Microstructural and compositional variation in pacu and piranha teeth related to diet specialization (Teleostei: Serrasalmidae)

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Abstract

In any vertebrate group, tooth shape is known to fit with a biological function related to diet. However, little is known about the relationships between diet and tooth microstructure and composition in teleost fishes. In this work, we describe the external morphology, internal microstructure and elemental composition of the oral teeth of three representative species of the family Serrasalmidae having different feeding habits (herbivorous vs. omnivorous vs. carnivorous). We used backscattered-electron imaging and low vacuum environmental scanning electron microscope to compare the organization and mineralization of tooth layers as well as energy dispersive X-ray microanalysis and Raman microspectrometry to investigate the elemental composition, Ca/P ratio and mineralogy of the most superficial layers. Oral teeth of each serrasalmid species have the same internal organization based on five distinctive layers (i.e. pulp, dentine, inner enameloid, outer enameloid and cuticle) but the general tooth morphology is different according to diet. Microstructural and compositional variation of the cuticle and iron-enrichment of superficial layers were highlighted between herbivorous and carnivorous species. Iron is more concentrated in teeth of the herbivorous species where it is associated with a thicker cuticle explaining the more intense red-pigmentation of the cutting edges of oral teeth. The iron-enrichment is interpreted as a substitution of Ca by Fe in the hydroxyapatite. These traits are discussed in the light of the evolutionary history of the family. Further considerations and hypotheses about the formation and origin of the mineralized tooth layers and especially the iron-rich superficial layers in teleost fishes are suggested.

Introduction

In vertebrates, teeth have been often regarded as a key innovation supporting the diversification process of jaws and are identified as the hardest biological material (Chen et al., 2008, Donoghue and Rücklin, 2016, Enax et al., 2012). Teeth considerably improve the efficiency of the jaws during feeding since they have extraordinary mechanical properties of resistance due to the hierarchical arrangement of their internal constituents (Berkovitz, 2013, Enax et al., 2012). Teeth have a well-conserved structure which consists of three main layers, from inner to outer layers: (1) the pulp, (2) the dentine and (3) the enameloid/enamel (Koussoulakou et al., 2009, Orvig, 1977, Poole, 1967). The pulp corresponds to the non-mineralized central part of the tooth which consists in highly vascularized connective tissue with collagen fibres and nerve endings. The dentine is the first mineralized tooth layer and generally contains ~45 vol% apatite (chemical formula: Ca5(PO4)3X, with X which could be OH, Cl- or F-) in the form of µm-sized prismatic crystals with a hexagonal cross-section shape, ~30 vol% of collagen fibres and ~25 vol% fluid (water, glycosaminoglycans and other organic molecules) (Chen et al., 2008, Meyers et al., 2008). The enameloid (so named in cartilaginous and bony fishes, and amphibian larvae) and the enamel (so named in remaining sarcopterygians) are different tissues and both represent the most external and mineralized tooth layer with a high mineral content of ~95 vol% apatite crystals (Chen et al., 2008, Sasagawa et al., 2009).

Histologically, the odontoblasts bordering the pulp (i.e. differentiated cells from the periphery of the mesenchymal dental papilla) are responsible for the synthesis of the organic matrix and mineralization of the dentine (Sasagawa et al., 2009). Although it is still under debate, they are also involved in the elaboration of at least a part of the enameloid in bony fishes (Sasagawa et al., 2009, Suga et al., 1989). On the opposite, the ameloblasts (i.e. differentiated cells of the inner dental epithelium from the enamel organ) synthetize and mineralize the entire enamel in tetrapods but might also be involved in the mineralization of the enameloid in bony fishes (Sasagawa, 1998). Consequently, the enameloid is morphologically and functionally similar to the enamel but both tissues are recognized to have different origins. The enameloid has epithelial and mesenchymal origins whereas enamel is secreted only by cells from the inner dental epithelium (Sasagawa, 2002a, Sasagawa et al., 2006).

With more than 30,000 species, teleost fishes are undeniably the most diverse vertebrate clade (Nelson et al., 2016). The diversity of this taxon is supported by an overwhelming diversity of tooth shapes (canine, conic, crenulate, flat, incisiform, molariform, monocuspid, multicuspid, pad, sawed, sharp, spatulate, villiform, etc.), tooth-bearing bones (dentary, premaxillary, maxillary, palatine, ectopterygoid, branchial arches, pharyngeal jaws, vomer, etc.), tooth arrangement (one vs. several rows, homodonty vs. heterodonty, etc.), tooth attachment (complete ankylosis, collagen ring, bone mineralization, etc.) and many more tooth conditions identified in the literature (e.g. Berkovitz et al., 2017, Fink, 1981, Huysseune and Witten, 2018, Kubota et al., 1971, Lauder, 1983, McAllister, 1970, Sparks et al., 1990, Streelman et al., 2003, Trapani, 2001) giving information about the phylogeny and ecology.

During feeding, tooth microstructure and composition are as important as shape itself for the function of teeth (mechanical properties) but the literature is early and limited on the subject. Previous studies demonstrated that there are more complex tooth organizations in teleosts than a three-layer structure. A subdivision of the enameloid layer into two sublayers on the basis of the collagen fibre organization has been reported in different families such as Anguillidae, Esocidae and Gadidae (Herold, 1975, Shellis and Miles, 1976). In addition, different species from various families (e.g. Acanthuridae, Aracanidae, Balistidae, Chaetodontidae, Cichlidae, Esocidae, Helostomatidae, Ostraciidae, Tetraodontidae) also possess a thin superficial layer, commonly named the cuticle, covering the enameloid and often red-pigmented (Garant, 1969, Herold, 1975, Motta, 1987, Suga et al., 1989). The red-pigmentation has been described as an iron-rich or iron oxide deposit and its presence is mainly thought to be phylogenetic rather than environmental (Suga et al., 1991, 1989; Suga and Taki, 1992).

