DOI: 10.5061/dryad.t76hdr88s
title: Data from: Tagging very small fish: Two effective and low impact methods

description: Identifying individuals over time and across contexts is essential in many
 scientific fields. There are a variety of well-established methods for
 uniquely marking individuals (e.g., paint, visible implant elastomer tags,
 barcodes, passive integrated transponders); however, for some species,
 life history stages, and/or experiments, existing methods are not
 sufficient. Here, we describe two methods – a tattoo method and a piercing
 method – for tagging juveniles of the African cichlid fish, Astatotilapia
 burtoni, which are too small for the methods used with adult A. burtoni or
 other known methods. The tattoo method is appropriate for the youngest,
 smallest A. burtoni juveniles and injects tattoo ink into the dorsal
 muscle. Different colors and injection locations can be used to
 distinguish among individuals. The ink is observable to the naked eye and
 lasts 3 weeks under our conditions. Because fish this young and small are
 sensitive to handling and injection, we also include physiological data
 showing that fish recover well from anesthetization and tagging. The
 piercing method is appropriate for fish of all sizes, including the
 youngest, smallest juveniles. It uses very thin fishing line threaded
 through the dorsal muscle and tied into a loop. Unique colors and patterns
 can be used to distinguish among individuals. These tags are visible to
 the naked eye and on video, and they last multiple months, at least. We
 expect these effective and inexpensive methods to be useful for a variety
 of small species and will facilitate early-life, developmental, and
 longitudinal research.
description: This dataset includes physiological and behavioral data from very
 young, very small juvenile fish of the African cichlid species,
 Burton's Mouthbrooder (<em>Astatotilapia burtoni</em>), 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 an 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"). These behavior
 variables include how long fish spend in different zones of an aquarium,
 and how frequently they move into each zone. For both kinds of tags --
 tattoo and piercing -- unique colors, color combinations, or tagging
 locations can unqiuely identify individuals.
description: # Data for: Methods for Tagging Very Small Fish 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.