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Salinity tolerance of the Seminole killifish, Fundulus seminolis, a candidate species for marine baitfish aquaculture
Aquaculture 293 (2009) 74–80
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Salinity tolerance of the Seminole killifish, Fundulus seminolis, a candidate species for marine baitfish aquaculture Matthew A. DiMaggio a,b,⁎, Cortney L. Ohs a,b, B. Denise Petty a,c a School of Forest Resources and Conservation, Program in Fisheries and Aquatic Sciences, Institute of Food and Agricultural Sciences, University of Florida, 7922 NW 71st St., Gainesville, Florida 32653, USA b Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, 2199 South Rock Rd., Fort Pierce, Florida 34945, USA c Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Institute of Food and Agricultural Sciences, University of Florida, P.O. Box 100136, Gainesville, Florida 32610, USA
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Article history: Received 12 January 2009 Received in revised form 13 April 2009 Accepted 14 April 2009 Keywords: Fundulus seminolis Seminole killifish Baitfish Salinity Tolerance
a b s t r a c t Aquaculture of marine baitfish species is still in its relative infancy and the increasing value of coastal property is forcing marine aquaculture inland. Fundulus seminolis, a freshwater killifish species endemic to Florida, has shown economic potential for use as a marine baitfish, with a small number of commercial operations currently in production. The objectives of this study were to determine both acute and gradual salinity tolerances as well as an upper lethal salinity tolerance for the species. Two separate acute acclimation experiments, natural seawater and sodium chloride, were carried out to determine if survival was influenced by the salinity source. F. seminolis were able to tolerate acute transfer to 0, 8, and 16 g/L using both salinity sources but only those in natural seawater were able to survive in 24 g/L. No survival was observed regardless of salinity source after acute transfer to 32 g/L. A gradual acclimation using natural seawater was also investigated to examine survival at various acclimation rates. A survival rate of 100% was achieved regardless of acclimation rate when salinity was changed from 0 to 32 g/L in 24, 48, 72, and 96 h treatment groups. An upper lethal salinity determination yielded an LC-50 of 60 g/L with a maximum salinity tolerance of 78 g/L. Results of these experiments provide information pertinent to the successful culture of this rarely studied species. Additionally, experimental outcomes will help to shape the marketing and distribution strategies for F. seminolis as a marine baitfish. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The Seminole killifish, Fundulus seminolis (Girard), is an endemic Florida killifish with a geographic range within peninsular Florida from the St. Johns and New River drainage basins to just south of Lake Okeechobee (Page and Burr, 1991). Relatively little is known regarding the life history of F. seminolis, with only one publication by DuRant et al. (1979) devoted entirely to the species. It has been referenced anecdotally or as a component in a larger study or survey in several publications (McLane, 1955; Phillips and Springer, 1960; Tabb and Manning, 1961; Gunter and Hall, 1963; Gunter and Hall, 1965; Foster, 1967; Griffith, 1974a; Nordlie, 2006). This species, as well as several other members of the Fundulus genus are commonly referred to as a
⁎ Corresponding author. School of Forest Resources and Conservation, Program of Fisheries and Aquatic Sciences, Institute of Food and Agricultural Sciences, University of Florida, 7922 NW 71st St., Gainesville, Florida 32653, USA. Tel.: +1 772 468 3922ext134; fax: +1 772 468 3973. E-mail addresses: mdimaggi@ufl.edu, [email protected] (M.A. DiMaggio). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.04.009
“bullminnow” or “mudminnow”. The Seminole killifish is one of the largest members of the genus, reaching total lengths of 20 cm (Hoyer and Canfield, 1994). Its popularity as a local freshwater baitfish for largemouth bass, Micropterus salmoides, and other piscivorous game fish has generated interest in this species as a potential candidate for aquaculture. With previous data placing the upper salinity tolerance of F. seminolis at 23.4 g/L (Griffith, 1974a), culture of this species for use as a saltwater baitfish warrants further investigation. If the species is able to acclimate to full strength seawater, it could be produced exclusively in freshwater ponds or recirculation systems only needing to be acclimated to saline water prior to marketing and distribution. Additionally, with coastal property values at a premium and limited access to seawater, baitfish producers would be able to utilize inland resources for the culture of a marine baitfish. Determination of salinity tolerance following gradual and acute transfer is necessary to evaluate the species’ physiological limitations which will influence culture and marketing practices. Similar studies have been conducted by Lotan (1971), Griffith (1974a), Stanley and Fleming (1977), Chervinski (1983), Nordlie (1987), Crego and Peterson (1997), Nordlie (2000) and Fuller (2008) on salinity tolerance of various members of the order cyprinodontiformes. For
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the most recent review of cyprinidontoid salinity tolerance consult Nordlie (2006). The previous studies examined multiple salinity ranges utilizing various salt sources (seawater and synthetic sea salts) and freshwater dilutions to achieve experimental salinities both hypo and hypersaline to natural seawater (32–35 g/L). Distinct salinity thresholds defined by survival and select hematological indices were then characterized for the species in question. Experimental salinity determinations such as these may be a more accurate representation of the organism's true salinity tolerance than maximum reported field salinities (Kefford et al., 2004). This can be seen in Phillips and Springers' (1960) record of F. seminolis inhabiting waters with a salinity of 13.5 g/L, the highest recorded salinity from published field observations. Griffith's (1974a) subsequent experimental salinity determination for this species placed the upper salinity tolerance much higher, with an experimental salinity range of 19.3–33.4 g/L. However, Griffith (1974a) reported high mortalities in maintaining this species in captivity and his sample size for salinity tolerance determinations was only four individuals. Although the lower lethal temperature has yet to be established for this species, the experimental water temperature of 15 °C might have exacerbated the physiological stress caused by the acclimation if this temperature impaired the functionality of essential enzymes. Taken together, these circumstances call into question the accuracy of the salinity range reported by Griffith (1974a). Investigations into the salinity tolerance of freshwater species have shared a predominant ecological motivation. Investigations by Bringolf et al. (2005) and Schofield et al. (2006) assessed salinity tolerances as barriers to invasion for the flathead catfish, Pylodictis olivaris, and goldfish, Carassius auratus, respectively. While the impetus for the current study is the evaluation of F. seminolis as an aquaculture candidate; investigations into its salinity tolerance should provide valuable information about the species' ability to handle osmoregulatory stressors and could further be extrapolated for ecological applications. Salinity tolerance is an important consideration in the culture of marine and freshwater organisms. It provides information about basic husbandry requirements necessary for the species to thrive in captivity as well as potential applications for the cultured organisms. Additionally, economic considerations associated with culture of marine or brackish water species make low saline or freshwater culture an attractive alternative. Research into low salinity aquaculture of marine species is common, but few studies have been conducted on acclimation of freshwater species to seawater. Experiments examining abrupt transfer of black sea bass, Centropristis striata, to low salinities have helped to identify a salinity threshold for the successful culture of this species (Young et al., 2006). Similarly, gradual acclimation experiments with Nile tilapia, Oreochromis niloticus, and blackchin tilapia, Sarotherodon melanotheron, (Lemarie et al., 2004) as well as larval salinity tolerance experiments with striped mullet, Mugil cephalus, thick-lipped grey mullet, Chelon labrosus (Hotos and Vlahos, 1998), and cobia, Rachycentron canadum (Faulk and Holt, 2006), have provided valuable evidence regarding the osmoregulatory ability of a species for use in conventional aquaculture conditions. The purpose of this study was to characterize the salinity tolerance of F. seminolis, a potential candidate for marine baitfish aquaculture. Abrupt and gradual salinity acclimations were evaluated as well as salinity sources (sodium chloride vs. seawater). A maximum lethal salinity resulting in 100% mortality was also investigated by means of gradual acclimation with a corresponding determination of the salinity concentration which resulted in 50% mortality (LC-50). This investigation represents the first comprehensive study focused on the salinity tolerance of F. seminolis. 2. Methods F. seminolis were collected by seine net from the eastern shore of Lake George, in Volusia County Florida and transported to the University of
