Dose–response tests and semi-field evaluation of lethal and sub-lethal effects of slow release pyriproxyfen granules (Sumilarv^®0.5G) for the control of the malaria vectors Anopheles gambiae sensu lato

The study was conducted at the International Centre of Insect Physiology and Ecology-Thomas Odhiambo Campus (icipe-TOC) in Mbita (0° 26΄ 06.19” S; 34° 12΄ 53.13” E) close to Lake Victoria, Western Kenya (altitude 1,137 m). Here, the major malaria vectors are An. arabiensis with a small number of An. gambiae s.s. and Anopheles funestus[58]. The area is characterized by a tropical climate with an average annual minimum temperature of 16°C and an average maximum temperature of 28°C (icipe-TOC meteorological station data for 2010 to 2012). The area experiences two major rainy seasons, the long rains between March and June and the short rains between October and December. The average annual rainfall for 2010 to 2012 was 1,150 mm (icipe-TOC meteorological station).

Both laboratory and standardized field tests used insectary-reared third instar larvae of An. arabiensis and An. gambiae s.s. (Mbita strains). Larvae were reared in round plastic tubs (diameter 60 cm) filled with water (5 l, 5 cm high) from Lake Victoria filtered through a charcoal-sand filter. Mosquito larvae were fed with fish food (Tetramin©Baby) twice daily. Third instar mosquito larvae were selected from different tubs so that the larvae were of a similar range in size in each tub tested [59]. Mosquito larvae were reared at ambient climate and light conditions in a netting-screened greenhouse with an average daily temperature of 27°C, an average 76% relative humidity and a natural 12 hours of dark and 12 hours of light cycle.

Sumilarv^®0.5G was provided by the manufacturer Sumitomo Chemicals Company, Japan, for all tests. It is a granular formulation containing 0.5% active ingredient (weight: weight).

Tests were done in the shade, under ambient climate and light conditions in a netting-screened greenhouse. Prior to the dose–response tests, range-finding tests were implemented by exposing test larvae to a wide range of test concentrations and a control. This served to find the activity range of the insecticide for each test species. Concentrations between 10 parts per million (ppm) active ingredient (ai) and 0.0000001 ppm ai were tested. After determining the emergence inhibition (EI) of the larvae in the wider range, nine concentrations were chosen, yielding between 10% and 95% EI in the range-finding tests in order to determine the EI₅₀, EI₉₀ and EI₉₉ in dose response bioassays. The following concentrations were tested: 0.005 ppm ai, 0.001 ppm ai, 0.0005 ppm ai, 0.0001 ppm ai, 0.00007 ppm ai, 0.00004 ppm ai and 0.00001 ppm ai, 0.000005 ppm ai, 0.000001 ppm ai.

A stock solution was prepared by grinding the granular formulation into a very fine powder following the procedure of Sihuincha and others [49]. Using a pestle and mortar, 5 g of Sumilarv^®0.5G (25 mg ai) was ground and added to 500 ml of non-chlorinated tap water. This gave a stock solution of 10,000 ppm Sumilarv^®0.5G (50 ppm ai). The mouth of the vial was covered with aluminium foil and the solution left to agitate for one hour on a shaker. Since Sumilarv^®0.5G is a slow release formulation the mixture was left overnight to allow the active ingredient to be released into solution. In the morning the mixture was again agitated on a shaker for 30 minutes to prepare a homogenous mixture since some of the inert ingredients of the formulation (potentially still containing some active ingredient) had settled overnight. Serial dilutions were made immediately after shaking in non-chlorinated tap water to produce the test concentrations.

Anopheles arabiensis and An. gambiae s.s. were evaluated in parallel. Each test concentration and a control were replicated four times per round per mosquito species. Two hundred ml of each test solution was set up in 300 ml plastic cups. Three rounds of tests were implemented. Separate batches of 25 insectary-reared third instar larvae of both test species were introduced into each test concentration and the control (non-chlorinated tap water). Thus in total 300 larvae of each species were tested per test concentration and control (total of 3000 larvae). Larvae were fed with Tetramin© Baby fish food every 24 hours and cups covered with netting to prevent any emerging adults from escaping. The number of live and dead larvae, pupae and adults was recorded every 24 hours for 10 days. Live pupae from each cup were transferred into a separate cup with approximately 20 ml of water from the respective cup of collection. These cups were covered with netting and pupae monitored for emergence. Separate pipettes were used to collect pupae from treated and control cups to avoid cross-contamination.

