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Im et al. Fisheries and Aquatic Sciences (2017) 20:21 DOI 10.1186/s41240-017-0066-8 RESEARCH ARTICLE Open Access Evaluation of visible fluorescent elastomer tags implanted in marine medaka, Oryzias dancena 1 2 2* 2 2 3 Jae Hyun Im , Hyun Woo Gil , In-Seok Park , Cheol Young Choi , Tae Ho Lee , Kwang Yeol Yoo , 4 4 Chi Hong Kim and Bong Seok Kim Abstract: The aim of this study was to assess visible implant fluorescent elastomer (VIE) tagging and stress response in marine medaka, Oryzias dancena. The experimental fish were anesthetized individually and marked with red, yellow, or green elastomer at each of the following three body locations: (1) the abdomen, (2) the back, and (3) the caudal vasculature. During 12 months, the accumulated survival rates of fish in the experimental treatments were not different among red, yellow, and green elastomers. The experimental fish retained > 85% of the tags injected in the back, > 70% of the tags injected in the caudal vasculature, and > 60% of the tags injected in the abdomen (P< 0.05). An important observation was that the abdomen site was associated with poor tag retention. For all injected sites, the red and green tags were able to be detected more easily than the yellow tags when observed under both visible and UV lights. Tag readability was lower for the abdomen site than for the other sites (back and caudal vasculature). Thus, VIE tags were easy to apply to marine medaka (< 1 min per fish) and were readily visible when viewed under UV light. Keywords: Marine medaka, Oryzias dancena, Readability, Visible fluorescent elastomer tag Background gonadogenesis, sexual differentiation, early ontogenesis, The marine medaka, Oryzias dancena, is nonindigenous embryogenesis, and exceptional capacity for hyperos- to South Korea and is a bony fish with high tolerance to moregulation and hypoosmoregulation. In addition, Kim salinity because of its salinity adaptation mechanisms et al. (Song et al., 2009) suggested that this species has a (Inoue and Takei, 2003). In addition to the studies of this short interval between generations with spawning euryhaline species, under various salinity conditions, it possible only for 60 days after hatching. A study of the has been the subject of extensive ecotoxicogenomic re- effects of clove oil and lidocaine HCl on the species by search; this should extend the use of the marine medaka Park et al. (2011) has contributed to the safe laboratory as a laboratory model for assessing its responses to salin- handling of this fish, which is required in many studies. ity changes. Its viability under conditions of maximum The research discussed above has demonstrated that the tolerable salinity has been measured, and incubation marine medaka has the ideal characteristics for an time of fry was assessed by its ability to adapt to various experimental animal (Song et al., 2009; Nam et al., 2010; salinity (Cho et al., 2010). This species was recently se- Park et al., 2011). lected by iMLMO (Institute of Marine Living Modified Identification of individuals is essential in studies of fish Organisms, Pukyong National University, Busan 608- growth, migration, and mortality and in stock identification 737, Korea) for use in a project to evaluate living modi- and stock selectivity for tracing particular fish populations fied organisms. Consistent with this purpose, detailed in- (Crossland, 1980). Although short-term tag retention may formation on its biology is being collected (Song et al., suffice for some experiments, the effect of a tag on fish sur- 2009; Nam et al., 2010), particularly related to its early vival, behavior, growth, and recognition and the costs of the tagging technique need to be considered. However, trad- * Correspondence: firstname.lastname@example.org itional external tags (such as spaghetti or dart tags) are Division of Marine Bioscience, College of Ocean Science and Technology, commonly lost soon after deployment (Crossland, 1980; Korea Maritime and Ocean University, 727 Taejong-ro, Yeong do-gu, Busan Bergman et al., 1992) and can affect growth or survival 49112, South Korea Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Im et al. Fisheries and Aquatic Sciences (2017) 20:21 Page 2 of 10 (Crossland, 1976; Tong, 1978; McFarlane and Beamish, 2001; Redding and Schreck, 1983). These changes can 1990; Serafy et al., 1995). Furthermore, these types of tags increase disease susceptibility leading to increased mor- can only be read by recapturing the fish. tality and subsequent economic losses (Schreck et al., Devices that are located internally but are readable 2001; Redding and Schreck, 