Principal Investigator:
Upendra Thapa Shrestha
Assistant professor
Universal Science College, Department of Biochemistry
Pokhara University
Research Assistants:
Udaya Prakash Bhandari and Santosh Khanal
Supervisor:
Prof. Dr. Vishwanath Prasad Agrawal
Director
Universal Science College
A. INTRODUCTORY SECTION
A.1. INTRODUCTION
Over half a century of synthetic pesticide applications had led to the emergence and spread of resistance in agricultural pest and vectors of human diseases and to the environmental degradation. The very properties that made these chemicals useful - long residual actions and toxicity to a wide spectrum of organisms - have brought about serious problems. An urgent need has thus emerged for environment friendly pesticides to reduce contamination and the likelihood of insect resistance (Ben-Dov et al., 1997) Human attempts at insect control have changed over time from natural methods to Synthetic chemical control and again now looking to natural methods as it is safe and free from pollution and other undesirable side affects (Neppl, 2000)
One such alternative is the use of microbial insecticides – insecticides that contain microorganisms or their by-products. Microbial insecticides are especially valuable because their toxicity to non target animals and humans is extremely low. Compared to other commonly used insecticides they are safe for both the pesticides user and consumers of treated crops (Neppl, 2000). The soil bacterium B. thuringiensis fulfills the requisites of a microbiological control agent against agricultural pest and vectors of diseases that lead to it’s wide spread commercial applications (Ben-Dov et al.,1999).
The main target pest of B. thuringiensis insecticides include various lepidopterous (butterfly), dipterous (flies and mosquitoes), and individual coleopterons (Beatle) species. Some strains have also been found to kill off nematodes (Edward et al.,1988) .Conventional B. thuringiensis preparations such as those registered in Germany but also worldwide are mostly derived from the highly potent strain B. thuringiensis var. kurstaki HD1, which was isolated in sixties ( Dulmage,1970).
Nepal being an agriculture based country is no exception regarding these problems. Research work shows that the use of pesticides in Nepal is increasing in total amounts. The generally used chemical pesticides in Nepal are organochlorine such as dichlorobiphenyl trichloroethane (DDT), hexachlorocyclohexane, Malathion, organophosphates, carbamates pyrethroids, fungicides as well as rodenticides. These pesticides are non-biodegradable, which have long term toxicity, accumulate in the food chain and pollute the environment. Surface run off from agricultural lands contaminates nearly all the rivers, lakes, ponds, wells etc. Pesticides residues in water may reach human through drinking water. It may also reach human through vegetables, fruits etc, if they are not properly cleaned. DDT residue has been detected in many food items including rice, wheat, meat, vegetables and milk in Nepal. Some of these products show the residual level above the safety limits of Food and Agricultural Organization (FAO) /World Health Organization (WHO). The Central Food Research Laboratory, Kathmandu has also detected pesticides residues in many food samples: among the sample examined 87.5% were the milk sample that were tested and found to contain DDT (Bhattarai, 2002). WHO has estimated that one million pesticides poisoning cases occur every year causing around 20,000 deaths per year globally. Pesticide poison may lead to severe human diseases like asthma and skin disorders, enlargement of liver, cancer, reproductive problems, tumors, and spontaneous abortion psychological problems and even to some extent degeneration of nerves often resulting in paralysis. Dinitrophenols are toxic to liver, kidney and nervous system (Subedi, 1999)
The characterizations of the strains from different niches provide useful information on the ecological patterns of distribution of B. thuringiensis and opportunities for the selection of strains to develop novel bioinsecticidal products.
A.2. BACKGROUND (Previous Results obtained)
Shrestha et al. (2006 and 2007) isolated more than hundred B. thuringiensis isolates from the soil samples of Khumbu region including Sagarmatha National Park and Phereche. All the isolates were found gram positive spore forming rods. Out of 109 isolates 86 were found to have crystal protein. Two isolates, one from Sagamatha National Park and one from Pheriche were contained high mosquitocidal activity. Preliminary experiments suggested that these two isolates survived in waste water for 30 days and retained the mosquitocidal activity.
A.3. PROBLEM
Open drainage system and stagnant water body are the main sites for mosquito breeding. All mosquitoes have one common requirement--they need water to complete their life cycle. Mosquitoes lay individual eggs on the stagnant water bodies. These eggs can lay dormant for several years. These eggs hatch in 24-48 hours releasing larvae that are commonly called "wrigglers". Generally, the larvae feed on microorganisms and organic material in the water, but some mosquitoes prey on the larvae of other mosquito species too. These larvas will be changed into the pupal or "tumbler" stage within 7-10 days in preparation for adult life which are vectors for many diseases. Thus the stagnant waste water bodies containing higher amount of organic matter are the best mosquito breeding sites from where numerous mosquitoes can be generated within a very short period. Therefore the main problem of mosquito breeding is the increasing number of stagnant waste water bodies.
