Dfc295

Individuals of B. manatorum were counted in 37 turtles. The 95% confidence interval (95% CI) for prevalence (i.e., percent infected turtles) was calculated with Sterne’s exact method [27 (link)], and for mean values of intensity (i.e., mean number of individuals per infected turtle) with the bias-corrected and accelerated bootstrap method using 20,000 replications [28 (link)]. Calculations were performed using the free software Quantitative Parasitology v.3 [29 ]. It is important to note that the hatchlings had been subject to a first treatment round against B. manatorum with freshwater a week before we counted them.
From the samples conserved in 70% ethanol we randomly selected 4 ovigerous females from 5 turtles (i.e., 20 specimens in total) to count the number of eggs and calculate egg diameter. Pairs of egg sacs were separated from each female under a magnifying glass (8-40x). In each of five females, one egg sac was photographed with a digital microscope color camera Leica DFC295 using Leica Application Suite (LAS), and diameters of five eggs from the sac (total n = 25) were measured from pictures using the software Image J 1.48v [30 ]. The egg sacs from the 20 ovigerous females were then dissected with fine forceps and the number of eggs counted. To calculate clutch size per female, the number of eggs from the two sacs were summed up.

The liver and adipose tissues collected from each group were fixed with 4% formalin and embedded in paraffin. The tissues were cut into 4‐mm sections. The sections were stained with haematoxylin and eosin (H&E) for histological examination. Images were acquired with a Leica microscope (Leica DFC295).

Immunohistochemical staining was performed using the UltraSensitive™ S-P kit (Wuhan Boster Bio-engineering Co, Ltd, Wuhan, China) according to the manufacturer’s instructions. Anti-endothelin receptor A and B (Sigma-Aldrich) were used as primary antibodies at a 1:100 dilution. Images from the entire sections were acquired using a digital camera system (Leica DFC295; Leica, Solms, Germany). Confocal images were then transferred to a personal computer using the NIH Image software package for image analysis, as previously reported (11 (link)) and with minor modifications.

The colon tissues were fixed with 10% paraformaldehyde and embedded in paraffin. For histopathological analysis, tissue samples were sectioned (5 µm) and stained with H&E to observe the extent of the severity of mucosal injury and crypt damage. The stained slides were observed with a Leica microscope (Leica DFC 295, Wetzlar, Germany) and photographed (Leica Application Suite, version 4.8.0, Heerbrugg, Aargau, Switzerland) [8 (link)].

Fully differentiated 3T3‐L1 cells were washed twice with phosphate‐buffered saline and fixed with 10% formalin for 1 hour. After fixation, the cells were washed thrice with distilled water and then stained with 0.4% Oil Red O solution in 60:40 (v/v) isopropanol/ H₂O for 2 hour at room temperature. The cells were washed with distilled water and observed under a Leica microscope (Leica DFC295). The Oil Red O dye was eluted using 100% isopropanol and the absorbance was measured at 490 nm using a microplate reader (Biotek).

Transverse sections of the testis were taken to investigate differences in spermatogenic activity across experimental treatments. Immediately following collection of the final spermic urine sample (12 h PA), a random subset of five individuals from the 20-IU hCG injection, 1-μg GnRH-a injection, 100-μg GnRH-a topical treatment and 0-μg GnRH-a (control) treatments were euthanized via double pithing and preserved in 70% ethanol. These hormone treatments were selected as they represent the most effective dose administered via injection and topical application for each hormone type, with a control treatment provided for reference. Preserved specimens were later dissected, and the right testis from each individual was removed, embedded in paraffin wax and serially sectioned. The tissue was stained with Gill’s hematoxylin and eosin and permanently mounted on pathology-grade glass microscope slides (Universal Choice Pty Ltd, Turrella, Australia). Stained testis sections were photographed using a digital camera mounted to a C-mount compound microscope (Leica DFC 295 and Leica 750; Leica Microsystems Pty Ltd, North Ryde, Australia).

Thirty-six ovigerous females were randomly selected from the culture of B. manatorum and individually separated in Petri dishes 40x12 mm with 3 ml of filtered seawater. Each Petri dish was checked every half hour until hatching of nauplii occurred. The hatching process was videotaped (n = 3) under a magnifying glass (20- 40x) with a digital microscope color camera Leica DFC295 operating at 25 frames per second and a resolution of 3.1 megapixels. After hatching, individual clutches (n = 5) were put in Petri dishes as described above and all nauplii (ca. 250 individuals in total) were examined every 6 h to record behavior until the last individual died.