Metamorphosing juvenile sea lamprey were captured in the Fort River, MA, during September 23–27, 2013, from stream sediment using backpack electrofishers (Model ABP-2, ETS Electrofishing Solutions, Madison WI) following US Fish and Wildlife Service standard collection protocol (slow pulse = 3 pps; fast pulse = 30 pps; duty cycle = 25%; burst = 3.1; voltage = 125; ). Sea lamprey were temporarily held at the Conte Anadromous Fish Research Laboratory, Turners Falls, MA, in 2.0 m diameter × 1.5 m depth (4000 L) circular flow-through tanks fed with water from the Connecticut River, with 10–15 cm deep river sediment covering the bottom. Fort River sea lamprey were transported to the Rubenstein Ecosystem Science Laboratory on October 10, 2013. Juvenile sea lamprey were also captured during outmigration from two tributaries of Lake Champlain: Morpion Stream, QC, Canada, and Mallets Creek, Colchester, VT, between October 28 and December 2, 2013; these lamprey were held in-stream in cylindrical mesh cages (29 cm × 42 cm) and transported to the Rubenstein Laboratory on December 6, 2013. Fort River and Lake Champlain sea lamprey were held in separate tanks.
Sea lamprey were housed in either round (observation) tanks (1.2 m × 0.8 m polyethylene) or a rectangular (holding) tank (0.42 m × 0.66 m × 0.28 m plastic). All tanks were fed with recirculating dechlorinated water from a single 568-L head tank with a 1/8 hp (93.3 W) pump at a rate of approximately 20 L per minute. Water temperature was maintained between 3.7 and 6.5 °C for the duration of the study except two events during which water was warmed to >10 °C (February 13–14 and February 24–26) and then rapidly cooled to <5 °C during a 24-h period. Water temperature was maintained using a 1-hp (746 W) inline chiller (Delta Star, Aqua-Logic, Inc.). Beach sand 2–10 cm deep covered the bottom of all tanks. A simulated fall photoperiod with 11 h of light and 13 h of darkness was maintained using overhead fluorescent lights set on a timer. The rectangular holding tank was maintained at 0.22 m water depth (74 L capacity). The circular tanks were maintained at either 0.42 or 0.76 m water depth (340 or 620 L capacity, respectively). Counterclockwise flow was maintained at a rate of 0.05–0.15 m/s, measured with a portable, Marsh–McBirney flow meter (model 201D), around a center column placed in both circular tanks using 38-L/min pumps. Water depth, temperature, and circulation velocity were varied in the two circular tanks as part of a separate behavioral observation study.
Lamprey were anesthetized by immersion in a 0.026 mL/L concentration of AQUI-S 20E (AQUI-S, New Zealand). When lamprey became unresponsive, they were removed from the anesthetic, weighed to the nearest 0.01 g, and measured to the nearest 1 mm. A sterile scalpel was used to make a 2–3-mm incision on the left lateral side approximately 20 mm posterior to the gill pores. A half-duplex PIT tag (12 mm × 2.12 mm, 83 mg, Oregon RFID) was inserted by hand and guided posteriorly into the body cavity away from the incision. Implanted tags have been observed to move posteriorly during healing ; therefore, we inserted tags posteriorly through the incision to prevent potential reopening of the wound and tag shedding as the tag moved past the incision. Tagged lamprey were placed in an aerated bucket with fresh water until they became mobile and then transferred to a 38-L observation tank set in a chilled water bath (held at same temperature as the larger holding tanks) for 24 h to monitor short-term survival and tag retention. Lamprey were then stocked into a 1.2-m circular tank described above. Untagged control lamprey were measured after the observation period, to avoid stress related to handling; controls were a random subset of the lamprey from each site and therefore assumed to have a similar length range to the tagged lamprey in their respective groups.
Short-term survival after tagging
Two groups of sea lamprey from the Fort River were used for direct comparison of survival for tagged and untagged (control) animals. Group 1 sea lamprey (n = 20 tagged; 23 control) were tagged November 19, 2013, and group 2 sea lamprey (n = 20 tagged; 15 control) were tagged December 6, 2013; each group was housed in a separate observation tank. Tanks were monitored daily Monday to Friday, and sea lamprey were removed if dead, or generally unresponsive and showing obvious signs of impairment (i.e. minimal reaction to stimulus, large protruding viscera, etc.), or exhibiting signs of fungal growth. Any visible symptoms were recorded at the time of removal. The effect of tagging on survival was evaluated by comparing Kaplan–Meier survival curves of tagged and control sea lamprey from groups 1 and 2 at 24 h post-tagging and on January 22, 2014, 64 day (group 1) and 47 day (group 2) post-tagging using a log-rank analysis.
Long-term survival and tag retention
After conclusion of the tagged versus control observation period, all tagged sea lamprey from groups 1 and 2 were stocked into a common observation tank and held for long-term assessment. Additionally, 110 sea lamprey from Lake Champlain tributaries, hereafter referred to as group 3, were tagged February 4, 2014, and stocked into a separate observation tank; for these lamprey, survival during the 60 day prior to tagging was monitored for comparison with tagging mortality. The long-term survival of all tagged sea lamprey was monitored until March 17, 2014.
Initial total length of sea lamprey was compared among tagging groups using ANOVA, and length differences between groups were identified using Tukey–Kramer HSD test. A logistic regression analysis using a likelihood ratio test (LRT) was used to determine whether total length influenced survival. All analyses were conducted in JMP ® Pro 11 (SAS Institute Inc., Cary, NC).
All tagged sea lamprey were scanned for tags using a PIT reader, and incisions were inspected on March 4, 2014. Percent tag retention was calculated for each tagging group. The proportion of sea lamprey in each tagging group with wounds sufficiently healed to no longer be vulnerable to tag loss was calculated. Incisions with healed skin, healed muscle, and the internal muscle appearing closed were considered sufficiently healed to prevent tag loss while incisions that remained open or with bulging muscle were considered still vulnerable to tag loss.