1983). So, analysis of stress externally, such as acoustic tags, are often limited by response can roughly examine the cause of mortality by short battery life or retention (Ralston and Horn, 1986), tagging. The steroid hormone cortisol is widely accepted and sample sizes are limited by the expense involved. as an indicator of stress in fish, generally increasing after Problems associated with biological compatibility, reli- exposure to physical stressors (Schreck et al., 2001). ability of identification, fouling of the tag by algae (Jones, Circulating cortisol levels are typically measured to de- 1987; Barrett, 1995), tag retention (Crossland, 1976; termine the stress status of an individual fish (Redding Parker, 1990), and external visibility of such devices have and Schreck, 1983). Alternatively, whole-body cortisol reduced confidence in the interpretation of results of in levels have been used to assess the stress responses of situ studies of reef fish ecology. A less frequently used the developing salmonids and flatfish because their approach is intrinsic identification, whereby cohorts are blood volumes are insufficient to allow for the measure- identified by size (Jones, 1987; Forrester, 1990) and indi- ments of circulating cortisol (Redding and Schreck, viduals are recognized by variation in natural markings 1983). Similarly, whole-body corticosteroids have been (Thompson and Jones, 1980; Connell and Jones, 1991) measured in smaller adult fish, including the three- or wound scarring. spined stickleback, Gasterosteus aculeatus (Pottinger et The latter approach has cost advantages, so does not al., 2002), and the zebrafish, Danio rerio (Pottinger and influence behavior, but is subject to potentially substan- Calder, 1995). In this study, we compared various tag tial levels of observer error. Furthermore, many fish colors for readability under visible and UV lights and species lack unique natural markings and cannot be assessed the likelihood of tag- or handling-related mor- recognized without an artificial means of verifying iden- tality, the retention rates of VIE tags placed, and the tity. Passive integrated tag (PIT) method is the most stress response in various body sites of marine medaka. commonly used. However, the body size of marine So, suitability of VIE tag method in marine medaka was medaka is similar to that of PIT chips, so marine medaka investigated by analysis of readability, mortality, and is unsuitable for tagging PIT chips. The visible implant stress response. fluorescent elastomer (VIE) tag was developed primarily for tagging large batches of small or juvenile fish. The Methods VIE system comprises a viscous liquid elastomer that The fish used in this experiment were adult marine me- sets to a pliable solid over a period of hours following daka, O. dancena (mean body length ± SD 35.1 ± 3.42 mm; application. The elastomer can be injected into transpar- mean body weight ± SD 54.4 ± 1.83 mg; age 10 months ent or translucent tissues to form a permanent biocom- after hatched). Injection of the VIE tags into the treatment patible mark. When exposed to UV light and viewed fish, and handling of the control fish, occurred on 16 through an amber filter, the compound fluoresces February 2012. Following to the method of Park et al. brightly. The tag size can easily be varied according to (2011), all fish were anesthetized in 800 ppm lidocaine the requirements of the researcher and the size of the hydrochloride/NaHCO at a water temperature of 10 °C. fish to be tagged. Thus far, the system has been used for The fish were sedated until they were completely immobile the identification of groups or cohorts of juvenile reef and then individually removed from the anesthetic solution, fish (Frederick, 1997) and salmonids, but is also proving rinsed in fresh water, and placed on a flat surface for potentially effective in controlled laboratory studies of tagging. adult blue gills (Dewey and Zigler, 1996). As an exter- Per group of 50, the fish were individually tagged with nally visible but sub-dermally situated marking system, yellow, red, or green elastomer (Northwest Marine VIE tags are potentially able to eliminate many of the Technology Inc., Shaw Island, Washington, USA) at problems associated with other methods. three body locations (Figs. 1 and 2a): (1) the surface of Tagging, weighing, measuring standard length, prepar- the abdomen, (2) the inside surface of the back, and (3) ing fish for live shipment and transport, injecting the surface of the caudal vasculature, and all experimen- vaccines and antibiotics, and collecting blood are causes tal groups were triplicated. Control fish (50) were