2.1. Preparation of culture vials The experiments were conducted in the Animal Biotechnology Laboratory of the Department of Genetic Engineering and Biotechnology at the Shahjalal University of Science and Technology (SUST), Sylhet, Bangladesh. Some vials and glass bottles (205 mm×180 mm, the mouth of the bottle is 100 mm diameter) were used for the experiments which are the best approach for Drosophila culture in the laboratory. These bottles are stronger than other glass bottles and also safe for cooking old media and reusing. These glass bottles are also transparent for observing the growth and development of the fruit fly. Some test tubes were also used for lifespan experiments. All vials and bottles were decontaminated very well by washing with tap water, distilled water, detergent, 70% ethanol and using the sterilizer.
2.2. Collection and maintenance of flies Tomato, lemon, banana, etc. were used as food for fly trapping. Fruits and vegetables were cut into the slice and kept on the dining table near the kitchen. When fruits and vegetables were about to rotten, fruit flies were flying around the rotting fruits and gobbled up the rotten fruits. The flies were collected with a water glass by inverting it carefully upon the flies so that they could not escape. Fruit flies always moves so the glass mouth was closed by hand to prevent the flies from escaping. Then, the flies were transferred to the bottle by keeping the mouth of the bottle to the mouth of glass inversely. This time the bottle was positioned at the top of the opening of glass and the glass remained on the ground. Before starting the present study, flies were maintained at room temperature on 12 hr light/12 hr dark cycle at 70%-90% relative humidity, with standardized nourishment prepared with agar (Wako Pure Chemical Industries Ltd., Japan), yeast extract (Wako Pure Chemical Industries Ltd., Japan), yellow cornmeal (Quaker Oats Company, USA), jackfruit, water and Natamycin fungicide (Sigma-Aldrich Corporation, USA) for several generations.
2.3. Identification of the experimental species The experimental fruit fly, D. melanogaster is one of the most common flies available in Bangladesh and easily identifiable than other fruit flies such as D. funebris and D. hydei considering different morphometric characteristics. Male and female D. melanogaster was confirmed by studying its life cycle considering different previous observations.
2.5. Experimental set up for the analysis of phenotypic characteristics Five vials were set up for reproduction and life cycle experiment and six vials for lifespan experiment and labeled all of them with a marker pen for each with the respective concentration of iron. Genders, generations, and dates of the beginning of the experiments were added to the label upon requirement. Prepared modified jackfruit fortified media were poured about 100 ml into the clean and dry labeled bottles. A funnel was used for transferring the media into bottles so that no media was clung to the side of mouth of the bottles. A cotton plug was put on the top of the culture bottles and kept it untouched until the media was solidified. After the media was solidified, a few grains of baker’s yeast were added to the surface. From the stock of D. melanogaster, some newly emerged (2 days of inoculation) were then transferred to an ice pad and put under anesthesia. Flies were then sorted by gender and separated into males and females group. Five unmated males and five virgin females were added per bottle for life cycle and reproduction experiment. Lifespan experiment was carried out with only one sex considering eight individuals. The bottles were kept in a definite place and each experiment was repeated 3 times.
2.7. Microscopic analysis of fruit flies Flies should be anesthetized using least harmful and simple cooling method before observing under the biological microscope (Model: CX31, Olympus Corporation, Japan). Culture bottle was placed in the freezer for 8-10 minutes until the flies are not moving. Then the flies were placed on the top of a Petri dish and the bottom of the Petri dish was placed on the top of a zip-lock type icebox. According to a previous report, keeping flies in this cooling system will help them to remain chilled long enough for further experiments. After each experiment, the flies were simply placed back into the culture vial. Observation of growth, development and all the stages of D. melanogaster was made out regularly. All development stages such as egg, larvae, pupae and adult fly as well as male and female of D. melanogaster was examined under the microscope with 4X/0.10 power magnification lens. Photographs of all stages of D. melanogaster with adult male and female were taken cautiously. 2.8. Statistical analysis Quantitative data was analyzed statistically considering mean, standard error, and significance t-test, etc.