Polypterus Senegalus Pectoral Fins Trina Y
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© 2020. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2020) 223, jeb217554. doi:10.1242/jeb.217554 RESEARCH ARTICLE Terrestrial acclimation and exercise lead to bone functional response in Polypterus senegalus pectoral fins Trina Y. Du* and Emily M. Standen ABSTRACT Mechanical loading is one of the most important determinants of The ability of bones to sense and respond to mechanical loading is bone phenotype (Biewener and Bertram, 1994; Carter et al., 1996). a central feature of vertebrate skeletons. However, the functional For example, observations in paralysed organisms (Hall and demands imposed on terrestrial and aquatic animals differ vastly. Herring, 1990) and mutants with disrupted muscle development The pectoral girdle of the basal actinopterygian fish Polypterus (Nowlan et al., 2010) have demonstrated that bone development is senegalus was previously shown to exhibit plasticity following significantly impaired by the absence of loading from muscle terrestrial acclimation, but the pectoral fin itself has yet to be activity. Gravitational loading is also important for normal bone examined. We investigated skeletal plasticity in the pectoral fins of maintenance and growth (Carmeliet and Bouillon, 2001), with – P. senegalus after exposure to terrestrial loading. Juvenile fish were astronauts losing as much as 1 2% of bone mass per month in space divided into three groups: a control group was kept under aquatic (Lang et al., 2004). Mechanical loading that leads to increased or conditions without intervention, an exercised group was also kept in novel deformation of bone tissue can also cause dynamic bone water but received daily exercise on land, and a terrestrial group modelling and remodelling through a process called bone functional was kept in a chronic semi-terrestrial condition. After 5 weeks, the adaptation (Chamay and Tschantz, 1972; Lanyon and Rubin, 1985; pectoral fins were cleared and stained with Alcian Blue and Alizarin Burr et al., 2002). This ability of bones to sense and respond to a Red to visualize cartilage and bone, allowing measurements of mechanical stimulus allows the skeleton to accommodate changes bone length, bone width, ossification and curvature to be taken for in functional demands throughout the lifetime of an organism. the endochondral radial bones. Polypterus senegalus fin bones The specific impacts of mechanical loading on bone development responded most strongly to chronic loading in the terrestrial are complex, depending on factors such as species, sex, disease, condition. Fish that were reared in a terrestrial environment had nutrition, which bone and which region of bone is being loaded, and significantly longer bones compared with those of aquatic properties of the loading regimen (Iwamoto et al., 1999; Hsieh et al., controls, wider propterygia and metapterygia, and more ossified 2001; Hamrick et al., 2006; Ruff et al., 2006; Skerry, 2006; metapterygia and medial radials, and they showed changes in Plochocki et al., 2008). However, some general rules of bone propterygial curvature. Exercised fish also had longer and functional adaptation have become apparent after decades of more ossified medial radials compared with those of controls. experimental study in mammals and birds (Turner, 1998; Burr Polypterus senegalus fin bones exhibit plasticity in response to et al., 2002). Increasing strain beyond a habitual level typically leads novel terrestrial loading. Such plasticity could be relevant for to increased bone formation, whereas decreasing strain leads to bone transitions between water and land on evolutionary scales, but key resorption. Dynamic and cyclically applied loads, in particular, are differences between fish and tetrapod bone make direct the primary drivers of functional adaptation, whereas static loading comparisons challenging. typically has no or even negative effects (Lanyon and Rubin, 1984; Robling et al., 2001). Additionally, the number and frequency of KEY WORDS: Amphibious fishes, Mechanical loading, Pectoral loading events, as well as the duration, pattern and gradient of strains radial bones, Skeletal plasticity also affect bone adaptation (Gross et al., 1997; Judex et al., 1997; Hsieh and Turner, 2001; Warden and Turner, 2004). INTRODUCTION The vast majority of studies examining the effects of mechanical The vertebrate skeleton provides structural support for organs and loading on bone development have been undertaken in tetrapod other soft tissues, and much of its form and structure reflect its models. However, interest in fish bone is increasing, both because of mechanical environment. In most vertebrates, the skeleton is the emerging applications of fish models for human