Antipredator responses of Indosylvirana indica tadpoles do not match the level of predation risk.

Predators are the key regulators of prey populations in different environments. However, they are not always present but often come and go. Moreover, the level of predation risk varies based on habitat complexity, resource availability and other ecological factors. Hence, it is adaptive for prey animals to match their antipredator responses with the level of predation risk as such responses are costly to produce and maintain, and there exists a trade-off between fitness-enhancing and fitness-reducing activities. To test whether larval Indosylvirana adjust their antipredator responses based on the level of predation risk, we designed an experiment in which tadpoles of Indosylvirana indica were exposed to nil, low, moderate or high levels of predation risk to assess the effect of risk on growth, behaviour, morphology and life-history traits. We also determined the whole-body corticosterone levels to assess the physiological changes associated with the level of predation risk. Our results show that the growth rate of tadpoles experiencing varying levels of predation risk was similar although there was a trend towards a higher growth rate at moderate and high risks. Surprisingly, tadpoles experiencing predation risk did not reduce their activity. However, the activity of tadpoles experiencing differential predation risk was comparable. Similarly, the use of shelter was comparable among tadpole groups, with an overall higher level of activity in the afternoon compared to other times. Although a few morphological traits were different among tadpole groups, there was no trend or pattern. Moreover, these morphological alterations did not contribute to overall tadpole shape changes. Physiologically, tadpoles experiencing predation risk had significantly lower levels of corticosterone compared to those without risk. Interestingly, corticosterone titres among tadpoles facing varying levels of predation risk were similar. Metamorphic traits of individuals in the various predation risk groups were significantly different. Tadpoles experiencing moderate and high risks emerged at a larger size, and those experiencing the highest level of risk metamorphosed at the earliest. The results of our study thus show that the antipredator responses of larval I. indica do not match the level of predation risk, indicating that physiological and ecological constraints could limit the extent to which prey species respond to predation risk and its intensity.

Interrelationship among annual cycles of sex steroids, corticosterone and body condition in Nyctibatrachus humayuni.

Synergism between extrinsic and intrinsic factors is crucial for the seasonality of reproduction. Environmental factors such as photoperiod and temperature activate the hypothalamus-pituitary-gonadal axis leading to the secretion of steroid hormones that are crucial for reproduction. Sex steroids are not only essential for the maturation of gonads, but also for development of secondary sexual characters in males and reproductive behaviour of both the sexes. In the present study, we quantified the urinary testosterone (UTM) and corticosterone (UCM) metabolites in males and urinary estradiol metabolites (UEM) and UCM in females of Nyctibatrachus humayuni for two consecutive years to determine annual and seasonal variation in the levels of sex steroids, corticosterone and body condition index (BCI). The results show that sex steroids were highest during the breeding season and lowest during the non-breeding season in both the sexes. An increase in UTM and UEM was observed in males and females respectively during the breeding season. Testicular histology showed the presence of all stages of spermatogenesis throughout the year indicating that spermatogenesis is potentially continuous. Ovarian histology showed the presence of vitellogenic follicles only during the breeding season indicating that oogenesis is strictly seasonal. In males, UCM levels were highest during the breeding season, while in females their levels were highest just prior to the breeding season. In males, BCI was highest during the pre-breeding season, declined during the breeding season to increase again during the post-breeding season. In females, BCI was comparable throughout the year. In males, UTM levels were positively correlated with UCM levels but negatively correlated with BCI. Interestingly, UEM, UCM and BCI were not correlated in females. These results indicate that N. humayuni exhibits an associated pattern of reproduction. Quantification of urinary progesterone metabolites (UPM) during the breeding season showed UPM levels were higher in post-spawning females, suggesting the significance of progesterone in ovulation. Further, non-invasive enzyme immunoassay has been successfully standardized in N. humayuni for the quantification of urinary metabolites of steroid hormones.

Interrelationship among steroid hormones, energetics and vocalisation in the Bombay night frog (Nyctibatrachus humayuni).

In vertebrates, the increase in plasma androgens and corticosteroids is essential for the expression of reproductive behaviour. In male anurans, the interaction between hypothalamus-pituitary-gonadal and hypothalamus-pituitary-interrenal axes plays a pivotal role in calling behaviour and energy mobilisation through the secretion of testosterone and corticosterone respectively. To explain the association among body condition, testosterone, corticosterone and calling behaviour the energetic-hormone-vocalisation (EHV) model has been proposed. The model predicts that with continued participation in chorus activity within and across nights, levels of circulating androgens, corticosterone and vocal effort tend to increase and should be positively correlated in calling males. Consequently, decreasing energy reserves should be inversely correlated with corticosterone level in calling males. Depleted energy reserves lead to the peaking of circulating corticosterone, which suppresses androgen production and calling behaviour. In the present study, we used Nyctibatrachus humayuni with unique reproductive behaviour to test the model by quantifying calling behaviour and urinary metabolites of testosterone and corticosterone. We also computed the body condition index (BCI) to assess the association among energetics, levels of testosterone, corticosterone and calling behaviour. The results show that calling males had higher levels of urinary testosterone metabolites (UTM) than non-calling males indicating the importance of testosterone in controlling the calling behaviour. Surprisingly, urinary corticosterone metabolite (UCM) levels were comparable between calling and non-calling males. Further, calling males had higher body condition estimates than non-calling males. The vocal effort was neither associated with UTM, UCM nor BCI. However, a positive association was observed between UTM and UCM levels in calling males indicating the requirement of higher energy for advertisement. Analysis of UTM and UCM levels throughout the breeding season revealed that breeding basal of UTM was significantly higher than that of UCM. Interestingly, UCM levels were maintained at a lower threshold during the breeding season. These observations are in line with some of the predictions of EHV model.

