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Table of Contents
RESEARCH ARTICLE
Year : 2022  |  Volume : 59  |  Issue : 3  |  Page : 246-252

Field evaluation of biosurfactants, surfactin and di-rhamnolipid produced by Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (VCRC B426) against immature stages of the urban malaria vector Anopheles stephensi


1 ICMR-National Institute of Malaria Research, Field Unit, DHS, Campal, Panaji, Goa; ICMR-Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry, India
2 ICMR-National Institute of Malaria Research, Field Unit, DHS, Campal, Panaji, Goa, India
3 ICMR-Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry, India

Date of Submission26-Nov-2020
Date of Acceptance18-Feb-2022
Date of Web Publication08-Dec-2022

Correspondence Address:
I Geetha
ICMR-Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry 605006
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.342401

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  Abstract 

Background & objectives: Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (B426) produce mosquitocidal biosurfactant, surfactin and di-rhamnolipid. The objective of the study was to carry out a small-scale field evaluation of the two biosurfactants to determine the efficacy, application dosage, residual activity and frequency of application against Anopheles stephensi immatures in selected sites in Goa, India.
Methods: Surfactin (VCRC B471) and di-rhamnolipid (VCRC B426) were formulated as aqueous suspensions (5% AS), and were applied at the dosages of 34, 51 and 68 mL/m2 and 27, 41 and 54 mL/m2 respectively. Two experiments were carried out with the two formulations.
Results: Surfactin (VCRC B471) formulation was effective at all the dosages and there was sustained reduction (>80%) in immature density in the treated sites up to 18 days in experiment 1 and up to 15 days in experiment 2. No pupae were found in the treated sites throughout the study. Di-rhamnolipid (VCRC B426) formulation was also found to reduce the immature density in the treated sites up to 14 days in experiment 1 and up to 15 days in experiment 2.
Interpretation & conclusion: For VCRC B471, the optimum application dosage determined was 51 mL/m2 and for VCRC B426, 27mL/m2. The formulations are to be applied fortnightly for effective control of Anopheles. The application dosage determined in the present study can be used for large scale field evaluation to assess their suitability for use in public health programmes for the control of Anopheles mosquitoes vectoring malaria.

Keywords: Biosurfactant; surfactin; di-rhamnolipid; malaria; Anopheles; Bacillus subtilis; Pseudomonas fluorescens


How to cite this article:
Kumar A, Kumar H, Manonmani A M, Prabakaran G, Vijayakumar B, Mathivanan A, Geetha I, Jambulingam P. Field evaluation of biosurfactants, surfactin and di-rhamnolipid produced by Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (VCRC B426) against immature stages of the urban malaria vector Anopheles stephensi. J Vector Borne Dis 2022;59:246-52

How to cite this URL:
Kumar A, Kumar H, Manonmani A M, Prabakaran G, Vijayakumar B, Mathivanan A, Geetha I, Jambulingam P. Field evaluation of biosurfactants, surfactin and di-rhamnolipid produced by Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (VCRC B426) against immature stages of the urban malaria vector Anopheles stephensi. J Vector Borne Dis [serial online] 2022 [cited 2023 Feb 2];59:246-52. Available from: http://www.jvbd.org//text.asp?2022/59/3/246/342401


  Introduction Top


Mosquito-borne diseases pose a major challenge to public health in terms of control and prevention of their transmission. In 1960, World Health Organisation (WHO) initiated a collaborative programme on the evaluation and testing of new insecticides for public health use[1]. Since then, more than thousand molecules have been screened and handful of them have been developed as insecticides[2]. Insecticide resistance is now widespread in a number of mosquito vector species[3],[5] and therefore there is a growing need for novel, cost effective, and reliable mosquito control agents[6],[7][8]. The use of biological control agents is an environment-friendly approach to mosquito control wherein natural enemies of mosquitoes are employed to actively control mosquito population. Most of the biological control agents, however, will at best be used to complement other vector control methods[9]. Many biological control agents are themselves living organisms governed by environmental and other factors for their survival and action on the target organism[2]. Microbial toxins or metabolites can be better ‘biological agents’ as they only need to withstand the aquatic parameters, like water temperature, pH, and salinity.

