The Suitability of Calcein to Mark Poeciliid Fish and a New Method of Detection

Abstract

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 timing and size at sexual maturity of least killifish Heterandria formosa and present a new method for detecting calcein. To test the assumption that marked individuals have unaltered viability, juvenile least killifish were immersed 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 calcein. All individuals were then removed and reared individually in separate containers. Upon examination under an epifluorescent microscope, all calcein-treated fish showed fluorescent green marks on their scales and fin rays, whereas controls did not. The calcein treatment had no significant effect on growth and survival through 9 weeks nor on the age and size at maturity. We also designed a portable detector (uses InGaN blue LEDs as a light source) 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 specimens for up to 6 years, provided specimens were stored in the dark.