Age and Growth of Elasmobranchs and Applications to Fisheries Management and Conservation in the Northeast Pacific Ocean.

In addition to being an academic endeavour, the practical purpose of conducting age and growth studies on fishes is to provide biological data to stock assessment scientists and fisheries managers so they may better understand population demographics and manage exploitation rates. Age and size data are used to build growth models, which are a critical component of stock assessments. Though age determination of elasmobranchs in the northeast Pacific Ocean (NEP) began in the 1930s, the field has evolved substantially in recent years, allowing scientists to incorporate age data into assessments for more species than ever before. Owing to the highly diverse biology of this group of fishes, each species has its own set of challenges with regard to age determination. Age determination methods typically rely on semicalcified hard structures that form regular growth patterns; however, the structure selected and preparation method used is often species specific. New staining techniques have improved the ability to assess age and improve ageing precision for some species, and advances in microchemical methods have allowed for independent means of estimating age and validating age determination accuracy. Here we describe current age determination methods for NEP elasmobranchs. While the library of available techniques is increasing, there are still some NEP species for which reliable ageing methods have yet to be defined; we discuss these challenges and potential avenues of future research. Finally, we conclude by describing how age estimates are used in growth models and subsequently in stock assessments of selected NEP elasmobranchs.

Dentition of the apron ray Discopyge tschudii (Elasmobranchii: Narcinidae).

The present study provides quantitative and qualitative analyses of the dentition of Discopyge tschudii. Overall, 193 individuals (99 males and 94 females) of D. tschudii were collected on scientific trawl surveys conducted by the National Institute for Fisheries Research and Development (INIDEP) and commercial vessels in Argentina. Discopyge tschudii has rhombic-shaped teeth, arranged in a semipavement-like dentition; each tooth has an erect cusp slightly inclined posteriorly and holaulachorized root. Mature males have greater tooth lengths than females and immature specimens. Discopyge tschudii exhibits dignathic homodonty and gradient monognathic heterodonty where teeth of the commissural row are shorter than those of the symphyseal and internal rows.

Elements and elasmobranchs: hypotheses, assumptions and limitations of elemental analysis.

Quantifying the elemental composition of elasmobranch calcified cartilage (hard parts) has the potential to answer a range of ecological and biological questions, at both the individual and population level. Few studies, however, have employed elemental analyses of elasmobranch hard parts. This paper provides an overview of the range of applications of elemental analysis in elasmobranchs, discussing the assumptions and potential limitations in cartilaginous fishes. It also reviews the available information on biotic and abiotic factors influencing patterns of elemental incorporation into hard parts of elasmobranchs and provides some comparative elemental assays and mapping in an attempt to fill knowledge gaps. Directions for future experimental research are highlighted to better understand fundamental elemental dynamics in elasmobranch hard parts.

Early development of rostrum saw-teeth in a fossil ray tests classical theories of the evolution of vertebrate dentitions.

In classical theory, teeth of vertebrate dentitions evolved from co-option of external skin denticles into the oral cavity. This hypothesis predicts that ordered tooth arrangement and regulated replacement in the oral dentition were also derived from skin denticles. The fossil batoid ray Schizorhiza stromeri (Chondrichthyes; Cretaceous) provides a test of this theory. Schizorhiza preserves an extended cartilaginous rostrum with closely spaced, alternating saw-teeth, different from sawfish and sawsharks today. Multiple replacement teeth reveal unique new data from micro-CT scanning, showing how the 'cone-in-cone' series of ordered saw-teeth sets arrange themselves developmentally, to become enclosed by the roots of pre-existing saw-teeth. At the rostrum tip, newly developing saw-teeth are present, as mineralized crown tips within a vascular, cartilaginous furrow; these reorient via two 90° rotations then relocate laterally between previously formed roots. Saw-tooth replacement slows mid-rostrum where fewer saw-teeth are regenerated. These exceptional developmental data reveal regulated order for serial self-renewal, maintaining the saw edge with ever-increasing saw-tooth size. This mimics tooth replacement in chondrichthyans, but differs in the crown reorientation and their enclosure directly between roots of predecessor saw-teeth. Schizorhiza saw-tooth development is decoupled from the jaw teeth and their replacement, dependent on a dental lamina. This highly specialized rostral saw, derived from diversification of skin denticles, is distinct from the dentition and demonstrates the potential developmental plasticity of skin denticles.

Perspectives on elasmobranch life-history studies: a focus on age validation and relevance to fishery management.