health and for composed of bone, a highly dynamic tissue that undergoes studying the evolution and diversity of bone mechanosensory growth, resorption and replacement during an individual’s systems in general (Apschner et al., 2011; Witten and Hall, 2015; lifetime. Although genetics determine a bone’s basic shape and Hall and Witten, 2018). Like those of tetrapods, fish skeletons have properties, much of skeletal development is influenced by a high capacity to respond to changes in mechanical loading. epigenetic factors, including mechanical loading, nutrition, Exercise generally promotes bone formation in fish, with swim- temperature and pathology (Hall, 2005). training leading to greater osteoblastic activity, earlier ossification and higher mineral content than in non-exercised fish (Deschamps et al., 2009; Totland et al., 2011; Fiaz et al., 2012; Grünbaum et al., Department of Biology, University of Ottawa, Gendron Hall, 30 Marie Curie, Ottawa, ON, Canada K1N 6N5. 2012). Other examples of loading-induced skeletal plasticity in fish include diet-induced plasticity in teeth and jaw bones (Meyer, 1987; *Author for correspondence ([email protected]) Huysseune et al., 1994; Albertson et al., 2003; Gunter et al., 2013; T.Y.D., 0000-0003-2193-0795; E.M.S., 0000-0002-3025-1957 Gunter and Meyer, 2014), and plasticity in vertebral columns of fish afflicted with spinal deformities, such as scoliosis and lordosis Received 1 November 2019; Accepted 25 April 2020 (Kranenbarg et al., 2005; Witten et al., 2005; Cardeira et al., 2015). Journal of Experimental Biology 1 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb217554. doi:10.1242/jeb.217554 Vertebrates that live on land must have skeletons that resist the force of gravity as well as stresses induced by weight-bearing activities such as running and jumping. In contrast, most aquatic Propterygium organisms experience relative weightlessness in the water and the majority of forces experienced by their skeletons are generated by muscle contractions. In this context, the skeletons of amphibious Medial radials vertebrates may provide new opportunities to understand the relationship between skeletal plasticity and loading, because moving between water and land imposes a wide range of * functional demands. Amphibious fish, in particular, play a key Mesopterygium part in understanding the role of bone functional adaptation across the fin-to-limb transition and the evolution of weight-bearing * appendages. The mechanical responsiveness of the tetrapod skeleton is often thought to have evolved in response to the increased gravitational loading experienced by the first tetrapods (Clack, 2012). However, recent experiments rearing amphibious fishes on land have begun to demonstrate that the fish skeleton is capable of responding to weight bearing in an adaptive manner, Metapterygium similar to tetrapods (Standen et al., 2014; Turko et al., 2017). In this study, we examined skeletal plasticity in the pectoral fins Fin rays of Polypterus senegalus, an African freshwater fish from the family 0.5 mm Polypteridae, notable for their phylogenetic placement as the most basal extant actinopterygians. The capacity of P. senegalus to Fig. 1. Example of a cleared and stained Polypterus senegalus left pectoral fin. Major skeletal elements are labelled, including the three basal tolerate terrestrial conditions for prolonged periods of time (Standen radials (propterygium, mesopterygium and metapterygium), the fan of medial et al., 2014; Du and Standen, 2017) makes it a useful system to study radials and the fin rays. The fin is shown in lateral view, with the head to the left the effects of terrestrial weight bearing on fish skeletons and their and tail to the right. Cartilaginous tissue is stained blue and ossified tissue is evolutionary implications. Notably, previous work in P. senegalus stained red. Note the fused and damaged medial radials indicated with demonstrated that terrestrial acclimation leads to plastic asterisks. These types of abnormal bones were excluded from the analysis. modification of the pectoral girdle bones, resembling the morphology of fossil stem tetrapods, suggesting that phenotypic loading regimes. In the first condition, fins experienced primarily plasticity could have played a facilitating role during the fin-to-limb static loading from terrestrially induced hypergravity; and in the transition (Standen et al., 2014). Girdle bones, such as the cleithrum, second, fins experienced primarily dynamic loading from terrestrial were the focus of this prior study because of their shared presence in exercise. fossil proto-tetrapods. However, their dermal origin and indirect involvement in aquatic or terrestrial locomotion limit their ability to MATERIALS AND METHODS inform us about the evolutionary