Protocol for Big Bend National Park Amphibian Monitoring Field Crews

Techniques

Population estimates of tadpoles are difficult to obtain.  One way to estimate populations of tadpoles is to use capture-recapture techniques.  Dyeing tadpoles using Neutral Red Dye has been used in the past by various researchers (Herreid and Kinney 1966, Guttman and Creasey 1973, Travis 1981, Sinsch 1997).  We used this technique at Big Bend for Scaphiopus couchii and Hyla arenicolor.

We used a timed search which consisted of dipnetting an isolated pool for 10 minutes recording each 1 meter long dipnet sweep in sequence and the number of tadpoles captured per sweep.  In the future, we will use a specified number of sweeps for both sampling periods (i.e., 60 sweeps for the initial capture period and 60 sweeps for the recapture period).  This removal sampling data can be used to estimate population size using Program Capture (scroll down to Capture). After counting tadpoles in each sweep, remove and place the tadpoles in a bucket of water.

Weigh 0.05 g of Neutral Red Dye (Fisher Scientific, N129-25, 25 g, $49.60) and place it in a small sandwich baggy.  Bring along 1 liter of clear, aged (over 24 hrs) tap water to add to the dye and also to transfer tadpoles into for easier viewing and counting as the pool water is often very murky making it hard to see the tadpoles.  When dyeing tadpoles we found it easiest to pour small amounts of water, from the liter of water we were using for the dye solution, into the baggy containing the dye and then slush the water around saturating the dye powder.  Repeat several times until all the dye is used (it’s important to use somewhat small amounts of water to saturate the dye as it takes several "slushes" before all the dye is used up and you only have one liter to play with).

Hyla arenicolor tadpoles

Soaking Hyla arenicolor tadpoles in a 0.005% dye solution (0.05 g dye per 1 liter of water) for 15 minutes noticeably stained tadpoles for up to 30 hours.  After collecting and dyeing tadpoles, we released them into the pool in which they had been captured and re-sampled the pool two hours later.  On 24 July at Boot Spring, we captured and dyed 61 tadpoles and recaptured 36 tadpoles along with 32 tadpoles that were not dyed.  Using a Lincoln-Peterson estimate of population size (number dyed/proportion of marked tadpoles in recapture), the population estimate was 115 tadpoles in the sampled pool.  On 11 September at Boot Spring, we conducted dye capture-recapture at two pools.  In Pool 1, we captured and dyed 98 tadpoles and recaptured 60 tadpoles and 51 new tadpoles, yielding a population estimate of 181 tadpoles.  In Pool 2, we captured and dyed 70 tadpoles and recaptured 27 tadpoles along with 57 new tadpoles, yielding a population estimate of 218 tadpoles. 

We also took dyed tadpoles back to the laboratory to record dye duration. Dyed tadpoles were visibly red 30 hours after dyeing, but rapidly faded soon after.  After 15 minutes in 0.005% dye solution, tadpoles were noticeably affected by the dye and were very sluggish after being returned to the pool in which they were captured.  However, upon recapture two hours later, there were no noticeable behavioral differences between dyed and not dyed tadpoles. We suggest conducting the recapture within 24 hours of the first capture effort or testing out dye durations at higher concentrations and/or longer soaking periods to see if varying concentrations and soaking times result in tadpoles retaining dye longer.  The average length of successfully dyed Hyla arenicolor tadpoles was approximately 50 mm.  Don’t dye tadpoles that have all four limbs (i.e. newly metamorphosing individuals) because they tend not to survive.

Scaphiopus couchii tadpoles

We tried dyeing Scaphiopus couchii tadpoles at concentrations of 0.005% Neutral Red Dye for 15, 25, 30, and 45 minutes. We could not reliably detect dyed tadpoles even after 45 minutes of soaking.  A large percentage of the tadpoles that soaked for 45 minutes died.  However, this may have been because we used tap water which had not been aged to get rid of the chlorine.  We suggest testing higher concentrations and/or longer soaking times to see if Scaphiopus couchii tadpoles can be successfully dyed.  The average size of dyed Scaphiopus couchii was 12 mm.  

Other species

Try varying soaking lengths and dye concentrations with other species (we only dyed S. couchii and H. arenicolor), such as Rana berlandieri, Gastrophryne olivacea, and perhaps Bufo species.

Important things to remember

It is very important to use either water from the site or water that has been aged at least 24 hours to remove chlorine.  When conducting tests on various dye concentrations and soaking periods be sure to have a control group (not dyed) for comparison.

Equipment

1) Two plastic tubs (one to place captured tadpoles in and one for the dye bath).
2) Dipnets (D-Frame Aquatic Dipnet 241, Ben Meadows) for sweeping.
3) Neutral Red Dye (weighed and placed in a plastic baggy).
4) Clear aged tap water for the dye bath (if needed).
5) One liter bottle for dye water.