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Florida Indian River Research and Education Center in Fort Pierce. Fish were assessed for pathogens and treated accordingly to ensure healthy research specimens for the subsequent salinity experiments. The acute salinity tolerance of F. seminolis to varying concentrations of sodium chloride (NaCl) and natural seawater (NSW) were investigated as well as survival following gradual NSW acclimation. A maximum lethal salinity investigation was also conducted. 2.1. Sodium chloride acute salinity tolerance Thirty five fish were transferred from a 6900 L recirculating system to 85 L glass aquaria with one fish per aquarium during the entire acclimation and experimental periods. Specimen's weight and total length (TL) were recorded prior to transfer. Length and weight ranges of 120–145 mm and 17.0–29.4 g were recorded with means of 131.2 ± 6.3 mm and 23.2 ± 3.6 g, respectively. Aquarium systems were maintained at b1 g/L salinity well water and recirculated through biofilter media during the 96 h acclimation period. Dissolved oxygen (DO), pH, temperature, salinity, total ammonia nitrogen (TAN), and nitrite were recorded daily during the acclimation and experimental periods with total alkalinity and total hardness recorded on days one and three of acclimation and daily during the course of the experiment. DO and temperature were measured using a YSI 550A meter (YSI Inc., Yellow Springs, Ohio). Salinity was determined using a handheld refractometer, pH was measured using a Hach sensION1 portable pH meter and total alkalinity and total hardness were determined using standardized titration techniques (Hach Co., Loveland, Colorado). TAN and nitrite were evaluated spectrophotometrically using a Hach DR 4800 spectrophotometer (Hach Co., Loveland, Colorado). Aquaria were held at ambient temperature with a range of 19.9–26.5 °C and a mean temperature of 22.7 °C during the experimental period. Temperature differences among aquaria never exceeded 2 °C. An ambient photoperiod of 11 L: 13 D was used during the experiment. Fish were fed once a day to satiation on days two and three of acclimation and food was withheld on days one and four of acclimation and during the entire 96 h experimental period. Following the 96 h freshwater acclimation, aquaria were made static and individual aquariums were randomly assigned to one of five treatments with seven replicates per treatment. Differences among treatment group's lengths and weights were not significant (F4,30 =0.68, p = 0.610; F4,30 = 0.20, p = 0.939, respectively). Treatment salinities examined were 0 (control), 8, 16, 24, and 32 g/L. Salinities were abruptly changed by removing the appropriate amount of fresh water and adding a predetermined volume of a concentrated brine solution made by dissolving 99.5% NaCl (Morton White Crystal Solar Salt, Morton International Inc., Chicago, IL) into well water. Control aquaria had a predetermined volume of fresh water removed and subsequently replaced to maintain similar treatment of control and experimental groups. Tanks were aerated to thoroughly mix the water, then salinities were remeasured to confirm the desired concentrations were attained. Individual biofilters which had been preconditioned to treatment salinities were placed in each tank to control nitrogenous wastes. Aquaria were examined for mortalities as follows: once every hour from 0–12 h, once every 6 h from 12–48 h, and once every 12 h from 48–96 h. A final weight was recorded upon discovery of a mortality or upon the termination of the 96 h exposure period. Mortality as used throughout this investigation was defined as loss of opercular movement and no response to physical stimulus. 2.2. Natural seawater acute salinity tolerance Methods for the NSW acute salinity toxicity trial adhere to the previous methods listed in Section 2.1. with exceptions as noted. Treatment salinities were abruptly changed by removing a predetermined volume of fresh water and adding a known volume of natural seawater collected from the Atlantic Ocean and filtered through a 1 micron cartridge filter.
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Control aquaria were treated as previously stated. Aquaria were held at ambient temperature with a range of 20.5–25.6 °C and a mean temperature of 23.4 °C during the experimental period. Temperature differences among aquaria never exceeded 2 °C. A ambient photoperiod of 11.5 L: 12.5 D was used during the experiment. Length and weight ranges of 122–146 mm and 16.6–31.2 g were recorded with means of 129.7±5.8 mm and 21.9±3.5 g, respectively. Differences among treatment group's lengths were not statistically significant (F4, 30 =1.12, p=0.368). Differences among treatment group's weights were statistically significant (p =0.010) but were not considered to be biologically significant for the purpose of this experiment with a mean treatment weight range of 18.7–23.8 g. Differences among treatment weights did not violate any assumptions of subsequent survival analysis. 2.3. Natural seawater gradual acclimation survival Sixty-four fish were transferred from a 6900 L recirculating system to 32, 85 L glass aquaria, divided in half by aquarium partitions (TDSU, Penn-Plax Inc., Happauge, NY USA) with one fish per aquarium subdivision during the entire acclimation and experimental periods. Fish weight and TL were recorded prior to transfer. Length and weight ranges of 124–152 mm and 21.7–38.3 g were recorded with means of 136.3 ± 6.3 mm and 27.5 ± 4.5 g, respectively. Aquarium systems were maintained at b1 g/L salinity well water and recirculated through biofilter media during the 96 h acclimation period. DO, pH, temperature, salinity, TAN, and nitrite were recorded daily during the acclimation and experimental periods with total alkalinity and total hardness recorded on days one and three of acclimation and at the initiation and cessation of experimental treatment periods. Methods for water quality analysis adhere to previously stated techniques (Section 2.1.) with the exception of salinity, which was measured using a YSI 30 salinity/conductivity meter (YSI Inc., Yellow Springs, OH USA). Aquaria were held at ambient temperature with a range of 17.5–27.2 °C and a mean temperature of 22.6 °C during the experimental period. Temperature differences among aquaria never exceeded 2 °C. An ambient photoperiod of 13 L: 11 D was used during the experiment. Fish were fed once a day to satiation on days two and three of acclimation and food was withheld on days one and four of acclimation and during the entire experimental period. Following the 96 h freshwater acclimation, aquaria were made static and individual tanks were randomly assigned to one of eight treatments with eight replicates per treatment. Differences among treatment group's lengths and weights were not significant (p = 0.784; F7,56 = 0.38, p = 0.908, respectively). Among the treatment groups, four underwent a gradual salinity change from 0 to 32 g/L over predetermined time periods with the remaining four treatments serving as corresponding controls. Acclimation times of 24, 48, 72, and 96 h were chosen with approximate salinity increases of 5.3, 2.7, 1.7, and 1.3 g/L, respectively, every 4 h. The final salinity of 32 g/L was achieved 4 h preceding the termination of the treatment group in question. Salinities were gradually changed every 4 h via addition of NSW equally dispersed between aquaria subdivisions until the desired salinity was achieved. Excess water during salinity changes was allowed to flow out of the aquarium's standpipe. A single air stone within each subdivision provided adequate mixing within and between subdivisions ensuring a homogeneous environment within each aquarium. Control aquaria had corresponding volumes of freshwater added in the same fashion as their saline counterparts. Aquaria were examined for mortalities as follows: once every hour from 0–12 h and once every 4 h from 12–96 h. A final weight was recorded upon discovery of a mortality or upon the termination of the acclimation exposure period. 2.4. Maximum lethal salinity determination Twenty four fish were transferred from a 6900 L recirculating system to 12, 85 L glass aquaria, divided in half by aquarium partitions (TDSU,
Penn-Plax Inc., Happauge, NY USA) with one fish per aquarium subdivision during the entire acclimation and experimental periods. Fish weight and TL were recorded prior to transfer. Length and weight ranges of 120–145 mm and 16.4–30.4 g were recorded with means of 129.0 ± 5.5 mm and 21.1 ± 3.4 g, respectively. Aquarium systems were maintained at 2 g/L salinity well water to aid in osmoregulation and inhibit nitrite toxicity. Water was recirculated through biofilter media during the 96 h acclimation period. DO, pH, temperature, TAN, and nitrite were recorded on day four of the acclimation period. DO, pH, and temperature were recorded daily and TAN and nitrite recorded once weekly during the experimental period. Salinity was measured twice weekly during salinity changes. Methods for water quality analysis adhere to Section 2.1. with the exception of salinity, which was measured using a YSI 30 salinity/conductivity meter (YSI Inc., Yellow Springs, OH USA). Aquaria were held at ambient temperature with a range of 19.0– 31.3 °C and a mean temperature of 26.3 °C during the experimental period. Temperature differences among aquaria never exceeded 2 °C. An ambient photoperiod of 11.25 L: 12.75 D was used during the experiment. Fish were fed to satiation three times a week during both the acclimation and experimental period to control nitrogenous wastes. Following the 96 h acclimation, aquaria were made static and individual tanks were assigned to one of two treatments with 12 replicates per treatment. Differences among treatment group's lengths and weights were not significant (F 1,22 = 0, p = 0.973; F 1,22 = 0.58, p = 0.454, respectively). Between the treatment groups, one underwent a gradual salinity change from 2 g/L until 100% mortality was observed for all replicates. The additional treatment served as a control and was maintained at 2 g/L salinity for the duration of the experiment. Salinities were increased by 6 g/L twice a week (every Monday and Thursday) with controls treated in a similar fashion. Salinity was increased to 30 g/L via the addition of NSW. Beyond this concentration, salinities were augmented by addition of synthetic sea salts (HW Marine Mix Professional, Hawaiian Marine Imports Inc. Houston, TX) to aquaria water to make a concentrated brine solution and then added back to the aquaria to achieve the desired concentration. A single air stone within each subdivision provided adequate mixing within and between subdivisions ensuring a homogeneous environment within each aquarium. Control aquaria had corresponding volumes of 2 g/L water added in the same fashion as their saline counterparts. Aquaria were examined for mortalities daily until 100% mortality was noted in the salinity treatment. 2.5. Statistical analysis Survival was estimated using a Kaplan-Meier product limit estimator (Kaplan and Meier, 1958). Log-rank tests were then performed to compare generated survivorship curves among treatments and within treatments between experiments. Length, weight, and water quality data were analyzed using a one-way ANOVA with a Tukey's HSD means separation test. Nonparametric data were analyzed with a KruskalWallis test. Kaplan-Meier analysis and subsequent log rank tests were performed in SPSS® version 12.0 (SPSS Inc. Chicago, IL USA). All other statistical analyses were performed in SAS® version 8.02 (SAS Institute Inc., Cary, NC USA) All numerical data are represented as the mean± SD
Table 1 Kaplan-Meier survival analysis for NaCl acute transfer. Treatment (g/L NaCl)
Mean survival (h ± SE)
Survival (%)
(Control) 0 8 16 24 32
⁎ ⁎ ⁎
100 100 100 0 0
6±1 2
⁎ Statistic cannot be calculated for comparison of treatments with 100% survival for both.