Standardized field tests [57] were carried out in an open field with grass approximately 3 cm in height between October 2011 and March 2012. Thirty artificial ponds were set up in an open field by sinking enamel-coated bowls (diameter 42 cm, depth 10 cm) into the ground (Figure 1A). Ponds were arranged 2 m apart in six rows. Each bowl was filled with 8 l of non-chlorinated tap water. Into each pond 2 l of soil collected from the surrounding field was added and mixed well to resemble a natural habitat. Batches of 50 insectary-reared third instar larvae were introduced into each pond. Sumilarv^®0.5G treatment was applied after introduction of larvae. Treatment of the ponds was allocated randomly using a lottery system. In each treatment round, 10 of the ponds served as untreated controls; in five of them An. arabiensis were introduced and in the other five An. gambiae s.s. Two application rates of Sumilarv^®0.5G were tested per mosquito species. The application rate was based on the surface area of the water, which was 0.14 m² per pond. Sumilarv^®0.5G was spread evenly over the entire water surface by hand. Five ponds were treated with 1 mg ai per m² (equalling 0.018 ppm ai considering the volume of 8 l of water) while five other ponds were treated with 5 mg ai per m² (or 0.09 ppm ai) per mosquito species. A netting-covered emergence trap was placed on top of each pond to prevent wild mosquitoes from laying eggs in the sites and to prevent the escape of any emerging adult mosquitoes (Figure 1B). The residual activity of Sumilarv^®0.5G was evaluated by introducing new batches of 50 insectary-reared third instar larvae into each pond at weekly intervals. After one week all the larvae had either emerged as adults or died. The efficacy of Sumilarv^®0.5G was evaluated for six weeks. This experiment was implemented three times (referred to as rounds in the analyses).

To assess larval mortality, the number of larvae present in each habitat was counted daily. First, the emergence trap over each pond was assessed for the presence of any newly emerged adults and any adults collected with an aspirator and placed into a disposable cup covered with netting. Any pupae in the ponds were transferred into plastic cups holding 50 ml of the water from the respective pond. Pupae collections were done in the morning and evening so that any emergence or emergence inhibition could be recorded daily in the laboratory.

To monitor environmental parameters that may influence the efficacy of the insecticide, daily data on turbidity and pH of water in each pond was collected. Ponds were visually categorized into clear (ground visible) or turbid ponds. The water pH was measured using a pH meter (Phywe International, Germany).

Tests to assess the impact of sub-lethal doses of Sumilarv^®0.5G were carried out under ambient conditions in a netting-screened greenhouse. The number of eggs laid and the number of eggs hatched (number of offspring produced) per adult mosquito that emerged from treated ponds were compared to that of the adults that emerged from the untreated ponds in standardized field tests. All pupae used in these tests were collected from the ponds in week six of each test round. Emerged adults were maintained with 6% glucose solution ad libitum. When the adults were two to four days old they were blood-fed twice on a human arm on two successive days. A single gravid mosquito was introduced into each cage with an oviposition cup (diameter = 7 cm) containing 100 ml of non-chlorinated tap water. The number of eggs laid by each mosquito overnight and the number of eggs hatched over one week were counted. Sub-lethal effects of the treatment dosage of 1 mg ai per m² were tested with 20 individual females per round of semi-field test for An. arabiensis and An. gambiae s.s., respectively (total 3 × 20 = 60 females per species). Due to the persistent high immature mortality of the 5 mg ai per m² treatment only 10 females per species and round could be tested (total 3 × 10 = 30 females per species).

Data analyses were done with SPSS statistical software version 19. All data from the replicates of the dose–response tests were pooled by doses for each mosquito species for the estimation of the EI₅₀, EI₉₀ and EI₉₉ values using the log dosage-probit regression analysis with the test dosages as covariates and species as factors in the model. Relative median potency estimates were used to compare the susceptibility of the two species. Generalized estimating equations (GEE) were used to estimate the overall emergence inhibition of the two Sumilarv^®0.5G dosages for the six weeks treatment period in standardized field tests. The number of successful emerged adults was the dependent variable and was fitted to a negative binomial distribution with a log-link function and an exchangeable correlation matrix. The treatments, test rounds, mosquito species, water turbidity (clear, turbid), water pH (grouped in two categories: pH < 8, pH ≥8) and the occurrence of rain during the test week (no rain, rain) were added to the model as fixed factors. Since the same pond was evaluated repeatedly for larval mortality over the six-week period, the unique pond ID was included as the repeated measures variable. Interaction terms were included in the model between treatments and turbidity, treatments and pH, and treatments and rain. GEE models were also used to estimate the impact of sub-lethal concentrations on the number of eggs laid and the number of eggs that hatched from emerged An. gambiae s.s. adults. The parameter estimates of the GEE models were used to calculate the weekly mean adult emergence, mean number of eggs laid per female and mean number of laid eggs that hatched into larvae and the associated 95% confidence intervals (CIs) by removing the intercept from the models. For the calculation of percent reduction the weekly emergence inhibition in the treated ponds was corrected using Abbott’s formula based on emergence in the untreated ponds as denominator [60]. Percent reduction was therefore calculated as follows:

