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The use of alizarin red S and alizarin complexone for immersion marking Japanese flounder Paralichthys olivaceus (T.)
Fisheries Research 98 (2009) 67–74
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The use of alizarin red S and alizarin complexone for immersion marking Japanese flounder Paralichthys olivaceus (T.) Q. Liu a , X.M. Zhang a,∗ , P.D. Zhang a , S.A. Nwafili a,b a b
The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, 266003, PR China Department of Fisheries, Delta State Ministry of Agriculture and Natural Resources, P.M.B. 5023, Asaba, Delta State, Nigeria
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Article history: Received 11 November 2008 Received in revised form 28 March 2009 Accepted 30 March 2009 Keywords: Paralichthys olivaceus Alizarin red S (ARS) Alizarin complexone (AC) Otoliths Scales Fin rays
a b s t r a c t Alizarin red S from 200 to 400 mg/l and alizarin complexone from 50 to 300 mg/l were used to mark Japanese flounder, Paralichthys olivaceus. Immersion for 24 h produced detectable marks in sagittae, ctenoid, cycloid scales, fin rays (dorsal, anal, pectoral, caudal and ventral fin rays) and parts of dissected asteriscus after 60 days in culture. All treatment concentrations had good marks on otoliths. Violet marks were visible under normal light after marking with 200–400 mg/l ARS and 300 mg/l AC. Scales and fin rays showed acceptable fluorescent marks at higher concentrations (400 mg/l ARS, 300 mg/l AC and 250–400 mg/l ARS, 300 mg/l AC, respectively). The best results were obtained after marking with ARS at 400 mg/l and AC at 300 mg/l. There was no significant difference on survival and growth of marked fish relative to controls throughout experiment (P > 0.05). © 2009 Elsevier B.V. All rights reserved.
1. Introduction Japanese flounder Paralichthys olivaceus is one of the most important commercial species along the coastal areas of China, southwest of the Korean peninsula and Japan (Zhu et al., 1963; Cheng and Zheng, 1987). Japanese flounder has declined significantly in recent years due to overexploitation. Since 1996, a hatchery-produced juvenile release program has been implemented in Shandong province. In 2007 alone, approximately, 9 million P. olivaceus juveniles were released into Shandong inshore waters. The success of this mass-releasing program of stock enhancement remains to be evaluated. To assess the effectiveness of the stock enhancement strategies for fisheries management (Brown et al., 2002; Taylor et al., 2005; Baer and Rösch, 2008), marking and recapture programs need to be carried out. Various methods have been developed to mark fish at different stages of the life cycle. However, fish in their early stage are too small to be marked with external marks (e.g. T-bar, plate disc and fin clipping) or internal tags (e.g. passive integrated transponder, PIT). Additionally, individual handling could involve large effort. Genetic tags are not suitable for recapture process due to high operating costs. Considering metamorphosis during the early stage of development and
∗ Corresponding author at: College of Fisheries, Ocean University of China, 5# Yushan Road, Qingdao, 266003, PR China. Tel.: +86 0532 82032076; fax: +86 0532 82032076. E-mail address: [email protected] (X.M. Zhang). 0165-7836/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fishres.2009.03.014
depressiform body structure of P. olivaceus, using coded wire tag seems difficult. Furthermore, thermal marking has been widely used for marking hatchery-produced salmons (e.g. Hagen et al., 1995; Volk et al., 1999) due to long period of egg incubation. However, P. olivaceus has relatively short incubation period when it could be thermally marked. Therefore, as far as authors know, chemical marks as internal–external marks are the most suitable tools for evaluation of large-scale stocking of small juveniles in contrast to other tagging methods (Brown et al., 2002; Simon and Dörner, 2005; Baer and Rösch, 2008). This method must be suitable for application to large numbers of small fish, handling stress should be kept at a minimum, markers should be retained for several months, marking process and recovery should be easy to carry out (Skov et al., 2001). Fluorochrome labeling dyes can produce detectable marks in otoliths, scales and other bony structures based on differential staining of chemical dyes. Various techniques for introducing marker have been investigated both in teleosts and elasmobranchs (Gelsleichter et al., 1997). The choice of technique depends on life history stage, environment (marine or fresh water) and experiment condition (Lagardère et al., 2000), for example, injection (Monaghan, 1993), dietary intake (Honeyfield et al., 2006) and immersion. Chemical marks mainly include: oxytetracycline (OTC) hydrochloride, calcein, alizarin red S (ARS) and alizarin complexone (AC). In recent years, alizarin red S (ARS) and alizarin complexone (AC) have been substituted for the other chemical dyes to produce clearly readable marks, causing less negative
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effect on survival and without availability limitation such as OTC in increasing salinity (Taylor et al., 2005) (e.g. Tsukamoto, 1988; Tsukamoto et al., 1989; Walt and Faragher, 2003; Baer and Rösch, 2008). However, no controlled laboratory experiment on the mortality and mark retention induced by AC and ARS has been conducted during relatively long periods on P. olivaceus, although Yamashita et al. (1994) used fixed 80 mg/l AC for 24 h in field experiment. There was little detail on evaluation of AC and without reference to ARS on this species. Therefore, the aims of this study were to evaluate growth, mortality and mark quality in otoliths, scales and fin rays of P. olivaceus marked with AC and ARS. 2. Methods 2.1. Experimental fish Juveniles (20–30 mm total length) were transported from a commercial hatchery company located at Wendeng City and kept in a 2 m3 recirculatory fiberglass tank for 27 days (1.4 individuals/l). During this acclimation period, fish were fed twice daily with commercial pellets (Shengsuo, Shandong, China) to satiation. Water quality was constantly monitored throughout the holding period and maintained (temperature, 20 ± 0.5 ◦ C; salinity, 31.0 ± 1.0; dissolved oxygen (DO), 5.26–5.32 mg/l; pH, 7.6–8.0; photoperiod, 14 L/10 D). Water was exchanged at the daily rate of 15% tank volume. 2.2. Immersion marking ARS and AC were dissolved in distilled water at the concentrations of 4000 mg/l ARS and 1000 mg/l AC, respectively as stock solution with salinity adjusted to 31. Then treatment concentrations of 0, 200, 250, 300, 400 mg/l ARS were prepared and water aerated strongly in order to raise pH. The fish were starved for 24 h before immersion. About 70 sixty-day-old P. olivaceus was randomly allocated to each treatment at a density of 3.5 individuals/l in ARS solution (20 L volume) for 24 h. Fish immersed in seawater without ARS served as control group. They were kept covered in dark environment to avoid stress throughout the immersion period. Following completion of the immersion period, juveniles were retrieved from the dye solutions, rinsed and then transferred to separate containers with fresh seawater for 4 h to completely wash away any remnant of dye. Lastly, fish were transferred into five aquaria for 72 h to determine the degree of acute mortality caused by ARS. PH was checked regularly during the immersion period in order to maintain a tolerable condition (7.23–7.86). The same procedure was followed in the application of AC. Fish were immersed in AC of concentrations 0, 50, 100, 150, 200 and 300 mg/l for 24 h. Fish treated in seawater without AC served as control group. For each solution, about 40 P. olivaceus at a density of 4 individuals/l was immersed in AC (10 L volume). Fish acute mortality due to AC was determined 72 h post-immersion. 2.3. Growth experiment To examine the effects of ARS and AC on survival and growing condition following immersion, 60 and 33 juveniles, respectively, were randomly selected per treatment concentration. For ARS marking, each treatment consisted of three replicates of 20 individuals (0.22 individuals/l) in a recirculatory system comprising 184 L storage aquarium and 8 titanium alloy columniform tanks (D:H = 65 cm × 64 cm). The tanks were continuously aerated and constant water quality parameters maintained (DO, 5.26–5.32 mg/l; salinity, 31.0 ± 1.0; photoperiod, 14 L/10 D; ammonia-N, 0.10–0.20 mg/l; pH, 7.6–8.0; temperature, 20 ± 0.5 ◦ C).