M.A. DiMaggio et al. / Aquaculture 293 (2009) 74–80 Table 2 Log-Rank analysis among treatments for NaCl acute transfer. Treatment
0 g/L
8 g/L 16 g/L 24 g/L 32 g/L
⁎ ⁎
8 g/L ⁎
p b 0.0001 p b 0.0001
p b 0.0001 p b 0.0001
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Table 4 Kaplan-Meier survival analysis for NSW acute transfer.
16 g/L
p b 0.0001 p b 0.0001
24 g/L
p b 0.0001
⁎ Statistic cannot be calculated for comparison of treatments with 100% survival for both.
unless otherwise stated. Statistical differences were considered significant when p ≤ 0.05. 3. Results 3.1. Sodium chloride acute salinity tolerance Survival was 100% at salinities of 0 (control), 8, and 16 g/L throughout the entire 96 h experimental period (Table 1). Acute transfer to higher salinities yielded 100% mortality with mean survival times of 6 h (95% Confidence Interval [CI], 5–7 h) at 24 g/L and 2 h (95% CI, 2–2 h) at 32 g/L (Table 1). Despite the complete mortality observed in these two treatments, survival times were significantly different (p ≤ 0.0001) from each other as well as from all other treatments examined (p b 0.0001) (Table 2). No biologically significant differences among treatments in the measured water quality parameters were observed throughout the course of this experiment (Table 3). 3.2. Natural seawater acute salinity tolerance Survival was 100% at salinities of 0 (control), 8, 16, and 24 g/L throughout the entire 96 h experimental period (Table 4). Acute transfer to 32 g/L yielded 100% mortality with a mean survival time of 13 h (95% CI, 11–16 h) (Table 4). This group was significantly different (p = 0.0002) from all other treatments (Table 5). No biologically significant differences among treatments in the measured water quality parameters were detected throughout the
Treatment (g/L NSW)
Mean survival (h ± SE)
Survival (%)
(Control) 0 8 16 24 32
⁎ ⁎ ⁎ ⁎
100 100 100 100 0
13 ± 1
⁎ Statistic cannot be calculated for comparison of treatments with 100% survival for both.
Table 5 Log-Rank analysis among treatments for NSW acute transfer. Treatment
0 g/L
8 g/L
16 g/L
8 g/L 16 g/L 24 g/L 32 g/L
⁎ ⁎ ⁎
⁎ ⁎ ⁎
⁎
p = 0.0002
p = 0.0002
p = 0.0002
24 g/L
p = 0.0002
⁎ Statistic cannot be calculated for comparison of treatments with 100% survival for both.
course of this experiment with the exception of total hardness. Due to experimental dilutions of seawater, total hardness varied by treatment salinity (Table 3). 3.3. Sodium chloride vs. natural seawater When survival was analyzed within treatment salinity between salt source, NSW and NaCl were significantly different (p b 0.0001) in the 24 and 32 g/L salinity treatments. 3.4. Natural seawater gradual acclimation survival For all treatment groups, survival was 100%. Therefore, no analysis of the data was required. With the exception of total hardness, no biologically significant differences among treatments in the measured water quality parameters were observed throughout the course of the experiment (Table 3).
Table 3 Acclimation and experimental water quality data collected from the NaCl acute salinity tolerance, NSW acute salinity tolerance, NSW gradual acclimation and maximum lethal salinity experiments. DO (mg/L)
pH
TAN (mg/L)
Nitrite (mg/L)
Alkalinity (mg/L CaCO3)
Hardness (mg/L CaCO3)
NaCl acute salinity tolerance - 96 h acclimation NaCl acute salinity tolerance - 96 h experimental NSW Acute Salinity Tolerance - 96 h acclimation NSW acute salinity tolerance - 96 h experimental
7.11 ± 0.42 (6.38–7.57) 7.73 ± 0.33 (7.14–8.35) 7.13 ± 0.41 (6.66–7.90) 7.87 ± 0.19 (7.50–8.24)
8.43 ± 0.06 (8.33–8.52) 8.43 ± 0.08 (8.28–8.58) 8.37 ± 0.11 (8.19–8.53) 8.36 ± 0.13 (8.06–8.63)
0.01 ± 0.01 (0.00–0.02) 0.03 ± 0.03 (0.00–0.09) 0.02 ± 0.02 (0.00–0.06) 0.03 ± 0.02 (0.00–0.10)
0.0025 ± 0.0008 (0.0006–0.0039) 0.0753 ± 0.0565 (0.0125–0.3102) 0.0011 ± 0.0017 (0.0000–0.0043) 0.0107 ± 0.0074 (0.0018–0.0356)
139.65 ± 6.98 (136.80–153.90) 135.80 ± 9.80 (119.70–153.90) 171.00 ± 18.73 (153.90–205.20) 144.00 ± 9.80 (119.70–171.00)
NSW gradual acclimation - 96 h acclimation NSW gradual acclimation - 96 h experimental
7.63 ± 0.29 (7.23–7.98) 7.86 ± 0.19 (7.56–8.35)
8.29 ± 0.11 (8.03–8.41) 8.31 ± 0.07 (8.12–8.46)
0.01 ± 0.01 (0.00–0.02) 0.03 ± 0.02 (0.00–0.07)
0.0025 ± 0.0010 (0.0011–0.0036) 0.0040 ± 0.0022 (0.0014–0.0200)
180
Maximum lethal salinity determination - acclimation and experimental
6.66 ± 0.91 (4.80–7.97)
8.36 ± 0.20 (7.80–8.86)
0.17 ± 0.19 (0.00–0.86)
0.2077 ± 0.2882 (0.0000–1.0731)
–
202.35 ± 19.99 (188.10–239.40) 193.90 ± 18.90 (171.00–256.50) 190.95 ± 33.19 (153.90–239.40) 0–182.60 ± 21.40 (153.90–222.30) 8–1557.10 ± 300.00 (1250.00–2570.00) 16–2860.00 ± 95.10 (2700.00–3090.00) 24–4282.10 ± 295.40 (3830.00–5580.00) 32–5808.60 ± 117.80 (5700.00–6060.00) 199.50 ± 8.83 (188.10–205.20) 0–205.20 32–5717.50 ± 116.02 (205.20–5840.00) –
170.00 ± 10.1 (160.00–180.00)
Bolded numbers indicate salinity concentration. Values are reported as the mean ± SD. Parameter ranges are listed below the mean in parentheses.
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Fig. 1. Observed survival (%) over a 46 day maximum lethal salinity experiment, culminating in 100% mortality for the treatment group which underwent gradual salinity increases of 6 g/L twice a week. The control group was maintained at 2 g/L throughout the entire experiment.
