Structural and immunohistochemical characterization of skeletal muscle in molly fish (Poecilia sphenops)

Skeletal muscle in teleost fish is traditionally regarded as a contractile tissue supporting locomotion; however, increasing evidence in vertebrates suggests that muscle also participates in immune regulation, stress adaptation, and tissue homeostasis. Despite this, the baseline spatial distribution of immune- and stress-associated proteins in teleost skeletal muscle remains insufficiently characterized. Here, we applied an integrated multiscale imaging approach combining histology, histochemistry, immunohistochemistry, semithin sectioning, and transmission electron microscopy to define the structural organization and molecular landscape of skeletal muscle in the molly fish (Poecilia sphenops). Light microscopy revealed highly organized W-shaped myomeres separated by connective myosepta, while ultrastructural analysis demonstrated well-defined sarcomeric architecture with abundant mitochondria, sarcoplasmic reticulum, and glycogen deposits, consistent with high metabolic activity. Immunohistochemical analysis demonstrated constitutive localization of proteins associated with inflammatory signaling (IL-1β, NF-κB, iNOS), oxidative stress regulation (Nrf2), growth modulation (myostatin, TGF-β), calcium-binding and signaling (S100), and developmental regulation (SOX9) within skeletal muscle fibers. These findings establish the presence of a broad group of immune- and stress-related molecular components in teleost skeletal muscle under physiological conditions. Collectively, this study provides a comprehensive structural and molecular baseline map of P. sphenops skeletal muscle and introduces a reproducible multiscale imaging framework for comparative, environmental, and experimental investigations of muscle biology in teleosts.