Water was exchanged at the daily rate of 15% tank volume and recirculated with composite sand filter, foam filter, biochemical filter balls and ultraviolet sterilization. Fish treated with AC were held in another recirculatory system, which consisted of 18 glass aquaria (L × W × H = 35 cm × 25 cm × 33 cm) equipped with filter bed and biochemical filter balls. Water volume was about 180 L. 33 P. olivaceus juveniles were randomly selected from each treatment concentration. This was then replicated three times consisting of 11 individuals (0.34 individuals/l) per sub-treatment and kept in glass aquarium. Water quality was maintained daily as described for ARS treatments. Water was exchanged at the daily rate of 30% tank volume. At the beginning of the growth experiments, all fish were starved for 24 h before total length and wet mass were measured to the nearest 0.1 mm and 0.01 g, respectively. Fish were measured from different batches of AC and ARS on 20th, 40th and 60th day to assess the extended effect of chemical marks on growth. 2.4. Sampling and mark analyses The sagittae and some scales (ctenoid scale from backside, cycloid scale from abdomen) of all fish were removed and 2 individuals per sub-treatment (6 individuals per concentration) were taken randomly for fin rays (dorsal, anal, pectoral, caudal and ventral fin ray) dissection at the base of the pterygiophores after 60 days post-marking. Parts of asteriscus were also dissected. They were all freed from adherent tissues and rinsed with water. All samples were checked directly without resin and polishing (e.g. Champigneulle and Cachera, 2003). To prevent fading of fluorescent marks, they were stored in sample envelopes avoiding high temperature and direct sunshine (Bashey, 2004). Marks were observed with an epifluorescence microscope (OLYMPUS BX51) equipped with a 10× objective lens and fitted with an Olympus DP70 high resolution digital camera (Table 1) and Stereoscopic Zoom Microscope (Nikon SMZ800) equipped with a 1× objective lens. Mark quality was assessed using a scale of 0–5 (0, no mark visible under fluorescence microscope; 1, poor mark under fluorescence microscope; 2, mark easily visible under fluorescence microscope; 3, mark shining brightly under fluorescence microscope; 4, mark visible in transmitted light; 5, mark distinct in transmitted light). Mark was identified by analyzing both sagittae, dissected parts of asteriscus, at least 20 cycloid and ctenoid scales of all fish and more than 4 pieces of fin rays per position of subsample fish. In general, mark quality on otoliths, scales and fin rays per specimen was analyzed separately by two researchers and where the presence of a mark was doubtful, it was determined by a third researcher. Mark quality ≥2 was judged an acceptable good mark as it could be readily detected in the structure (Taylor et al., 2005). Marks with high scores require less time for analyses and can be easily applied during mass-marking programs. 2.5. Data analyses Analysis of variance (ANOVA) with Turkey’s honestly significant difference (HSD) test (SPSS13.0) was used to assess the significance Table 1 Filter wavelengths for visualizing ARS and AC marks. Light source
WB WG UV
Wavelength (nm) Excitation filter
Barrier filter
490 545 365
515 590 420
Both of AC and ARS marks were detected using same filters.
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Fig. 1. Mean total length and wet mass of ARS (A and C) and AC treatments (B and D). Data were collected from 0th, 20th, 40th and 60th day (vertical bars show standard error, one-way ANOVA, P > 0.05).
of differences in survival, total length and wet mass exposed to different chemical concentrations of AC and ARS, respectively. A 0.05 significant level was used in the analysis. 3. Results 3.1. Growth and survival There was no mortality except one fish from 300 mg/l ARS and one from 300 mg/l AC during the 24 h immersion period. Post 72 h immersion mortality was 0% in all treatments. After 60 days post-marking, no mortality was found in AC growth experiment while 6.33% mortality was recorded in ARS treatment (19 of 300 Japanese flounders died). One-way ANOVA test showed no significant difference in survival between the control and ARS marked groups (d.f. = 4, P > 0.05). There was no evidence to suggest that fluorochrome label caused any mortality. The mortalities we recorded must have been caused by fish jumping out from holding tanks and bacterial disease (Yellow Sea Fisheries Research Institute Fish Disease Laboratory, Personal Communication). No significant difference was found in total length and wet mass of juvenile P. olivaceus both in ARS and AC treatment during the 60-day growth experiment (d.f. = 4, error d.f. = 10, P > 0.05 for 2 ARS treatments; d.f. = 5, error d.f. = 12, P > 0.05 for 2 AC treatments) (Fig. 1). 3.2. Mark quality There was no evidence of any fluorescent mark in control groups in both ARS and AC treatments. Marks were observed under fluorescence microscope with all the filters (WB, WG and UV). Using
green laser as emission wavelength, the intensity of red mark was stronger than violet mark produced by UV laser wavelength. Redorange mark produced with blue laser was the most obvious mark (Fig. 2). By inspecting otolith sections under fluorescence microscope, all concentrations of ARS and AC treatments had 100% marks in sagittal otoliths (score ≥2) (Fig. 6). Optimal marking concentrations were 200–400 mg/l ARS and 300 mg/l AC for 24 h when violet marks were clearly visible by transmitted light to naked eye (score ≥4) (Fig. 6). Marks were also detected in dissected parts of asteriscus (Fig. 3). In the present study, ctenoid and cycloid scales showed ring marks both of ARS and AC treatments with blue and green laser (Fig. 4). Fluorescent marks were detected at the concentrations of 250–400 mg/l ARS and 300 mg/l AC. Dosages of 400 mg/l ARS and 300 mg/l AC showed good marks on scales (score = 2) (Fig. 7). Marks in fin rays (Fig. 5) (dorsal, anal, pectoral, caudal and ventral fin ray) were more obvious at 250–400 mg/l and 300 mg/l for ARS and AC (score ≥2), respectively, despite high autofluorescence of fin rays (Fig. 8). The sagittal otoliths (Fig. 6) provided the best visible marks in comparison with fin rays and scales under different ARS and AC treatments. However, fluorochrome label on scales (Fig. 7) and fin rays (Fig. 8) showed acceptable marks (score ≥2) with relatively high concentrations, which were clearly visible under fluorescence microscope. Fin rays and scales were obtained non-lethally and the process under field condition was less time-consuming. In general, the optimum concentrations were 400 mg/l ARS and 300 mg/l AC, respectively since otolith marks were obvious to the naked eye. Further, all scales and fin rays had good marks without killing fish. The results verified marks would be retained and detected at least 60 days without the need for sacrificing and preparing otolith samples.