3.5. Maximum lethal salinity determination The maximum lethal salinity determination experiment lasted for 46 days until a lethal salinity of 78 g/L was reached resulting in 100% mortality for the salinity treatment group with an LC-50 of 60 g/L (Figs. 1 and 2). Survival in the control group was 91.67% at the end of the 46 day trial. No biologically significant differences among treatments in the measured water quality parameters were observed throughout the course of the acclimation or experimental periods (Table 3). 4. Discussion Survival analysis for NaCl and NSW acute salinity tolerance experiments revealed F. seminolis can tolerate direct transfer from freshwater to salinities up to 16 and 24 g/L, respectively. Daily water quality analysis during the 96 h experimental periods helped to eliminate potentially confounding variables in an effort to identify salinity as the driving force behind observed mortality. Upon completion of the 96 h experimental exposure in acute transfer experiments, surviving fish were left in their respective aquaria and monitored for 2 weeks. No mortalities were observed during this time period for any surviving treatments regardless of salinity source. This is evidence that results of 96 h survival experiments are useful indicators of a species' ability to survive long term in selected treatment conditions (DiMaggio, personal communication). Additionally, although 100% survival was noted at 16 g/L in both NaCl and NSW experiments, fish in the NSW treatment generally appeared healthier. F. seminolis at 16 g/L in NaCl exhibited multiple areas of hyperemia, overall pallor, decreased activity, and increased mucus production. Excessive mucus has been previously reported in Fundulus kansae exposed to low calcium “artificial seawater” (Potts and Fleming, 1970). Differences between survival due to salinity source were evident in 24 and 32 g/L treatments (Tables 1 and 4). Due to lack of physiological data it is only possible to speculate as to the underlying causes for the recorded differences. The role of calcium in osmoregulation and ionic transport has been well studied (Potts and Fleming, 1970; Carrier and Evans, 1976; Isaia and Masoni, 1976; Pic and Maetz, 1981; Hunn, 1985). Although calcium was not measured directly, total hardness levels (mg/L CaCO3) were recorded throughout both experiments. Signifi-
cant differences (p b 0.0001) in hardness were noted among NSW treatment salinities of 8, 16, 24, and 32 g/L. Additionally, hardness values from these treatment groups were also significantly different (p b 0.0001) from all NaCl treatment groups and the NSW control. It can be inferred from this data that calcium and other divalent ions were in a greater abundance in the NSW treatment groups than their NaCl counterparts. Low external calcium in an environment hyperosmotic to fish has been shown to increase ion efflux in Lagodon rhomboides (Carrier and Evans, 1976) and alter net fluxes of both ions and water in Anguilla anguilla (Isaia and Masoni, 1976). Potts and Fleming (1970) reported a 35% reduction in the gill permeability of F. kansae in NSW when compared with fresh water, thereby decreasing ion efflux and drinking rate. Conversely they also reported F. kansae to have an increased drinking rate and water exchange rate when transferred to low calcium synthetic seawater. Although not directly tested, findings from the aforementioned studies support the hypothesis that the mortality observed in the NaCl experiment relative to the NSW experiment was a result of loss of hydro-mineral regulation due to lower environmental ion concentrations, particularly calcium. However, environmental deficiencies of other divalent ions such as magnesium, should not be dismissed because these ions also play a functional role in salinity acclimation (Isaia and Masoni, 1976). Results of gradual NSW acclimation were encouraging for this marine baitfish candidate. F. seminolis was able to easily acclimate to 32 g/L in all four time intervals with no mortalities in any treatment. Modest lethargy in most replicates of the 24 h treatment group was the only outward sign of physiological stress observed among any of the treatments upon completion of the acclimation process. The maximum lethal salinity of 78 g/L recorded in this study was more than twice the reported maximum salinity of 33.4 g/L reported by Griffith (1974a). The first mortality recorded from the salinity treatment group at day 25 (Fig. 1) and 48 g/L (Fig. 2) is well above natural ocean salinities in the Atlantic. These results provide further evidence of the ability of F. seminolis to successfully acclimate to salinities characteristic of NSW as well as hypersaline environments given appropriate acclimation conditions. It should be noted that animals which underwent salinity change actively fed throughout the experimental period and did not exhibit any gross signs of stress when compared with their controls. Salinity tolerance of the predominantly euryhaline Fundulus genus has been extensively investigated yet almost no experimental
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Fig. 2. Observed survival (%) at treatment salinities during the entire experimental period. Gradual salinity increases of 6 g/L were carried out twice a week until 100% mortality was observed.
evidence exists regarding F. seminolis until the present study. Griffith's (1974a) seminal investigation on salinity tolerance of the genus is the only study that has evaluated this emerging aquaculture candidate, but due to confounding experimental factors these data are potentially unreliable. An additional study by Griffith (1974b) attempted to elucidate the role of pituitary control in the freshwater acclimation of the genus, but only one F. seminolis was used, again limiting the usefulness of the data. Salinity tolerance results from this experiment confirm F. seminolis can acclimate to the upper end of Griffith's (1974a) reported range and beyond. Survival by F. seminolis after acute transfer to 24 g/L indicates superior salinity tolerance when compared to Fundulus nottii's acute tolerance of 17 g/L (Crego and Peterson, 1997). With a maximum lethal salinity of 78 g/L (Fig. 2) determined in this experiment, it is unlikely F. seminolis would tolerate salinities of 95+ g/L commonly reported for F. grandis (Nordlie, 2000), F. heteroclitus (Griffith, 1974a), F. confluentus (Griffith, 1974a; Nordlie, 2000), F. similis (Nordlie, 2000), F. parvipinnis (Feldmeth and Waggoner, 1972), Aphanius dispar (Lotan, 1971), and Adenia xenica (Nordlie, 1987). F. seminolis is widely considered to be a freshwater killifish, with upper field salinities reported at 2.4 (Gunter and Hall, 1963), 7.3 (Gunter and Hall, 1965), and 13.5 g/L (Phillips and Springer, 1960). Results from our experiments clearly indicate F. seminolis is capable of tolerating brackish, full strength seawater, and hypersaline environments for extended periods of time. Experimental results substantiate the potential of F. seminolis as a candidate for marine baitfish aquaculture following seawater acclimation. Ability to tolerate acute transfer from 0–24 g/L and gradual acclimation to 32 g/L NSW over a broad time period allows for flexibility in the development of a salinity acclimation protocol. Additionally, a mean survival time of 13 h after acute transfer to full strength seawater allows for marketing and use of freshwater acclimated fish in the marine environment if so desired. NSW acclimated fish provide an additional option for bait retailers utilizing saline holding facilities as well as anglers who wish to store their bait in saline livewells. Demands of bait retailers and sportsman will ultimately dictate the acclimation regime for this new marine baitfish species. Acknowledgements The authors wish to thank Greg Harris and Edsel E. Redden for their assistance in procurement of the research specimens as well as Dr. Andrew Rhyne, Scott Grabe, Shawn DeSantis, and John Marcellus for
their continued help throughout the course of numerous experiments. The authors also wish to recognize Dr. Pamela Schofield for her statistical assistance and Dr. Jeff Hill for his editorial contributions. This research was funded by the Florida Department of Agriculture and Consumer Services Division of Aquaculture. References Bringolf, R.B., Kwak, T.J., Cope, W.G., Larimore, M.S., 2005. Salinity tolerance of the Flathead Catfish: implications for dispersal of introduced populations. Transactions of the American Fisheries Society 134, 927–936. Carrier, J.C., Evans, D.H., 1976. The role of environmental calcium in the freshwater survival of the marine teleost Lagodon rhomboides. Journal of Experimental Biology 65, 529–538. Chervinski, J., 1983. Salinity tolerance of the mosquito fish, Gambusia affinis (Baird and Girard). Journal of Fish Biology 22, 9–11. Crego, G.J., Peterson, M.S., 1997. Salinity tolerance of four ecologically distinct species of Fundulus (Pisces: Fundulidae) from the northern Gulf of Mexico. Gulf of Mexico Science 1997, 45–49. DuRant, D.F., Shireman, J.V., Gasaway, R.D., 1979. Reproduction, growth and food habits of seminole killifish, Fundulus seminolis, from two central Florida Lakes. American Midland Naturalist 102, 127–133. Faulk, C.K., Holt, G.J., 2006. Responses of cobia Rachycentron canadum larvae to abrupt or gradual changes in salinity. Aquaculture 254, 275–283. Feldmeth, C.R., Waggoner III, J.P., 1972. Field measurements of tolerance to extreme hypersalinity in the California killifish, Fundulus parvipinnis. Copeia 1972 (3), 592–594. Foster, N.R. 1967. Comparative studies on the biology of killifishes (Pisces, Cyprinodontidae). Ph.D. Dissertation, Cornell University 369 p. Fuller, R.C., 2008. A test for a trade-off in salinity tolerance in early life-history stages in Lucania goodei and L. parva. Copeia 2008 (1), 154–157. Griffith, R.W., 1974a. Environment and salinity tolerance in the genus Fundulus. Copeia 1974, 319–331. Griffith, R.W., 1974b. Pituitary control of adaptation to fresh water in the teleost genus Fundulus. Biological Bulletin 146, 357–376. Gunter, G., Hall, G.E., 1963. Biological investigations of the St. Lucie estuary (Florida) in connection with Lake Okeechobee discharges through the St. Lucie canal. Gulf Research Reports 1, 189–307. Gunter, G., Hall, G.E., 1965. A biological investigation of the Caloosahatchee estuary of Florida. Gulf Research Reports 2, 1–71. Hotos, G.N., Vlahos, N., 1998. Salinity tolerance of Mugil cephalus and Chelon labrosus (Pisces: Mugilidae) fry in experimental conditions. Aquaculture 167, 329–338. Hoyer, M.V., Canfield Jr., D.E., 1994. Handbook of common freshwater fish in Florida lakes. University of Florida/Institute of Food and Agricultural Sciences. Gainesville, Florida. Spec. Pub., vol. 160. Hunn, J.B., 1985. Role of calcium in gill function in freshwater fishes. Comparative Biochemistry and Physiology 82A (3), 543–547. Isaia, J., Masoni, A., 1976. The effects of calcium and magnesium on water and ionic permeabilities in the sea water adapted eel, Anguilla anguilla L. Journal of Comparative Physiology 109, 221–233. Kaplan, E.L., Meier, P., 1958. Nonparametric estimation from incomplete observations. Journal of the American Statistical Association 53, 457–481.