The Neotropical family Serrasalmidae (Teleostei: Characiformes) is a monophyletic group of freshwater fishes from South America having various feeding habits ranging from the predominantly herbivorous and frugivorous pacus to the omnivorous and primarily carnivorous piranhas (Correa et al., 2007). All species have a specific tooth morphology that mirrors their diet specialization (Calcagnotto et al., 2005, Goulding, 1980, Huby et al., 2019, Ortí et al., 2008, Thompson and Silva, 2013). Carnivorous species have a single row of highly specialized triangular and sharp teeth on each jaw to slice pieces of fleshy preys (Jégu, 2003, Shellis and Miles, 1976). In contrast, herbivorous and omnivorous species have one and two rows of large specialized incisiform-to-molariform teeth on the lower and upper jaws, respectively, to cut pieces of plants or crush hard fruits and seeds (Goulding, 1980, Goulding and Carvalho, 1982, Jégu, 2003). At the microscopic level, teeth of serrasalmid fishes such as the carnivorous Pygocentrus nattereri have two main mineralized components: the inner layer identified as orthodentine (i.e. a very compact and tubular form of dentine) and the outer layer qualified as enameloid with two main subdivisions (Kubota et al., 1971, Shellis and Berkovitz, 1976).

Furthermore, herbivorous species have a superficial red-pigmentation on the cutting edges of their oral teeth compared to carnivorous species (Fig. S1). Curiously, Farina and collaborators (1999) had previously reported the presence of an iron-rich cuticle covering the enameloid in the carnivorous piranha Pygocentrus nattereri. However, no similar study has been done for their herbivorous relatives until today. Do they also have an iron-rich cuticle? If yes, is it thicker or richer in iron? Apart from the tooth arrangement and shape, we expect other possible changes in tooth features between divergent feeding habits. In teleosts, the relationships between tooth microstructure and composition with diet has been rarely examined as well as the function, microstructure, composition and formation of the cuticle (but see Farina et al., 1999, Motta, 1987).

Serrasalmid fishes give us the opportunity to explore all these outstanding issues. In the present study, we investigated the relationships between the microstructural organization (e.g. thickness) and constitution (e.g. mineralization) of tooth layers and feeding habits with the objectives of providing answers to the following questions: Do hard-eating species necessarily have thicker tooth layers than soft-eating species? Do hard-eating species require hyper-mineralized tooth layers compared to soft-eating species? Then, we examined the elemental composition of each tooth layer, and specifically the cuticle, to compare and relate it with the red-pigmentation and diet while answering these questions: Is the cuticle and its composition related to diet? Is this additional layer a modification of the enameloid or an external deposit? How does the iron-enrichment occur and what is its (organo-)mineral nature?

Section snippets

Biological material

Three representative species of Serrasalmidae were chosen for their position in the phylogenetic tree (Fig. S2) (Thompson et al., 2014) and their feeding habits: the herbivorous red-bellied pacu Piaractus brachypomus (Cuvier, 1818), the omnivorous lobe-toothed piranha Pygopristis denticulata (Cuvier, 1819) and the carnivorous red-bellied piranha Pygocentrus nattereri Kner, 1858. Three subadult specimens of each species (mean standard length ± standard deviation: 112 ± 23 mm; 82 ± 27 mm;

External morphology

In the herbivorous Piaractus brachypomus, oral teeth are broad, gradually red-pigmented toward the cusp cutting edges (Fig. 1A, D and S1B) and separated from each other by small spaces (Fig. 2A). In frontal section, they appeared tricuspid showing a well-developed primary cusp with broad edges and two poorly developed secondary cusps (Fig. 2A). A red-pigmented border is present at their surface and seems to gradually increase in thickness on the cusps (Fig. S4A). The omnivorous Pygopristis

Discussion

The present study focuses on the buccal teeth of three representative species of the Neotropical family Serrasalmidae with different feeding habits highlighted preserved histological traits in the tooth structure within the group as well as adaptive characteristics related to diet. Microstructural and compositional analyses pointed out an iron-enrichment and an original fibre organization in the outermost layers, i.e. enameloid and cuticle. Specializations were evidenced between hard-feeding

Conclusion

Our study points out that microstructural and compositional variation in tooth layers can occur in closely related teleost species with different feeding habits, but dependent on the same “biting” feeding mode. In serrasalmid fishes, tooth microstructure is preserved in five distinctive layers and the presence of a terminal cuticle is not related to diet. It should rather be considered as an ancestral character within the family. In addition, our results suggest two adaptive strategies for

CRediT authorship contribution statement

Yann Delaunois: Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. Alessia Huby: Conceptualization, Resources, Writing - original draft, Writing - review & editing, Visualization, Funding acquisition. Cédric Malherbe: Resources, Writing - review & editing. Gauthier Eppe: Resources, Writing - review & editing. Éric Parmentier: Conceptualization, Resources, Writing - review & editing, Funding acquisition. Philippe Compère: Conceptualization,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was funded by the Belgian National Fund for Scientific Research (F.R.S. – FNRS, Research Project no. 23625340), which allowed the design of the study, acquisition of specimens, data analysis and writing of the manuscript. A.H. is supported by a doctoral Research Fellowship from F.R.S.-FNRS. We thank Sarah Smeets for technical assistance.

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