Does atrazine induce changes in predator recognition, growth, morphology, and metamorphic traits of larval skipper frogs (Euphlyctis cyanophlyctis)?

Atrazine, an info disruptor, interferes with the olfaction of aquatic organisms by impairing the chemosensory system. Consequently, it affects behavior, physiology, and growth increases mortality and infections, and suppresses the immune system of aquatic animals. In this study, we wanted to determine the sensitivity of larval Euphlyctis cyanophlyctis to different concentrations of atrazine by assessing their antipredator behavior, growth, morphology, and metamorphic traits. The results indicate that exposure to atrazine did not affect the survival of tadpoles. However, it caused retarded growth at higher concentrations. Interestingly, the antipredator behavior of tadpoles toward conspecific alarm cues decreased in a dose-dependent manner with an increase in the concentration of atrazine. Tadpoles exposed to low concentrations of atrazine had deeper, wider bodies and tails while those exposed to higher concentrations had shallower and narrower bodies with shallower tail muscles. However, at low and moderate concentrations atrazine did not affect size at metamorphosis, it extended the larval duration at higher concentrations.

Can embryonic skipper frogs (Euphlyctis cyanophlyctis) learn to recognise kairomones in the absence of a nervous system?

In this study, we used larval Euphlyctis cyanophlyctis to determine the predator recognition mechanism. We conducted a series of experiments to determine if larval E. cyanophlyctis have the innate ability to recognise predatory odour (kairomones) as a threat or if they learn to do so during ontogeny. In the case of learning, we wanted to determine the developmental window during which learning is accomplished. Further, we tested the antipredator response of predator- naive as well as predator-experienced tadpoles to chemical cues of different origins in order to assess if they exhibit differential responses. Our results clearly indicate that predator-nai¨ve tadpoles of E. cyanophlyctis do not reduce their activity against predatory cues of dragonfly nymphs, suggesting that they lack the innate ability to recognise kairomones. However, they could learn to do so when trained to perceive kairomones simultaneously along with alarm cues. Surprisingly, larval E. cyanophlyctis could learn to recognise kairomones through association during embryonic stages even before the development of a nervous system. Although larval E. cyanophlyctis lack the innate ability to recognise kairomones, they were able to recognise conspecific alarm cues on the first encounter, indicating that they have the innate ability to recognise alarm cues as a potential threat.

Sexual dimorphism in baseline urinary corticosterone metabolites and their association with body-condition indices in a peri-urban population of the common Asian toad (Duttaphrynus melanostictus).

Field endocrinology research through the quantification of glucocorticoids or stress hormones in free-living wildlife is crucial for assessing their physiological responses towards pervasive environmental changes. Urinary corticosterone metabolite (UCM) enzyme-immunoassay (EIA) has been validated for numerous amphibian species as a non-invasive measure of physiological stress. Body-condition indices (BCIs) have also been widely used in amphibians as an indirect measure of animal health. Field endocrinology research on amphibian species in Asia is limited. In this study, we validated a UCM EIA in a peri-urban sub-population of the common Asian toad (Duttaphrynus melanostictus) in Pune, Maharashtra, India. We determined the baseline levels of UCMs in male (n=39) and female (n=19) toads. Secondly, we used a standard capture handling protocol to quantify changes in UCMs during short-term captivity. We also determined BCIs in the male and female toads using Fulton's index (K) and residual condition index (RCI). The results showed that mean baseline levels of UCMs were significantly higher in male toads than in females. There was no significant change in mean levels of UCMs of males and females between capture and captivity (0-12h). This highlights plausible habituation of the species to the peri-urban environment. Associations between UCMs with BCIs (K and R) were positive in male toads but negative in females. In conclusion, our UCMs EIA can be applied with BCIs to assess health of the Asian toads. We also suggest that direct fitness parameters such as sperm and oocyte quality, reproductive ecology and immunocompetence measurements should be applied in combination with these conservation physiology tools to quantify the fitness consequences of pervasive environmental changes on native amphibians.

Determining sensitive stages for learning to detect predators in larval bronzed frogs: importance of alarm cues in learning.

