Sindre Grotmol

Pinealectomy induces malformation of the spine and reduces the mechanical strength of the vertebrae in Atlantic salmon, Salmo salar

Abstract:  This study describes the long‐term effects of surgical ablation of the pineal gland on the spine of 3‐yr‐old Atlantic salmon (Salmo salar L.) with a mean weight of 3.2 kg. Radiographic examinations showed that 82% of the pinealectomized fish developed marked lateral (scoliosis) and dorso‐ventral spinal curvatures. The proportions of the individual vertebral bodies and their mechanical properties were also altered. The stiffness, yield limit and resilience of the vertebral bodies, as measured by compression in the cranio‐caudal direction, were significantly lower in the pinealectomized than in the sham‐pinealectomized group. Calcium, phosphorous and total mineral content of the vertebral bodies were also significantly lower in the pinealectomized fish, while these parameters were similar in scales in the two groups. Alterations of the spinal curve accompanied by changes in the proportions, mechanical strength and mineral content of the vertebral bodies of the pinealectomized salmon indicate that melatonin has several functions related to vertebral bone growth. As the lesions found in salmon are similar to the spinal malformations observed in avian species and mammals after pinealectomy, this study strengthens the hypothesis of a phylogenetically conserved function of the pineal gland related to skeletal development.

Notochord segmentation may lay down the pathway for the development of the vertebral bodies in the Atlantic salmon

This study indicates that the development of the vertebrae in the Atlantic salmon requires the orchestration of two sources of metameric patterning, derived from the notochord and the somite rows, respectively. Before segmentation of the salmon notochord, chordoblasts exhibit a well-defined cell axis that is uniformly aligned with the cranio-caudal axis. The morphology of these cells is characterised by a foot-like basal projection that rests on the notochordal sheath. Notochordal segments are initially formed within the chordoblast layer by metameric change in the axial orientation of groups of chordoblasts. This process results in the formation of circular bands of chordoblasts, with feet perpendicular to the cranio-caudal axis, the original chordoblast orientation. Each vertebra is defined by two such chordoblast bands, at the cranial and caudal borders, respectively. Formation of the chordoblast segments closely precedes formation of the chordacentra, which form as calcified rings within the adjacent notochordal sheath. Sclerotomal osteoblasts then differentiate on the surface of the chordacentra, using them as foundations for further vertebral growth. Thus, the morphogenesis of the rudiments of the vertebral bodies is initiated by a generation of segments within the chordoblast layer. This dual segmentation model for salmon, in which the segmental patterns of the neural and haemal arches are somite-derived, while the vertebral segments seem to be notochord-derived, contrasts with current models for avians and mammals.

The salmon vertebral body develops through mineralization of two preformed tissues that are encompassed by two layers of bone

The teleost backbone consists of amphicoelous vertebrae and intervertebral ligaments, both of which include notochord‐derived structures. On the basis of a sequential developmental study of the vertebral column of Atlantic salmon (Salmo salar L.) from the egg stage up to early fry stage (300–2500 day‐degrees) we show that the vertebral body consists of four layers or compartments, two of which are formed through mineralization of preformed collagenous tissue (the notochordal sheath and the intervertebral ligament) and two of which are formed through ossification. The three inner layers have ordered lamellar collagen matrixes, which alternate perpendicularly from layer to layer, whereas the outer layer consists of cancellous bone with a woven matrix. The bone layers also differ in osteocyte content. In this study we describe the structural details of the layers, and their modes of formation. The results are compared with previous descriptions, and possible phylogenetic implications are discussed.

A segmental pattern of alkaline phosphatase activity within the notochord coincides with the initial formation of the vertebral bodies

This study shows that segmental expression of alkaline phosphatase (ALP) activity by the notochord of the Atlantic salmon (Salmo salar L.) coincides with the initial mineralization of the vertebral body (chordacentrum), and precedes ALP expression by presumed somite‐derived cells external to the notochordal sheath. The early expression of ALP indicates that the notochord plays an instructive role in the segmental patterning of the vertebral column. The chordacentra form segmentally as mineralized rings within the notochordal sheath, and ALP activity spreads within the chordoblast layer from ventral to dorsal, displaying the same progression and spatial distribution as the mineralization process. No ALP activity was observed in sclerotomal mesenchyme surrounding the notochordal sheath during initial formation of the chordacentra. Our results support previous findings indicating that the chordoblasts initiate a segmental differentiation of the notochordal sheath into chordacentra and intervertebral regions.

Stepwise enforcement of the notochord and its intersection with the myoseptum: an evolutionary path leading to development of the vertebra?

The notochord constitutes the main axial support during the embryonic and larval stages, and the arrangement of collagen fibrils within the notochord sheath is assumed to play a decisive role in determining its functional properties as a fibre‐wound hydrostatic skeleton. We have found that during early ontogeny in Atlantic salmon stepwise changes occur in the configuration of the collagen fibre‐winding of the notochord sheath. The sheath consists of a basal lamina, a layer of type II collagen, and an elastica externa that delimits the notochord; and these constituents are secreted in a specific order. Initially, the collagen fibrils are circumferentially arranged perpendicular to the longitudinal axis, and this specific spatial fibril configuration is maintained until hatching when the collagen becomes reorganized into distinct layers or lamellae. Within each lamella, fibrils are parallel to each other, forming helices around the longitudinal axis of the notochord, with a tangent angle of 75–80° to the cranio‐caudal axis. The helical geometry shifts between adjacent lamellae, forming enantiomorphous left‐ and right‐handed coils, respectively, thus enforcing the sheath. The observed changes in the fibre‐winding configuration may reflect adaptation of the notochord to functional demands related to stage in ontogeny. When the vertebral bodies initially form as chordacentra, the collagen lamellae of the sheath in the vertebral region are fixed by the deposition of minerals; in the intervertebral region, however, they represent a pre‐adaptation providing torsional stability to the intervertebral joint. Hence, these modifications of the sheath transform the notochord per se into a functional vertebral column. The elastica externa, encasing the notochord, has serrated surfaces, connected inward to the type II collagen of the sheath, and outward to type I collagen of the mesenchymal connective tissue surrounding the notochord. In a similar manner, the collagen matrix of the neural and haemal arch cartilages is tightly anchored to the outward surface of the elastic membrane. Hence, the elastic membrane may serve as an interface between the notochord and the adjacent structures, with an essential function related to transmission of tensile forces from the musculature. The interconnection between the notochord and the myosepta is discussed in relation to function and to evolution of the arches and the vertebra. Contrary to current understanding, this study also shows that notochord vacuolization does not result in an increased elongation of the embryo, which agrees with the circular arrangement of type II collagen that probably only enables a restricted increase in girth upon vacuolization, not aiding elongation. As the vacuolization occurs during the egg stage, this type of collagen disposition, in combination with an elastica externa, also probably facilitates flexibility and curling of the embryo.

Sustained swimming increases the mineral content and osteocyte density of salmon vertebral bone

This study addresses the effects of increased mechanical load on the vertebral bone of post‐smolt Atlantic salmon by forcing them to swim at controlled speeds. The fish swam continuously in four circular tanks for 9 weeks, two groups at 0.47 body lengths (bl) × s−1 (non‐exercised group) and two groups at 2 bl × s−1 (exercised group), which is just below the limit for maximum sustained swimming speed in this species. Qualitative data concerning the vertebral structure were obtained from histology and electron microscopy, and quantitative data were based on histomorphometry, high‐resolution X‐ray micro‐computed tomography images and analysis of bone mineral content, while the mechanical properties were tested by compression. Our key findings are that the bone matrix secreted during sustained swimming had significantly higher mineral content and mechanical strength, while no effect was detected on bone in vivo architecture. mRNA levels for two mineralization‐related genes bgp and alp were significantly upregulated in the exercised fish, indicating promotion of mineralization. The osteocyte density of the lamellar bone of the amphicoel was also significantly higher in the exercised than non‐exercised fish, while the osteocyte density in the cancellous bone was similar in the two groups. The vertebral osteocytes did not form a functional syncytium, which shows that salmon vertebral bone responds to mechanical loading in the absence of an extensive connecting syncytial network of osteocytic cell processes as found in mammals, indicating the existence of a different mechanosensing mechanism. The adaptive response to increased load is thus probably mediated by osteoblasts or bone lining cells, a system in which signal detection and response may be co‐located. This study offers new insight into the teleost bone biology, and may have implications for maintaining acceptable welfare for farmed salmon.

