Cold Temperature Tolerance of Albino Rainbow Shark (Epalzeorhynchos Frenatum), a Tropical Fish with Transgenic Application in the Ornamental Aquarium Trade

Canadian Journal of Zoology Cold temperature tolerance of albino rainbow shark (Epalzeorhynchos frenatum), a tropical fish with transgenic application in the ornamental aquarium trade Journal: Canadian Journal of Zoology Manuscript ID cjz-2018-0208.R1 Manuscript Type: Note Date Submitted by the 23-Sep-2018 Author: Complete List of Authors: Leggatt, Rosalind; Department of Fisheries and Oceans, CAER Is your manuscript invited for Draft consideration in a Special Not applicable (regular submission) Issue?: COLD HARDINESS < Discipline, GENETIC ENGINEERING < Discipline, Keyword: TEMPERATE < Habitat, FRESHWATER < Habitat, FISH < Taxon, ANIMAL IMPACT < Discipline, TEMPERATURE < Discipline https://mc06.manuscriptcentral.com/cjz-pubs Page 1 of 14 Canadian Journal of Zoology 1 1 2 3 4 5 Cold temperature tolerance of albino rainbow shark (Epalzeorhynchos frenatum), a 6 tropical fish with transgenic application in the ornamental aquarium trade 7 8 R.A. Leggatt 9 Centre for Aquaculture and the Environment, Centre for Biotechnology and Regulatory 10 Research, Fisheries and Oceans Canada 11 4160 Marine Drive, WestDraft Vancouver, BC, Canada, V7V 1N6 12 [email protected] 13 Tel: 1-604-666-7909; Fax: 1-604-666-3497 14 15 https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 2 of 14 2 16 Cold temperature tolerance of albino rainbow shark (Epalzeorhynchos frenatum), a 17 tropical fish with transgenic application in the ornamental aquarium trade 18 19 R.A. Leggatt 20 21 Abstract: Application of fluorescent protein transgenesis has commercial use for the ornamental 22 aquarium trade by creating new colour phenotypes in various species. To determine the potential 23 for transgenic ornamental aquarium fish to overwinter in Canada, the minimum temperature 24 tolerance of albino rainbow shark (Epalzeorhynchos frenatum Fowler 1934) was estimated, to 25 complement a previous study examining cold tolerance of zebrafish (Danio rerio Hamilton, 26 1822), black tetra (Gymnocorymbus ternetziDraft Boulenger, 1895), and tiger barb (Puntius tetrazona 27 Bleeker, 1855) (Leggatt et al. 2018). Rainbow shark had higher low temperature tolerance limits 28 (LD50 = 10.7 ± 0.1 ºC) than surveyed winter water temperatures in Canada. There was a 29 significant negative correlation between condition factor and temperature at loss of equilibrium, 30 suggesting fish with lower social status may be more susceptible to cold temperature than 31 dominant fish. These results indicate transgenic E. frenatum are not expected to persist over 32 winter in Canadian waters. 33 34 Keywords: 35 rainbow shark, Epalzeorhynchos frenatum, thermal minimum, temperature, fluorescent protein, 36 transgene, risk assessment, survival, overwinter, social, aquarium trade; lower lethal temperature 37 38 https://mc06.manuscriptcentral.com/cjz-pubs Page 3 of 14 Canadian Journal of Zoology 3 39 Manuscript 40 In a recent study, the cold temperature tolerance of three wild-type tropical fish species 41 (zebrafish, black tetra, tiger barbs) with application as fluorescent transgenic fish in the 42 ornamental aquarium trade was determined (Leggatt et al. 2018). Chronic cold tolerance of these 43 fish was examined in the context of minimum recorded surface water temperatures in Canada to 44 determine the potential for fluorescent transgenic tropical fish used in the ornamental pet trade to 45 establish in Canadian freshwater systems. The paper concluded a lack of potential for the 46 examined fish to establish in Canadian waters, as minimum cold temperature tolerance of 47 examined lines and species was higher than measured winter water temperatures in Canada 48 (Leggatt et al. 2018). Since this study was published, an additional transgenic freshwater fish has 49 been commercialized within the United DraftStates ornamental aquarium trade, specifically two lines 50 of fluorescent transgenic rainbow shark (Epalzeorhynchos frenatum Fowler 1934). The rainbow 51 shark is native to Cambodia, Lao People’s Democratic Republic, Thailand and Viet Nam where 52 it lives in freshwater rivers, marshlands and floodplains (Vidthayanon 2012). However, the cold 53 tolerance of this species, and hence its ability to persist in Canadian waters, is not known. In this 54 note, the chronic cold temperature tolerance was examined for the base morph of the novel 55 transgenic fish, specifically the albino form of E. frenatum. 