General experimental procedures
This study used 3-year-old commercially reared chub mackerel (Table 4). Mean body mass of experimental fish was 459 ± 64 g. The fish were caught by commercial purse seine fisheries in the coastal area around Kyushu, Japan when they were 0-year-old fish, and were grown to maturity in a net cage. We carefully checked fish conditions prior to the experiments and used only healthy fish in the experiments.
The fish were anaesthetized with 2-phenoxyethanol (200–275 ppm solution), measured for fork length (cm) and body mass (g), and were held on the platform for the tag attachment. Seawater was circulated through the mouth of the fish with a water pump to aid in respiration during tag attachment. To identify all fish, we inserted a Passive Integrated Transponder (PIT) tag (BIO12B.01, BIOMARK Inc., Idaho, USA) into muscular tissue near the dorsal fin before applying the experimental treatment.
Experiments were conducted in a round tank ~5 m diameter and ~1 m in depth (i.e. ~20 t of water). The tank was exposed to ambient light and temperature (i.e., light and temperature were not regulated). The experimental system was confirmed to be appropriate for small pelagic fishes, including chub mackerel, in previous studies [e.g. 26]. During the experiments, we observed fish each day and fed dried pellets to the fish two or three times a day. During feeding, if diseased or moribund fish were found, they were caught, identified, and their fork length and body mass were measured. Subsequently, necropsies were conducted on these fish.
Due to a technical difficulty, we could not measure the body mass of 12 fish (seven from the Implantation-A group and five from the Implantation-C group) at the beginning of the experiment at the cold temperature. Body mass (g) of these fish was therefore estimated by the power function of fork length (cm) from other individuals in the same lot [Body mass = 0.0062 × Fork length3.2446, R
2 = 0.7264, n = 59, P < 0.0001].
Experiment at warm temperature
This experiment was conducted from 30 September to 15 November 2013 (46 days). Water temperature at the start of the Experiment I was ~24 °C and subsequently dropped to ~19 °C. Changes in the daily water temperature were 0.30 ± 0.06 °C (n = 44).
Five archival tags and 40 dummy tags were used. LAT2800 (LOTEK Wireless Inc., Ontario, Canada), a relatively small tag, was used in this study. Few case studies of small scombrids have been carried out, and none of them specify an optimal size for tags. Because the LAT2800 is formed by mixing an epoxy resin and an amine-based epoxy hardener, the dummy tags were prepared using similar materials. All tags were 11 mm in diameter and 38 mm in length, and weighed 6.1 ± 0.2 g in air (n = 45). The mean ratio of tag mass to fish body mass was 1.4 ± 0.2 % (n = 45).
There were three tagged groups (Implantation-A, External-1, and External-2) and three untagged groups (Untagged, Implant-sham, and External-sham) with 15 fish in each group in the experiment. In the Implantation-A group, a ~20 mm incision was made through the skin and muscle of the lower abdominal region of the fish with a scalpel, and a sterile tag was implanted into the peritoneal cavity. The incision was sutured with a round needle with a circular cross section (17 mm in length) and two stitches of a thread made from absorbable polyglycolic acid (Opepolyx N, Alfresa Co., Ltd., Tokyo, Japan). The incision was then sealed with an α-cyanoacrylate-based quick-setting adhesive (Aron Alpha, Toagosei Co. Ltd., Tokyo Japan). In the External-1 group, we pitted two tiny holes (<2 mm in diameter) near the dorsal fin using stainless steel needles, and the tag was fixed using two plastic cable ties (<2 mm in width) through the holes. This method is commonly used in field studies of marine fish and allows long-term monitoring over months [27–29]. In the External-2 group, we made one tiny hole near the dorsal fin and the tag was towed using a silicon-coated stainless steel wire (1.5 mm in diameter).
We did not conduct any treatment in the Untagged group (i.e., we only inserted the PIT tag). The Implant-sham and External-sham groups were used to examine the effects of the surgical operation itself. In the Implant-sham group, the surgical operation was similar to that in the Implantation-A group, but no tag was implanted into the peritoneal cavity. In the External-sham group, we made one 2-mm hole near the dorsal fin, but neither the tag and plastic cable nor the stainless steel wire was attached.
Experiment at cold temperature
This experiment was conducted from 4 December 2013 to 23 January 2014 (50 days) and began with water temperature at ~15 °C, which subsequently fell to ~10 °C. Changes in the daily water temperature were 0.47 ± 0.05 °C (n = 49). No significant difference in the rate of change in the daily water temperature was noted between the warm and cold conditions (t test, t = 1.6296, df = 91, P = 0.1066).
Because low survival rates were observed in the External-1 and External-2 groups under warm-temperature conditions, external attachment groups were not included in this experiment. This experiment included three treatment groups (Implantation-A, Implantation-B, Implantation-C) and one untagged group (Untagged). In the Implantation-A group, tags were identical to those used at warm temperatures. In the Implantation-B group, the tags were wrapped in paraffin film (Parafilm, Pechiney Plastic Packaging, Menasha, WI, USA), which might have some benefits in terms of biological compatibility (Y. Makiguchi, personal communication). In the Implantation-C group, tags that were half the length and weight of standard tags were used, in order to reduce drag. The small tags were also wrapped in paraffin film. The mean ratios of tag mass to fish body mass were 1.32 ± 0.08 % for the Implantation-A group (n = 7), 1.29 ± 0.10 % for the Implantation-B group (n = 8), and 0.67 ± 0.08 % for the Implantation-C (n = 7) group. To reduce the possible number of fish lost, the sample size of each group was reduced in comparison to sample sizes in the warm-water experiment.
Specific Growth Rate (SGR) was calculated using the following equation: SGR = (lnM
1 − lnM
2)/Δd × 100 where M
1 and M
2 are body mass at the start and the end of the experiment, and Δd is the number of days in the experiment.
The survival rate of each group was estimated using the Kaplan–Meier method. Data from cases in which a tag was expelled from an individual, or in which an individual survived until the end of each experiment, were treated as censored data. Differences in survival among groups were examined using the log-rank test. The Cox regression model (proportional hazards analysis) with a likelihood ratio test was fitted to determine which components of tagging affected fish survival. To determine whether water temperature alters the effect of tag deployment on fish survival, two Cox regression models (one with and one without temperature as a factor) were compared . To fit these models, we used the data of the Implantation-A groups at both temperatures and the untagged groups at both temperatures, because no other variables (i.e., tag size, tag coating, surgery type, etc.) in these groups changed between the warm-condition experiment and the cold-condition experiment.
The cutoff for statistical significance was set at α = 0.05 for all statistical analyses. In cases where multiple comparisons were performed, a Bonferroni correction was used to keep the overall probability of a Type I error at α = 0.05.