anes- to increasing stress (Dewey and Zigler, 1996). Stress thetized but not marked. We used the VIE hand responses can include physiological changes such as oxy- injection Master Kit (Northwest Marine Technology gen uptake and transfer, metabolic and hematological Inc., Shaw Island, Washington, USA) for tagging the fish. changes, mobilization of energy substrates, reallocation Following the kit protocol, the elastomer and curing of energy away from growth and reproduction, and agent were mixed at a ratio of 10:1 and the prepared suppressive effects on immune functions (Schreck et al., elastomer was injected as a liquid (0.3 mL per site). The Im et al. Fisheries and Aquatic Sciences (2017) 20:21 Page 3 of 10 intervals. The tanks were checked daily for dead fish, which if present were removed and fixed in 10% neutral formalin solution. The marking with the various colors at the three sites was observed visually from a distance of 30 cm under ambient visible light and UV light and measured the differentiation rate of each group after 1 month. Tag retention data for the dead fish were used Fig. 1 Elastomer injection locations (red dotted lines) on marine to calculate the percent tag retention up to the date that medaka, Oryzias dancena: (1) the surface of the abdomen; (2) the the fish died, but were not used in the subsequent calcu- inside surface of the back; and (3) the surface of the caudal vasculature lations (Zerrenner et al., 1997). To observe the effects of stress on the whole-body cortisol, glucose, and lactic acid levels of fish under VIE instruments used are shown in Fig. 2b. Tagged fish were tag injection, we injected the VIE tag in the abdomen, divided into different tag colors and injection sites in the back, and the caudal vasculature, respectively, and tanks. The fish were held in 18 flow-through fiber- 90 samples were used in each site. The stress responses reinforced plastic tanks (50 × 20 × 20 cm; water volume of the experimental fish were measured at 0, 1, 6, 12, 24, 2 L) supplied with filtered seawater. The bottom of each and 48 h. Fifteen samples were used in each measured tank was fitted with a black sheet to facilitate for obser- time. Control fish were not injected VIE tag, but their vation of the tag. The flow rate was 2 L/min/tank, and cortisol levels were measured. For these measurements, the mean water temperature was 26 ± 2.5 °C. A common 150 fish were used in each experimental group, and no day–night cycle was established, and all tanks were distinction was made between male and female fish. We covered with netting to retain the fish in the tanks. measured the whole-body cortisol, glucose, and lactic Throughout the 12-month trial, the fish were fed daily acid levels of the control fish before the experiment. In- to satiation using a dry commercial flounder feed (Agri- dividual fish were blotted onto paper towels to remove brand Furina Korea Co., Korea) that was alternated with excess water, immediately frozen in liquid nitrogen for a formulated Artemia diet. The food was placed on the 10–30 s, and placed in individual 5.0 mL plastic screw aquarium floor so that it could be eaten within 2 h. cap centrifuge tubes. The samples were stored at − 80 °C The survival rate, tag retention, and detection of tags until we extracted the cortisol, glucose, and lactic acid. (under visible and UV lights) were recorded at 2-month The term “whole-body cortisol” is used to describe the portion of corticosteroid extracted and measured with a cortisol-specific radioimmunoassay (Pottinger et al., 2002). Whole-body glucose concentration was analyzed according to the methodology of Raabo and Terkildsen (1960) (Kit 510, Sigma, St Louis, MO, USA), where the production of H O by glucose oxidase in the presence 2 2 of o-dianisidine was evaluated as an absorbance increase at 450 nm. The lactic acid concentrations were analyzed using blood automatic analysis (Boehringer Mannheim Reflotron, Germany). All measured data were induced by triplicate experi- ments from all experimental samples. Differences in survival rate between control and experimental groups were assessed using the t test (Cody and Smith, 1991), and the tag retention rate (%) among tagging sites was assessed using a one-way ANOVA and Duncan’s mul- tiple range test (Duncan, 1955). The differences were considered to be significant at a probability of 0.05. Results Fig. 2 External morphology of marine medaka, Oryzias dancena, Table 1 shows the retention rate of the VIE tags at each tagged with visible implant fluorescent elastomer (VIE), showing the yellow tags at each tagging site under UV light (a) and (b) the VIE site for marine medaka, O. dancena. During the experi- kit (Northwest Marine Technology, Shaw Island, Washington) ment, there was no difference in tag retention among including the UV lamp (1), mixing beaker (2), silicon (3), mixing stack the various tag colors for the abdomen site, but the tag (4), colored elastomer in a syringe (5), and the injection syringe (6) retention rate for this site was different from that of the Im et al. Fisheries and Aquatic Sciences (2017) 20:21 Page 4 of 10 Table 1 Tagging rate using the naked eye and the UV lamp of Table 2 Differentiation rate using the naked eye of visible visible implant fluorescent elastomer (VIE) tags in each site of marine implant fluorescent elastomer (VIE) tags in each sites of marine medaka, Oryzias dancena, from 0 to 12 months after VIE tagging medaka Oryzias dancena, from 0 to 12 months after VIE tagging Month Color Tagging rate (%) Month Color Differentiation rate (%) Abdomen Back Caudal vasculature Abdomen Back Caudal vasculature a b b c a a 1 Red 57.4 ± 3.26 99.3 ± 0.16 99.0 ± 0.22 1 Red 60.0 ± 4.21 100.0 100.0 a b c a c a Green 77.6 ± 1.11 94.7 ± 0.85 99.3 ± 0.10 Green 84.0 ± 3.67 96.0 ± 2.11 100.0 a b b b b b Yellow 74.3 ± 1.48 94.9 ± 0.80 95.4 ± 0.67 Yellow 73.9 ± 4.55 97.8 ± 4.34 96.3 ± 3.11 a b c c a a 3 Red 60.6 ± 2.32 93.5 ± 1.03 99.4 ± 0.05 3 Red 59.4 ± 3.56 97.0 ± 1.55 100.0 a b c a c b Green 76.4 ± 0.87 93.8 ± 0.58 95.6 ± 0.57 Green 82.0 ± 5.07 93.2 ± 4.74 95.6 ± 4.12 a b b b b c Yellow 73.2 ± 1.75 94.9 ± 0.99 92.9 ± 2.10 Yellow 77.3 ± 3.85 96.2 ± 2.65 94.3 ± 3.01 a b c c a a 6 Red 57.3 ± 3.74 92.0 ± 1.52 96.5 ± 0.60 6 Red 59.8 ± 4.21 94.4 ± 2.51 97.4 ± 2.33 a b b a a b Green 80.7 ± 2.10 92.6 ± 0.83 94.9 ± 0.81 Green 80.3 ± 6.47 92.3 ± 5.78 96.9 ± 2.11 a b b b a b Yellow 75.1 ± 1.68 95.4 ± 1.00 92.2 ± 1.88 Yellow 75.7 ± 4.68 97.9 ± 0.91 96.3 ± 2.97 a b c c b b 9 Red 64.4 ± 3.19 93.4 ± 0.67 97.5 ± 0.80 9 Red 62.4 ± 3.93 93.7 ± 2.86 98.6 ± 3.12 a b c a b a Green 79.0 ± 1.46 89.8 ± 1.79 99.1 ± 0.09 Green 84.4 ± 5.41 93.7 ± 0.74 99.5 ± 2.69 a b b b a c Yellow 75.9 ± 2.00 95.5 ± 0.73 94.4 ± 1.73 Yellow 74.8 ± 5.23 98.3 ± 1.99 94.3 ± 3.79 a b c c c a 12 Red 67.2 ± 3.05 90.5 ± 1.31 96.6 ± 0.66 12 Red 65.2 ± 4.41 92.1 ± 4.61 98.8 ± 0.72 a b c a b a Green 79.5 ± 1.96 94.4 ± 0.74 97.2 ± 0.38 Green 85.9 ± 2.38 94.0 ± 1.68 98.1 ± 1.08 a b b b a b Yellow 70.4 ± 1.21 94.6 ± 0.94 95.6 ± 0.86 Yellow 68.5 ± 5.53 97.3 ± 0.23 96.3 ± 2.89 Samples tagged with VIE were investigated. Values are mean ± S.E. (n = 50). Samples tagged with VIE were investigated. Both dead samples and The experiment was performed in triplicate. The values in each column that eliminated VIE tags were excluded from the analysis. Values are mean ± S.E. do not share a common superscript are significantly different from one (n = 50). The experiment was performed in triplicate. The values in each another (P < 0.05) column that do not share a common superscript are significantly different from one another (P < 0.05) back and caudal vasculature sites. For the abdomen site, Table 3 Differentiation rate using the UV lamp of visible the retention (%) of the elastomer at 1 month were implant fluorescent elastomer (VIE) tags in each site of marine 57.4 ± 3.26 for red, 77.6 ± 1.11 for green, and medaka, Oryzias dancena, from 0 to 12 months after VIE tagging 74.3 ± 1.48 for yellow but at 6 months were 57.3 ± 3.74 Month Color Differentiation rate (%) (red), 80.7 ± 2.10 (green), and 75.1 ± 1.68 (yellow). And Abdomen Back Caudal vasculature in this term, the green and yellow values were signifi- b a a 1 Red 88.0 ± 2.61 100.0 100.0 cantly higher than the red value. However, the retention a a a Green 92.0 ± 1.73 100.0 100.0 rate of the abdomen site was not affected by color. In c a a summary, the retention rate for red was 67.2 ± 3.05, for Yellow 86.9 ± 3.11 100.0 100.0 b a a green was 79.5 ± 1.96, and for yellow was 70.4 ± 1.21. 3 Red 89.8 ± 1.73 100.0 100.0 For the back site at 1 month, the retention rate for red a b a Green 90.3 ± 2.22 99.4 ± 0.03 100.0 was 99.3 ± 0.16 and for green was 94.7 ± 0.85. The re- c a a Yellow 84.7 ± 2.83 100.0 100.0 tention rate for yellow was 94.9 ± 0.80. After 9 months, c a b 6 Red 85.9 ± 3.85 100.0 99.4 ± 0.01 the values had declined to 93.4 ± 0.67, 89.8 ± 1.79, and a c c Green 91.8 ± 2.89 97.8 ± 1.01 98.9 ± 1.07 95.5 ± 0.73 along red, green, and yellow, respectively. b b a These values show that the VIE was removed from the Yellow 87.5 ± 3.10 99.5 ± 0.20 100.0 c a a tagged site by the time in each site. In conclusion, at 9 Red 87.5 ± 2.81 100.0 99.1 ± 0.04 12 months, the tag retention rates for the back were a c b Green 91.3 ± 2.10 97.1 ± 0.78 98.2 ± 1.10 90.5 ± 1.31, 94.4 ± 0.74, and 94.6 ± 