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Fig. 2. Photographs of a frontal section of sagittae after 60 days culture. Fish were treated with AC at a concentration of 300 mg/l (A–C). Green light (A); blue light (B); UV light (C). A pair of sagittates showed visible violet marks by normal transmitted light (D). Mark was produced with 300 mg/l ARS. A section of unmarked sagittae was shown by green light (E) and blue light (F). The sagittates were neither ground nor polished. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
4. Discussion 4.1. Marking quality and application Marking of fish by immersion involves a compromise between cost, concentration, immersion period, salinity, mortality, growing condition and retention time to produce the best mark (Taylor et al., 2005). Using both ARS and AC, we obtained 100% marking success. Despite variations in the quality of marks, fluorescent marks were detected in all treatments and in all the body structures analyzed. This study has shown that juveniles can be successfully marked and sampled non-lethally. Our results further agree with several studies showing that ARS and AC are effective for immersion marking, which can be widely used in biological research and evaluation of stock enhancement programs (e.g., Sánchez-Lamadrid, 2001; Skov et al., 2001; Baer and Rösch, 2008). In our experiments, there was little mortality during the immersion period (one from ARS treatments and one from AC treatments) and no mortality 72 h post-marking. The growth experiments demonstrated that both dyes with 200–400 mg/l and 50–300 mg/l of ARS and AC, respectively following long immersion time does not
affect growth and survival during 60 days of laboratory experiment. In other studies similar results were obtained (e.g. Tsukamoto, 1988; Lagardère et al., 2000; Bashey, 2004). Nevertheless, Beckman and Schulz (1996) observed that immersion in 400 mg/l ARS (equivalent to our 400 mg/l ARS concentration) or higher concentrations resulted in excessive mortalities and reduced marking success. This discrepancy may be attributed to the life stage of fish more than the high concentration of dyes and the immersion period. The otolith mark retention in the present investigation was 100% after 60 days in all treatments. However, longer retention time of fluorescent marks had been demonstrated (Tsukamoto et al., 1989; Taylor et al., 2005; Walt and Faragher, 2003). Thus, it appears that otolith mark retention will not be a problem to mark P. olivaceus with ARS and AC. The different retention time may be caused by individual metabolic difference and polishing operation of the otolith. Bashey (2004) suggested that it might be caused by mark detection method, taxa related differences or environmental factors (i.e. high water temperature and direct sunshine). In the present investigation, the same immersion method, equal mark effect and lower price of ARS in comparison with AC increase feasibility of its
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Fig. 3. Photographs of an asteriscus after 60 days culture. Fish were treated for 24 h in 400 mg/l ARS solution. Green light (A); blue light (B); UV light (D). Mark was obvious by normal transmitted light (C). An unmarked sagittae was shown by green light (E) and blue light (F). The asteriscus was neither ground nor polished. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
use and appears to be a promising technique for large-scale marking program in assessing stock enhancement. 4.2. Visible otolith mark In our study, visible mark under normal light was clearly observed at concentrations of 200–400 mg/l ARS and 300 mg/l AC. This must have been due to, firstly, the fairly stable pH of dye solutions during alizarine marking in saline water because pH was maintained within a narrow range by natural buffer system. The change in pH may help enhance the transport of dyes into fish. Secondly, the best life stage for marking with chemical dyes appears to be during the period of rapid growth development. Eckmann (2003) reported that chemical dyes formed complexes with calcium and these were deposited in the bone as the fish grows. In order to assess whether life stage was an essential factor, another experiment was conducted after fish were kept for >6 months
in the laboratory. About sixty 240-day-old juveniles P. olivaceus (Lf = 166.77 ± 26.65 mm, mean ± S.D.) were immersed for 24 h at a concentration of 400 mg/l ARS. Following immersion, all fish survived. Both sagittae were immediately removed from samples. Mark quality was identified as described above. Based on the same concentration of ARS and immersion time, violet marks were clearly poorer (score = 4) than that of prior test (score = 5). Visible mark was observed on all samples. However, retention time of visible mark on otolith seemed not as long as in the first experiment due to low luminary. Thus, it appears safe to conclude that the most appropriate life stage to mark P. olivaceus is at sizes of 50–60 mm, a period of rapid growth during which bright fluorescent marks are formed. Similar visible otolith marks were obtained when AC solutions of 150–350 mg/l for 10–12 h were used to mark 9–14-day-old larval Schizothorax davidi (He et al., 2008). Lastly, this study suggests that high dosage and 24 h immersion times are essential to form visible otolith mark. However, without the fluorescence microscope, marks
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Fig. 4. Scales (ctenoid scale from backside, cycloid scale from abdomen) subject to ARS 400 mg/l solution for 24 h (A–F). Photographs of a marked ctenoid scale (A–C) and a cycloid scale (D–F) after 60 days treatment. An unmarked ctenoid scale was shown as control group (G and H). Green light (A, D and G); blue light (B, E and H); normal transmitted light (C and F). The fluorescent mark was not produced under normal light.
in fin rays and scales are not visible to the unaided eye. Thus, otoliths could provide the best mark quality in mass-marking program. 4.3. Mark on fin rays and scales The major disadvantage of the otolith mark is that fish must be sacrificed to collect the structures (Bart et al., 2001). In this study, fin rays and scales with high concentration of dyes provided good marks (score ≥2), which allows non-lethal detection of marks (Brown et al., 2002; Leips et al., 2001; Frenkel et al., 2002; Honeyfield et al., 2006); removes the effort in preparing otolith (Barker and Mckaye, 2004) and marks were retained for a relatively long time. Much longer retention time in other fluorescent mark-
ers have already been reported (Leips et al., 2001; McFarlane and Beamish, 1995). Using fluorescent marks on hard tissues, large samples can be analyzed in a comparatively short time and it is more applicable in large-scale releases of hatchery-reared fish. Bashey (2004) found that ARS marking could produce good fluorescent mark in guppies Poecilia reticulate scales. However, as far as we know, no reference about fin or scale marks with AC existed before now. 4.4. Limitation of fluorescent mark Chemical batch marking provided convenient methods for largescale marking of fish (Sánchez-Lamadrid, 2001). However, general
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Fig. 5. Details of a section of anal fin ray after 60 days treatment (A–C). Fish treated in 300 mg/l AC for 24 h. An unmarked anal fin ray was shown as control group (D and E). The fin rays were dissected from tissue without grinding or polishing. Normal transmitted light (A); green light (B and D); blue light (C and E). The fluorescent mark was not produced under normal light.
