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Larval rearing of zebrafish at suboptimal temperatures
Journal of Thermal Biology 74 (2018) 170–173
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Larval rearing of zebrafish at suboptimal temperatures Thomas A. Delomas, Konrad Dabrowski
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T
School of Environment and Natural Resources, The Ohio State University, Columbus, OH 43210, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Temperature-sensitive Conditional mutant Zebrafish Screen Husbandry
Temperature-sensitive mutants have been widely utilized in single-cell and invertebrate model systems, particularly to study the function of essential genes. Few temperature-sensitive mutants have been identified in zebrafish, likely due to the difficulty of raising zebrafish at low temperatures. We describe a novel rearing protocol that allows rapid growth of larval and juvenile zebrafish at 23 °C compared to previous data in the literature. Embryos collected from four breeding pairs were maintained at 28.5 ± 0.5 °C until 5 days postfertilization (dpf) – the onset of exogenous feeding. Larvae were then divided to six tanks and three tanks were cooled to 23 ± 0.2 °C. Fish were fed a live diet (marine rotifers Brachionus plicatilis and Artemia nauplii) and maintained under a set of environmental parameters shown to increase growth rate: continuous light, low salinity (3ppt), and algal turbidity. Mean total length and weight of fish at 21dpf were 12.7 ± 0.3 mm and 20.5 ± 1.5 mg for the 23 °C treatment and 18.5 ± 0.4 mm and 67.3 ± 3.4 mg for the 28.5 °C control. By 35 dpf, the fish raised at 23 °C had reached a mean length and weight of 18.9 ± 0.7 mm and 76.4 ± 6.7 mg, approximately the size control fish reached at 21 dpf. At 35 dpf, water temperature was raised to 28 °C and fish were reared to maturity (75 dpf) under standard conditions (freshwater, 13 L:11D photoperiod, dry diet, no added algal turbidity). Sex ratio and fertility were assessed and compared between temperature groups. There were no significant differences in sex ratio, fertilization rate, embryo viability at 1 dpf, clutch size, or relative fecundity. This rearing protocol will allow for efficient utilization of temperature-sensitive mutations in the zebrafish model system.
1. Introduction Zebrafish were the first vertebrate species to be used in a large-scale genetic screen. These screens identified loss-of-function mutants for multiple genes involved in embryonic development (Driever et al., 1996; Haffter et al., 1996), and success has continued with screens for complex traits, such as behavioral phenotypes (Chiu et al., 2016). The functions of many essential genes are difficult to analyze in loss-offunction mutants due to mortality of the embryo. In other model systems, such as yeast, this has been overcome by screening for conditional mutants, in which the mutated gene is functional under one set of environmental conditions but not fully functional under other conditions. The most frequent examples of conditional mutants are temperature-sensitive mutants. Screens for temperature-sensitive yeast mutants have most notably identified essential genes involved in cell-cycle control (Hartwell et al., 1970; Nurse et al., 1976) and currently there are temperature-sensitive mutant strains available for close to half of all essential genes in Saccharomyces cerevisiae (Li et al., 2011). Temperature-sensitive mutants have also been used to study development throughout the life cycle and cell-cycle control in Caenorhabditis elegans
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Corresponding author. E-mail address: [email protected] (K. Dabrowski).
https://doi.org/10.1016/j.jtherbio.2018.03.017 Received 8 February 2018; Received in revised form 2 March 2018; Accepted 18 March 2018 Available online 19 March 2018 0306-4565/ © 2018 Elsevier Ltd. All rights reserved.
(Hirsh and Vanderslice, 1976; O’Connell et al., 1998). Zebrafish can tolerate (based on loss of equilibrium) water temperatures as low as 6.2 and 10.6 °C when acclimated to 20 and 30 °C, respectively (Cortemeglia and Beitinger, 2005) which allows temperature shifts to be used as an experimental tool in this vertebrate model system (López-Olmeda and Sánchez-Vázquez, 2011). A small number of temperature-sensitive mutants have been found in zebrafish. Temperature-sensitive mutations affecting fin regeneration have been identified (Johnson and Weston, 1995; Nechiporuk et al., 2003) with a permissive temperature of 25 °C, slightly below the optimum temperature for growth of zebrafish (28 °C), and a restrictive temperature of 33 °C. Additionally, one temperature-sensitive mutation in nodal-related 2 (ndr2), also known as cyclops, a nodal-related signaling factor involved in floor-plate specification, was found (Tian et al., 2003). This allowed precise determination of the timing of ndr2 action during embryonic development through temperature shift experiments. Temperature shift experiments were not performed on juvenile fish to investigate the function of ndr2 after the completion of embryonic development, likely due to the difficulty of raising zebrafish at the permissive temperature (22 °C).
