Long-term effects and recovery from surgical implantation of dummy transmitters in two marine fishes

Journal of Experimental Marine Biology and Ecology 351 (2007) 243 – 254 www.elsevier.com/locate/jembe

Long-term effects and recovery from surgical implantation of dummy transmitters in two marine fishes Mary C. Fabrizio ⁎, Jeffrey P. Pessutti NOAA-National Marine Fisheries Service, Northeast Fisheries Science Center, 74 Magruder Road, Highlands, NJ 07732 USA Received 11 May 2006; received in revised form 1 February 2007; accepted 30 June 2007

Abstract We surgically implanted black sea bass and summer flounder with dummy transmitters and monitored recovery, survival, and growth during an 11-month post-operative period. We also examined transmitter retention rates as neither species had been previously implanted with transmitters. Recovery time from surgery and anesthesia was significantly greater than recovery time from anesthesia alone for black sea bass, but this relation was not observed for summer flounder. Summer flounder recovery times were highly variable, but in general, smaller fish had longer recovery times. All black sea bass and summer flounder retained their surgically implanted transmitter at least 11 months and had high survival rates in laboratory trials (black sea bass survival, 97.9%; summer flounder survival, 94.6%). Nonparametric analyses of covariance using initial size as the covariate indicated that black sea bass exhibited no significant detrimental growth effects after 11 months, but significantly slower growth was observed for summer flounder (this was especially pronounced in the larger [N800 g] fish). Surgical implantation of acoustic transmitters in these species can be used to conduct long-term field studies of habitat use and movements because fish exhibited high survival rates and 100% retention of transmitters. © 2007 Elsevier B.V. All rights reserved. Keywords: Acoustic transmitters; Black sea bass; Clove oil; Growth; Summer flounder; Surgical implantation

1. Introduction Monitoring the behavior of individual fish in the wild can enhance our understanding of fish-habitat relations. Such individualized information may be obtained from tagging studies with acoustic tags (i.e., transmitters) that permit simultaneous tracking of multiple fish. In the marine environment, ultrasonic tagging experiments have been used to study movements, habitat use, and home range of a variety of fish species (Hooge and Taggart, ⁎ Corresponding author. Current address: Department of Fisheries Science, Virginia Institute of Marine Science, PO Box 1346, Gloucester Point, VA 23062 USA. E-mail address: [email protected] (M.C. Fabrizio). 0022-0981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2007.06.031

1998; Arendt et al., 2001; Cote et al., 2003; Lowe et al., 2003; Parsons et al., 2003; Heupel et al., 2004). In studies that track individuals over substantial periods of time, transmitter retention must be assured, and retention rates should be known prior to conducting experiments in the field. Transmitter expulsion occurs when fish encyst and eject transmitters implanted in the peritoneal cavity either through the incision, the abdominal body wall, or via the intestines (Summerfelt and Mosier, 1984; Chisholm and Hubert, 1985; Helm and Tyus, 1992; Baras and Westerloppe, 1999; Walsh et al., 2000; Lacroix et al., 2004; Gosset and Rives, 2005). In general, larger transmitters are associated with significantly increased transmitter expulsion rates (Marty and Summerfelt, 1986; Lacroix et al., 2004) and higher

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mortality rates (Lacroix et al., 2004). Winter (1996) recommended that transmitter weight should not exceed 2% of the fish's weight in air, but this commonly used guidance is not well supported by empirical data (Mulcahy, 2003; Zale et al., 2005). Appropriate transmitter size is best determined by considering the objectives of the study, the attachment or implantation method, and the species under study, as well as other factors (Jepsen et al., 2005). Transmitter retention rates can be improved by ensuring proper transmitter characteristics (weight, shape, volume, roughness), appropriate implantation methods, and proficiency of the surgeon. For compressiform fishes, transmitter shape may be an important determinant of retention rates. Although commercially available ultrasonic transmitters are cylindrical, flat elongated transmitters have been suggested for peritoneal implantation in laterally compressed fish (Jepsen et al., 2002). Because transmitter retention, mortality rates, and behavioral effects vary by species and with size of fish, such rates and effects must be determined experimentally prior to conducting ultrasonic tagging experiments in the field. In this study, we examined the long-term effects and recovery from transmitter implantation in black sea bass Centropristis striata and summer flounder Paralichthys dentatus. We selected these species because we planned to conduct a field study of habitat associations of black sea bass and summer flounder in coastal waters using ultrasonic transmitters. Both species support commercial and recreational fisheries in the mid-Atlantic region and are locally abundant. Black sea bass and summer flounder are demersal species, but they differ in body shape: black sea bass exhibit a typical fusiform body, whereas summer flounder are compressiform fishes. Both species were to receive peritoneal implantation of transmitters involving anesthesia, surgery, and resuscitation. We conducted a series of laboratory experiments to ascertain the effects of transmitter implantation and clove oil1 anesthesia in black sea bass and summer flounder. We examined short-term recovery from surgical implantation as well as long-term growth and mortality, and long-term effects of clove oil anesthesia alone. To our knowledge, no studies have been published on the effects of clove oil 1 Although clove oil is “generally recognized as safe” when used as a direct food additive, it is not approved for use as a fish anesthetic by the USDA Center for Veterinary Medicine (FDA, 2002). Nevertheless, researchers have experimented with clove oil as a fish anesthetic (e.g., Peake, 1998; Taylor and Roberts 1999; Schreer et al., 2001; Woody et al., 2002). Our laboratory work with clove oil was performed in anticipation of the availability of a zero-withdrawal fish anesthetic based on isoeugenol, an active compound in clove oil; such a product is currently under investigation and testing (Schnick, 2006).