0.94 for the red, b b b Yellow 88.1 ± 1.08 98.9 ± 1.00 98.5 ± 0.74 green, and yellow elastomers, respectively. For the cau- b a a 12 Red 87.2 ± 3.02 100.0 98.9 ± 0.03 dal vasculature, there were no significant differences a c b Green 95.0 ± 2.97 96.9 ± 1.61 97.9 ± 0.69 (P< 0.05) among the elastomer colors (red, 99.0 ± 0.22; c b a green, 99.3 ± 0.10; yellow, 95.4 ± 0.67), but at the end of Yellow 84.0 ± 2.11 98.3 ± 0.57 98.2 ± 0.42 the experiment, the values were 96.6 ± 0.66, 97.2 ± 0.38, Samples tagged with VIE were investigated. Both dead samples and eliminated VIE tags were excluded from the analysis. Values are mean ± S.E. and 95.6 ± 0.86, respectively. Among the three colors, (n = 50). The experiment was performed in triplicate. The values in each the tag retention rate for the back was the highest column that do not share a common superscript are significantly different (P< 0.05), followed by that of the caudal vasculature from one another (P < 0.05) Im et al. Fisheries and Aquatic Sciences (2017) 20:21 Page 5 of 10 tags. The abdomen tag retention rate was the lowest the red tags (%) was 98.9 ± 0.03, for the green was among the tagging sites. 97.9 ± 0.69, and for the yellow was 98.2 ± 0.42, indicat- We measured the visual differentiation of the tags using ing that red and yellow tags were more easily detected two detection methods (visible and UV lights) (Tables 2 than green tags in the caudal vasculature. In conclusion, and 3). During 1 min, the VIE tags were initially observed by UV lamp, the tag readability for the back and caudal using visible light at 30-cm distance from the experimental vasculature sites were significantly greater than those for fish. Table 2 shows the dead fish and those that eliminated the abdomen site (P< 0.05; Table 3). Regardless of site, the VIE tags were excluded from the analysis. For the abdo- all color tags under UV light were more easily detected men site, the capacity to detect for the red and green tags than all color tags under visible light. was significantly greater than that for the yellow tags During experimental period (12 months), accumulated (P< 0.05). At 1 month, the detection rate of the red tags survival rates of back, abdomen, and caudal vasculature (%) was 60.0 ± 4.21, for the green tags was 84.0 ± 3.67, and groups were not significantly different among red, green, for the yellow tags was 73.9 ± 4.55. After 12 months, the and yellow, respectively (Table 4, P > 0.05). However, values were 65.2 ± 4.41, 85.9 ± 2.38, and 68.5 ± 1.53, accumulated survival rates of each color were affected respectively, indicating that the red and green colors were by the tagging site (Table 4, P < 0.05). During 12 months, more easily detected (P< 0.05). For the back site at the accumulated survival (%) of the control group was 1 month, the detection rate (%) of red tags was 100, for the highest (P < 0.05) and the reduction ratio of the green was 96.0 ± 2.11, and for yellow was 97.8 ± 4.34. After accumulated survival in the control group was the most 12 months, tag detection were 92.1 ± 4.61 and 94.0 ± 1.68 gradual. However, the accumulated survival (%) of the for the red and green tags, respectively, and for the yellow abdomen group in each color were the lowest (P < 0.05), tags was 97.3 ± 0.23. For the caudal vasculature at 1 month, and the reduction ratio were the most dramatic in each the detection rates (%) of red and green tags were 100, color (Table 4). respectively, but for the yellow tags was 96.3 ± 1.11. At 6 months, the detection rates (%) were 97.4 ± 2.33, 96.9 ± 2.11, and 96.3 ± 1.97, respectively (P< 0.05). At the Table 4 Accumulated survival rate using the UV lamp of visible end of the experiment, the detection rates (%) were implant fluorescent elastomer (VIE) tags in each site of marine medaka, Oryzias dancena, from 0 to 12 months after VIE tagging 98.8 ± 1.72, 98.1 ± 1.08, and 96.3 ± 2.89, respectively. Table 2 shows that abdomen tags were less well detected Month Color Accumulated survival rate (%) than back and caudal vasculature tags. Control Abdomen Back Caudal (no injection) vasculature Table 3 shows the results for tag differentiation using a a a a 0 Red 100.0 100.0 100.0 100.0 the UV light for detection of the VIE tags at each site in a a a a the experimental fish. The observation protocol was as Green 100.0 100.0 100.0 100.0 described above. For the abdomen site, the detection of a a a a Yellow 100.0 100.0 100.0 100.0 the red and green tags was significantly greater than for a c a b 1 Red 100.0 85.7 ± 0.71 99.3 ± 0.24 97.5 ± 0.41 the yellow tags (P< 0.05). At 1 month, the detection rate a c a b Green 100.0 85.0 ± 1.14 99.0 ± 0.59 97.9 ± 0.87 (%) for the