Croaking in amphibians is an adaptive behaviour directed at enhancing mating success. To attract receptive females, males produce acoustic signals which advertise their sexual maturity and mating readiness (Kelley, 2004; Wells and Schwartz, 2006). In seasonally breeding anurans, the croaking behaviour of the male frogs is confined to the reproductive season (Wijayathilaka and Meegaskumbura, 2016). A range of factors such as temperature, humidity, photoperiod, light intensity, rainfall and geographic location are known to influence croaking (Bonnefond et al., 2020; Sun et al., 2020; Krishna et al., 2019; Ulloa et al., 2019). The correlation between acoustic communication and an increase in plasma corticosterone and androgens in frogs is well established (Joshi et al., 2019; Wilczynski and Burmeister, 2016; Arch and Narins, 2009; Remage-Healey and Bass, 2005). However, the information of the central mechanisms mediating croaking behaviour is limited.

In anurans, croaking is induced by electrical stimulation in acoustic centers of the brain specifically in the preoptic area (POA), ventral hypothalamus (vHy) and the anteroventral tegmental nucleus (AV) (Allison, 1992; Urano and Gorbman, 1981; Knorr, 1976; Schmidt, 1973). Intraperitoneal injection of neuropeptide arginine vasotocin (AVT) induced vocalizations in male Xenopus tropicalis in the presence of a female (Miranda et al., 2015). Intraperitoneal injections of arginine vasopressin and oxytocin activated silent males to release advertisement calls in the territorial frog, Eleutherodactylus coqui (Ten Eyck and Ten Eyck, 2017). Similarly, AVT injections to the gray treefrog, Hyla versicolor increased call duration and number of pulses in each call (Klomberg and Marler, 2000). Intracerebroventricular infusion of luteinizing hormone (LH) stimulated croaking in Xenopus laevis (Yang et al., 2007). The effect of acoustic stimulation could be modulated by interplay between the circulating hormones and central neuropeptides via the thalamic nuclei (Kelley, 2004).

The involvement of CART in reproductive physiology has been studied across different vertebrate species. Reproductive phase-related changes in cocaine- and amphetamine-regulated transcript (CART) have been investigated in brain of catfish (Barsagade et al., 2010) and night migratory bird, redheaded buntings (Singh et al., 2020). CART activity was correlated to the photoperiod and seasonal variations during migration and reproduction (Barsagade et al., 2010; Singh et al., 2020). The role of CART in reproduction of mammals has been suggested (Ahmadian-Moghadam et al., 2018; Subhedar et al., 2014). Female odors stimulated CART production in the brain of inexperienced male rats (Cavalcante et al., 2006, Cavalcante et al., 2011). CART-containing axon terminals were observed on GnRH-containing neurons in the hypothalamus of the Siberian hamster and rats (Rondini et al., 2004; Leslie et al., 2001). We have previously reported, the role of CART in reproduction of anuran, Microhyla ornata. We observed upregulation of CART during breeding season in the brain areas (vHy, AV, pineal and pituitary gland) associated with reproduction (Hadawale et al., 2022).

Microhyla nilphamariensis (earlier Microhyla ornata) is distributed throughout south Asia (Howlader et al., 2015) and although its reproductive cycle coincides with the monsoon, the precise breeding activity depends on geographical location (Supplementary Fig. 1). While different species of frogs display territorial, warning, distress, rain and mating calls (Bezerra et al., 2021; Muscat et al., 2020; Xie et al., 2018) the vocalization in M. nilphamariensis is advertorial in nature (Wijayathilaka and Meegaskumbura, 2016). Earlier studies from our laboratory have shown the occurrence CART peptide in the acoustic centers (POA, vHy, AV and Ra) along with pineal and pituitary gland during breeding season (Hadawale et al., 2022). These data suggest that CART might have a role in mediating the reproductive behaviour inclusive of croaking. In this background, we try to find out the correlation, if any, between croaking behaviour and expression of CART in the brain of male M. nilphamariensis.