Collection of botanicals and extracts preparation: The fresh plant leaves of custard apple (Annona reticulata), thorn apple (Datura stramonium), neem (Azadirachta indica) and tobacco (Nicotiana tabacum) were collected from the HSTU campus and surrounding area. They were kept in the laboratory for 7 days of air drying followed by one day sun drying before making powder. They were powdered separately by an electric grinder in the laboratory and passed through a 60-mesh sieve. Hundred gram of every plant powders were taken separately in a 500 ml conical flask and mixed with 300 ml of methanol solvent. Then the mixture was stirred for 30 minutes and then allowed to shake in the shaker machine for 24 hours. The mixture was then filtered through a filter paper (Whatman no. 1) and allowed to evaporate the solvents in the vacuum rotary evaporator (Lab Tech EV311H Rotary Evaporator, Manufactured in China) for obtained crude extract. The crude extracts were then preserved in tightly corked vials and stored in a refrigerator (4°C) for future experimental use.
Mass culture of tested insect: Adult of Sitophilus oryzae were collected from naturally infested wheat grains of the local market of Dinajpur town. The adult weevils were mass cultured in separate glass jar (500 ml) with the food medium under ambient laboratory conditions (28 ± 2°C and 75 ± 5% RH). Two hundred weevils were released in each glass jar containing 500 gm of wheat seeds. Then the jars were covered with a piece of white muslin cloth tightly fixed with the help of rubber bands to avoid possible escape of the weevils. The jar were left undisturbed for a maximum period of 7 days for oviposition. After oviposition, the weevils were carefully separated from the seeds by sieving and seeds along with eggs were left in the jar for emergence of adult. After emergence, the newly emerged adults were collected and again released in new seeds allowed in different jars for oviposition to continue and maintained the stock culture. Only 1 to 3 days old adult of S. oryzae were used for the experimental purposes.
Dose preparation: The crude extracts were weighted in the electronic balance (Mettler Toledo MS-TS Analytical Balances) and dissolved in methanol solvent for making different doses (7.07, 3.53, 1.76, 0.88, 0.44 mg/cm2 ) along with control. Prior to conducting study, a pilot experiment was done to obtain the appropriate dose.
Insect bioassays (mortality test): Direct toxicity test of botanical extracts against rice weevil were performed in the laboratory with ambient conditions (28 ± 2°C, and 75 ± 5% RH)following the residual film method (Busvine 1971). One ml liquid of each dose (7.07, 3.53, 1.76, 0.88, 0.44 mg/cm2 ) of plant extracts was dropped separately on the Petridishes (60 mm) with the help of micro pipette. Then the plant extracts were covered uniformly to the whole area of the Petri dishes internally and kept open for 15/30 min. to evaporate the solvent. Two days old 10 weevils were released in each Petridish. Control Petri dishes were treated with methanol solvent only. Three replications were made for each concentration of plant extracts including control treatment. The Petridishes were kept without food for mortality tested of plant extracts. Insect mortality was recorded at 24, 48 and 72 hours after treatments (HAT).
Repellent test: The repellent activities of plant extracts were evaluated using the filter paper impregnation method. For this, the filter paper (Whatman no. 1) wascut into two half, and 1 ml solution of each three concentrations were applied to one half uniformly with the help of micropipette. The treated and control papers were then air dried for 20 minutes to evaporate the solvent. The treated half of the paper was attached with the untreated half one in the way that attachment did not interfere with the free movement of weevils from one half to another. The treated and control paper were placed in a glass Petri dish (90 mm) and 5 pair of 5-days old weevils were released at the centre of the filter paper. The Petridish was covered with its lid and kept in undisturbed in the laboratory. Each treatment was replicated thrice and the number of insects on each portion of filter paper was counted at hourly intervals upto the 5th hour with control treatment. The data was expressed as percentage of repulsion (PR) using the following formula: PR= (Nc-50) x 2 where, Nc = % of insects present in the control half. Positive values were expressed repellency while negative attractancy. The average values were categorized as class 0: repellency 0 > 0.01 to 0.1%, class I: repellency 0.1 to 20.0%, class II: repellency 20.1 to 40.0%, class III: repellency 40.1 to 60.0%, class IV: repellency 60.1 to 80.0% and class V: repellency 80.1 to 100.0% (McDonald et al. 1970).
Statistical analysis: The data were statistically analyzed by MSTAT computer program. The significance of the mean differences was tested by DMRT. The observed mortality was also subjected to probit analysis. All the graphical works were done by the Microsoft excel program.