Competing interest statement
Conflict of interest: the authors declare no potential conflict of interest.
Vector-borne diseases affect two third of the world population as reported to kill millions of people every year; these diseases are directly associated with economic growth and costs limit of several undeveloped and developing countries.
Mosquitoes are still being the most important vectors spreading life-threatening human diseases. It is well documented that the synthetic insecticides dramatically reduce the risk of vector born diseases. However, the indiscriminate use of chemical pesticides like organophosphates and pyrethroids has led to the development of resistance in mosquito populations (McGaughey, 1985; Huang & Ottea, 2004).
Similarly, the eco-friendly and safe bioinsecticide Bacillus thuringiensis is being used worldwide as an effective mosquito control agent. The intensive use of Bacillus thuringiensis has led to the development of resistance in natural mosquito population (Boyer et al., 2012). However, some reports suggest a cross development against Bt, due to the increase in detoxifying activity of enzyme in xenobiotic reaction, decreasing activity of mid gut protease and a modification of specific receptor of mid gut (Jurat-Fuentes et al., 2004; Boyer et al., 2007; Saengwiman et al., 2011).
Despite that, majority of health and agriculture pest, which were brought under control by various pesticides, are now on rebound. The major vector pests-mosquitoes are well known for transferring dengue and malaria and are becoming resistant to most of the available pesticides.
To fight against the resistance problem, various methods were advocated like the use of different pesticides especially in combination with different chemical class/family that has a different mode of actions against the pest. This strategy definitely leads to prevent or to delay the development of resistance against pesticides.
The combined use of biological and chemical pesticides, having different mode of action, is one of the promising economic and eco-friendly strategies to fight the resistance problem.
However, the applicability and compatibility of this perspective is a real challenge. The incompatibility of pesticides of bio and synthetic origin may lead to a loss of efficacy of pest control, as well as economic loss and environmental hazards.
Compatibility is the capacity to mix different pesticides without physical or chemical interactions or changes, leading to the enhancement of their biological efficiency. The majority of researchers extensively studied the mode of action and resistance development mechanisms of biological and chemical pesticide in the past. This study aims to test the combination between Bt and chemical pesticides and its synergistic impact on larvicidal efficiency and mosquito larvae metabolism.
Materials and Methods
Chemicals and reagents
Nutrient broth, α-napthyl acetate, β-napthyl acetate, α-naphthol, β-naphthol, ethyl acetate were procured from Hi-Media (Mumbai, India) and temephos (EC 50%), chloropyrifos (EC 20%), cypermethrin (EC 25%), cyhalothrin (EC 5%), malathion (EC 50%), spinosad (EC 45%), azadirachtin (3000 ppm) was obtained from M/S Gharda chemicals limited (India).
For the laboratory trial, early fourth instars larvae of Ae. aegypti and An. stephensi were maintained as Patil et al., 2012 In brief, the larvae were maintained in plastic enamel trays containing dechlorinated tap water and all the experiments were carried out at 28±2°C and 75-85% relative humidity under 14:10 light and dark cycles. Larvae were fed with a diet of finely ground brewer’s yeast and dog biscuits (3:1).
Pesticide tolerance tests for B. thuringiensis SV2
The pesticide tolerance test was performed with previously isolated B. thuringiensis SV2 (BtSV2) strain (HM854748) (Patil et al., 2012) using solid and liquid media. The compatibility and tolerance of BtSV2 isolate was tested at 5 ppm concentration of temephos, chloropyrifos, cypermethrin, cyhalothrin, malathion, spinosad and azadirachtin in nutrient agar and nutrient broth respectively at 28±2°C for 24 hrs.
The pesticide accumulation was studied by inoculating BtSV2 spore suspension (2×105/mL) in nutrient broth containing various pesticides at their highest concentrations as per WHO recommendations i.e. temephos (1 ppm), chloropyrifos (2.5 ppm), cypermethrin (20 ppm), cyhalothrin (25 ppm), malathion (15 ppm), spinosad (0.5 ppm) and azadirachtin (50 ppm) and incubated at 28±2°C for 48 hrs. Growth pattern of BtSV2 was observed by measuring OD spectrophotometrically at 600 nm in comparison to control (Harley & Prescott., 1996; Li et al., 2012)
Sample preparation for bioassay
The pesticides grown lyophilized culture of BtSV2 was dissolved in distilled water to prepare test concentrations and used to make different combinations with temephos. The lyophilized BtSV2 biomass without pesticide was dissolved in distilled water, which served as a control. The solutions were homogenized by stirring on shaker for 30 min at 120 rpm and used for mosquito larvicidal assay.
The larvicidal assay was performed by individual lyophilized BtSV2 isolate, temephos and combined formulation of BtSV2 (TBT) and temephos at sub lethal concentrations of both temephos and BtSV2 i.e. A (1:1), B (1:2), C (1: 3) and D (1:4).
