the trap. 13

3.6 Physiological experiments The effect of DMTS on developed eggs was tested. Eight cages of 30 * 30 * 30 cm were provided with 15 gravid females, 48 hours after blood feeding. Females were blood fed once before the start of the experiment, and did not receive another blood meal during the experiment. A control cup (containing 30 ml of the solvent in a concentration of 10-11 ml / ml) was placed in four of the eight cages. The same was done with four cups containing 30 ml of a DMTS solution with a concentration of 5.5 * 10-11 ml / ml. A bottle with a 6% sugar solution was added to the cage, as an additional food source. Every morning, the number of eggs oviposited was counted. 2, 4, 6 and 8-d after the start of the experiment, all mosquitoes of one cage containing a control cup and of one cage containing a treated cup were dissected. The number of retained eggs was counted, using a binocular microscope. Data between the treatment and control was analyzed with the Mann Whitney U test. Data within the treatment or control was analyzed with the Kruskal Wallis test. 3.7 Semi-field experiments The effects of DMTS 5.5 * 10-11 ml / ml and nonane 5.5 * 10-11 ml / ml on oviposition behaviour were investigated against their controls, in a semi-field situation (mosquito spheres) where there was free movement of air, rainfall, sunlight and variable relative humidity. The objective was to scale up the exploration into a semi-field situation and compare results from the laboratory with semi-field results. Three mosquito spheres facilities (11.4 * 7.1 * 5.0 m, Figure 2) exist at Amani Medical Research Centre, and were used in this study. In each sphere there was a small traditional Tanzanian house, there were some banana trees and low vegetation (Figure 3). During the experimental period (September 19th – October 16th, 2008) the average temperatures in the spheres ranged from a minimum of 16oC during the night to a maximum of 37oC during the day. The average RH ranged from a minimum of 40% to a maximum of 100%. In each sphere, two holes were symmetrically dug in the ground at the centre of the sphere. The holes were located with a distance of 3 m from each other (Figure 2). A green plastic bowl with a diameter of 26 cm and a height of 10 cm, was placed in each hole as an artificial breeding site. The bowls were placed in a way that the rim of the bowls was at the surface level (Figure 3). The bowls had a capacity of 5 litres and were filled with 3 litres of the solutions.

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240 mosquitoes (Anopheles gambiae, R70-strain) were given an opportunity to blood feed twice, on day 3 and day 5 after emergence, and were released on day 6. Mosquitoes were released one hour before dusk. Eggs were counted in the first and second morning after releasing mosquitoes and the solutions were replaced after the first night. The results were analyzed with the Wilcoxon signed rank test. Experiments with both chemicals were repeated for six times.

Figure 2: Schematic top view of one of the three mosquito spheres.

Figure 3: Mosquito sphere, containing a traditional Tanzanian house, a banana tree and some low vegetation. Two holes were dug with a distance of 3 meters from each other, containing green bowls filled with 3 liters of either the treated or control solution. Mosquitoes were released from the center of the sphere, one hour before dusk.

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4.

Results

4.1 Dose response Six concentrations (ranging from 5.5 * 10-7 to 5.5 * 10-12 ml / ml) of the four chemicals were tested, of which the most interesting concentrations were chosen for further experiments. Where mean values are presented, these are accompanied by standard errors of the mean. No significant differences were found between the different concentrations and between the different concentrations and the control (P> 0.05, Friedman test) for all chemical compounds. In the first set of experiments with DMDS (5.5 * 10-7 to 5.5 * 10-9 ml / ml) the cup that received the fewest eggs was the concentration of 5.5 * 10 -7 ml / ml. On average, the number of eggs oviposited in this cup was 8 times as low as in the control cup. The concentration of 5.5 * 10-9 ml / ml received more than twice as many eggs as the control, 226 ± 57 over 101 ± 74. The remaining concentration (5.5 * 10-8 ml / ml) did not differ much from the control (Figure 4a). Although two of the concentrations tested in the second set of experiments (5.5 * 10-10 to 5.5 * 10-12 ml / ml) received fewer eggs than the control, these differences were not as large as in the first set (Figure 4b). Because the largest effects were observed with the concentrations of 5.5 * 10-7 and 5.5 * 10-9 ml / ml, these were used in subsequent experiments (see below). In the second set of concentrations of DMTS (5.5 * 10-10 to 5.5 * 10-12 ml / ml) two of the three cages did not contain a single egg after a period of 36 hours. In the third cage, fewest eggs were oviposited in the cup containing a solution of 5.5 * 10-11 DMTS ml / ml (Figure 4d). This concentration was considered most interesting, as well as the concentration of 5.5 * 10-9 ml / ml from the first set of experiments. The cup containing this concentration received the fewest eggs, on average less than half the number of eggs oviposited in the control cup (Figure 4c). The use of 2,4-pentanedione did not cause many differences in the number of eggs laid between the cups with the different concentrations, except for 5.5 * 10-10 ml / ml (Figures 4e and 4f). The cups containing this concentration received more than twice as many eggs as compared to the control cups (230 ± 42 compared to 93 ± 68), but this was not significant (P= 0.101, Wilcoxon signed rank test). For nonane, the differences in amount of eggs found on the oviposition papers of the different cups were small. The most interesting concentration was found to be 5.5 *

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10-11 ml / ml, which received 1.5 more eggs than the control (Figures 3g and 3h), but this was not significant (P= 0.593, Wilcoxon signed rank test). The control experiments, in which all four cups contained water, showed no significant difference between the number of eggs laid in each cup (P= 0.659, Friedman). Between the four different chemicals and water, a difference was found in the total number of eggs oviposited by the mosquitoes per cage (P= 0.005, Kruskal Wallis). The total number of eggs laid by the females was lowest with DMTS. The difference in the total number of eggs oviposited per cage with DMTS was significant, when compared to the total number of eggs laid per cage with any of the other compounds (P= 0.025, P= 0.006, P= 0.004 and P= 0.010 for the differences in the total number of eggs per cage between DMTS and DMDS, DMTS and Nonane, DMTS and 2,4-pentanedione and DMTS and water, respectively; Mann Whitney U test). In the cages containing 2,4-pentanedione more eggs were laid than in the control cages containing water (P= 0.030, Mann Whitney U test) and in the cages with nonane, mosquitoes tended to oviposit more than in cages with water only (P= 0.078, Mann Whitney U test). 4.2 Dual choice None of the concentrations of the chemicals tested showed a significant difference in the number of eggs oviposited in the treated or control cup. The average number of eggs in the control and treatment cup was very similar, except for nonane in a concentration of 5.5 * 10-10 ml / ml. Twice as many eggs were laid in the control cup than in the treatment cup. This difference was not significant, however (Table 1). The percentage of mosquitoes that had developed eggs, but did not oviposit was higher in DMDS in a concentration of 5.5 * 10-7 ml / ml and DMTS in a concentration of 5.5 * 10-11 ml / ml, than with 2,4-pentanedione, nonane and water. Consecutively, the number of eggs retained in the abdomen of the mosquitoes was higher with DMDS in a concentration of 5.5 * 10-7 ml / ml and DMTS in a concentration of 5.5 * 10-11 ml / ml than with 2,4-pentanedione, nonane and water (table 1). A significant difference was found in the number of eggs retained, when this number of retained eggs in mosquitoes exposed to these four chemicals and water was tested against each other (P= 0.002, Kruskal Wallis). When tested individually, differences were found between DMDS and 2,4pentanedione (P= 0.010, Mann Whitney U), DMDS and nonane (P= 0.027, Mann

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Whitney U), DMDS and water (P= 0.018, Mann Whitney U) DMTS and 2,4-pentanedione (P= 0.008, Mann Whitney U), DMTS and nonane (P= 0.014, Mann Whitney U) and DMTS and water (P= 0.007, Mann Whitney U). In general, when a mosquito oviposited, no retained eggs were found, with exception of a few mosquitoes exposed to water or 2,4-pentanedione, of which all mosquitoes oviposited, but still retained some eggs. The blends did not show significant differences in the number of eggs oviposited between the control and treatment cups, for all concentrations (Table 2). Although the differences were not significant, a reduction in concentration of Blend R seems to make it more attractive as oviposition site (the OAI increases from -0.38 in the blend to +0.28 when the blend is diluted by a factor one hundred and to +0.65 when diluted by a factor one thousand). The same was shown with Blend A (OAI increases from -0.43 to -0.18 when diluted a hundred times to +0.17 when diluted a thousand times). The blend of DMDS and DMTS in the same concentrations as used in the dual choice experiments caused an increase in egg retention when compared to other concentrations, the blend of nonane and 2,4-pentanedione and the control. Mosquitoes exposed to blend A, for all three concentrations, oviposited all eggs, no retained eggs were found.

