|Year : 2017 | Volume
| Issue : 6 | Page : 714-721
Fly ash-based water dispersible powder formulation of Bacillus thuringiensis var. israelensis: Development & laboratory evaluation against mosquito immatures
Saravanan Tamilselvan1, Arulsamy Mary Manonmani2, Purushothaman Jambulingam3
1 Department of Biotechnology, Pondicherry University, Puducherry, India
2 Department of Microbiology & Molecular Biology; ICMR-Vector Control Research Centre, Puducherry, India
3 ICMR-Vector Control Research Centre, Puducherry, India
|Date of Submission||29-Apr-2015|
|Date of Web Publication||13-Apr-2018|
Dr. Arulsamy Mary Manonmani
Department of Microbiology & Molecular Biology, ICMR-Vector Control Research Centre, Indira Nagar, Puducherry 605 006
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background & objectives: Bacillus thuringiensis var. israelensis (Bti) formulations are presently being used for insect control. In this study, a water dispersible powder (WDP) formulation using fly ash (FA) as a carrier material was developed and studied for its activity against the larval stages of major mosquito vector species.
Methods: An indigenous isolate Bti (Vector Control Research Centre B17) was mass produced using a 100 l fermentor in soya-based medium. The bacterial biomass was mixed with lignite FA and made into WDP formulations. The most effective formulation was used for determining 50 per cent lethal concentration (LC50) against the larval stages of major mosquito vector species, effect on non-target organisms and mammalian systems using standard protocols.
Results: Sixteen types of WDP formulations were prepared, of which the formulation containing bacterial biomass, FA and carboxymethyl cellulose was found to be the most effective. The LC50values of the formulation against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi larvae were 0.0417, 0.0462 and 0.1091 mg/l, respectively. The formulation was found to be safe to non-target organisms found associated with the mosquito larval stages and also to mammalian systems.
Interpretation & conclusions: The study shows that FA can be effectively used to replace commercially available carrier materials used in biopesticidal formulations.
Keywords: Bacillus thuringiensis var. israelensis - fly ash - laboratory evaluation - mosquito biolarvicides - water dispersible powder formulation
|How to cite this article:|
Tamilselvan S, Manonmani AM, Jambulingam P. Fly ash-based water dispersible powder formulation of Bacillus thuringiensis var. israelensis: Development & laboratory evaluation against mosquito immatures. Indian J Med Res 2017;146:714-21
|How to cite this URL:|
Tamilselvan S, Manonmani AM, Jambulingam P. Fly ash-based water dispersible powder formulation of Bacillus thuringiensis var. israelensis: Development & laboratory evaluation against mosquito immatures. Indian J Med Res [serial online] 2017 [cited 2021 Aug 2];146:714-21. Available from: https://www.ijmr.org.in/text.asp?2017/146/6/714/230141
Mosquitoes play a predominant role in the transmission of malaria, dengue fever, yellow fever, filariasis and several other diseases . Effective control of aquatic mosquito larvae has been reported to be achieved using Bacillus thuringiensis var. israelensis (Bti). Although Bti has been in use for more than two decades, no resistance has been detected in target insects exposed to this biolarvicide. Bti is highly selective for use against members belonging to the family Culicidae and Simuliidae. It offers an additional advantage of not affecting non-target species of vertebrates and invertebrates, thereby ensuring the safety of its prolonged use on a large scale, without damaging the environment . The effectiveness of Bti is dependent on the bioavailability of the material in treated areas, which in turn depends on the design of the formulation. An ideal mosquito larvicidal formulation comprises the active ingredient (Bti), additives and carrier material. A number of additives such as carboxymethyl cellulose (CMC), gum Acacia, Xanthan gum, guar gum, pectin, starch, polyethylene glycol (PEG), sodium alginate, paraffin, gelatin and lignin have been used in biopesticidal formulations ,,. In India, coal and lignite are the most economic and easily available raw materials for power generation, but its utilization is faced with several environmental constraints, the main being production of fly ash (FA) in enormous quantities (120 million tonnes per annum) which needs to be disposed of in an appropriate manner.
