Low Mortality of Larval Lampreys from Electrofishing, Suction-pumping, Anesthesia, and Handling

Fisheries conservation and management often involves monitoring and research requiring capturing and handling of live fish. Ideally, capture and handling of live fish is done in a manner that minimizes stress. The failure to do so can lead to reduced survival and increased susceptibility to other stressors such as pathogens or predators (Kelsch and Shields 1996). Anesthetics, commonly buffered tricaine methanesulfonate (MS-222; Ross and Ross 2008), are often used to mitigate the stress response of fish. Researchers and managers need to consider the effect on the target fish, their conservation status, and the goals and objectives of the research or management action when developing the sampling and handling techniques in the study design. Furthermore, for fishes with a conservation status (e.g., endangered or threatened; US Endangered Species Act [ESA 1973, as amended]) the process of acquiring the required permits for research or management can be affected by knowledge of how the target fish respond to the collection techniques.

The widespread decline of the anadromous Pacific Lamprey Entosphenus tridentatus has prompted conservation action, most notably in the Columbia River Basin (Close et al. 2002; Luzier et al. 2011). The status of the co-occurring Lampetra spp. (i.e., Western Brook Lamprey L. richardsoni and River Lamprey L. ayresii) are largely unknown. Furthermore, worldwide declines in lamprey populations have created conservation needs (Renaud 1997). Larval lamprey (Figure 1), which live-burrow in fine stream sediments, are typically targeted at the freshwater larval stage for research and assessment (Moser et al. 2007; Cowx et al. 2009). Although they are detectable using standard electrofisher settings that are typically employed in salmonid surveys (Dunham et al. 2013), prolonged exposure to electrofishing can produce narcosis and prohibit the emergence of burrowed larvae; thus, specialized techniques are preferred for studies targeting larval lamprey (Bergstedt and Genovese 1994; Pajos and Weise 1994; Moser et al. 2007). Specifically, backpack electrofishing has been adapted to target these larvae through the delivery of 2-stage electrical current (Weisser and Klar 1990).

Larval lamprey also burrow in fine sediments in relatively deeper water areas that are not accessible to backpack electrofishing. A boat-mounted electrofisher and suction pump combination (Figures 2A, 2B) has been used in large, mainstem rivers (i.e., Columbia River, Washington [Harris and Jolley 2017]; Willamette River, Oregon [Jolley et al. 2012]; St. Marys River, Michigan [Fodale et al. 2003]) to collect larval lamprey from these habitats. The deepwater electrofisher consists of a covered, electrified grid connected to a pump (Figure 2). The electric field under the cover irritates larvae and causes them to emerge from the substrate, where they are pumped to the surface and screened through a collection basket. Although the effect of sampling and handling on larval lamprey has not been explicitly assessed, anecdotal and observational evidence suggests that lamprey are resilient to these activities (Jolley et al. 2013; Kurath et al. 2013; Jolley et al. 2015).

We evaluated the effect of typical collection protocols (backpack or deepwater electrofishing, suction-pumping, anesthesia, and handling) on the survival of larval Pacific Lamprey and Lampetra spp. Specifically, we determined the survival of larval lamprey subjected to 1) handling only (i.e., control trial), 2) backpack electrofishing and anesthesia, 3) backpack electrofishing without anesthesia, 4) deepwater electrofishing and suction-pumping with anesthesia, and 5) deepwater electrofishing and suction-pumping without anesthesia. Our expectation through anecdotal and observational experience was that these activities would have a minimal impact on larval lamprey short-term survival. We also evaluated whether hematocrit levels reflect increases in stress by examining unstressed (i.e., handling only) compared with supposedly highly stressed (electrofishing and suction-pumping without anesthesia) larvae. We expected hematocrit to be higher in larvae that were stressed relative to unstressed fish.