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ns

ns

a

b

ns

ns

c

d

Figure 4a-d: Boxplots showing number of eggs oviposited in cups containing different concentrations of dimethyl disulfide (4a and 4b) and dimethyl trisulfide (4c and 4d). Boxplots show the median (fat line), first and third quartiles and the maximum and minimum of the number of eggs oviposited. Ns indicates a significant difference is not found, tested with Friedman test, N=3.

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ns

ns

e

f

ns

ns

g

h

Figure 4e-h: Boxplots showing number of eggs oviposited in cups containing different concentrations of 2,4pentanedione (4e and 4f) and nonane (4g and 4h). Boxplots show the median (fat line), first and third quartiles and the maximum and minimum of the number of eggs oviposited. Ns indicates a significant difference is not found, tested with Friedman test, N=3.

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Table 1: Data of the dual-choice experiments of the tested concentrations of the single compounds dimethyl disulfide (DMDS), dimethyltrisulfide (DMTS), 2,4-pentanedione and nonane, and the control experiments with distilled water. The table shows the number of replicates (N), the P-value of the Wilcoxon test between the number of eggs oviposited in the cups containing the control and the treatment, the oviposition active index (OAI), average number of eggs oviposited in each cup ±SE and the average number of eggs retained in the abdomen of the mosquitoes ±SE, and the number and percentage of mosquitoes that developed eggs and had oviposited. Differences between the number of eggs oviposited in control or treated cup were considered significant with P< 0.05, chemicals were considered attractive with an OAI > +0.30 and repelling with an OAI < -0.30. Mean number of retained eggs indicated with an * differed significantly from control (P< 0.05, Mann Whitney U test). DMDS

N P-value AOI Mean nr eggs control Mean nr eggs treatment Mean nr retained eggs Nr mosquitoes oviposited % mosquitoes oviposited

DMTS

2,4-pentanedione Nonane Water (Control) 5.5*10-7 5.5*10-9 5.5*10-9 5.5*10-11 5.5*10-10 5.5*10-11 17 8 8 16 14 17 10 0.674 0.866 1.000 0.799 0.861 0.147 0.241 -0.15 0.05 -0.05 -0.31 -0.05 -0.33 18.12 ± 7.51 54.43 ± 24.75 11.88 ± 6.97 8.88 ± 5.45 36.43 ± 9.47 40.20 ± 10.31 19.90 ± 9.62 13.41 ± 5.66 59.57 ± 23.73 10.75 ± 8.69 4.69 ± 2.48 32.86 ± 10.32 20.40 ± 8.91 48.30 ± 15.59 52.06* ± 13.43 37.13* ± 9.02 15.40 ± 15.40 28.71 ± 13.83 6.92 ± 6.92 15.00 ± 10.36 2.20 ± 2.20 8 of 17 7 of 8 5 of 8 7 of 16 14 of 14 15 of 17 10 of 10 47.06 87.50 62.50 43.75 100.00 88.24 100.00

Table 2: Data of the dual-choice experiments of the tested blends, in most effective concentrations determined in previous experiments and after a dilution of a hundred and a thousand times. The table shows the number of replicates (N), the P-value of the Wilcoxon test between the number of eggs oviposited in the cups containing the control and the treatment, the oviposition active index (OAI), average number of eggs oviposited in each cup ±SE and the average number of eggs retained in the abdomen of the mosquitoes ±SE, and the number and percentage of mosquitoes that developed eggs and had oviposited. Differences between the number of eggs oviposited in control or treated cup were considered significant with P< 0.05, chemicals were considered attractive with an OAI > +0.30 and repelling with an OAI < -0.30. Mean number of retained eggs indicated with an * differed significantly from control (Table 1, P< 0.05, Mann Whitney U test).

N P-value OAI Mean nr eggs control Mean nr eggs treatment Mean nr retained eggs Nr mosquitoes oviposited % mosquitoes oviposited

Blend R Blend R100 Blend R1000 8 10 6 0.499 0.386 0.225 -0.37 +0.28 +0.65 52.13 ± 21.31 15.60 ± 7.60 7.17 ± 4.93 24.00 ± 14.57 27.60 ± 12.58 33.67 ± 21.59 41.00 ± 26.87 1.25 ± 1.25 16.80 ± 16.80 7 of 8 10 of 10 5 of 6 7 87.5 100 83.33333

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Blend A Blend A100 Blend A1000 7 6 6 0.397 0.753 0.917 -0.43 -0.18 +0.17 45.14 ± 20.42 30.67 ± 18.46 39.00 ± 12.84 18.14 ± 7.06 21.50 ± 11.17 55.17 ± 19.64 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 of 7 6 of 6 6 of 6 100 100 100

4.3 Sticky-screen bioassay Significantly more mosquitoes were caught in the control cups, when mosquitoes were give a choice between a control cup and a cup containing DMDS (P= 0.037, Wilcoxon test). The same tendency was shown with DMTS, but the difference was not significant. The average number of mosquitoes caught in the dual choice with 2,4-pentanedione was equal in both cups. More mosquitoes were trapped in the cup treated with nonane, than in the control, but this difference was not significant (Table 3). When mosquitoes were provided two control cups, there was no difference in the number of mosquitoes trapped.

Table 3: Data of the sticky screen bioassay of dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), 2,4-pentanedione, and nonane in the concentrations determined in the previous experiments. Data shows the number of replications (N), the P-value of the Wilcoxon test between the control and treated cups, and the average number of mosquitoes caught in the control and the treatment ±SE. As a control, two cups containing water was tested.