Effect of corticosterone on larval growth, antipredator behaviour and metamorphosis of Hylarana indica.

Corticosterone (CORT), a principal glucocorticoid in amphibians, is known to regulate diverse physiological processes including growth and metamorphosis of anuran tadpoles. Environmental stressors activate the neuroendocrine stress axis (hypothalamus-pituitary-interrenal axis, HPI) leading to an acute increase in CORT, which in turn, helps in coping with particular stress. However, chronic increase in CORT can negatively affect other physiological processes such as growth and metamorphosis. Herein, we studied the effect of exogenous CORT on larval growth, antipredator behaviour and metamorphic traits of Hylarana indica. Embryonic exposure to 5 or 20µg/L CORT did not affect their development, hatching duration as well as larval growth and metamorphosis. Exposure of tadpoles to 10 or 20µg/L CORT throughout larval development caused slower growth and development leading to increased body mass at stage 37. However, body and tail morphology of tadpoles was not affected. Interestingly, larval exposure to 5, 10 or 20µg/L CORT enhanced their antipredator response against kairomones in a concentration-dependent manner. Further, larval exposure to increasing concentrations of CORT resulted in the emergence of heavier froglets at 10 and 20µg/L while, delaying metamorphosis at all concentrations. Interestingly, the heavier froglets had shorter hindlimbs and consequently shorter jump distances. Tadpoles exposed to 20µg/L CORT during early, mid or late larval stages grew and developed slowly but tadpole morphology was not altered. Interestingly, exposure during early or mid-larval stages resulted in an enhanced antipredator response. These individuals metamorphosed later but at higher body mass while SVL was unaffected.

Does corticosterone mediate predator-induced responses of larval Hylarana indica?

Prey-predator interactions have been studied extensively in terms of morphological and behavioural responses of prey to predation risk using diverse model systems. However, the underlying physiological changes associated with morphological, behavioural or life historical responses have been rarely investigated. Herein, we studied the effect of chronic predation risk on larval growth and metamorphosis of Hylarana indica and the underlying physiological changes in prey tadpoles. In the first experiment, tadpoles were exposed to a caged predator from Gosner stage 25-42 to record growth and metamorphosis. Further, whole body corticosterone (CORT) was measured to determine the physiological changes underlying morphological and life historical responses of these prey tadpoles. Surprisingly, tadpoles experiencing continuous predation risk grew and developed faster and metamorphosed at a larger size. Interestingly, these tadpoles had significantly lower CORT levels. In the second experiment, tadpoles were exposed to predation risk (PR) or PR+CORT from stage 25-42 to determine the role of CORT in mediating predator-induced responses of H. indica. Tadpoles facing continuous predation risk grew and developed faster and metamorphosed at a larger size reinforcing the results of the first experiment. However, when CORT was administered along with predation risk, tadpoles grew and developed slowly leading to delayed metamorphosis. Interestingly, growth and metamorphic traits of tadpoles exposed to PR+CORT were comparable to those of the control group indicating that exogenous CORT nullified the positive effect of predation risk. Apparently, CORT mediates predator-induced morphological responses of H. indica tadpoles by regulating their physiology.

Are sex steroids essential for gonadal differentiation of the ornate frog, Microhyla ornata?

Knowledge about sensitivities and responses of amphibian larvae to sex steroids and the chemicals alike is the first step towards understanding and assessing the effect of diverse chemicals that interfere with gonadal development and other endocrine functions. Herein, we used Microhyla ornata to determine the role of sex steroids on its gonad differentiation and sex ratio. Our results show that the exposure to increasing concentrations of estradiol-17ß throughout larval development did not affect gonad differentiation resulting in 1:1 sex ratio at metamorphosis. But, females emerging from estradiol-17ß treatment had larger ovaries with larger sized follicles. Further, testes of some males contained lumens, the number of which was dose dependent. Similarly, exposure to testosterone propionate had negligible effects on gonad differentiation. However, the mean diameter of the largest follicles was smaller in treated ovaries. Treatment of tadpoles with tamoxifen had no effect on gonad differentiation and ovary development while testicular development was accelerated at the highest concentration. Similarly, treatment of tadpoles with cyproterone acetate had little effect on gonad differentiation as well as development, hence the sex ratios at the end of metamorphosis. Further, in tadpoles exposed to increasing concentrations of formestane, gonad differentiation was normal resulting in 1:1 sex ratio. Thus, in M. ornata, both estradiol and testosterone are essential for the development of ovaries and testes respectively but, they are not critical to gonadal differentiation. Hence, the effects of sex steroids and other endocrine disrupting chemicals could be species-specific; different species may have differential sensitivities to such chemicals.

