Neobenedenia infection

Trujillo-Gonzalez ́, Alejandro, Constantinoiu, Constantin C., Rowe, Richard & Hutson, Kate S., 2015, Tracking transparent monogenean parasites on fish from infection to maturity, International Journal for Parasitology: Parasites and Wildlife 4 (3), pp. 316-322 : 317

2.3. Neobenedenia infection of L. calcarifer over time

Fish were infected with fluorescent oncomiracidia and examined at 10 different time intervals to determine parasite distribution on the host body surface over its development. Fifty L. calcarifer were each infected with 50 ± 3 CFSE-labelled oncomiracidia, and held in individual aquaria (20 × 15 × 15 cm) in sea water (35 ppt; 25 ± 2.5 ǫC). A pilot study showed that parasite sampling and detection on the fish body surface took an average of 30 min for each individual fish. Thus, to enable precisely timed sampling, fish were infected over the course of five days, with ten randomly selected fish infected with labelled oncomiracidia each day. Each of the ten fish corresponded to one of ten time periods (15, 30, 60, 120 min, 24, 48, 96 h, eight, 12 and 16 d post-infection). Five replicates were made for each time period. Each fish was euthanised with a dose of Aqui-S aquatic anaesthetic (25 mL L –1 for 15 min), which does not cause parasite detachment ( Sharp et al., 2004; Trujillo-Gonźalez et al., 2014). Immediately following euthanasia, each fish was placed under an epifluorescence stereomicroscope (Olympus BX51) and both sides of the body surface (alternating left hand side first) were carefully examined for live parasites ( Fig. 1A ). The gills, buccal folds, buccal cavity and nasal chamber were not examined. Parasite location was recorded using an XY coordinate system based on a gridded translucent sheet of plastic (25 dots/ cm 2) placed over the fish. The tip of the mandible of each fish was placed on a marked location on the translucent grid to maintain a consistent coordinate origin. Scaled photographs were taken of each fish and of representative parasites attached to fish in each time period.

Figures

Fig. 1. Lates calcarifer microhabitat terminology (A) and body surface regions (B) used for statistical analysis. af = anal fin; cf = caudal fin; cp = caudal peduncle; dhf = dorsal hard fin; dsf = dorsal soft fin; e = eye; h = head; m = mandible; mb = middle body; op = operculum; plf = pelvic fin; ptf = pectoral fin; ub = upper body; vb = ventral body. B = body; F = fins; H = head. Terminology is based on Helfman et al. (2009) and Roberts and Ellis (2012).