The present study reports the development of water dispersible powder (WDP) formulations using FA generated from the neighbouring Neyveli Lignite Corporation, Neyveli, Tamil Nadu, India, and an indigenous isolate of Bti (VCRC B17). The best formulation was taken up for the evaluation under laboratory conditions against the larval stages of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti, vectors of filariasis, malaria, chikungunya and dengue, respectively.
| Material & Methods|| |
Bacterial strain: Bti (VCRC B17), an indigenous isolate , was obtained from the microbial culture collection of ICMR-Vector Control Research Centre, Puducherry, India. The strain was revived from the lyophilized spores and streaked onto modified nutrient yeast salt medium (NYSM) agar slants . The slants were incubated at 30°C for 48 h and then stored at 4°C for further use.
Inoculum preparation:The seed inoculum was produced using shake flasks. The first-stage seed was prepared by inoculating 10 ml of NYSM broth with one loop full of a slant culture and incubating at 30°C on a rotary shaker (200 rpm) for 6 h. The second-stage seed was prepared by inoculating 2 per cent v/v of first-stage seed into 600 ml of NYSM medium in a 2 l Erlenmeyer flask and incubating on a rotary shaker as done earlier.
Pilot sale fermentation:The second-stage seed in log phase was used to inoculate a 100 l bioreactor at 2 per cent (v/v). Fermentation experiments were conducted in a pilot scale bioreactor (Bioengineering, Wald, Switzerland) filled with 60 l production medium (2.0% soya powder) as describedearlier , except for the stirrer speed which was adjusted to 200 rpm. Fermentation was terminated after completion of spore-crystal complex formation as confirmed by microscope (Motic Microscope Model DM143, Germany). The fermentation was repeated on three different days.
Continuous flow centrifugation:The sporulated culture broth was harvested by centrifugation using a continuous flow centrifuge (CEPA Z41, Germany) as described elsewhere . The bacterial biomass deposited in the form of a cake on the rotor was scooped out and used for the preparation of WDP formulation.
Processing of carrier material: The lignite FA was obtained from Neyveli Lignite Corporation Limited, Neyveli, Tamil Nadu, India. It was powdered using a ball mill and the FA powder was sieved through 25-μ mesh, to obtain particles of size ≤25 μ to suit the feeding size range of mosquito larvae. This FA material was sterilized and used in the preparation of formulations. The elemental analysis of FA was done using scanning electron microscope (SEM) (Model JSM-6510LV, JEOL, USA) and energy dispersive X-ray spectroscopy (EDS) (INCAPentaFETx3 Model 8129, Oxford Instruments, England).
Development of formulation:Sixteen types of WDP formulations were prepared using a mixture of bacterial biomass: FA (4:5) and various quantities of organic, plant-based and synthetic polymer additives, namely CMC or Acacia gum or soluble starch or PEG or Xanthan gum purchased from HiMedia, India, respectively [Table 1]. The formulations were dried at 40°C, ground to a fine powder, sieved to a size of ≤25 μ and stored after confirming the final moisture content to be 5 per cent. The final product of these formulations was greyish fine-sized powder that dispersed readily when mixed with water.
|Table 1: Composition of fly ash-based water dispersible powder formulations|
Click here to view
Laboratory evaluation: The WHO standard procedure  was followed to determine the efficacy of the WDP formulations against late third instars of Cx. quinquefasciatus, obtained from the rearing and colonization facilities of our institute. Suspensions of the WDP formulations were made by suspending 10 mg of each formulation in 10 ml of sterile distilled water and mixed well using glass homogenizer. Range-finding bioassays were performed using a wide range of concentrations ranging from 1 to 10 μg. Each concentration had four replicates each, along with appropriate number of controls which contained only plain water. Larval mortality was scored after 24 h. The experiment was done at least three times on different days with freshly prepared suspensions.
Statistical analysis: Larval mortality in control (5-20%) was corrected according to the Abbott's formula . The corrected mortality was subjected to mortality-concentration regression analysis  to calculate 50 and 90 per cent lethal concentration (LC50 and LC90) values as well as their 95 per cent fiducial limits (95% FL) using log-probit analysis software (SPSS Statistics ver. 21, IBM Corporation, NY, USA). The LC50 and LC90 values obtained for WDP formulations were subjected to one-way ANOVA followed by Tukey's honest significant difference (HSD) multiple comparison test to determine the differences in formulations.
