Current methods of fish epithelial injury detection are limited to gross macroscopic examination that has a subjective bias as well as an inability to reliably quantify the degree of injury. Fluorescein, a presumptive test for blood, has been shown to have the capability to detect and quantify fish epithelial injury. However, there are several other presumptive tests for blood (Bluestar©, phenolphthalein, and Hemastix®) that may have benefits over the use of fluorescein, particularly for field research on wild fish. This study investigated the capabilities of these four tests to detect and quantify a variety of injuries commonly encountered by fish (abrasion, cuts, fin frays, and punctures) using the freshwater bluegill Lepomis macrochirus as a model. Fluorescein was consistently found to be the most reliable (i.e., detected the highest proportion of true positive results and rarely detected false positive reactions) of the four presumptive tests for blood compared. Further testing was conducted to examine the reliability of fluorescein. By 24 h after an injury was inflicted, the injury was no longer detectable by fluorescein, and when fluorescein was applied to an injured fish, the fluorescein was no longer detectable 3 h after application. In a comparison of two common anaesthetics used in fisheries research, there was no significant difference in the proportion of injury detected when 3-aminobenzoic acid ethyl ester methanesulfate (tricaine) was used compared with a clove oil and ethanol (1∶9) solution. In summary, fluorescein was the most reliable presumptive test for blood examined in this study for the detection and quantification of recent (hours) fish epithelial injury.
Bluegill were angled from Opinicon Lake, located in southeastern Ontario, Canada, using size 8 barbless hooks and standard angling gear. Fish within the total length range of 120–190 mm were collected; fish outside this size range were released. Bluegill that were deeply hooked or exhibited any macroscopic evidence of injury also were released. Fish were handled only by gently, but firmly, gripping them by the lower lip using padded pliers. Fish were immediately placed in a water bath (4 L) in a plastic cooler with rounded edges containing 50 ppm clove oil anaesthetic (clove oil emulsified in ethanol, 1∶9; Sigma-Aldrich, Toronto, Ontario) and remained there until fish reached stage 4 of anaesthesia noted by a loss of equilibrium and coordinated fin movements (Summerfelt and Smith 1990). Fish were then removed from the anaesthetic bath by grasping them by the lower lip with the padded pliers, and those fish to be treated with fluorescein and Bluestar were dragged across a 5-mm nylon mesh net (18 cm in length) three times on their right side. All fish were then inflicted with standard injuries on their left side (Figure 1A). The upper or lower lobe of the caudal fin was frayed by cutting with a scalpel 30 times in the anterior–posterior plane from the base of the fin to the end. A puncture wound was made in the left cheek and posterior and dorsal to the left eye using a dental pick. A scalpel scrape (1 × 3 cm) was induced above or below the lateral line to simulate a large section of mucous loss. Finally, a shallow cut was made from the posterior end of the soft dorsal fin to the posterior end of the anal fin, across the left side of the caudal peduncle using a scalpel to simulate a deep wound that may occur due to a predation attempt (e.g., animal bite). These injuries were inflicted to replicate injuries that might occur in the natural environment or after interaction with anthropogenic structures. Fish were then treated with one of the four presumptive tests for blood, as outlined below.
Fluorescein. Immediately after injury infliction, fish were sprayed with a 0.2 mg/mL solution of fluorescein (fluorescein, disodium salt; Aldon Corp., Avon, NY) in distilled water; the solution was left unrinsed for 6 min (Noga and Udomkusonsri 2002). Fish were then placed in an anaesthetic bath containing 120 ppm clove oil (clove oil emulsified in ethanol at 1∶9) for 6 min to euthanize them. Fish were then photographed using a digital SLR ELIXIM Pro EX-F1 camera (Casio Computer Co., Ltd., Tokyo, Japan) under white light, long UV light (365 nm), or short UV light (254 nm) under various exposures (10, 13, 15, and 20 s) and at an ISO of 100. Fish were photographed in complete darkness, against a black background, with the camera positioned 42 cm directly above the fish and the UV light source (Mineralight® UVGL-48; UVP Inc., Upland, CA) at a 45° angle to the fish, 22 cm above, so that the entire organism was illuminated by the UV light. It was determined that short UV light and 20-s exposure were the best settings for examining the injury patterns; thus, these settings were used for the remainder of the study with fluorescein.
Bluestar. Immediately after injury infliction, fish were sprayed with Bluestar solution (Bluestar Forensic Mini Kit; Bluestar, Monte Carlo, Monaco) that was prepared following the instructions from the manufacturer. Because the Bluestar reaction is time sensitive, fish were immediately photographed in complete darkness with a digital camera (SLR ELIXIM Pro EX-F1; Casio Computer ) positioned 42 cm directly above the fish, with an exposure of 30 s, and following the photography directions outlined by the manufacturer. Fish were then euthanized as described above.
Hemastix. A moistened cotton swab was applied to 1-cm2 predetermined areas of the fish, representing areas that were inflicted with injury and areas that were not (Figures 1B and 1C). Each cotton swab was then touched to the pretreated reagent pad of the Hemastix (Hemastix Reagent Strips for Urinalysis; Bayer HealthCare LLC, Elkhart, IN), and the color change reaction was compared with a scale provided by the manufacturer; the result was recorded after 60 s. Fish were then euthanized as described above.
Phenolphthalein. A moistened cotton swab was applied to 1-cm2 predetermined areas of the fish, representing areas that were inflicted with injury and areas that were not (Figures 1B and 1C). A series of reagents were then applied to each cotton swab, following the phenolphthalein manufacturer's instructions (WARD'S Phenolphthalein Blood Test Kit; WARD'S Natural Science Establishment, LLC, Rochester, NY). The reaction results were recorded after 60 s and 4 min. Fish were then euthanized as described above.