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.

Experimental evaluation of calcein and alizarin red S for immersion marking grass carp Ctenopharyngodon idellus

“…Immersion marking with fluorochrome dyes involves a compromise between cost, concentration, immersion time, salinity, mortality, growing conditions, and mark retention to produce the best mark [19]. In the present study, juvenile grass carp were successfully marked by immersion in 50-200 mg/l CAL or 150-300 mg/l ARS for 24 h. With the exception of non-lateral line scales from the 150 mg/l ARS treatment group and lateral line scales Our results further agree with other similar studies demonstrating that CAL and ARS are effective chemical markers for immersion marking which can be used in biological research and for the evaluation of stock enhancement programs (e.g., Baer and Rösch [27]; Leips et al [30]; Walt and Faragher [32]). …”

“…Immersion marking fish with fluorochrome dyes is widely used to evaluate stock enhancement programs (e.g., Barker and Mckaye, [12]; Lagardère et al [21]); however, the dye concentrations and immersion times that yield optimal results differs between species [30]. In this study, the methods of mark detection were similar in the sagittae, scales (including non-lateral line and lateral line scales), and fin rays.…”

“…In view of previous studies, a variety of fluorochrome labeling dyes, such as oxytetracycline (OTC) [12], tetracycline hydrochloride (TC) [29], calcein (CAL) [30], alizarin red S (ARS) [31], and alizarin complexone (ALC) [32], have proven effective in producing fluorescent or pigmented marks in calcified or bony structures with few negative effects on survival. Beckman and Schulz [33] also observed that much higher concentrations of dyes (e.g., ARS and ALC) can result in excessive mortalities and even reduce mark success.…”

Evaluation of marking efficiency of different alizarin red S concentrations on body fish structures in <i>Oreochromis niloticus</i> (Perciformes: Cichlidae) juveniles

“…In this study, mortality resulted greater than in other similar studies (Leips et al 2001, Bashey 2004, Crook et al 2007, Crook et al 2009). Size of fish could be an explanation of these results since, in those studies, fish were smaller and we observed that small fish were less vulnerable to the osmotic immersion shock.…”

Growth rate variation of the stalked barnacle Pollicipes pollicipes (Crustacea: Cirripedia) using calcein as a chemical marker

“…Calcein (C 30 H 26 N 2 O 13 ) is a fluorochrome that binds to calcium and becomes incorporated in calcified structures of growing animals when exposed to this chemical agent, and has been successfully used in ecological studies of several marine species (Moran 2000). Immersion in a calcein solution leaves a fluorescent mark in calcified structures, which is visible under epifluorescent microscopy and has been used to identify marked individuals and to estimate growth rates in several organisms, including fishes (Wilson et al 1987, Leips et al 2001, Frenkel et al 2002, ascidians (Lambert and Lambert 1996), molluscs (Kaehler and McQuaid 1999, Moran 2000, Van der Geest et al 2011, echinoderms Urbaniak 2004, Ebert et al 2008) and crustaceans (Helms 2004, Kilada et al 2012.…”