Canadian Journal of Zoology

Cold temperature tolerance of albino rainbow ( 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 (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 . 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 ( 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 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 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 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)]3100. 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. Gilmour et al. 2005). This would concur with other studies that

114 found lower social status resulted in decreased tolerance to high temperatures (LeBlanc et al.

115 2011), impaired hypoxia tolerance (Thomas and Gilmour 2012), impaired defense against

116 bacterial infection (Peters et al. 1988), and impaired capacity to respond to acute stress (Jeffrey

117 et al. 2012) in rainbow trout (Oncorhynchus mykiss Walbaum 1792), possibly due to higher

118 energy demands and chronic stress of subordinateDraft versus dominant fish (Sloman and Armstrong

119 2002, Jeffrey et al. 2012, Thomas and Gilmour 2012). However, the low correlation factor of

120 LOE and condition factor (R2 = 0.418) suggests that while poor condition may play a role in

121 temperature tolerance, it is not a primary factor driving overall temperature tolerance.

122

123 Fish in the control tank remained active and fed normally throughout the experiment with no

124 mortalities or loss of equilibrium. These fish also had a large range in weight (4.3-fold difference

125 between smallest and largest), length (1.4-fold difference) and condition factor (1.60-fold

126 difference, see Table 1), and observed dominance hierarchy-associated behaviour. While ideally

127 the control group should have had equal number of fish as the treatment tanks, the lack of

128 morbidity or mortality in the control group demonstrates the loss of equilibrium observed in the

129 treatment tanks was due to the decreased temperature and not an underlying issue in the overall

https://mc06.manuscriptcentral.com/cjz-pubs Page 7 of 14 Canadian Journal of Zoology

7

130 population. There was no significant difference among the three tanks in weight (P = 0.388),

131 length (P = 0.091), or condition factor (P = 0.185).

132

133 This is the first report of cold tolerance and response to cold temperatures for rainbow shark. All

134 rainbow sharks lost equilibrium at temperatures higher than those measured for minimum water

135 temperatures in Canada (Leggatt et al. 2018). While the dominance hierarchies formed may have

136 diminished the cold tolerance of subordinate fish, with potential overestimation of LD50, even

137 the most cold tolerant fish had LOE temperature (minimum 9.6 ºC) at least 5 °C above that of the

138 majority of winter water temperatures (≤ 4 ºC), and at least 3 °C above the warmer freshwater

139 systems in Canada (≤ 6 ºC, see Leggatt et al. 2018). These data indicate that rainbow sharks

140 cannot survive Canadian winter water temperatures.Draft There could potentially be isolated pockets

141 of warm water in Canada (e.g. thermal springs) with temperatures in the required range for

142 rainbow sharks persistence. Other biotic and abiotic requirements for survival and reproduction

143 are not well characterized for rainbow shark, and it is not known if these requirements would be

144 met in isolated pockets of warmer waters in Canada. Tuckett et al. (2017) found escaped rainbow

145 sharks adjacent to an ornamental aquaculture facility in Florida, but not downstream of the

146 facility, suggesting even in warmer climates rainbow sharks are not expected to persist and

147 spread if released. This is further supported by lack of reported occurrences of escaped E.

148 frenatum in the USA (US Geological Survey of Nonindigenous Aquatic Species database,

149 https://nas.er.usgs.gov/default.aspx), and Hill et al. (2017) rated E. frenatum as non-invasive,

150 based on a medium risk score using the Fish Invasiveness Screening Kit. While the effect of

151 fluorescent transgenesis on cold tolerance was not examined in this species, fluorescent zebrafish

152 (Cortemeglia and Beitinger 2005, 2006; Cortemeglia et al. 2008; Leggatt et al. 2018) and tetras

https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 8 of 14

8

153 (see DFO 2018) have equal or diminished cold-tolerance relative to wild-type fish. Consequently

154 fluorescent transgenesis is not expected to improve cold tolerance of rainbow sharks. Overall, the

155 current results support that fluorescent transgenic E. frenatum used in the aquarium trade are not

156 expected to overwinter and persist in Canadian freshwater environments, should they be

157 released.

158

159 Acknowledgements

160 This project was funded by the Canadian Regulatory System for Biotechnology. Thank you to

161 Robert H. Devlin for review of this manuscript. Christine MacWilliams and Christy Thompson

162 were responsible for diagnostics work on experimental fish, and Athena Csuzdi, Harrison Tadey

163 and Brendan Yates assisted in fish care Draftand monitoring.

164

165 References

166 Cortemeglia, C., and Beitinger, T.L. 2005. Temperature tolerances of wild-type and red

167 transgenic zebra . Trans. Am. Fish. Soc. 134: 1431-1437.

168 Cortemeglia, C., and Beitinger, T.L. 2006. Projected US distributions of transgenic and wildtype

169 zebra danios, Danio rerio, based on temperature tolerance data. J. Therm. Biol. 31: 422-428.

