The following materials used and methods during the study period are described below.
2.1 Experimental fish Tilapia (Oreochromis niloticus), were selected as the experimental fish for the study, as this fish are widely cultured in the Patuakhali district.
2.2 Study area: The study was conducted in Dumki upazilla and Bauphal upazilla of Patuakhali district.
2.3 Sample collection A total 80 live or freshly dead host sample fishes were collected from the study area. The samples were collected from four distinct sources; i) Taranga Tonu pond of (PSTU), ii) Nilkomol pond of PSTU, iii) Pirtala fish market of Dumki upazilla, and iv) Chanchal hatchery in Bauphal upazilla of Patuakhali district. Samples were collected by cast net from ponds in PSTU and hatchery; and purchased from bazar. Samples were collected from different sources were transported using oxygenated polythene bags with water in live condition or freshly dead for investigation. Among the 80 samples, twenty (20) samples were collected from Taranga Tonu pond, twenty (20) samples from Nilkomol pond, twenty (20) samples from the Hatchery, and twenty (20) samples from the pirtala fish market. The sampling was done from August 2016 to February 2017 and transported to the Laboratory of Faculty of Fisheries, Patuakhali Science and Technology University for detailed investigation 2.4 Size of host fishes The entire eighty host fishes were almost of the same sizes, 8- 10 inches for every individual.
2.5 Examination of host fishes Foremost, the external surface of the host body including scales, fins, skin, fin base etc. was examined under a Microscope (optical microscopes Italy) for ectoparasites or any kind of lesions. Then scrapping of the skin was done by an unshaped scalpel to collect the mucus in a glass slides for microscopic examination. Next, gills were removed from the branchial cavity by a sharp scissors and placed on a glass slide for microscopic examination for each fishes and also gills slime was collected and incorporated into the slides. After examination of the external surface, the organs were dissected to search the internal parasites. Considerable attention was given to the internal organs, viz., and heart, Liver, Intestine, spleen, gonads and kidneys. Peritoneum and mesenteries were also observed for parasites. After that Parasites were collected from the infected area for gross observation and identified by Microscope (optical microscopes Italy). Cestode parasites were observed under microscopes in a slide and small nematodes and helminthes were collected by a hairbrush. Protozoan parasites were collected from the mucus or body fluid by needle in a slide for microscopic observation.
2.6 Identification of parasites Parasites were then placed under a compound microscope (Optical microscopes, Italy) and then observed. The printed parasites figures were being used to identify them from the respective published papers. After that a digital camera were used to capture the parasitic image.
2.7 Statistical analysis All calculations will be calculated by using Microsoft Excel 2010 and other appropriate statistical analytical program was used.
The following statistical analyses will carried out after Margolis et al. (1982) [8]: 2.7.1 Prevalence Prevalence (%) = (Number of infected host) ÷ (Total number of host examined) ×100 2.7.2 Abundance Abundance (%) = (Number of parasites) ÷ (Total number of host examined) ×100 2.7.3 Mean intensity Mean intensity (%) = (Number of parasites) ÷ (Total number of infected host) ×100.