We used Pacific Lamprey that were larvae captive-housed at Eagle Creek National Fish Hatchery for the control trial (see Jolley et al. 2015 for description of rearing configuration). We removed 10 larvae from their rearing vessels on 7 July 2015 and placed them in buckets of hatchery water supplied with oxygen for transport to the laboratory at the Columbia River Fish and Wildlife Conservation Office where all survival monitoring trials occurred. We used 15 lamprey larvae for each of the remaining trials. We collected wild, larval lamprey individuals from North Fork Eagle Creek near the confluence of the mainstem of Eagle Creek (Clackamas River Drainage, Clackamas County, Oregon) on 7 July 2015 (water temperature was 19.7°C, conductivity was 50.1 μS/cm). We collected larvae using an AbP-2 backpack electrofisher (ETS Electrofishing, Verona, WI). The AbP-2 electrofisher produces two distinct pulse frequencies (or outputs); the primary output is pulsed direct current in a 3:1 pulse pattern (i.e., every fourth pulse deleted), which appears to effectively stimulate the emergence of lamprey larvae from their burrows without inducing muscle tetany and immobilizing them in the substrate (Weisser and Klar 1990; Moser et al. 2007). The secondary output is a higher frequency, standard direct current pulse (30 Hz) that is activated to induce muscle tetany and to aid the dip-netting capture of emergent larvae in the water column (Weisser and Klar 1990; Hintz 1993; Bowen et al. 2003). We chose lamprey randomly for anesthesia or nonanesthesia trials. We gently handed lamprey in the nonanesthesia trials and placed them in buckets of creek water supplied with oxygenators for transport (<60 min transport time) to the laboratory. We immobilized lamprey in the anesthesia trials with MS-222 (1–4 min; 150 mg/L), measured them (total length), and placed them in recovery buckets of creek water as described above for transport to the laboratory. We also collected wild, larval lamprey from the Wind River near the confluence of the Columbia River within Bonneville Reservoir (Skamania County, Washington) on 15 July 2015 (water temperature was 22.9°C, conductivity was 95.5 μS/cm). We collected larvae using a deepwater electrofisher described in Jolley et al. (2012). We again randomly assigned larvae to anesthesia or nonanesthesia trials and processed them as described above.

We placed larvae that were returned to the laboratory for monitoring of survival in covered, plastic containers (61.5 × 41.2 × 22.4 cm), supplied them with 1–2 rocks (approximate diameter 9–18 cm) sourced from the North Fork of Eagle Creek as refugia. We supplied containers with supplemental oxygen and 38 L of water from the source of lamprey collection (i.e., Eagle Creek National Fish Hatchery, North Fork Eagle Creek, or Wind River). We maintained temperature at 11°C. We examined larvae every 24 h for survival, with observations beginning immediately upon larvae arrival at the laboratory. We judged any larvae observed actively moving to be alive and exhibiting normal behavior. We very gently prodded larvae that were lying motionless on the bottom of the container for evidence of active movement. We closely examined larvae that were not moving and unresponsive to stimuli and subsequently judged them to be dead if we observed no sign of respiration. We made observations for 96 h, at which time we terminated the trials.

We evaluated mean total length (when collected) for differences among trials using 1-way analysis of variance (ANOVA). We evaluated differences in survival rates among trials using Fisher's Exact Tests multivariate permutation technique (Brown and Fears 1981). We evaluated mean hematocrit levels between nonstressed and stressed fish using a t-test. We examined mean hematocrit for differences over time (i.e., control, 0, 10, 30 min) using a 1-way ANOVA. We conducted all statistical analyses with the Statistical Analysis System (SAS Institute 2012) at a significance level of α = 0.05. When fish were anesthetized or euthanized, we identified them to genus using pigmentation patterns on the caudal fin (Goodman et al. 2009; Docker et al. 2016). We did not identify other fish to genus; therefore, Pacific Lamprey and Lampetra spp. results are largely pooled.

To evaluate hematocrit levels, we used captive larvae from Eagle Creek National Fish Hatchery as nonstressed control fish to establish baseline hematocrit levels. We removed fish from their rearing vessels and immediately euthanized them in an overdose of MS-222. We measured total length in millimeters. We removed the posterior one-third of the body and extracted blood from the vessels using heparinized microhematocrit tubes. We centrifuged blood samples at 5,000 × g for ≥5 min and recorded the percent of red blood cells in the whole blood (i.e., hematocrit) with a hematocrit reader (Clay-Adams Company, Inc., New York, NY). We also measured hematocrit for fish that were stressed through collection, handling, and anesthesia with a deepwater electrofisher (from the Wind River). The response time of larval hematocrit to stress was unknown, so we measured hematocrit as described above for groups of five larvae at approximately 0 (i.e., immediate), 10, and 30 min after capture.

