Overall summary: This dataset includes physiological and behavioral data from very young, very small juvenile fish of the African cichlid species, Burton's Mouthbrooder (Astatotilapia burtoni), a model system in social neuroscience. The manuscript describes two novel methods for uniquely identifying individuals using a visual tag. The first is a tattoo method that injects nanoliter quantities of tattoo ink (~100 nL) into the dorsal muscle of juvenile fish using a pulled glass capillary tube needle. Because fish this young and small are sensitive to handling and injection, we also include physiological data showing fish recover well from anesthetization and tagging (see file "AnesthesiaRecovery_A.burtoni.juveniles.csv". The second method is a piercing method that threads very thin fishing line through the dorsal muscle using a minutien pin or insulin needle. The thread is then tied into a barbell or loop. We also include open field exploration behavior data because a physical tag might impede normal movement in very small fish (see file "OpenFieldBehavior_A.burtoni.juveniles.csv"). Unique colors, color combinations, or tagging locations can uniquely identify individuals.

“AnesthesiaRecovery_A.burtoni.juveniles.csv”: Anesthesia and recovery with tattooed fish

Summary: We anesthetized juvenile fish in tricaine methanesulfonate (MS-222) at a dose of 0.0003 g / mL aquarium water, buffered with sodium hydroxide to pH 7-7.5. During anesthetization, we recorded the time it took for body movement to stop; loss of equilibrium, when the fish’s dorsal side first rolled away from an upright orientation; and when breathing stop, indicated by movement of the opercula stopping fully. We removed fish from the MS-222 immediately after breathing stopped. After tagging (see above), the fish was placed in a 200 mL plastic beaker of fresh aquarium water to recover. We immediately began resuscitating the fish using a plastic pipette to gently push water over the gills. We recorded the time it took for the fish to first move its body; first move the opercula; breathe independently without resuscitation; initial regaining of equilibrium, when the dorsal side of the fish first returned to an upright position; and sustained equilibrium, when the fish was permanently upright. We also recorded when the fish looked fully recovered.

The anesthesia and recovery processes were observed live. Missing values (blanks) occurred when the observer missed a particular step in anesthetization or recovery, or not all fish go through each step. This could be physiological or circumstantial (e.g., a fish that goes under anesthesia while propped against the side of the beaker may not show loss of equilibrium).

Descriptions of variables:

• Date: The day that fish underwent the anesthetization, tagging, and recovery procedure.
• ID: Unique identification number given to each fish.
• Injected: "yes" indicates that the fish was anesthetized and tagged. "no" indicates the fish was a control. It was anethetized but not tagged.
• Into_MS222: The clock started when fish were placed in the anesthetic (MS-222). All values are 0, and the clock ran continuously throughout anesthetization and recovery.
• LossEquilibrium: (seconds) The time when the fish’s dorsal side first rolled away from an upright orientation.
• StopBreathing: (seconds) The time when movement of the opercula stopped fully.
• StopMoving: (seconds) The time when body movement stopped fully.
• Out_MS222: (seconds) The time a fish was removed from the MS-222.
• IntoRecoveryCup: (seconds) The time a fish was placed into a 200 mL plastic beaker of clean aquarium water to recover.
• StartBreathing_Resus: (seconds) The time a fish first moved its opercula while the researcher was still resuscitating.
• IndependentBreathing: (seconds) The time a fish first started breathing independently, without resuscitation.
• BodyMovement: (seconds) The time a fish first moved its body.
• InitialRegainedEquilibrium: (seconds) The first time the dorsal side of the fish returned to an upright position.
• SustainedRegainedEquilibrium: (seconds) The time when the fish was first permanently upright.
• Recovered: (seconds) We also recorded when the fish looked fully recovered, which tended to happen suddenly. Fully recovered fish swam smoothly and breathed steadily, with no wobbles in equilibrium.

“OpenFieldBehavior_A.burtoni.juveniles.csv”: Open field exploration behavior test following tagging with a fishing line piercing.

Summary: A physical tag may be more likely to affect locomotion and behavior, particularly in the smallest juveniles. We used an open field exploration to compare between juveniles that were anesthetized and tagged and those that were anesthetized but not tagged. This test is commonly used across species to assess movement, activity, and anxiety-like behavior. We tagged the juveniles less than 2 hrs after being removed from the mother’s buccal cavity, and the open field test was carried out the next day (~20 hrs later). The time it took to complete each tagging surgery was recorded (average 257 +/- 22 s), and each control fish was kept out of water and manipulated with forceps for the same amount of time as one of the tagged fish. Additional MS-222 (or water) was applied to the fish during the procedure, as needed.

For the open field exploration, we placed fish individually in a small, novel aquarium (22.9 x 15.2 x 15.2 cm) without a cover for 30 min. For analysis, aquaria were divided into 4 zones: the territory zone, which contained a small terracotta pot territory / shelter, the close zone, the far zone, and the investigate zone. Video cameras recorded behavior from above, and we used BORIS to score the number of times fish entered each zone of the aquarium, and how long fish spent in each zone of the tank during a 10 min observation (minutes 20-30 of the test).

Descriptions of variables:

• BORIS.ID: unique identifier for scoring the behavior videos. Used in the software BORIS (Behavioral Observation Research Interactive Software).
• Treatment: Whether the fish was anesthetized and tagged with a loop of fishing line through the dorsal muscle, or control fish were anesthetized only, not tagged.
• Date: The date of the behavior test.
• TankID: A unique identifier for each test aquarium.
• Close_Duration: The amount of time (s) spent in the close zone in a 10 min observation.
• Close_Frequency: The number of times a fish crossed into the close zone in a 10 min observation.
• Far_Duration: The amount of time (s) spent in the far zone in a 10 min observation.
• Far_Frequency: The number of times a fish crossed into the far zone in a 10 min observation.
• Investigate_Duration: The amount of time (s) spent in the investigate zone in a 10 min observation.
• Investigate_Frequency: The number of times a fish crossed into the investigate zone in a 10 min observation.
• Territory_Duration: The amount of time (s) spent in the territory zone in a 10 min observation.
• Territory_Frequency: The number of times a fish crossed into the territory zone in a 10 min observation.