Susceptibility of Anopheles stephensi and Aedes aegypti:The most effective WDP formulation from the test with Cx. quinquefasciatus was selected and the dose required for inciting LC50 and LC90 for the larvae of other two major mosquito vectors, namely, An. stephensi and Ae. aegypti, were determined using the WHO procedure . The LC50 values obtained for different mosquito species were compared using one-way ANOVA and post hoc tests (Tukey HSD) were performed to determine the difference in the susceptibility among species.
Toxicity against non-target organisms:The safety of the selected WDP formulation to non-target organisms that are commonly found in association with mosquito larvae in aquatic habitats, namely Ostracods, Cyclops, Daphnia sp., Notonecta sp., Diplonychus sp. and fish, was studied . These organisms were collected from the aquatic environments where the biopesticides are applied and tested in laboratory at the dose of 1.54 mg/l (10 times the LC90 value obtained for An. stephensi which was the species found to be most tolerant to the formulation) in quadruplicate. The fauna was observed for one week for mortality if any.
Mammalian toxicological studies: The safety of this WDP formulation on mammalian systems was done at the International Institute of Biotechnology and Toxicology (IIBAT), Padappai, Tamil Nadu, India. The tests done were acute oral toxicity/pathogenicity in Wistar rats and acute dermal toxicity/pathogenicity and primary skin irritation in New Zealand white rabbits, respectively.
| Results|| |
Nature and properties of fly ash (FA):The SEM observation of FA sample revealed greater number of hollow glass spheres called cenospheres [Figure 1], which are hard, rigid, lightweight and inert largely made of silica and alumina. This property prompted us to use FA as carrier in our formulation. The size of the FA particles used in the formulation ranged between 1 and 25 μ. The SEM-EDS analysis of FA revealed the presence of only macro- and micro-nutrients such as Si, Al, Ca, Fe together with Mg, S, Na and Cu. It did not contain any toxic heavy metals such as Pb, Cd, Ni, As, Hg, Se and Cr and radionuclides such as U, Ra and Th. [Figure 2].
|Figure 1: Scanning electron micrograph of fly ash showing cenospheres in different sizes (×2000).|
Click here to view
|Figure 2: (A) Energy dispersive X-ray spectroscopy image showing the major elements in the fly ash. (B) Scanning Electron Micrograph (Energy dispersive X-ray spectroscopy) showing the peaks of major and minor elements and its actual proportion in fly ash.|
Click here to view
Evaluation of the water dispersible powder (WDP) formulations:Larvicidal efficacy of the various WDP formulations is given in [Table 2]. Among the 16 formulations tested against Cx. quinquefasciatus, WDPA2 was found to be the most active, with an LC50 value of 0.0419 mg/l and LC90 of 0.0753 mg/l. This formulation contained FA and technical grade Bti in the ratio 4:5 with one per cent CMC as a binding agent. The ANOVA test showed that the LC50 and LC90 values among the WDP formulations were significantly different at P<0.001. The post hoc tests indicated that the activity of WDPA2 formulation was significantly different and required lesser concentration when comparing its LC50 and LC90 values with that of all the other formulations (P<0.01). The formulation WDPAA without binder showed less activity than WDPA2 with the LC50 and LC90 of 0.0646 mg/l (0.0606-0.0686) and 0.1062 mg/l (0.0999-0.1142), respectively [Table 2]. The formulation which showed the least activity was WDPB3 with LC50 of 0.0830 mg/l (0.0779-0.0883) and LC90 of 0.1378 mg/l (0.1287-0.1496).