Fig. 6. Mark quality was detected in sagittae by immersion batches using ARS (A) and AC (B). No marks were detected in control groups both in ARS and AC treatments (standard error = 0).
Fig. 7. Mark quality was detected in ctenoid and cycloid scales by batches of ARS (A) and AC (B) treatment. No marks were detected in control groups between ARS and AC treatments (vertical bars show standard error).
Fig. 8. Variations in mark quality subjected to ARS treatments (A) and AC treatments (B). No marks were detected in control groups both in ARS and AC treatments (vertical bars show standard error).
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limitations to use of fluorescent dyes include limited tag code that could not differentiate among individual samples and the difficulty in distinguishing stocks from different hatcheries even at the cost of double chemical marking. Moreover, this may not be true to verify spawning locality by recapturing marked fish (Iglesias and Rodriguez-Ojea, 1997). Fluorescent marks could only show the recapture information rather than the change in fish behavior, physiology and ecology in the wild. Hence, suitable marking dyes in combination with other tagging methods appear to be more suitable. Besides evaluating the mortality and growing condition of marked fish, it is essential to quantify fluorescent dyes residues in tissues for evaluation of tag method. Brown et al. (2002) reported that OTC was depleted in adult yellow perch Perca flavescens (M.) muscle tissue and reached acceptable tolerance level by about 2 h post-immersion. However, there is no reference on detecting ARS and AC residues and more efforts should be made in this direction in future research. In conclusion, the results suggest marking juvenile P. olivaceus by immersion in 400 mg/l ARS and 300 mg/l AC provide two methods for mass-marking operations under field conditions. Further, marks in fin scales or rays may allow non-lethal detection to get mark information. Use of ARS marking is cheaper and recommended. Both chemical dyes as powerful tool allow fishery managers to evaluate the effectiveness of stock enhancement. Acknowledgements We wish to express thanks to Shuhan Lei, Yuan Huang, Liming Jiao, Zhengyong Wu, Yue Wang and Hongtan Zhang for their assistance during laboratory work, especially for skillful dissection and preparation samples. Funding was provided by the National Key Technology R & D Program of China during the 11th FiveYear Plan Period (No. 2006BAD09A15) and the Key Laboratory of Mariculture of Ministry of Education, Ocean University of China. References Baer, J., Rösch, R., 2008. Mass-marking of brown trout (Salmo trutta L.) larvae by alizarin: method and evaluation of stocking. J. Appl. Ichthyol. 24, 44–49. Barker, J.M., Mckaye, K.R., 2004. Immersion marking of juvenile midas cichlids with oxytetracycline. Trans. Am. Fish Soc. 24, 262–269. Bart, A.N., Kindschi, G.A., Ahmed, H., Clark, J., Young, J., Zohar, Y., 2001. Enhanced transport of calcein into rainbow trout, Oncorhynchus mykiss, larvae using cavitation level ultrasound. Aquaculture 196, 189–197. Bashey, F., 2004. A comparison of the suitability of alizarin red S and calcein for inducing a nonlethally detectable mark in juvenile guppies. Trans. Am. Fish Soc. 133, 1516–1523. Beckman, D.W., Schulz, R.G., 1996. A simple method for marking fish otoliths with alizarin compounds. Trans. Am. Fish Soc. 125, 146–149.
Brown, M.L., Powell, J.L., Lucchesi, D.O., 2002. In-transit oxytetracycline marking, nonlethal mark detection, and tissue residue depletion in yellow perch. N. Am. J. Fish Manag. 22, 236–242. Champigneulle, A., Cachera, S., 2003. Evaluation of large-scale stocking of early stages of brown trout, Salmo trutta, to angler catches in the French-Swiss part of the River Doubs. Fish Manag. Ecol. 10, 79–85. Cheng, Q.T., Zheng, B.S., 1987. Systematic Synopsis of Chinese Fishes. Science Publishing (in Chinese) p. 492. Eckmann, R., 2003. Alizarin marking of whitefish, Coregonus lavaretus otoliths during egg incubation. Fish Manag. Ecol. 10, 233–239. Frenkel, V., Kindschi, G., Zohar, Y., 2002. Noninvasive, mass marking of fish by immersion in calcein: evaluation of fish size and ultrasound exposure on mark endurance. Aquaculture 214, 169–183. Gelsleichter, J., Cortés, E., Maniré, C.A., Hueter, R.E., Musick, J.A., 1997. Use of calcein as a fluorescent marker for elasmobranch vertebral cartilage. Trans. Am. Fish Soc. 126, 862–865. Hagen, P., Munk, K., Alen, B.V., White, B., 1995. Thermal mark technology for inseason fisheries management: a case study. Alaska Fish Res. Bull. 2, 143–155. He, Z.L., Fu, Z.D., Yan, T.M., Song, Z.L., 2008. Otolith marking of larval Schizothorax davidi with fluorescent substances. Sichuan J. Zool. 3, 331–334 (in Chinese). Honeyfield, D.C., Ostrowski, C.S., Fletcher, J.W., Mohler, J.W., 2006. Dietary calcein marking of brook trout, Atlantic salmon, yellow perch, and coho salmon scales. N. Am. J. Fish Manag. 26, 431–437. Iglesias, J., Rodriguez-Ojea, G., 1997. The use of alizarin complexone for immersion marking of the otoliths of embryos and larvae of the turbot Scophthalmus maximus (L.): dosage and treatment time. Fish Manag. Ecol. 4, 405–417. Lagardère, F., Thibaudeau, K., Bégout Anras, M.L., 2000. Feasibility of otolith markings in large juvenile turbot, Scophthalmus maximus, using immersion in alizarin-red S solutions. ICES J. Mar. Sci. 57, 1175–1181. Leips, J., Baril, C.T., Rodd, F.H., Reznick, D.N., Bashey, F., Visser, G.J., Travis, J., 2001. The suitability of calcein to mark poeciliid fish and a new method of detection. Trans. Am. Fish Soc. 130, 501–507. McFarlane, G.A., Beamish, F.W.H., 1995. Production. In: Secor, D.H. (Ed.), Recent Developments in Fish Otolith Research. University of South Carolina Publications, pp. 319–329. Monaghan Jr., J.P., 1993. Comparison of calcein and tetracycline as chemical markers in summer flounder. Trans. Am. Fish Soc. 122, 298–301. Sánchez-Lamadrid, A., 2001. The use of alizarin complexone for immersion marking of otoliths of larvae of gilthead sea bream Sparus aurata L. Fish Manag. Ecol. 8, 279–281. Skov, C., Grønkjær, P., Nielsen, C., 2001. Marking pike fry otoliths with alizarin complexone and strontium: an evaluation of methods. J. Fish Biol. 59, 745–750. Simon, J., Dörner, H., 2005. Marking the European eel with oxytetracycline, alizarin red and coded wire tags: an evaluation of methods. J. Fish Biol. 67, 1486–1491. Taylor, M.D., Fielder, D.S., Suthers, I.M., 2005. Batch marking of otoliths and fin spines to assess the stock enhancement of Argyrosomus japonicus. J. Fish Biol. 66, 1149–1162. Tsukamoto, K., 1988. Otolith tagging of ayu embryo with fluorescent substances. Nippon Suisan Gakkaishi 54, 1289–1295. Tsukamoto, K., Kuwada, H., Hirokawa, J., Oya, M., Sekiya, S., Fujimoto, H., Imazumi, K., 1989. Size dependent mortality of red sea bream, Pagrus major, juveniles released with fluorescent otolith-tags in New Bay, Japan. J. Fish Biol. 35 (Suppl. A), 59–69. Volk, E.C., Schroder, S.L., Grimm, J.J., 1999. Otolith thermal marking. Fish Res. 43, 205–219. Walt, B.V.D., Faragher, R.A., 2003. Otolith marking of rainbow trout fry by immersion in low concentrations of alizarin complexone. N. Am. J. Fish Manag. 