RESEARCH ARTICLE The mechanics of air breathing in African clawed frog tadpoles, Xenopus laevis (Anura: Pipidae) Jackson R. Phillips* ,§ , Amanda E. Hewes ‡ , Molly C. Womack* and Kurt Schwenk ABSTRACT Frog larvae (tadpoles) undergo many physiological, morphological and behavioral transformations throughout development before metamorphosing into their adult form. The surface tension of water prevents small tadpoles from breaching the surface to breathe air (including those of Xenopus laevis), forcing them to acquire air using a form of breathing called bubble sucking. With growth, tadpoles typically make a behavioral/biomechanical transition from bubble sucking to breaching. Xenopus laevis tadpoles have also been shown to transition physiologically from conforming passively to ambient oxygen levels to actively regulating their blood oxygen. However, it is unknown whether these mechanical and physiological breathing transitions are temporally or functionally linked, or how both transitions relate to lung maturation and gas exchange competency. If these transitions are linked, it could mean that one biomechanical breathing mode (breaching) is more physiologically proficient at acquiring gaseous oxygen than the other. Here, we describe the mechanics and development of air breathing and the ontogeny of lung morphology in X. laevis throughout the larval stage and examine our findings considering previous physiological work. We found that the transitions from bubble sucking to breaching and from oxygen conforming to oxygen regulation co-occur in X. laevis tadpoles at the same larval stage (Nieuwkoop–Faber stages 53–56 and 54–57, respectively), but that the lungs do not increase significantly in vascularization until metamorphosis, suggesting that lung maturation, alone, is not sufficient to account for increased pulmonary capacity earlier in development. Although breach breathing may confer a respiratory advantage, we remain unaware of a mechanistic explanation to account for this possibility. At present, the transition from bubble sucking to breaching appears simply to be a consequence of growth. Finally, we consider our results in the context of comparative air-breathing mechanics across vertebrates. KEY WORDS: Anura, Functional morphology, Physiology, Behavior, Respiration, Lungs, Development INTRODUCTION Vertebrate lungs and other air-filled organs serve a diverse range of functions across different lineages and life stages (Carrier, 1987; Liem, 1988; Graham, 1997; Brainerd, 1999; Graham and Wegner, 2010; Hsia et al., 2013). For example, air breathing can serve purely for gas exchange in mammalian lungs (Schmidt-Nielsen, 1997), for hydrostatic control in the gas bladders of physostomous fishes (Scholander, 1956; Alexander, 1990; Uotani et al., 2000; Smith and Croll, 2011), or for both gas exchange and hydrostatic control in the gas bladders of air-breathing physostomous fishes (Graham, 1997; Hedrick and Jones, 1999). In amphibians that retain a biphasic lifestyle, the biological role of air breathing can differ substantially between larvae and adults. For most anurans (frogs and toads), the lungs are important both for buoyancy (hydrostatic) control and gas exchange in aquatic larvae (tadpoles), while the hydrostatic function is lost in most adults (Wassersug and Seibert, 1975; Burggren and West, 1982; Burggren and Mwalukoma, 1983; Gee and Waldick, 1995; Gee and Rondeau, 2012). The function of the lungs also shifts within the tadpole phase, from a nearly exclusive hydrostatic role after hatching to a much greater role in gas exchange later in development (Burggren and West, 1982; Hastings and Burggren, 1995; Phillips et al., 2020). The degree to which the lungs function in gas exchange may be reflected both in the biomechanics of air breathing and in the morphology of the lungs (Phillips et al., 2020). Tadpole air breathing has been studied both physiologically (e.g. Burggren and West, 1982; Hastings and Burggren, 1995) and biomechanically (Wassersug and Yamashita, 2000; Schwenk and Phillips, 2020; Phillips et al., 2020), but there has been little integration of these disciplines. The African clawed frog, Xenopus laevis, is perhaps the species whose larval air-breathing physiology is best understood, making it an excellent candidate for cross- disciplinary study. Xenopus laevis tadpoles are midwater suspension feeders with an unusual head-down swimming posture and derived extensions of the lungs called ‘dorsal diverticulae’, which are believed to function hydrostatically to maintain this posture (Bles, 1906; Weisz, 1945a,b; Van Bergeijk, 1959; Fejtek et al., 1998). The lungs also play an important role in larval X. laevis respiration – air- breathing rates increase as oxygen partial pressure decreases or the branchial (gill) surfaces become covered with food (Bles, 1906; Feder and Wassersug, 1984; Feder et al., 1984). However, Hastings and Burggren (1995) found that only late-stage X. laevis larvae were able to extract significant oxygen via air breathing. They showed that at Nieuwkoop–Faber (NF) stages 54–57 (Nieuwkoop and Faber, 1994), X. laevis tadpoles transition from having blood oxygen determined passively by ambient oxygen levels (oxygen conforming) to actively regulating their blood oxygen to concentrations above ambient levels (oxygen regulating). They speculated that this transition was a result of lung maturation, and that older tadpoles were able to survive hypoxia by supplementing cutaneous and branchial respiration with air breathing. Hastings and Burggren (1995) suggested that X. laevis tadpoles transition to oxygen regulation because of a shift from non-respiratory to respiratory air breathing at ∼NF stages 54–57. Notably, however, those authors did not examine lung development in detail. Received 23 September 2021; Accepted 13 April 2022 Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269-3043, USA. *Present address: Department of Biology, Utah State University, Logan, UT 84322, USA. ‡ Present address: Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA. § Author for correspondence ( jack.phillips@usu.edu) J.R.P., 0000-0001-9831-6609; A.E.H., 0000-0001-7706-633X; M.C.W., 0000- 0002-3346-021X; K.S., 0000-0002-0767-3940 1 © 2022. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2022) 225, jeb243102. doi:10.1242/jeb.243102 Journal of Experimental Biology