The Tagging Efficiency and Physiological Responses of Passive Integrated Transponder Tags in Black Rockfish, Sebastes schlegeli

The Tagging Efficiency and Physiological Responses of Passive Integrated Transponder Tags in Black Rockfish, Abstract Passive integrated transponder (PIT) technology has been used for the identification of individual animals. PIT tags are inserted in the dorsal muscle, abdominal cavity, sham dorsal muscle, and sham abdominal cavity of the black rockfish, Sebastes schlegeli . During the experimental period, the tag retention rate and readability rate were greater in dorsal tags than in abdominal cavity tags in black rockfish ( P < 0.05). Plasma cortisol and plasma glucose as indicators of stress were measured according to the passage of time after tagging. During 192 hrs, the tag insertion group showed higher plasma cortisol and plasma glucose levels compared to the sham group, and this similar pattern was seen in the abdominal cavity insertion group compared to the dorsal muscle insertion group ( P < 0.05). While inserting PIT tags, stress can be minimized by insertion in the dorsal muscle of a fish.

concentration rises by stress is the second-order reaction by hormone rise reaction caused by stress". Although PIT tags are used through insertion in the fish body, fish can be affected by its presence. The effects of these tags can differ in fish of different regions. Many fish species have transparent tissues that are suitable for tagging, including the abdominal cavity, head region, dorsal muscle, and tail muscle [10]. Sites suitable for retaining tags differ among species [10]. The aim of this study was to insert PIT tags into black rockfish of two regions in order to assess the stress that occurs in black rockfish through the insertion of PIT tags, and to investigate their physiological responses for determining the most suitable tagging site.

Materials and Methods
In September 2018, black rockfish, Sebastes schlegeli were The populations were divided into five experimental groups: one in which a PIT tag was inserted in the dorsal muscle and another in which a PIT tag was inserted in the abdominal cavity, two sham groups in which each underwent pierce injection only, and a control group (Figure 1). Twenty-four fish were used for each group, except the control group, which was composed of five fish. The blood physiology response experiment began on January 2019. For the fish blood physiology response experiment, the food supply was disrupted 24 hrs prior to sampling. A blood physiology response experiment of PIT tags for each tagging region  Co., Japan) prior to analysis. The plasma cortisol concentration was measured using 1470 WIZARD Automatic Gamma Counter (Cobra, Packard Co., USA) after the antigen antibody response was derived using Coat-A-count TKCO Cortisol RIA Kit (DPC, USA) according to the [14] method. The plasma glucose concentration was analyzed using a Chemistry System (Hitachi 7180, Hitachi, Japan). The differences among groups were analyzed by one-way ANOVA using the SPSS Statistics Package (SPSS 9.0, SPSS Inc., USA), and multiple comparisons were performed using Duncan's multiple range test [15].

Results
During the experiment periods, no samples were died from the stress of syringe injection. (Table 1) lists the survival rates in each of the three experimental groups, as well as the tag retention and readability rates in black rockfish, Sebastes schlegeli tagged with PITs in the abdominal cavity or dorsal muscle. One month after tagging (September 2018), the tag retention rate of abdominal group was lower than dorsal group (October 2018), the survival rate was lower in the abdominal group than in the other groups.
The tag retention rate was significantly lower in the abdominal group than in dorsal groups (P < 0.05). However, the tag readability rate was not significantly different among the experimental groups (P > 0.05). Two months after tagging, the survival rate was not significantly different among each group (P > 0.05). Although the cumulative retention rate of abdominal group was significantly lower than dorsal groups (P < 0.05), the tag readability rate was not significantly different among the three experimental groups. Three months after tagging, the survival rate in the abdominal group was significantly lower than the control group (P < 0.05). The trends in the tag retention and readability rates at 3 months were similar to those at 2 months.
As shown in Table 1, four months after tagging, the survival rate in the abdominal group was lower than the dorsal and control groups. The trends in tag retention and readability rates at 4 months were similar to those at 2 and 3 months. Five months after tagging, the survival rates were not significantly different among the experimental groups (P > 0.05). The cumulative retention rate of abdominal group was lower than that of dorsal groups. Six months after tagging, the trends in survival and tag retention rates were similar to those at 5 months. The tag retention and readability rates of abdominal group were lower than dorsal groups. The number of surviving fish, retained tags, and readable tags were the lowest in the abdominal group. The tag retention and readability rates in the dorsal group were significantly higher than those of abdominal groups throughout the experimental period. The plasma cortisol and plasma glucose concentration in As shown in (Figures 3a & 3b), the plasma glucose concentration of fish was 53.2±8.67 mg/dL at the beginning of the experiment.
The plasma cortisol concentration tended to be increased with the passage of time after tagging at 1 hr, and decreased from 6 hrs; the control group showed a similar tendency after 12 hrs. From the results that compared the four experimental groups in each time, groups in which a tag had been inserted generally displayed higher values at 1, 3 and 9 hrs compared to the sham groups (P < 0.05), and the abdominal cavity insertion group generally displayed higher values than the dorsal muscle insertion group at 1, 3 and 6 hrs (P < 0.05).