anesthesia on black sea bass or summer flounder, and only three studies reported the results of clove oil as an anesthetic with strictly marine fishes (coral reef species, Munday and Wilson, 1997; intertidal rockpool fishes of Australia, Griffiths, 2000; black sea bass, King et al., 2005). Because surgical implantation may require that fish remain anesthetized (and therefore, exposed to the anesthetic) for periods greater than 5 min, and because fish exposed to clove oil for periods greater than 5 min may have higher mortality (Woody et al., 2002), we conducted overexposure trials with black sea bass and monitored long-term growth and mortality effects. Thus, our three treatment groups included: fish anesthetized with clove oil and surgically implanted with dummy transmitters, fish anesthetized with clove oil and exposed for varying lengths of time after full induction, and control fish which were neither exposed to clove oil nor surgically altered. The overexposure trials with black sea bass provided an indication of how much longer we could expose fish without incurring mortalities or prolonging recovery from anesthesia. In addition, we examined transmitter retention rates among the surgically altered fish, as neither species had been previously implanted with ultrasonic transmitters. 2. Field collections Adult black sea bass were collected during May and June 2002 from fish traps deployed at 21-25 m and using hook and line techniques in 7–9 m waters off the coast of New Jersey. Most of the trap-captured fish had inflated swim bladders, and some exhibited stomach evulsion. We deflated swim bladders by puncturing the abdominal wall with a hollow needle and exerting gentle pressure on the abdominal area (Collins et al., 1999). Fish captured by hook and line did not exhibit decompression trauma because they were taken from shallower water. Fish that survived handling and transport to the laboratory (size range: 224–445 mm total length [TL] and 192–1044 g) were used in subsequent experiments. We collected adult summer flounder (size range: 281–509 mm TL and 197–1618 g) from NJ coastal waters from June to August 2002 using hook and line techniques, and held fish in the laboratory for later work. In November 2002, we acquired 18 summer flounder (size range: 313–509 mm TL and 330–1318 g) from an aquaculture facility and transported the fish to the laboratory; hereafter these fish are referred to as the ‘hatchery’ fish. We began experimental trials on hatchery fish after all surviving hatchery fish (N = 17) had begun feeding.

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3. Experimental methods All fish were held in laboratory aquaria (either 1500L circular, 2400-L circular or 9000-L rectangular tanks) supplied with flow-through seawater and auxiliary aeration; aquaria were covered with netting (about 2.5 cm mesh) to minimize fish escapement. Fish were fed to satiation every other day with a mixture of fresh or frozen prey items including chopped squid Loligo pealeii, whole Atlantic silversides Menidia menidia, young-of-the year menhaden Brevoortia tyrannus, and young-of-the-year bluefish Pomatomus saltatrix. Photoperiod and salinity in the laboratory followed the natural daily and seasonal cycles in New Jersey. Similarly, water temperatures followed natural seasonal temperatures but were capped between 10° and 21 °C to prevent thermal stress (D. Nelson, NOAA-NMFS, pers. com.). Fish for each of the trials described below were removed from the holding tanks with rubber-coated dip nets; although not a strict random selection, we avoided pre-selection of fish. We constructed replicas of a 30 mm long, 9 mm diameter transmitter weighing 5 g in air (3.1 g in water) because we planned to use this size transmitter in a subsequent field application. Dummy transmitters were constructed in a mold using polypropylene glue uniformly embedded with stainless steel. Each dummy transmitter was coated with a thin layer of one-hour epoxy and dipped in melted beeswax, which provided an inert and smooth coating. We coated transmitters with beeswax because beeswax-coated transmitters are

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expelled at a lower rate than those coated with paraffin or silicone (Helm and Tyus, 1992). We used rank-transformed data to fit nonparametric regression models and to conduct analyses of covariance (ANCOVA) because in some cases, sample sizes were small (b10) and the assumptions of parametric tests may not be met. Nonparametric comparisons of two or more samples were conducted using Wilcoxon's two-sample rank-sum test (we report the asymptotic P-values corresponding to the normal (Z) approximation of the Wilcoxon test statistic) and the Kruskal–Wallis test (we report the χ2 approximation). In cases where the Kruskal–Wallis test indicated a significant difference, Dunn's procedure for unequal sample sizes was used to conduct multiple comparison tests (Hollander and Wolfe, 1973). All statistical analyses were performed using SAS and with an α level of 0.05. 4. Black sea bass 4.1. Clove oil trials Clove oil solutions were prepared by dissolving clove oil in 95% ethanol and adding the resultant solution to a seawater bath. We exposed individual fish to 40 mg clove oil/L at a median temperature of 15.9 °C and median salinity of 26.5‰ (Table 1). The overexposure trials were conducted at 20.9 °C and 24.3‰ salinity (median values). For all trials, the time to full induction (stage 5 anesthesia) was defined as the minimum exposure time needed to elicit no opercular