N P-value Mean number of mosquitoes caught in control cup Mean number of mosquitoes caught in treated cup

DMDS -7 5.5*10 12 0.037

DMTS -11 5.5*10 12 0.234

2,4-pentanedione -10 5.5*10 6 0.414

Nonane -11 5.5*10 6 0.891

Water

4.33

±0.56

3.58

±0.47

2.33

±0.67

2.67

±0.61

3.33

±0.61

2.58

±0.36

2.50

±0.53

2.33

±0.61

3.50

±0.76

3.83

±0.75

6 0.679

4.4 Physiological experiment After two and four days, mosquitoes in the cage containing a control cup on average laid approximately three times more eggs than the mosquitoes in the cage containing a cup with DMTS. However, this difference in number of eggs laid by the mosquitoes was not significant (P= 0.149 after two days, P= 0.127 after four days, Mann Whitney U test). After four days, mosquitoes ceased ovipositing, in the cages containing a control cup as well as the mosquitoes containing a cup with DMTS. The number of eggs oviposited over time did not differ significantly within the control and treatment (P= 0.115 for the control and P= 0.194 for the treated cups, Kruskal Wallis; Figure 5a). The number of eggs that were retained, was higher in the group of mosquitoes with DMTS, than in the group with a control cup. After the first two days, the number of eggs retained by the mosquitoes in the cages with DMTS was lower than in the control

22

group, i.e. in six days, only one mosquito retained a small number of eggs. However, these differences were not significant (P= 0.305 for day two, P= 0.197 for day four, P= 0.272 for day six and P= 0.071 for day eight). After two days, there was a sharp significant decrease in number of retained eggs within the experimental group (P= 0.001, Kruskal Wallis) which was not observed in the control group (P= 0.504, Kruskal Wallis; Figure 5b). Generally, when eggs were retained the number of retained eggs was relatively high. Figure 5c shows the potential to oviposit of mosquitoes in the physiological experiment. The oviposition potential consists of the average of the number of eggs oviposited plus the number of eggs retained per mosquito in one cage. Whereas the oviposition potential is relatively constant for the control group, the oviposition potential of the treated group decreases sharply after two days.

a

b

c

Figure 5a-c: Figure 5a shows the average number of eggs ± S.E oviposited by mosquitoes in a cage containing an oviposition cup with water (control) and a cage containing a cup with DMTS (N= 4 for 2-d, N=3, N=2 and N=1 for 4, 6 and 8 days, respectively). Figure 5b shows the average number of eggs retained ± S.E by mosquitoes exposed to oviposition cups containing a control and DMTS for 2, 4, 6 and 8 days, respectively (N=12-15). Figure 5c shows the potential to oviposit, i.e. the average 23 of eggs retained per gravid mosquitoes exposed of the number of eggs oviposited plus the number to a control and treated cup for 2, 4, 6 and 8 days, respectively.

4.5 Semi-field experiments The bowls containing DMTS and the bowls containing nonane both received more eggs than the control bowls, indicated by the positive OAI. For DMTS, this difference was not significant (P= 0.144, Wilcoxon test). For nonane, however, there was a significant difference in the average number of eggs that were laid in the treated bowls compared to the control (P= 0.043, Wilcoxon test). The bowl containing nonane received, on average, over 2.5 times more eggs than its control (Table 4). Table 4: Data of the semi-field experiments of dimethyl trisulfide (DMTS) and nonane in the concentrations determined in the previous experiments. Table shows the number of replications (N), the P-value of the Wilcoxon test between the control and treated bowls, the oviposition active index (OAI) and the average number of eggs oviposited in the control and the treatment ±SE.

N P-value OAI Mean nr eggs control Mean nr eggs treatment

DMTS 5.5 * 10-11 6 0.144 +0.31 206.67 ±79.51 392.33 ±117.72

24

Nonane 5.5 * 10 -11 6 0.043 +0.46 248.50 ±104.02 637.50 ±168.14

5.

Discussion

5.1

DMDS and DMTS: Negative effects on oviposition

Based on the results with the dose response experiments with DMDS, this compound was further studied with the concentrations of 5.5 * 10-7 ml / ml and 5.5 * 10-9 ml / ml. With these concentrations the number of eggs oviposited in the cups were the lowest. With the latter concentration offered in a dual choice experiment, the number of eggs oviposited was not different from the control. Furthermore, mosquitoes exposed to this concentration did not retain many eggs, and the majority of the mosquitoes oviposited. In a concentration of 5.5 * 10-7 ml / ml, DMDS showed some interesting results in the dual choice test. Although the repelling/deterring effects caused by fourth instars (Mwingira et al, in prep.; Schoelitsz, 2008) were not seen in the dual choice experiments with this concentration of DMDS, the average number of eggs oviposited was low, when compared to the lower concentration tested and compared to 2,4-pentanedione, nonane and water. This low number of eggs oviposited is the result of inhibition or suppression of oviposition, because more than half of the mosquitoes did not oviposit, resulting in a high number of retained eggs four days after blood-feeding. The sticky-screen bioassay suggests that DMDS in this concentration is repellent because more mosquitoes were caught in the control cups than treated cups. It seems that mosquitoes are able to assess the abundance of DMDS from a distance and respond to it, by visiting the other oviposition cup. Mosquitoes are not only repelled by DMDS in a concentration of 5.5 * 10-7 ml / ml, they may also be inhibited to oviposit when they are exposed to this semiochemical in this concentration. The effects of DMDS in this concentration in a semi-field situation need to be tested. Inhibition or suppression of oviposition was also observed with DMTS. In the dose response experiments, mosquitoes exposed to cups containing a concentration range from 5.5 * 10-10 to 5.5 * 10-12 ml / ml did not lay any egg in two of the three cages. From both concentration ranges, the cups with the concentration that received fewest eggs was chosen for further experiments, i.e. 5.5 * 10-9 ml / ml and 5.5 * 10-11 ml / ml. In the dual choice experiments, there was no difference in number of eggs oviposited in both cups, but the exposure of both concentrations to the mosquitoes inhibited oviposition. Mosquitoes did not have a preference for the control cup and often did not oviposit at all. The proportion of mosquitoes that did not oviposit was high for both concentrations, but especially for the lowest concentration, where more than half of the

25

mosquitoes that had developed eggs did not oviposit in two nights. Like with DMDS, the number of eggs retained was high with both concentrations. Volatile induced suppression and inhibition of oviposition in Anopheles is reported for the first time by Dhar et al. (1996). Ovipostion by gravid females of An. stephensi was suppressed when mosquitoes were exposed to neem volatiles for a short-term period (90 minutes) and was confirmed by the high number of retained eggs when exposed to the volatiles. The number of eggs retained by mosquitoes exposed to cups containing DMDS in a concentration of 5.5 * 10 -7 and DMTS in a concentration of 5.5 * 10-11 ml / ml was significantly higher than with nonane, 2,4-pentanedione or the control, emphasizing the negative effect of these compounds on oviposition. The concentration of DMTS of 5.5 *10-11 ml / ml was tested in further experiments, because of the results obtained. The low number of eggs oviposited when exposed to DMTS was also seen in the experiments studying the effects of DMTS on the physiological properties of the gonotrophic system of mosquitoes. The differences were not found to be significant, however, probably because of the low number of replicates. The average number of eggs oviposited in the cup with DMTS was approximately three times lower than in the control cup after two and four days since the start of the experiment. This suggests that mosquitoes detecting DMTS are triggered to retain their eggs more, compared to mosquitoes that do not detect this chemical in the oviposition site. It should be noted that based on the other experiments performed it seems that when a mosquito oviposits it lays all of its eggs. As a result of the lower number of eggs oviposited, the number of eggs that are retained after two days in mosquitoes exposed to DMTS is higher than in the control. The same result was expected for the number of eggs retained during the remaining days of the experiment, but this was not the case. After 4 days the number of eggs retained showed a strong decrease. The difference in the number of retained eggs by mosquitoes with the treated cups between the consecutive days differed significantly, whereas this was not different in the control group. The oviposition potential, determined as the average of the total number of eggs oviposited and retained per mosquito, is expected to be constant when DMTS would not affect the gonotrophic cycle of the mosquitoes, as is seen in the control group. This was not the case, mosquitoes seemed to have resorbed the eggs. Magnarelli (1983) showed the phenomenon of naturally occurring resorption of oocytes in a late developmental stage and retained eggs by mosquitoes from wild populations of several Aedes species. Several studies have shown an effect of insecticides on egg development in insects (Cabral et al., 2008; Friesen et