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Kefford, B.J., Papas, P.J., Metzeling, L., Nugegoda, D., 2004. Do laboratory salinity tolerances of freshwater animals correspond with their field salinity? Environmental Pollution 129, 355–362. Lemarie, G., Baroiller, J.F., Clota, F., Lazard, J., Dosdat, A., 2004. A simple test to estimate the salinity resistance of fish with specific application to O. niloticus and S. melanotheron. Aquaculture 240, 575–587. Lotan, R., 1971. Osmotic adjustment in the euryhaline teleost Aphanius dispar (Cyprinodontidae). Zeitschrift fur Vergleichende Physiologie 75, 383–387. McLane, W.M. 1955. Fishes of the St. Johns River System. Ph.D. Dissertation, University of Florida, 361p. Nordlie, F.G., 1987. Salinity tolerance and osmotic regulation in the diamond killifish, Adinia xenica. Environmental Biology of Fishes 20 (3), 229–232. Nordlie, F.G., 2000. Salinity responses in three species of Fundulus (Teleostei: Fundulidae) from Florida salt marshes. Proceedings - International Association of Theoretical and Applied Limnology 27, 1276–1279. Nordlie, F.G., 2006. Physicochemical environments and tolerances of cyprinodontoid fishes found in estuaries and salt marshes of eastern North America. Reviews in Fish Biology and Fisheries 16, 51–106. Page, L.M., Burr, B.M., 1991. A field guide to freshwater fishes of North America North of Mexico. Houghton Mifflin Co., NY. 432 p.
Phillips, R.C., Springer, V.G., 1960. A report on the hydrology, marine plants, and fishes of the Caloosahatchee river area, Lee County, Florida. Special Scientific Report No. 5, Marine Laboratory, Florida Board of Conservation, St. Petersburg, FL. Pic, P., Maetz, J., 1981. Role of external calcium in sodium and chloride transport in the gills of seawater-adapted Mugil capito. Journal of Comparative Physiology 141, 511–521. Potts, W.T.W., Fleming, W.R., 1970. The effects of prolactin and divalent ions on the permeability to water of Fundulus kansae. Journal of Experimental Biology 53, 317–327. Schofield, P.J., Brown, M.E., Fuller, P.L., 2006. Salinity tolerance of goldfish Carassius auratus L., a non-native fish in the United States. Florida Scientist. 69 (4), 258–268. Stanley, J.G., Fleming, W.R., 1977. Failure of seawater acclimation to alter osmotic toxicity in Fundulus kansae. Comparative Biochemistry and Physiology 58A, 53–56. Tabb, D.C., Manning, R.B., 1961. A checklist of the flora and fauna of northern Florida Bay and adjacent brackish waters of the Florida mainland collected during the period July, 1957 through September, 1960. Bulletin of Marine Science of the Gulf and Caribbean 11, 552–649. Young, S.P., Smith, T.I.J., Tomasso, J.R., 2006. Survival and water balance of black sea bass held in a range of salinities and calcium-enhanced environments after abrupt salinity change. Aquaculture 258, 646–649.
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Salinity tolerance of the Seminole killifish, Fundulus seminolis, a candidate species for marine baitfish aquaculture Matthew A. DiMaggio a,b,⁎, Cortney L. Ohs a,b, B. Denise Petty a,c a School of Forest Resources and Conservation, Program in Fisheries and Aquatic Sciences, Institute of Food and Agricultural Sciences, University of Florida, 7922 NW 71st St., Gainesville, Florida 32653, USA b Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, 2199 South Rock Rd., Fort Pierce, Florida 34945, USA c Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Institute of Food and Agricultural Sciences, University of Florida, P.O. Box 100136, Gainesville, Florida 32610, USA
a r t i c l e
i n f o
Article history: Received 12 January 2009 Received in revised form 13 April 2009 Accepted 14 April 2009 Keywords: Fundulus seminolis Seminole killifish Baitfish Salinity Tolerance
a b s t r a c t Aquaculture of marine baitfish species is still in its relative infancy and the increasing value of coastal property is forcing marine aquaculture inland. Fundulus seminolis, a freshwater killifish species endemic to Florida, has shown economic potential for use as a marine baitfish, with a small number of commercial operations currently in production. The objectives of this study were to determine both acute and gradual salinity tolerances as well as an upper lethal salinity tolerance for the species. Two separate acute acclimation experiments, natural seawater and sodium chloride, were carried out to determine if survival was influenced by the salinity source. F. seminolis were able to tolerate acute transfer to 0, 8, and 16 g/L using both salinity sources but only those in natural seawater were able to survive in 24 g/L. No survival was observed regardless of salinity source after acute transfer to 32 g/L. A gradual acclimation using natural seawater was also investigated to examine survival at various acclimation rates. A survival rate of 100% was achieved regardless of acclimation rate when salinity was changed from 0 to 32 g/L in 24, 48, 72, and 96 h treatment groups. An upper lethal salinity determination yielded an LC-50 of 60 g/L with a maximum salinity tolerance of 78 g/L. Results of these experiments provide information pertinent to the successful culture of this rarely studied species. Additionally, experimental outcomes will help to shape the marketing and distribution strategies for F. seminolis as a marine baitfish. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The Seminole killifish, Fundulus seminolis (Girard), is an endemic Florida killifish with a geographic range within peninsular Florida from the St. Johns and New River drainage basins to just south of Lake Okeechobee (Page and Burr, 1991). Relatively little is known regarding the life history of F. seminolis, with only one publication by DuRant et al. (1979) devoted entirely to the species. It has been referenced anecdotally or as a component in a larger study or survey in several publications (McLane, 1955; Phillips and Springer, 1960; Tabb and Manning, 1961; Gunter and Hall, 1963; Gunter and Hall, 1965; Foster, 1967; Griffith, 1974a; Nordlie, 2006). This species, as well as several other members of the Fundulus genus are commonly referred to as a
⁎ Corresponding author. School of Forest Resources and Conservation, Program of Fisheries and Aquatic Sciences, Institute of Food and Agricultural Sciences, University of Florida, 7922 NW 71st St., Gainesville, Florida 32653, USA. Tel.: +1 772 468 3922ext134; fax: +1 772 468 3973. E-mail addresses: mdimaggi@ufl.edu, [email protected] (M.A. DiMaggio). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.04.009
“bullminnow” or “mudminnow”. The Seminole killifish is one of the largest members of the genus, reaching total lengths of 20 cm (Hoyer and Canfield, 1994). Its popularity as a local freshwater baitfish for largemouth bass, Micropterus salmoides, and other piscivorous game fish has generated interest in this species as a potential candidate for aquaculture. With previous data placing the upper salinity tolerance of F. seminolis at 23.4 g/L (Griffith, 1974a), culture of this species for use as a saltwater baitfish warrants further investigation. If the species is able to acclimate to full strength seawater, it could be produced exclusively in freshwater ponds or recirculation systems only needing to be acclimated to saline water prior to marketing and distribution. Additionally, with coastal property values at a premium and limited access to seawater, baitfish producers would be able to utilize inland resources for the culture of a marine baitfish. Determination of salinity tolerance following gradual and acute transfer is necessary to evaluate the species’ physiological limitations which will influence culture and marketing practices. Similar studies have been conducted by Lotan (1971), Griffith (1974a), Stanley and Fleming (1977), Chervinski (1983), Nordlie (1987), Crego and Peterson (1997), Nordlie (2000) and Fuller (2008) on salinity tolerance of various members of the order cyprinodontiformes. For