Secondary metabolites of two bacteria viz. Bacillus subtilis subsp. subtilis (VCRC B471), and Pseudomonas fluorescens (VCRC B426) isolated by Vector Control Research Centre (VCRC) at Puducherry, India, were found to be lethal to immatures (larvae and pupae) of mosquitoes, particularly the pupal stages, in the laboratory[10],[11]. These mosquitocidal toxins were characterized as biodegradable biosurfactants, viz, surfactin (VCRC B471), a lipopeptide and di-rhamnolipid (VCRC B426), a glycolipid [12,[13]. These two biosurfactants are the first ever pupicides to be reported and the immatures of Anopheles stephensi are more susceptible to these two. Upstream and downstream processes for production of these biosurfactants were standardized in our laboratory and Indian patents were granted for both the technologies[14],[15]. Acute oral toxicity/pathogenicity tests conducted on Wistar rats with crude surfactin from B. snbtilis and di-rhamnolipid from P. fluorescens showed no adverse effect on body weight, food consumption, rectal temperature, or the haematological and biochemical parameters of the treated rats. No dermal reactions were observed at 24 h or 72 h following the application of crude biosurfactants, and this confirms that it is non-irritant to the skin of rabbits in the study. The result of the toxicity studies indicated that mosquitocidal biosurfactants are non-toxic to mammals (VCRC, Unpublished data). Data on the efficacy of these biosurfactants in natural conditions is vital to transfer the technology for operational use in the vector control programme. A small scale field evaluation of VCRC B471 containing surfactin and VCRC B426 containing di-rhamnolipid was carried out to determine the efficacy, application dosage, residual activity and frequency of application against Anopheles stephensi immatures in selected construction sites in Goa, India.


  Material & Methods Top


Production of biosurfactants and laboratory evaluation B. subtilis (VCRC B471) and P. fluorescens (VCRC B426) were cultured in liquid medium and biosurfactants were separated from the culture supernatants. Many batches of culture were produced to obtain sufficient quantity of the biosurfactants for field evaluation. Briefly, in the case of VCRC B471, the production strain was cultured in 5 L Erlenmeyer flasks containing 1.5 L of Sugar Urea Mineral Salt Medium containing Urea (3g), Sugar (60g), Sodium Chloride (5g), Citric acid (4g), Ferrous sulphate (0.138g), Manganese sulphate (0.0017g), Magnesium sulphate (0.6g), Calcium chloride (0.001g), Potassium dihydrogen phosphate (1.36g) per L-1) and incubated at 250 rpm for 72 h on a rotary shaker (New Brunswick Scientific, NJ, USA) at 28± 2°C. Bacterial cells were removed from the culture by centrifugation at 9000 × g for 25 min at 4°C in a Hitachi Hispeed refrigerated entrifuge, GR22GIII (Hitachi Koki Co. Ltd., Japan). The cell-free supernatant was used to detect biosurfactants produced by VCRC B471 by determining the surface tension with a Kruss EasyDyne tensiometer (Hamburg, Germany) using the plate method. The surface tension of water (70 mN m-1) was used as a reference. The biosurfactant present in the culture supernatant was precipitated with 6NHCl and collected by centrifugation at 9000 × g for 25 min at 4°C. The precipitate was re-suspended in distilled water, adjusted to pH 7.0 using 1N NaOH and lyophilized (Freeze Dryer Modulyo Edwards; B.O.C. Ltd, Crawley, UK). MALDI-TOF mass spectra were recorded for the precipitate and the analyses showed that surfactin is the chief component of the lipopeptide biosurfactant produced by VCRC B471[16]. The biosurfactant containing surfactin was used for field evaluation.

In the case of VCRC B426, SS Medium containing soya (2%) and sunflower oil (2%) was used for the production of biosurfactant. Production was carried out in 2L flasks containing 600ml SSM, inoculated with 2% inoculum and incubated on a rotary shaker at 30°C, 250 rpm for a period of 72 h. The pupicidal metabolites produced by P. fluorescens was harvested after 72 h by centrifugation at 10,000 rpm for 15 min at 4°C and the cell free supernatant was collected. The supernatant was mixed thoroughly with equal volume of ethyl acetate and the mixture was shaken on a rotary shaker at 180 rpm at 30°C for 2 h. Extraction with ethyl acetate was repeated three times. Then, the aqueous layer was discarded and the solvent layer was transferred to a glass beaker and was allowed to evaporate on a water bath. After evaporation the viscous brown-colored residue left behind was subjected to MALDI-TOF and mass spectra showed that the predominant component produced by P fluorescens was di-rhamnolipid[13].

Using the biosurfactants surfactin and di-rhamnolip-id, five percent aqueous suspension (5% AS) formulations were prepared and tested against pupae of An. stephensi by laboratory bioassay in Vector Control Research Centre (VCRC). The bioassays were conducted separately for the two formulations following standard protocols[17]. Surface tension of the water used for bioassay before and after adding the biosurfactant formulation was measured by a Kruss EasyDyne Tensiometer (Hamburg, Germany) using plate method. Surface tension of distilled water was used as the reference.