170 Cortemeglia, C., Beitinger, T.L., Kennedy, J.H., and Walters, T. 2008. Field confirmation of

171 laboratory-determined lower temperature tolerance of transgenic and wildtype zebra danios,

172 Danio rerio. Am. Midl. Nat. 160(2): 477-479.

173 DFO. 2018. Environmental and indirect human health risk assessment of the Glofish® Electric

174 Green® Tetra and the Glofish® Long-Fin Electric Green® Tetra (Gymnocorymbus ternetzi):

175 a transgenic ornamental fish. DFO Can. Sci. Advis. Sec. Sci. Advis. Rep 2018/027.

https://mc06.manuscriptcentral.com/cjz-pubs Page 9 of 14 Canadian Journal of Zoology

9

176 Gilmour, K.M., DiBattista, J.D., and Thomas, J.B. 2005. Physiological causes and consequences

177 of social staus in salmonid fish. Integr. Comp. Biol. 45: 263-273.

178 Hill, J.E., Tuckett, Q.M., Hardin, S., Lawson, L.L.J., Lawson, K.M., and Ritch, J.L. 2017. Risk

179 screen of freshwater tropical ornamental fishes for the conterminous United States. Trans.

180 Am. Fish. Soc. 146: 927-938.

181 Jeffrey, J.D., Esbaugh, A.J., Vijayan, M.M., and Gilmour, K.M. 2012. Modulation of

182 hypothalamic-pituitary-interrenal axis function by social status in rainbow trout. Gen. Comp.

183 Endocrinol. 176(2): 201-210.

184 LeBlanc, S., Middleton, S., Gilmour, K.M., and Currie, S. 2011. Chronic social stress impairs

185 thermal tolerance in the rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 214(10): 1721-

186 1731. Draft

187 Leggatt, R.A., Dhillion, R.S., Mimeault, C., Johnson, N., Richards, J.G., and Devlin, R.H. 2018.

188 Low-temperature tolerances of tropical fish with potential transgenic applications in relation

189 to winter water temperatures in Canada. Can. J. Zool. 96: 253-260.

190 Peters, G., Faisal, M., Lang, T., and Ahmed, I. 1988. Stress caused by social-interaction and its

191 effect on susceptibility to Aeromonas hydrophila infection in rainbow trout Salmo gairdneri.

192 Dis. Aquat. Org. 4(2): 83-89.

193 R Core Team. 2018. R: A language and environment for statistical computing. R Foundation for

194 Statistical Computing, Vienna, Austria. Available from https://www.r-project.org/.

195 Sloman, K.A., and Armstrong, J.D. 2002. Physiological effects of dominance hierarchies:

196 laboratory artefacts or natural phenomena? J. Fish Biol. 61(1): 1-23.

197 Thomas, J.B., and Gilmour, K.M. 2012. Low social status impairs hypoxia tolerance in rainbow

198 trout (Oncorhynchus mykiss). J. Comp. Physiol. B 182(5): 651-662.

https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 10 of 14

10

199 Tuckett, Q.M., Ritch, J.L., Lawson, K.M., and Hill, J.E. 2017. Landscape-scale survey of non-

200 native fishes near ornamental aquaculture facilities in Florida, USA. Biol. Invasions, 19:

201 223-237.

202 Venables, W.N., and Ripley, B.D. 2002. Modern Applied Statistics with S. Springer, New York.

203 Vidthayanon, C. 2012. Epalzeohynchos frenatum. The IUCN Red List of Threatened Species

204 2012. e.T181093A1697683. http://dx.doi.org/10.2305/IUCN.UK.2012-

205 1.RLTS.T181093A1697683.en. Downloaded on 24 July 2018.

206

207 Draft

https://mc06.manuscriptcentral.com/cjz-pubs Page 11 of 14 Canadian Journal of Zoology

11

208 Table 1: Body measurements of rainbow sharks (Epalzeohynchos frenatum) from experimental

209 and control tanks at the completion of the experiment.

210

Experimental Fish (n = 31) Control fish (n = 9)

Weight (g)

average ± SEM 0.74 ± 0.04 0.85 ± 0.16

maximum 1.67 2.04

minimum 0.48 0.47

Fork Length (cm)

average ± SEM 4.03 ± 0.06 4.13 ± 0.17

maximum 5.0 Draft 5.3

minimum 3.6 3.6

Condition factor

average ± SEM 1.11 ± 0.02 1.12 ± 0.06

maximum 1.84 1.37

minimum 0.73 0.86

211

212

https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 12 of 14

12

213 Figure 1: Survival of rainbow sharks (Epalzeohynchos frenatum) during cold temperature trials

214 where temperature was dropped by 1 ºC per day, starting at 20.5 ± 0.5 ºC. Arrows indicate at

215 what temperature fish showed a detectable decrease in activity (e.g. active swimming) and

216 feeding, as well as when they stopped feeding and activity.

217

218 Figure 2: Relationship between temperature at loss of equilibrium (ºC) and weight (g, black

219 diamonds), length (cm, grey squares), or condition factor (white triangles) in rainbow shark

220 (Epalzeohynchos frenatum) exposure to 1 ºC drop in water temperature daily.

221 Draft

https://mc06.manuscriptcentral.com/cjz-pubs Page 13 of 14 Canadian Journal of Zoology

100

80 activity/feeding Decrease stop￿feeding stop￿activity Draft 60

40 %￿survival

20

0 19 14 9 Temperature

https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 14 of 14

5

4

3 Draft

2

1

0 9 11 13 15 Temperature￿at￿loss￿of￿equlilbrium

https://mc06.manuscriptcentral.com/cjz-pubs