Table 1.
A brief summary of the chronic unpredictable stress (CUS) protocol used in the present study (adapted from48).
| CUS days | Specific CUS stress procedures | Behavioral tests and sampling |
|---|---|---|
| 1 | Tank change → local hypothermia* | |
| 2 | Net chasing (10 min) → novel objects added to the hometank **(6 h) | |
| 3 | Crowding (2 h) → Air exposure *** | |
| 4 | Darkness (24 h) | |
| 5 | Food deprivation (24 h) | |
| 6 | Water change → noise exposure in unfamiliar tank **** (4 h) | |
| 7 | Alarm pheromone ***** → remixing cohorts | Week 1 NTT/LDT |
| 8 | Bright light (4 h) → novel objects (8 h) | ST |
| 9 | Crowding (2 h) + remixing cohorts → net chasing (15 min) | ZTI |
| 10 | Noise exposure in unfamiliar tank **** (5 h) → water change | Euthanasia, sampling |
| 11 | Darkness (24 h) | |
| 12 | Food deprivation (24 h) | |
| 13 | Air exposure → local hypothermia | |
| 14 | Bright light (6 h) → alarm pheromone | Week 2 NTT/LDT |
| 15 | Noise exposure in an unfamiliar tank (6 h) → remixing cohorts | ST |
| 16 | Novel objects (10 h) → shallow water (8 h) + water change | ZTI |
| 17 | Crowding (3 h) + remixing cohorts → alarm pheromone | Euthanasia, sampling |
| 18 | Darkness (24 h) | |
| 19 | Food deprivation (24 h) | |
| 20 | Net chasing (20 min) → local hypothermia | |
| 21 | Bright light (8 h) → alarm pheromone | Week 3 NTT/LDT |
| 22 | Overcrowding (12 h) → remixing cohorts | ST |
| 23 | Exposure to air → shallow water (10 h) + water change | ZTI |
| 24 | Noise exposure in unfamiliar tank (8 h) → alarm pheromone | Euthanasia, sampling |
| 25 | Darkness (24 h) | |
| 26 | Food deprivation (24 h) | |
| 27 | Bright light (12 h) → shallow water (12 h) | |
| 28 | Tank change + remixing cohorts → air exposure | Week 4 NTT/LDT |
| 29 | Food deprivation (24 h) → local hypothermia | ST |
| 30 | Bright light (14 h) → net chasing (30 min) | ZTI |
| 31 | Alarm pheromone → novel objects (18 h) | Euthanasia, sampling |
| 32 | Darkness (24 h) | |
| 33 | Food deprivation (24 h) | |
| 34 | Noise exposure in unfamiliar tank (10 h) → overcrowding (12 h) | |
| 35 | – | Week 5 NTT/LDT |
| 36 | – | ST |
| 37 | – | ZTI |
| 38 | – | Euthanasia, sampling |
Note that fish undergoing 3-day behavioral testing on specific weeks 1–5 were separated from stress cohort, excluded from CUS procedures after behavioral assays, and euthanized for sampling next day after the last behavioral test of the battery (ZTI) was administered.
NTT the novel tank test, LDT the light/dark test, ST shoaling test, ZTI the zebrafish tail immobilization test.
*Adding 0.5 L of ice to the hometank.
**Adding 20 novel plastic children kinder toys to the hometank.
***Simultaneous exposure of all cohort to air in the net (30 s, followed by a 1-min rest, repeated 5 times).
****Exposure to loud white noise sound using the 40-W sound speakers.
*****Exposure to 5 mL alarm pheromone (per L of water) extracted from additional intact fish as in40, 68.