Table 1 Description of the experimental treatments applied to black sea bass and summer flounder Clove oil exposure Full induction

Overexposure

Surgical implantation

Control

Black sea bass Number of fish Temperature (°C) Salinity (‰) Total length (mm) Weight (g) Observation period (d)

7 15.9 (15.9–19.4) 26.5 (24.2–26.5) 287 (245–345) 296 (192–572) 316 (240–343)

9 20.9 (20.9–21.0) 24.3 (24.3–24.4) 300 (230–360) 401 (305–701) 309 (12–309)

47 16.3 (15.1–21.2) 27.1 (26.1–27.2) 325 (224–445) 425 (202–1044) 357 (66–368)

4 –a –a 277 (235–290) 264 (197–351) 359 (359–359)

Summer flounder Number of fish Temperature (°C) Salinity (‰) Total length (mm) Weight (g) Observation period (d)

5 15.1 (15.1–20.7) 22.0 (22.0–27.1) 398 (322–460) 770 (386–1009) 223 (135–310)

– – – – – –

37 17.1 (14.8–19.3) 24.6 (22.0–25.0) 383 (281–508) 692 (197–1618) 344 (3–450)

3 –a –a 480 (420–509) 1040 (701–1317) 352 (352–352)

a

Temperature and salinity data were recorded at time of treatment, therefore control fish have no data. Temperature, salinity, length, weight, and observation period length are presented as medians; values in parentheses are ranges. Overexposure refers to exposure of fish for 5, 10, or 15 min post full induction.

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movement (after Summerfelt and Smith, 1990). We selected 40 mg/L because preliminary trials with clove oil in our laboratory indicated that this concentration allowed fish to achieve full induction in less than 5 min and recovery in less than 10 min (Fabrizio et al., 2005). Summerfelt and Smith (1990) note that an ideal anesthetic has an induction time less than 15 min, but preferably less than 3 min, and a short recovery time (i.e., ≤5 min). Each batch of anesthetic solution was used to anesthetize up to 4 fish (this was done to ensure a fairly constant concentration of anesthetic). Once a fish was anesthetized, we recorded total length (TL) and weight, identified sex based on external morphology, and tagged the fish with an individually numbered T-bar anchor tag. Fish were transferred to a recovery tank where they were resuscitated via ram ventilation. We recorded recovery time which occurred when the fish regained equilibrium and swam in a forward direction (minimum of 3 fin strokes) in response to gentle prodding in the peduncle area. Seven fish with a median size of 287 mm and 296 g were assigned to the 0-min overexposure trial, that is, they were removed from the anesthetic bath as soon as they achieved full induction (stage 5). These fish were held about 316 days (10.4 months) to observe for postexposure effects (Table 1). Nine black sea bass with a median size of 300 mm and 401 g were exposed to clove oil for an additional 5, 10, or 15 min after induction to stage 5 anesthesia. We held these fish for about 309 days (10.2 months) post recovery (Table 1). We tested the null hypothesis of no difference in the distribution of recovery times among fish exposed to clove oil for 0, 5, 10, or 15 min after fullinduction using the Kruskal–Wallis test. Although we hypothesized that recovery time may be related to fish size, fish weights were not significantly different among individuals from the four treatment groups (Kruskal– Wallis test, χ2 = 3.941, P N 0.05), nor did we find weight to be a significant predictor of recovery time (nonparametric regression, F = 1.95; P N 0.05). 4.2. Surgical trials To perform the surgery, we exposed black sea bass to 40 mg/L clove oil at a median temperature of 16.3 °C and median salinity of 27.1‰ (Table 1). We implanted 47 black sea bass (20 males, 27 females) with dummy transmitters using surgical protocols modified from methods described in Summerfelt and Smith (1990) and Wooster et al. (1993). Once anesthetized, a fish was placed dorsal side down in a surgical cradle lined with wet foam and a moist chamois cloth. Anesthetic solution

