A Comparison of the Suitability of Alizarin Red S and Calcein for Inducing a Nonlethally Detectable Mark in Juvenile Guppies

—Mark–recapture studies are an important component of fisheries research. A diversity of marks is needed to meet the demands of experimental designs and to overcome species-specific variation in marking success. Suitable marks must not alter the viability of marked individuals, must be easy to detect, and must be retained for an appropriate period of time. I compared the effect of alizarin red S and calcein on the individual growth and mortality rates of guppies Poecilia reticulata via short-term experiments (,14 d) conducted both in environments where alizarinand calcein-marked fish were allowed to interact with unmarked fish and in environments where fish were segregated by mark. Neither mark affected the growth or mortality of marked individuals. Both marks were easily applied, did not affect the appearance of fish, and could be detected on the skeleton of live, anesthetized fish or ethanol-preserved specimens without the additional preparation (or lethality) involved in detecting marks on otoliths. However, both marks were subject to fading with time and when fish were exposed to high water temperatures or to direct sunlight. Thus, pilot experiments should be conducted under field conditions before marks are used for longterm mark–recapture studies. I also present an inexpensive and portable technique for detecting alizarin that uses a green laser pointer as the excitation source. This alizarin detector performed as well as a portable calcein detector (Leips et al. 2001) and could easily be modified for improved performance. Alizarin red S and calcein fluoresce at different wavelengths, so both marks can be used simultaneously in studies examining multiple treatment groups or cohorts. The use of alizarin red S may be preferred to that of calcein because its red fluorescence is more easily distinguished from the autofluorescence of bone. Mark–recapture studies are an important component of fisheries research (Hilborn et al. 1990). Mass-marking is often preferred over individual tagging methods as it is more rapidly applied, reduces handling stress, and is often the only successful way of marking eggs, larvae, or small ju* E-mail: [email protected] 1 Present address: 1001 East 3rd Street, 142 Jordan Hall, Department of Biology, Indiana University, Bloomington, Indiana 47405, USA. Received April 9, 2003; accepted May 11, 2004 veniles. Several chemicals are available for massmarking of fish by immersion: calcein, tetracycline, alizarin, strontium, and lanthanides. These chemical markers have the advantage that they provide no visible change in the appearance of the fish, so they should not affect risk of predation by visually foraging predators or influence visually dependent behaviors (e.g., Mohler et al. 2002). Strontium and lanthanides are calcium analogues readily deposited in bones and fin rays creating a traceable tag, but have the disadvantage that they require mass spectrometry or scanning electron microscopy to be detected (Ennevor and Beames 1993; Clear et al. 2000). Tetracycline fluoresces when exposed to ultraviolet or blue light (Brooks et al. 1994), but it is an antibiotic and concern has arisen over its use because of its potential to alter fish behavior, growth, and mortality (e.g., Monaghan 1993) and due to its potential environmental impacts (Blom et al. 1994). Calcein fluoresces when exposed to blue light, has proven to be an effective chemical marker in several species, and can be detected on live fish (Leips et al. 2001). Nevertheless, a diversity of chemical marking agents is often necessary to meet the demands of different experimental designs and to compensate for species-specific variability in marking success (e.g., Hernaman et al. 2000). Alizarin compounds have also been used successfully in mass-marking experiments. Alizarin complexone has been used to estimate the survival and growth of marked fish in the wild (Tsukamoto et al. 1989; Secor and Houde 1995). Alizarin red S has also proven to be a good alternative to alizarin complexone, at only a fraction of the cost (Blom et al. 1994; Nagiec et al. 1995; Beckman and Schultz 1996; Eckmann et al. 1998; Lagardere et al. 2000). These studies have demonstrated that otolith marks can remain highly readable for long durations when fish are reared in the laboratory (up to 842 d) and have been successfully detected on fish recaptured after 128 d in the field (Nagiec et al. 1995). However, none of these studies have