Study area

The field evaluation was carried out by National Institute for Malaria Research (NIMR), Field Unit, Goa, in building construction sites and the upcoming apartments in a suburban area, Durgabhat, Ponda, which is about 25 km from Panaji, Goa, India. The area has been endemic for malaria. The major vector breeding habitats are rainwater collections and stagnant waters maintained for curing purposes in the construction sites.

Study design

Evaluation of the two biosurfactant formulations was carried out as per the standard protocol[17]. Water collections in the building construction sites were surveyed for the presence of immatures of Anophelines. Density of larvae and pupae in the sites was recorded before the treatment (Day 2, 1, 0) by taking samples using the dipper method. From each site, five dipper samples were taken and the larval instars and pupae collected from each dipper sample were counted and recorded stage-wise and returned to the site.

The treatment dosage was fixed based on the LC90 dose determined in the laboratory bioassay against pupae of An. stephensi. Three dosages were used for evaluation: 4 times, 6 times and 8 times of LC90 dose. For each dosage five replicates of water accumulations in the construction sites were selected for treatment. Five replicates were kept as control (untreated) for comparison. Allocations for treatment or control were made with comparable pre-treatment immature densities. To determine the final quantity of AS formulations to be applied, the actual surface area of water collections having an average water depth of 17 cm was used. Three dosages were used to determine the optimum application dosage for control of Anophelines. The dosages used for B471 AS were 34 mL, 51 mL and 68 mL/m2; B426 AS was used at 27 mL, 41 mL and 54 mL/m2. On the site, the aqueous suspension formulations were shaken thoroughly, measured and applied directly on the surface of the water so that the biosurfactant spread evenly. Two experiments were conducted in September 2017 and January–February 2018.

Density of immatures in the treated sites was monitored on day 1, 2, 3 and 7 post-treatment and then weekly. Monitoring was stopped when the density of IV instar larvae in the treated sites returned to pre-treatment level or comparable to the density in the control sites. Temperature and pH of water in the pits were recorded every day before taking dipper samples using a thermometer and a handheld pH meter.

Data analysis

The laboratory bioassay data were subjected to probit regression analysis with SPSS 10.0 for windows software and LC50 and LC90 as well as their 95% fiducial limits were determined. In the field application study, the mean number of pupae and larvae collected per dip from the control and treated sites was calculated for each sampling day and for each replicate. The reduction of density of the immatures was estimated by comparing the pre-and post-treatment densities in the treated habitats with the corresponding densities in the untreated habitats using Mulla’s formula[18]. The difference between the dosages was compared using two-way ANOVA with dosage and day as the main factors after transforming percentage reduction (of immature density) to arcsine values[19] using the software STATA 14.2 (Texas, USA). The effect of treatment over days was compared by the interaction effect of dosage and day. Pairwise comparison of dosages was done using a post hoc test based on the least significant difference (LSD). The mean arcsine values were back-transformed to percent values and presented in the text. The optimum application dosage was determined for both the biosurfactant formulations on the basis of effective duration for each of the three dosages. The effective duration of efficacy for the formulation is the day post-treatment up to which the lower limit of the 95 per cent confidence interval for the mean per cent reduction of density will be ≥80 per cent.

Ethical statement: Not applicable


  Results Top


Effect ofbiosurfactant surfactin (VCRC B471) on An. stephensi

In the laboratory bioassays, LC50 and LC90 dosage determined for VCRC B471 (5% AS) were found to be 2.1 and 5 μl/100 mL. The LC90 dosage was multiplied by factor 2 and three dosages i.e., 34 mL, 51 mL and 68 mL/m2, were applied to the sites. The impact of treatment was monitored for 18 days in experiment 1. Pupae were not observed in the treated sites throughout the study period. The efficacy of VCRC B471 formulation in terms of reduction in immature density on different days of post treatment is presented in [Figure 1]. Mean percentage of reduction of immatures in the treated sites obtained was statistically significant, between dosages (F (2,96) =13.773, p<0.001). The interaction effect of dosages by days was not significant (p> 0.05). The mean percentage reduction for the three different dosages used i.e., 34, 51 and 68 mL/m2 was 88.8 [95% CI 82.5 - 93.9], 98.7 [95% CI 95.8 - 99.9] and 95.3 [95% CI 90.7 - 98.4] respectively. Pairwise comparison of dosages showed significant reduction in immature density in all the pairs of dosages. The VCRC B471 formulation was effective at all the dosages and showed a sustained reduction (>80%) in immature density in the treated sites up to 18 days in experiment 1 and the effective duration for dosage 1 was 14 days and for dosage 2 and 3, the duration was 18 days.
Figure 1: Effect of surfactin (VCRC B471) (5% AS mL/m2) on Anopheles stephensi breeding in the application sites in experiment 1.