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the most recent review of cyprinidontoid salinity tolerance consult Nordlie (2006). The previous studies examined multiple salinity ranges utilizing various salt sources (seawater and synthetic sea salts) and freshwater dilutions to achieve experimental salinities both hypo and hypersaline to natural seawater (32–35 g/L). Distinct salinity thresholds defined by survival and select hematological indices were then characterized for the species in question. Experimental salinity determinations such as these may be a more accurate representation of the organism's true salinity tolerance than maximum reported field salinities (Kefford et al., 2004). This can be seen in Phillips and Springers' (1960) record of F. seminolis inhabiting waters with a salinity of 13.5 g/L, the highest recorded salinity from published field observations. Griffith's (1974a) subsequent experimental salinity determination for this species placed the upper salinity tolerance much higher, with an experimental salinity range of 19.3–33.4 g/L. However, Griffith (1974a) reported high mortalities in maintaining this species in captivity and his sample size for salinity tolerance determinations was only four individuals. Although the lower lethal temperature has yet to be established for this species, the experimental water temperature of 15 °C might have exacerbated the physiological stress caused by the acclimation if this temperature impaired the functionality of essential enzymes. Taken together, these circumstances call into question the accuracy of the salinity range reported by Griffith (1974a). Investigations into the salinity tolerance of freshwater species have shared a predominant ecological motivation. Investigations by Bringolf et al. (2005) and Schofield et al. (2006) assessed salinity tolerances as barriers to invasion for the flathead catfish, Pylodictis olivaris, and goldfish, Carassius auratus, respectively. While the impetus for the current study is the evaluation of F. seminolis as an aquaculture candidate; investigations into its salinity tolerance should provide valuable information about the species' ability to handle osmoregulatory stressors and could further be extrapolated for ecological applications. Salinity tolerance is an important consideration in the culture of marine and freshwater organisms. It provides information about basic husbandry requirements necessary for the species to thrive in captivity as well as potential applications for the cultured organisms. Additionally, economic considerations associated with culture of marine or brackish water species make low saline or freshwater culture an attractive alternative. Research into low salinity aquaculture of marine species is common, but few studies have been conducted on acclimation of freshwater species to seawater. Experiments examining abrupt transfer of black sea bass, Centropristis striata, to low salinities have helped to identify a salinity threshold for the successful culture of this species (Young et al., 2006). Similarly, gradual acclimation experiments with Nile tilapia, Oreochromis niloticus, and blackchin tilapia, Sarotherodon melanotheron, (Lemarie et al., 2004) as well as larval salinity tolerance experiments with striped mullet, Mugil cephalus, thick-lipped grey mullet, Chelon labrosus (Hotos and Vlahos, 1998), and cobia, Rachycentron canadum (Faulk and Holt, 2006), have provided valuable evidence regarding the osmoregulatory ability of a species for use in conventional aquaculture conditions. The purpose of this study was to characterize the salinity tolerance of F. seminolis, a potential candidate for marine baitfish aquaculture. Abrupt and gradual salinity acclimations were evaluated as well as salinity sources (sodium chloride vs. seawater). A maximum lethal salinity resulting in 100% mortality was also investigated by means of gradual acclimation with a corresponding determination of the salinity concentration which resulted in 50% mortality (LC-50). This investigation represents the first comprehensive study focused on the salinity tolerance of F. seminolis. 2. Methods F. seminolis were collected by seine net from the eastern shore of Lake George, in Volusia County Florida and transported to the University of
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Florida Indian River Research and Education Center in Fort Pierce. Fish were assessed for pathogens and treated accordingly to ensure healthy research specimens for the subsequent salinity experiments. The acute salinity tolerance of F. seminolis to varying concentrations of sodium chloride (NaCl) and natural seawater (NSW) were investigated as well as survival following gradual NSW acclimation. A maximum lethal salinity investigation was also conducted. 2.1. Sodium chloride acute salinity tolerance Thirty five fish were transferred from a 6900 L recirculating system to 85 L glass aquaria with one fish per aquarium during the entire acclimation and experimental periods. Specimen's weight and total length (TL) were recorded prior to transfer. Length and weight ranges of 120–145 mm and 17.0–29.4 g were recorded with means of 131.2 ± 6.3 mm and 23.2 ± 3.6 g, respectively. Aquarium systems were maintained at b1 g/L salinity well water and recirculated through biofilter media during the 96 h acclimation period. Dissolved oxygen (DO), pH, temperature, salinity, total ammonia nitrogen (TAN), and nitrite were recorded daily during the acclimation and experimental periods with total alkalinity and total hardness recorded on days one and three of acclimation and daily during the course of the experiment. DO and temperature were measured using a YSI 550A meter (YSI Inc., Yellow Springs, Ohio). Salinity was determined using a handheld refractometer, pH was measured using a Hach sensION1 portable pH meter and total alkalinity and total hardness were determined using standardized titration techniques (Hach Co., Loveland, Colorado). TAN and nitrite were evaluated spectrophotometrically using a Hach DR 4800 spectrophotometer (Hach Co., Loveland, Colorado). Aquaria were held at ambient temperature with a range of 19.9–26.5 °C and a mean temperature of 22.7 °C during the experimental period. Temperature differences among aquaria never exceeded 2 °C. An ambient photoperiod of 11 L: 13 D was used during the experiment. Fish were fed once a day to satiation on days two and three of acclimation and food was withheld on days one and four of acclimation and during the entire 96 h experimental period. Following the 96 h freshwater acclimation, aquaria were made static and individual aquariums were randomly assigned to one of five treatments with seven replicates per treatment. Differences among treatment group's lengths and weights were not significant (F4,30 =0.68, p = 0.610; F4,30 = 0.20, p = 0.939, respectively). Treatment salinities examined were 0 (control), 8, 16, 24, and 32 g/L. Salinities were abruptly changed by removing the appropriate amount of fresh water and adding a predetermined volume of a concentrated brine solution made by dissolving 99.5% NaCl (Morton White Crystal Solar Salt, Morton International Inc., Chicago, IL) into well water. Control aquaria had a predetermined volume of fresh water removed and subsequently replaced to maintain similar treatment of control and experimental groups. Tanks were aerated to thoroughly mix the water, then salinities were remeasured to confirm the desired concentrations were attained. Individual biofilters which had been preconditioned to treatment salinities were placed in each tank to control nitrogenous wastes. Aquaria were examined for mortalities as follows: once every hour from 0–12 h, once every 6 h from 12–48 h, and once every 12 h from 48–96 h. A final weight was recorded upon discovery of a mortality or upon the termination of the 96 h exposure period. Mortality as used throughout this investigation was defined as loss of opercular movement and no response to physical stimulus. 2.2. Natural seawater acute salinity tolerance Methods for the NSW acute salinity toxicity trial adhere to the previous methods listed in Section 2.1. with exceptions as noted. Treatment salinities were abruptly changed by removing a predetermined volume of fresh water and adding a known volume of natural seawater collected from the Atlantic Ocean and filtered through a 1 micron cartridge filter.
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Control aquaria were treated as previously stated. Aquaria were held at ambient temperature with a range of 20.5–25.6 °C and a mean temperature of 23.4 °C during the experimental period. Temperature differences among aquaria never exceeded 2 °C. A ambient photoperiod of 11.5 L: 12.5 D was used during the experiment. Length and weight ranges of 122–146 mm and 16.6–31.2 g were recorded with means of 129.7±5.8 mm and 21.9±3.5 g, respectively. Differences among treatment group's lengths were not statistically significant (F4, 30 =1.12, p=0.368). Differences among treatment group's weights were statistically significant (p =0.010) but were not considered to be biologically significant for the purpose of this experiment with a mean treatment weight range of 18.7–23.8 g. Differences among treatment weights did not violate any assumptions of subsequent survival analysis. 