26

al., 2003; Fujiwara, 2002; Dugravot et al., 2002). Furthermore, Dhar et al. (1996) have shown that the gonotrophic cycle of An. stephensi and An. culicifaces was impaired by constant and long exposure to neem volatiles. And Hopwood et al. (2001) showed that malaria-induced resorption of developing follicles in An. stephensi and An. gambiae is preceded by apoptosis of the follicular epithelial cells. Whether this is also the case with developed eggs is not known, but these examples indicate that oviposition by mosquitoes that have developed eggs and are exposed to DMTS is not only suppressed, but mosquitoes do also resorb the produced eggs and DMTS affects the gonotrophic cycle of the mosquitoes. In this study, the effect of DMTS is shown on developed eggs. It would be very interesting to study the effect of DMTS when exposed during the development of the eggs, directly after blood feeding. When it would prove that DMTS inhibits the development of eggs instead of oviposition, a new field of applications may be possible. By, for example, spraying walls of houses with DMTS, as is done with DDT at the moment. Mosquitoes resting on the wall after feeding will be exposed to DMTS and egg development will be inhibited. The life history of the mosquito is interrupted at an important stage. This will not directly reduce the chance of transmitting the malaria parasite by killing the adult, on which most intervention rely (Mboera et al., 2007), but target the suppression of the productivity of the mosquito. The effect of DMDS on the development of the eggs needs to be tested. Both DMDS and DMTS are emitted by a broad range of natural sources; they are produced, for example, by bacteria (Khoga et al., 2002), DMDS can be found in human faeces (Moore et al, 1987) and both compounds are known to be emitted by plants (Stensmyr et al., 2002; Soler et al., 2007; Du and Millar, 1999). Whereas DMTS is produced by Helicodiceros muscivorus to attract flies (Stensmyr et al., 2002), insecticidal and repelling properties of both DMDS and DMTS are also described, repelling insects from damaged plants (Soler et al., 2007). DMDS has been shown to be an effective insecticide against termites (Auger et al., 2004), a coleopteran species (Dugravot et al., 2002) and a cockroach species (Dugravot et al., 2003), and the effect of DMDS on developed eggs may be comparable to that of DMTS. Whether the mosquitoes in the semi-field are affected by DMTS in the same way as the mosquitoes in the laboratory cannot be said, yet. There was no significant difference in number of eggs between the control and treatment in the semi-field, although the bowl with DMTS received most eggs, which is in contradiction with the

27

hypothesis that DMTS is a repellent/deterrent. The only other comparison that can be made is between the total number of eggs laid in the semi-field with nonane as treatment. The total number of eggs oviposited in mosquito spheres containing nonane as treatment was twice as high as the total number of eggs oviposited in mosquito spheres containing DMTS as treatment. More data is needed to see whether DMTS inhibits oviposition in the semi-field. The blend of DMDS (5.5 * 10-7 ml / ml) and DMTS (5.5 * 10-11 ml / ml) showed a high average of egg retention, the percentage of ovipositing mosquitoes however, was fairly high and there was no difference in number of eggs oviposited between the two cups. It should be noted that the effects of DMDS and DMTS are most probably caused due to a spatial effect of the compounds. The abundance of one of these chemicals in a cup affected many mosquitoes in a way in which they did not oviposit in the control cup either, as shown by the reduced oviposition. 5.2

Nonane and 2,4-pentanedione: Positive effects on oviposition

Although in the dose response experiments no effects were caused by nonane, in the semi-field experiments bowls containing this compound received significantly more eggs than the control. Also, in the sticky-screen test, more mosquitoes were trapped in the treatment than in the control cup and in the dose response experiments, the concentration of 5.5 * 10-11 ml / ml received slightly more eggs than the control, although differences in both experiments were not significant. In the dual choice experiments, however, mosquitoes oviposited more in the control cup than in the treatment. Although the difference was not significant, this trend was not expected, because nonane was assumed to be attractive. This assumption was based on the fact that nonane was present in both the headspace of L1 and L4 larvae, and that L1 larvae were attractive and this attractive effect was cancelled out by DMDS and/or DMTS in L4 larvae (Schoelitsz, 2008). The inconsistency in results between the different experiments may be caused by the size of the cages in the laboratory settings. Especially in the dose response experiments, the distance between the different concentrations and control was small (20 – 25 cm). It is possible that the cages were saturated with the chemical, because of limited air movement, and mosquitoes were no longer able to assess the differences between the oviposition sites. Furthermore, the possibility that nonane is stimulating and

28

not attracting is not excluded. When nonane is stimulating and the cages are saturated, mosquitoes will sense nonane everywhere in the cage and will be stimulated to oviposit in the first oviposition site they encounter, which may also be the control. This goes for both the dose response as the dual choice experiments. Therefore, this setting may not be suitable for dose response experiments. Furthermore, it is possible that all concentrations tested in the second dose response experiments of nonane are attractive/stimulating, this his difficult to detect in this set up, and should be tested in another setting. In the semi-field experiments, the distance between the control and treated bowl is larger and there is a movement of air. The odour plume may be distinguishable by the mosquitoes, which is less likely in the laboratory cages, and detection of nonane may stimulate the mosquitoes to oviposit in the bowl containing nonane. Possibly, because that bowl was closest to the plume, or because the mosquitoes use the odour plume to lead them towards its source. In terms of attractiveness, 2,4-pentanedione in a concentration of 5.5 * 10-10 ml / ml showed the most promising results of the four chemicals in the different concentrations, where attraction of mosquitoes was concerned. Although this compound did not cause a significant difference in oviposition choice compared to the controls, mosquitoes exposed to this concentration laid most eggs. The cages containing this compound in the dose response experiments received significantly more eggs than the cages containing cups with water only, suggesting a stimulating effect on oviposition. In the further experiments no significant differences were found in the choices the mosquitoes made between cups containing this compound or the control. In the dual choice experiments however, one hundred percent of the mosquitoes that developed eggs and were exposed to this chemical oviposited, suggesting a stimulating effect on oviposition again. Only few records were found in which 2,4-pentanedione is assumed to affect insects. It has a moderately toxicity when a broad range of aquatic species is concerned, including a chironomid species (Devillers et al., 1992). Furthermore, it is found in the wet faeces of larvae of different species of butterflies of the Pieris genus. The faeces is known to attract the parasitoid of these species (Agelopous et al., 1995). More data concerning the effect of this chemical on oviposition is not found, and it may be interesting to test the effect of 2,4-pentanedione in greater detail, especially concerning the stimulation of oviposition. At least, the semi-field experiments with the chemical need

29

to be performed. It will be interesting to see whether the results will show the same trend as nonane did. The blends of nonane and 2,4-pentanedione in the dual choice experiments did not affect the choice for an oviposition site, there were no significant differences between the control and the treatment. But, the combination of these two compounds in three different concentrations resulted in the oviposition of every egg that was developed. Not a single retained egg was to be found in 18 mosquitoes exposed to these combinations of volatiles, whereas exposure to water only, egg retention was observed. This suggests that the blend of the volatiles found in both L1 and L4 larvae have a strong stimulating effect on the gravid females. More experiments have to be performed, however, to study this in greater detail. 5.3

Comparison between chemicals and larvae

The results obtained in the experiments with the semiochemicals present in the headspace of water with larvae are different from the results obtained with the larvae themselves. Whereas the larvae give clear results between treatment and control in dual choice experiments, in terms of number of eggs oviposited, the chemicals in the tested concentrations do not. The effects of the chemicals are comparable to the effects of larvae, however. It was expected that DMDS and DMTS would give the same results as L4 larvae, repellent or deterrent. But DMDS (5.5 * 10-7 ml / ml) and DMTS (5.5 * 10-11 ml / ml) inhibit or suppress oviposition, and DMTS in this concentration seems to have a poisonous effect which causes mosquitoes to resorb the eggs. Both L4 larvae and DMDS and DMTS have a negative effect on oviposition by Anopheles gambiae. Furthermore, it was expected that nonane and 2,4-pentanedione, which are present in headspace collected from L1 as well as L4 larvae, would receive more eggs than the controls. This was not the case, but these compounds seem to have a positive effect on oviposition, and are therefore comparable to L1 larvae. The fact that the compounds showing a positive effect on oviposition are found in both young and old larvae, but only young larvae have a positive effect on oviposition, and DMDS and DMTS are only present in L4 larvae, suggests that these compounds mask the positive effects in L4 larvae. This is a strong argument in favour of this hypothesis, which was postulated earlier (Schoelitsz, 2008).

