2.4 Sampling procedure
Information on the infection of E. sardella fish hosts by the parasite L. intestinalis was collected over a period of 10 years, from 2005 to 2013 and then in 2015 in Wissman Bay. In the period 2005-2013 data was generated from fish caught by local fishermen from sites of Matema, Mwaya, Kafyofyo and Kiwira within Lake Nyasa. E. sardella were caught using an open water seine net, locally known as ‘Ndaturu’, with 10 mm mesh size at a depth of about 100 m during the dark moon phase days. In 2015, fish were caught by our research team, using the same traditional fishing method as was used in the period 2005-2013.
The fishing procedure involved nine crew members using two dug-out canoes and one large plank-boat. On the fishing ground, one of the dug-out canoes was equipped with pressurized paraffin lamps (between one to three) and was stationed with one crew member away from the remaining vessels. The artificial light was used to concentrate the fish into the given area. This process took several hours. After a sufficient amount of fish had been attracted, the other unlit fishing vessels simultaneously deployed a net in a semicircular shape around the concentrated fish, and this was hauled by hand into the plank-boat. Sampling was carried out in both lake habitats (i.e. littoral and pelagic zones) as well as throughout different seasons (i.e. dry and wet seasons) and a total of 3,488 E. sardella were sampled (Table 1). Upon landing, the total length and weight of each E. sardellawere measured to the nearest 5 mm and 0.01 g respectively. The fish were later dissected for parasite determination. L. intestinalis was identified according to the protocol by Dobben (1952). The sex ofE. sardella was determined using a stereomicroscope (Wild Heerbrugg M5) at 6.4X magnification. Gonad maturity was assessed on a seven-stage maturity scale (Table 2), modified from Holden and Raitt (1974). For seven years of the ten years (i.e. 2005, 2006, 2010, 2011, 2012, 2013 and 2015) the maturity stages of E. sardella was determined and recorded by the same investigator (N.P.Gabagambi). Therefore, we were able to maintain a good level of consistency and accuracy in the determination of maturity stage across our sampling period. In 2007, 2008 and 2009 maturity determination was carried out by trained research technicians (E.J.Magesa and J.M.Masore), following the same seven-stage maturity scale as was applied in all other sampling years.
Gonads were weighed to the nearest 0.01 g (wet weight) using sensitive precision balances (vwrTM-model ECN 611-2315 and EndelTM- model WPS) and fecundity for infected and non-infected female E. sardella was determined through gravimetric methods (Holden and Raitt, 1974) by counting the advanced yolked oocytes present in ripe and gravid E. sardella . The complete ovary was taken out and preserved in modified Gilson’s fluid (100 ml 60% alcohol, 800 ml water, 15 ml 80% nitric acid, 18 ml glacial acetic acid, 20 g mercuric chloride) for 24 hours. Thereafter, the ovaries were shaken periodically to help loosen the eggs from connecting ovarian tissues. After the eggs were liberated from the ovarian tissues, they were washed thoroughly, spread on blotting paper, and allowed to dry at ambient temperature ranging between 25 and 30 °C. Thereafter, the total numbers of eggs were weighed to the nearest 0.01 g using sensitive precision balance to have a total weight of eggs. Afterwards we collected a random sub-sample of the eggs, which were weighed and counted out on petri dish sub-sections using a stereomicroscope (Wild Heerbrugg M5) at 6.4X magnification. The total number of eggs (i.e. fecundity) in the ovaries was calculated following the formula given by Holden and Raitt (1974) as follows; F= nG/g where; n=number of eggs in sub-sample, G=total weight of eggs from the ovary, g= weight of the sub-sample. Fish somatic weight was determined by subtracting the gonad weight from the total weight of the fish.