(40 mg/L clove oil) was continuously circulated across the gills via a flexible tube inserted through the mouth and into the gill cavity. Individual scales were removed from a small area posterior to the pectoral fins and along the ventral midline, and a small incision (about 2.5 cm long) was made in the descaled area. A sterilized, beeswax-coated dummy transmitter, representing about 1.2% (median; range: 0.5–2.4%) of the body weight of these fish, was inserted into the peritoneal cavity through the incision. We used nonabsorbable monofilament nylon sutures (Ethilon® 3-0 and 4-0 with FS-1 cutting needle, Ethicon, Somerville, NJ) in a simple interrupted suture pattern (3 knots per stitch) to close the incision, and both the incision and sutures were covered with a small amount of cyanoacrylate (VetBond™, 3M, St. Paul, MN), a tissue adhesive. Monofilament sutures, which are recommended for fish surgical procedures (Mulcahy, 2003), are associated with significantly less tissue damage when applied in an interrupted pattern (Wagner et al., 2000). To reduce the likelihood of infection, antibiotic ointment (bacitracin, neomycin, and polymixin-B sulfate) was swabbed over the sutured area. At the completion of surgery, we recorded surgery time, size (TL and weight) and sex; tagged the fish with an individually numbered anchor tag; transferred the fish to a recovery tank; and recorded recovery time. Fish in the surgical trials had a median size of 325 mm and 425 g; they were held about 357 days (11.7 months) and monitored for post-implantation effects and transmitter loss (Table 1). 4.3. Controls During the same time period (June 2002–June 2003) we also maintained a small group of black sea bass (N = 4, median size: 277 mm, 264 g) that had not been exposed to clove oil, but were tagged with individually numbered anchor tags and otherwise handled in a manner similar to that used for treatment fish. We recorded TL and weight, and noted sex at the beginning of the observation period. These fish were held 359 days (11.8 months; Table 1). 4.4. Post-treatment observations Prior to terminating the experiment and releasing fish, we obtained length and weight data from each fish to permit calculation of specific growth rates for the subset of fish that retained their anchor tag (some fish shed their tag and we could not determine their identity conclusively). The weight of the dummy transmitter was

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subtracted from the initial and final weights of implanted fish. Specific growth in weight (GW, % day− 1) was estimated by GW ¼ 100  ðloge Wfinal  loge Winitial Þ=t where weight (W) data were recorded at the beginning and end of the observation period and t is the number of days in the observation period. These estimated specific growth rates represent an instantaneous daily rate during the course of an approximate year. Specific growth rates of surgically treated fish were compared with those from clove-oil exposed fish and control fish using a nonparametric ANCOVA with initial length as the covariate. (In these comparisons, clove-oil exposed fish included fish exposed to the anesthetic until full induction as well as fish exposed for 5–15 min post-induction.) We used initial length as the covariate in the ANCOVA because initial weight was used in the calculation of GW; using initial weight as a covariate violates the assumption of independence. 5. Summer flounder 5.1. Clove oil trials Clove oil solutions were prepared as described for black sea bass treatments except we used 80 mg clove oil/L to anesthetize summer flounder. Based on preliminary trials with summer flounder, this concentration provided full induction in less than 5 min and recovery in about 5 min (Fabrizio et al., 2005). We exposed 5 adult fish (median size 398 mm, 770 g; Table 1) to clove oil, and once the fish achieved full induction, it was transferred to a recovery tank where we used ram ventilation to resuscitate the fish. We recorded time to recovery, which we defined as the time when a fish swam in a forward direction in response to gentle prodding in the peduncle area. Summer flounder exposed to clove oil were observed for about 223 days (7.3 months). Overexposure trials were not conducted for summer flounder.

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resented about 0.7% (median; range: 0.3–2.5%) of fish weight. Perhaps more important than weight considerations was size of the transmitter, as flatfish have a small peritoneal cavity (Paukert et al., 2001; Mulcahy, 2003). To perform the surgery, we exposed summer flounder to 80 mg/L clove oil at a median temperature of 17.1 °C and median salinity of 24.6‰ (Table 1). Each batch of anesthetic solution was used to expose up to 3 fish, one at a time in the bath. Once anesthetized, the ocular side of the fish was placed on a padded surgical cradle and anesthetic solution was continuously circulated across the gills. A 2.5-cm long incision was made on the nonocular side about halfway between the pectoral and pelvic fins, but posterior to the pectoral fin insertion. A sterilized dummy transmitter coated in beeswax was inserted into the peritoneal cavity through the incision, which was oriented from anterior to posterior (i.e., parallel to the long axis of the body). To close the incision, we used nonabsorbable monofilament nylon sutures (Ethilon® 3-0 and 4-0 with FS-1 cutting needle, Ethicon, Somerville, NJ) in a simple interrupted suture pattern (3 knots per suture). The incision was closed with 4 sutures, covered with a small amount of cyanoacrylate, and swabbed with antibiotic ointment (bacitracin, neomycin, and polymixin-B sulfate). Each fish was then weighed, it's length recorded, and an individually numbered T-bar anchor tag was inserted below the dorsal fin. Recovery was as described previously. For these fish, we recorded surgery and recovery times, and monitored post-implantation effects for about 344 days (11.3 months). 5.3. Controls We maintained a small group of summer flounder (N = 3) that had not been exposed to clove oil, but were tagged with individually numbered anchor tags. We recorded TL and weight (medians: 480 mm, 1040 g) and held these fish for 352 days (11.6 months; Table 1). 5.4. Post-treatment observations

5.2. Surgical trials We implanted 37 summer flounder with dummy transmitters using surgical protocols similar to those developed for black sea bass. Summer flounder in the surgery trials had a median size of 383 mm and 692 g (Table 1) and included both wild captured (N = 25) and hatchery fish (N = 12). As before, we used 30-mm-long and 9-mm-diameter dummy transmitters weighing 5 g in air. In these surgical trials, dummy transmitters rep-