30

It should be noted that a large range of concentrations has been tested, but the concentration of the chemicals released by the larvae are not known. It is possible that there are more effective concentrations that have not been tested in this study. 5.4

Application and further research

Application of these chemicals in the field needs to be tested in the semi-field, before field experiments can be conducted. This has been done for DMTS and nonane, in which nonane attracted gravid mosquitoes to oviposit. When nonane could be implemented in a trap, it is possible to catch gravid mosquitoes before they get a chance to oviposit. The effect of DMDS in the semi-field is yet to be tested. Would it prove to be a repellent in the semi-field, the application of nonane in combination with DMDS would be interesting. By providing DMDS in a village, mosquitoes can be repelled from the houses. By placing a trap with nonane in a short range from DMDS, the mosquitoes may be lured to the nearby trap containing nonane. A push-pull system is created. The effects of DMTS on eggs during the development of eggs have to be tested, when it inhibits development, DMTS may prove a strong chemical in the reduction of the productivity of the mosquito. But the effectiveness of such systems is also dependent on the residual effect of the chemicals. When the effect of the chemicals is wearing off too quickly, application in the field may prove to be futile. Further studies will have to reveal the possibilities of application of these chemicals in field situations. Furthermore, it should be tested whether these concentrations of tested compounds affect other vector species, like Anopheles arabiensis Giles and Anopheles funestes Patton. Together, these three mosquitoes are considered the major vectors of malaria in Africa (Mouchet, 1998) and the effectiveness of such methods would be increased greatly when not one, but all three of these species are affected.

5.5

Acknowledgement

I would like to thank Prof. Takken for giving me this opportunity, Dr. Mboera and Dr. Magesa for having me work at NIMR, Victor Mwingira for the succesfull cooperation, Greek, Ben, Mr. Miyamba and everyone else at Ubwari for their help and support. And last but certainly not least, Aza Kimambo and Mama K. and their family for taking care of me and showing me the Tanzanian way of living.

31

6.

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Du, Y., J. G. Millar, 1999. Electroantennogram and oviposition bioassay responses of Culex quinquefasciatus and Culex tarsalis (Diptera: Culicidae) to chemicals in odors from Bermuda grass infusions. J. Med. Entomol. 36(2): 158166 (1999). Dugravot, S., A. Sanon, E. Thibout, J. Huignard, 2002. Susceptibility of Callosobruchus Dinarmus

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Khoga, J. M., E. Tóth, K. Márialigeti, J. Borossay, 2002. Fly-attracting volatiles produced by Rhodococcus fascians and Mycobacterium aurum isolated from myiatic lesions of sheep. Journal of Microbiological Methods 48 (2002) 281–287. Kramer, W.L., M. S. Mulla, 1979. Oviposition attractants and repellents of mosquitoes: Oviposition responses of Culex mosquitoes to organic infusions. Environmental Entomology, Vol 8, Nr 6, 1979 , pp. 1111-1117(7).

Magnarelli, L. A., 1983. Resorption of retained eggs and follicular degeneration in mosquitoes (Diptera: Culicidae). Journal of Medical Entomology, Vol. 20, no. 1, 106-107, 1983.

Mboera, L. E. G., W. Takken, K. Y. Mdira, G. J. Chuwy, J. A. Pickett, 2000a. Oviposition and behavioural responses of Culex quinquefasciatus to skatole and synthetic oviposition pheromone in Tanzania. Journal of Chemical Ecology, Vol. 26, No. 5, 2000.

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Millar, J. G., J. D. Chaney, J. W. Beehler, M. S. Mulla, 1994. Interaction of the Culex quinquefasciatus egg raft pheromone with a natural chemical associated with oviposition sites. Journal of the American Mosquito Control Association, 10(3): 374-9, 1994. Moore, J. G., L. D. Jessop, D. N. Osborne, 1987. Gas-chromatographic and massspectrometric analysis of the odor of human feces. Gastroenterology, 93(6):1321-9, 1987.

Mouchet, J., S. Manguin, J. Sircoulon, S. Laventure, O. Faye, A. W. Onapa, P. Carnevale, J. Julvez, D. Fontenille. Evolution of malaria in Africa for the past 40 years: Impact of climatic and human factors. Journal of the American Mosquito Control Association, 14(2): 121-130, 1998.

Mwingira, V.S., J. Spitzen, L.E.G. Mboera, J.L. Torres-Estrada, W. Takken, in prep. The influence of larval stage and density on oviposition site-selection behaviour of the Afro-tropical malaria mosquito Anopheles gambiae Giles sensu stricto.

Otieno, W. A., T. O. Onyango, M. M. Pile, B. R. Laurence, G. W. Dawson, L. J. Wadhams, J. A. Pickett, 1988. A field trial of the synthetic oviposition pheromone with Culex quinquefasciatus Say (Diptera: Culicidae) in Kenya. Bulletin of Entomological Research 78(3) p. 463-478.

Perich, M. J., A. Kardec, I. A. Braga, I. F. Portal, R. Burge, B. C. Zeichner, W. A. Brogdon, R. A. Wirtz, 2003. Field evaluation of a lethal ovitrap against dengue vectors in Brazil. Medical and Veterinary Entomology (2003) 17, 205–210.

Schoelitsz, B., 2008. The effects of larval age composition and female body size on the oviposition behaviour of Anopheles gambiae s.s.. MSc thesis report, Wageningen University, 2008.

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Soler, R., J. A. Harvey, A. F. D. Kamp, L. E. M. Vet, W. H. Van der Putten, N. M. Van Dam, J. F. Stuefer, R. Gols, C. A. Hordijk, T. M. Bezemer, 2007. Root herbivores influence the behaviour of an aboveground parasitoid through changes in plant-volatile signals. Oikos, 116: 367-376, 2007. Sumba, L. A., C. B. Ogbunugafor, A. L. Deng, A. Hassanali, 2008. Regulation of oviposition in Anopheles gambiae s.s.: Role of inter- and intra-specific signals. Journal of Chemical Ecology (2008) 34:1430–1436.

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7.

