MATERIAL AND METHOD 28 Chapter III Material and Method 3.1 Line of Work: Initially for six months from July 2007 to December 2007 different localities of North Maharashtra were visited to design line of work. North Maharashtra includes four districts namely; Nashik, Dhule, Jalgaon and Nandurbar. Sites were selected randomly from each district such that the sites represent the entire area of the district (Table 3). Total 44 sites were selected from entire study area. Tables: Line of work designed to cover entire study area Name of Number Area per Names of selected Survey site District of sites sq. km Nashik city, Trimbakeshwar, Igatpuri, Sinnar, Saptashrungi gadh, Lasalgaon 15,530 Vinchur, Dindori Kalwan, Satana, Nashik 16 (ISTehsils) Jaikhedan, Malegaon, Ankai, Peint, Surgana, Andarsul Dhule city, Lambkhani, Sakri, Varse, 8,061 Chhadwel, Indva, Sindkheda, Shirpur, Dhule 09 (04 Tehsils) Boradi. Chalisgaon, Mehunbare, Pachora, 11,765 Parola, Amalner, Chopda, Jalgaon city, Jalgaon 12 (15 Tehsils) Yaval, Bhusawal, Jamner, Edalabad, Raver. Navapur, Nandurbar city, Shahada, 5,034 Taloda, Dhadgaon, Toranmal, Nandurbar 07 (06 Tehsils) Akkalkuwa Total sites 44 29 Visits During year 2008 Table 4: Visit during year 2008 2"" week 3"" week 4'" week Sr. Jan.2008 1"' week visit visit visit visit No. -Dec. 2008 Nashik Dhuie Jalgaon Nandurbar Nashik city, 1. January Dhule city Chalisgaon Navapur Trimbakeshwar Nandurbar 2. February Igatpuri, Sinnar Lambkhani Mehunbare city Saptashrungi 3. March Sakri Pachora Shahada gadh Lasalgaon 4. April Varse, Parola Taloda Vinchur 5. May Dindori Chhadwel Amalner Dhadgaon 6. June Kalwan. Satana Indva Chopda Toranmal, 7. July Jalkhedan Sindkheda Jalgaon city Akkalkuwa 8. August Malegaon, Shirpur Yaval Shahada 9. September Ankai Boradi Bhusawal Taloda 10. October Peint Dhule city Jamner Toranmal, 11. November Surgana Sakri Edalabad Akkalkuwa Nandurbar 12. December Andarsul Shirpur Raver city 30 Visits during Year 2009 Table 5: Visit during year 2009 Sr. Jan.2009 1"' week visit 2"" week 3'" week 4'" week No. -Dec. Nashik visit visit visit 2009 Dhule Jalgaon Nandurbar 1. January Peint Dhule city Jamner Toranmal, 2. February Surgana Sakri Edalabad Akkalkuwa 3. March Andarsul Shirpur Raver Nandurbar city 4. April Nashik city, Dhule city Chalisgaon Navapur Trimbakeshwar 5. May Igatpuri, Sinnar Lambkhani Mehunbare Nandurbar city 6. June Saptashrungi Sakri Pachora Shahada gadh 7. July Lasalgaon Varse Parola Taloda Vinchur 8. August Dindori Chhadwel Amalner Dhadgaon 9. September Kalwan. Satana Indva Chopda Toranmal, 10. October Jaikhedan Sindkheda Jalgaon Akkalkuwa city 11. November Malegaon, Shirpur Yaval, Shahada 12. December Ankai Boradi Bhusawal Taloda 31 Visits during Year 2010 Table 6: Visit during year 2010 1** week visit 2"" week 3"* week Sr. Jan.2010- 4"" week visit Nashik visit visit No June 2010 Nandurbar Dhule Jalgaon 1. January Jaikhedan Sindkheda Jalgaon city ~ 2. February Malegaon, Shirpur Yaval Dhadgaon 3. March Ankai Boradi Bhusawal Toranmal, 4. April Peint Dhule city Jamner Akkalkuwa 5. May Surgana Lambkhani Edalabad Shahada 6. June Andarsul Sakri Raver Taloda 7. July Nashik city, Chalisgaon Nashik city, Trimbakeshwar Trimbakeshwar 8. August Igatpuri, Sinnar ~ Mehunbare ~ 9. September Saptashrungi Pachora gadh 10. October Lasalgaon Varse Parola Navapur Vinchur 11. November Dindori Chhadwel Amalner Nandurbar city 12. December Kalwan. Satana Indva Chopda Saptashrungi gadh 32 Visits during Year 2011 Table 7: Visit during year 2011 Sr. Jan.2011 1'* week visit 2"" week 3'" week 4"" week No. -June Nashik visit visit visit 2011 Dhule Jalgaon Nandurbar 1. January Lasalgaon Parola Vinchur 2. February Dindori Sakri Amalner Toranmal, 3. March Kalwan. Satana Varse Chopda Akkalkuwa 4. April Jaikhedan Chhadwel Jalgaon Shahada city 5. May Malegaon, Indva Yaval, Taloda 6. June Ankai ~ Bhusawal Navapur 7. July Peint Sindkheda Jamner Nandurbar city 8. August Surgana Shirpur Edalabad ~ 9. September Andarsul Boradi Raver ~ 10. October Nashik city, Dhule city Chalisgaon Trimbakeshwar 11. November Igatpuri, Sinnar Lambkhani Mehunbare Dhadgaon 12. December Saptashrungi Pachora gadh 33 3.2 Sampling methods Data was collected through two sampling methods, Random Survey and Line Transact Method. Random survey was done to record butterfly species and line Transact method was used to study diversity of butterflies. 