In the field of fish structural biology, the present research contributes to the characterization of the vertebrae of the Common carp. To date, to our knowledge, there has been no study that integrates radiography, micro-CT, differential collagen staining, and semi-quantitative elemental analysis to describe these structures. This study provides valuable knowledge in terms of the macroscopic and microscopic anatomy of the vertebrae, as well as offering insights into the ontogeny and factors that affect bone biomechanics, such as the composition of collagen and the elements constituting the extracellular matrix and bone structure. Although variability in the number of vertebrae among the specimens of Common carp examined was observed, it is noteworthy that such variation did not necessarily correlate with differences in body length, challenging the notion that vertebration has a direct impact on the size or morphology of the body. Conversely, in taxonomically unrelated species such as the seabass, it has been reported that certain nutritional factors, including vitamin C deficiency, can induce skeletal malformations and affect vertebral count (Berillis, 2015). Additional studies in teleosts suggest that environmental conditions—including temperature, salinity, and oxygen—impact vertebral configuration (Fjelldal et al., 2013). Understanding the determinants of the number of vertebrae in Common carp remains a challenge that requires further research. Future studies, focused on specific populations and genetic and environmental variables, are imperative to elucidate the underlying causes of the observed variability and their implications on the physiology and evolution of these fish. These findings underscore the need to continue exploring the biological complexity that defines vertebral morphology in different fish species.
The vertebral centrum of the Common carp aligns with the general morphological pattern observed in teleosts (Sakashita et al., 2019; Schultze & Arratia, 2013; Barak et al., 2024). It is crucial to recognize that the macroscopic structure of the vertebrae is not homogeneous and, as confirmed by histological studies, neither are the microscopic characteristics. The shape variation between abdominal and caudal vertebrae, particularly in the hemal and neural arches, is undoubtedly linked to their respective functions—the articulation with the head for abdominal vertebrae and participation in the caudal peduncle for caudal vertebrae. Although all vertebrates share endoskeletal elements that constitute the vertebral axis, the vertebrae themselves are not structurally homologous across different classes of vertebrates and evolve through diverse patterning mechanisms (Arratia et al., 2001). The vertebral bodies exhibit an amphicoelous hourglass shape common among vertebral segments, which is typical in fish (Nordvik et al., 2005). Nonetheless, the pattern of the longitudinal bony trabeculae in the vertebral body varies between segments, as has been observed in other species (Laerm, 1976; Arratia et al., 2001; Eastman et al., 2014; Sakashita et al., 2019). These traits reflect an evolutionary adaptation aimed at optimizing the function and biomechanical efficiency of the spine in Common carp. From a radiological perspective, the integrity of vertebral mineralization is critical for biomechanical function and structural stability, and these images provide evidence of the overall health and robustness of the skeleton in Common carp.
Radiography of the abdominal, transitional, and caudal vertebrae of the Common carp provided evidence of both shared characteristics and distinctive variations in their bone structure. The common amphicoelous morphology, representative of the hourglass shape across all vertebrae, highlights an adaptation for flexibility and force dissipation during swimming, essential for the fish's undulatory movements. Studies suggest that the internal shape of vertebrae can predict swimming modes and ecological adaptations, emphasizing the significance of vertebral shape in swimming biomechanics (Donatelli et al., 2021). The patterns of trabecular bone tissue, present in each vertebral segment, suggest structural support optimized for load management and mechanical stress. The bone marrow spaces, consistent throughout the spine, vary in size and shape, potentially reflecting functional differences in hematopoiesis. The trabecular architecture of the vertebrae in Common carp reflects a specific adaptation of its skeleton for biomechanical function and structural integrity, as demonstrated by the lacunarity analysis in trabecular bone tissue (Dougherty and Henebry, 2002). Furthermore, the close relationship between form and function of trabecular bone underscores the relevance of this structure in resistance and load management (Smit et al., 1997). Previous studies have elucidated how mechanical forces are essential for adapting and maintaining the structure of trabecular bone, potentially reflecting the tissue's ability to adjust to varying levels of stress and preserve its functionality (Huiskes et al., 2000). The variability in bone marrow spaces along the spine, indicating differences in hematopoiesis, adds to the complex functionality of the vertebrae, maintaining both structural strength and the metabolic and hematopoietic capacities necessary for the organism.
However, the robustness of the vertebral bodies and the prominence of the neural and hemal arches differ among segments. The abdominal vertebrae are notable for their greater robustness and more pronounced arches compared to the transitional and caudal vertebrae. Additionally, there is significant variability in the length and angulation of the spinous processes, particularly elongated in the caudal vertebrae. This pattern may be related to their role in supporting the fins and participating in caudal propulsion. Barak et al. (2024) analyzed the morphology of the vertebrae of the grass carp, providing data that can be extrapolated to understand the vertebral structure in the Common carp. They report that the vertebral bodies exhibit an hourglass shape and asymmetry between the cranial and caudal ends, with the caudal end being more robust, suggesting a specific adaptation to better withstand the forces generated during active swimming and caudal propulsion. In the context of the Common carp, this pattern of increased robustness in the caudal vertebrae may be related to their support function for the caudal fins and their critical role in propulsion during swimming. The more robust vertebrae, with elongated and angled spinous processes in the caudal region, would help manage mechanical stress during rapid tail movements, facilitating efficient and powerful swimming movements. The caudal vertebrae, while sharing the amphicoelous morphology, are distinguished by their greater articular complexity and the prominence of the spinous processes, suggesting an evolutionary specialization linked to the biomechanical functions of the anal fins. Together, these observations reflect how the overall structural coherence of the vertebral column is modulated by adaptive variations in each segment, reaffirming the divergent evolution of bone structures in response to specific mechanical demands within the axial skeleton (Moran et al., 2016). Radiographic findings also indicated the presence of vertebral compressions and fusions in mild degrees, implying alterations in the intervertebral space rather than damage to the vertebral bodies per se. These anomalies suggest that altered mechanical load could cause the replacement of intervertebral tissue by cartilaginous tissues, resulting in vertebral compression and fusion. It remains unclear whether intervertebral tissue damage in wild conditions could be due to elevated biomechanical demands or excessive mechanical stress (Witten et al., 2005).
In this study, we analyzed the intricate 3D internal structure of vertebral bodies from specimens using micro-CT scans, identifying two common structural units: lamellar trabeculae and internal hollow spaces, which have been previously described in teleosts (Barak et al., 2024). Nordvik et al. (2005) observed that in Atlantic salmon, trabeculae in the lateral portion of each vertebral body radiate and branch from the center, with densely assembled osteoblasts covering the tips. They hypothesized that vertebral bodies grow outward by selectively adding bone at the trabeculae ends. Our findings confirm that such trabecular structures are typical in Common carp vertebrae. While some fish species exhibit internal hollow spaces, these do not depend on the arrangement of laminar trabeculae; rather, their presence varies among species and even among closely related species (Atkins et al., 2015). The determinants of these hollow spaces remain unclear and warrant further investigation. However, their presence in Common carp vertebral bodies can be a useful parameter for species identification. Notably, the walls of these hollow spaces often display an alveolar pattern due to multiple Howship lacunae, indicating osteoclastic activity (Francillon-Vieillot et al., 1990). The prevalence of trabeculae in the abdominal vertebrae suggests bone erosion at the vertebral center, as depicted in volume-rendered computed tomography images (Fig. 2C). This erosion uncovers a radiated bone architecture around the spinal canal, highlighting its mechanical properties. Our study implies ongoing vertebral bone turnover in the Common carp, with resorption predominating, a phenomenon that further studies should confirm. This dominance of bone resorption might result from accelerated osteoclastic processes or a reduction in basic bone formation, as described in adult eels (López & Martelly-Bagot, 1971; López, 1973). Additionally, Sakashita et al. (2019) suggest that bone tissue adapted to withstand compression and tension is preferable, particularly in bones with a higher trabeculae proportion, like those in abdominal vertebrae. The higher surface-volume ratio of trabecular bone allows for extensive interaction with surrounding tissues, enhancing biomechanical responses such as increased bone remodeling and strength, observed in our histological analysis. The substantial presence of trabeculae also enhances the bone's capacity to distribute forces evenly, reducing the likelihood of fractures (Baxter et al., 2022; Sakashita et al., 2019). This trabecular architecture functions effectively under mechanical stress, distributing forces throughout the bone structure and providing a superior adaptation to diverse environmental conditions.
