Laboratory experiments were conducted at the Laboratory of Entomology of Wageningen University & Research in The Netherlands and at the Amani Research Centre of the National Institute for Medical Research, Muheza, Tanzania. The semi-field study was conducted at the Amani Research Centre in Tanzania.
Insects and Rearing Procedures
We used Anopheles coluzzii originating from Suakoko, Liberia, previously known as An. gambiae s.s. M form (Coetzee et al. 2013) that was reared at the Laboratory of Entomology, Wageningen University & Research, The Netherlands. Larvae were raised under standardized conditions (water surface >2 cm2 per larva), in a climate-controlled chamber at 28 °C and 80% relative humidity, with a 12:12 h L:D photoperiod. Larvae were reared in 2.5 l plastic trays filled with acclimatized tap water and were fed 0.003 g/larva Tetramin® fish food (Tetra Werke, Germany per day). Pupae were collected daily and placed in small cups inside a 30 × 30 × 30 cm Bugdorm® cage (https://www.shop.bugdorm.com) for emergence. Adults (males and females) were kept in a Bugdorm® cage with ad libitum access to a 6% glucose solution. When 3–5 days old, females were fed blood by offering a human arm. Gravid mosquitoes from this group were used to study response to volatiles produced by larvae in the laboratory. Ethical approval for blood feeding was not requested as this method of blood feeding is not subject to the Dutch Act of Medical Research involving Human Subjects (WMO). In our anopheline mosquito cultures, no experimental infections took place and mosquitoes were free of any parasite.
At the Amani Research Centre, adult An. gambiae s.s. (originating from Ifakara, southern-central Tanzania) were kept in a 30 × 30 × 30 cm metal framed cage covered with netting. Larvae were reared in round aluminium pans with a diameter of 27 cm, filled with filtered tap water to a depth of 2 cm. Larvae were fed on Tetramin® fish food (Tetra Werke, Germany) and were kept in a 12:12 h L:D light regime. The temperature in the insectarium was 29 °C. Pupae were removed from the trays daily and were placed in the mosquito cages for emergence. Male and female mosquitoes were kept in the same cages. For blood feeding, 3–5 days old females were offered a human arm. An approval involving human subjects in blood feeding mosquitoes was obtained from the Medical Research Coordinating Committee of the National Institute for Medical Research in Tanzania. The same volunteer donated blood to all batches of mosquitoes throughout the study, and mosquitoes were fed blood only once during their lifetime. Gravid mosquitoes from this group were used to study the response to infochemicals in the laboratory and semi-field experiments.
Oviposition in Response to Larvae
Experiments concerning the oviposition behavior in response to the presence of first or fourth instars were performed at the Laboratory of Entomology in Wageningen. The aim of this experiment was to investigate if the concept that larval habitats of An. coluzzii emit chemical cues that mediate oviposition behavior in conspecific adults. Nine-day old female An. coluzzii were fed blood on a human arm for ten minutes, two days before the start of the experiment and were kept as described above.
Early-stage larvae (L1) were collected two days after oviposition using a glass pipette. Water drops with larvae were placed on the bottom of a white, dry rearing tray and larvae were counted. Late-stage larval instars (L3/L4) were collected with a plastic pipette (5 ml) and were counted in a similar way as L1 larvae. A total of 100 larvae of the same developmental stage were placed in a plastic oviposition cup (5.25 cm diameter × 3 cm height). The volume of rearing water was removed to a minimum before the transfer of larvae and after the transfer of the larvae cups were filled with tap water to a volume of 30 ml.
Wet filter paper, 125 mm in diameter, Whatman® (Whatman International Ltd., Maidstone, England) was placed over the cup, serving as an oviposition site for the mosquitoes thus preventing stimulation by visual stimuli. To prevent drying out of the oviposition paper, a cylinder made of filter paper was placed in the cups (Fig. 1, left panel). This cylinder ensured that when the water level in the cup decreased, the oviposition paper remained wet. Moreover, because of the cylinder, the oviposition paper did not have to be in contact with the liquid, which would decrease the area of the water surface for the larvae to breathe. Larvae were placed within and outside of the cylinder. As a control the cups were filled with 30 ml tap water.
Gravid mosquitoes were held solitary in a 30 × 30 × 30 cm Bugdorm® cage for 48 h – two oviposition periods (Fritz et al. 2008), under the circumstances described above. Each mosquito was given a choice between ovipositing in a treated cup, with either 100 early-stage larvae (L1) or 100 late-stage larvae (L4), and a control cup. Cups were placed diagonally in corners as far from each other as possible, at a distance of approximately 30 cm. Eggs were counted after 24 and 48 h and the total number of eggs after 48 h was taken as the response of the mosquitoes. Each treatment was repeated 17 times.