23, 141–148. Yamashita, Y., Nagahora, S., Yamada, H., Kitagawa, D., 1994. Effects of release size on survival and growth of Japanese flounder Paralichthys olivaceus in coastal waters off Iwate Prefecture Northeastern Japan. Mar. Ecol-Prog. Ser. 105, 269–276. Zhu, Y.D., Zhang, C.L., Cheng, Q.T., 1963. The Fauna of East China Sea Fishes. Science Publishing (in Chinese) pp. 511–512.
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The use of alizarin red S and alizarin complexone for immersion marking Japanese flounder Paralichthys olivaceus (T.) Q. Liu a , X.M. Zhang a,∗ , P.D. Zhang a , S.A. Nwafili a,b a b
The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, 266003, PR China Department of Fisheries, Delta State Ministry of Agriculture and Natural Resources, P.M.B. 5023, Asaba, Delta State, Nigeria
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Article history: Received 11 November 2008 Received in revised form 28 March 2009 Accepted 30 March 2009 Keywords: Paralichthys olivaceus Alizarin red S (ARS) Alizarin complexone (AC) Otoliths Scales Fin rays
a b s t r a c t Alizarin red S from 200 to 400 mg/l and alizarin complexone from 50 to 300 mg/l were used to mark Japanese flounder, Paralichthys olivaceus. Immersion for 24 h produced detectable marks in sagittae, ctenoid, cycloid scales, fin rays (dorsal, anal, pectoral, caudal and ventral fin rays) and parts of dissected asteriscus after 60 days in culture. All treatment concentrations had good marks on otoliths. Violet marks were visible under normal light after marking with 200–400 mg/l ARS and 300 mg/l AC. Scales and fin rays showed acceptable fluorescent marks at higher concentrations (400 mg/l ARS, 300 mg/l AC and 250–400 mg/l ARS, 300 mg/l AC, respectively). The best results were obtained after marking with ARS at 400 mg/l and AC at 300 mg/l. There was no significant difference on survival and growth of marked fish relative to controls throughout experiment (P > 0.05). © 2009 Elsevier B.V. All rights reserved.
1. Introduction Japanese flounder Paralichthys olivaceus is one of the most important commercial species along the coastal areas of China, southwest of the Korean peninsula and Japan (Zhu et al., 1963; Cheng and Zheng, 1987). Japanese flounder has declined significantly in recent years due to overexploitation. Since 1996, a hatchery-produced juvenile release program has been implemented in Shandong province. In 2007 alone, approximately, 9 million P. olivaceus juveniles were released into Shandong inshore waters. The success of this mass-releasing program of stock enhancement remains to be evaluated. To assess the effectiveness of the stock enhancement strategies for fisheries management (Brown et al., 2002; Taylor et al., 2005; Baer and Rösch, 2008), marking and recapture programs need to be carried out. Various methods have been developed to mark fish at different stages of the life cycle. However, fish in their early stage are too small to be marked with external marks (e.g. T-bar, plate disc and fin clipping) or internal tags (e.g. passive integrated transponder, PIT). Additionally, individual handling could involve large effort. Genetic tags are not suitable for recapture process due to high operating costs. Considering metamorphosis during the early stage of development and
∗ Corresponding author at: College of Fisheries, Ocean University of China, 5# Yushan Road, Qingdao, 266003, PR China. Tel.: +86 0532 82032076; fax: +86 0532 82032076. E-mail address: [email protected] (X.M. Zhang). 0165-7836/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fishres.2009.03.014
depressiform body structure of P. olivaceus, using coded wire tag seems difficult. Furthermore, thermal marking has been widely used for marking hatchery-produced salmons (e.g. Hagen et al., 1995; Volk et al., 1999) due to long period of egg incubation. However, P. olivaceus has relatively short incubation period when it could be thermally marked. Therefore, as far as authors know, chemical marks as internal–external marks are the most suitable tools for evaluation of large-scale stocking of small juveniles in contrast to other tagging methods (Brown et al., 2002; Simon and Dörner, 2005; Baer and Rösch, 2008). This method must be suitable for application to large numbers of small fish, handling stress should be kept at a minimum, markers should be retained for several months, marking process and recovery should be easy to carry out (Skov et al., 2001). Fluorochrome labeling dyes can produce detectable marks in otoliths, scales and other bony structures based on differential staining of chemical dyes. Various techniques for introducing marker have been investigated both in teleosts and elasmobranchs (Gelsleichter et al., 1997). The choice of technique depends on life history stage, environment (marine or fresh water) and experiment condition (Lagardère et al., 2000), for example, injection (Monaghan, 1993), dietary intake (Honeyfield et al., 2006) and immersion. Chemical marks mainly include: oxytetracycline (OTC) hydrochloride, calcein, alizarin red S (ARS) and alizarin complexone (AC). In recent years, alizarin red S (ARS) and alizarin complexone (AC) have been substituted for the other chemical dyes to produce clearly readable marks, causing less negative