The dose–response tests showed that Sumilarv^®0.5G affected adult mosquito emergence in An. arabiensis and An. gambiae s.s. at very low and over a very wide range of concentrations (0.000001-0.005 ppm ai). Data from the three rounds of dose–response tests showed similar trends in emergence inhibition for each species and were, therefore, pooled per dose (Figure 2) to estimate emergence inhibition (EI) rates; EI ₅₀, EI₉₀ and EI₉₉ (Table 1). The minimum dosage that completely inhibited adult emergence was estimated to be between 0.01-0.03 ppm ai (Table 1). Anopheles arabiensis and An. gambiae s.s. were equally susceptible to Sumilarv^®0.5G.

There was no difference in adult emergence from treated ponds between An. arabiensis and An. gambiae s.s. (p=0.3) and data for both species were pooled for analysis. The weekly adult emergence per round from the treated and untreated ponds is shown in Figure 3 and emergence inhibition calculated in Table 2. Complete emergence inhibition was observed for two weeks in rounds one and three of the high treatment dose of 5 mg ai per m² (0.09 ppm ai). However at the lower dosage of 1 mg ai per m² (0.018 ppm ai) which corresponded with the minimum effective dosage established in the dose–response tests complete emergence inhibition was only observed in week one in round one and three. Ponds treated at 5 mg ai per m² provided better residual impact than the lower treatment dosage of 1 mg ai per m² (Figure 3 and Table 2). Adjusting for other factors the GEE model estimated that Sumilarv^®0.5G inhibited 85% of adult emergence over a period of six weeks at an application dose of 1 mg ai per m² and 97% at a dose of 5 mg ai per m² compared to emergence from untreated ponds (Table 3). The overall impact of 5 mg ai per m² on inhibiting emergence was significantly higher than the impact of 1 mg ai per m² (p<0.001). Despite consistent rainfall during the first round of the standardized field tests and occasional rainfall during the following two rounds (Figure 4), rain did neither affect the emergence of adults from control and treatment ponds nor the impact of the treatments (Table 3). There were also no main effects of water turbidity or pH on adult emergence but interactions were identified between the treatments and water turbidity, and the treatments and water pH. Turbid water and high pH reduced the impact of the treatments leading to slightly higher adult emergence from treatment ponds under these conditions (Table 3). The impact of the interactions can be calculated by multiplication of the odds ratios [61]. This means for example emergence inhibition was 85% at 1 mg ai per m² when ponds were clear and had a pH <8, emergence inhibition was reduced to 79% when the same treatment pond was turbid with a pH <8 and to 74% when the same treatment pond was turbid and had a pH ≥8. Similarly for the 5 mg ai per m² ponds in round one, overall emergence inhibition is 97% when treatment ponds are clear with pH <8, emergence inhibition is reduced to 95% when the treatment ponds are turbid with pH <8 and further reduced to 90% when the treatment ponds are turbid and with pH ≥8.

The impact of sub-lethal effects could not be evaluated for An. arabiensis that emerged from pupae since neither females from untreated ponds nor females from treated ponds laid eggs, possibly due to unsuitable mating conditions provided for this species [62]. Exposure of An. gambiae s.s. to both Sumilarv^®0.5G dosages during the larval stage resulted in: (i) a reduced probability of the adult female laying eggs; (ii) reduced mean number of eggs laid per female; and, (iii) reduced mean number of eggs that hatched into larvae (Table 4). Treatment rounds were not significantly different (p=0.687), and data for all rounds for An. gambiae s.s. were pooled for analysis. Mosquitoes that emerged from treated ponds were 65-68% less likely to lay eggs compared to mosquitoes that emerged from untreated ponds. The mean number of eggs laid per female An. gambiae s.s. was reduced by 47% from females emerging from ponds treated at 1 mg ai per m² and by 74% from females emerged from ponds treated at 5 mg ai per m² compared to that in the untreated controls (Table 4). The impact of the higher dosage was twice the impact measured from the lower dosage (odds ratio (OR) 2.1, 95% CI 1.2-3.7, p=0.02). Furthermore, it was 90% less likely for an egg to hatch that was laid by a female exposed to the higher Sumilarv^®0.5G dosage compared to eggs laid by females that emerged from low dosage ponds (OR=0.10, 95% CI 0.04-0.23, p<0.0001). The probability of an egg hatching was reduced by 77% for eggs laid by a female exposed to the lower treatment dosage and 98% for eggs laid by a female exposed to the higher dosage as compared to eggs in females that emerged from the untreated control ponds.