Journal of Thermal Biology 74 (2018) 170–173
T.A. Delomas, K. Dabrowski
Table 1 Survival and growth of fish raised at standard (control) and low temperatures during larval and early juvenile stages. All values are given as tank mean ± SD. *calculated with an initial weight of 0.25 mg based on bulk weighing of 25 larvae at 5 days post-fertilization (dpf). Age (dpf)
21
Temperature group Sex
Low Juvenile
Survival (%) Weight (mg) Length (mm) SGR (% day−1)
86.1 20.5 12.7 27.5
± ± ± ±
Control
2.7 1.5 0.3 0.5 *
90.0 67.3 18.5 35.0
± ± ± ±
3.0 3.4 0.4 0.3 *
35
62
Low
Control Male
85.8 ± 2.9 76.4 ± 6.7 18.9 ± 0.7 9.4 ± 0.5
75
Female
86.9 ± 2.8 428 ± 10 589 ± 74 36.4 ± 0.4 38.4 ± 1.7 4.5 ± 0.1 5.3 ± 0.3
Low Male
434 ± 18 36.7 ± 0.4 4.4 ± 0.1
Female 82.1 ± 6.2 572 ± 46 37.8 ± 0.8 5.0 ± 0.3
of exogenous feeding. This allowed us to directly compare the effects of low temperatures on growth after yolk absorption and the physiological transition to exogenous feeding.
Unlike yeast, there has not been widespread screening for temperature-sensitive mutations of essential genes in zebrafish. This is presumably due to the difficulty of raising zebrafish at low temperatures. Even at optimum temperatures, there is wide variation between laboratories in growth and survival of zebrafish (Dabrowski and Miller, 2018; Lawrence, 2011). Recently, a new method of rearing zebrafish that results in high survival and faster growth compared to traditional protocols was developed (Dabrowski and Miller, 2018; Delomas and Dabrowski, 2018). This method utilizes continuous access to live food, low salinity, algal turbidity, and continuous light during the larval and early juvenile stages to increase growth rate. The low salinity allows marine food organisms to stay alive for 24 – 48 h (Conte et al., 1972; Walker, 1981), continuous light allows continuous foraging, as zebrafish do not forage in the dark (Carrillo and McHenry, 2016; McElligott and O’malley, 2005), and the algal turbidity increases contrast of the live food organisms, decreases aggressive interactions between larvae, and acts as a food source for the marine live food (McEntire et al., 2015; Naas et al., 1992; Reitan et al., 1997; Rieger and Summerfelt, 1997). We assessed whether (1) this novel rearing method could be applied at temperatures close to the minimum for growth, (2) result in high survival (3) acceptable growth rate and metamorphosis to juveniles, and (4) sexual maturation after returning to optimum temperatures.
2.2. Rearing larvae and juveniles Fish were raised based on the method described by Dabrowski and Miller (2018) and Delomas and Dabrowski (2018). During the larval and early juvenile stages, fish were maintained in static water containers at low salinity (3 ppt), moderate turbidity (3 – 10 NTU) maintained with Nannochloropsis algae paste (Nanno 3600, Reed Mariculture, Campbell, CA, USA), and 24 L:0D photoperiod. The CO group was fed marine rotifers (Brachionus plicatilis) until 10 dpf, when the water volume in the tank was increased to 6 L (20 fish/L) and the diet was switched to Artemia nauplii. The CO group was maintained under these conditions until 21 dpf. At 21 dpf, 10 – 20 fish from each tank (both CO and LT groups) were measured and the CO group was stocked in a freshwater recirculating system at a density of 2 fish/L and with a 13 L:11D photoperiod. Diet for the CO group was transitioned to dry feed (Otohime B1/B2) supplemented with Artemia nauplii. The CO group was maintained under these conditions for the remainder of the experiment. As fish development and metamorphosis are dependent upon growth (McMenamin et al., 2016), we standardized changes in husbandry based upon size and not time (age). The LT group was maintained under the larval and early juvenile conditions (low salinity, turbidity, continuous light) until 35 dpf, when they had attained approximately the same size as the CO group at 21 dpf (Table 1). Additionally, the LT group was fed marine rotifers until 13 dpf, when they had attained the same mean length as the CO group had at 10 dpf (8.7 ± 0.1 mm, n = 10 fish measured from the CO group at 10 dpf and from the LT group at 13 dpf), and then fed Artemia nauplii until 35 dpf. At 35 dpf, 20 fish from each of the LT tanks were measured and the water temperature was raised to 28.5 ± 0.5 °C over 8 h. Fish were then stocked in a freshwater recirculating system at a density of 2 fish/L and with a 13 L:11D photoperiod. Diet was transitioned to dry feed (Otohime B1/B2) supplemented with Artemia nauplii. The LT group was maintained under these conditions for the remainder of the experiment.