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effect on survival and without availability limitation such as OTC in increasing salinity (Taylor et al., 2005) (e.g. Tsukamoto, 1988; Tsukamoto et al., 1989; Walt and Faragher, 2003; Baer and Rösch, 2008). However, no controlled laboratory experiment on the mortality and mark retention induced by AC and ARS has been conducted during relatively long periods on P. olivaceus, although Yamashita et al. (1994) used fixed 80 mg/l AC for 24 h in field experiment. There was little detail on evaluation of AC and without reference to ARS on this species. Therefore, the aims of this study were to evaluate growth, mortality and mark quality in otoliths, scales and fin rays of P. olivaceus marked with AC and ARS. 2. Methods 2.1. Experimental fish Juveniles (20–30 mm total length) were transported from a commercial hatchery company located at Wendeng City and kept in a 2 m3 recirculatory fiberglass tank for 27 days (1.4 individuals/l). During this acclimation period, fish were fed twice daily with commercial pellets (Shengsuo, Shandong, China) to satiation. Water quality was constantly monitored throughout the holding period and maintained (temperature, 20 ± 0.5 ◦ C; salinity, 31.0 ± 1.0; dissolved oxygen (DO), 5.26–5.32 mg/l; pH, 7.6–8.0; photoperiod, 14 L/10 D). Water was exchanged at the daily rate of 15% tank volume. 2.2. Immersion marking ARS and AC were dissolved in distilled water at the concentrations of 4000 mg/l ARS and 1000 mg/l AC, respectively as stock solution with salinity adjusted to 31. Then treatment concentrations of 0, 200, 250, 300, 400 mg/l ARS were prepared and water aerated strongly in order to raise pH. The fish were starved for 24 h before immersion. About 70 sixty-day-old P. olivaceus was randomly allocated to each treatment at a density of 3.5 individuals/l in ARS solution (20 L volume) for 24 h. Fish immersed in seawater without ARS served as control group. They were kept covered in dark environment to avoid stress throughout the immersion period. Following completion of the immersion period, juveniles were retrieved from the dye solutions, rinsed and then transferred to separate containers with fresh seawater for 4 h to completely wash away any remnant of dye. Lastly, fish were transferred into five aquaria for 72 h to determine the degree of acute mortality caused by ARS. PH was checked regularly during the immersion period in order to maintain a tolerable condition (7.23–7.86). The same procedure was followed in the application of AC. Fish were immersed in AC of concentrations 0, 50, 100, 150, 200 and 300 mg/l for 24 h. Fish treated in seawater without AC served as control group. For each solution, about 40 P. olivaceus at a density of 4 individuals/l was immersed in AC (10 L volume). Fish acute mortality due to AC was determined 72 h post-immersion. 2.3. Growth experiment To examine the effects of ARS and AC on survival and growing condition following immersion, 60 and 33 juveniles, respectively, were randomly selected per treatment concentration. For ARS marking, each treatment consisted of three replicates of 20 individuals (0.22 individuals/l) in a recirculatory system comprising 184 L storage aquarium and 8 titanium alloy columniform tanks (D:H = 65 cm × 64 cm). The tanks were continuously aerated and constant water quality parameters maintained (DO, 5.26–5.32 mg/l; salinity, 31.0 ± 1.0; photoperiod, 14 L/10 D; ammonia-N, 0.10–0.20 mg/l; pH, 7.6–8.0; temperature, 20 ± 0.5 ◦ C).
Water was exchanged at the daily rate of 15% tank volume and recirculated with composite sand filter, foam filter, biochemical filter balls and ultraviolet sterilization. Fish treated with AC were held in another recirculatory system, which consisted of 18 glass aquaria (L × W × H = 35 cm × 25 cm × 33 cm) equipped with filter bed and biochemical filter balls. Water volume was about 180 L. 33 P. olivaceus juveniles were randomly selected from each treatment concentration. This was then replicated three times consisting of 11 individuals (0.34 individuals/l) per sub-treatment and kept in glass aquarium. Water quality was maintained daily as described for ARS treatments. Water was exchanged at the daily rate of 30% tank volume. At the beginning of the growth experiments, all fish were starved for 24 h before total length and wet mass were measured to the nearest 0.1 mm and 0.01 g, respectively. Fish were measured from different batches of AC and ARS on 20th, 40th and 60th day to assess the extended effect of chemical marks on growth. 2.4. Sampling and mark analyses The sagittae and some scales (ctenoid scale from backside, cycloid scale from abdomen) of all fish were removed and 2 individuals per sub-treatment (6 individuals per concentration) were taken randomly for fin rays (dorsal, anal, pectoral, caudal and ventral fin ray) dissection at the base of the pterygiophores after 60 days post-marking. Parts of asteriscus were also dissected. They were all freed from adherent tissues and rinsed with water. All samples were checked directly without resin and polishing (e.g. Champigneulle and Cachera, 2003). To prevent fading of fluorescent marks, they were stored in sample envelopes avoiding high temperature and direct sunshine (Bashey, 2004). Marks were observed with an epifluorescence microscope (OLYMPUS BX51) equipped with a 10× objective lens and fitted with an Olympus DP70 high resolution digital camera (Table 1) and Stereoscopic Zoom Microscope (Nikon SMZ800) equipped with a 1× objective lens. Mark quality was assessed using a scale of 0–5 (0, no mark visible under fluorescence microscope; 1, poor mark under fluorescence microscope; 2, mark easily visible under fluorescence microscope; 3, mark shining brightly under fluorescence microscope; 4, mark visible in transmitted light; 5, mark distinct in transmitted light). Mark was identified by analyzing both sagittae, dissected parts of asteriscus, at least 20 cycloid and ctenoid scales of all fish and more than 4 pieces of fin rays per position of subsample fish. In general, mark quality on otoliths, scales and fin rays per specimen was analyzed separately by two researchers and where the presence of a mark was doubtful, it was determined by a third researcher. Mark quality ≥2 was judged an acceptable good mark as it could be readily detected in the structure (Taylor et al., 2005). Marks with high scores require less time for analyses and can be easily applied during mass-marking programs. 2.5. Data analyses Analysis of variance (ANOVA) with Turkey’s honestly significant difference (HSD) test (SPSS13.0) was used to assess the significance Table 1 Filter wavelengths for visualizing ARS and AC marks. Light source
WB WG UV
Wavelength (nm) Excitation filter
Barrier filter
490 545 365
515 590 420
Both of AC and ARS marks were detected using same filters.