|Table 2: Toxicity of fly ash-based water dispersible powder formulations against late third instar Culex quinquefasciatus larvae|
Click here to view
Susceptibility of different species of mosquito larva:Among the three species of mosquito larvae tested, the LC50 and LC90 values of the WDPA2 formulation against Cx. quinquefasciatus, Ae. aegypti and An. stephensi larvae were 0.0417, 0.0462 and 0.1091 mg/l and 0.0755, 0.0928 and 0.1541 mg/l, respectively [Table 3]. The ANOVA test showed that the LC50 and LC90 values among the mosquito larvae of different species were significantly different at P<0.05. The post hoc tests indicated that the LC50 and LC90 values were significantly lower at P<0.06 and P<0.05 for Cx. quinquefasciatus compared to that of Ae. aegypti and An. stephensi (P<0.05), respectively. The test further indicated that the LC50 and LC90 values for Ae. aegypti larvae were significantly (P<0.05) lower than that for An. stephensi.
|Table 3: Susceptibility of III instar larvae of different mosquito species to water dispersible powder A2 formulation|
Click here to view
Effect of ingredients (individual/combined) of the WDPA2 formulation on mosquito-larvicidal activity:Among the components tested, the Bti with FA+CMC resulted in mortality rate of 51.7 per cent. The percentage mortality due to FA and CMC was only 1.3 and 2.3 per cent, which was well below the allowed mortality levels in control experiments. The ANOVA results showed that there was significant difference among percentage mortality due to ingredients (P<0.001). The post hoc tests further indicated that the percentage mortality in Bti+ FA+CMC (WDPA2) was significantly different from FA and CMC tested alone (P<0.001). However, the mortality in FA alone did not differ significantly from CMC. Hence, FA and CMC as carrier and additive did not have any significant effect on the larval population and the larvicidal activity of the WDPA2 formulation was only due to the presence of Bti.
Tests against non-target organisms and mammalian systems:When tested at 10 times the concentration of WDPA2 formulation used for obtaining 90 per cent kill in the least susceptible mosquito larvae of species An. stephensi, the WDPA2 formulation was found to be safe to Crustaceans, namely Ostrocods, Cyclops and Daphnia, insects, namely Notonectids and Diplonychus, and fish, namely Poecilia [Table 4]. Acute oral toxicity conducted on rats and acute dermal toxicity and primary skin irritation tests conducted on rabbits showed that the WDPA2 formulation was non-toxic and non-irritant on mammalian systems.
|Table 4: Toxicity of water dispersible powder A2 formulation to non-target organisms|
Click here to view
| Discussion|| |
The trend to use biological control agents in mosquito control programmes has gained widespread importance in recent years due to detrimental effects of chemical insecticides on the environment and human health. One of our indigenously isolated mosquito-larvicidal agents Bti (VCRC B17) was found to be highly effective against larvae of different mosquito species in various aquatic habitats ,. Mosquito larvae (especially late instars) being filter feeders are reported to selectively feed on particles of colloidal to 50 μ in size ,,. Hence, incorporation of FA as a carrier material has enhanced the chances of ingestion of this formulation by the mosquito larvae. The SEM analysis of FA used for making WDP formulations contains only macro- and micro-nutrients which is beneficial when applied in freshwater bodies such as paddy fields. Dutta et al revealed that the leaching of toxic elements/heavy metals from FA was negligible when the p H of the water body was alkaline or nearly neutral and mosquito larval-breeding habitats are always reported to be alkaline in nature.
This study showed that the LC50 values were lower for Culicines than for Anophelines. The susceptibility pattern of the larval stages of the three vector species to this biopesticide was of the following order: Cx. quinquefasciatus<Ae. aegypti<An. stephensi. This was in agreement with many other reports with this bacterial species ,. The relative lower efficacy of Bti formulation against Anopheles species might be due to their reduced filtration rates as has been reported by Aly et al. Further, this variation in the susceptibility has been attributed to variation in the gut p H of these insect species which is known to play a major role in the activation of the endotoxins of this bacterium . Differences in the feeding behaviour of these three species are also known to be responsible for this varied susceptibility .
The formulation was found to be safe to non-target organisms found in association with mosquito larvae in the aquatic habitats. This observation reinforces the results of several earlier studies conducted with Bti,,. This study showed that the carrier material and additive, namely FA and CMC used in this formulation, as well as the active ingredient, Bti, were safe on mammalian systems. Hence, FA can be effectively used to replace commercially available carrier materials in the preparation of biopesticidal formulations. FA has earlier been successfully used as such or as carriers for the production of biofertilizers and biopesticides for agriculture ,. Furthermore, powder formulations are reputed for their long shelf life, miscibility in water compared to technical grade materials, microgels and aqueous suspensions .