Life-history (age, growth, age validation, reproduction and demography) studies of elasmobranchs date back to the middle of the last century with major early contributions made by British fishery scientists. As predicted by Holden in the early 1970s, many sharks and rays can be vulnerable to fishery mortality because they grow slowly, mature late in life, reproduce infrequently, have relatively low fecundities and can have relatively long life spans. As has now been found, however, not all species exhibit these traits. Also, ageing structures (neural arches and caudal thorns), other than vertebrae and spines, have since been evaluated. Various methods for validating age and growth estimates have been developed and tested on numerous species of elasmobranchs. These include tag-recapture analyses, oxytetracycline injections, centrum or spine edge and marginal increment analyses, and bomb radiocarbon dating of calcified structures. Application of these techniques has sometimes not only validated relatively slow growth and long life span estimates, but also has produced other results. A brief historical perspective on the applications and limitations of these techniques for elasmobranchs is provided, along with a discussion of selected species for which these techniques worked well, did not work at all or have produced variable and conflicting results. Because many fishery management techniques utilize age or stage-specific information, often through demographic analyses, accurate information on the life histories of fished populations, especially age validation, is extremely important for the fishery management of these cartilaginous fishes.

Age and growth parameters of shark-like batoids.

Estimates of life-history parameters were made for shark-like batoids of conservation concern Rhynchobatus spp. (Rhynchobatus australiae, Rhynchobatus laevis and Rhynchobatus palpebratus) and Glaucostegus typus using vertebral ageing. The sigmoid growth functions, Gompertz and logistic, best described the growth of Rhynchobatus spp. and G. typus, providing the best statistical fit and most biologically appropriate parameters. The two-parameter logistic was the preferred model for Rhynchobatus spp. with growth parameter estimates (both sexes combined) L(∞) = 2045 mm stretch total length, LST and k = 0·41 year⁻¹. The same model was also preferred for G. typus with growth parameter estimates (both sexes combined) L∞ = 2770 mm LST and k = 0·30 year⁻¹. Annual growth-band deposition could not be excluded in Rhynchobatus spp. using mark-recaptured individuals. Although morphologically similar G. typus and Rhynchobatus spp. have differing life histories, with G. typus longer lived, slower growing and attaining a larger maximum size.

Complementary redescription of Anacanthobatis ori (Wallace, 1967) and its assignment to Indobatis n. g. (Elasmobranchii, Anacanthobatidae), with comments on other legskates.

Anacanthobatis ori is one of the least known species of the family Anacanthobatidae with only four juvenile specimens reported. The species remained assigned to the genus Anacanthobatis sensu lato due to the lack of an adult male as external and skeletal clasper characters are the essential diagnostic features for the differentiation of genera and subgenera within the family Anacanthobatidae. Since an adult male of A. ori became available, along with an adult female and six further juveniles, the authors reinvestigated the species and present its so far unknown diagnostic characters of clasper morphology and skeleton and scapulocoracoid. The clasper turned out to be the most complex one of all known anacanthobatids as the external components flag, slit, pseudosiphon-like cavity, pecten, and two sentinas are not known from any other anacanthobatid species. Furthermore, a dorsal terminal 1 cartilage is present but displaced proximally of the terminal clasper skeleton, the outer edge of dorsal terminal 2 is deeply serrated, the ventral terminal has a very long, curved, strap-like process, and the proximal part of accessory terminal 1 is embedded in the cavity of the baseball-glove-like head of accessory terminal 2. Due to the strong differences in external and internal clasper characters to all other known anacanthobatid species, A. ori is placed in its own, newly erected genus, Indobatis. 

Biology, ecology and conservation of the Mobulidae.

The Mobulidae are zooplanktivorous elasmobranchs comprising two recognized species of manta rays (Manta spp.) and nine recognized species of devil rays (Mobula spp.). They are found circumglobally in tropical, subtropical and temperate coastal waters. Although mobulids have been recorded for over 400 years, critical knowledge gaps still compromise the ability to assess the status of these species. On the basis of a review of 263 publications, a comparative synthesis of the biology and ecology of mobulids was conducted to examine their evolution, taxonomy, distribution, population trends, movements and aggregation, reproduction, growth and longevity, feeding, natural mortality and direct and indirect anthropogenic threats. There has been a marked increase in the number of published studies on mobulids since c. 1990, particularly for the genus Manta, although the genus Mobula remains poorly understood. Mobulid species have many common biological characteristics although their ecologies appear to be species-specific, and sometimes region-specific. Movement studies suggest that mobulids are highly mobile and have the potential to rapidly travel large distances. Fishing pressure is the major threat to many mobulid populations, with current levels of exploitation in target fisheries unlikely to be sustainable. Advances in the fields of population genetics, acoustic and satellite tracking, and stable-isotope and fatty-acid analyses will provide new insights into the biology and ecology of these species. Future research should focus on the uncertain taxonomy of mobulid species, the degree of overlap between their large-scale movement and human activities such as fisheries and pollution, and the need for management of inter-jurisdictional fisheries in developing nations to ensure their long-term sustainability. Closer collaboration among researchers worldwide is necessary to ensure standardized sampling and modelling methodologies to underpin global population estimates and status.

High mercury concentrations reflect trophic ecology of three deep-water chondrichthyans.

The relative contributions of proximity to mercury sources and trophic ecology to realized axial muscle mercury concentrations were explored for three deep-water chondrichthyans (Etmopterus princeps, Centroscymnus coelolepis, and Hydrolagus affinis), two species of which are harvested for human consumption. Samples were taken at three North Atlantic Ocean locations: the Azores, the Charlie Gibbs Fracture Zone, and the Bear Seamount. Despite the long distances between anthropogenic sources and the sampling locations, all species from all locations had muscle mercury concentrations exceeding the United States human health screening value of 0.3 mg/kg wet weight. Proximity to anthropogenic sources was not an obvious determinant of these elevated concentrations. Generally, mercury concentrations appeared to increase with increased dependence on benthic versus pelagic food sources (as indicated by interspecies differences in δ(13)C), and with higher position in the trophic web (as indicated by differences in δ(15)N).

Age and growth of Neotrygon picta, Neotrygon annotata and Neotrygon kuhlii from north-east Australia, with notes on their reproductive biology.

Vertebral band formations were used to define age and growth in three Neotrygon species caught regularly as by-catch in prawn trawl fisheries in north-east Australia. Centrum edge and marginal increment ratio analyses were used to validate annual band formations. Age estimates ranged from 1 to 18 years, with the von Bertalanffy growth function considered to have the best fit to Neotrygon picta (males, W(D∞) = 271 mm, k = 0·12; females, W(D∞) = 360·5 mm, k = 0·08) and Neotrygon kuhlii (males, W(D∞) = 438·6 mm, k = 0·08; females, W(D∞) = 440·6 mm, k = 0·08) disc width (W(D))-at-age data. The Gompertz growth function had the best fit to Neotrygon annotata W(D)-at-age data (males, W(D∞) = 230·4 mm, k = 0·20; females, W(D∞) = 265·5 mm, k = 0·31). Age at sexual maturity ranged from 3 to 6 years, with N. picta having the smallest size at birth (100 mm W(D)), smallest W(D) at 50% maturity (W(D50): male, 172 mm, female, 180·7 mm) and lowest age at sexual maturity (3-4 years). This study helps redefine and improve the accuracy of fisheries-based risk assessments for these small species with relatively conservative life-history variables.

First determination of the levels of platinum group metals in Manta birostris (manta ray) caught along the Ghanaian coastline.

Tissues from Manta birostris caught by fishermen from Dixcove in the western part of Ghana were analyzed for their Platinum, palladium and rhodium concentrations (PGM). The use of chondrichthyan fish has permitted the study of trace levels of Platinum group metals (PGMs) which have travelled very far into the sea. The analysis showed that Ghana's coastline is fairly polluted with these platinum group metals (PGMs). PGM concentration in manta ray recorded a range of (0.15-0.85) microg/g for Pt, (0.033-0.67) microg/g for Pd and (0.007-0.145) microg/g for Rh. Comparing these values to the UK dietary intake of 0.2 microg/day for Pt and Rh and 1.0 microg/day for Pd, its indicates that the values obtained from the analysis for Pt was above the required level. This is the first study to show the accumulation of PGM in chondrichthyan fish, although the sources of this pollution are not clear as manta birostris is migratory and therefore need to be investigated further. The presence of the PGM is very significant, since manta ray meat is consumed in Ghana. This may presents a health risk, due to a possible accumulation of PGMs in humans.

Ontogeny of the tessellated skeleton: insight from the skeletal growth of the round stingray Urobatis halleri.