Collection and Identification of Chemicals
Three procedures were conducted at the Laboratory of Entomology, Wageningen University & Research: (i) a proof of concept for emission of volatile chemicals from larval habitats, (ii) collection of volatile chemicals from larval habitats by headspace techniques and (iii) identification of entrapped chemicals by GC-MS.
Volatile compounds released by water containing either no larvae, early stage or late stage larvae of An. coluzzii were collected from cups filled with 30 ml of tap water placed in separate air-tight cuvettes.
Volatiles were collected using the “purge and trap” approach on an adsorbing polymer: Tenax-TA 20/35 (Alltech, USA). To reduce background volatiles, air was sucked into the cuvette through a carbon filter and a cartridge containing 100 mg Tenax-TA. Headspace volatiles were trapped at a flow rate of 100 ml/min for 24 h on a second cartridge containing 100 mg Tenax TA connected to the outlet of the cuvette. Samples were released from the adsorbent using a thermodesorption unit (Ultra 50:50 TD, Markes, Llantrisant, UK) while re-collected in an electrically cooled cold trap (Unity, Markes) and followed by gas chromatography (Trace GC Ultra) and mass spectrometry (Trace DSQ quadrupole mass spectrometer), both from Thermo (Thermo Fisher Scientific, Waltham, USA).
The program for thermal desorption consisted of dry purging for 3 min and pre-purging for 1 min using helium (residual oxygen removal) at 30 °C. This was followed by tube desorption at 250 °C for 3 min and the volatiles were focused on a cold trap at 0 °C. Injection onto the analytical column was achieved by heating of the cold trap at the maximum heating acceleration (> 60 °C per second) to 250 °C in a split mode at a split ratio of 1:6. The transfer line between the cold trap and the GC was kept at 160 °C throughout the analysis.
A 30 m × 0.25 mm ID × 1.0 μm F.T. capillary GC column (Rtx-5 MS, Restek, USA) with helium (5.0 grade) as carrier gas at a flow rate of 1.0 mL/min was used for separation of volatile compounds. The GC temperature was programmed as follows: 45 °C for 3 min, followed by a ramp of 8 °C/min to 280 °C and was held at 280 °C for 2 min. The transfer line between the GC and MS was set to 275 °C. MS spectra were recorded by ionization of the column effluent by electron impact (EI) ionization at 70 eV, scanning in positive mode from 35 to 300 m/z with a speed of 5 scans per second. The ion source temperature was set to 250 °C and the filament was switched off from 13.6–13.8 min because of a high background peak. Peak identification was performed by comparing the obtained spectra with those in the NIST library (version 2.0 d), experimentally calculated retention indices and using the retention times of authentic synthetic reference compounds.
Chemicals
The synthetic chemicals dimethyl disulfide (DMDS, ≥ 99.0%), dimethyl trisulfide (DMTS, ≥ 98.0%), nonane (≥ 99.0%) and 2,4-pentanedione (2,4-PD), which is also known as acetylacetone (ReagentPlus®, ≥ 99.0%), all from Sigma Aldrich (Sigma Aldrich, Chemie BV, Zwijndrecht, The Netherlands), were used for testing the oviposition response. Since all of these chemicals were insoluble in water, they were dissolved in methanol and Tween20, in the following ratios: 55 g (test chemical) + 40 ml Methanol +5 ml Tween20. Hereafter, the chemicals were dissolved and diluted in distilled water to make 1 l of diluted chemicals and dilution process continued until the required concentrations for bioassay was reached. The final concentrations of all chemicals ranged from 10−7 to 10−12 M.
Oviposition Bioassays
Identified chemicals were tested for effects on oviposition behavior at the Amani Research Centre, Muheza, Tanzania, using An. gambiae s.s mosquitoes (Ifakara strain). Two experiments were conducted: laboratory experiments were performed under the same conditions and with the same materials as was done in Wageningen, with the aim to select and confirm effective doses for each chemical. Semi-field experiments were designed to verify potential attractive/repellent effects of these compounds under natural ambient conditions.
Dose Response Effects on Oviposition
DMDS, DMTS, 2,4-PD and nonane were each tested at six different doses in a four cups choice set up against controls. Gravid An. gambiae s.s (48 h post blood feeding) were placed in a 30 × 30 × 30 cm cage. In each cage cups containing 30 ml of a solution of the chemical in concentrations of 10−7 M, 10−8 M, 10−9 M and control, or in concentrations of 10−10 M, 10−11 M, 10−12 M and control were placed. Each of the four oviposition cups was placed in a corner of the cage. Mosquitoes were given a 6% glucose solution as an additional food source. The determination of the most effective concentration was based on the total number and percentage of eggs found in both control and treated cups after 36 h (two nights).