Type I collagen, predominantly found in the extracellular matrix of the vertebral bodies studied, plays a crucial role in maintaining spinal rigidity in wild fish, while its colocalization with non-aligned type II collagen adds viscoelasticity, essential for preserving flexibility and range of motion during swimming. This collagen not only reinforces the spinal column by distributing compressive forces and resisting tension but also facilitates bone mineralization by serving as a substrate for calcium salt deposition, thereby increasing spinal rigidity (Yamada et al., 2021). The composition and proportion of collagen types in Common carp vertebrae have significant implications for both fish ecology and their nutritional value as a supplementary food source for humans and other animals. The presence of a higher proportion of aligned collagen in the vertebral centrum suggests that this area is well-suited to withstand bending and torsional forces commonly experienced during swimming and propulsion, enhancing the stability and damage resistance of the Common carp's vertebral column, thereby maintaining its swimming performance (Donatelli et al., 2021). Furthermore, since aligned collagen is involved in regulating bone mineralization (Yamada et al., 2021), it may indicate that the centrum has enhanced mineralization capacity, potentially leading to a more rigid and mineralized bone structure. However, the precise morphofunctional implications of aligned collagen concentration and proportion in the centrum of Common carp remain unclear, as they cannot be directly extrapolated from other species due to potential variations among species and environmental conditions. Thus, further research is necessary to fully understand these effects.
Our study utilized, for the first time, an Energy Dispersive X-ray Spectroscopy (EDX) detector to assess elemental composition in the bones of a wild fish, showcasing its significant potential in investigating bone mineralization and chemical makeup. The EDX facilitated a precise and non-destructive evaluation of elemental distribution within the vertebral body tissue, including essential elements such as calcium, phosphorus, magnesium, and trace elements. The most prevalent elements identified were oxygen, carbon, and calcium, crucial for the structural and functional integrity of bones. Oxygen and carbon are primary components of organic tissue, whereas calcium plays a vital role in bone mineralization. Variations in the concentrations of these elements across different vertebral segments might indicate local adaptations to the specific biomechanical demands of each region (Lall & Kaushik, 2021). Additionally, phosphorus, nitrogen, and magnesium are essential for key biological processes. Phosphorus is a critical component of bone tissue and central to bone matrix construction and calcium regulation. Nitrogen suggests the presence of organic compounds such as proteins and nucleic acids, essential for tissue growth and repair. While present in smaller quantities, magnesium is crucial for numerous enzymatic reactions and the metabolism of calcium and phosphorus (Kim & Jung, 2007). The analysis also detected traces of other elements like sodium, potassium, and chlorine, important for osmotic regulation and neuromuscular function. The presence of aluminum and silicon, albeit in minor amounts, could relate to the ingestion of sediments or particles from the fish's natural habitat, as these elements are common in aquatic and terrestrial environments (Wetzel & Likens, 2000).