Appendix

7.1

Dose response

Tables with raw data of the dose response experiments, showing the number of eggs found in the cups containing different cups, after one and two nights, including the total amount and the average and standard error for the tested chemicals and water. DMDS Treatment Control

Concentration 1

5.5*10 -7 Concentration 2

5.5*10

-8

Concentration 3

5.5*10 -9

Number of eggs Replicate

Total

Average

S.e

59 245 0 0 0 37

59 245 0 0 0 37

101.3333

73.82487

12.33333

12.33333

1 2 3

0 0 0

158 103 36

158 103 36

99

35.27511

1 2 3

0 0 0

250 310 118

250 310 118

226

56.70979

Number of eggs

Treatment Control

Concentration 1 -10

Concentration 2

5.5*10 -11 Concentration 3

5.5*10

36 hours 0 0 0 0 0 0

DMDS

5.5*10

12 hours

1 2 3 1 2 3

-12

Replicate

12 hours

36 hours

Total

Average

S.e

1 2 3 1 2 3

0 0 0 0 0 0

88 64 108 21 5 74

88 64 108 21 5 74

86.66667

12.71919

33.33333

20.85133

1 2 3 1 2 3

0 0 0 0 0 0

91 0 2 42 20 196

91 0 2 42 20 196

31

30.00556

86

55.36545

37

Number of eggs

DMTS Treatment Control

Concentration 1

5.5*10 -7 Concentration 2

5.5*10

-8

Concentration 3

5.5*10

-9

DMTS Treatment

Replicate 12 hours 36 hours Total 1 72 0 2 0 1 3 25 11

72 1 36

Average S.e 36.33333 20.49661

1 2 3 1 2 3

66 38 0 0 0 0

74 0 0 43 0 27

140 38 0 43 0 27

59.33333

41.79846

23.33333

12.54769

1 2 3

0 12 0

3 12 21

3 24 21

16

6.557439

Number of eggs Replicate

12 hours

36 hours

Total

Average

S.e

Control

1 2 3

0 0 21

0 0 0

0 0 21

7

7

Concentration 1

1 2 3

0 0 56

0 0 0

0 0 56

18.66667

18.66667

1 2 3

0 0 15

0 0 0

0 0 15

5

5

1 2 3

0 0 0

0 0 40

0 0 40

13.33333

13.33333

5.5*10

-10

Concentration 2

5.5*10

-11

Concentration 3

5.5*10 -12

38

Nonane Treatment Control

Concentration 1

5.5 * 10

-7

Concentration 2

5.5 * 10

-8

Concentration 3

5.5 * 10 -9

Number of eggs Replicate

Total

Average

130 114 0 1 3 123

130 224 105 1 17 167

1 2 3

0 160 28

140 12 0

1 2 3

0 0 107

266 129 81

S.e

153

36.22614

61.66667

52.86881

140 172 28

113.3333

43.65521

266 129 188

194.3333

39.67507

Number of eggs

Treatment Control

Concentration 1 -10

Concentration 2

5.5 * 10 -11 Concentration 3

5.5 * 10

36 hours

0 110 105 0 14 44

Nonane

5.5 * 10

12 hours

1 2 3 1 2 3

-12

Replicate

12 hours

36 hours

Total

Average

S.e

1 2 3

0 0 0

37 63 101

37 63 101

67

18.58315

1 2 3

0 0 36

20 105 32

20 105 68

64.33333

24.60578

1 2 3 1 2 3

0 0 0 0 0 70

164 79 69 0 79 69

164 79 69 0 79 139

104

30.13857

72.66667

40.2506

39

2,4-pentanedione Treatment Control

Number of eggs Replicate

12 hours

36 hours

Total

Average

S.e

1 2 3 1 2 3

98 104 35 0 82 164

19 21 33 45 15 3

117 125 68 45 97 167

103.3333

17.81697

103

35.34591

Concentration 2 5.5 * 10-8

1 2 3

0 0 300

0 45 311

118.6667

97.04008

45 11

Concentration 3 5.5 * 10-9

1 2 3

135 0 132

79 69 0

214 69 132

138.3333

41.97751

Concentration 1 5.5 * 10-7

Number of eggs

2,4-pentanedione Treatment

Replicate

12 hours

36 hours

Total

Average

S.e

Control

1 2 3

14 0 32

215 19 0

229 19 32

93.33333

67.93706

Concentration 1 5.5 * 10-10

1 2 3

238 0 257

0 153 42

238 153 299

230

42.33596

Concentration 2 5.5 * 10-11

1 2 3 1 2 3

0 0 21 198 0 19

5 49 82 49 125 0

5 49 103 247 125 19

52.33333

28.33922

130.3333

65.87193

Concentration 3 5.5 * 10-12

40

Number of eggs

Water Cup 1

2

3

4

Replicate 12 hours 36 hours Total Average S.e 1 55 0 55 98.66667 26.18609 2 138 0 138 3 71 0 71 4 12 0 12 5 128 0 128 6 188 0 188 1 2 3 4 5

18 70 27 37 34

0 0 0 0 34

18 70 27 37 68

6 1 2 3 4 5

53 12 49 179 89 0

0 0 0 43 0 0

53 12 49 222 89 0

6 1 2 3 4 5 6

19 123 12 39 45 134 21

0 0 13 38 0 16 0

19 123 25 77 45 150 21

41

45.5

8.819486

65.16667

33.97589

73.5

21.7834

7.2

Dual choice

Data of dual choice experiments of DMDS (2 concentrations), DMTS (2 concentrations), nonane, 2,4-pentanedione and water (control experiment). Tables show the number of eggs oviposited by a single mosquito in the control or treatment and the number of eggs each mosquito retained. Mosquitoes that did not develop eggs, or did not oviposit and data of egg retention was not available were excluded from the analysis and are excluded in these tables.

DMDS

5.5*10

-7

# eggs Control Replicate 1 3 5 6 7 8

Date

12 h

Treatment 36 h

17-18-sept 17-18-sept 17-18-sept 17-18-sept 17-18-sept 17-18-sept

0 0 0 0 0 100

10 17-18-sept 11 11-12-oct 12 13 14 15 16

Total

12 h

36 h

0 0 0 0 0 0

0 0 0 0 0 100

0 0 0 0 0 0

36

0

36

0

29

29

11-12-oct 11-12-oct 11-12-oct 11-12-oct 11-12-oct

0 0 0 0 0

37 0 0 0 0

17 11-12-oct

0

83

18 11-12-oct

0

0

19 11-12-oct

0

23

20 11-12-oct

0

0

0

Total

# Retained eggs

0 0 0 0 0 0

0 0 0 0 0 0

158 65 66 59 112 0

41

0

41

0

0

17

17

0

37 0 0 0 0

0 0 56 66 0

0 0 0 0 0

0 0 56 66 0

0 123 0 0 98

83

0

0

0

0

0

0

0

0

100

23

48

0

48

0

0

0

0

104

Average

18.11765

13.41176

52.05882

S.E.

7.510719

5.658192

13.42831

42

DMDS 5.5*10

-9

# eggs Control 12 h

Treatment

Replicate

Date

1 2 3 4 5 6

17-18-sept 17-18-sept 17-18-sept 17-18-sept 17-18-sept 17-18-sept

0 88 25 0 0 0

36 h 0 0 0 0 0 0

Total

9

17-18-sept

101

0

12 h

36 h

Total

0 88 25 0 0 0

144 0 121 0 0 104

0 0 0 0 48 0

101

0

0

# Retained eggs 144 0 121 0 48 104

0 0 ************ 77 ************ ************

0

0

Average

35.66667

69.5

19.25

S.E.

19.09392

25.50523

19.25

DMTS

5.5*10

-9

# eggs Control Replicate 1 2 4 5 7 8

Date 19-20-sept 19-20-sept 19-20-sept 19-20-sept 19-20-sept 19-20-sept

9 10 Average S.E.

12 h

Treatment 36 h

Total

12 h

36 h

Total

# Retained eggs

0 0 0 0 0 0

0 0 9 49 37 0

0 0 9 49 37 0

0 0 0 0 0 0

0 0 9 0 0 0

19-20-sept

0

0

0

0

71

71

0

19-20-sept

0

0

0

0

6

6

0

11.875 6.973003

43

0 59 0 60 9 0 0 0 0 ************ 0 82

10.75 8.694723

28.71429 13.83012

DMTS

5.5*10

-11

# eggs Control Replicate

Date 3 4 5 6 7 8

Treatment

12 h

36 h

Total

12 h

36 h

Total

# Retained eggs

19-20-sept 19-20-sept 19-20-sept 19-20-sept 19-20-sept 19-20-sept

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

51 62 45 88 50 86

9 19-20-sept

0

0

0

0

0

0

56

10 19-20-sept

0

0

0

0

0

0

92

11-12-oct 11-12-oct 11-12-oct 11-12-oct 11-12-oct

2 49 76 1 0

0 0 0 0 0

2 49 76 1 0

18 0 0 37 7

0 0 0 0 0

18 0 0 37 7

0 0 0 0 2

18 11-12-oct

0

14

14

5

2

7

0

19 11-12-oct

0

0

0

6

0

6

0

20 11-12-oct

0

0

0

0

0

0

62

11 13 14 16 17

Average S.E.