The twenty numbers of fourth instars larvae were introduced in 100 mL of the test medium containing a particular concentration of pesticide against the tap water control. All containers were maintained at room temperature with naturally prevailing photoperiod in the laboratory. Larval mortality was checked after 24 hrs of incubation. Each treatment was performed in triplicates. In all assays, the mortality of larvae was recorded and calculated by Abbott formula (Abbott, 1925). The larvicidal activity of BtSV2, temephos and TBT at various concentrations was subjected to probate regression analysis. The lethal concentrations in ppm (LC50, LC90) were calculated and recorded.
Preparation of whole body homogenates
Physiological and biochemical changes in larvae were analysed by biochemical experiments using the procedures outlined by WHO (WHO, 1998).
Preparation of larval extracts
For each replicate, twenty early fourth instars larvae treated with LC30 after 12 hrs were washed with distilled water and the adhering water was completely removed from the body surface by blotting with tissue paper and chilled in ice. The larvae then pooled and homogenized in eppendorf tubes (held in ice) using a Teflon hand homogenizer, homogenized using a vortex for 1 minute and centrifuged at 15,000 g for 10 min at 4°C. The clear supernatants from larvae of the same biological replicate were kept at 4°C, and further used for biochemical assays like protein, phosphatase, esterase, protease etc.
Total protein estimation
The proteins in the larval homogenates of TBT(A) treated larvae were first precipitated by 80% ethanol (Subhashini & Ravindranath, 1980) and the protein concentration (Lowry et al., 1951) was estimated and used for further enzyme profile analysis.
Carboxyl esterase assay
Carboxyl esterase activity in the treated larval homogenates was measured with appropriate modifications (Van Asperen, 1962). Larval homogenate (200 µL) was mixed with 2 mL of the alpha and beta naphthyl acetate solution, reaction was allowed for 30 min at room temperature. After incubation, 500 μL of the fast blue SDS reagent was added (22.5 mg fast blue salt in 2.25 mL distilled water and 5% SDS in 0.2 M phosphate buffer (pH 7.2). Colour was allowed to develop for 15 min at RT. The optical density of the sample was read at 588 nm in the spectrophotometer against the respective reagent blank.
Phosphatases in the treated larval homogenate were determined by the method of Powell & Smith, 1954. The reaction mixture consists of 1 mL carbonate buffer (pH 10.4) in 1 mL of 0.01 M disodium phenyl phosphate (substrate), and 100 µL of larval homogenate then incubated for 30 min at 37°C. At the end of incubation period, 0.8 mL of 0.5 N NaOH was added in 1.2 mL of 0.5 N NaHCO3, followed by 1 mL of 4-aminoantipyrine solution and 1 mL of potassium ferricyanide. The produced colour was measured immediately by spectrophotometer at 510 nm. The enzymatic activity was expressed as μM/mg protein/min.
General proteases assay
Proteolytic activity was assayed by using azocasein (Sigma System, Marlborough USA) as a substrate. Briefly, 120 μL of enzyme solution was added to 480 μL of azocasein (1%, w/v) in reaction buffer (Tris buffer containing 5 mM MgCl2) and the mixture was incubated at 30°C for 30 min. The reaction was terminated by adding 600 μL of 10% (w/v) trichloroacetic acid and incubated for 30 min at 4°C. Then reaction mixture was centrifuged at 15,000 g, at 4°C for 10 min. The 800 μL of resulting supernatant was removed and neutralized by adding 200 μL of 1.8 N NaOH, and the absorbance was measured at 420 nm using a spectrophotometer (Shimadzu, Kyoto, Japan). One unit of enzyme activity was calculated as the amount product produced which leads to an increase in absorbance at 420 nm of 0.01 in 30 min at 30°C (Oppenoorth & Van Asperen, 1960; Elpidina et al., 2001).
Synergistic effect of different combination was determined by the methods of Sun & Johnson (1960) and Clausing & Bieleke (1980). According to the formula reported by these authors, if CTC value >100, the effect is synergistic, V<0.8 corresponds to antagonism action, V from 0.8 to 1.5 corresponds to additive action and V>1.5 means potentiation.
Each experiment was performed with five determinations using samples from different preparations. The difference in the levels of various biochemical parameters between control and experimental larvae were tested for statistical significance using mean difference Student’s t-test (MINITAB Software package). The acceptance level of statistical significance was P≤0.05 in all instances.
Results and discussion
On nutrient agar, B. thuringiensis SV2 grew as typical off-white coloured colonies with slightly raised elevation and regular margins in presence of malathion, temephos, cyhalothrin, spinosad, azadirachtin and cypermethrin supplemented with respective pesticide each at 5 ppm with zones of clearance were observed on solid medium after 8 days of incubation.
However, in nutrient broth it was found that, in presence of malathion and temephos, BtSV2 grows well without degrading pesticide but it degrades cyhalothrin, spinosad, azadirachtin, cypermethrin.
These results confirm that temephos and malathion have no inhibitory effect on growth of BtSV2 in broth culture (Figure 1). Hence temephos and malathion are the most suitable pesticides for combination. As per the WHO recommendations, temephos is used as larvicide while malathion as adulticide; so, temephos and BtSV2 combination was evaluated and recorded as a potent larvicidal formulation.