56 57 The experiment was reviewed by the Pacific Region Animal Care Committee and conducted 58 under an institutional animal care permit (AUP18-007) meeting guidelines established by the 59 Canadian Council on Animal Care (Ottawa, Ontario, Canada). Albino rainbow sharks were 60 purchased from a local aquarium wholesaler (Little Fish Co., Surrey, BC, Canada) and reared in 61 triplicate 37 L aquarium. The fish were reared in static dechlorinated municipal water (West https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 4 of 14 4 62 Vancouver, BC, Canada), aerated and filtered, and held at 24 ºC on 10 h light: 14 h dark to 63 mimic winter light cycles. Tank environments were enriched with gravel, two 4 inch diameter 64 PVC pipe “caves” and one plastic plant per tank. Fish were fed a mixed diet of commercial 65 flakes, algae wafers, bottom-feeder wafers, frozen blood worms and brine shrimp, 1-2 times per 66 day. One week after arrival at the research facility, mortalities were noted in the population and 67 diagnostics revealed an external monogean trematode infection. Three treatments of 1 hour 160 68 ppm formalin baths were administered to the fish over 1 week to combat the infection, and 3 ppt 69 Instant Ocean aquarium salts (Spectrum Brands, Blacksburg, VA, USA) were added to the 70 rearing tanks for the remainder of the experiment. Two weeks after the last formalin treatment, 71 fish were reallocated among tanks so that two tanks contained 15-16 fish each (experimental 72 tank) and one tank contained 9 fish (controlDraft tank). The two experimental tanks were connected to 73 DS-3 in-line chillers (AquaLogic Inc. San Diego, CA, USA) via Laguna Statuary 2 pumps 74 (Hagen Inc., Montreal, QC, Canada). Temperature in the two experimental tanks was dropped to 75 20.5 ºC over 2 weeks and held at 20.5 ± 0.5 ºC for 1 week prior to starting the cold temperature 76 trial. Throughout the trial the temperature in the experimental tanks was dropped rapidly by 1 ºC 77 at approximately 8:30am each day, and maintained at this temperature (± 0.5ºC) for 24 h. 78 Temperature was logged every minute by Tidbit or Pendant monitors (Onset Computer 79 Corporation, Bourne, MA, see Supplemental Figure S1 for temperature traces during the 80 experiment). Fish were monitored 2 times a day for activity level, feeding behaviour, and ability 81 to maintain equilibrium until fish stopped eating, then monitored 4 times per day. Once fish 82 started losing equilibrium, fish were monitored a minimum of every 15 min during the day. 83 When a fish lost equilibrium, it was removed and euthanized by 400 mg/L buffered tricaine 84 methanesulfonate (Syndel Canada, Nanaimo, BC, Canada) at current tank temperature, time and https://mc06.manuscriptcentral.com/cjz-pubs Page 5 of 14 Canadian Journal of Zoology 5 85 temperature recorded, and fish weight and length recorded. Condition factor was calculated as 86 [weight (g)]/[length (cm)]3100. Total ammonia, pH and nitrite levels were monitored 87 throughout the experiment: pH was stable at 7.5 - 7.6, while ammonia and nitrite were not 88 detectable. 89 90 The rainbow sharks in the experimental tanks began decreasing feeding and activity at 91 approximately 17 ºC, stopped feeding at approximately 13 ºC, and stopped swimming activity at 92 approximately 12 ºC. There was no difference in average temperature at loss of equilibrium 93 (LOE) between the two experimental tanks (P = 0.776) as analyzed using the lm and anova 94 functions in R (R Core Team 2018), and data from the two tanks were subsequently pooled. Fish 95 lost equilibrium between 16.0 and 9.6 ºC,Draft and the majority of fish (80%) lost equilibrium within 96 a 1.8 ºC temperature range from 11.4 to 9.6 ºC (see Figure 1). The lethal dose for 50 % of the 97 population (LD50) was calculated as 10.7 ± 0.1 ºC using the dose.p function of the MASS 98 package (Venables and Ripley 2002) in R (R Core Team 2018). 99 100 Rainbow sharks are known to be territorial and aggressive to conspecifics. In the approximately 101 2 months from the fish arriving at the facility to the start of the experimental treatment, fish 102 formed dominance hierarchies, with 1-2 larger fish dominating each tank. Weight, length and 103 condition factor of experimental fish are given in Table 1 and fish fell into a large range of 104 weight (3.5-fold difference between smallest and largest fish), length (1.39-fold difference) and 105 condition factor (1.84-fold difference). The large dominant fish were some of the final fish to 106 lose equilibrium, and there was a weak but significant negative correlation between temperature 107 at LOE and condition factor (P < 0.001, R2 = 0.418, see Figure 2), but not with weight (P = https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 6 of 14 6 108 0.056, R2 = 0.006) or length (P = 0.671, R2 = 0.121). It is reasonable to speculate that subordinate 109 fish experienced stress due to negative social interactions (e.g. Gilmour et al. 2005, Jeffrey et al. 110 2012) than may have contributed to loss of equilibrium at higher temperatures. Indeed, of the 111 four fish that lost equilibrium before the 1.8 ºC range of the majority of fish, all had condition 112 factor less than 1.0 (see Figure 2), suggesting low social status, secondary infection and/or poor 113 overall health of these fish (e.g.

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