red tags was 88.0 ± 2.61, for green was a c a b Yellow 100.0 85.9 ± 1.06 98.8 ± 0.67 96.8 ± 0.91 92.0 ± 1.73, and for yellow was 86.9 ± 3.11, indicating a c a b 3 Red 97.5 ± 1.88 80.4 ± 1.73 97.3 ± 1.89 93.6 ± 1.41 that the red and green tags were more readily detected a c a b than the yellow tags (P< 0.05). After 6 months, this had Green 97.5 ± 1.88 79.1 ± 1.92 97.2 ± 1.14 94.1 ± 0.92 not changed significantly, and at the end of the experi- a c a b Yellow 97.5 ± 1.88 81.1 ± 1.88 97.8 ± 0.91 93.6 ± 1.10 ment (12 months), the detection (%) of the red, green, a d b c 6 Red 96.1 ± 2.84 77.1 ± 3.24 93.3 ± 1.57 86.4 ± 2.14 and yellow tags were 87.2 ± 3.02, 95.0 ± 2.97, and a d b c Green 96.1 ± 2.84 76.7 ± 2.88 92.6 ± 1.25 87.0 ± 1.55 84.0 ± 2.11, respectively, showing that the yellow tags a d b c Yellow 96.1 ± 2.84 76.9 ± 3.10 93.4 ± 1.09 86.8 ± 3.81 were least detectable when observed by the UV light a d b c 9 Red 93.8 ± 1.55 71.2 ± 3.44 90.7 ± 2.48 81.1 ± 4.39 (P< 0.05). For the back site, the tag detection rate was a d b c 100% for the three colors, while at 12 months, for the Green 93.8 ± 1.55 70.4 ± 4.05 90.5 ± 3.24 80.1 ± 3.81 red tags was 100.0, for the green was 96.9 ± 1.61, and a d b c Yellow 93.8 ± 1.55 71.2 ± 2.12 89.9 ± 1.85 81.1 ± 2.58 for the yellow was 98.3 ± 0.57 (P< 0.05). These results a d b c 12 Red 90.4 ± 2.88 64.2 ± 4.32 86.9 ± 3.14 74.1 ± 2.89 indicate that red tags were significantly more readily de- a d b c Green 90.4 ± 2.88 63.8 ± 4.75 86.7 ± 2.99 74.6 ± 3.09 tected than green and yellow tags (P< 0.05). In addition, a d b c Yellow 90.4 ± 2.88 64.3 ± 4.02 87.4 ± 3.02 73.5 ± 2.77 the back tags were detected more easily relative to those Samples tagged with VIE were investigated. Values are mean ± S.E. (n = 50). in the abdomen site. On the caudal vasculature, the The experiment was performed in triplicate. The values in each column that detection rate (%) of all the color tags were 100%. After do not share a common superscript are significantly different from one the 12 months of the experiment, the detection rate for another (P < 0.05) Im et al. Fisheries and Aquatic Sciences (2017) 20:21 Page 6 of 10 In three colors of VIE tagging groups, the accumulated cortisol concentration (P> 0.05), and the change of survival rates of the three sites and the control group whole-body cortisol concentration according to exposure were 100% at the initiation of experiment (Table 4). was seen compared to that at pre-experiment and the cor- Accumulated survival (%) of the control group declined tisol concentration was the highest at 6 h in the control gradually to 90.4 ± 2.88 during 12 months. In addition, group. However, the cortisol concentration was the high- accumulated survival (%) of the back group in three est at 12 h in the three experimental groups. colors declined gradually during 12 months. However, The whole-body glucose and lactic acid concentration the abdomen group declined drastically to 64.2 ± 4.32 in variations of tagged marine medaka during 48 h are red, 63.8 ± 4.75 in green, and 64.3 ± 4.02 in yellow dur- shown in Figs. 4 and 5. The whole-body glucose and lactic ing 12 months, respectively. Accumulated survival (%) of acid concentrations of the control groups were 25 mg/dL the caudal vasculature group in three colors declined and 0.8 mmol/L, respectively, and have been rapidly in- gradually to 93.6 ± 1.41 in red, 94.1 ± 0.92 in green, and creased to 55 mg/dL and 1.48 mmol/L in 12 h (P< 0.05). 93.6 ± 1.10 in yellow until 3 months after injection and At 48 h, it rather decreased to 38 mg/dL and 1.0 mmol/L declined drastically to 74.1 ± 2.89 in red, 74.6 ± 3.09 in (P< 0.05). The whole-body glucose concentrations of green, and 73.5 ± 2.77 in yellow until 12 months after in- three experimental groups were increased rapidly from 12 jection. In summary, the experimental fish of three colors to 24 h and decreased drastically from 24 to 48 h. The survived > 85% of the tags injected in the back, > 70% of whole-body glucose concentrations of the three experi- the tags injected in the caudal vasculature, and > 60% of mental groups were the highest at 24 h. The lactic acid the tags injected in the abdomen (Table 4, P< 0.05). concentrations of the three experimental groups were The whole-body cortisol concentration variations of increased rapidly from 24 to 48 h (P< 0.05). The lactic the tagged group during 48 h are shown in Fig. 3. The acid concentrations of the three experimental groups were whole-body cortisol concentration of the control groups the highest at 48 h. The lactic acid concentrations of the was 0.9 μg/dL and has been increased to 1.20 μg/dL in three tagged groups were not observed