Following surgery, we investigated patterns of recovery and healing in 34 summer flounder by inspecting for inflammation and redness along the incision and suture sites. These post-surgical observations were conducted 7 times, about every 2 weeks from mid-September 2002 to mid-February 2003. An eighth assessment was conducted at the end of March 2003 and a final assessment was conducted in mid-November 2003. The median time interval from surgery to final assessment was

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310 days (10.2 months) but ranged up to 373 days (12.3 months). Because the experiment was terminated on the same calendar date, the time interval varied depending on the surgery date. Post-surgery visual inspections included observations on redness or inflammation along the incision and suture sites, percent closure of the incision, and whether the closure was partial or robust. Partial closure occurs when the inner layer of muscle tissue closes completely, but the outer layer and skin remain unclosed. A robust closure was defined as 100% closure through all tissue layers. Prior to releasing experimental fish, we obtained length and weight information to calculate GW for fish that retained their anchor tag (N = 34). These data were obtained from summer flounder from all three treatment groups (control, clove oil exposed, and surgically altered). We compared recovery times of summer flounder from the surgery and clove oil trials using a nonparametric ANCOVA with weight as the covariate. Specific growth rates of surgically treated fish and fish exposed to clove oil were compared with those from control fish using a nonparametric ANCOVA (with initial length as the covariate). In addition, a subset of fish (N = 5) was euthanized to permit examination of internal organs and inspection for tissue damage. 6. Results 6.1. Black sea bass 6.1.1. Recovery from anesthetic exposure Recovery times of black sea bass exposed to clove oil for 0, 5, 10, and 15 min post-induction varied significantly with duration of exposure (χ2 = 8.849, P b 0.05). Although median recovery times of black sea bass increased as exposure times increased (medians: 3.8, 4.8, 5.7, and 6.6 min for 0-, 5-, 10-and 15-min exposures), Dunn's multiple comparison test failed to identify significant pairwise comparisons with an experiment-wise error rate of 0.05. All fish recovered and no mortalities were observed due to over-exposures up to 15 min. All black sea bass exposed to clove oil survived until we terminated the experiment. 6.1.2. Recovery from surgery The median surgery time for black sea bass was 4.5 min (range: 2.7–9.8 min), indicating that implanted fish were exposed to clove oil for an additional 4.5 min after achieving full induction. To investigate the possible additive effects of surgery on recovery time, we compared recovery times of black sea bass from the surgical trials (N = 46) with those of fish exposed to

clove oil for 0-or 5-min post-induction (N = 10). Because fish weights in the two groups were not significantly different (Z = − 1.542, P N 0.05) and size was not a good predictor of recovery time (nonparametric regression: F = 2.43, P N 0.05), we used the Wilcoxon rank-sum test for this comparison. We hypothesized no difference in recovery times of fish from the surgical trials and fish exposed to clove oil for up to 5 min. Recovery times of black sea bass from the surgical trials differed significantly from those of fish exposed to clove oil for up to 5 min (Z = −2.311, P b 0.05; mediansurgery = 5.3 min; medianclove oil = 4.1 min). We note that surgical times varied between 2.7 and 9.8 min so some fish may have experienced significantly longer exposures to clove oil during the surgery (15 fish had surgery times that exceeded 5 min). Omitting these 15 fish from the analysis did not affect our results or conclusion: recovery times of fish surgically implanted in 5 min or less remained significantly different from recovery times of fish exposed to clove oil and no surgery (Z = −2.038, P b 0.05; mediansurgery = 4.8; medianclove oil = 4.1). The difference in recovery times may arise from the additional stress associated with surgery. 6.1.3. Transmitter retention and mortality All 47 black sea bass recovered from surgery and were actively feeding within a few days. In addition, these fish retained the dummy transmitter for at least 11 months. Sutures in black sea bass were shed after about 7–8 weeks; shedding occurred after the incision was fully closed and healed. We observed 14 mortalities among the fish in the surgical trials, only 1 of which may have been associated with the surgical treatment; this single fish died of unknown causes, though the only observed gross abnormality in this fish was the presence of ‘pop-eye’ (bulging eyes). Thus, we calculated the overall mortality rate of surgically implanted black sea bass as 2.1% (1/47). The remaining 13 mortalities were due to fish escaping from the aquaria (N = 3) or accidental loss of control over seawater temperature or aeration (N = 10) of the aquaria. Necropsies on the 14 fish that died during the course of the experiment confirmed the lack of transmitter rejection or other postsurgical complications. 6.1.4. Specific growth rates Only individuals for which we obtained a final size measurement (N = 47) were included in the analysis of differences in GW among surgery fish, clove-oil exposed fish (pooled across the 0, 5, 10 and 15 min postinduction treatments), and control fish. Fish with no final measurement had either shed their tag or died and

