The suitability of calcein to mark poeciliid fish and a new method of detection

The suitability of calcein as a marker for population studies depends on (1 ) the assumption that marked individuals have unaltered viability, (2) the fidelity of the calcein label, and (3) the facility with which calcein can be used. We examined the effects of calcein on survival, growth, and the ti ming and size at sexual maturity of least killifish Heterandria formosa and present a new method for detecting calcein. To test the assumption that ma rked individuals have unaltered viability, juvenile least killifish were im mersed for 24 h in a 250-mg/L solution of calcein. A control group of same- aged juveniles was immersed in the same volume of water for 24 h without ca lcein. All individuals were then removed and reared individually in separat e containers. Upon examination under an epifluorescent microscope, all calc ein-treated fish showed fluorescent green marks on their scales and fin ray s, whereas controls did not. The calcein treatment had no significant effec t on growth and survival through 9 weeks nor on the age and size at maturit y. We also designed a portable detector (uses InGaN blue LEDs as a light so urce) for distinguishing calcein-marked individuals; using either this new detector or a standard epifluorescent microscope, the fluorescent mark was visible on the test fish for up to 5 weeks in the laboratory, although some individuals required remarking (due to fading) at 2-3 weeks postimmersion. The calcein tag was also visible in the vertebrae of ethanol-preserved spe cimens for up to 6 years, provided specimens were stored in the dark.