8.875

4.6875

37.125

5.445851

2.479447

9.015207

Nonane 5.5*10-11 # eggs Control Replicate Date 2 3 4 6 7 8

12 h

Treatment 36 h

Total

12 h

36 h

Total

# Retained eggs

17-18-sept 17-18-sept 17-18-sept 17-18-sept 17-18-sept 17-18-sept

46 0 19 72 141 1

0 0 38 0 0 0

46 0 57 72 141 1

0 0 0 0 10 0

0 146 18 0 0 0

0 146 18 0 10 0

0 0 0 0 0 0

9 17-18-sept

0

0

0

0

0

0

120

10 17-18-sept

109

0

109

0

0

0

0

11 13 14 15 16

28-29-sept 28-29-sept 28-29-sept 28-29-sept 28-29-sept

13 80 110 6 0

0 0 1 0 0

13 80 111 6 0

0 0 0 94 29

0 0 0 0 0

0 0 0 94 29

0 0 0 0 0

17 28-29-sept

49

0

49

77

0

77

0

18 28-29-sept

0

0

0

0

0

0

150

19 28-29-sept

28

0

28

0

0

0

0

20 28-29-sept

91

0

91

34

0

34

0

Average

47.29412

24

15.88235

11.29151

10.27382

10.94971

44

2,4-pentanedione

5.5*10

-10

# eggs Control Replicate 1 2 3 5 6 7

12 h

Treatment 36 h

Total

12 h

36 h

Total

# Retained eggs

21-22 sept 21-22 sept 21-22 sept 21-22 sept 21-22 sept 21-22 sept

22 119 61 47 58 0

0 0 0 0 0 0

22 119 61 47 58 0

22 15 75 56 6 9

0 0 0 0 0 0

8 21-22 sept

0

4

4

0

0

0

90

9 21-22 sept

0

0

0

117

0

117

0

0 65 0 24 62

0 0 0 0 0

0 65 0 24 62

93 6 12 0 0

0 0 0 0 0

93 6 12 0 0

0 0 0 0 0

48

0

48

49

0

49

0

10 11 14 15 17

21-22 sept 11-12 oct 11-12 oct 11-12 oct 11-12 oct

20 11-12 oct

22 0 15 0 75 0 56 0 6 0 9 ************

Average

36.42857

32.85714

6.923077

S.E.

9.471017

10.3222

6.923077

Water # eggs Cup A Replicate

Date

1 2 3 4 5 6 7 8 9 10 Average S.E.

24-25-dec 24-25-dec 24-25-dec 24-25-dec 24-25-dec 24-25-dec 24-25-dec 24-25-dec 24-25-dec 24-25-dec

12 h 81 5 0 0 0 44 0 0 0 0

Cup B 36 h

Total 0 9 0 0 0 0 60 0 0 0

81 14 0 0 0 44 60 0 0 0 19.9 9.622254

45

12 h 7 0 0 41 65 0 53 160 56 3

36 h 0 87 11 0 0 0 0 0 0 0

Total

# Retained eggs

7 87 11 41 65 0 53 160 56 3 48.3 15.58849

0 0 0 0 0 0 0 0 0 22 2.2 2.2

The data of dual choice experiments of the blends in the different concentrations are shown below. Mosquitoes that did not develop eggs, or did not oviposit and data concerning egg retention was not available were excluded from analysis and these tables. DMDS 5.5*10-7 + DMTS 5.5*10 -11

Blend R

# eggs Control Replicate 1 2 3 4 5 6

Date

Treatment

12 h

36 h

Total

12 h

36 h

Total

# Retained eggs

24-25-sept 24-25-sept 24-25-sept 24-25-sept 24-25-sept 24-25-sept

0 0 0 0 0 77

6 89 0 0 0 2

6 89 0 0 0 79

0 0 2 85 0 96

5 0 4 0 0 0

5 0 6 85 0 96

171 0 0 0 157 0

7 24-25-sept

163

0

163

0

0

0

0

9 24-25-sept

80

0

80

0

0

0

0

Average S.E. Blend R 100 x dilluted

52.125

24

41

21.31016

14.5737

26.87338

-9

DMDS 5.5*10 + DMTS 5.5*10

-13

# eggs Control Replicate 1 2 3 4 5 6

Date

Treatment

12 h

36 h

Total

12 h

36 h

Total

# Retained eggs

3-4-Oct 3-4-Oct 3-4-Oct 3-4-Oct 3-4-Oct 3-4-Oct

0 0 22 0 0 0

0 1 0 0 0 0

0 1 22 0 0 0

0 0 52 0 124 0

10 0 0 37 0 2

7 3-4-Oct 8 3-4-Oct

0

75

75

0

3

3

0

0

29

29

0

48

48

0

9 3-4-Oct

0

26

26

0

0

0

0

10 3-4-Oct

0

3

3

0

0

0

0

Average S.E.

15.6 7.602923

46

10 0 0 ************ 52 0 37 0 124 10 2 ************

27.6 12.58235

1.25 1.25

Blend R 1000 x dilluted

DMDS 5.5*10-10 + DMTS 5.5*10-14 # eggs

Control Replicate 1 2 4 7 8 9

Date

Treatment

12 h

5-6-Oct 5-6-Oct 5-6-Oct 5-6-Oct 5-6-Oct 5-6-Oct

36 h 0 0 0 14 0 29

Total 0 0 0 0 0 0

12 h 0 0 0 14 0 29

36 h

22 0 137 0 0 39

Total 0 4 0 0 0 0

# Retained eggs

22 ************ 4 0 137 0 0 0 0 84 39 0

Average

7.166667

33.66667

16.8

S.E.

4.928939

21.59424

16.8

Blend A

Nonane 5.5*10-11 + 2,4-pentanedione 5.5*10-10 # eggs

Control Replicate 1 2 3 4 5

Date

Treatment

12 h

36 h

Total

12 h

36 h

Total

# Retained eggs

24-25-sept 24-25-sept 24-25-sept 24-25-sept 24-25-sept

133 55 1 0 97

4 0 0 2 0

137 55 1 2 97

0 8 30 0 0

15 0 0 1 0

15 8 30 1 0

0 0 0 0 0

7 24-25-sept

0

0

0

0

20

20

0

8 24-25-sept

24

0

24 45.14286 20.42341

53

0

53 18.14286 7.062404

0

Average S.E.

Blend A 100 x dilluted

0 0

Nonane 5.5*10-13 + 2,4-pentanedione 5.5*10-12 # eggs

Control Replicate 3 4 5 6 7 Average S.E.