2. Materials and methods 2.1. Broodstock care and reproduction Broodstock zebrafish were from an AB/TL hybrid line and were kept in a freshwater recirculating system (Thoren Aquatic Systems, Hazleton, PA, USA) maintained at 28 ± 1 °C with a 13 L:11D (light: dark) photoperiod. Fish were fed Artemia nauplii supplemented with dry feed (Otohime B2, Reed Mariculture, Campbell, CA, USA). Four breeding pairs were naturally spawned on the same day. Pairs were placed in a tank with a perforated false bottom overnight and allowed to spawn naturally the following morning. Oocytes were collected from all four pairs and incubated in mesh baskets suspended in a recirculating system (Pentair Aquatic Ecosystems, Apopka, FL) maintained at 28.5 ± 0.5 °C. At 5 days post-fertilization (dpf), swim-up larvae were collected and combined. Larvae were distributed randomly into six static water tanks with a water volume of 4 L and 120 larvae per tank (30 fish /L). Three tanks were maintained at 28.5 ± 0.5 °C (control group, CO) while three tanks were passively cooled to 23 ± 0.5 °C (low temperature group, LT) over four hours. The LT group was maintained at 23 °C, as this was reported to be the lower thermal limit for embryonic development in zebrafish (Schirone and Gross, 1968) and therefore is the lowest temperature that could be applied from fertilization to adulthood (egg to egg) in a screen for temperature-sensitive mutations. However, more recent work has shown that zebrafish embryos can survive temperatures as low as 22 °C (Scott and Johnston, 2012). The previously mentioned studies have clearly demonstrated that zebrafish embryos survive at 22 – 23 °C; therefore, we chose to begin the low temperature treatment at the onset
2.3. Sex identification and fertility testing Once fish were large enough to determine their sex by examining external morphology (Parichy et al., 2009) (62 dpf for the CO group and 75 dpf for the LT group), the numbers of males and females in each tank were counted. Five males and five females from each tank were measured. Fertility tests were then performed to determine if the low temperature during early gonadal development had any effect on reproductive performance later in life. A sample of males and females (three and four fish of each sex from CO and LT tanks, respectively) from each tank was tested for fertility. Each male and female was spawned separately with a test fish. Test fish were unrelated to the experimental fish and had previously been proven to have high fertility (over 90% fertilization rate when spawned 171
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amongst themselves). The same group of test fish was used for assessing both the CO and LT groups. Spawning between the experimental fish and the test fish was performed in the same manner as described above in Section 2.1 for the original broodstock fish. Fertilization rate (at the two- to four-cell stage), 1 dpf viability (number alive at 1 dpf/number fertilized), clutch size, and post-spawn weight and length were recorded. Pairs which did not spawn were attempted again one to two days later. All fish spawned within three attempts.