We used both Pacific Lamprey E. tridentatus and Lampetra spp. in the trials. Fish from Eagle Creek National Fish Hatchery (survival control group and baseline hematocrit) were all Pacific Lamprey (Jolley et al. 2015) and had been undisturbed for >1 y. Fish from the North Fork of Eagle Creek were predominantly Pacific Lamprey while fish from the Wind River were largely Lampetra spp. (Table 1; Data S1, Supplemental Material). Larvae ranged in total length from 68 to 165 mm. The group of larvae used for baseline hematocrit measurements (from Eagle Creek National Fish Hatchery) were longer than fish used in the backpack and deepwater electrofishing trials that were anesthetized, while total length was similar among all survival trials (F = 12.13, df = 3, P < 0.05; Table 1).

One lamprey died during the trials. This individual was from the backpack electrofishing trial without anesthesia (93% survival) and was found 22 h after initiating the trial; it was observed to have an injury to the branchiopore region of the head. Survival was 100% in all other trials and did not differ among trials (Fisher's Exact Test; P = 0.99; Data S1, Supplemental Material). Daily observations revealed larvae actively swimming. Hematocrit ranged from 13 to 38% and did not differ between the baseline and stressed fish (t = 0.48, df = 29, P = 0.63). Hematocrit was not different when measured at 0, 10, or 30 min after electrofishing (F = 0.74, df = 3, P = 0.54; Figure 3) nor was any trend apparent over time.

After conclusion of the trials, approximately 5 d after collection, four additional mortalities were observed (one from the control trial, three from the backpack electrofishing + anesthesia trial). All dead larvae had fungus on the head, mouth, and tail and showed signs of internal hemorrhaging. Two individuals were found to have Aeromonas hydrophila (assessed by K. Lujan, U.S. Fish and Wildlife Service).

The survival rate of Pacific Lamprey and Lampetra spp. larvae when subjected to backpack and deepwater electrofishing, with or without anesthesia, was high in our study. We observed only one mortality in the study, which suggests that larval lamprey are resilient to these collection types. We noted one larvae to be injured after collection from North Fork Eagle Creek; trauma occurring during collection likely led to the mortality of this fish during the experiment. Furthermore, hematocrit of lamprey collected through electrofishing and suction (assumed highly stressful event) pumping did not differ from a nonstressed control group. Electrofishing, suction-pumping, and anesthesia showed no short-term negative effects on larval lamprey and these techniques provided efficient and safe methods for collecting and surveying larval lamprey.

The stress of the capture and handling techniques we used did not appear to cause hematocrit to increase in the larval lamprey. This was evident by similar hematocrit levels in all test groups. Hematocrit is an indicator of stress (Iwama et al. 1997), and the secondary stress response (Pickering 1981) has been employed in other fish studies (Gaulke et al. 2014; Cho et al. 2015). In addition, the levels of hematocrit we observed were predominantly between 20 and 30%, within the typical range reported for larval lamprey (see Reis-Santos et al. 2008). Given that the response of hematocrit levels in fish to a stressor is also a function of time, it is possible the larvae were stressed and we did not capture the peak hematocrit over the time period examined, although similar time frames have been used by others (Reis-Santos et al. 2008; Ferreira-Martins et al. 2016). Hematocrit levels are generally expected to climb relatively quickly in response to stress (Krise and Binkowski 1996; Sopinka et al. 2016). Researchers often evaluate stress by comparing treatment with control animals, similar to our methods (Fazio et al. 2015). It is also possible that this was not a stressful event for larvae and therefore hematocrit levels did not spike or that the hematocrit response was difficult to interpret (Sopinka et al. 2016). Finally, it is also possible that the collection techniques were not stressful. Additional investigations on the time-response of larval hematocrit to stress or more conclusive techniques (e.g., cortisol) to document stress are warranted and should not be overlooked when feasible.