of reduction while 1 h and became 5.10 μg/dL in 6 h. After 12 h, it rather at 48 h. Tagging sites were not affected significantly in decreased to 1.26 μg/dL a bit and became 0.90 μg/dL in whole-body glucose and lactic acid concentration 24 h and 0.86 μg/dL in 48 h. The whole-body cortisol (P> 0.05). The change of whole-body glucose concentra- concentrations of caudal vasculature, abdomen, and back tion according to exposure was seen compared to that at tagged groups were 0.81, 0.92, and 1 μg/dL, respectively, pre-experiment, and the whole-body glucose and lactic and has been rapidly increased to 14.76, 15.60, and acid concentrations were the highest at 12 h in the control 15.49 μg/dL in 1 h and increased drastically in 6 h group. However, the times observed when the highest glu- (P< 0.05). The whole-body cortisol concentrations of cose and lactic acid concentrations of the three groups the three experimental groups were the highest at 12 h, were delayed were 24 and 48 h. and became 29.43, 29.80, and 30.43 μg/dL, respectively. In 24 h, the whole-body cortisol concentrations of the Discussion three groups decreased rapidly until 48 h (P< 0.05). The In assessing the tagging sites for fish, it is important to es- tagging sites were not affected significantly in whole-body tablish the effect of the tag, including the tag retention at Fig. 3 The whole-body cortisol concentration variations of the tagged marine medaka, Oryzias dancena, during 48 h Im et al. Fisheries and Aquatic Sciences (2017) 20:21 Page 7 of 10 Fig. 4 The whole-body glucose variations of the tagged marine medaka, Oryzias dancena, during 48 h the tagging site, the rate of tag detection following the tag- are lower, greater attenuation of red light may occur (Willis ging site,and thesurvivalrateof the tagged fish (Frederick, and Babcock, 1998). In direct sunlight, red tags were clearly 1997; Dewey and Zigler, 1996; Park et al., 2013; Willis and detectable at up to 5 m distant in clear water (Pottinger Babcock, 1998). Statistically significant differences were and Calder, 1995). found among tag colors and sites, with red and green tags As shown in Fig. 3, the VIE tag affected the survival of being easier to detect and distinguish under visible and UV marine medaka in the laboratory (P< 0.05). In conclusion, lights than yellow tags in marine medaka, O. dancena.In survival was significantly higher in the control group than contrast, Park et al. (2013) reported that red and orange in any of the experimental groups. Among the experimen- were easier to detect and identify than green and yellow tal groups, fish tagged in the abdomen site showed the when viewed under UV light, but green and yellow were lowest survival. Therefore, skilled injection of the elasto- easily detected in visible light in a greenling, Hexagrammos mer is crucial for keeping the mortality low, as suggested otakii.However,as inthe current study,red tags were more by the decrease in mortality of marked fish during the easily detected than green or yellow tags (Willis and laboratory experiment (Frederick, 1997). In previous Babcock, 1998). In deeper water, where natural light levels study, the primary causes of mortality among the tagged Fig. 5 The whole-body lactic acid concentration variations of the tagged marine medaka, Oryzias dancena, during 48 h Im et al. Fisheries and Aquatic Sciences (2017) 20:21 Page 8 of 10 samples were internal damage and infection, as a result of activity of the gluconeogenesis enzyme; also, this gas bladder rupture, and infection from anatomical increase is the result of a second reaction to the first trauma caused by handling (Willis and Babcock, 1998). reaction (response of hormone) to stress (Barton and The causes of mortality among experimental groups were Iwama, 1991). not determined in this study, and histological observations Buckley et al. (1994) found that in juvenile reef fish, of post-mortem samples are necessary for investigating Sebastes spp., the VIE tags could be detected visually in the causes of mortality. The results of this experiment are situ for up to 258 days using underwater UV lights. In similar to those of the previous studies showing high response to concerns about amphibian declines, Jung et retention of VIE (Dewey and Zigler, 1996; Willis and al. (2000) evaluated and validated amphibian monitoring Babcock, 1998) in the marine medaka, which had > 90% techniques using VIE tags in studies in the Shenandoah tag retention for the back tagging site (Table 1), with the and Big Bend national parks, USA. Godin et al. (1995) caudal vasculature and the abdomen having lower tag re- found that to identify populations of shrimp, Penaeus