4. Discussion In comparison to non-vertebrate model organisms, there has been a lack of temperature-sensitive mutations identified in zebrafish. We describe a novel rearing protocol that results in high survival and acceptable growth at low temperatures (23 °C), comparable or higher than previously reported for zebrafish at 35 dpf at 27 °C (Uusi-Heikkilä et al., 2015). Despite being cultured at temperatures close to the lower thermal limit for embryonic development, there was no difference in survival between the LT and CO groups. The survival of the LT group at 21 dpf (86.1 ± 2.7%) was higher than that reported by authors using dry feed as a sole food source (Carvalho et al., 2006; Goolish et al., 1999) and comparable to results of other authors utilizing live food during larval and early juvenile stages (Aoyama et al., 2015; Best et al., 2010; Dabrowski and Miller, 2018). Growth rate at 23 °C was expectedly slower than at 28.5 °C. However, growth rate at 23 °C using this method was similar to that reported with other commonly utilized protocols at 28.5 °C. Best et al. (2010) described a protocol for first-feeding larvae with marine rotifers and maintaining low levels of salinity (5 ppt), then transitioning to a freshwater environment and feeding Artemia nauplii at 10 dpf. Utilizing this method at 28.5 °C, the authors reported a mean length of 13 mm at 23 dpf. Carvalho et al. (2006) fed larvae a diet of Artemia nauplii at 28 °C and at 26 dpf the fish had obtained a mean length of 14.3 mm. The current protocol yielded a mean length at 21 dpf of 12.7 ± 0.3 mm for the LT group and 18.5 ± 0.4 mm for the CO group (Table 1). This demonstrates that application of this protocol at low temperatures will allow temperature-sensitive mutations to be identified without reducing growth rate below currently accepted levels. Culturing zebrafish at low temperatures also raises the question of whether the low temperature has an effect on sex determination. Temperature-dependent sex determination has been observed in several fish species (Roemer and Beisenherz, 1996; Strüssmann et al., 1996), with higher temperatures generally causing an increased proportion of males and lower temperatures causing an increased proportion of females. While extreme high temperatures (above 35 °C) have been shown to cause an increase in the proportion of males in zebrafish (Uchida et al., 2004), this results in high mortality and is outside the range of environmental conditions generally experienced in the wild. Therefore, it has been suggested that this is not an evolved response (Ospina-Álvarez and Piferrer, 2008). In this study, low temperature was applied from the beginning of exogenous feeding until fish reached a mean length of 18.9 ± 0.7 mm. Based on the observation of gonad morphology in juvenile zebrafish, sex determination is thought to occur between 12 and 23 mm (Maack et al., 2003), meaning that fish in the low temperature group were exposed to low temperatures through the majority of the sex determination period. There was no difference in sex ratio between the LT and CO groups suggesting that low temperature does not affect sex determination in zebrafish. There were no significant differences in fertility parameters (Table 2) between the LT and CO groups, when female size did not
2.4. Statistical analysis Specific growth rate (SGR) was calculated as lnfinal weight − lninitial weight 100* and was calculated from the last weighing. days Fertility parameters were compared between LT and CO groups using linear mixed models. In all models, temperature treatment was a fixed effect and tank was included as a random effect (random intercept) nested within temperature treatment. For testing each fertility parameter, a t-test was applied to determine if the coefficient for temperature treatment was significantly different from zero. Survival rate and sex ratio were compared between treatments using a Student's t-test with Tank considered to be the experimental unit. F-tests demonstrated that data were homoscedastic between treatments. Variables that were expressed as proportions derived from counts (survival, sex ratio, fertilization rate, viability at 1 dpf) were Arcsin transformed prior to modeling and hypothesis testing. A type I error rate of 0.05 was used for all statistical tests. Statistical tests and modeling were performed using R statistical software ver. 3.3.2 (R Core Team, 2017) and the lme4 (Bates et al., 2015) and lmerTest (Kuznetsova et al., 2017) packages. All values are given as mean ± SD.
3. Results Survival was high in both groups (CO and LT) throughout the life cycle (Table 1). There was no significant difference in survival between the CO and LT groups at any time point. Growth was expectedly more rapid at 28.5 °C than at 23 °C with SGR during the first 21 dpf of 35.0 ± 0.3% day−1 and 27.5 ± 0.5% day−1, respectively (Table 1). After the LT group was transitioned to 28.5 °C, growth rate increased, and was not different from that of the CO group (Table 1). Sex ratio was not significantly different between the two temperature treatments. The mean percent male in the CO and LT groups were 41 ± 5% and 55 ± 12%, respectively. There were no significant differences in fertility parameters (fertilization rate, viability at 1 dpf, clutch size, or relative fecundity) between the temperature groups (Table 2). All fish spawned on the first attempt except for two females, one from the LT group and one from the CO group. The LT female spawned on the second attempt and the CO female spawned on the third attempt.
Table 2 Fertility of fish raised at standard (control) and low temperatures during larval and early juvenile stages. All values are given as mean ± SD. dpf, days postfertilization. Temperature Group
Weight (mg) Length (mm) Age (dpf) Fertilization rate (%) Viability at 1 dpf (%) Clutch size (oocytes/female) Relative fecundity (oocytes/mg)
Females
Males
Low (n = 12)
Control (n = 9)
Low (n = 12)
Control (n = 9)
647 ± 95 39.5 ± 1.6 80 ± 3 82 ± 13 88 ± 10 327 ± 140 0.51 ± 0.21
592 ± 51 39.3 ± 1.4 67 ± 4 81 ± 15 92 ± 6 275 ± 85 0.47 ± 0.14
447 ± 60 37.2 ± 1.6 83 ± 2 83 ± 14 95 ± 7 – –
437 ± 64 37.3 ± 2.6 68 ± 4 91 ± 9 92 ± 6 – –
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differ, suggesting that low temperatures during early development do not affect later reproductive function in zebrafish.