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Fig. 1. Mean total length and wet mass of ARS (A and C) and AC treatments (B and D). Data were collected from 0th, 20th, 40th and 60th day (vertical bars show standard error, one-way ANOVA, P > 0.05).
of differences in survival, total length and wet mass exposed to different chemical concentrations of AC and ARS, respectively. A 0.05 significant level was used in the analysis. 3. Results 3.1. Growth and survival There was no mortality except one fish from 300 mg/l ARS and one from 300 mg/l AC during the 24 h immersion period. Post 72 h immersion mortality was 0% in all treatments. After 60 days post-marking, no mortality was found in AC growth experiment while 6.33% mortality was recorded in ARS treatment (19 of 300 Japanese flounders died). One-way ANOVA test showed no significant difference in survival between the control and ARS marked groups (d.f. = 4, P > 0.05). There was no evidence to suggest that fluorochrome label caused any mortality. The mortalities we recorded must have been caused by fish jumping out from holding tanks and bacterial disease (Yellow Sea Fisheries Research Institute Fish Disease Laboratory, Personal Communication). No significant difference was found in total length and wet mass of juvenile P. olivaceus both in ARS and AC treatment during the 60-day growth experiment (d.f. = 4, error d.f. = 10, P > 0.05 for 2 ARS treatments; d.f. = 5, error d.f. = 12, P > 0.05 for 2 AC treatments) (Fig. 1). 3.2. Mark quality There was no evidence of any fluorescent mark in control groups in both ARS and AC treatments. Marks were observed under fluorescence microscope with all the filters (WB, WG and UV). Using
green laser as emission wavelength, the intensity of red mark was stronger than violet mark produced by UV laser wavelength. Redorange mark produced with blue laser was the most obvious mark (Fig. 2). By inspecting otolith sections under fluorescence microscope, all concentrations of ARS and AC treatments had 100% marks in sagittal otoliths (score ≥2) (Fig. 6). Optimal marking concentrations were 200–400 mg/l ARS and 300 mg/l AC for 24 h when violet marks were clearly visible by transmitted light to naked eye (score ≥4) (Fig. 6). Marks were also detected in dissected parts of asteriscus (Fig. 3). In the present study, ctenoid and cycloid scales showed ring marks both of ARS and AC treatments with blue and green laser (Fig. 4). Fluorescent marks were detected at the concentrations of 250–400 mg/l ARS and 300 mg/l AC. Dosages of 400 mg/l ARS and 300 mg/l AC showed good marks on scales (score = 2) (Fig. 7). Marks in fin rays (Fig. 5) (dorsal, anal, pectoral, caudal and ventral fin ray) were more obvious at 250–400 mg/l and 300 mg/l for ARS and AC (score ≥2), respectively, despite high autofluorescence of fin rays (Fig. 8). The sagittal otoliths (Fig. 6) provided the best visible marks in comparison with fin rays and scales under different ARS and AC treatments. However, fluorochrome label on scales (Fig. 7) and fin rays (Fig. 8) showed acceptable marks (score ≥2) with relatively high concentrations, which were clearly visible under fluorescence microscope. Fin rays and scales were obtained non-lethally and the process under field condition was less time-consuming. In general, the optimum concentrations were 400 mg/l ARS and 300 mg/l AC, respectively since otolith marks were obvious to the naked eye. Further, all scales and fin rays had good marks without killing fish. The results verified marks would be retained and detected at least 60 days without the need for sacrificing and preparing otolith samples.
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Fig. 2. Photographs of a frontal section of sagittae after 60 days culture. Fish were treated with AC at a concentration of 300 mg/l (A–C). Green light (A); blue light (B); UV light (C). A pair of sagittates showed visible violet marks by normal transmitted light (D). Mark was produced with 300 mg/l ARS. A section of unmarked sagittae was shown by green light (E) and blue light (F). The sagittates were neither ground nor polished. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
4. Discussion 4.1. Marking quality and application Marking of fish by immersion involves a compromise between cost, concentration, immersion period, salinity, mortality, growing condition and retention time to produce the best mark (Taylor et al., 2005). Using both ARS and AC, we obtained 100% marking success. Despite variations in the quality of marks, fluorescent marks were detected in all treatments and in all the body structures analyzed. This study has shown that juveniles can be successfully marked and sampled non-lethally. Our results further agree with several studies showing that ARS and AC are effective for immersion marking, which can be widely used in biological research and evaluation of stock enhancement programs (e.g., Sánchez-Lamadrid, 2001; Skov et al., 2001; Baer and Rösch, 2008). In our experiments, there was little mortality during the immersion period (one from ARS treatments and one from AC treatments) and no mortality 72 h post-marking. The growth experiments demonstrated that both dyes with 200–400 mg/l and 50–300 mg/l of ARS and AC, respectively following long immersion time does not
affect growth and survival during 60 days of laboratory experiment. In other studies similar results were obtained (e.g. Tsukamoto, 1988; Lagardère et al., 2000; Bashey, 2004). Nevertheless, Beckman and Schulz (1996) observed that immersion in 400 mg/l ARS (equivalent to our 400 mg/l ARS concentration) or higher concentrations resulted in excessive mortalities and reduced marking success. This discrepancy may be attributed to the life stage of fish more than the high concentration of dyes and the immersion period. The otolith mark retention in the present investigation was 100% after 60 days in all treatments. However, longer retention time of fluorescent marks had been demonstrated (Tsukamoto et al., 1989; Taylor et al., 2005; Walt and Faragher, 2003). Thus, it appears that otolith mark retention will not be a problem to mark P. olivaceus with ARS and AC. The different retention time may be caused by individual metabolic difference and polishing operation of the otolith. Bashey (2004) suggested that it might be caused by mark detection method, taxa related differences or environmental factors (i.e. high water temperature and direct sunshine). In the present investigation, the same immersion method, equal mark effect and lower price of ARS in comparison with AC increase feasibility of its
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Fig. 3. Photographs of an asteriscus after 60 days culture. Fish were treated for 24 h in 400 mg/l ARS solution. Green light (A); blue light (B); UV light (D). Mark was obvious by normal transmitted light (C). An unmarked sagittae was shown by green light (E) and blue light (F). The asteriscus was neither ground nor polished. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
use and appears to be a promising technique for large-scale marking program in assessing stock enhancement. 4.2. Visible otolith mark In our study, visible mark under normal light was clearly observed at concentrations of 200–400 mg/l ARS and 300 mg/l AC. This must have been due to, firstly, the fairly stable pH of dye solutions during alizarine marking in saline water because pH was maintained within a narrow range by natural buffer system. The change in pH may help enhance the transport of dyes into fish. Secondly, the best life stage for marking with chemical dyes appears to be during the period of rapid growth development. Eckmann (2003) reported that chemical dyes formed complexes with calcium and these were deposited in the bone as the fish grows. In order to assess whether life stage was an essential factor, another experiment was conducted after fish were kept for >6 months
in the laboratory. About sixty 240-day-old juveniles P. olivaceus (Lf = 166.77 ± 26.65 mm, mean ± S.D.) were immersed for 24 h at a concentration of 400 mg/l ARS. Following immersion, all fish survived. Both sagittae were immediately removed from samples. Mark quality was identified as described above. Based on the same concentration of ARS and immersion time, violet marks were clearly poorer (score = 4) than that of prior test (score = 5). Visible mark was observed on all samples. However, retention time of visible mark on otolith seemed not as long as in the first experiment due to low luminary. Thus, it appears safe to conclude that the most appropriate life stage to mark P. olivaceus is at sizes of 50–60 mm, a period of rapid growth during which bright fluorescent marks are formed. Similar visible otolith marks were obtained when AC solutions of 150–350 mg/l for 10–12 h were used to mark 9–14-day-old larval Schizothorax davidi (He et al., 2008). Lastly, this study suggests that high dosage and 24 h immersion times are essential to form visible otolith mark. However, without the fluorescence microscope, marks
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Fig. 4. Scales (ctenoid scale from backside, cycloid scale from abdomen) subject to ARS 400 mg/l solution for 24 h (A–F). Photographs of a marked ctenoid scale (A–C) and a cycloid scale (D–F) after 60 days treatment. An unmarked ctenoid scale was shown as control group (G and H). Green light (A, D and G); blue light (B, E and H); normal transmitted light (C and F). The fluorescent mark was not produced under normal light.