In conclusion, with biopesticides gaining wide importance in recent times in the wake of maintaining a safe environment, use of FA will help not only in adding to its utilization as almost half of the formulated material contains FA but also in ensuring safety to the environment where it is applied as it has proved to be safe to non-target organisms and mammalian systems.
| Acknowledgment|| |
This work was supported by a research grant (EE-36, 2007-2011) from the Ministry of Coal, Government of India and Central Mine Planning and Designing Institute Limited (CMPDIL), Jharkhand, India. Authors thank Sarvshri K. Sathianathan, R. Jayabala and S. Panneerselvam for technical assistance. Authors acknowledge the officials of Centre for Applied Research and Development (CARD), NLC, Neyveli, Tamil Nadu, and Department of Entomology, Annamalai University, Chidambaram, Tamil Nadu, for their help in providing the fly ash material and powdering the same. The Senior Research Fellow provided by Coal S&T to the first author (TS) is gratefully acknowledged.
Conflicts of Interest: None.
| References|| |
Cheng SS, Huang CG, Chen YJ, Yu JJ, Chen WJ, Chang ST, et al.
Chemical compositions and larvicidal activities of leaf essential oils from two Eucalyptus
species. Bioresour Technol
Sharma SK, Upadhyay AK, Haque MA, Raghavendra K, Dash AP. Field evaluation of a previously untested strain of biolarvicide (Bacillus thuringiensis israelensis
H14) for mosquito control in an urban area of Orissa, India. J Am Mosq Control Assoc
Lacey LA, Mulla MS. Safety of Bacillus thuringiensis
(H-14) and Bacillus sphaericus
to non-target organisms in the aquatic environment. In: Laird M, Lacey LA, Davidson EW, editors. Safety of microbial insecticides
. Boca Raton: CRC Press Inc.; 1990. p. 169-88.
Bergeron V, Martin JY, Vovelle L. Use of polymers as sticking agents. United States Patent 6534563B1; 2003.
Aguilar-Meza O, Ramírez-Suero M, Bernal JS, Ramírez-Lepe M. Field evaluation against Aedes aegypti
larvae of aluminum-carboxymethylcellulose-encapsulated spore-toxin complex formulation of Bacillus thuringiensis
. J Econ Entomol
deChant P, Devisetty BN. Formulation and delivery of Bacillus thuringiensis
and Bacillus sphaericus
in combination and broad spectrum activity and management of resistance to biological mosquito larvicides. Valent Biosciences Corporation: United States Patent 8454983B2; 2013.
Vitekari HN, Talele AP, Mane RG, Gaikwad VS, Shah JV. Fly ash based biopesticides: A comprehensive review.Int J Pharm Biol Sci
Balaraman K, Hoti SL, Manonmani AM. An indigenous virulent strain of Bacillus thuringiensis
, highly pathogenic and specific to mosquitoes.Curr Sci
Myers PS, Yousten AA. Localization of a mosquito-larval toxin of Bacillus sphaericus
1593.Appl Environ Microbiol
Prabakaran G, Balaraman K, Hoti SL, Manonmani AM. A cost-effective medium for the large-scale production of Bacillus sphaericus
H5a5b (VCRC B42) for mosquito control. Biol Control
Prabakaran G, Hoti SL. Application of different downstream processing methods and their comparison for the large-scale preparation of Bacillus thuringiensis
after fermentation for mosquito control. Biologicals
World Health Organization. Guidelines for laboratory and field testing of mosquito larvicides
. WHO/CDS/WHOPES/GCDPP/2005.13. Geneva: Communicable Disease Control, Prevention and Eradication, WHO Pesticide Evaluation Scheme, WHO; 2005. p. 1-39.
Abbott WS. A method of computing the effectiveness of an insecticide.J Econ Entomol
Finney DJ. Probit analysis
. Cambridge: Cambridge University Press; 1952.