tention rates. The various characteristics of this species vannamei, individuals could be tagged internally using must be investigated to determine the greater loss of VIE an externally visible elastomer. Basic considerations in tag retention for the caudal vasculature and the abdomen the use of tags in fisheries management or research in- sites during the experimental period. clude the effects of the tags on animal survival, behavior, The use of VIE tagging in small fish, marine medaka, growth, permanency, and recognition and the cost of the is advocated as a practical and reliable method for fish marking technique (McFarlane and Beamish, 1990; Park identification and monitoring, but it may cause negative and Lee, 2001). VIE tags are made of non-toxic medical effects on growth and mortality. Foreign materials such grade fluorescent elastomer material and have been used as tags can lead to stress and may cause changes of the successfully to identify fish, amphibians, and decapod blood reactions of fish. When stress is induced, the fish crustaceans (Willis and Babcock, 1998; Jerry et al., 2001; consume energy, which drives a response of excess Bailey, 2004). The retention rate was 92% for visual im- secretion of catecholamine and cortisol, and has a con- plant elastomer (VIE) tags in juvenile crayfish, Cherax siderable influence on the maintenance of homeostasis destructor, and 100% for VIE tags in lobsters, Homarus (Schreck et al., 2001). Plasma cortisol and plasma glu- gammarus (Jerry et al., 2001; Uglem et al., 1996). As cose are recognized as useful indicators of stress in fish with VIE tags, passive inductive transponder (PIT) tags (Schreck et al., 2001). In our study, whole-body physio- are sometimes used in experiments. However, in a study logical responses of marine medaka from each tagging involving injection of small, mid-sized, and large tags region, in the form of high whole-body cortisol, whole- into four small Cyprinidae fish species, Carassius gibelio body glucose, and lactic acid values, were generally ob- langsdorfi, Hypophthalmichthys molitrix, Pseudorasbora served in tagged groups in which a tag had actually been parva, and Phoxinus phoxinus, Jang et al. (2007) re- inserted compared to the responses seen in control ported that PIT tags caused high mortality. The larger groups. This finding indicates that the actual insertion of and heavier PIT tags can affect the swimming ability of a tag rather than just a pierce injection can result in small fish, including marine medaka. Thus, Jang et al. added stress, and this result shows that tagging sites (2007) concluded that PIT tags are inappropriate for were not affected significantly in stress response. small individuals. So, the VIE tags are small, light, and The plasma cortisol levels induced by stress appear to made of non-toxic medical grade fluorescent elastomer increase at various speed and time according to the spe- material and are therefore more appropriate for small in- cies of fish (Pickering and Pottinger, 1989). The plasma dividuals and species, including marine medaka, and are cortisol concentration after stress is usually reported to considered effective for laboratory experiments and increase to a peak value within 1 ~ 3 h and normally aquaculture facilities. Unfortunately, the relationship recovers within 6 h (Willis and Babcock, 1998). As a among decreasing survival rate, spawning behavior, and whole, the whole-body cortisol values for the tagged VIE tag was not determined by the previous studies. group were similar to the values seen in the control Thus, future investigation will focus on the relationship group after 48 h. Therefore, the time required for the among reduced survival rates, spawning behavior, and black rockfish to adapt after the insertion of a tag is ap- VIE tag. proximately 48 h. The whole-body cortisol concentra- tions showed peak values before 48 h in this study. The Conclusions trends in cortisol and glucose observed in this experi- During 12 months, the accumulated survival rates of ment indicated generalized stress reactions. Glucose for- marine medaka, Oryzias dancena, in the experimental mation was increased simultaneously as the cortisol treatments were not different among red, yellow, and quantity increased. Elevated cortisol secretion under green elastomer. The experimental fish retained >85% of stress increases the activation of plasma glucose by the the tags injected in the back, >70% of the tags injected Im et al. Fisheries and Aquatic Sciences (2017) 20:21 Page 9 of 10 in the caudal vasculature, and >60% of the tags injected Barton BA, Iwama GK. 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