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Larval rearing of zebrafish at suboptimal temperatures Thomas A. Delomas, Konrad Dabrowski
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T
School of Environment and Natural Resources, The Ohio State University, Columbus, OH 43210, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Temperature-sensitive Conditional mutant Zebrafish Screen Husbandry
Temperature-sensitive mutants have been widely utilized in single-cell and invertebrate model systems, particularly to study the function of essential genes. Few temperature-sensitive mutants have been identified in zebrafish, likely due to the difficulty of raising zebrafish at low temperatures. We describe a novel rearing protocol that allows rapid growth of larval and juvenile zebrafish at 23 °C compared to previous data in the literature. Embryos collected from four breeding pairs were maintained at 28.5 ± 0.5 °C until 5 days postfertilization (dpf) – the onset of exogenous feeding. Larvae were then divided to six tanks and three tanks were cooled to 23 ± 0.2 °C. Fish were fed a live diet (marine rotifers Brachionus plicatilis and Artemia nauplii) and maintained under a set of environmental parameters shown to increase growth rate: continuous light, low salinity (3ppt), and algal turbidity. Mean total length and weight of fish at 21dpf were 12.7 ± 0.3 mm and 20.5 ± 1.5 mg for the 23 °C treatment and 18.5 ± 0.4 mm and 67.3 ± 3.4 mg for the 28.5 °C control. By 35 dpf, the fish raised at 23 °C had reached a mean length and weight of 18.9 ± 0.7 mm and 76.4 ± 6.7 mg, approximately the size control fish reached at 21 dpf. At 35 dpf, water temperature was raised to 28 °C and fish were reared to maturity (75 dpf) under standard conditions (freshwater, 13 L:11D photoperiod, dry diet, no added algal turbidity). Sex ratio and fertility were assessed and compared between temperature groups. There were no significant differences in sex ratio, fertilization rate, embryo viability at 1 dpf, clutch size, or relative fecundity. This rearing protocol will allow for efficient utilization of temperature-sensitive mutations in the zebrafish model system.
1. Introduction Zebrafish were the first vertebrate species to be used in a large-scale genetic screen. These screens identified loss-of-function mutants for multiple genes involved in embryonic development (Driever et al., 1996; Haffter et al., 1996), and success has continued with screens for complex traits, such as behavioral phenotypes (Chiu et al., 2016). The functions of many essential genes are difficult to analyze in loss-offunction mutants due to mortality of the embryo. In other model systems, such as yeast, this has been overcome by screening for conditional mutants, in which the mutated gene is functional under one set of environmental conditions but not fully functional under other conditions. The most frequent examples of conditional mutants are temperature-sensitive mutants. Screens for temperature-sensitive yeast mutants have most notably identified essential genes involved in cell-cycle control (Hartwell et al., 1970; Nurse et al., 1976) and currently there are temperature-sensitive mutant strains available for close to half of all essential genes in Saccharomyces cerevisiae (Li et al., 2011). Temperature-sensitive mutants have also been used to study development throughout the life cycle and cell-cycle control in Caenorhabditis elegans
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Corresponding author. E-mail address: [email protected] (K. Dabrowski).
https://doi.org/10.1016/j.jtherbio.2018.03.017 Received 8 February 2018; Received in revised form 2 March 2018; Accepted 18 March 2018 Available online 19 March 2018 0306-4565/ © 2018 Elsevier Ltd. All rights reserved.
(Hirsh and Vanderslice, 1976; O’Connell et al., 1998). Zebrafish can tolerate (based on loss of equilibrium) water temperatures as low as 6.2 and 10.6 °C when acclimated to 20 and 30 °C, respectively (Cortemeglia and Beitinger, 2005) which allows temperature shifts to be used as an experimental tool in this vertebrate model system (López-Olmeda and Sánchez-Vázquez, 2011). A small number of temperature-sensitive mutants have been found in zebrafish. Temperature-sensitive mutations affecting fin regeneration have been identified (Johnson and Weston, 1995; Nechiporuk et al., 2003) with a permissive temperature of 25 °C, slightly below the optimum temperature for growth of zebrafish (28 °C), and a restrictive temperature of 33 °C. Additionally, one temperature-sensitive mutation in nodal-related 2 (ndr2), also known as cyclops, a nodal-related signaling factor involved in floor-plate specification, was found (Tian et al., 2003). This allowed precise determination of the timing of ndr2 action during embryonic development through temperature shift experiments. Temperature shift experiments were not performed on juvenile fish to investigate the function of ndr2 after the completion of embryonic development, likely due to the difficulty of raising zebrafish at the permissive temperature (22 °C).
