, Southern Shark Age Validation: Part 1 - Project Overview, Vertebral Structure and Formation of Growth-Increment Bands Used fo r Age Determination Final Report to Fisheries Research and Development Corporation (FRDC Project 91/037) T. I. Walker, R. A. Officer, J. G. Clement, and L. P. Brown May 1995 Southern Shark Age Validation: Part 1 - Project Overview, Vertebral Structure and Formation of Growth-Increment Bands Used for Age Determination Final Report to Fisheries Research and Development Corporation (FRDC Project 911037) T. I. Walker,I R. A. Officer,2 J. G. Clement ,3 and L. P. Brown I May 1995 1Victorian Fisheries Research Institute Department of Conservation and Natural Resources PO Box 114, Queenscliff, Victoria 3225 2 Z9ology Department University of Melbourne Parkville Victoria 3052 30ral Medicine and Surgery Research Unit School 'of Dental Science University of Melbourne Parkville Victoria 3052 Non Technical Summary • Gummy sharks and school sharks are aged by counting growth-increment bands on the articular faces of vertebrae stained with alizarin red. The age data are required for application of age-structured fishery assessment models used by the Southern Shark Fishery Assessment Group for stock advice to the Southern Shark Fishery Management Advisory Committee. The Southern Shark Age Validation Project had five successfullycomple ted tasks and the three following objectives. • Objective 1 was to validate the assumption that the bands of alizarin red stain are annual and provide reliable estimates of age. This objective could not be met completely by the proposed method of injecting captive sharks with vertebra-marking tissue-dyes. The objective is being further addressed as part of the current FRDC funded Southern Shark Tagging Project. • Objective 2 was to investigate the fe asibility of adopting a microradiographic method as a more accurat� and more cost-effective method than the alizarin red staining method. This objective was met completely. • Objective 3 was to investigate whether growth rates of gummy sharks have changed in response to fishing by testing the hypothesis for the Phenomenon of Apparent Change in Growth Rate. This objective was met completely. • The project demonstrated that gummy sharks can be held captive in land-based tanks for long periods, that survival rates in captive sharks can be improved with appropriate feeding regimes and treatment with antibiotics, and that growth rates of captive sharks can be increased over those found in the wild. The study also demonstrated that school sharks are difficult to hold in captivity. • Application of light microscopy, microradiography, electron microscopy and injection of live sharks (held captive or tagged and released. in the wild) with vertebra-marking tissue-dyes showed that growth-increment bands stained with alizarin red on the articular face of a whole vertebra centrum and visible on microradiographs of a sectioned centrum arise from patterns of varying mineral density. • It is the bands of high mineral density that stain with alizarin red on whole vertebrae and give radio-opaque bands in microradiographs of sectioned vertebrae. These bands tend to be deposited during the winter months when overall growth · of the sharks is lowest. • Additional growth-increment bands, 'disturbance check marks', were found to form in captive sharks. These are assumed to be caused by the trauma of initial capture of the shark from the wild and unavoidable handling in captivity. • Age estimates made fromthe alizarin staining method are similar to those made from the microradiographic method. Whilst there is no bias between the methods, the microradiographic method gives marginally better precision. Experienced readers using the alizarin red method provide more precise and less biased age estimates than do inexperienced readers. • Observed differences in von Bertalanffy growth curves for gummy shark between two separate periods and two separate regions have been explained by the Phenomenon of Apparent Change of Growth Rate caused by length-selective fishing mortality. Three pieces of evidence are presented to support this conjecture. 