Experiments were carried out in large netting-screened semi-field cages (10.8 m long × 6.7 m wide × 2.4 m high) at the International Centre of Insect Physiology and Ecology, Thomas Odhiambo Campus (icipe-TOC), located on the shore of Lake Victoria in Mbita, Homa Bay county, western Kenya (geographic coordinates 0⁰ 26’ 06.19” S, 34⁰ 12’ 53.13” E; altitude 1,137 m above sea level). The cages had a sand floor and did not contain any vegetation. Mbita is characterized by tropical climate with an annual average minimum temperature of 16 ºC and maximum temperature of 29 ºC. The area experiences two rainy seasons; the long rains between March and June and the short rains between October and December.
An experimental formulation of dust, with particles 12μm diameter, containing 10% of pyriproxyfen (PPF) (Sumilarv®, Sumitomo Chemical Company) was used in all experiments.
Anopheles gambiae s.s. (Mbita strain) larvae and adults used in this study were obtained from the mosquito insectaries at icipe-TOC. Immature stages were reared in a semi-field cage at ambient conditions with average daily temperature of 25-28 ºC, relative humidity of 68-75% and natural lighting. Mosquito larvae were reared in round plastic tubs (diameter 60 cm) filled with 5 L water (5 cm deep) from Lake Victoria filtered through a charcoal-sand filter. Mosquito larvae were fed with a pinch of fish food (Tetramin©Baby) twice daily. Third (late) instar mosquito larvae were randomly selected from different tubs to ensure that cohorts of larvae used in experiments were a representative sample of the size distribution of the experimental larval population. Adult mosquitoes were held in mosquito-netting covered cages (30 cm x 30 cm x 30 cm) in a holding room with ambient climate conditions and provided with 6% glucose solution ad libitum. Three-day old females were allowed to feed on a human arm on two consecutive nights. Gravid mosquitoes were used for experiments in this study.
Development of a bait-station
Contamination of adult An. gambiae s.s. with PPF. Water vapour has been shown to attract gravid malaria vectors (38) and hence it was considered essential to include water in the bait-station. Females were prevented from accessing the water to lay eggs using fly gauze (black fibre-glass netting gauze, mesh size 1 mm x 1 mm). To determine the best method to treat netting surfaces with PPF for efficient contamination of mosquitoes, preliminary cage tests were conducted in small-sized cages (30 cm x 30 cm x 30 cm). Two methods of applying PPF on the netting gauze that served as the dissemination platforms were compared. First, the netting gauze (diameter 7 cm) was treated with 1 g of PPF dust applied with a soft brush to ensure uniform spreading of the PPF over the netting surface. Second, 1 g of PPF dust was mixed with 2 ml of cooking oil and applied to the netting gauze with a soft brush and left to dry in the air for 30 minutes. The rationale for this was to test a formulation that would be easier to apply and less likely to be distributed by wind.
Each experimental cage was provided with two glass cups (Pyrex®, capacity 100 ml, diameter 7 cm) and the cups were placed at the diagonal corners of the cage, approximately 26 cm apart. The first cup in each cage was filled with 100 ml non-chlorinated tap water and left open for gravid mosquitoes to lay eggs. The second cup, serving as the bait-station, was filled with six-day old soil infusion previously shown to attract gravid An. gambiae s.s. (39). Soil infusion was prepared by incubating 15 L of non-chlorinated tap water with 2 kg of soil collected from a known breeding habitat of An. gambiae sensu lato (39) which was dry at the time of collection. Infusions were prepared in round plastic tubs (diameter 0.42 m) and left for six days before use in experiments as described in detail previously (39). The top of the bait-station in the control cages was covered with untreated netting gauze while in the test cages it was covered with netting gauze treated with either PPF dust or PPF dust formulated in oil. In each cage five gravid An. gambiae s.s. were released at 18:00 h and left overnight. The following morning, open oviposition cups were assessed for the presence of eggs. To confirm the transfer of PPF in test cages, 10 insectary-reared late instar An. gambiae s.s. larvae were introduced into oviposition cups in all cages and monitored for adult emergence. Introduced larvae were fed daily on a pinch of Tetramin®Baby fish food. Pupae that developed were transferred with a small volume of water from the oviposition cup into plastic cups (diameter 7 cm) and monitored for adult emergence. It took 6-7 days for all larvae introduced into the oviposition cups to develop into adults or die. These experiments were conducted over three rounds on separate dates. There were five replicate cages per treatment in each experimental round, thus in total there were 15 cages with untreated bait-stations, 15 cages with bait-stations treated with PPF dust and 15 cages with bait-stations treated with PPF dust formulated in oil. Oviposition cups were randomly allocated to one of the four corners in the cages.
Luring gravid An. gambiae s.s. to a pond. Preliminary experiments were conducted in a semi-field cage (Figure 1) to compare attractiveness of two substrates that attract gravid An. gambiae s.s.: a six-day old soil-infusion (39) and the sesquiterpene alcohol, cedrol (Cedrol ≥99.0% (sum of enantiomers, GC, Sigma-Aldrich, Steinheim, USA) (40). The two substrates were evaluated separately on different dates. Four artificial ponds were created by digging holes in the sand at the four corners of the cage, and each hole was filled with a round enamel tub (diameter 0.42 m, depth 8 cm). The tubs were placed 1 m away from the nearest wall. During each experimental round, three of the tubs were filled with 7 L of non-chlorinated tap water as oviposition habitats while the fourth tub was filled with a test substrate to attract gravid females introduced into the cage. The test substrate consisted of either 7 L of six-day old soil infusion or 7 L of non-chlorinated tap water treated with cedrol. Two concentrations of cedrol were tested sequentially: 5 ppm and 20 ppm. Cedrol was prepared in ethanol by first preparing a stock solution of 10,000 ppm by dissolving 150 mg of cedrol to 15 ml of absolute ethanol (≥99.8% (GC), Sigma Aldrich). Dilutions were made by adding the appropriate volume of stock solution to water in the pond. For instance, 5 ppm cedrol was prepared by adding 3.5 ml of stock solution into 7 L of water. Similarly, 20 ppm cedrol was prepared by adding 14 ml of stock solution to the 7 L of water in the artificial pond.
A small wooden hut (1.78 m long x 1.73 wide x1.80 m high) was set up in the centre of the semi-field cage (Figure 1) to simulate the natural indoor environment where female An. gambiae s.s. take a blood-meal and rest till they are gravid (41). The hut had a door and two windows that were shut when the experiment was in progress. Two open eaves (1.70 m x 0.18 m) located at opposite sides of the hut served as the only exit points for the mosquitoes released in the hut. In each experimental night 200 gravid An. gambiae s.s. were released at 18:00 h inside the hut.
To measure the number of mosquitoes visiting the ponds, the top of each pond was covered by a black fibre-glass netting gauze cut to size (diameter 0.42 m) on which a fine film of insect glue was sprayed (Oeco insect spray, Oecos, UK) to trap the gravid females as they searched for oviposition substrates. At 6:00 h the following morning the number of females trapped on the sticky screens was counted. Each of the test substrates was evaluated over 12 replicate nights with fresh oviposition substrates and fresh batches of mosquitoes used each night. The four ponds (three ponds filled with non-chlorinated water and the pond containing the test substrate) were randomly allocated in all four corners of the semi-field cage using a randomized complete block design.
Line 278: The four ponds [meaning untreated, soil infusion or cedrol?] were randomly allocated in all four corners of the semi-field cage using a randomized complete block design.
Evaluation of the auto-dissemination of PPF by gravid An. gambiae s.s. from the bait-station to larval habitats
These experiments were conducted in three identical semi-field cages which contained a wooden hut at the centre and four enamel tubs used to create artificial ponds at the corners of each cage as described above (Figure 2). The experiments were done under standardized conditions without vegetation. In the first semi-field cage, three ponds were filled with 7 L of non-chlorinated tap water each and left open for mosquito oviposition, while the fourth pond serving as the bait-station contained 7 L of water treated with 20 ppm cedrol as described above. A netting gauze (diameter 0.42 m) treated with 3.5g PPF dust (average 20.3 g PPF/m2 retained on gauze on weighing) was placed on top of the cedrol-treated pond. The three open ponds were 4.4 m, 8.4 m and 10.3 m away from the bait-station (Figure 2). The set-up in the second semi-field cage was the same as the first, except that no mosquitoes were released in the cage. The aim here was to investigate if PPF might be distributed by air movement to neighbouring ponds, rather than mosquitoes. In the third semi-field cage, mosquitoes were released but the netting gauze placed on top of the bait-station was left untreated. This set-up served to investigate natural adult mosquito emergence rates from ponds when no insecticide was present in the semi-field cage. Two hundred gravid An. gambiae s.s. were released at 18.00 h per experimental night inside the hut and allowed to disperse through the open eaves.