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Table 1 Survival and growth of fish raised at standard (control) and low temperatures during larval and early juvenile stages. All values are given as tank mean ± SD. *calculated with an initial weight of 0.25 mg based on bulk weighing of 25 larvae at 5 days post-fertilization (dpf). Age (dpf)
21
Temperature group Sex
Low Juvenile
Survival (%) Weight (mg) Length (mm) SGR (% day−1)
86.1 20.5 12.7 27.5
± ± ± ±
Control
2.7 1.5 0.3 0.5 *
90.0 67.3 18.5 35.0
± ± ± ±
3.0 3.4 0.4 0.3 *
35
62
Low
Control Male
85.8 ± 2.9 76.4 ± 6.7 18.9 ± 0.7 9.4 ± 0.5
75
Female
86.9 ± 2.8 428 ± 10 589 ± 74 36.4 ± 0.4 38.4 ± 1.7 4.5 ± 0.1 5.3 ± 0.3
Low Male
434 ± 18 36.7 ± 0.4 4.4 ± 0.1
Female 82.1 ± 6.2 572 ± 46 37.8 ± 0.8 5.0 ± 0.3
of exogenous feeding. This allowed us to directly compare the effects of low temperatures on growth after yolk absorption and the physiological transition to exogenous feeding.
Unlike yeast, there has not been widespread screening for temperature-sensitive mutations of essential genes in zebrafish. This is presumably due to the difficulty of raising zebrafish at low temperatures. Even at optimum temperatures, there is wide variation between laboratories in growth and survival of zebrafish (Dabrowski and Miller, 2018; Lawrence, 2011). Recently, a new method of rearing zebrafish that results in high survival and faster growth compared to traditional protocols was developed (Dabrowski and Miller, 2018; Delomas and Dabrowski, 2018). This method utilizes continuous access to live food, low salinity, algal turbidity, and continuous light during the larval and early juvenile stages to increase growth rate. The low salinity allows marine food organisms to stay alive for 24 – 48 h (Conte et al., 1972; Walker, 1981), continuous light allows continuous foraging, as zebrafish do not forage in the dark (Carrillo and McHenry, 2016; McElligott and O’malley, 2005), and the algal turbidity increases contrast of the live food organisms, decreases aggressive interactions between larvae, and acts as a food source for the marine live food (McEntire et al., 2015; Naas et al., 1992; Reitan et al., 1997; Rieger and Summerfelt, 1997). We assessed whether (1) this novel rearing method could be applied at temperatures close to the minimum for growth, (2) result in high survival (3) acceptable growth rate and metamorphosis to juveniles, and (4) sexual maturation after returning to optimum temperatures.
2.2. Rearing larvae and juveniles Fish were raised based on the method described by Dabrowski and Miller (2018) and Delomas and Dabrowski (2018). During the larval and early juvenile stages, fish were maintained in static water containers at low salinity (3 ppt), moderate turbidity (3 – 10 NTU) maintained with Nannochloropsis algae paste (Nanno 3600, Reed Mariculture, Campbell, CA, USA), and 24 L:0D photoperiod. The CO group was fed marine rotifers (Brachionus plicatilis) until 10 dpf, when the water volume in the tank was increased to 6 L (20 fish/L) and the diet was switched to Artemia nauplii. The CO group was maintained under these conditions until 21 dpf. At 21 dpf, 10 – 20 fish from each tank (both CO and LT groups) were measured and the CO group was stocked in a freshwater recirculating system at a density of 2 fish/L and with a 13 L:11D photoperiod. Diet for the CO group was transitioned to dry feed (Otohime B1/B2) supplemented with Artemia nauplii. The CO group was maintained under these conditions for the remainder of the experiment. As fish development and metamorphosis are dependent upon growth (McMenamin et al., 2016), we standardized changes in husbandry based upon size and not time (age). The LT group was maintained under the larval and early juvenile conditions (low salinity, turbidity, continuous light) until 35 dpf, when they had attained approximately the same size as the CO group at 21 dpf (Table 1). Additionally, the LT group was fed marine rotifers until 13 dpf, when they had attained the same mean length as the CO group had at 10 dpf (8.7 ± 0.1 mm, n = 10 fish measured from the CO group at 10 dpf and from the LT group at 13 dpf), and then fed Artemia nauplii until 35 dpf. At 35 dpf, 20 fish from each of the LT tanks were measured and the water temperature was raised to 28.5 ± 0.5 °C over 8 h. Fish were then stocked in a freshwater recirculating system at a density of 2 fish/L and with a 13 L:11D photoperiod. Diet was transitioned to dry feed (Otohime B1/B2) supplemented with Artemia nauplii. The LT group was maintained under these conditions for the remainder of the experiment.
