Effects of Antidiuretic Hormone on Water Absorption in the American Tree Frog, Hyla Cinerea

Effects of Antidiuretic Hormone on Water Absorption in the American Tree Frog, Hyla cinerea Poupak Fakhrai and Julanar Selman Department of Biological Sciences Saddleback College Mission Viejo, CA 92692

Terrestrial anurans absorb water through their pelvic region, which is affected by the antidiuretic hormone, arginine vasotocin (AVT). It was predicted that AVT would increase the rate of absorption in frogs subject to an isotonic medium and decrease depletion in a hypertonic solution. Prior to measurements, frogs were dehydrated to 90% of initial weight and bladders emptied. Control frogs were injected with 0.1 mL/g bodyweight of amphibian Ringer’s and treatment condition were given 0.01025 μg AVT per 0.1 mL administered. Frogs were placed into petri dishes with hypertonic (425 mOsm) Ringer’s solution, taken out at 10 minute intervals, blotted dry and weighed for 30 minutes. Thereafter, dishes were filled with isotonic (204 mOsm) Ringer’s, and measured for an additional 30 minutes. Control frogs lost water to the hypertonic medium, at a mean rate of 9.9 μg/minute (n=6), while AVT treated frogs gained water at a mean rate of 0.5 μg/minute (n=6). These results were significant (p= 0.040 one-tailed paired t-test). In isotonic solution, treatment frogs gained water at a significantly faster rate, 9.7μg /minute than did control, 6.3 μg/minute (p= 0.035 one-tailed paired t-test). These data suggest that AVT regulates rate of water absorption and depletion.

Introduction

All animals function as aqueous systems with water as the primarily channel for biochemical pathways. (Consider reversing- water is the primary…) Amphibians adapted to land (to ) face (the) particularly demanding osmotic challenges (associated with living on land) . , (Amphibians are known to loose) loosing (delete ) many times more water than a comparatively sized reptile (significantly more water than any other comparative reptile). Terrestrial amphibians from order Anura have evolved (in order to develop) a number of physiological adaptations to help combat water loss and limited water availability. One such adaptation is the storage of water as dilute urine, McClanahan et al., (1994) found many species to deposit as much as 25% of their bodyweight in their urinary bladder. Hence, when faced(delete…replace with, When Amphibians are faced) with limited hydration sources, terrestrial anurans prolong their activity (prolong activity of what… dehydration?) by reabsorbing water from their bladders (Hillyard et al., 1998). Furthermore,(delete) terrestrial and arboreal species do not typically drink water but rather maintain osmotic balance by soaking up water through their specialized pelvic region labeled as their “seat patch”. The skin on this ventral surface is thinner and highly vascularized, generating more blood flow when in contact with a hydration source (Viborg and Hillyard, 2005). A supporting network of two different categories of aquaporin’s has also been identified in the pelvic region (Ogushi et al., 2010). These membrane proteins act as water channels and facilitate rapid water absorption into cells. Arginine vasotocin (AVT), the amphibian antidiuretic hormone has been shown to regulate these aquaporin’s by translocation from the cytoplasm to the outermost region of the plasma membrane (Hasegawa et al., 2003). AVT is released by the pituitary gland in response to dehydration (is there a certain point of dehydration?) and serves to decrease water loss by urine and alters the water potential of the frog, promoting absorption (Tracy, 1976). The present study (This study) aimed to examine the suggested nature of AVT and its mechanism of decreasing water potential in tree frogs, thereby aiding in water absorption. It was hypothesized that AVT would increase the rate of water absorption in an isotonic medium and decrease the rate of water depletion in hypertonic solution of dehydrated tree frogs.

Materials and Methods

Six tree frogs of species Hyla cinerea were purchased from the Reptile Zoo in Fountain Valley, CA. They (the frogs or test subjects) were initially housed in a ten-gallon aquarium filled with branches and greenery to simulate their natural habitat. Temperature was maintained at 27 °C during the day and 21°C at night (explain how maybe…) . Bottled mineral water was provided and aquarium was misted daily, as well as live crickets administered three times a week. During the experiments each frog was placed in separate holding tanks and numbered accordingly. All experiments were carried out in the laboratory at room temperature 24 ± 2°C. To minimize individual variances in assessments, each frog served as its own control, and experiments were carried out on alternate days with approximately 48 hours of rest in between (reword to explain … Frogs served as own control by conducting experiments on alternate days to minimize individual variances) . All measurements were taken after frogs were dehydrated to 90% of their standard weight; namely a fully hydrated frog with an empty urinary bladder (general comments). Forceps were inserted into frog’s cloaca and urinary bladder emptied by applying light pressure. Standard weight was recorded and frogs were placed in ventilated areas of the laboratory and allowed to dehydrate for approximately 1 hour. An isotonic (204 mOsm) amphibian Ringer’s solution was prepared (Wright, 2006) and amount administered was measured to correspond to dosage of AVT. At 90% of standard weight, frogs were injected with 0.1 mL/g of bodyweight of Ringer’s solution into their dorsal lymph sacs, and placed into individual petri dishes filled with 14 mL of a hypertonic (425 mOsm) Ringer’s solution.( maybe explain what a ringer solution is first) Frogs were contained within the hypertonic medium and taken out at 10 minute intervals, blotted dry and weighed for a total of 30 minutes. Thereafter, petri dishes were filled with 14 mL of the isotonic Ringer’s solution and frogs were measured for an additional thirty minutes according to the aforementioned procedure. Following the control, frogs were given 48 hours of rest before carrying out the treatment portion of experiment. To examine the effect of antidiuretic hormone on water absorption, above-mentioned (sounds weird) procedures were repeated with injections of arginine vasotocin. A solution was prepared using isotonic Ringer’s, such that each frog was injected with 0.1 mL/g of body weight, containing 0.01025 μg AVT per 0.1 mL administered. All data collected was transferred to MS Excel (Microsoft Corporation, Redmond, Washington) where statistical analysis was completed.