Lacey LA, Merritt RW. The safety of bacterial microbial agents used for black fly and mosquito control in aquatic environments. In: Hokkanen HT, Hajek A, editors. Environmental impacts of microbial insecticides
. Netherlands: Springer; 2003. p. 151-68.
Balakrishnan N, Pillai PK, Kalyanasundaram M, Balaraman K. Efficacy of a slow release formulation of Bacillus thuringiensis
H 14 against mosquito larvae. Indian J Med Res
Jambulingam P, Kuriakose KM, Gunasekaran K, Manonmani AM. Field efficacy of Bacillus thuringiensis
H-14 formulations against mosquito larvae in casuarina & coconut garden pits. Indian J Med Res
Clements AN, Kerkut GA. The physiology of mosquitoes. In: International series of monographs on pure and applied biology
. New York: Pergamon Press, Elsevier; 1963. p. 393.
Dadd RH. Effects of size and concentration of particles on rates of ingestion of latex particulates by mosquito larvae.Ann Entomol Soc Am
Wallace JB. Filter feeding ecology of aquatic insects.Annu Rev Entomol
Dutta BK, Khanra S, Mallick D. Leaching of elements from coal fly ash: Assessment of its potential for use in filling abandoned coal mines.Fuel
Balaraman K, Balasubramanian M, Manonmani LM. Bacillus thuringiensis
H-14 (VCRC B-17) formulation as mosquito larvicide. Indian J Med Res
Mulla MS. Activity, field efficacy, and use of Bacillus thuringiensis israelensis
against mosquitoes. In: de Barjac H, Sutherland DJ, editors. Bacterial control of mosquitoes & black flies: Biochemistry, genetics and applications of Bacillus thuringiensis israelensis and Bacillus sphaericus
. New Brunswick, New Jersey: Rutgers University Press; 1990. p. 134-60.
Aly C, Mulla MS, Xu BZ, Schnetter W. Rate of ingestion by mosquito larvae (Diptera
) as a factor in the effectiveness of a bacterial stomach toxin. J Med Entomol
Faust RM. Toxins of Bacillus thuringiensis
: Mode of action. In: Briggs JD, editor. Biological regulation of vectors: The saprophytic and aerobic bacteria and fungi
. Washington: U.S. Department of Health, Education, and Welfare; 1977. p. 31-48.
Sun CN, Georghiou GP, Weiss K. Toxicity of Bacillus thuringiensis
to mosquito larvae variously resistant to conventional insecticides.Mosq News
Boisvert M, Boisvert J. Effects of Bacillus thuringiensis
on target and nontarget organisms: A review of laboratory and field experiments.Biocontrol Sci Technol
Arteaga ME, Mancebo A, Molier T, Gómez D, González C, Bada AM, et al.
Dermal toxicity, eye and dermal irritation and skin sensitization evaluation of a new formulation of Bacillus thuringiensis
SH-14. Regul Toxicol Pharmacol
Arivazhagan K, Ravichandran M, Dube SK, Mathur VK, Khandakar RK, Yagnanarayana K, et al
. Effect of coal fly ash on agricultural crops: Showcase project on use of fly ash in agriculture in and around thermal power station areas of National Thermal Power Corporation Ltd., India. World of Coal Ash (WOCA) Conference; 2011 May 9-12; Denver, CO, USA; 2011. Available from: http://www.flyash.info/2011/016-Arivazhagan-2011.pdf
, accessed on November 30, 2017.
Manonmani AM, Prabakaran G, Hoti SL. Retention of mosquito larvicidal activity of lyophilized cells and WDP formulation of Bacillus thuringiensis
on long-term storage. Acta Trop
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]
|This article has been cited by|
||Carlina oxide from Carlina acaulis root essential oil acts as a potent mosquito larvicide
| ||Giovanni Benelli,Roman Pavela,Riccardo Petrelli,Franks Kamgang Nzekoue,Loredana Cappellacci,Giulio Lupidi,Luana Quassinti,Massimo Bramucci,Stefania Sut,Stefano Dall’Acqua,Angelo Canale,Filippo Maggi |
| ||Industrial Crops and Products. 2019; 137: 356 |
|[Pubmed] | [DOI]|