2.3. Natural seawater gradual acclimation survival Sixty-four fish were transferred from a 6900 L recirculating system to 32, 85 L glass aquaria, divided in half by aquarium partitions (TDSU, Penn-Plax Inc., Happauge, NY USA) with one fish per aquarium subdivision during the entire acclimation and experimental periods. Fish weight and TL were recorded prior to transfer. Length and weight ranges of 124–152 mm and 21.7–38.3 g were recorded with means of 136.3 ± 6.3 mm and 27.5 ± 4.5 g, respectively. Aquarium systems were maintained at b1 g/L salinity well water and recirculated through biofilter media during the 96 h acclimation period. DO, pH, temperature, salinity, TAN, and nitrite were recorded daily during the acclimation and experimental periods with total alkalinity and total hardness recorded on days one and three of acclimation and at the initiation and cessation of experimental treatment periods. Methods for water quality analysis adhere to previously stated techniques (Section 2.1.) with the exception of salinity, which was measured using a YSI 30 salinity/conductivity meter (YSI Inc., Yellow Springs, OH USA). Aquaria were held at ambient temperature with a range of 17.5–27.2 °C and a mean temperature of 22.6 °C during the experimental period. Temperature differences among aquaria never exceeded 2 °C. An ambient photoperiod of 13 L: 11 D was used during the experiment. Fish were fed once a day to satiation on days two and three of acclimation and food was withheld on days one and four of acclimation and during the entire experimental period. Following the 96 h freshwater acclimation, aquaria were made static and individual tanks were randomly assigned to one of eight treatments with eight replicates per treatment. Differences among treatment group's lengths and weights were not significant (p = 0.784; F7,56 = 0.38, p = 0.908, respectively). Among the treatment groups, four underwent a gradual salinity change from 0 to 32 g/L over predetermined time periods with the remaining four treatments serving as corresponding controls. Acclimation times of 24, 48, 72, and 96 h were chosen with approximate salinity increases of 5.3, 2.7, 1.7, and 1.3 g/L, respectively, every 4 h. The final salinity of 32 g/L was achieved 4 h preceding the termination of the treatment group in question. Salinities were gradually changed every 4 h via addition of NSW equally dispersed between aquaria subdivisions until the desired salinity was achieved. Excess water during salinity changes was allowed to flow out of the aquarium's standpipe. A single air stone within each subdivision provided adequate mixing within and between subdivisions ensuring a homogeneous environment within each aquarium. Control aquaria had corresponding volumes of freshwater added in the same fashion as their saline counterparts. Aquaria were examined for mortalities as follows: once every hour from 0–12 h and once every 4 h from 12–96 h. A final weight was recorded upon discovery of a mortality or upon the termination of the acclimation exposure period. 2.4. Maximum lethal salinity determination Twenty four fish were transferred from a 6900 L recirculating system to 12, 85 L glass aquaria, divided in half by aquarium partitions (TDSU,
Penn-Plax Inc., Happauge, NY USA) with one fish per aquarium subdivision during the entire acclimation and experimental periods. Fish weight and TL were recorded prior to transfer. Length and weight ranges of 120–145 mm and 16.4–30.4 g were recorded with means of 129.0 ± 5.5 mm and 21.1 ± 3.4 g, respectively. Aquarium systems were maintained at 2 g/L salinity well water to aid in osmoregulation and inhibit nitrite toxicity. Water was recirculated through biofilter media during the 96 h acclimation period. DO, pH, temperature, TAN, and nitrite were recorded on day four of the acclimation period. DO, pH, and temperature were recorded daily and TAN and nitrite recorded once weekly during the experimental period. Salinity was measured twice weekly during salinity changes. Methods for water quality analysis adhere to Section 2.1. with the exception of salinity, which was measured using a YSI 30 salinity/conductivity meter (YSI Inc., Yellow Springs, OH USA). Aquaria were held at ambient temperature with a range of 19.0– 31.3 °C and a mean temperature of 26.3 °C during the experimental period. Temperature differences among aquaria never exceeded 2 °C. An ambient photoperiod of 11.25 L: 12.75 D was used during the experiment. Fish were fed to satiation three times a week during both the acclimation and experimental period to control nitrogenous wastes. Following the 96 h acclimation, aquaria were made static and individual tanks were assigned to one of two treatments with 12 replicates per treatment. Differences among treatment group's lengths and weights were not significant (F 1,22 = 0, p = 0.973; F 1,22 = 0.58, p = 0.454, respectively). Between the treatment groups, one underwent a gradual salinity change from 2 g/L until 100% mortality was observed for all replicates. The additional treatment served as a control and was maintained at 2 g/L salinity for the duration of the experiment. Salinities were increased by 6 g/L twice a week (every Monday and Thursday) with controls treated in a similar fashion. Salinity was increased to 30 g/L via the addition of NSW. Beyond this concentration, salinities were augmented by addition of synthetic sea salts (HW Marine Mix Professional, Hawaiian Marine Imports Inc. Houston, TX) to aquaria water to make a concentrated brine solution and then added back to the aquaria to achieve the desired concentration. A single air stone within each subdivision provided adequate mixing within and between subdivisions ensuring a homogeneous environment within each aquarium. Control aquaria had corresponding volumes of 2 g/L water added in the same fashion as their saline counterparts. Aquaria were examined for mortalities daily until 100% mortality was noted in the salinity treatment. 2.5. Statistical analysis Survival was estimated using a Kaplan-Meier product limit estimator (Kaplan and Meier, 1958). Log-rank tests were then performed to compare generated survivorship curves among treatments and within treatments between experiments. Length, weight, and water quality data were analyzed using a one-way ANOVA with a Tukey's HSD means separation test. Nonparametric data were analyzed with a KruskalWallis test. Kaplan-Meier analysis and subsequent log rank tests were performed in SPSS® version 12.0 (SPSS Inc. Chicago, IL USA). All other statistical analyses were performed in SAS® version 8.02 (SAS Institute Inc., Cary, NC USA) All numerical data are represented as the mean± SD
Table 1 Kaplan-Meier survival analysis for NaCl acute transfer. Treatment (g/L NaCl)
Mean survival (h ± SE)
Survival (%)
(Control) 0 8 16 24 32
⁎ ⁎ ⁎
100 100 100 0 0
6±1 2
⁎ Statistic cannot be calculated for comparison of treatments with 100% survival for both.
M.A. DiMaggio et al. / Aquaculture 293 (2009) 74–80 Table 2 Log-Rank analysis among treatments for NaCl acute transfer. Treatment
0 g/L
8 g/L 16 g/L 24 g/L 32 g/L
⁎ ⁎
8 g/L ⁎
p b 0.0001 p b 0.0001
p b 0.0001 p b 0.0001
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Table 4 Kaplan-Meier survival analysis for NSW acute transfer.
16 g/L
p b 0.0001 p b 0.0001
24 g/L
p b 0.0001
⁎ Statistic cannot be calculated for comparison of treatments with 100% survival for both.
unless otherwise stated. Statistical differences were considered significant when p ≤ 0.05. 3. Results 3.1. Sodium chloride acute salinity tolerance Survival was 100% at salinities of 0 (control), 8, and 16 g/L throughout the entire 96 h experimental period (Table 1). Acute transfer to higher salinities yielded 100% mortality with mean survival times of 6 h (95% Confidence Interval [CI], 5–7 h) at 24 g/L and 2 h (95% CI, 2–2 h) at 32 g/L (Table 1). Despite the complete mortality observed in these two treatments, survival times were significantly different (p ≤ 0.0001) from each other as well as from all other treatments examined (p b 0.0001) (Table 2). No biologically significant differences among treatments in the measured water quality parameters were observed throughout the course of this experiment (Table 3). 3.2. Natural seawater acute salinity tolerance Survival was 100% at salinities of 0 (control), 8, 16, and 24 g/L throughout the entire 96 h experimental period (Table 4). Acute transfer to 32 g/L yielded 100% mortality with a mean survival time of 13 h (95% CI, 11–16 h) (Table 4). This group was significantly different (p = 0.0002) from all other treatments (Table 5). No biologically significant differences among treatments in the measured water quality parameters were detected throughout the
Treatment (g/L NSW)
Mean survival (h ± SE)
Survival (%)
(Control) 0 8 16 24 32
⁎ ⁎ ⁎ ⁎
100 100 100 100 0
13 ± 1
⁎ Statistic cannot be calculated for comparison of treatments with 100% survival for both.
Table 5 Log-Rank analysis among treatments for NSW acute transfer. Treatment
0 g/L
8 g/L
16 g/L
8 g/L 16 g/L 24 g/L 32 g/L
⁎ ⁎ ⁎
⁎ ⁎ ⁎
⁎
p = 0.0002
p = 0.0002
p = 0.0002
24 g/L
p = 0.0002
⁎ Statistic cannot be calculated for comparison of treatments with 100% survival for both.
course of this experiment with the exception of total hardness. Due to experimental dilutions of seawater, total hardness varied by treatment salinity (Table 3). 3.3. Sodium chloride vs. natural seawater When survival was analyzed within treatment salinity between salt source, NSW and NaCl were significantly different (p b 0.0001) in the 24 and 32 g/L salinity treatments. 3.4. Natural seawater gradual acclimation survival For all treatment groups, survival was 100%. Therefore, no analysis of the data was required. With the exception of total hardness, no biologically significant differences among treatments in the measured water quality parameters were observed throughout the course of the experiment (Table 3).