2. Materials and methods 2.1. Broodstock care and reproduction Broodstock zebrafish were from an AB/TL hybrid line and were kept in a freshwater recirculating system (Thoren Aquatic Systems, Hazleton, PA, USA) maintained at 28 ± 1 °C with a 13 L:11D (light: dark) photoperiod. Fish were fed Artemia nauplii supplemented with dry feed (Otohime B2, Reed Mariculture, Campbell, CA, USA). Four breeding pairs were naturally spawned on the same day. Pairs were placed in a tank with a perforated false bottom overnight and allowed to spawn naturally the following morning. Oocytes were collected from all four pairs and incubated in mesh baskets suspended in a recirculating system (Pentair Aquatic Ecosystems, Apopka, FL) maintained at 28.5 ± 0.5 °C. At 5 days post-fertilization (dpf), swim-up larvae were collected and combined. Larvae were distributed randomly into six static water tanks with a water volume of 4 L and 120 larvae per tank (30 fish /L). Three tanks were maintained at 28.5 ± 0.5 °C (control group, CO) while three tanks were passively cooled to 23 ± 0.5 °C (low temperature group, LT) over four hours. The LT group was maintained at 23 °C, as this was reported to be the lower thermal limit for embryonic development in zebrafish (Schirone and Gross, 1968) and therefore is the lowest temperature that could be applied from fertilization to adulthood (egg to egg) in a screen for temperature-sensitive mutations. However, more recent work has shown that zebrafish embryos can survive temperatures as low as 22 °C (Scott and Johnston, 2012). The previously mentioned studies have clearly demonstrated that zebrafish embryos survive at 22 – 23 °C; therefore, we chose to begin the low temperature treatment at the onset
2.3. Sex identification and fertility testing Once fish were large enough to determine their sex by examining external morphology (Parichy et al., 2009) (62 dpf for the CO group and 75 dpf for the LT group), the numbers of males and females in each tank were counted. Five males and five females from each tank were measured. Fertility tests were then performed to determine if the low temperature during early gonadal development had any effect on reproductive performance later in life. A sample of males and females (three and four fish of each sex from CO and LT tanks, respectively) from each tank was tested for fertility. Each male and female was spawned separately with a test fish. Test fish were unrelated to the experimental fish and had previously been proven to have high fertility (over 90% fertilization rate when spawned 171
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amongst themselves). The same group of test fish was used for assessing both the CO and LT groups. Spawning between the experimental fish and the test fish was performed in the same manner as described above in Section 2.1 for the original broodstock fish. Fertilization rate (at the two- to four-cell stage), 1 dpf viability (number alive at 1 dpf/number fertilized), clutch size, and post-spawn weight and length were recorded. Pairs which did not spawn were attempted again one to two days later. All fish spawned within three attempts.
4. Discussion In comparison to non-vertebrate model organisms, there has been a lack of temperature-sensitive mutations identified in zebrafish. We describe a novel rearing protocol that results in high survival and acceptable growth at low temperatures (23 °C), comparable or higher than previously reported for zebrafish at 35 dpf at 27 °C (Uusi-Heikkilä et al., 2015). Despite being cultured at temperatures close to the lower thermal limit for embryonic development, there was no difference in survival between the LT and CO groups. The survival of the LT group at 21 dpf (86.1 ± 2.7%) was higher than that reported by authors using dry feed as a sole food source (Carvalho et al., 2006; Goolish et al., 1999) and comparable to results of other authors utilizing live food during larval and early juvenile stages (Aoyama et al., 2015; Best et al., 2010; Dabrowski and Miller, 2018). Growth rate at 23 °C was expectedly slower than at 28.5 °C. However, growth rate at 23 °C using this method was similar to that reported with other commonly utilized protocols at 28.5 °C. Best et al. (2010) described a protocol for first-feeding larvae with marine rotifers and maintaining low levels of salinity (5 ppt), then transitioning to a freshwater environment and feeding Artemia nauplii at 10 dpf. Utilizing this method at 28.5 °C, the authors reported a mean length of 13 mm at 23 dpf. Carvalho et al. (2006) fed larvae a diet of Artemia nauplii at 28 °C and at 26 dpf the fish had obtained a mean length of 14.3 mm. The current protocol yielded a mean length at 21 dpf of 12.7 ± 0.3 mm for the LT group and 18.5 ± 0.4 mm for the CO group (Table 1). This demonstrates that application of this protocol at low temperatures will allow temperature-sensitive mutations to be identified without reducing growth rate below currently accepted levels. Culturing zebrafish at low temperatures also raises the question of whether the low temperature has an effect on sex determination. Temperature-dependent sex determination has been observed in several fish species (Roemer and Beisenherz, 1996; Strüssmann et al., 1996), with higher temperatures generally causing an increased proportion of males and lower temperatures causing an increased proportion of females. While extreme high temperatures (above 35 °C) have been shown to cause an increase in the proportion of males in zebrafish (Uchida et al., 2004), this results in high mortality and is outside the range of environmental conditions generally experienced in the wild. Therefore, it has been suggested that this is not an evolved response (Ospina-Álvarez and Piferrer, 2008). In this study, low temperature was applied from the beginning of exogenous feeding until fish reached a mean length of 18.9 ± 0.7 mm. Based on the observation of gonad morphology in juvenile zebrafish, sex determination is thought to occur between 12 and 23 mm (Maack et al., 2003), meaning that fish in the low temperature group were exposed to low temperatures through the majority of the sex determination period. There was no difference in sex ratio between the LT and CO groups suggesting that low temperature does not affect sex determination in zebrafish. There were no significant differences in fertility parameters (Table 2) between the LT and CO groups, when female size did not
2.4. Statistical analysis Specific growth rate (SGR) was calculated as lnfinal weight − lninitial weight 100* and was calculated from the last weighing. days Fertility parameters were compared between LT and CO groups using linear mixed models. In all models, temperature treatment was a fixed effect and tank was included as a random effect (random intercept) nested within temperature treatment. For testing each fertility parameter, a t-test was applied to determine if the coefficient for temperature treatment was significantly different from zero. Survival rate and sex ratio were compared between treatments using a Student's t-test with Tank considered to be the experimental unit. F-tests demonstrated that data were homoscedastic between treatments. Variables that were expressed as proportions derived from counts (survival, sex ratio, fertilization rate, viability at 1 dpf) were Arcsin transformed prior to modeling and hypothesis testing. A type I error rate of 0.05 was used for all statistical tests. Statistical tests and modeling were performed using R statistical software ver. 3.3.2 (R Core Team, 2017) and the lme4 (Bates et al., 2015) and lmerTest (Kuznetsova et al., 2017) packages. All values are given as mean ± SD.
3. Results Survival was high in both groups (CO and LT) throughout the life cycle (Table 1). There was no significant difference in survival between the CO and LT groups at any time point. Growth was expectedly more rapid at 28.5 °C than at 23 °C with SGR during the first 21 dpf of 35.0 ± 0.3% day−1 and 27.5 ± 0.5% day−1, respectively (Table 1). After the LT group was transitioned to 28.5 °C, growth rate increased, and was not different from that of the CO group (Table 1). Sex ratio was not significantly different between the two temperature treatments. The mean percent male in the CO and LT groups were 41 ± 5% and 55 ± 12%, respectively. There were no significant differences in fertility parameters (fertilization rate, viability at 1 dpf, clutch size, or relative fecundity) between the temperature groups (Table 2). All fish spawned on the first attempt except for two females, one from the LT group and one from the CO group. The LT female spawned on the second attempt and the CO female spawned on the third attempt.
Table 2 Fertility of fish raised at standard (control) and low temperatures during larval and early juvenile stages. All values are given as mean ± SD. dpf, days postfertilization. Temperature Group
Weight (mg) Length (mm) Age (dpf) Fertilization rate (%) Viability at 1 dpf (%) Clutch size (oocytes/female) Relative fecundity (oocytes/mg)
Females
Males
Low (n = 12)
Control (n = 9)
Low (n = 12)
Control (n = 9)
647 ± 95 39.5 ± 1.6 80 ± 3 82 ± 13 88 ± 10 327 ± 140 0.51 ± 0.21
592 ± 51 39.3 ± 1.4 67 ± 4 81 ± 15 92 ± 6 275 ± 85 0.47 ± 0.14
447 ± 60 37.2 ± 1.6 83 ± 2 83 ± 14 95 ± 7 – –
437 ± 64 37.3 ± 2.6 68 ± 4 91 ± 9 92 ± 6 – –
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differ, suggesting that low temperatures during early development do not affect later reproductive function in zebrafish.
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