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In the second experiment, the impact of application of the biosurfactant formulation was assessed for 22 days and no pupae were found in the treated sites. The mean percentage of reduction was significantly different between dosages (F (2,107) =10.661, p< 0.001). Percent reduction of immature also varied significantly over the period (F (8,107) =15.166, p< 0.05). But, the interaction effect of dose and day was not statistically significant (F (16,107) =1.024,p> 0.05), as in the case of the first experiment. The mean percentage reduction (with 95% CI) for the three different dosages used i.e., 34 mL, 51 mL and 68 mL/m2 was 84.1 [77.3–89.9], 91.2 [85.7–95.5] and 95.2 [90.9–98.2] respectively. Pairwise comparison of dosages showed significant reduction in immature density in all the pairs. In experiment 2, the effectiveness of the formulation lasted for up to 15 days and the effective duration for the dosages 1, 2 and 3 was 8, 15 and 15 days respectively [Figure 2]. Based on the two experiments, the optimum application dosage determined was dosage 2 i.e., 51 mL/m2 i.e., the lowest dosage and duration of the residual activity was for 15 days i.e., the shortest duration.
Figure 2: Effect of surfactin (VCRC B471) (5% AS mL/m2) on Anopheles stephensi breeding in the application sites in experiment 2.

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Effect of biosurfactant di-rhamnolipid (VCRC B426) on An, stephensi

The LC50 and LC90 determined in the laboratory for the VCRC B426 formulation were 1.4 and 4 μl/100mL and three dosages i.e., 27 mL, 41 mL and 54 mL/m2 were applied to the sites. The impact of treatment was monitored for 18 days and no pupae were seen in the treated sites. The efficacy of VCRC B426 formulation in the first experiment is presented in [Figure 3]. Mean percentage of reduction was statistically significant between dosage (F (2,93) =8.011, p<0.001) and the pairs of dosage 1 and 2, and 2 and 3 were statistically significant (p<0.05). Percentage of reduction of immatures was not statistically significant over the period (F (7,93) =0.729, p>0.05) and interaction effect of dose and day also was also not statistically significant (F (14,93) =0.824, p>0.05). The mean percentage reduction obtained for the three different dosages i.e., 27 mL, 41 mL and 54 mL/m2 was 84.6 [77.3–90.6], 97.6 [94.0–99.6] and 86.7 [79.8–92.3] respectively. Pairwise comparison of dosages showed significant reduction in immature density between dosage 1 and 2 and dosage 2 and 3. The efficacy was observed up to 14 days in experiment 1 and effective duration for the dosages 1 and 2 was 14 days. For dosage 3, the effective duration was 18 days.
Figure 3: Effect of di-rhamnolipid (VCRC B426) (5% AS mL/m2) on Anopheles stephensi breeding in the application sites in experiment 1.

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In experiment 2, the mean percentage of reduction was statistically significant between the dosages (F (2,108) =3.602, P< 0.05). When pair-wise comparison of dosage was carried out, significant (p<0.05) difference was obtained only between the dosage 2 and 3. Reduction of immature density in the treated sites also varied over the period and it was also statistically significant (F (8,108) =34.855, p<0.05), but interaction effect of dosage and day was not statistically significant [F (16,108) = 1.477, (p> 0.05]. The mean percentage reduction with 95% CI for the three different dosages used i.e., 27 mL, 41 mL and 54 mL/m2 was 87.5 [81.3–92.6], 91.3 [85.8–95.5] and 85.8 [79.3–91.3] respectively. Pairwise comparison of dosages showed significant reduction in immature density between dosage 2 and 3. The efficacy was observed up to 15 days in experiment 2 and the effective duration for the dosages 1 and 2 was 15 days. For dosage 3, the effective duration was only 8 days [Figure 4]. No pupae were found in the treated sites throughout the study period. Based on the two experiments, the optimum application dosage determined for VCRC B426 was the lowest dosage tested i.e., 27 mL/m2 and the effective duration of activity determined was 14 days, the shortest duration.
Figure 4: Effect of di-rhamnolipid (VCRC B426) (5% AS mL/m2) on Anopheles stephensi breeding in the application sites in experiment 2.