Dual Choice Experiments with Selected Doses
Based on the results from the dose-response test, dual choice experiments were performed with single compounds. The following concentrations of single compounds were tested against respective controls:
- (a)
DMDS: 10−7 M and 10−9 M
- (b)
DMTS: 10−9 M and 10−11 M
- (c)
2,4-PD: 10−10 M
- (d)
nonane: 10−11 M
Determination of Oviposition Activity and Egg Retention
To ascertain the effect of emitted infochemicals as either attractive or repellent, an oviposition activity index (OAI) was calculated using the formula OAI = (Nt-Nc)/(Nt + Nc) (Kramer and Mulla 1979), with Nt = number of eggs laid in the egg cup with larvae or test compound, and Nc = number of eggs oviposited in the cup with control materials. Individual gravid Anopheles coluzzii females were exposed to emanations of either 100 first or 100 fourth instars; individual gravid females of An. gambiae s.s. were exposed to nonane (10−11 M), 2,4-DP (10−10 M), DMDS (10−7 M) or DMTS (10−9 M), respectively. Each treatment was replicated 17 times.
At the end of the dual-choice experiments in the laboratory, females were killed and the status of their ovaries was examined for egg retention by dissection of the ovaries. The abdomen of the female was placed on a glass slide, opened with fine surgical forceps and the ovaries were gently pulled out and placed in a drop of physiological saline. The ovaries were examined at 400x magnification under a dissecting microscope. The number of mature eggs present per female were counted (Takken et al. 2013).
Semi-Field Oviposition Experiments
The effects of DMTS at a concentration of 10−11 M, DMDS at 10−7 M, nonane at 10−11 M and 2,4-PD at 10−10 M on oviposition response were investigated against their controls (distilled water+methanol+Tween20) in a dual choice assay in a semi-field situation (mosquito spheres) at Muheza in Tanzania, under natural ambient conditions (Knols et al. 2002). The objective was to scale up the exploration into a field situation and compare laboratory with semi-field results. Three mosquito spheres (11.4 × 7.1 × 5.0 m) were used in this study (Fig. 2). During the experimental period, the average temperatures in the spheres ranged from a minimum of 16 °C during the night to a maximum of 32 °C during the day. The average relative humidity (RH) ranged from a minimum of 40% to a maximum of 100%.
Two symmetrical holes were dug in the ground at the centre of each sphere, and were located 3 m apart. A green plastic bowl (diameter 26 cm, height 10 cm) was placed in each hole as an artificial breeding site. The bowls were placed in such a way that the rim of the bowls was at ground level. The bowls had a capacity of 5 l and were filled with 3 l of the test solutions of the concentrations mentioned above or with distilled water.
A total of 240 mosquitoes (An. gambiae s.s.) were given an opportunity to blood feed twice, on day 3 and day 4 after emergence, and were released on day 5, when eggs had matured (Takken et al. 1998). Mosquitoes were released one hour before dusk (at about 18:00 h), from the centre of the sphere between the bowls. Eggs were counted on the first and second morning after releasing mosquitoes and the solutions were replaced after every experiment. The total number of eggs after two nights was taken as the oviposition response. Each pair in this experiment was replicated 17 times.
Data Analysis
Differences in oviposition preferences of mosquitoes were analysed using the Wilcoxon matched-pairs signed rank test and Mann-Whitney test for matched-pairs. This non-parametric test was used because the data were not normally distributed. To compare more than two paired groups, like with the dose response test, the Friedman test was used.
Analysis of OAI data was done by comparing the response value with zero. When the OAI values differed significantly from zero with positive or negative values, the treatment was considered to have significant attractant or repellent effect, respectively, on oviposition behavior of gravid females. Oviposition preference of gravid females was determined by OAI values using the Wilcoxon signed rank test (α = 0.05, two-sided). The OAI was also used to compare behavioral assays involving larvae and chemical assays involving identified infochemicals.
The amount of volatiles quantified in headspace collections was analysed using the Kruskal-Wallis test. Differences in egg retention were analysed using the Mann-Whitney U-test.
All tests were performed in SPSS, version 20 (IBM, Armonk, NY, USA).
Ethical Clearance
The study was conducted according to Standard Operating Procedures approved by the Medical Research Coordinating Committee (MRCC) of the National Institute for Medical Research (NIMR), Tanzania. It received a research permit from MRCC with reference number NIMR/HQ/R.8a/Vol. IX/573 and a permit from the Tanzania Commission for Science and Technology with reference number CST/RCA 138/225/2008. In the Netherlands, ethical approval for blood feeding was not requested as this method of blood feeding is not subject to the Dutch Act of Medical Research involving Human Subjects (WMO).