Mosquito larvicidal potential of individual Bt, temephos and their combinations were studied against the fourth instars larvae of Ae. aegypti and An. stephensi. It was observed that in combination, the larvicidal potential of TBT (A) was increased significantly as compared to individual applications of both pesticides.
In TBT the LC50 value against the Ae. aegypti and An. stephensi decrease by 30.68 and 22.36%. These results indicate that the reduction in LC50 values is due to synergistic action of both. For further combination studies, major three preparations were designed i.e. temephos plus Bt A (1:1), B (1:2), C (1:3) and D (1:4) to find out type of interaction taking place during the combined effect of temephos and BtSV2 at different ratio against Ae. aegypti and An. stephensi. Similarly co-toxicity indexes (CTC) for each combination were analysed. In all combinations CTC value was always above 100, indicating synergistic action of combined pesticide. In case of An. stephensi TBT(A) preparation (1:1), showed highest CTC value i.e. 1021.5 (Table 1) while in case of Ae. aegypti, TBT(C) preparation (1:3) with highest CTC value i.e. 1434.75 (Table 2).
The biochemical changes in treated larvae were monitored for BtSV2 and its combinations. It was observed that in both larval treatment TBT caused reduction in protein content as compared to control; on the contrary, in BtSV2 treatment the protein content was slightly increased as compared to control. In an enzymatic study with TBT treatment, the activity of some enzymes were found to be decreased significantly i.e. α-esterase, acetylcholine esterase, while β-esterase and acid phosphatase were found to remain unchanged in any treatment and alkaline phosphatase level was found to be increased. The altered physiological condition in TBT treatment may have contributed to larval toxicity. Total protease level remained unchanged in TBT treatment as compared to untreated control while in BtSV2 treatment it was found to be slightly increased (Figure 2).
Gut proteases, modified receptor and other detoxifying enzymes like esterases, monooxygenase etc. have previously been reported for their major role in mechanism for insect resistance to Bt cry proteins (Rahman et al., 2004; Bravo et al., 2007; 2011). It was observed that in combination study, level of α carboxyl esterase and acetylcholine esterase was decreased than control, and enzymes like total proteases level remain unchanged. So with the use of combined formulation we may expect to have decreased chances of resistance generation against Bt.
In a previous report, the expression of several proteases has been reported in Bt resistant Ae. aegypti (Silva-Filha et al., 2014), but in our observation during TBT treatment protease neither increased or decreased. The level of proteases might have been maintained fixed, due to the contradicting effect of the individual components of the formulation. In organism on one side, Bt toxin might induce activity of proteases as a part its natural protection mechanism while on the other side temephos may be working to decrease activity of proteases. Therefore in the treated larvae finally the activity of proteases seems to remain unaltered. This is also supported by the increased protease activity in BtSV2 treatment.
In mixture treatment the level of acetylcholine was found to be decreased, similar reduction in acetylcholine esterase was previously reported by Koodalingam et al. (2012).
Although the mixture contains chemical pesticide i.e. temephos as well as BtSV2, it was observed that in combination treatment acetylcholine esterase and α-esterase levels were decreased. Previous report advocates rise of esterase level in chemical pesticide exposed insect pest or higher levels of esterase mainly concern with chemical pesticide resistance in mosquitoes and other insects e.g. Culex quinquefasciatus (Sahgal et al., 1994), Blattella germanica (Anspaugh et al., 1994; Polson et al., 2011). Similarly the major role of carboxyl esterase has been reported for its detoxification of organophosphates in resistant pest (Rossiter et al., 2001; Yang et al., 2004).
Besides these, level of alkaline phosphatase (ALP) was found to be increased in mixture treatment than untreated and BtSV2 exposed larvae. The justification for this may lie in fact that, ALP plays vital role in cry toxicity, which is already evaluated in Ae. aegypti. It is well known that ALP found in regions of microvilli where cry toxin binds to microvilli of gastric caeca. Similarly it was also reported that the ALP expression was significantly reduced in Cry1 resistance in insects (Jurat-Fuentes & Adang, 2004; Jurat-Fuentes et al., 2011; Flores-Escobar et al, 2013). In addition, ALPs have also been observed to bind a number of Cry toxins (Jiménez et al., 2012), as in Manduca sexta, Heliothis virescens and Spodoptera frugiperda (McNall & Adang, 2003; Jurat-Fuentes & Adang, 2004; Flores-Escobar et al., 2013).
Although during treatment (mixture treatment, untreated and BtSV2) there was no alteration of acid phosphatase activity in treated larvae. Decreased level of esterases, constant level of gut proteases and increased level of ALP is the ideal physiology reported in susceptible or sensitive pest, such ideal physiology was found to be maintained by combined formulation of temephos and BtSV2.
The combined preparation of Bt and temephos leads to biochemical changes in mosquito larvae, which eventually contributes to increase their toxicity. The formulation may have its implications on the use of reduced quantity of chemical pesticides, which is a major cause of resistance development and ecological pollution. Similarly, due to the differential mode of action, this combination may also contribute to prolong the generation of resistance in pest. Significantly, the cumulative strategy may improve performance of integrated pest management programs.