Delayed mortality occurred after the 96-h study, with four individuals developing fungus and two confirmed to be infected with A. hydrophila. How our sampling, study design, or holding configuration may have contributed to these results is unclear and beyond the scope of our study. Aeromonas hydrophila (the causative agent of hemorrhagic septicemia) and Saprolegnia spp. (fungus) are ubiquitous in the aquatic environment (Daskalov 2006; van West 2006) and have been documented from healthy larval lamprey collected from these areas (Jolley et al. 2011). Aeromonas hydrophila has also been documented as one of the predominant bacterium of the gut biome of larval Pouched Lamprey Geotria australis (Rogers et al. 1980), and Aeromonas spp. were also present in the Sea Lamprey Petromyzon marinus gut biome (Tetlock et al. 2012). Water temperature is well-known to affect the prevalence of fish-borne diseases, and our study occurred at or near the peak of summer when surface-water temperatures were nearly 20°C at some collection locations. Mueller et al. (2006) noted fungal infections of larval Pacific Lamprey that were tagged with Passive Integrated Transponders were a source of mortality at warmer water temperatures. We recommend using best practices of disinfecting sampling equipment prior to each use. The prevalence and ecology of lamprey pathogens remains a strong area of interest and is poorly documented and understood in lamprey species (Maitland et al. 2015).

The typical electrofisher settings for larval lamprey (i.e., low-pulse, low-frequency) are likely relatively benign in the short term, and suction-pumping and handling showed no harmful effects. Long-term effects such as delayed mortality, behavioral changes, and growth effects that could lead to secondary mortality remain unstudied. Current sampling techniques (electrofishing and suction-pumping) for sampling larval lamprey are reasonable, do not cause adverse effects on larval lamprey over short time periods, and are beneficial relative to conservation and monitoring goals. Capture and handling of live fish is necessary for fisheries conservation and management, and our techniques minimize mortality and stress to native larval lampreys of the Pacific Northwest.

Please note: The Journal of Fish and Wildlife Management is not responsible for the content or functionality of any supplemental material. Queries should be directed to the corresponding author for the article.

Data S1. Survival data for larval Pacific Lamprey Entosphenus tridentatus and Lampetra spp. exposed to five treatments from the Pacific Northwest in 2015.

Found at DOI: http://dx.doi.org/10.3996/052017-JFWM-046.S1 (16 KB XLSX).

Reference S1. Jolley JC, Silver GS, Whitesel TA, Telles L. 2011. Captive rearing of Pacific lamprey. Vancouver, Washington: U.S. Fish and Wildlife Service.

Found at DOI: http://dx.doi.org/10.3996/052017-JFWM-046.S2; also available at https://www.fws.gov/columbiariver/publications/captive_lamprey2010final.pdf (1.2 MB PDF).

Reference S2. Jolley JC, Silver GS, Whitesel TA. 2013. Captive rearing of Pacific lamprey. Vancouver, Washington: U.S. Fish and Wildlife Service.

Found at DOI: http://dx.doi.org/10.3996/052017-JFWM-046.S3; also available at https://www.fws.gov/columbiariver/publications/EC_lamprey_AR_2012_final.pdf (1.9 MB PDF).

Reference S3. Luzier CW, Schaller HA, Brostrom JK, Cook-Tabor C, Goodman DH, Nelle RD, Ostrand K, Streif B. 2011. Pacific lamprey (Entosphenus tridentatus) assessment and template for conservation measures. Portland, Oregon: U.S. Fish and Wildlife Service.

Found at DOI: http://dx.doi.org/10.3996/052017-JFWM-046.S4; also available at https://www.fws.gov/columbiariver/publications/USFWS_Pacific_Lamprey_Assessment_and%20_Template_for_Conservation_Measures_2011.pdf (5.2 MB PDF).

We thank S. Hansen and B. Silver (U.S. Fish and Wildlife Service) for field and technical assistance. S. Hillman and J. Podrabsky (Portland State University) provided histological equipment. C. Peterschmidt (U.S. Fish and Wildlife Service) provided space and resources to house larval lamprey at Eagle Creek National Fish Hatchery. K. Lujan (U.S. Fish and Wildlife Service) performed evaluations of lamprey mortalities. L. Beck and two anonymous reviewers provided comments on earlier drafts. References to trade names do not imply endorsement by the U.S. Government.

Any use of trade, product, website, or firm names in this publication is for descriptive purposes only and does not imply endorsement by the U.S. Government.