Patuxent Wildlife Research Center

Marking larval anurans is particularly problematic. We were studying predation risk of European waterfrogs while BA was on staff at the University of Zurich.  We needed a method that would not affect the predation risk experienced by tadpoles and therefore rejected fin clips (Turner 1960), and vital stains (Travis 1981), as being unlikely candidates. We were unsuccessful in our attempts to mark tadpoles by injecting organic dyes (Seale and Boraas 1974) and acrylic polymers (Cecil and Just 1976). We found that the mark just diffused away. We were also concerned that the pigments might be toxic (eg. cadmium).  We developed and tested methods for marking larval anurans (Anholt et al. 1998) which we summarize here.

We first anaesthetised the tadpoles in 1:5000 TMS (MS-222).  All animals were immobile within 4 min and all recovered after about 30 min.  We marked nearly 1000 tadpoles of Rana lessonae and R. esculenta that had been anaesthetized this way.

We injected the tadpoles under the skin, above the musculature of the tail with a tiny amount  of VIE using a 1 ml tuberuclin syringe and No. 20 needle. Injection was done while observing through a stereomicroscope at 6.5X. The procedure can mark animals as small as 8 mm snout-vent length and takes less than 10 s for a single mark. Multiple marks take longer because of the care that must be taken to avoid damaging the tail fin by creating multiple injection wounds. With larger animals it is possible to slide the needle from the tail well up onto the back of the tadpoles. We found this preferable because it was easier to read the marks and we felt that the marks were less likely to be lost from the back than the tail. Although flexible, the elastomer is not as flexible as a tadpole's tail and can be forced out through the skin. VIE marks injected below the tail musculature were all lost.

Retention of single marks is high but not 100%. In one experiment, of 160 surviving animals, 136 (85%) were still identifiably marked after 8 days. We believe that mark retention would have been even higher if we had marked all animals on the back and rejected animals with doubtful marks.  In a second experiment we marked 12 lots of 60 animals with a combination of three marks with the constraint that adjacent marks had to be different colors. With five available colors this gave us 80 (5X4X4) unique combinations. Although many animals lost marks, the remaining marks and electrophoretic information allowed us to resolve the identity of almost every surviving animal at the end of the experiment. In treatments without predators, survival was up to 100% after 5 weeks, and some animals had metamorphosed.  Metamorphs could not be identified alive because the skin had become opaque.  However, the marks were retained through metamorphosis.  Dissections of metamorphs showed that the marks were lined up in the depression between the back muscles directly over the spine.

We have also conducted additional experiments to test whether the elastomer changes the behavior or survival of tadpoles in the presence of the predators. In no case were we able to detect an effect of the marking procedure.

I have been happy enough with the procedure to now have my student, Purnima Price use it for marking tadpoles in the wild to estimate populations sizes and survival rates from mark-recapture studies.  So far this year (1998) she has been experiencing good recapture rates.
 

Literature Cited

Anholt, B.R. ,  S. Negovetic and C. Som. 1998. Methods for anaesthetizing and marking larval anurans. Herpetological Review (in press).
 
Cecil, S. G. and J. J. Just 1976. Use of acrylic polymers for marking of tadpoles (Amphibia, Anura). J.Herpetol. 12:95-96.
 
Seale, D. and M. Boraas. 1974. A permanent mark for amphibian larvae. Herpetologica 30:160--162.
 
Travis, J. 1981. The effect of staining on the growth of Hyla gratiosa tadpoles. Copeia 1981:193--196.
 
Turner, F. B. 1960. Population dynamics of the spotted frog, Rana p. pretiosa Baird and Girard, in Yellowstone Park, Wyoming. Ecol. Monogr. 30:251--278.