Successful survival and reproduction of prey organisms depend on their ability to detect their potential predators accurately and respond effectively with suitable defences. Predator detection can be innate or can be acquired through learning.We studied prey-predator interactions in the larval bronzed frogs (Sylvirana temporalis), which have the innate ability to detect certain predators. We conducted a series of experiments to determine if the larval S. temporalis rely solely on innate predator detection mechanisms or can also learn to use more specific cues such as conspecific alarm cues for the purpose. The results of our study clearly indicate that larval S. temporalis use both innate and learned mechanisms for predator detection. Predator-naive tadpoles could detect kairomones alone as a potential threat and responded by reducing activity, suggesting an innate predator detection mechanism. Surprisingly, predator-naive tadpoles failed to detect conspecific alarm cues as a potential threat, but learned to do so through experience. After acquiring the ability to detect conspecific alarm cues, they could associate novel predator cues with conspecific alarm cues. Further, post feeding stages of larval S. temporalis are sensitive for learning to detect conspecific alarm cues to label novel predators.

Occurrence of parotoid glands in tadpoles of the tropical frog, Clinotarsus curtipes and their role in predator deterrence.

Tadpoles of the tropical bicolored frog, Clinotarsus curtipes are unique in having parotoid glands secreting a white viscous fluid and are structurally similar to granular glands from other amphibians. To ascertain the involvement of these glands and their secretion in predator deterrence, it was tested against a predatory fish, Clarias gariepinus, using a paired choice behavioral assay. The results showed that the fish avoid eating C. curtipes tadpoles when paired with tadpoles of a sympatric species, Sylvirana temporalis. While the fish fed on C. curtipes tadpoles whose parotoid glands were surgically removed, did not touch those with intact glands, suggesting a role for the parotoid gland secretion in predator deterrence. Histochemical and biochemical analyses of the gland secretion revealed the presence of high concentrations of proteins, lipids, and alkaloids. SDS-PAGE showed the presence of proteins with prominent bands at 17 and 50kDa. The presence of other small molecules (950-2000amu) as detected by LC-MS showed the presence of five major peaks. Peaks 1 and 2 are probably tetrodotoxin and/or its analogs. Peaks 3 and 5 are possibly bufalin and argininosuccinic acid, respectively while peak 4 remains unidentified. Thus, secretion of parotoid glands of larval C. curtipes contains chemicals which, either alone or in combination, might be responsible for deterring predators.

Gonadal sex differentiation, development up to sexual maturity and steroidogenesis in the skipper frog, Euphlyctis cyanophlyctis.

Gonadal sex differentiation, development up to sexual maturity and steroidogenesis were studied in the Indian skipper frog, Euphlyctis cyanophlyctis. In stage 25 tadpoles, gonads contained a few yolk laden germ cells and somatic cells. Ovarian differentiation occurred at stage 27 with the initiation of meiosis. Interestingly, meiosis preceded the formation of a central lumen that was discernible at stage 28. Folliculogenesis in the developing ovary was observed at stage 29. Vitellogenesis was observed in the 3 months old frogs and the females attained sexual maturity around 4 months. Testicular differentiation occurred indirectly through an ovarian phase. In some animals, from stage 37 onwards, oocyte degeneration was observed that was completed around metamorphic climax. Concurrently, large numbers of mesonephric cells were invading the gonads. Around metamorphosis, reorganization of the germ and somatic cells into testicular cords was observed. Following metamorphosis, the formation of seminiferous tubules was observed in the 2 weeks old males. Meiosis in the developing testes was observed in 1.5 months old males. In 3 months old males, the testes contained all stages spermatogenesis including spermatozoa. Steroidogenesis in the developing gonads was studied by immunohistochemical localization of 3ß-HSD enzyme. At stage 26, a few immunoreactive cells were seen in the kidneys (interrenal cells). However, during and after differentiation, gonads failed to show positive immunoreaction. In the developing ovary at stage 37, follicular cells surrounding the oocytes were positive for 3ß-HSD immunoreactivity. In the ovaries of 3 months old females, follicular cells surrounding the vitellogenic oocytes and stromal cells were positive for 3ß-HSD immunoreaction. E. cyanophlyctis exhibits undifferentiated type of gonadal differentiation, in which gonadal differentiation precedes steroidogenesis.