Experiments were carried out in large netting-screened semi-field cages (10.8 m long × 6.7 m wide × 2.4 m high) on the Thomas Odhiambo Campus of the International Centre of Insect Physiology and Ecology (icipe-TOC), located on the shore of Lake Victoria in Mbita, Homabay county, western Kenya (geographic coordinates 0⁰ 26’ 06.19” S, 34⁰ 12’ 53.13”E; altitude 1,137 m above sea level). Mbita is characterized by tropical climate with an annual average minimum temperature of 16 ºC and maximum temperature of 29ºC. The area experiences two rainy seasons; the long rains between March and June and the short rains 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 sensu stricto (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 25-28ºC, 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 instar mosquito larvae were randomly selected from different tubs to ensure that those introduced into experimental ponds were the same size. 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. Only gravid female mosquitoes were used for experiments in this study.
Development of a bait-station
Contamination of adult An. gambiae s.s. with PPF. Since water vapour is a general attractant for malaria vectors (34), we considered it was essential to have water in our bait station. Females were prevented from accessing the water to lay eggs using fly gauze (black fibre-glass netting gauze (1.7x1.5 mm) treated with PPF. Two methods of applying the PPF to the netting material were tested in cage experiments. 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 PPF over the netting surface. The amount of PPF on netting gauze was 1.3 g/m2 after weighing. Second, PPF was mixed with cooking oil (Ufuta Pure Vegetable Cooking Oil, Bidco Africa) and applied to the netting. Here 1 g of PPF dust was mixed in 2 ml of oil and this formulation applied to a netting gauze with a brush. The netting treated with PPF served as the dissemination station. The control netting gauze was untreated and was used in control cages.
These experiments were conducted in mosquito-netting covered cages (30 cm x 30 cm x 30 cm) to determine which of the two methods of treating the netting gauze contaminated gravid An. gambiae s.s. with lethal doses of PPF for transfer to water. Each cage was provided with two glass cups (Pyrex®, 100 ml, diameter 7 cm). The first cup in each cage was filled with 100 ml non-chlorinated tap water while the second cup that served as the bait station was filled with 100 ml of six-day old soil infusion that has been shown previously to attract gravid females in cages (35) to lure gravid females. The top of the cup that served as the bait-station in the control cages was covered with untreated netting while in the test cages it was covered with netting gauze treated with either PPF dust or PPF dust formulated in oil. The top of the other cup filled with water was left open in all cages to allow for egg-laying by gravid females.
In each cage five gravid An. gambiae s.s. were released at 18:00 h and left overnight. The following morning the number of eggs in each open cup was counted. To confirm the transfer of PPF in test cages, 10 insectary-reared late instar An. gambiae s.s. larvae were introduced into all open cups with water in all cages and monitored for adult emergence. Larvae were fed daily on a pinch of Tetramin®Baby fish food. Because PPF does not produce acute toxicity on mosquito larvae but prevents emergence of adults from exposed pupae (36), any pupae that developed were transferred into plastic cups (diameter 7 cm) and monitored for emergence. It took 6-7 days for all larvae introduced into the cups to develop into adults or die. These experiments were conducted in 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 netting gauze, 15 cages with netting gauze treated with PPF dust and 15 cages with netting treated with PPF dust formulated in oil. Both cups were randomly allocated to one of the four corners in the first cage. The positions of the cups in subsequent cages were rotated to the next corner in a clockwise direction relative to the starting position.
Soil infusion was prepared by incubating 15 L of non-chlorinated tap water with 2 kg of soil collected from a known An. gambiae s.l. aquatic habitat. Infusions were prepared in round plastic tubs (diameter 0.42 m) and left for six days before use in experiments. During the six-day incubation period tubs were covered with mosquito netting and kept in sheds, to protect them from rain.
Luring gravid An. gambiae s.s. to a pond. These experiments were conducted in a semi-field cage (Figure 1). Four artificial ponds were created by digging down round enamel tubs (diameter 0.42 m, depth 8 cm) at the four corners of the semi-field cage. The tubs were dug 1 m away from the nearest wall. During each experimental round three of the ponds were filled with 7 L of non-chlorinated tap water while the fourth pond was filled with a test substrate to attract gravid females.
Two test substrates were tested based on previous published work that showed their potential in attracting gravid female An. gambiae s.s.: a six-day old soil infusion (35) and the sesquiterpene alcohol, cedrol (Cedrol ≥99.0% (sum of enantiomers, GC, Sigma-Aldrich, Steinheim, USA) (37). Both substrates were evaluated separately on different dates. Thus, during the experiments the test pond was either filled with 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 in the dug down tub. Similarly, 20 ppm cedrol was prepared by adding 14 ml of stock solution into 7 L of water in tub.
