The intricate process of anuran development entails the change from an aquatic larval to an adult terrestrial stage. In India, during the post monsoon season (October to December), most of the ephemeral and transitory water bodies such as small puddles and ponds dry up subjecting the anuran larvae to pond desiccation. Pond desiccation leads to challenging environmental conditions such as decreased oxygen levels (hypoxia), increased salinity, high larval density, rivalry for food and slower growth resulting in battling for survival. Anurans lack homeostatic mechanism to cope up with environmental challenges (Navas and Otani, 2007; Kreiman et al., 2019). Hence, to escape stress induced mortality they exhibit phenotypic-developmental plasticity in response to puddle drying (Denver et al., 1998; Enriquez-Urzelai et al., 2013). Environmental stress activates neuroendocrine axis that serves as an interface between the central nervous system and the endocrine system (Kinlein et al., 2015). The neuroendocrine i.e. hypothalamic–pituitary–interenal/thyroid axis in turn accelerate metamorphosis (Denver, 1997, Denver, 1998; Crespi and Denver, 2005; Gomez-Mestre et al., 2013).

Neurohormone corticotropin-releasing factor (CRF) translates environmental stress signals into the endocrine response thereby accelerating the rate of metamorphosis (Denver, 1997). Elevated expression of hypothalamic CRF was observed in the tadpoles of the western spadefoot toad Spea hammondii when exposed to simulated habitat desiccation that exhibited accelerated metamorphosis (Denver, 1993). Like CRF, neuropeptide cocaine and amphetamine regulated transcript peptide (CARTp) in the brain also regulates energy homeostasis and respond to stress (Lau et al., 2016). Interestingly, we have observed elevated expression of CARTp in the POA and hypothalamus of anuran tadpoles during laboratory simulated thermal stress (Shewale et al., 2015). High densities of CARTp and CRF positive cells are reported in the hypothalamic and extra hypothalamic regions of the mammalian brain as well (Koylu et al., 2006; Aguilera and Liu, 2012). In anuran tadpoles although CARTp and CRF exist in neuroendocrine axis that responds to environmental stress their implications in term of puddle desiccation induced accelerated metamorphosis is understudied. In the present study we have investigated response of CARTp and CRF immunoreactivity in the brain of anuran tadpoles to natural puddle desiccation using immunohistochemistry.

Indian skipper frog- Euphlyctis cyanophlyctis tadpoles were chosen for this study as these anurans lay eggs in the temporary puddles formed during monsoon. Due to temperate climate, the day temperature gradually increases during post monsoon. This desiccates the puddles and restricts the tadpole's area of movement and the density per cm2. In the present study, anuran larvae of prometamorphic stage were fixed on field during the post monsoon season (October) from a natural temporary puddle on the verge of drying from Bhimashankar, Maharashtra, India. The controls were collected from the nearby ponds (∼500 L water) from the same regions.