The following morning, all open ponds in the three semi-field cages were visually assessed for the presence of eggs laid. Eggs were not counted since an exact estimate would have required removing the eggs from the ponds using a sieve or similar tools potentially interfering with the amount of PPF transferred. To ensure sufficient replication of the experiment, the impact of PPF was not assessed by monitoring the development of eggs laid in ponds by exposed mosquitoes. That would have taken approximately two weeks to complete per replicate and, therefore, over half a year to complete 12 replicates (42). Instead, the possible transfer of PPF by females to the ponds was assessed by monitoring the adult emergence of 50 insectary-reared late instar An. gambiae s.s. larvae that were introduced into each open pond in all three experimental set-ups. The larvae were introduced into the ponds in the morning after the gravid females were released. Introduced larvae were fed daily with a pinch of Tetramin®Baby Fish food. Any pupae that developed in the three ponds were transferred with a small volume of water from the pond into 200 ml plastic cups (diameter 7 cm) and monitored for adult emergence. It took 6-7 days for all introduced larvae to develop into adults or die. Thereafter the ponds and hut were cleaned and all remaining flying adult mosquitoes in cages aspirated using a motorized backpack aspirator (John W. Hock Company, USA). A new round of replicates was set-up with fresh oviposition substrates and fresh batches of gravid mosquitoes and mosquito larvae. The experiment was replicated over 12 rounds with each round lasting seven days. The four ponds were randomly allocated in all four corners of the three semi-field cages in a randomized complete block design. To avoid contamination, specific semi-field cages were dedicated to the test and controls.
Liquid-chromatography-mass spectrometry (LC-MS) quantification of the amount of PPF carried by an individual mosquito and transferred to a water sample
An enamel bowl (diameter 0.42 m) filled with 7 L of non-chlorinated tap water was introduced into a 60 x 60 x 60 cm cage (BugDorm-2120F; MegaView Science Taiwan). The top of the bowl was covered with black fibre-glass netting gauze treated with 3.5g PPF dust (average 20.3 g PPF/m2 retained on gauze on weighing) as described above. Gravid An. gambiae s.s. were introduced into the cage and monitored for contact with the PPF-treated netting gauze. At any time, there were only two females in the cage. Females that contacted the PPF-treated netting material at least twice were gently aspirated from the cage into holding containers.
Two different tests were conducted with females that contacted the PPF-treated netting. First, 200 potentially contaminated females were individually transferred into 1.5 ml Eppendorf tubes and frozen at -70 °C until they were used to quantify the amount of PPF on their body. Secondly, 30 potentially contaminated females were used to determine the amount of PPF that a single mosquito transfers to water during oviposition. For this, bioassays were conducted by introducing these females individually into 15 cm x 15 cm x 15 cm cages provided with a glass cup (Pyrex®, 100 ml, diameter 7 cm) filled with 100 ml of non-chlorinated tap water. The females were left overnight in the cages to lay eggs. The following morning the glass cups were assessed for presence of eggs laid.
To confirm the transfer of PPF into the oviposition water, 10 insectary-reared late instar An. gambiae s.s. larvae were introduced into all cups in which females had laid eggs and monitored for adult emergence as described above. When all introduced larvae had died or emerged as adults, the water from the cups was transferred into 50 ml glass jars. The water samples were frozen at -70 °C awaiting chromatographic quantification of PPF in the samples. Comparisons were made to a control group of gravid females that were unexposed to PPF. Thirty replicates of test and control cages were done.
For quantification of the amount of PPF that contaminates a gravid mosquito when she makes contact with a PPF-treated netting material, PPF was washed off the body of individual mosquitoes using 1.5 ml methanol (Sigma Aldrich, 99.9% HPLC grade) in Eppendorf tubes. The content of the Eppendorf tubes was agitated in a sonicator (Branson 2510 Ultrasonic cleaner, Eagle Road, Danbury) at 25 ºC for 5 minutes. It was then centrifuged at 13,000 revolutions per minute (rpm) for 5 minutes in a microcentrifuge (PRISMTM). The supernatant was transferred into 2 ml glass vials and used for detection of PPF by liquid chromatography-mass spectrometry (LC-MS).
To detect PPF in water samples used in bioassays, the samples were first pooled into groups of 10 before extraction (10 x 50 ml). Thus, there were six pools of water samples in which females that contacted PPF laid eggs and another six pools of water samples in which females unexposed to PPF laid eggs. Each pool of water sample (500 ml) was extracted in 200 ml chloroform (Sigma Aldrich, 99.9% HPLC grade) to separate the aqueous and organic layers. The organic layer, where PPF was expected to dissolve, was concentrated by evaporating it to dryness in a rotary evaporator (Heidolph Instruments, Germany). The residue was dissolved in 1 ml methanol and stored at 4 °C awaiting analysis. To assist in quantification of PPF, a known concentration (0.00002 µg) of 4-benzylbiphenyl (99%, Sigma Aldrich) was added into each extracted water sample as internal standard just before the LC-MS run. The LC-MS run was performed using electron spray ionization (LC/ESI-MS). First, the standards of pure 10% PPF and 4-benzylbiphenyl were initially run separately in the LC-MS system to confirm the retention times of PPF and the internal standard. PPF used as standard was prepared by dissolving 40 mg of PPF (10%) in 1.5 ml ethanol in a 2 ml glass vial. This was agitated in a sonicator at 25 ºC for 5 minutes. The mixture was centrifuged at 13,000 rpm for 5 minutes. The supernatant was transferred into 2 ml glass vials ready for detection of PPF. The peaks of PPF and 4-benzylbiphenyl at the retention times were identified based on the molecular masses of their individual ions (molecular masses of PPF-322 and 4-benzylbiphenyl-247).
The LC/ESI-MS used consisted of a quaternary LC pump (Model 1200) coupled to Agilent MSD 6120-Single quadruple MS with electrospray source (Palo Alto, CA). The MS component of the system was used to verify the peak assigned to PPF or 4-benzylbiphenyl as the active ingredients based on the identification of molecular masses of the ions. The system was controlled using ChemStation software (Hewlett-Packard). Reverse-phase liquid chromatography was performed using an Agilent Technologies 1200 infinite series LC, equipped with a Zorbax Eclipse Plus C18 column, 4.6 x 100 mm x 3.5 µm (Phenomenex, Torrance, CA). A gradient using A (5% formic acid in LC-grade ultra pure H2O) and B (LC-grade methanol) (Sigma, St. Louis, MO) was used; 0-5 min, 95-100% B; 5-10 min, 100% B; 100-5 min. The mobile phase liquid was acetonitrile (Sigma Aldrich). The flow rate was held constant at 0.7 mL min-1. The sample injection volume was 100 μl, and data were acquired in a full-scan positive-ion mode using a 100 to 500 m/z scan range. The dwell time for each ion was 50 ms. Other parameters of the mass spectrometer were as follows: capillary voltage, 3.0 kV; cone voltage, 70 V; extract voltage, 5 V; RF voltage, 0.5 V; source temperature, 110 ºC; nitrogen gas temperature for desolvation, 350ºC; and nitrogen gas flow for desolvation, 400 L/h.
Data were analysed in R statistical software package version 2.13. Generalized estimating equations (GEE) were used to analyse all data with experimental round/night included as repeated measure in the models (43,44). Data collected in cage and semi-field experiments to determine the transfer of PPF to water were analysed as proportions. Proportions were analysed by fitting a binomial distribution with a logit function and an exchangeable correlation matrix. Preliminary cage bioassays testing the two PPF formulations were analysed by including treatment (cage with untreated bait-station, bait-station treated with PPF dust or PPF dust formulated in oil) as fixed factor with the control cage (cage with untreated bait-station) used as the reference (45). For the analysis of the semi-field experiments, the open pond ID identified by its distance from the bait-station was used as the fixed factor with the pond closest to the bait-station used as the reference. Count data evaluating the number of mosquitoes visiting ponds treated with soil infusion or cedrol were fitted to a Poisson distribution with a log link function. Here the ponds were included in the model as fixed factors with the bait-station used as reference. All means (proportions or counts) per treatment and their corresponding 95% confidence intervals (CIs) were modelled as the exponential of the parameter estimated for the individual models with no intercept included.