These findings are particularly crucial as they establish a baseline for future research aimed at understanding the relationship between nutrition, bone mineralization, and the health of the Common Carp, as well as identifying patterns and factors influencing bone mineralization and health. Calcium, detected in the vertebrae of Common Carp, is primarily sourced from its environment, such as the water and food it consumes. The presence of calcium and phosphorus was identified in the Common Carp vertebrae, with a relative ratio of approximately 2:1, indicative of typical mineralization of mature and functional bone in areas critical for the structural integrity and biomechanics of the vertebrae. This is consistent with findings by Drábiková et al. (2021), where phosphorus deficiency in the diet was linked to spinal deformities in Atlantic salmon, including areas of unmineralized bone in the vertebrae, highlighting the importance of phosphorus in proper bone mineralization. Nordvik et al. (2005) describe how preformed bone tissues in salmonids mineralize through specific processes, resulting in a complex structural organization that supports specific biomechanical functionalities. This includes the formation of different layers in the vertebral structure, where the inner layers have an orderly laminar collagen matrix similar to that observed in this study, crucial for structural integrity, while the outer layers are made of spongy bone with a more woven matrix, also playing a role in impact absorption and structural support. A deficiency in calcium or phosphorus can lead to bone weakness, osteoporosis, and bone deformities (Cotti et al., 2020), which can impact the fish’s ability to swim, feed, and reproduce. Based on our results, we did not observe skeletal alterations in any of the specimens studied. Therefore, we can infer that while the appropriate (or normal) proportion of calcium and phosphorus is unknown in the Common Carp, our data could be considered a baseline for further studies. We found no trace elements such as iron and zinc in the vertebral bone tissue.
The pioneering study of the mineral composition of Common Carp vertebrae using EDX adds a practical dimension crucial for understanding bone health in widespread and wild fish species like the Common Carp. Since bone growth and mineralization are inherently linked to the availability and absorption of essential microelements such as calcium and phosphorus, this analysis provides a solid foundation for assessing environmental impacts on the skeletal integrity of these fish. By identifying specific concentrations of these elements in the vertebrae, it is possible to correlate the quality of aquatic habitats with the bone health of carp populations, offering an invaluable tool for conservation efforts and fisheries management. This approach not only enhances our understanding of the biological processes affecting the Common Carp but also sets a precedent for similar studies in other species of wild fish.
Limitations of the study. Research efforts have often been impeded by the destruction of anatomical samples from dissections of wild species, such as the Common Carp. To address this, micro-CT has been adopted as a non-invasive, non-destructive method to study vertebral anatomy. The 3D reconstruction from micro-CT scan proved invaluable for visualizing the spatial configuration of vertebral trabecular tissue and has enhanced the sharing of species information in digital libraries and museums. However, a notable limitation of 3D reconstruction is the poor visibility of certain structures, complicating the delineation of some vertebrae and the identification of specific arches, spines, and trabeculae due to image shading. This study confirms that the morphology of Common Carp vertebrae aligns with the species’ ecology and phylogeny, allowing comparisons with similarly habituated and closely related animals. The use of radiographic and tomographic imaging for observing complete skeletons was found to be complementary, suggesting that the findings could significantly support further research and serve as a valuable reference for identifying wild fish.
In conclusion, the morphological study of the vertebrae of the Common Carp using micro-CT and EDX has revealed a complex and heterogeneous vertebral structure, characterized by a series of lamellar trabeculae and internal hollow spaces. These structural features are consistent across the studied vertebral regions, with each vertebra displaying a distinctive amphicoelous body that reflects its primary structural function. Histologically, an internal architecture was observed, which includes radially oriented bone trabeculae around a central core, providing a spongy texture crucial for the biomechanical functionality of the spine. Furthermore, a predominant pattern of aligned type I collagen was identified, essential for vertebral rigidity and bone mineralization, thereby facilitating the deposition of calcium salts that increase vertebral stiffness. Analysis using SEM and EDX allowed for a precise and non-destructive evaluation of the elemental composition in the bone tissues of the vertebrae, revealing the presence of essential elements such as calcium, phosphorus, magnesium, as well as other trace elements. These elements play a vital role in the structural and functional integrity of the bones, indicating adequate mineralization without signs of active bone pathologies. This in-depth analysis of vertebral morphology and elemental composition provides a detailed understanding of the bone structure in the Common Carp, highlighting the sophistication of its vertebral anatomy.