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were dessicated when found. Initial weight of black sea bass differed significantly in the three groups (χ2 = 7.12, P b 0.05). Therefore, we tested equality of specific growth rates using a nonparametric ANCOVA with initial length as the covariate. The interaction term (initial length × treatment group) was non-significant (F = 0.14, P N 0.05), indicating equality of slopes for the relation of specific growth rate with initial length of fish among the treatment groups. The effect of initial size on growth rate of black sea bass was significant (F = 3.98, P b 0.05), and when initial size was taken into account, the observed differences in GW among treatment groups were not significant (F = 0.26, P N 0.05). The overall median GW for black sea bass was 0.217% d− 1. In addition to overall specific growth rates, we estimated sex-specific growth rates among surgically implanted fish using a nonparametric ANCOVA with initial length as the covariate (insufficient sample size did not permit sex-based investigations for the other treatment groups). Though significantly lighter in weight than males at the beginning of the surgical trial (Z = 3.13, P b 0.05), specific growth rates of female fish did not differ significantly from those of male fish (F = 0.49, P N 0.05; nonsignificant interaction term, F = 0.17, P N 0.05). 6.2. Summer flounder 6.2.1. Recovery from anesthetic exposure Summer flounder exposed to clove oil had highly variable recovery times (median = 1.4 min; range: 0.5– 5.2 min). Because our sample size for the clove oil trial was limited, we investigated the observed variation in

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recovery time using recovery time data from the surgical trials. Duration of surgery was not a significant predictor of recovery time (nonparametric regression: F = 0.16, P N 0.05), but recovery time was significantly shorter for hatchery fish than for wild fish (Z = − 3.947, P b 0.05; Fig. 1). In addition, recovery time was negatively related to weight (Fig. 1) such that smaller summer flounder had significantly longer recovery times than larger fish (nonparametric regression: F = 12.52, P b 0.05). Therefore, we used a nonparametric ANCOVA with weight as the covariate to test for equality of recovery times among fish exposed to clove oil and clove-oil exposed fish that were surgically altered. Taking into account the effect of size, we found no significant difference in recovery times of fish exposed to clove oil only and fish surgically altered after clove oil exposure (F = 1.36, P N 0.05; nonsignificant interaction term, F = 0.45, P N 0.05); the median recovery time for all fish exposed to clove oil was 3.0 min. All summer flounder exposed to clove oil survived until we terminated the experiment. Exposure to clove oil at 80 mg/L did not cause short-or long-term mortality in summer flounder. 6.2.2. Recovery from surgery The median time to perform surgery on summer flounder was 6.3 min (N = 37) but varied from 4.3 to 13.4 min. All 37 fish recovered from surgical implantation and were actively feeding soon after surgery (some individuals began feeding as early as one day post-surgery). Post-surgery observations indicated that summer flounder heal and recover quickly from surgery. Two

Fig. 1. Relation of summer flounder size and recovery time from transmitter implantation surgery. Filled circles are wild fish; open circles are hatchery fish.

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weeks after surgery, at least 1 suture was shed in 69.7% of fish, and muscle layers at the incision site were completely closed in 90.3% of fish; however, in some of these fish, the external tissue layers remained incompletely closed. After 1 month, all tissue layers were completely closed in 81.5% of fish. Four months after surgery all summer flounder had complete closure of internal and external tissues layers at the incision site (Fig. 2A), though only 60.0% of fish had shed 1 or more sutures. Inflammation of the incision area was evident in 21.9% of fish 2 weeks after surgery; after 2 months, the inflammation was visible in less than 5% of fish (Fig. 2B). We noted redness at the incision site in 68.8% of fish 2 weeks after surgery; the frequency of this condition decreased rapidly such that by the end of the first month, 44.8% of fish exhibited redness at the incision site. However, incision site redness was present 347 days (11.4 months) after surgery in 28.6% of the summer flounder (Fig. 2B). Among the fish observed for 310 days (10.2 months) or more, 71.4% retained at least 2 sutures, and 14.3% retained all 4 sutures. Bet-

Fig. 2. (A) Percent of summer flounder exhibiting full closure of the incision site (open circles, dotted line) and closure of both internal and external layers at the incision site (closed circles, solid line). (B) Percent of summer flounder exhibiting redness at the suture puncture sites (open circles, dotted line), redness at the incision site (x and dashed line), or inflammation at the incision site (closed circles, solid line).

ween 61.9 and 79.2% of fish exhibited redness at the needle puncture (suture) sites throughout the post-surgical observation period (Fig. 2B). The degree of redness at the incision site varied during holding time and may be a function of handling effects, type of substrate in the tank (we used coarse sand), temperature, and number of animals in the holding tank. Necropsies on five fish revealed that the presence of redness did not compromise tissue closure or penetrate beneath the dermal tissues. We found no signs of adhesions, hemorrhaging, necrotic tissue or other damage; dummy transmitters were free and intact. Summer flounder tolerated the implantation of dummy transmitters, although they exhibited redness or inflammation at the incision and suture sites. Overall, we found no deleterious effects of surgery (as determined by gross morphological inspection). 6.2.3. Transmitter retention and mortality None of the summer flounder rejected the transmitters and we achieved a retention rate of 100% up to 373 days (12.3 months) post-surgery. Five of the 37 implanted summer flounder died in the period following surgery. One fish died 3 days after surgery from the accidental nicking of the intestine by the surgeon's scalpel, and another died 10 days after surgery with an incompletely closed incision. These fish were the 2 smallest fish we attempted to surgically implant (281 mm, 209 g; and 286 mm, 197 g). A third fish died 16 days after surgery; a necropsy revealed full closure of the incision, no redness or inflammation near the incision, no apparent damage or inflammation of internal organs, and no apparent infection. In addition, this fish had fed the day before it died. We do not know the cause of death. A fourth fish died 41 days after surgery when it escaped from the aquarium, and a fifth fish died 166 days after surgery, with no apparent inflammation or infection; we do not know the cause of death of this fish. Surgical treatment of summer flounder larger than 286 mm resulted in 5.4% mortality (2/37), but it is unclear if the observed mortality was related to implantation of the transmitter. 6.2.4. Specific growth rates Because we were interested in long-term growth effects on summer flounder, only those fish that were observed for 310 days (10.2 months) or more (N = 37) were included in the comparison of GW from the three treatment groups (control, clove-oil exposed, and surgery). Thus, 8 (out of 45) fish were omitted from this analysis because they either shed their tag or died. We tested for differences in GW using a nonparametric ANCOVA with initial length as the covariate. The

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interaction term (initial length × treatment group) was non-significant (F = 0.44, P N 0.05), indicating equality of slopes for the relation of specific growth rate with initial length of fish. The effect of initial length on summer flounder GW was significant (F = 17.13, P b 0.05), and taking initial size into account, specific growth rates of fish from the three groups were significantly different (F = 8.82, P b 0.05). Dunn's multiple comparison test indicated that specific growth rates of surgically altered fish did not differ significantly from those of clove-oil exposed fish (zsurgery vs. clove oil = 5.839, zcritical = 18.895), but growth rates of control fish were significantly greater than those for fish in the other two groups (zcontrol vs. clove oil =27.515, zcritical =23.666; zcontrol vs. surgery =21.676, zcritical =15.653). Thus, summer flounder which had been exposed to either anesthetic alone or anesthetic plus surgery had lower specific growth rates than control fish. Six summer flounder from the surgically altered group had negative specific growth rates; these fish were among the larger fish we implanted (6 of 10 fish greater than 800 g). Slower growth rates may have resulted from reduced feeding by transmitter-implanted fish, but we have no estimates of individual consumption because summer flounder were fed ad libitum. It should be noted that in all other respects, all fish were handled similarly during the post-surgical assessments so handling stress was equal among surgery, clove oil-exposed, and control fish. 7. Discussion Our study of long-term effects and recovery from surgical implantation of transmitters in black sea bass and summer flounder provides evidence that this surgical technique can be used in telemetry studies to investigate fish movements and habitat use. Long-term (11 months) transmitter retention was 100%, indicating that both fusiform and compressiform fishes can retain an internal transmitter for long periods of time, thereby further facilitating long-term telemetry studies. Few other studies have monitored survival or transmitter retention in fish over long periods: Cote et al. (1999) examined growth, mortality, and retention rates for 220 days in juvenile Atlantic cod, and Lacroix et al. (2004) studied survival and retention rates in juvenile Atlantic salmon for 316 days. As the availability of smaller transmitters with a longer battery life increases, we anticipate the duration of field studies to also increase allowing observations on the movements and site fidelity of fish over periods lasting more than one year. Thus, long-term effects of surgical implantation should be examined.

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Some of the factors necessary to ensure retention include selection of an appropriately sized transmitter and aseptic surgical procedures that minimize inflammation and adverse effects (Wagner and Cooke, 2005). The dummy transmitters we implanted weighed less than 2.5% of the body weight of our study animals, and although it has been shown that some species may not be affected by larger (heavier) transmitters, we suggest that researchers select the smallest possible transmitter to minimize possible negative effects. For black sea bass, consideration of the weight of the transmitter seems appropriate; however, for summer flounder, consideration should be given to the volume occupied by the transmitter. Laterally compressed peritoneal cavities have little free volume, but suitably small (dimension) transmitters are readily accommodated with little to no effects on recovery; long-term growth may decrease, especially among larger fish. Necropsies of the summer flounder that died accidentally indicated no adverse effects of the transmitter on internal organs or tissues, that is, we observed no hemorrhaging, inflammation, or infection. However, a small proportion (28.6%) of the summer flounder continued to exhibit redness of the skin at the incision site almost one year after surgery. We believe that this was most likely associated with the additional handling (net capture) of the fish after surgery when they were assessed for patterns of healing. Some individual fish exhibited no redness during one or more assessments, but later exhibited significant redness. This pattern supports the notion of an acute disturbance (such as capture or handling during an assessment), rather than a gradual progression of healing or a chronic condition among individual fish. The median time to perform surgery was 4.5 min for black sea bass and 6.3 min for summer flounder, which compares favorably with times reported for surgical implantation of transmitters. Prince and Powell (2000) reported an average surgery time of 5.8 min (range: 4.0– 7.4 min), and Cooke et al. (2003) reported an average of 4.2 min for an expert surgeon and 6.0 min for a novice surgeon performing similar surgery. Surgery duration times for summer flounder were longer than those for black sea bass and reflect the extra care necessary when working with a laterally compressed fish. Long-term mortality associated with implantation of transmitters was insignificant: black sea bass exhibited 2.1% mortality and summer flounder greater than 286 mm TL exhibited a mortality rate of 5.4%. Implantation surgery on summer flounder smaller than 286 mm should be avoided because delicate tissues proved difficult to suture properly and because of the increased risk for accidental damage of internal organs

