Anuran larvae (i.e., tadpoles) have been a mainstay of vertebrate developmental biology studies on metamorphosis since the early 20^th century. A pollywog’s small size and robust response to chemically induced metamorphosis make these animals ideal for the undergraduate teaching lab 1. Metamorphosis is the process where tadpoles transform into adult frogs. This process includes the gradual development of forelimbs and hindlimbs, loss of the tail by apoptosis and reabsorption, the shortening of the intestine to accommodate a carnivorous lifestyle, and the loss of gills and development of lungs in preparation for the transition from an aquatic environment to a terrestrial habitat (234, reviewed in 1).

Thyroxine (T₄) and tri-iodothyronine (T₃) are hormones produced by the thyroid gland that regulate metabolic rate, growth, and development of various organs 5. During tadpole metamorphosis, the concentration of circulating T₄ and T₃ increases. These hormones bind to thyroid receptors resulting in activation of gene expression, thus driving the transformation of the tadpole into an adult frog. Iodine is a requisite micronutrient for the synthesis of thyroid hormones 6. During metamorphosis, the tadpole's thyroid gland starts producing more thyroxine requiring an adequate supply of iodine. On the other hand, actinomycin D inhibits RNA synthesis; therefore, in tadpole metamorphosis, the synthesis of specific proteins, such as thyroxine, required for the transformation from tadpole to frog, would be impeded 17.

Immersion of American bullfrog tadpoles, R. catesbeiana, in high concentrations of thyroxine solution (e.g., 10^-5 M) precociously induces metamorphosis but also leads to physiological stress and early lethality 389. Exogenous exposure to elemental iodine will also induce metamorphosis above pond-water-only controls 910. In contrast, actinomycin D completely inhibits metamorphosis in tadpoles as T₄ and T₃ cannot be synthesized 10. In the teaching laboratory, we house R. catesbeiana tadpoles in circular glass dishes having a surface area (SA) of 182 cm² (15.24 cm diameter glass bowl) while they are being immersed in pond water, actinomycin D, or increasing concentrations of thyroxine and iodine (Experimental Procedures) in order for students to study metamorphosis during amphibian development. We have observed over the four academic years this lab was performed (2014, 2016, 2018, and 2022) significant mortality of our experimental tadpoles, especially those exposed to high concentrations of thyroxine. Furthermore, we have noted aggressive behavior between experimental animals perhaps due to competition for space in the experimental vessel. In this study, we investigated whether increasing the surface area of the glass containers, by using square dishes, to 413 cm² (20.32 cm x 20.32 cm glass dish) reduced mortality of the experimental animals while immersed in experimental solution.

In our undergraduate teaching lab, we have historically used round dishes having a surface area of 182.4 cm² to house tadpoles and have successfully observed metamorphosis with the exogenous application of thyroxine and iodine (Henderson, J.O. unpublished data). However, we have consistently observed that the tadpoles tend to be aggressive towards each other, especially large ones to smaller ones (data not shown). We hypothesized that housing the tadpoles in larger square dishes with a surface area of 413 cm² would give each tadpole more space, decreasing aggression and therefore increase overall survivability during the experimental timeframe.

We ran two independent experiments using square dishes and inducing metamorphosis as described in Experimental Procedures1. This protocol was used previously in the teaching lab as a pedagogical tool for observing frog development (Henderson, J.O. unpublished data). We confirmed the presence of precocious metamorphosis with increasing concentrations of thyroxine (10^-7 to 10^-5 M) and iodine (1 x 10^-6 M and 2 x 10^-6 M) and the absence of metamorphosis in tadpoles submerged in a solution of 2.5 μg/mL actinomycin D through the collection of data on body length from snout to tip of tail, body length from snout to base of tail, hind-limb length, and overall body morphology (e.g., pigmentation changes, movement of the eye to the top of the head; see also 9). As expected, we observed a shortening of the tail, limb development, movement of the eyes to the top of the head, and development of lungs in the thyroxine and iodine treated animals (data not shown). In animals treated with actinomycin D, there were no measurable differences in the rate of metamorphosis compared to the pond water control group.

We then compared the survival rate of our tadpoles (N = 80) from duplicate experiments using square dishes, performed Fall 2022, with historical survival data collected over four semesters in the same teaching lab space (N = 160 tadpoles; Spring 2014, 2016, 2018, and 2022) using SPSS. An omnibus Kaplan-Meier survival analysis demonstrated a statistically significant decrease in the mortality of the group of tadpoles reared in large dishes compared to those experimented on in small dishes (P < 0.05; Figure 1). These data indicate that a larger aquatic environment decreased overall mortality. We then analyzed each individual treatment group (e.g., pond water, 10^-7 M thyroxine etc.) comparing tadpoles experimented on in square dishes (N = 10) versus those maintained in round dishes (N = 20). As we hypothesized, tadpoles in pond water (i.e., control conditions) maintained in square dishes lived ~1.5x longer than those housed in the smaller round dishes (P < 0.01; Figure 2A). Similarly, those tadpoles exposed to iodine and living in the larger square dishes also lived ~1.5x longer than those experimented on in the smaller round dishes (P < 0.05; Figure 2F, Figure 2G). These data indicate that the environmental effect of a larger surface area was to decrease mortality, perhaps by decreasing stress caused by crowding. Anecdotally, we observed less aggression between tadpoles in the larger dishes compared to those in the smaller dishes, though this behavior was not quantified. Another possibility is that an increase in surface area allowed for greater oxygenation of the experimental solution decreasing the energy required to extract oxygen from the water before the development of lungs. Unfortunately, we did not measure dissolved oxygen levels under the two different housing conditions, but this would be an interesting avenue to pursue in the future. In the same vein, it would be appealing to test whether aerating the test solutions in combination with an increased surface area would have a positive synergistic effect on survivability. Future work to tease out how raising tadpoles during precocious metamorphosis in larger dishes decreases mortality could also include a formal analysis of tadpole behaviors in large versus smaller dishes to quantify aggressive behavior 12 and determine if this behavior is, in fact, significantly decreased in the larger dishes. Nevertheless, our data reveal that a larger surface area did allow an increase in longevity under our experimental environment for tadpoles in pond water alone and those undergoing only a mild increase to metamorphosis by exogenous exposure to iodine.

In contrast, tadpoles exposed to thyroxine of any concentration, even for as little as one week, died just as quickly when housed in large dishes as when maintained in small dishes (Figure 2B-E); therefore, the physiological stress and rapid mortality induced by exogenous exposure to 10^-7 M to 10^-5 M thyroxine could not be overcome by the change we made in surface area environment available to the tadpoles. Interestingly, we saw a statistically significant but moderate decrease in the mortality of tadpoles immersed in 2.5 μg/mL actinomycin D and maintained in large dishes compared to those tadpoles housed in small dishes (P < 0.05; Figure 2H). Actinomycin D inhibits mRNA synthesis, and thus protein production, thereby inhibiting metamorphosis by constricting the formation of endogenous thyroxine. Tadpoles maintained in actinomycin D are not expected to metamorphose; therefore, they are not subjected to the physiological stress of rapid metamorphosis like those tadpoles exposed to thyroxine. However, since these tadpoles are not able to synthesize new protein, they would be expected to have a rapid onset of mortality as cellular processes are quashed due to a lack of protein turnover (i.e., no new protein is made to replace those proteins undergoing normal degradation). We did observe the phenomenon of rapid mortality in our experiment (compare Figure 2H to Figure 2A) where tadpoles maintained in small dishes and immersed in actinomycin D start dying by day 16 compared to day 20 for those in pond water, with all tadpoles deceased by day 28 when immersed in actinomycin D compared to pond water where 20% of the tadpoles were alive when the experiment was ended on day 30. This rapid onset of mortality was ameliorated in tadpoles housed in large dishes while immersed in actinomycin D with onset of death at day 22 with all tadpoles deceased by day 35 (Figure 2H). The observation that these animals survived longer when housed in larger dishes while immersed in actinomycin D suggests that a larger surface area mitigates mortality. This increase in survivability of actinomycin D-immersed tadpoles while housed in large dishes was unexpected. We predicted that since protein turnover rates are intrinsic to the protein itself, we would observe no change in mortality. These data indicate that other stressors to the tadpoles while housed in smaller dishes exacerbate the effects of RNA synthesis inhibition. Avenues of investigation into this phenomenon parallel those listed above, including measuring dissolved oxygen levels, quantifying aggressive behavior, and analyzing metabolite concentrations (e.g., ammonia).

In conclusion, our results reveal that increased surface area has a substantial positive impact on the survival rate of tadpoles maintained in pond water and immersed in iodine, a mild inducer of precocious metamorphosis, in a teaching lab setting. However, increased surface area is unable to counteract the physiological stress and increased mortality induced by immersion in thyroxine at the concentrations used in this study. Therefore, these findings have positive implications for the optimization of environmental conditions for housing tadpoles in the teaching lab to enhance the educational experience and success of similar experiments provoking precocious metamorphosis in R. catesbeiana tadpoles.

Supplementary Table 1

Supplementary Figure 1

Conceptualization: K.E.M. and J.O.H.; Formal Analysis: A.K.P, S.S.W., D.W.H., and J.O.H.; Investigation: A.K.P and K.E.M.; Supervision: J.O.H.; Visualization: A.K.P., S.S.W., D.W.H., and J.O.H.; Writing – original draft: A.K.P. and J.O.H.; Writing – reviewing and editing: J.O.H.