Results

Control frogs were found to lose water in hypertonic medium, at a mean rate of 9.9 ± 0.18 μg/minute (± SEM n=6). Frogs treated with AVT gained water against the osmotic gradient, at a mean rate of 0.5 ± 0.13 μg/minute (± SEM n=6). The difference between the two conditions was found to be significant (p= 0.040 one-tailed paired t-test) and is presented in figure 1 and figure 2. In isotonic solution, control frogs gained water at a rate of 6.3 ± 0.8 μg/minute (± SEM n=6), while treated frogs absorbed water at a rate of 9.7 ± 0.8 μg/minute (± SEM n=6). These results are shown in figure 3 and figure 4. AVT injected frogs absorbed water at a significantly higher rate (p= 0.035 one-tailed paired t-test).

Figure 1. Water gained or lost by mean weight of frogs in hypertonic (425 mOsm) amphibian Ringer’s solution (n=6). AVT injected frogs displayed a net gain of water compared to control frogs that lost water (p= 0.040 one-tailed paired t-test). Error bars are mean ± SEM.

Figure 2. Water gained or lost by mean percentage of initial body weight of frogs in hypertonic (425 mOsm) amphibian Ringer’s solution (n=6). AVT injection resulted in a net gain in percent body weight (p= 0.0059 one-tailed paired t-test). Error bars are mean ± SEM.

Figure 3. Water absorption by mean weight of frogs in isotonic (204 mOsm) amphibian Ringer’s solution (n=6). AVT injected frogs gained water at a faster rate than did control frogs (p= 0.035 one-tailed paired t-test). Error bars are mean ± SEM.

Figure 4. Water gained by mean percentage of initial body weight of frogs in isotonic (204 mOsm) amphibian Ringer’s solution (n=6). Treated frogs absorbed water at a faster rate than did control frogs (p= 0.018 one-tailed paired t-test). Error bars are mean ± SEM.

Discussion

Terrestrial anuras face particularly demanding(delete) desiccation (insert) challenges on land in regards to desiccation(delete), when often roaming from aquatic to terrestrial habitats. As a result, various adaptations have evolved to ensure their continuous success. Among these is the dynamic skin on their seat patch with supporting aquaporin’s, with its permeability being modified depending on the substrate suitability. The permeability of their seat patch and osmotic balance has been shown to be (delete) is regulated by the neurohypophyseal hormone, arginine vasopressin (Duellman and Trueb, 1994). AVT is believed to merge vesicles holding aquaporin’s with membranes of water absorbing tissues (Hasegawa et al., 2003). Additionally, AVT is assumed to increase cutaneous blood flow in the pelvic region, facilitating rapid absorption (Malvin, 1993). Moreover, the response to the mechanism of AVT seems to correspond to the species habitat and has been used to determine phylogenetic relationships (Wells, 2007). (first paragraph in discussion does not discuss results) As an arboreal species, Hyla cincerea was predicted to respond to the action of AVT and decrease rate of dehydration in hypertonic solution. Results indicated not only an ability to withstand water loss but a net gain of water despite the osmotic gradient. When subject to the hypertonic medium, an initial rise in hydration levels cushioned against further depletion, resulting in a net gain of water. It is likely that this was made possible by relocating dilute fluids and ions to lower the osmotic potential of their seat patch, promoting water absorption. As the capacity of doing so was reached, water loss was imminent and increased over time. Nevertheless, the initial absorption buffered against a large fall in hydration levels. Desert toads have been known to utilize this strategy frequently by retaining urea from their stored urine (Cooke, 2004). In isotonic solution, AVT injection resulted in a faster rate of absorption compared to control. This is likely due to faster transportation of water into tissues by the fusion of aquaporin’s to their membranes. (why the new paragraph?)

Acknowledgments

We would like to thank professor Teh for his support and guidance throughout this experiment and also express our sincere gratitude to Saddleback’s Department of Biology for provision of arginine vasopressin obtained from Sigma-Aldrich Co. LLC.

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Review Form Department of Biological Sciences Saddleback College, Mission Viejo, CA 92692

Author (s): Poupak Fakhrai and Julanar Selman

Title- Effects of Antidiuretic Hormone on Water Absorption in the American Tree Frog, Hyla cinerea

Summary Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.

Terrestrial anurans absorb water through their pelvic region, which is affected by the antidiuretic hormone, arginine vasotocin (AVT). When you place one of the frogs in a hypertonic solution the frog will undergo dehydration while if placed in an isotonic solution the frog will hydrate. AVT is predicted to control the loss or gain of water by controlling what is known as the “seat patch”. After the experiment was conducted they saw that the control frogs actually gained water against the osmotic gradient in a hypertonic solution. As expected both control and AVT frogs gained water in a isotonic solution. The affect of AVT on water tension was found to be significant.

General Comments Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of knowledge.

Overall the paper is very strong with only a few minor things that can be easily be changed up. The introduction does a great job explaining how amphibians from order Anura retain water. I would add an explanation of concentration gradients and how they work. From here you can expand more on how AVT effects concentration gradients generally from species in Anura and then specifically how Hyla cinerea is affected. I think your results section is very strong with some very good visual representations to help explain your results and would not change much in that section. I also might be missing it but explain how the frogs in your graph start out at different weight? I would have assumed that this would be the same because it’s the same frogs.

Technical Criticism Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.

This paper was a final version This paper was a rough

Recommendation

 This paper should be published as is X This paper should be published with revision  This paper should not be published