Tagging very small fish: Two effective and low impact methods

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.

Methods
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 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.