Date

Treatment

12 h

36 h

Total

12 h

36 h

Total

3-4-Oct 3-4-Oct 3-4-Oct 3-4-Oct 3-4-Oct

26 0 4 0 0

2 0 116 22 0

28 0 120 22 0

0 59 5 0 0

4 0 5 0 54

8 3-4-Oct

0

14

14

0

2

30.66667 18.45836

47

# Retained eggs

4 59 10 0 ************ 54 2 21.5 11.17065

0 0 0 0 0 0 0

Blend A 1000 x dilluted

Nonane 5.5*10

-14

+ 2,4-pentanedione 5.5*10

-13

# eggs Control Replicate 1 2 3 5 7

Date

Treatment

12 h

36 h

Total

12 h

36 h

Total

5-6-Oct 5-6-Oct 5-6-Oct 5-6-Oct 5-6-Oct

53 68 4 0 36

0 0 0 0 0

53 68 4 0 36

20 53 0 139 70

0 0 0 0 0

10 5-6-Oct

73

0

73

49

0

Average S.E

39 12.84264

7.3

# Retained eggs

20 53 0 139 70

0 0 0 0 0

49

0

55.16667 19.6391

0 0

Sticky-screen bioassay

Tables showing the data of the sticky-screen bioassays are shown below. Data included are the number of mosquitoes that where trapped on cups containing a treatment of DMDS, DMTS, nonane and 2,4-pentanedione and water as a control. DMDS Replicate

Date

5.5 * 10

Control

Treatment 12

1 2 3 4 5 6 7 8 9 10 11

-7

36 Total

12

36 Total

7-8-Oct 7-8-Oct 7-8-Oct 7-8-Oct 7-8-Oct 7-8-Oct 13-14-Oct 13-14-Oct 13-14-Oct 13-14-Oct 13-14-Oct

0 1 2 0 1 0 5 3 3 4 4

6 3 4 1 2 2 2 1 2 3 0

6 4 6 1 3 2 7 4 5 7 4

0 1 0 0 0 1 3 3 1 3 4

0 1 2 2 3 1 0 0 1 0 0

0 2 2 2 3 2 3 3 2 3 4

12 13-14-Oct

2

1

3

1

4

5

Average SE

48

4.333333

2.583333

0.55505

0.35799

DMTS Replicate

Date

5.5 * 10

Control

Treatment 12

1 2 3 4 5 6 7 8 9 10 11

-11

36 Total

7-8-Oct 7-8-Oct 7-8-Oct 7-8-Oct 7-8-Oct 7-8-Oct 13-14-Oct 13-14-Oct 13-14-Oct 13-14-Oct 13-14-Oct

0 0 1 0 3 1 5 2 0 4 3

4 3 6 2 2 2 0 0 1 0 0

12 13-14-Oct

4

0

12

36 Total

4 3 7 2 5 3 5 2 1 4 3

0 2 0 1 0 1 2 4 2 4 1

4 4 0 2 0 2 0 0 0 0 0

4

1

0

4 6 0 3 0 3 2 4 2 4 1 1

Average

3.583333

2.5

SE

0.468045

0.529437

Nonane Replicate

Date

5.5 * 10

Control

Treatment 12

1 2 3 4 5

-10

36 Total

9-10-Oct 9-10-Oct 9-10-Oct 9-10-Oct 9-10-Oct

1 4 2 0 4

1 0 0 1 1

6 9-10-Oct

1

1

12

36 Total

2 4 2 1 5

3 4 1 1 1

1 1 1 2 0

2

5

1

4 5 2 3 1 6

Average

2.666667

3.5

SE

0.614636

0.763763

2,4-9 pentanedione 5.5 * 10 Replicate Date

Control

Treatment 12

1 2 3 4 5

36 Total

9-10-Oct 9-10-Oct 9-10-Oct 9-10-Oct 9-10-Oct

0 2 1 2 2

0 2 0 2 0

6 9-10-Oct

3

0

12

36 Total

0 4 1 4 2

0 1 3 0 0

0 2 1 3 3

3

1

0

0 3 4 3 3 1

Average

2.333333

2.333333

SE

0.666667

0.614636

49

Water Replicate Date

Cup 1

Cup 2 12

36 Total

12

36 Total

1 9-10-Oct 2 9-10-Oct 3 9-10-Oct

3 2 2

0 0 0

3 2 2

1 1 4

2 0 1

3 1 5

4 13-14-Oct 5 13-14-Oct

3 3

3 0

6 3

5 6

0 0

5 6

6 13-14-Oct

3

1

4

3

0

3

Average

3.333333

3.833333

SE

0.614636

0.749074

7.4

Physiological experiments

Tables below show the number of eggs that are oviposited in each cage in the physiological experiments. Furthermore there are tables showing the number of eggs that were retained by the mosquitoes. Experiments were done with DMTS in a concentration of 5.5 * 10-11 and the solvent in the same concentration as a control.

DMTS 5.5*10 -11

Cage Day

Nr of eggs oviposited

1

2

3

1

0

51

15

2

12

0

0

0

12

51

15

143

3

25

101

0

4

0

27

0

25

Total

Total

4 Average

128

0

5

0

0

6

0

0

0

0

Total 7

0

8

0

Total

0

50

S.E.

143 55.25

30.56244

51

39.17057

0

0

0

5.5*10

DMTS

-11

ml/ml

Cage

1

2

3

4

0 75 71 0 0 0 0 106 130 90 110 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 2 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

Average

44.76923

0

0.153846

0

S.E

14.53252

0

0.153846

0

# eggs retained

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Control

Cage Day

Nr of eggs

1

2

1

78

240

0

117

2

0

0

140

0

78

Total

3

4 Average

240

140

117

3

147

0

1

4

140

65

167

287

Total

65

168

5

0

0

6

0

0

0

0

Total 7

0

8

0

Total

0

51

S.E.

143.75

34.54074

173.3333

64.14134

0

0

0

Control Cage # eggs retained

1

2

3

4

0 56 0 0 87 0 75 0 0 0 49 69 0

0 0 50 0 104 0 0 0 0 0 0 0 0

90 0 0 50 0 0 0 0 0 98 0 0 0

0 0 0 0 12 55 0 0 0 0 1 0

14

0

0

15

0

1 2 3 4 5 6 7 8 9 10 11 12 13

Average

25.84615

10.26667

17

5.666667

S.E

9.742746

7.475335

9.422326

4.593034

7.5

Semi-field experiments

Following tables contain the data concerning the semi-field experiments with DMTS 5.5 * 10-11 and nonane 5.5 * 10-11. Tables show the number of eggs oviposited in the bowls containing the chemicals and the controls, i.e. the solvent in the same concentration as in the treatments. Further information about the set up are included (date of start of experiment, colour of the bowl, the sphere used, position of treatment and whether it rained during the nights of the experiments).

52

DMTS

5.5 * 10

Number of eggs Control Day1 Day2

Treatment Day 1

Total

Day2

Starting date

Total

Colour bowl

Sphere

Side of treatment

Rainfall

1 2 3 4 5

5 238 238 265 56

22 39 86 243 0

27 277 324 508 56

340 194 228 357 6

160 28 499 337 38

500 222 727 694 44

05-Oct 11-Oct 13-Oct 13-Oct 15-Oct

Green Green Green Green Green

A A B C A

Right Left Right Right Left

No Yes No No Yes

6

21

27

48

135

32

167

15-Oct Green

C

Left

Yes

206.6667

79.51338

Average and SE

Nonane

5.5 * 10

Number of eggs Control Day1 Day2

Average and SE

-11

392.3333

117.7174

-11

Treatment Day 1

Total

Day 2

Starting date

Total

1 2 3 4 5

58 187 140 686 23

36 197 46 17 5

94 384 186 703 28

505 487 151 974 847

115 239 185 223 63

620 726 336 1197 910

6

89

7

96

24

12

36

248.5

104.0166

637.5

53

Colour bowl

Sphere

Side of treatment

Rainfall

Green Green Green Green Green

A B C C A

Right Right Right Left Right

No No No Yes No

15-Oct Green

B

Left

Yes

19-Sep 05-Oct 05-Oct 11-Oct 13-Oct

168.1408