3.2.1 Line transacts method: This method was adapted from Pollard E. (1991) and Royer et al., 1998. This method is also called as Pollard Walk. Line transacts were walked at constant pace for approximately half an hour. Transact were walked between 8 am to 11 am when butterflies were more active. These transacts were walked during all site visits to record sighting of butterfly species along each transact. Thus total forty-four transects were selected for study (One transect at each site). At the sites where high activity of butterflies was seen, a pause was taken to record every individual in data sheet. Most of the butterfly species were readily identified in the field itself. Kunte (2007), Kehimkar, (2008) and Singh, A.P. (2011) were used for authentic identification. Diversity data of three years (2008, 2009 and 2010) was recorded from study area so as to analyze seasonal variation of butterflies from selected area. 3.2.2 Transact Flora: Plants belonging to families Cupressaceae, Menispermaceae, Fabaceae, Malvaceae, Solanaceae, Convolvulaceae, Tiliaceae, Euphorbiaceae, Mimosaceae, Ebenaceae, Sapotaceae, Sapindaceae, Brassicaceae, Asteraceae, Poaceae, Linaceae, Chenopodiaceae; with subsequent less proportion of plants from families Myrtaceae, Rutaceae, Rhizophoraceae, Periplocaceae, Combretaceae, Thymeliaceae, Fagaceae, Santalaceae were recorded along transact path. 3.3 Butterfly collection: Most of the Butterfly species were identified upto family level in the field itself with the help of keys. Some dead specimens and few live specimens were collected from the study area for reference with forest department's permission. For identification, the specimens were captured with the help of butterfly net, observed, photographed and released. 34 Butterflies which come under Wildlife Protection Act, were photographed and only dead specimens of these butterflies were collected from the field. Some voucher specimens which were difficult to identify in the field were brought to laboratory and preserved. Long handled butterfly net was used to catch butterflies (Fig. 13 b). This net is made up of a light metal pipe, light metal ring and soft, thin mosquito net. Butterflies were chased in the field and when they came in sweeping range, the net was rapidly brought down on the butterfly. Butterflies have habit to fly in upward direction when caught in the net, when butterfly get closed to sealed end of net, the rim was rapidly turned around to close the net. This net was lifted without letting the butterfly escape. If the butterfly is resting of vegetation, the net was either brought down upon it or swept in from the side. Most of the butterflies were released from the net after proper identification only two specimens of a species were collected for laboratory preservation. These butterflies were immobilized immediately after catching, because butterflies flutter and try to escape and in the process damage their wings. Butterflies were immobilized and killed by lightly pinching their abdomen. For Tigers, Crows, Awls and other powerful butterflies killing jars were used (Fig.12 a). Killing jars are containers with harmful chemicals. Ethyl acetate soaked in cotton was put in the container to scarify the butterflies. 3.3.1 Papering: Papering method was used to securely bring the specimens from field to laboratory for preservation. (Schauff, 1986) Specimens were placed with the wings folded together dorsally, in triangular envelopes (fig. 12 b) of butter paper. This ensured that the scales of butterflies remained intact. When collection was made from out of Nashik district, paper envelops with specimens were kept in airtight freezer to prevent drying of insect. However, if the butterfly was too dry to spread, it was kept in a decicator with a little water and ethyl acetate, so that the muscles of the butterfly relaxed and then it was spread in a conventional manner. 35 3.3.2 Butterfly trap: Butterfly trap was used to trap butterflies with the help of bait (Fig. 13 b). This trap is made up of light metal rings, and fine mosquito net of white colour. Pineapple pulp, banana, Chiku, dry fishes and crabs were used to lure the butterflies. Hanging trap was made at home with the help of frying pan (fig. 13 c). At the anterior end of this trap colourful plastic scrubber was kept. This scrubber was dipped in sugar solution. Butterflies got attracted towards it mistaking it for a colourful flower with sweet nectar. This hanging trap was helpful for photographing butterflies. 3.3.3 Pinning: Butterflies were pinned with white nickel plated entomological pins. Pin no. 3(41 X 0.42mm), pin no. 5 (30 x 0.40 mm) and pin no. 16(26 x 0.30 mm) were used for pinning insects. Long pin was inserted through the middle of the thorax. 3.3.4 Spreading: Pinned butterflies were spread on a spreading board (Fig. 12 c). Wooden spreading board or thermacol was used to spread butterflies. Wooden spreading board has a groove in the middle with two flat surfaces on either side of it. Adjustable spreading board whose groove width could be changed was used for this purpose. When thermocol was used for spreading, groove was made in it according to the size of the abdomen of the butterfly. Strips of butter paper were used to hold the wings in right position, and then they were set with pins. 3.3.5 Drying: An oven was used to dry butterfly specimens at 45 to 50 °C for at least 24 hrs. Drying is necessary step in preservation as it removes moisture from insect and evades chances of infection (Fig. 14 a). 3.3.6 Storing: After drying specimens were pinned in a wooden box. These boxes were completely closed from all sides and covered from top by glass.