2 Technical Summary • A vertebra from a gummy shark or school shark comprises a centrum, haemal arch and neural arch. The centum has two articular cups and fourstruts (intermedialia) arranged laterally and dorso-ventrally as buttresses between the articular cups. These structures are comprised of heavily mineralized cartilage which provides strength to the vertebra whilst the cavities behind the articular cups and between the struts are filledwith non­ calcified hyalin� cartilage. The vertebra is enclosed, along with the other vertebrae, in a fibrous tissue sheath (perichondrium) which extends the full length of the vertebral column. The perichondrium enables the vertebrae to grow by producing cells (chondroblasts) which mature into cartilage cells (chondrocytes) and secrete extracellular matrix which depending on location on the surface or marginal edge of the vertebra is either mineralized (i.e. becomes cemented) or non-mineralized. • Application of techniques in light microscopy, microradiography, electron microscopy and injection of vertebra-marking tissue-dyes showed that growth-increment bands visible on microradiographs of sectioned vertebrae (longitudinally through opposite intermedialia) arise from patterns of hyper-mineralized (radio-opaque) and hypo­ mineralized (radio-translucent) bands across a section. It was found that growth­ increment bands result from changes over time in the ratio of extracellular matrix volume to cell volume. When cells grow or proliferate slowly prior to cementation by the extracellular matrix, the ratio is high and results in a hyper-mineralized growth­ increment band. Conversely, when cells grow or proliferate quickly prior to cementation, the ratio is low and results in a hypo-mineralized growth-increment band. Mineralization of cartilage involves deposition of mineral salts, primarily of calcium, in the extracellular matrix. Sharks inhabit a calcium-rich environment and unlike terrestrial vertebrates do not have to store calcium forthe ir metabolic requirements. Hence, the growth processes in mineralized tissues of sharks differs from those of other vertebrates by not having to resorb calcium Once cemented, the mineralized tissue of sharks remains permanently deposited and subsequent growth can only occur by apposition, where new layers of cartilage are deposited over existing cartilage. • Temporal variation in ambient phosphorus may affect the rates at which sharks can deposit calcium phosphate in the mineralizing cartilages. High availability of calcium phosphate could facilitate rapid mineralization allowing the formation of hyper­ mineralized growth-increment bands; whereas low availability of calcium phosphate could retard mineralization allowing formation of hypo-mineralized growth-increment bands. Simulated three-dimensional reconstruction of vertebrae by computer-imaging corroborated results from light microscopy, microradiography and electron microscopy that growthwas appositional .on some parts of the vertebra surface. Husbandry methods were developed to hold sharks captive to enable serial injection with fluorescent tissue-dyes which mark vertebrae in the regions of growth at the time of injection to determine rates at which growth-increment bands form. With appropriate feeding regimes and procedures for care, survival rates in a 6.25-m diameter tank (27,000 litres) with flow-through natural seawater (temperature range I 0-21°C) were foundto be satisfactory. Survival rates were poor in smaller tanks . .., _) • School sharks were more difficult to hold than gummy sharks. School sharks need to continually swim to ventilate their gills, whereas gummy sharks can ventilate their gills while remaining stationary. Large school sharks developed snout damage, leading to necrosis of the snout and eventually death as a result of swimming into the sides of the tank. Hence only school sharks of 520-610 mm total length were introduced into the tank. A total of 53 gummy shark and 31 school shark surviving the first month in captivity were held forvary ing periods during the 32-month period fromAugust 1992 to March 1995. • Growth rates of gummy sharks held captive exceeded growth rates of those of sharks in the wild and growth rates slowed during the winter months when water temperatures were lowest. • Several tissue-dyes (alizarin red, alizarin complexone, calcein, oxytetracycline and xylenol orange) in,:ected into captive and tagged sharks in the wild were found to give distinct fluorescent marks in the vertebrae without affecting the growth rates and health of gummy sharks and school sharks. • The density of mii1eralized cartilage formed during winter, when overall growth of gummy shark is slowest, was found to be significantly higher than the density of mineralized cartilage formed during the other seasons. • Bands of alizarin red on the stained surfaces of the articular faces of whole centra were found to correlate with layers of hyper-mineralized bands deposited during winter in micro radio graphs of vertebral sections. • The number of hyper-mineralized bands formed in tagged gummy sharks while at liberty was found to be less than the number formed in sharks held captive for comparable periods. This difference and the positions of the bands indicated that the formation of growth-increment bands resulting from disturbance is not as common in wild sharks as it is in captive sharks. Inevitable