Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Efficacy of Bacillus Thuringiensis Var. Israelensis Against the Larvae of Aedes Aegypti

Murshida Begum
Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh

Homaira Akbar
Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh

Mihir Lal Saha
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Humayun Reza Khan
Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

Efficacy of aqueous suspension of Bacillus thuringiensis var. israelesis (Bti) was studied against the laboratory reared 3rd and 4th instar larvae of Aedes aegypti. Mortality rates as well as histological changes in the larval midgut due to toxic effect of Bti were investigated. Several microscopic techniques were also used to identify the toxic effect on the mosquito larvae. The highest mortality rate (96.66%) was found in case of 3rd instar larvae at l.µl/ml dose where LC₅₀ value was 0.0097. Larval mortalities increased significantly to A. aegypti as doses increased (p< 0.05). Histological study revealed that cellular layers of the midgut epithelium were intact in control sample, but in case of the Bti treated larvae none of these were found in the midgut, only bacterial spores were seen. Results of the microscopic studies indicated that, among the six different colonies found in bacterial cultures, Bti spore, sporangium and vegetative cells were confirmed from one colony by phase transition and fluorescent microscopy. The Cry (crystals) endotoxins and Bti spore were confirmed by the SEM.

Keywords: Aedes aegypti, Bacillus thuringiensis var. israelensis, Midgut, Phase transition microscopy, LCso, Scanning electron microscopy

Location: Department of Zoology, University of Dhaka

Start Date:

End Date:

Program Area: Pest Management

Commodity: Insects

Objectives:

To investigate the effect on toxicity and microbial activity of Bti against A. aegypti.

Methodology:

The selected strain of Bacillus used throughout the experiment was Bacillus thuringiensis var. israelensis (Bti). The aqueous suspension of Bti containing 1200 International unit/ml with 1.2% active ingredient and 98.8% inert ingredient was collected from the laboratory for Plant and food Science, School of Agriculture, food and Wine, University of Adelaide, Australia. One ml of Bti was dissolved in one liter of distilled water to prepare 1 µl/rnl solution. This solution was diluted 10 times to formulate 0.1 µl/rnl dose and 0.01 and 0.00I µl/ml Bti doses were prepared accordingly. A. aegypti was reared in the laboratory of The Department of Zoology, University of Dhaka in an ambient environmental condition at 28±6°C and 70-80% RH. The adult mosquitoes were kept in a rearing cage made of steel frame, covered with mesh net (30x30x30 cm in size). The larvae were kept in a water plastic bowl (7cm in diameter) covered with a piece of fine mesh net. The larvae were fed with cereals and adult female mosquitoes were fed with pigeon blood meal. Adult males were supplied with sugar solution soaked in wads of cotton wool. A batch of 20 healthy, laboratory reared 3rd and 4th instar larvae of A. aegypti were placed separately in beakers containing 200 ml Bti solution each containing 1.0, 0.1, 0.01 and 0.001 µI/ml concentration of bacterial doses to determine the mortality of this mosquito. One ml of Bti was dissolved in 999 ml of distilled water to make 1.0 µl/ml dose. Other doses of Bit were prepared likewise. A. aegypti larval mortalities after 24 hours were recorded. Besides the bacterial doses, whether other factors were involved in the mortality or not, a control replicate was also used. All bioassay experiments were repeated three times. The mean percent of mortalities of different doses of Bti was statistically analyzed using ANOVA. Multiple comparisons were done by Tukey's honest significance test. A probit analysis was also done by following EPA program, version 1.5 for calculating LC values of Bti against 3rd and 4th instar larvae at 95% confidence limit. Mean percent of mortality and LC values of 3rd and 4th instar larvae were compared by t-tests in each dose. Histological slides of the Bti treated 3rd instar larvae of A. aegypli in control and in 0.1µ/ml were prepared by longitudinal sectioning the tissues of the larval midgut region. Ethanol, Myer's albumin and Xyline were used as fixatives. Serial Longitudinal sections of the tissues were cut at 0.5 µm thickness with the help of a rotary microtome machine. The tissue sections were stained with eosin and Heidenhein's haematoxylene in the laboratory condition. Bti treated larval midgut samples were streaked onto a Petridish containing nutrient agar medium with a sterile platinum loop to find specific bacterial cultures. After overnight incubation at 37⁰C, six different bacterial colonies were found in the nutrient agar plate, Subcultures were also done for the six colonies distinguished as sample 1, 2, 3, 4, 5 and 6 separately in the same culture media using the same procedure. Each of the above samples was stained following simple staining procedure with Acridine orange and Crystal violet dye. Bacterial spore staining was done by malachite green dye to observe whether Bt spore available or not. All the stained samples were observed under a Nikon Microphot microscope in phase contrast condition. A Nikon optiphot florescent microscope was also used to observe the sample under UV light in bright field condition. Photographs were taken with a Nikon UFX - II camera in all cases. Scanning electron microscopic (SEM) studies were done from the six pre fixed bacterial sample slides. The samples were sputter coated by platinum and processed to observe under a Jeol JSM 6490 LA scanning electron microscope. This technique is necessary for the identification of bacterial spores and crystal (Cry) toxin.

Source of Information: J. Asiat. Soc. Bangladesh, Sci. 41(1): 33-11-11-, June 2015

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The results of histological examination have been made attention on microbial study of the treated larval midgut element by several microscopic techniques, whether Bti Cry-toxin identification could be possible or not. Different types of dyes were used in several microscopic studies. Vegetative cells, spores, Crystal toxin and paraposal Crystal formation of B. thuringiensis species was identified by phase contrast microscopy, fluorescent microscopy, scanning electron microscopy and electron' microscopy (Mendoza et al. 2012 and Noguera and Ibarra 2010). Crystal toxin and spores of B. thuringiensis var. thompsoni and B. sphaericus was identified by phase contrast microscopic study (Surendran and vennison 2011). Results of the present investigations are in close agreement with the upshots of research done by above authors. Thus, it may be inferred that the Bti is highly susceptible to the larvae of A. aegypti; it causes tissue disruption in the midgut of the larvae of this insect.

Knowledge Management: Journal