A.4. OBJECTIVES
A.4.1. GENERAL OBJECTIVE
To study of survivability and efficiency of Bacillus thuringiensis in waste water for the biological control of mosquito breeding
A.4.2. SPECIFIC OBJECTIVES
i. To revive B. thuringeinsis isolates from those master cultures preserved in RLABB.
ii. To test the efficiency of revived Bacillus thuringiensis isolates against mosquito larva
iii. To obtain some more potent B. thuringiensis isolates
iv. To inoculate potent B. thuringiensis in waste water in lab and count colonies (colony forming units=cfus) in each 3 days to find their multiplication rate.
v. To observe the survivability of B. thuringiensis in the waste water lab.
vi. To test the efficiency of these isolates against the mosquito larva in natural habitat.
A.5. HYPOTHESES
It is hypothesized that B. thuringiensis will survive and multiply in stagnant waste water and retain its potency so that their spray can be used in controlling mosquito breeding.
A.6. RATIONALE
Mosquitoes are recognized as a health and nuisance problem. Mosquitoes are important pests because their biting activity often interferes with outdoor activities and can transmit many diseases to people and domestic animals. In context of Nepal they are one of the major problems in transmitting vector borne diseases like Malaria, Filaria, Yellow fever, Dengue fever, Japanese encephalitis and many more viral diseases (en.wikipedia.org). The advancement in biological knowledge made available many new controlling methods of mosquito populations. The most important of them is use of microbes as killing agents for mosquito control in order to minimize the environmental side effects of insecticides. Besides it has many more advantages over the synthetic pesticides like
• It does not leave harmful residues
• It has substantially reduced impact on non-target species
• When locally produced, it may be cheaper than chemical pesticides
• In the long-term it may be more effective than chemical pesticides
Therefore the use of Bacillus thuringiensis may be the only alternative for biological control of mosquitoes instead of using synthetic pesticides.
A.7.1. BACILLUS THURINGIENSIS
Bacillus thuringiensis is a ubiquitous gram positive, spore forming bacterium that forms a parasporal crystal during the stationary phase of its growth cycle. B thuringiensis was initially characterized as insect pathogen, and its insecticidal activity was attributed largely or completely (depending upon the insects) to the parasporal crystals. This observation leads to the development of bioinsecticides based on B. thuringiensis for the control of certain insect species among the order Lepidoptera, Diptera, and Coleoptera. There are more recent reports of B. thuringiensis isolates active against other insects of order (Hymenoptera, Homoptera, Orthoptera, and Mallophaga) and against Nematodes, mites and protozoa. B. thuringiensis is already a useful alternative or supplement to the synthetic chemical pesticide application in commercial agriculture for pest management and mosquito control. It is also a key source of genes for transgenic expression to provide pest resistant in plants (Schnepf et al., 1998)
A.7.2. MECHANISM OF ACTION OF TOXIN PRODUCED BY BACILLUS THURINGIENSIS.
When a susceptible insect ingests B. thuringiensis, the protein toxin is activated by alkaline conditions and enzyme activity in the insect's gut. The toxicity of the activated toxin is dependent on the presence of specific receptor sites on the insect's gut wall. This necessary match between toxin and receptor sites determines the range of insect species killed by each B. thuringiensis subspecies and isolate. If the activated toxin attaches to receptor sites, it paralyzes and destroys the cells of the insect's gut wall, allowing the gut contents to enter the insect's body cavity and bloodstream. Poisoned insects may die quickly from the activity of the toxin or may die within 2 or 3 days from the effects of septicemia (blood poisoning). Although a few days may elapse before the insect dies, it stops feeding soon after ingesting B. thuringiensis. B. thuringiensis does not colonize or cycle (reproduce and persist to infect subsequent generations of the pest) in the environment in the magnitude necessary to provide continuing control of target pests. The bacteria may multiply in the infected host, but bacterial multiplication in the insect does not result in production of abundant spores or crystalline toxins. The usual result is that few or no infective units are released into the environment when a poisoned insect dies (Schnepf et al., 1998).
Figure 1: Mechanism of action of toxin in larval midgut.
B. METHODOLOGY
B.1. REVIVAL OF BACILLUS THURINGIENSIS
B. thuringiensis will be revived from the master cultures preserved in RLABB following Acetate selection method to avoid any contamination (Travers et al., 1987). And will be subculture on Nutrient Agar for further processing.
B.2. CHARACTERIZATION OF B. THURINGIENSIS FOR THEIR CRYSTAL PROTEIN FORMATION
Identification will be again carried out by gram staining, spore staining and crystal staining, and biochemical tests, (Claus & Berkeley 1986).
