Changes in body fluids of the cocooning fossorial frog Cyclorana australis in a seasonally dry environment Stephen J. Reynolds ⁎, Keith A. Christian, Christopher R. Tracy, Lindsay B. Hutley School of Environmental and Life Sciences, Charles Darwin University, Darwin, Northern Territory 0909, Australia abstract article info Article history: Received 9 May 2011 Received in revised form 29 June 2011 Accepted 30 June 2011 Available online 13 July 2011 Keywords: Burrowing frog Body fluid osmolality Cocoon Aestivation Hylid Seasonality Soil water potential We investigated changes in the lymph (equivalent to plasma) and urine of the cocooning frog Cyclorana australis during the dry season in monsoonal northern Australia. Frogs in moist soil for two days were fully hydrated (lymph 220 mOsm kg -1 , urine 49 mOsm kg -1 ). From five weeks onwards the soil was dry (matric potential b-8000 kPa). Aestivating frogs at three and five months formed cocoons in shallow (b 20 cm) burrows and retained bladder fluid (25–80% of standard mass). After three months, urine but not lymph osmolality was elevated. After five months, lymph (314 mOsm kg -1 ) and urine (294 mOsm kg -1 ) osmolality and urea concentrations were elevated. Urea was a major contributing osmolyte in urine and accumulated in lymph after five months. Lymph sodium concentration did not change with time, whereas potassium increased in urine after five months. Active animals had moderate lymph osmolality (252 mOsm kg -1 ), but urea concentrations remained low. Urine was highly variable in active frogs, suggesting that they tolerate variation in hydration state. Despite prolonged periods in dry soil, osmolality increase in C. australis was not severe. Aestivation in a cocoon facilitates survival in shallow burrows, but such a strategy may only be effective in environments with seasonally reliable rainfall. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Fossorial frogs use underground burrows as a refuge from desiccating conditions in arid and seasonally dry environments (Mayhew, 1965; Bentley, 1966; Pinder et al., 1992). Survival for prolonged periods in the soil is aided by capacious bladders, tolerance to dehydration and metabolic depression (Bentley, 1966; Seymour and Lee, 1974; Heatwole, 1984; Shoemaker, 1988; Warburg, 1997; Bentley, 2002). Development of a cocoon in some species, including all members of the genus Cyclorana, reduces rates of water loss to the air (Lee and Mercer, 1967; Withers, 1995; Christian and Parry, 1997; Withers, 1998) and to the soil (Reynolds et al., 2010). The osmoregulatory response of a burrowed frog will be affected by the characteristics of the organism and the properties of the soil. Soil type and texture and the depth of the burrow are important, but only as they affect the water potential of the soil in contact with the frog. Duration underground in dry soil, defined as soil with a water potential lower than that of the body fluids of the frog, will result in an increase in the osmolality of the body fluids, with con- comitant changes in the composition and concentration of osmolytes (McClanahan, 1972; Pinder et al., 1992; Cartledge et al., 2006a, 2008). Frogs have evolved three main strategies in response to drying of the surface soil. Several species burrow deep in sand and remain in a layer of moist soil (Roberts, 1990; Thompson et al., 2005; Cartledge et al., 2006a,b). Desert spadefoots (Scaphiopus/Spea) remain in equilibrium with the soil water by accumulating urea and thereby altering the osmolality of the body fluids (Ruibal et al., 1969; Shoemaker et al., 1969; McClanahan, 1972). The semi-fossorial species Bufo viridis also accumulates urea (Katz and Gabbay, 1986; Katz, 1989; Hoffman and Katz, 1991). A variety of species form cocoons (Cartledge et al., 2006a; Tracy et al., 2007; Cartledge et al., 2008) which provides insulation from the drying effects of the soil (McClanahan et al., 1976; Reynolds et al., 2010). Monsoonal northern Australia has a markedly seasonal rainfall regime (Taylor and Tulloch, 1985; McDonald and McAlpine, 1991). Each year during the dry season, Cyclorana australis aestivate for approximately six months in shallow (10–20 cm deep) burrows where they form a cocoon (Tracy et al., 2007). We investigated patterns of change in body fluids (lymph and urine) over time under natural air temperature and humidity conditions in the dry season. We hypothesised that prolonged periods in dry savanna soils would result in dehydration, elevation of body fluid osmolality, and osmoregulatory imbalance. 2. Methods 2.1. Seasonal moisture and frog activity The climate of the northern savannas is strongly influenced by the seasonal reversal of the north-west monsoon (Suppiah, 1992; Cook Comparative Biochemistry and Physiology, Part A 160 (2011) 348–354 ⁎ Corresponding author. Tel.: + 61 88946 6863. E-mail address: steve.reynolds@cdu.edu.au (S.J. Reynolds). 1095-6433/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2011.06.028 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa