Comparative Analysis of Intermuscular Bones in Five Characiform Species and Their Early Development in Tambaqui (Colossoma macropomum)

Intermuscular bones (IBs) are spicule-like, ossified structures located in the myosepta of muscles on either side of the vertebrae that are unique to several orders of teleost fishes, including Order Characiformes, for which research on the characteristics of IBs is limited. We investigated the morphologies and numbers of epineural and epipleural IBs in five characiform fishes: common wolffish/taíra Hoplias malabaricus (Family Erythrinidae), blackfin pacu/tambaqui Colossoma macropomum (Serrasalmidae), three-spot leporinus/piau-três-pintas Leporinus friderici (Anostomidae), flannel-mouthed characiform/grumatã Prochilodus vimboides (Prochilodontidae), and saguiru Cyphocharax modestus (Curimatidae). In addition, we characterized the ontogenetic formation of IBs in C. macropomum, examining cleared and stained juveniles at different developmental stages. The effects of species, intermuscular bone type, IB series, body side, and their interactions were assessed using a zero-inflated generalized linear model. The results revealed differences in the number, shape, and distribution of intermuscular bones (IBs) among species and morphological types. All seven previously reported types of IBs in characiforms were observed, along with three newly identified shapes. The total number of IBs ranged from 242 to 304 across the respective species. In juvenile C. macropomum, mineralized IBs were first detected at approximately 15 mm total length, after the main axial skeleton was already formed. Early IBs first appeared as simple elements and progressively acquired bifurcated morphologies during development, with “Y”-shaped IBs becoming evident in later juvenile stages.