B.3. INSECT BIOASSAY FOR THE SELECTION OF SOME MORE POTENT STRAINS
Mosquito larvae will be collected from the local areas where mosquito breeds for insect bioassay. Ten Mosquito larvae will be placed in containers with 20 ml of distilled water and 5 mg of larval diet, to which will be added whole or solubilized crystals of B. thuringiensis. The larvae will be kept at room temperature and the mortality rate will be recorded with interval of 24 hrs for 3 days if required few more days will be followed up. The isolates with strong mosquitocidal activities will be further preceded.
B.4. TEST FOR SURVIVABILITY AND EFFICIENCY OF B. THURINGIENSIS IN WASTE WATER
Water from Mosquito breeding sites will be collected and B. thuringiensis isolates will be inoculated in it. Quantitative measurement of B. thuringiensis isolates will be done by Plate count method after its isolation following the Acetate selection method (Travers et al., 1987). The survivability will be tested till 30 days or more. After survivability test, the bacteria were again inoculated in waste water containing mosquito larva and the death of mosquito will be recorded in 1, 2 and 3 days and will be recorded for upto 7 days if needed. Then the death of mosquito will be analyzed for whether the death is caused due to B. thuringiensis or not.
C. OBSERVATION/RESULTS
It is expected that B. thuringiensis strains will survive in waste water for 30 – 40 days retaining their mosquitocidal activity.
D. DISCUSSION, CONCLUSION AND RECOMMEDATION
It can be concluded that the potent strains will be really novel ones if they can survive, multiply and kill those mosquito larva in the natural habitat where mosquitoes breed. Therefore our prime focus will be on survivability test of B. thuringiensis isolates in waste water. If we get success in obtaining such potent strains which will show its activity in natural habitat then we will be successful in mosquito control by means of biological method.
E. REFERENCES
Ben-Dov E, Zaritsky A, Dahan E, Barak Z, Sinai R, Manasherob R, Khamraev A, Troitskaya E, Dubitsky A, Berezina N and Margalith Y (1997) Extended screening by PCR for even cry-group genes from field-collected strains of B. thuringiensis. Appl Environ. Microbiol.; 63: 4883-4890.
Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamraev A, Baizhanov M, Glupov V, Margalith Y (1999) Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Appl. Environ. Microbiol. 65: 3714-6.
Bhattarai S (2002) Insecticidal activities of Bacillus thuringiensis against Cules quinquefasciatus and Spodoptera litura. Central Department of Microbiology, Tribhuvan University.
Claus D and Berkeley R W C (1986) Genus Bacillus Cohn 1872. In Bergey’s Manual of Systematic Bacteriology Vol. 2. (Eds. Sneath, P.H.A) Baltimore: Williams & Wilkins. pp. 1105-1138
Dulmage HT (1970) Production of spore-delta-endotoxin complex by variants of Bacillus thuringiensis in two fermentation media. J. Invertebr Pathol 16: 385-389
Neppl CC (2000) Managing Resistance to Bacillus thuringiensis Toxins. Environmental Studies University of Chicago
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR and Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62: 775-806
Shrestha UT, Sahukhal GS, Pokhrel S, Tiwari KB, Singh A and Agrawal VP (2006) Delta-endotoxin immuno cross-reactivity of Bacillus thuringiensis isolates collected from Khumbu base camp of Mount Everest region. Journal of Food Science Technology Nepal. 2: 128-131.
Shrestha UT, Sahukhal GS, Pokhrel S, Tiwari KB, Singh A and Agrawal VP (2007) Strong mosquitocidal Bacillus thuringiensis from Mt. Everest. Our Nature 5: 67-69.
Subedi KR (1999) Insecticidal Activities and Immunology of Delta-endotoxins of Bacillus thuringiensis Isolated from Soil and Insect Samples of Nepal. Central Department of Microbiology, Tribhuvan University.
Travers RS, Martin PA and Reichelderfer CF (1987) Selective Process for Efficient Isolation of Soil Bacillus spp. Appl Environ Microbiol; 53: 1263-6.
http://en.wikipedia.org/wiki/Mosquito-borne_disease
BUDGET ESTIMATE
Table 1: Budget Estimation for our research work
S. no. Particulars Amount in rupees
1. Remuneration to Researches 10,000
2. Remuneration to Assistants 5,000
3. Transportation Cost 2,000
4. Stationary 2,000
5. Typing, Printing and Binding of Reports 2,000
6. Contingencies/Other Expences
Chemicals, media, glasswares 14,000
Total 35,000
F. DURATION OF THE STUDY
-Total six months
-1 month for reviving bacteria and their identification
-2 months for insect bioassay and survivability test in waste water
-3 months for efficiency test of bacteria in field
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