**ABSTRACT NOT FOR CITATION WITHOUT AUTHOR PERMISSION. The title, authors, and abstract for this completion report are provided below.  For a copy of the full completion report, please contact the author via e-mail at mwilkie@wlu.ca. Questions? Contact the GLFC via email at frp@glfc.org or via telephone at 734-662-3209.**

Environmental AND Physiological Determinants of Larval Sea Lamprey Tolerance AND

Resilience to TFM Exposure

¹Michael P. Wilkie, and ²Jeffrey Slade

¹Department of Biology and Laurier Institute for Water Science, Wilfrid Laurier University

Waterloo, Ontario, N2L 3C5.

²Ludington Biological Station, Ludington MI. Retired May 2014.

January 2017

ABSTRACT:

The lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), is typically applied to streams infested with larval sea lampreys at least once every 5 years, resulting in the eradication of multiple generations of these invasive species with a single treatment. However, treatment success can be confounded by “residual” larval sea lampreys, which survive treatment and eventually undergo metamorphosis, before migrating downstream where they parasitize/prey upon economically and culturally important fishes in the Great Lakes. The overarching goal of the present study was to identify the abiotic and biotic factors that can result in “treatment residuals” following TFM treatments. Accordingly, the objectives of our research were to: (I) establish if seasonal differences in water temperature explain the greater tolerance of larval lampreys to TFM in summer, (II) determine if sea lamprey recover more rapidly from TFM exposure in warmer waters, (III) explain how water pH influences the sensitivity of sea lampreys to TFM and their recovery from TFM exposure, and (IV) ascertain how body size, life stage and the physiological condition of lampreys influences TFM sensitivity. To determine how seasonal changes in temperature altered TFM sensitivity (Obj. I), larval sea lampreys were collected during the spring (May), summer (June, August) and fall (October) from the Au Sable River, MI and transported to the Hammond Bay Biological Station (HBBS), where acute toxicity tests were completed within 1-2 weeks of capture. These experiments confirmed that TFM tolerance was greatest in late summer (August), when the minimum lethal concentration of TFM fatal to 50% and 99.9% (LC50, LC99.9 over 12 h) of the population sampled was 2.7-3.0 fold greater compared to values measured in spring. Follow-up toxicity tests, conducted the following summer in cool (6ºC), moderate (12ºC) or warm (21ºC) waters at the HBBS, indicated that TFM tolerance increased in warmer waters, as illustrated by a strong linear (R2 > 0.98) increase in the 12-h LC50 with temperature, which was more than 2-fold greater at 21ºC compared to 6ºC. Potential indices of sea lamprey tolerance to TFM (Obj. IV), including whole body, brain and liver glycogen and lipid reserves, were only marginally affected by season (time of year) and temperature in untreated (no TFM) animals. We therefore conclude that the greater tolerance of sea lampreys to TFM in summer is mainly due to warmer water temperatures, and that the physiological condition of the animals plays a secondary role. HPLC analysis indicated the whole body concentrations of the TFM detoxification product TFM-glucuronide were just above levels of detection, showing no variation in concentration with season or temperature. The mRNA coding for the enzyme involved in TFM-glucuronide production, UDP-glucuronyltransferase (UDPGT), was detected, suggesting that the sea lamprey may have limited capacity to biotransform TFM to TFM-glucuronide. To determine how temperature influenced TFM loading and the capacity to recover from TFM exposure (Obj. II), radio-labelled TFM (14C-TFM) was used to track rates of TFM uptake (ṀTFM) and elimination. Water temperature markedly influenced ṀTFM, which increased linearly with temperature, and was 2.3-fold greater at 22ºC compared to 6ºC. Yet no differences were detected in rates of TFM elimination measured over 24 h following the injection of TFM, suggesting that a greater capacity for TFM-detoxification or other processes accounted for the relatively higher tolerance of larval sea lampreys to TFM at warmer temperatures. Similar experiments using 14C-TFM also demonstrated a clear inverse relationship between water pH and TFM uptake (Obj. III), indicating that the majority of TFM uptake across the gills took place by simple diffusion in its un-ionized, more lipophilic form (TFM-OH). ṀTFM was 4-5 fold greater at low pH (pH 6.5) compared to higher pH (pH 9.0), when there was virtually no TFM-OH in the water. However, ṀTFM was not eliminated at pH 9.0, suggesting that significant amounts of TFM were taken-up in its ionized (TFM-O-) form at pH 9.0. In contrast to uptake, TFM elimination was enhanced when sea lamprey, loaded with known amounts of 14C-labeled TFM, were introduced into more alkaline water. Combined with previous studies indicating that sea lampreys can rapidly recover from short-term TFM exposure, these findings suggest that even short-term interruptions of TFM treatments (e.g. due to mechanical failure, changes in water pH, altered water flow, behavioural avoidance), could enhance the probability of treatment residuals. Biotic factors also influenced TFM sensitivity (Obj. IV), but not completely as expected. As hypothesized, both oxygen consumption (ṀO2) and ṀTFM were inversely proportional to body mass, with respective rates of ṀO2 and ṀTFM being up to 5-fold and 15-fold in the smallest (< 0.5 g) compared to the largest larvae (~ 3.6 g). Each scaled allometrically to increases in body mass (M) as described by the respective allometric equations: ṀO2 = 1.89·M0.57 and ṀTFM = 6.47·M0.26. Surprisingly, no differences in ṀO2 or ṀTFM were observed between comparably sized larval and post-metamorphic juvenile sea lampreys. Neither body mass or length affected the 9-h LC50 or LC99.9 (MLC; Minimum Lethal Concentration) of TFM. However, larger animals survived TFM exposure for much longer than smaller larval sea lampreys. Notably, survival approached or exceeded 9 h in larval sea lampreys greater than 90 mm in length. These findings suggest that for typical 9 h treatments, in which the MLC is based on a population average, that larger larval sea lampreys are much more likely to be a source of residuals than smaller animals. We conclude that variation in abiotic and biotic factors can markedly influence the effectiveness of TFM treatments. It may therefore be prudent to consider the effects that variations in water temperature, pH and body size have on treatment success when planning and carrying out TFM treatments to further reduce numbers of residual sea lampreys.