Sex hormones alter sex ratios in the Indian skipper frog, Euphlyctis cyanophlyctis: Determining sensitive stages for gonadal sex reversal.

In amphibians, although genetic factors are involved in sex determination, gonadal sex differentiation can be modified by exogenous steroid hormones suggesting a possible role of sex steroids in regulating the process. We studied the effect of testosterone propionate (TP) and estradiol-17ß (E2) on gonadal differentiation and sex ratio at metamorphosis in the Indian skipper frog, Euphlyctis cyanophlyctis with undifferentiated type of gonadal differentiation. A series of experiments were carried out to determine the optimum dose and sensitive stages for gonadal sex reversal. Our results clearly indicate the importance of sex hormones in controlling gonadal differentiation of E. cyanophlyctis. Treatment of tadpoles with 10, 20, 40, and 80µg/L TP throughout larval period resulted in the development of 100% males at metamorphosis at all concentrations. Similarly, treatment of tadpoles with 40µg/L TP during ovarian and testicular differentiation resulted in the development of 90% males, 10% intersexes and 100% males respectively. Treatment of tadpoles with 10, 20, 40, and 80µg/L E2 throughout larval period likewise produced 100% females at all concentrations. Furthermore, exposure to 40µg/L E2 during ovarian and testicular differentiation produced 95% females, 5% intersexes and 91% females, 9% intersexes respectively. Both TP and E2 were also effective in advancing the stages of gonadal development. Present study shows the effectiveness of both T and E2 in inducing complete sex reversal in E. cyanophlyctis. Generally, exposure to E2 increased the larval period resulting in significantly larger females than control group while the larval period of control and TP treated groups was comparable.

Effect of sex steroids on gonadal differentiation and sex reversal in the frog, Rana curtipes.

The bicolored frog, Rana curtipes, is endemic to Western Ghats of Southern India, having a prolonged larval life. In this species, gonadal differentiation is of the semidifferentiated type. The gonads initially differentiate into ovaries in all the individuals at Gosner stage 25. Later, in genetic males, the oocytes degenerate and testicular differentiation occurs at stages 30-31. Exposure of R. curtipes tadpoles to 50 microg/L of testosterone (T) or estradiol-17beta (E(2)) during stages 24-26 or 29-32 did not affect gonadal sex differentiation and proportion of males and females at metamorphosis. In all the groups, the sex ratio was almost 1:1 as in the controls. Likewise, exposure of tadpoles to low concentration of steroids (12.5 microg/L T or E(2)) throughout larval development was ineffective in altering the sex ratio or the gonadal sex differentiation. On the other hand, exposure to higher concentrations of steroids (25 and 50 microg/L) throughout the larval development (stages 25-45) significantly skewed the sex ratio toward the male or female direction, depending upon the sex steroid used. Thus, exposure to T or E(2) throughout the larval period could produce 93% males or 79% females, respectively, indicating the ability of these steroids to cause sex reversal. This study shows a possible absence of a "critical stage" that is sensitive to sex steroids for gonadal sex reversal in this frog.

Pattern of gonadal sex differentiation, development, and onset of steroidogenesis in the frog, Rana curtipes.

Histomorphological changes and steroidogenic potential of the gonads during sexual differentiation and development were studied in Rana curtipes from tadpole stage 25 (Gosner) until maturity. In stage 25 tadpoles of smaller snout-vent length (SVL; 4-5 mm) the gonads were indifferent, containing a few somatic and germ cells, whereas in larger tadpoles (SVL > 7 mm) gonads were differentiated into ovaries with a central lumen. Onset of meiosis was seen in these ovaries. At stage 26, diplotene and first growth phase oocytes were found. With advancement in developmental stage and after metamorphosis the ovaries progressively enlarged due to increase in number and size of oocytes. Vitellogenesis began in the ovary of 4-month-old frogs. Females attained maturity 6 months after metamorphosis. The frogs showed amplexus and one frog spawned. Onset of testicular formation seen at stage 31 was associated with the degeneration of oocytes and infiltration of darkly stained somatic cells in the central region. By stage 35 all oocytes degenerated, leaving behind a large number of somatic and germ cells interspersed with each other. At stage 38, formation of seminiferous tubules enclosing spermatogonia and pre-Sertoli cells was seen. Initiation of meiosis was evident at metamorphic climax. Cysts of elongated spermatids associated with Sertoli cells were seen in 45-day-old frogs. Histochemically, delta(5)-3 beta-hydroxysteroid dehydrogenase activity was localized in the ooplasm, follicular cells, and interstitium of the ovary from stage 28 onward. The enzyme activity in the testis appeared in 45-day-old froglets. In R. curtipes gonadal differentiation is a semidifferentiated type since gonads initially differentiate into ovaries, and later, in the prospective males, the ovaries degenerate and transform into testes. The males attain maturity much earlier than the females. In R. curtipes gonadal sex differentiation precedes the onset of gonadal steroidogenesis.