To simulate the natural environment, where female An. gambiae s.s. take a blood-meal indoors and rest till they are gravid, we released gravid females inside a small wooden hut (1.78 m long x 1.73 wide x1.80 m high) that was set up in the centre of the semi-field cage (Figure 1).
The hut had a door and two windows that were shut when the experiment was in progress. The hut had two open eaves (1.70 m x 0.18 m) located at opposite sides which served as exit points for the gravid females. In each experimental night 200 gravid An. gambiae s.s. were released at 18:00 h in the centre of the hut. To measure the number of mosquitoes visiting a pond, 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 mosquitoes as they searched for oviposition substrate to lay eggs. At 6:00 h the following morning the number of mosquitoes trapped on the sticky screens placed on top of each pond was counted. Each of the test substrates were evaluated during 12 nights with fresh substrates and fresh batches of mosquitoes. The four ponds 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 a bait-station to larval habitats
The experimental set-up used in this test was modified from Lwetoijera et al. (31). These experiments were conducted in three identical semi-field cages which included a small wooden hut at the centre and four ponds in the corners (Figure 2). In the first semi-field cage, three ponds were filled with 7 L of non-chlorinated tap water and left open for mosquito oviposition, whilst the fourth pond served as the bait-station which consisted of 7 L of water treated with 20 ppm cedrol as described above. On top of the cedrol-treated pond a netting gauze of diameter 0.42 m was placed and treated with 3.5 g PPF (25.3 g PPF/m2). The three open ponds were 4.4 m, 8.4 m and 10.3 m from the bait-station (Figure 2). Two hundred gravid An. gambiae s.s. were released at 18:00 h per experimental night in the centre of the hut. The set-up in the second semi-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 pond serving as the bait station was untreated. This last set-up served to investigate natural adult mosquito emergence rates from ponds when no insecticide was present in the semi-field cage. The set-ups in the second and third semi-field cages thus served as controls.
The following morning the number of eggs laid in each open pond was counted. To ensure sufficient replication of the experiment the impact of PPF was not assessed by monitoring the development of eggs that were laid by the exposed females which would have taken approximately two weeks to complete one experiment (38). 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 the open ponds in all three set-ups in the morning after 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 into 200 ml plastic cups (diameter 7 cm) and monitored for 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 alive adult mosquitoes aspirated using a motorized backpack aspirator (John W. Hock Company, USA). A new set of experiments was set-up with fresh substrates, fresh batches of adult gravid mosquitoes and mosquito larvae. The experiments were conducted for 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, the semi-field cages in which the test and the two control experiments were conducted were not changed.
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). Above the water a netting treated with 3.5 g PPF dust (25.3 g PPF/m2 after weighing amount retained on netting) as described above was fixed. Two gravid An. gambiae s.s. were introduced at a time into the cage and observed. Two different tests were conducted with females that contacted the PPF-treated netting. Firstly, 200 females that contacted the PPF-treated netting were individually transferred into 1.5 ml Eppendorf tubes and frozen at -70°C until they were used for quantification of PPF on their bodies.
Secondly, 30 females that contacted PPF-treated netting in the BugDorm cage were used to determine the amount of PPF that a single mosquito transfers to water. For this, bioassays were conducted by introducing individual females into 15 x 15 x 15 cm cages containing 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 number of eggs laid by each female was counted. To confirm the transfer of PPF into the water in the cup, 10 late instar An. gambiae s.s. larvae were introduced and monitored for adult emergence as described above. Comparisons were made to a control group of gravid females that were unexposed to PPF. Thirty replicates of test and control cages were done. When all 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 until used for chromatographic quantification of PPF.
For quantification of the amount of PPF that contaminates a mosquito when a female makes contact with a PPF-treated netting material, PPF was washed off the body of an individual mosquito in an Eppendorf tube using 1.5 ml methanol (Sigma Aldrich, 99.9% HPLC grade). 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 samples was extracted separately. For each pool of water sample, approximately 500 ml of the sample 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). The following 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, 110ºC; 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. Data collected in cage and semi-field experiments that 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 assumed. In analysing data performed in cage experiments to determine if mosquito can get contaminated with PPF dust or PPF dust formulated in oil from treated netting, the cage (control or test) was included as fixed factor with the control cage used as the reference. In semi-field experiments to evaluate the potential of gravid female to transfer PPF to open ponds, the open pond ID identified by its distance from the bait station was used as the fixed factors 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 pond serving as the bait station used the 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.