Table 3 Acclimation and experimental water quality data collected from the NaCl acute salinity tolerance, NSW acute salinity tolerance, NSW gradual acclimation and maximum lethal salinity experiments. DO (mg/L)
pH
TAN (mg/L)
Nitrite (mg/L)
Alkalinity (mg/L CaCO3)
Hardness (mg/L CaCO3)
NaCl acute salinity tolerance - 96 h acclimation NaCl acute salinity tolerance - 96 h experimental NSW Acute Salinity Tolerance - 96 h acclimation NSW acute salinity tolerance - 96 h experimental
7.11 ± 0.42 (6.38–7.57) 7.73 ± 0.33 (7.14–8.35) 7.13 ± 0.41 (6.66–7.90) 7.87 ± 0.19 (7.50–8.24)
8.43 ± 0.06 (8.33–8.52) 8.43 ± 0.08 (8.28–8.58) 8.37 ± 0.11 (8.19–8.53) 8.36 ± 0.13 (8.06–8.63)
0.01 ± 0.01 (0.00–0.02) 0.03 ± 0.03 (0.00–0.09) 0.02 ± 0.02 (0.00–0.06) 0.03 ± 0.02 (0.00–0.10)
0.0025 ± 0.0008 (0.0006–0.0039) 0.0753 ± 0.0565 (0.0125–0.3102) 0.0011 ± 0.0017 (0.0000–0.0043) 0.0107 ± 0.0074 (0.0018–0.0356)
139.65 ± 6.98 (136.80–153.90) 135.80 ± 9.80 (119.70–153.90) 171.00 ± 18.73 (153.90–205.20) 144.00 ± 9.80 (119.70–171.00)
NSW gradual acclimation - 96 h acclimation NSW gradual acclimation - 96 h experimental
7.63 ± 0.29 (7.23–7.98) 7.86 ± 0.19 (7.56–8.35)
8.29 ± 0.11 (8.03–8.41) 8.31 ± 0.07 (8.12–8.46)
0.01 ± 0.01 (0.00–0.02) 0.03 ± 0.02 (0.00–0.07)
0.0025 ± 0.0010 (0.0011–0.0036) 0.0040 ± 0.0022 (0.0014–0.0200)
180
Maximum lethal salinity determination - acclimation and experimental
6.66 ± 0.91 (4.80–7.97)
8.36 ± 0.20 (7.80–8.86)
0.17 ± 0.19 (0.00–0.86)
0.2077 ± 0.2882 (0.0000–1.0731)
–
202.35 ± 19.99 (188.10–239.40) 193.90 ± 18.90 (171.00–256.50) 190.95 ± 33.19 (153.90–239.40) 0–182.60 ± 21.40 (153.90–222.30) 8–1557.10 ± 300.00 (1250.00–2570.00) 16–2860.00 ± 95.10 (2700.00–3090.00) 24–4282.10 ± 295.40 (3830.00–5580.00) 32–5808.60 ± 117.80 (5700.00–6060.00) 199.50 ± 8.83 (188.10–205.20) 0–205.20 32–5717.50 ± 116.02 (205.20–5840.00) –
170.00 ± 10.1 (160.00–180.00)
Bolded numbers indicate salinity concentration. Values are reported as the mean ± SD. Parameter ranges are listed below the mean in parentheses.
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Fig. 1. Observed survival (%) over a 46 day maximum lethal salinity experiment, culminating in 100% mortality for the treatment group which underwent gradual salinity increases of 6 g/L twice a week. The control group was maintained at 2 g/L throughout the entire experiment.
3.5. Maximum lethal salinity determination The maximum lethal salinity determination experiment lasted for 46 days until a lethal salinity of 78 g/L was reached resulting in 100% mortality for the salinity treatment group with an LC-50 of 60 g/L (Figs. 1 and 2). Survival in the control group was 91.67% at the end of the 46 day trial. No biologically significant differences among treatments in the measured water quality parameters were observed throughout the course of the acclimation or experimental periods (Table 3). 4. Discussion Survival analysis for NaCl and NSW acute salinity tolerance experiments revealed F. seminolis can tolerate direct transfer from freshwater to salinities up to 16 and 24 g/L, respectively. Daily water quality analysis during the 96 h experimental periods helped to eliminate potentially confounding variables in an effort to identify salinity as the driving force behind observed mortality. Upon completion of the 96 h experimental exposure in acute transfer experiments, surviving fish were left in their respective aquaria and monitored for 2 weeks. No mortalities were observed during this time period for any surviving treatments regardless of salinity source. This is evidence that results of 96 h survival experiments are useful indicators of a species' ability to survive long term in selected treatment conditions (DiMaggio, personal communication). Additionally, although 100% survival was noted at 16 g/L in both NaCl and NSW experiments, fish in the NSW treatment generally appeared healthier. F. seminolis at 16 g/L in NaCl exhibited multiple areas of hyperemia, overall pallor, decreased activity, and increased mucus production. Excessive mucus has been previously reported in Fundulus kansae exposed to low calcium “artificial seawater” (Potts and Fleming, 1970). Differences between survival due to salinity source were evident in 24 and 32 g/L treatments (Tables 1 and 4). Due to lack of physiological data it is only possible to speculate as to the underlying causes for the recorded differences. The role of calcium in osmoregulation and ionic transport has been well studied (Potts and Fleming, 1970; Carrier and Evans, 1976; Isaia and Masoni, 1976; Pic and Maetz, 1981; Hunn, 1985). Although calcium was not measured directly, total hardness levels (mg/L CaCO3) were recorded throughout both experiments. Signifi-
cant differences (p b 0.0001) in hardness were noted among NSW treatment salinities of 8, 16, 24, and 32 g/L. Additionally, hardness values from these treatment groups were also significantly different (p b 0.0001) from all NaCl treatment groups and the NSW control. It can be inferred from this data that calcium and other divalent ions were in a greater abundance in the NSW treatment groups than their NaCl counterparts. Low external calcium in an environment hyperosmotic to fish has been shown to increase ion efflux in Lagodon rhomboides (Carrier and Evans, 1976) and alter net fluxes of both ions and water in Anguilla anguilla (Isaia and Masoni, 1976). Potts and Fleming (1970) reported a 35% reduction in the gill permeability of F. kansae in NSW when compared with fresh water, thereby decreasing ion efflux and drinking rate. Conversely they also reported F. kansae to have an increased drinking rate and water exchange rate when transferred to low calcium synthetic seawater. Although not directly tested, findings from the aforementioned studies support the hypothesis that the mortality observed in the NaCl experiment relative to the NSW experiment was a result of loss of hydro-mineral regulation due to lower environmental ion concentrations, particularly calcium. However, environmental deficiencies of other divalent ions such as magnesium, should not be dismissed because these ions also play a functional role in salinity acclimation (Isaia and Masoni, 1976). Results of gradual NSW acclimation were encouraging for this marine baitfish candidate. F. seminolis was able to easily acclimate to 32 g/L in all four time intervals with no mortalities in any treatment. Modest lethargy in most replicates of the 24 h treatment group was the only outward sign of physiological stress observed among any of the treatments upon completion of the acclimation process. The maximum lethal salinity of 78 g/L recorded in this study was more than twice the reported maximum salinity of 33.4 g/L reported by Griffith (1974a). The first mortality recorded from the salinity treatment group at day 25 (Fig. 1) and 48 g/L (Fig. 2) is well above natural ocean salinities in the Atlantic. These results provide further evidence of the ability of F. seminolis to successfully acclimate to salinities characteristic of NSW as well as hypersaline environments given appropriate acclimation conditions. It should be noted that animals which underwent salinity change actively fed throughout the experimental period and did not exhibit any gross signs of stress when compared with their controls. Salinity tolerance of the predominantly euryhaline Fundulus genus has been extensively investigated yet almost no experimental
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Fig. 2. Observed survival (%) at treatment salinities during the entire experimental period. Gradual salinity increases of 6 g/L were carried out twice a week until 100% mortality was observed.
evidence exists regarding F. seminolis until the present study. Griffith's (1974a) seminal investigation on salinity tolerance of the genus is the only study that has evaluated this emerging aquaculture candidate, but due to confounding experimental factors these data are potentially unreliable. An additional study by Griffith (1974b) attempted to elucidate the role of pituitary control in the freshwater acclimation of the genus, but only one F. seminolis was used, again limiting the usefulness of the data. Salinity tolerance results from this experiment confirm F. seminolis can acclimate to the upper end of Griffith's (1974a) reported range and beyond. Survival by F. seminolis after acute transfer to 24 g/L indicates superior salinity tolerance when compared to Fundulus nottii's acute tolerance of 17 g/L (Crego and Peterson, 1997). With a maximum lethal salinity of 78 g/L (Fig. 2) determined in this experiment, it is unlikely F. seminolis would tolerate salinities of 95+ g/L commonly reported for F. grandis (Nordlie, 2000), F. heteroclitus (Griffith, 1974a), F. confluentus (Griffith, 1974a; Nordlie, 2000), F. similis (Nordlie, 2000), F. parvipinnis (Feldmeth and Waggoner, 1972), Aphanius dispar (Lotan, 1971), and Adenia xenica (Nordlie, 1987). F. seminolis is widely considered to be a freshwater killifish, with upper field salinities reported at 2.4 (Gunter and Hall, 1963), 7.3 (Gunter and Hall, 1965), and 13.5 g/L (Phillips and Springer, 1960). Results from our experiments clearly indicate F. seminolis is capable of tolerating brackish, full strength seawater, and hypersaline environments for extended periods of time. Experimental results substantiate the potential of F. seminolis as a candidate for marine baitfish aquaculture following seawater acclimation. Ability to tolerate acute transfer from 0–24 g/L and gradual acclimation to 32 g/L NSW over a broad time period allows for flexibility in the development of a salinity acclimation protocol. Additionally, a mean survival time of 13 h after acute transfer to full strength seawater allows for marketing and use of freshwater acclimated fish in the marine environment if so desired. NSW acclimated fish provide an additional option for bait retailers utilizing saline holding facilities as well as anglers who wish to store their bait in saline livewells. Demands of bait retailers and sportsman will ultimately dictate the acclimation regime for this new marine baitfish species. Acknowledgements The authors wish to thank Greg Harris and Edsel E. Redden for their assistance in procurement of the research specimens as well as Dr. Andrew Rhyne, Scott Grabe, Shawn DeSantis, and John Marcellus for
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