Hatchability of eggs from Atlantic cod, turbot and Atlantic halibut after disinfection with ozonated seawater

Abstract In aquaculture the risk of transmission of fish pathogens via eggs is reduced by disinfection in ozonated seawater, but this treatment may delay or reduce hatching. The objective of this study was to investigate the tolerance of Atlantic cod, turbot and Atlantic halibut eggs to ozonated seawater. Groups of eggs were treated with different concentrations and exposure times 2 days before hatching, and the effects on hatchability were observed. The groups of eggs of all three species that had been exposed to 2 mg O 3 /l for 2 min or less showed normal hatching. In the groups with high total exposure (4 mg O 3 /l for 1 min or higher), a clearly lower percentage of hatching was found. Interspecies differences in tolerance were observed, with turbot eggs displaying higher tolerance than eggs of either halibut or cod. Due to the interspecies differences, the tolerance of eggs to ozonated seawater should be carefully evaluated in controlled laboratory-scale experiments in order to establish a basis for disinfection protocols. Two milligrams O 3 per liter for 2 min and lower exposures ought to be sufficient to ensure an excess of oxidants for efficient inactivation of fish pathogens while avoiding negative effects on the hatchability of halibut, cod and turbot eggs.

A method for stable gene knock-down by RNA interference in larvae of the salmon louse (Lepeophtheirus salmonis)

The salmon louse (Lepeophtheirus salmonis), an ectoparasitic copepod of salmonid fish, is a major threat to aquaculture in Norway, Ireland, Scotland and Canada. Due to rise in resistance against existing pesticides, development of novel drugs or vaccines is necessary. Posttranscriptional gene silencing by RNA interference (RNAi), when established in a high throughput system is a potential method for evaluation of molecular targets for new medical compounds or vaccine antigens. Successful use of RNAi has been reported in several stages of salmon lice. However, when we employed a previously described protocol for planktonic stages, no reproducible down-regulation of target genes was gained. In the present study, we describe a robust method for RNAi, where nauplius larvae are soaked in seawater added double stranded RNA (dsRNA). In order to test for when dsRNA may be introduced, and for the efficacy and duration of RNAi, we performed a series of experiments on accurately age determined larvae, ranging from the hatching egg to the copepodid with a salmon louse coatomer and a putative prostaglandin E synthase gene. Presumptive knock-down was monitored by real time PCR. Significant gene silencing was obtained only when nauplius I larvae were exposed to dsRNA during the period in which they molted to nauplius II. A knock down effect could be detected 2days after soaking, and it remained stable until the last measurement, on day 12. Soaking nauplius I larvae, knock-down was verified for six additional genes with a putative role in molting. For one chitinase, a loss-of-function phenotype with abnormal swimming was obtained. Hence, RNAi, induced in the nauplius, may facilitate studies of the molecular biology of the louse, such as the function of specific genes in developmental processes and physiology, host recognition, host-parasite interaction, and, in extension, the engineering of novel medicines.

Spatial distribution of fiber types within skeletal muscle fascicles from Standardbred horses

Skeletal muscle fascicles from superficial and deep portions of semitendinosus (ST) and gluteus medius (GM) muscles from Standardbred trotters were analyzed with regard to muscle fiber type proportion (types I, IIA, and IIB) and spatial distribution. Muscle fibers within a fascicle were divided into four layers (L1–4) from the fascicle periphery toward the center. The observed proportions of fiber types among layers were found to be statistically significantly different from a random distribution of fiber types. Type IIB fibers predominated in the peripheral layer, type I fibers prevailed in the layer underneath, and proportions close to the mean of the whole fascicles were observed in the central layer. This pattern of spatial distribution of fiber types within the layers of the fascicles was observed at all four muscle sampling sites. The functional significance of the common pattern is unknown, but possible functional roles are discussed. Anat Rec 268:131–136, 2002.

Inspired by Sharks: A Biomimetic Skeleton for the Flapping, Propulsive Tail of an Aquatic Robot

The vertebral column is the primary stiffening element of the body of fish. This serially jointed axial support system offers mechanical control of body bending through kinematic constraint and viscoelastic behavior. Because of the functional importance of the vertebral column in the body undulations that power swimming, we targeted the vertebral column of cartilaginous fishes—sharks, skates, and rays— for biomimetic replication. We examined the anatomy and mechanical properties of shark vertebral columns. Based on the vertebral anatomy, we built two classes of biomimetic vertebral column (BVC): (1) one in which the shape of the vertebrae varied and all else was held constant and (2) one in which the axial length of the invertebral joint varied and all else was held constant. Viscoelastic properties of the BVCs were compared to those of sharks at physiological bending frequencies. The BVCs with variable joint lengths were then used to build a propulsive tail, consisting of the BVC, a vertical septum, and a rigid caudal fin. The tail, in turn, was used as the propeller in a surface-swimming robot that was itself modeled after a biological system. As the BVC becomes stiffer, swimming speed of the robot increases, all else being equal. In addition, stiffer BVCs give the robot a longer stride length, the distance traveled in one cycle of the flapping tail.