Discussion
Implanted tags caused no internal damage, presumably because their midventral location prevented most contacts with organs and dissipated the risk of erosion. The encapsulation of most tags by host tissue and the resulting limited migration of tags inside the cavity may also added to the reduction of this risk [16,17]. [18]'s study has demonstrated that PIT tagging in the abdominal cavity and the dorsal muscle in kelp grouper, Epinephelus bruneus has the potential to reliably identify individuals and is associated with high tag retention rates, readability and fish survival. Although mass gain of the dorsal muscle tagged group was slightly lower than in the abdominal cavity tagged group, no difference was established in all other factors. Based on the criteria and variables outlined in [18]'s study, the use of PIT tagging in the abdominal cavity of kelp grouper is advocated as a practical and reliable method for fish identification and monitoring, without negative effects on growth and mortality.
However, foreign materials such as tags can lead to stress, and may cause changes of the blood reactions of fish. When stress is induced, fish consume energy, which drives a response of excess secretion of catecholamine and cortisol and has a considerable influence on the maintenance of homeostasis [12,[19][20][21].
Plasma cortisol and plasma glucose are recognized as useful indicators of stress in fish [11]. Notable increases in plasma cortisol and plasma glucose levels were reported in the black rockfish, Sebastes schlegeli, after exposure to MS-222 and lidocaine-HCl anesthetic [22]. In this study, blood physiology responses of black The plasma cortisol levels induced by stress appear to increase at various speed and time according to the species of fish [12].
The plasma cortisol concentration after stress is usually reported to increase to a peak value within 1~3 hrs, and normally recovers within 6 hrs [23]. As a whole, plasma cortisol and plasma glucose values in the sham groups and the groups in which tags had been inserted were similar to the values seen in the control group after 6 hrs. Therefore, the time required for the black rock fish to adapt after the insertion of a tag is approximately 6 hrs. Also, the plasma cortisol and plasma glucose concentrations showed peak values before 6 hrs in this study; these values agree with those reported by [23]. The trends in cortisol and glucose observed in this experiment indicated generalized stress reactions. Glucose formation was increased simultaneously as the cortisol quantity increased.

Elevated cortisol secretion under stress increases the activation of plasma glucose by activity of the gluconeogenesis enzyme and this
increase is also the result of a second reaction to the first reaction (response of hormone) to stress [12,24].
In this study, groups in which a tag was inserted suffered from more stress than the sham groups. In addition, the group in which a tag was inserted in the abdominal cavity suffered from less stress than the group that experienced insertion of a tag in the dorsal muscle, but this difference was fairly minor. Therefore, the insertion of PIT tags into the dorsal muscles of fish will minimize stress compared to other sites of insertion. This study examined the most suitable site for the stress responses by PIT tags. As mentioned by [9,25], the tissue reaction to the implanted tag included an increased abundance of fibroblasts in the fibrous capsule, which represents chronic inflammation rather than acute inflammation.
So, future investigation needs to be focused on determining the most suitable site by the histological responses to PIT tags which were inserted into two sites in this species.

Conclusion
A variety of tagging products have been tried depending on the tagging aim. The passive integrated transponder (PIT) tag, have been shown to be a reliable and effective method for identification and monitoring [9,26,27] as well showing little or no effect on fish growth and survival [10,28]. The effects of PIT tags inserted in the dorsal muscle, abdominal cavity, sham dorsal muscle, and sham abdomind cavity of the black rockfish, Sebastes schlegeli on the tag retension rate, readability rate and physiological responses (plasma cortisol and plasma glucose as indicators of stress) was examined for 6 months. During the experimental period, the tag retension rate and readability rate were greater in dorsal tags than in abdominal cavity tags in black rockfish. While inserting PIT tags, stress can be minimized by insertion in the dorsal muscle of a fish.

Ethics Approval and Consent to Participate
The experiments performed in this study complied with the

Conflict of Interests
The author has no financial or personal conflicts of interests.