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Effect of abiotic factors on the efficacy of surfactin (VCRC B47) and di-rhamnolipid (VCRC B426)

The surface tension of water was reduced from pretreatment level of 70 mN/m to 27 mN/m after treatment with the biosurfactant formulations in the laboratory. During the field experimental period, pH of water ranged from 6.6 to 7.2 in the control sites and from 6.6 to 9.6 in the treated sites during pre-treatment. After treatment with either of the biosurfactants, the pH of water ranged from 6.6 to 11.8 in experiments. The biosurfactants increased the pH of the treated water by 1.1–1.7 times. The water temperature recorded from the breeding sites during the experimental period ranged from 21.4°C to 26°C.


  Discussion Top


The biosurfactant formulations containing surfactin and di-rhamnolipid were effective at all the applied dosages but the duration of effectiveness varied between the dosages. No pupae were found in the sites treated with the biosurfactant formulations throughout the study period. For VCRC B471, the optimum application dosage determined was 51 mL/m2 and for VCRC B426, the optimum application dosage determined was 27 mL/m2 and to be applied fortnightly for effective control of Anophelines. The initial pH of the breeding habitats varied marginally with each site and there was an increase in the pH of the treated water after the addition of VCRC B471 and VCRC B426. The increase in pH was marginal as the biosurfactant formulations were maintained at neutral pH and there was no shift in the pH to either acidic or alkaline in the breeding habitat. It is likely that the water temperature was only within the range of 24°C to 26°C as the breeding sites were in the construction area.

Earlier, an emulsifiable concentrate (EC) formulation (0.09%) of VCRC B426 was tested for efficacy against Culex quinquefasciatus larvae and pupae in cesspits and drains in Puducherry. The formulation was applied at the dosages of100, 200, 300 mL/m2 and it caused 100% elimination of larvae and pupae 24 h post treatments and >80% reduction for a maximum period of 11 days in cesspits and 10 days in U-shaped drains[20]. In the present study, the application dosage of VCRC B426 AS (5%) formulation was 3 times lower than that required for Cx, quinquefas-ciatus and the effective duration was also longer in the field conditions. The reduction in the application dosage and longer residual activity in the present study is because of the difference in the quality of water, mosquito species and formulation applied.

Surface tension (ST) plays a major role in the survival of aquatic stages of mosquitoes. Sing and Micks (1957) studied the effect of surface tension on pupae of Aedes aegypti, Culex fatigans, Culex molestas, Culex pipiens, and Anopheles quadrimaculatus and reported that none of the species can emerge when the surface tension is 41 or less, or 78 or more. Both surfactin and di-rhamnolipid are powerful biosurfactants and were found to reduce the ST of laboratory water from 70 to 27 mN/m. Therefore, in the application sites, the biosurfactant formulations are effective in controlling both larvae and pupae of An, stephensi. Among the different mosquitoes, Anopheles sp, are the least adaptable to change in ST. An, quadrimaculatus was reported as the least adaptable, having very limited range of ST i.e., 53–73.5[21]. In the laboratory, it was proved that Anopheles is highly susceptible to the biosurfactant formulations of VCRC B471 and VCRC B426 compared to other species. Among the immatures, pupae are more susceptible than larvae. Piper and Maxwell (1971) determined the relationship between surface tension reducing properties of non-ionic surfactants and mortality of pupae of Cx, quinquefasciatus. As pupal stages of mosquitoes are solely dependent on their trumpets for respiration, reduction in the surface tension caused by surfactants to a level that prevented the trumpets to retain their position at the water’s surface caused the pupae to lose contact with the air and ultimately death[22]. In the present evaluation, because of the reduction in ST by surfactin and di-rhamnolipid, no pupae were able to come to the water column and were not seen from the treated sites till the last day of observation i.e. Day 22.

Contemporary control of human disease vectors relies almost exclusively on insecticide-based interventions. Currently, to control/eliminate malaria in the face of insecticide resistance requires new tools to be operational within the next 5–10 years[23],[24]. Diverse alternative technologies such as toxic sugar baits, house screening/modification, endectocides, repellents, lethal ovitraps, mass trapping and genetic control strategies are all the subjects of the ongoing research[25]. At present, only a few biological agents are registered for public health use and vaccines are not available for most of the mosquito borne diseases and vector control is still an important strategy for disease control.


  Conclusion Top


The present study showed that the two biosurfactant formulations containing surfactin and di-rhamnolipid are effective in controlling the immatures of Anopheles and the residual activity lasts for two weeks in the treated sites. The application dosage of the formulations determined in the present study can be used for large scale field evaluation to assess their suitability for use in public health programmes for the control of Anopheles mosquitoes vectoring malaria. Thus, products based on biosurfactants containing surfactin and di-rhamnolipid can be added to the arsenal of biocontrol agents, to combat Anopheles mosquitoes, and subsequently, malaria, after large scale field trial or Phase III evaluation.

Conflict of interest: None





 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]



 

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