in fin rays and scales are not visible to the unaided eye. Thus, otoliths could provide the best mark quality in mass-marking program. 4.3. Mark on fin rays and scales The major disadvantage of the otolith mark is that fish must be sacrificed to collect the structures (Bart et al., 2001). In this study, fin rays and scales with high concentration of dyes provided good marks (score ≥2), which allows non-lethal detection of marks (Brown et al., 2002; Leips et al., 2001; Frenkel et al., 2002; Honeyfield et al., 2006); removes the effort in preparing otolith (Barker and Mckaye, 2004) and marks were retained for a relatively long time. Much longer retention time in other fluorescent mark-
ers have already been reported (Leips et al., 2001; McFarlane and Beamish, 1995). Using fluorescent marks on hard tissues, large samples can be analyzed in a comparatively short time and it is more applicable in large-scale releases of hatchery-reared fish. Bashey (2004) found that ARS marking could produce good fluorescent mark in guppies Poecilia reticulate scales. However, as far as we know, no reference about fin or scale marks with AC existed before now. 4.4. Limitation of fluorescent mark Chemical batch marking provided convenient methods for largescale marking of fish (Sánchez-Lamadrid, 2001). However, general
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Fig. 5. Details of a section of anal fin ray after 60 days treatment (A–C). Fish treated in 300 mg/l AC for 24 h. An unmarked anal fin ray was shown as control group (D and E). The fin rays were dissected from tissue without grinding or polishing. Normal transmitted light (A); green light (B and D); blue light (C and E). The fluorescent mark was not produced under normal light.
Fig. 6. Mark quality was detected in sagittae by immersion batches using ARS (A) and AC (B). No marks were detected in control groups both in ARS and AC treatments (standard error = 0).
Fig. 7. Mark quality was detected in ctenoid and cycloid scales by batches of ARS (A) and AC (B) treatment. No marks were detected in control groups between ARS and AC treatments (vertical bars show standard error).
Fig. 8. Variations in mark quality subjected to ARS treatments (A) and AC treatments (B). No marks were detected in control groups both in ARS and AC treatments (vertical bars show standard error).
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limitations to use of fluorescent dyes include limited tag code that could not differentiate among individual samples and the difficulty in distinguishing stocks from different hatcheries even at the cost of double chemical marking. Moreover, this may not be true to verify spawning locality by recapturing marked fish (Iglesias and Rodriguez-Ojea, 1997). Fluorescent marks could only show the recapture information rather than the change in fish behavior, physiology and ecology in the wild. Hence, suitable marking dyes in combination with other tagging methods appear to be more suitable. Besides evaluating the mortality and growing condition of marked fish, it is essential to quantify fluorescent dyes residues in tissues for evaluation of tag method. Brown et al. (2002) reported that OTC was depleted in adult yellow perch Perca flavescens (M.) muscle tissue and reached acceptable tolerance level by about 2 h post-immersion. However, there is no reference on detecting ARS and AC residues and more efforts should be made in this direction in future research. In conclusion, the results suggest marking juvenile P. olivaceus by immersion in 400 mg/l ARS and 300 mg/l AC provide two methods for mass-marking operations under field conditions. Further, marks in fin scales or rays may allow non-lethal detection to get mark information. Use of ARS marking is cheaper and recommended. Both chemical dyes as powerful tool allow fishery managers to evaluate the effectiveness of stock enhancement. Acknowledgements We wish to express thanks to Shuhan Lei, Yuan Huang, Liming Jiao, Zhengyong Wu, Yue Wang and Hongtan Zhang for their assistance during laboratory work, especially for skillful dissection and preparation samples. Funding was provided by the National Key Technology R & D Program of China during the 11th FiveYear Plan Period (No. 2006BAD09A15) and the Key Laboratory of Mariculture of Ministry of Education, Ocean University of China. References Baer, J., Rösch, R., 2008. Mass-marking of brown trout (Salmo trutta L.) larvae by alizarin: method and evaluation of stocking. J. Appl. Ichthyol. 24, 44–49. Barker, J.M., Mckaye, K.R., 2004. Immersion marking of juvenile midas cichlids with oxytetracycline. Trans. Am. Fish Soc. 24, 262–269. Bart, A.N., Kindschi, G.A., Ahmed, H., Clark, J., Young, J., Zohar, Y., 2001. Enhanced transport of calcein into rainbow trout, Oncorhynchus mykiss, larvae using cavitation level ultrasound. Aquaculture 196, 189–197. Bashey, F., 2004. A comparison of the suitability of alizarin red S and calcein for inducing a nonlethally detectable mark in juvenile guppies. Trans. Am. Fish Soc. 133, 1516–1523. Beckman, D.W., Schulz, R.G., 1996. A simple method for marking fish otoliths with alizarin compounds. Trans. Am. Fish Soc. 125, 146–149.
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