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during the incision or suturing procedures. We expect that similar, low mortality rates would be achieved in a field study with these species provided that handling and surgical methods are identical or similar to those used in this laboratory study. In recent years, fishery researchers have recognized the importance of proper surgical techniques and the need to establish and maintain aseptic conditions during surgery (Wagner and Cooke, 2005). Though appearing to be cumbersome or time-consuming, such techniques ensure the health and survival of surgically implanted fish. Unhealthy fish or fish that alter their behaviors in response to transmitter implantation provide unreliable data in a field setting. Both black sea bass and summer flounder recovered quickly from anesthetic exposure and surgery, though the response of the two species differed. The duration of the recovery period from the combination of anesthesia and surgery was significantly longer than that from anesthesia alone for black sea bass, but we could not detect a significant difference for summer flounder. This may have been partly due to the highly variable recovery times observed for summer flounder. Such variability within a species has been observed previously for induction times (e.g., Hoskonen and Pirhonen, 2004), but not recovery times. A portion of this variability may be associated with the origin of summer flounder, as we noted that wild fish had significantly slower recovery times than hatchery fish. Recovery from anesthesia takes longer in field-captured and lab-acclimated summer flounder than in fish reared in an aquaculture setting. Another factor contributing to the large variation in recovery rates among summer flounder is size. Small summer flounder required significantly longer recovery periods than larger fish. This may simply reflect the additional time needed by smaller fish to clear the anesthetic and regain equilibrium. We did not observe size-related recovery times in black sea bass, but we attribute this to the reduced variation in sizes of black sea bass compared with summer flounder. Although the range of fish lengths in this study was similar (228 mm for summer flounder and 221 mm for black sea bass), the range of weights was wider for summer flounder (1421 g) than for black sea bass (852 g). Without further study, we cannot be certain that recovery times for black sea bass are not related to size. The study of recovery times per se is not critical to implementing a field study, but such measures can reveal the limitations under which field studies should be conducted. For example, larger fish may require higher concentrations of anesthetic to ensure short recovery times (under 5 min) as recommended by Summerfelt and Smith (1990). Additionally, researchers may need to adjust anesthesia concentrations and ex-

posure times to accommodate the additional time some species may require to recover from the added effects of surgery. Implantation of dummy transmitters in black sea bass had no significant effect on growth observed over the course of 11 months; however, long-term growth appears to be reduced in summer flounder. The lack of an effect of surgical implantation on black sea bass growth was also observed in a similar species, the European sea bass Dicentrarchus labrax, though the post-surgical observation period was only 47 days (Bégout Anras et al., 2003). Atlantic cod exhibited no significant difference in growth among surgically implanted and nonimplanted fish after 220 days (Cote et al., 1999). Lack of growth effects has also been reported for rainbow trout Salmo gairdneri observed for 7 months (Lucas, 1989), bluegill Lepomis macrochirus observed for 10 weeks (Paukert et al., 2001); and striped bass Morone saxatilis recaptured after 1.2 years (Young and Isely, 2004). Slower growth rates were observed in summer flounder exposed to anesthetic alone, as well as in implanted fish, implying that anesthetic exposure may have contributed to the observed decline in growth rates. Taken together, the results of our investigation on longterm effects of transmitter implantation in black sea bass and summer flounder indicate that small transmitters (9 mm diameter, 30 mm long) can be implanted in fish without significant effects on survival of either species or on growth of black sea bass. Specific growth rates of implanted summer flounder, particularly larger fish (N 800 g), were significantly depressed. Long-term telemetry studies of summer flounder and black sea bass can be realized without concern for transmitter loss or mortality, but growth effects may occur for some summer flounder. Acknowledgments We are grateful to M. and J. Berko (F/V The Wizard), Lt S. Sirois, (R/V Gloria Michelle), and LtJG R. Haner (R/V Gloria Michelle) for vessel operations in support of fish collections. We thank C. Chambers (NOAA-NMFS, Highlands, NJ) for contributing fish and providing laboratory space. J. Kocik (NOAA-NMFS, Orono, ME), T. Shaheen (NOAA-NMFS, Woods Hole, MA), and the smolt tagging team in Maine generously shared with us their knowledge of surgical techniques. B. Dunnigan, D.V.M., and R. Williams, D.V.M. (NOAA-NMFS, Woods Hole, MA), provided guidance on general surgical procedures for fish. We thank A. Drohan, J. Manderson, A. Pollack, F. Morello, J. Rosendale, J. Hilbert, P. Shaheen, B. Phelan, and F. Scharf (NOAA-NMFS, Highlands, NJ) for assistance with laboratory experiments

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