The majority of the skeleton of elasmobranch fishes (sharks, rays and relatives) is tessellated: uncalcified cartilage is overlain by a superficial rind of abutting, mineralized, hexagonal blocks called tesserae. We employed a diversity of imaging techniques on an ontogenetic series of jaw samples to investigate the development of the tessellated skeleton in a stingray (Urobatis halleri). We compared these data with the cellular changes that characterize cartilage calcification in bony skeletons. Skeletal growth is characterized by the appearance of tesserae as well as changes in chondrocyte shape, arrangement and density. Yolk sac embryos (35-56 mm disc width, DW) have untessellated lower jaw tissue wrapped in perichondrium and densely packed with chondrocytes. Chondrocyte density decreases dramatically after yolk sac absorption (histotroph stage: 57-80 mm DW) until the formation of tesserae, which are first visible using our techniques as thin (approximately 60 microm), sub-perichondral plaques. During the histotroph stage, flattened chondrocytes align parallel to the perichondrium at the tissue periphery, where we believe they are incorporated into developing tesserae to form the cell-rich laminae observed within tesserae; in older animals peripheral cells in the uncalcified phase are rounder and less uniformly oriented. By parturition (approximately 75 mm DW), cell density and the number of adjoining chondrocyte pairs (an indicator of cell division) have dropped to less than a third of their initial values; these remain low and tesserae continue to grow in size. The tessellated skeleton is a simple solution to the conundrum of growth in an endoskeleton with external mineralization and no remodeling. Although we see parallels with endochondral ossification (e.g. chondrocytes decreasing in density with age), the lack of chondrocyte hypertrophy and the fact that mineralization is sub-perichondral (not the case in mammalian cartilage) suggest that the similarities end there.

DNA Barcode Reveals the Bycatch of Endangered Batoids Species in the Southwest Atlantic: Implications for Sustainable Fisheries Management and Conservation Efforts.

Today, elasmobranchs are one the most threatened vertebrate groups worldwide. In fact, at least 90% of elasmobranch species are listed in the International Union for Conservation of Nature (IUCN) Red List, while more than 40% are data-deficient. Although these vertebrates are mainly affected by unsustainable fishery activities, bycatch is also one of the major threats to sharks and batoids worldwide, and represents a challenge for both sustainable fishery management and for biodiversity and conservational efforts. Thus, in this study, DNA barcode methodology was used to identify the bycatch composition of batoid species from small-scale industrial fisheries in the southwest Atlantic and artisanal fisheries from southeast Brazil. A total of 228 individuals belonging to four Chondrichthyes orders, seven families, and at least 17 distinct batoid species were sequenced; among these individuals, 131 belonged to species protected in Brazil, 101 to globally threatened species, and some to species with trade restrictions provided by Appendix II of the Convention on International Trade in Endangered Species (CITES). These results highlight the impacts on marine biodiversity of bycatch by small-scale industrial and unmanaged artisanal fisheries from the southwest Atlantic, and support the implementation of DNA-based methodologies for species-specific identification in data-poor fisheries as a powerful tool for improving the quality of fisheries' catch statistics and for keeping precise bycatch records.

Global-scale predictions of community and ecosystem properties from simple ecological theory.

We show how theoretical developments in macroecology, life-history theory and food-web ecology can be combined to formulate a simple model for predicting the potential biomass, production, size and trophic structure of consumer communities. The strength of our approach is that it uses remote sensing data to predict properties of consumer communities in environments that are challenging and expensive to sample directly. An application of the model to the marine environment on a global scale, using primary production and temperature estimates from satellite remote sensing as inputs, suggests that the global biomass of marine animals more than 10(-5) g wet weight is 2.62 x 10(9)t (=8.16 gm(-2) ocean) and production is 1.00 x 10(10) tyr-1 (31.15 gm(-2)yr(-1)). Based on the life-history theory, we propose and apply an approximation for distinguishing the relative contributions of different animal groups. Fish biomass and production, for example, are estimated as 8.99 x 10(8)t (2.80 gm(-2)) and 7.91 x 108 t yr(-1) (2.46 gm(2)yr(-1)respectively, and 50% of fish biomass is shown to occur in 17% of the total ocean area (8.22 gm(-2)). The analyses show that emerging ecological theory can be synthesized to set baselines for assessing human and climate impacts on global scales.

New hypothesis helps explain elasmobranch "outburst" on Georges Bank in the 1980s.

Regime shifts are a feature of many ecosystems. During the last 40 years, intensive commercial exploitation and environmental changes have driven substantial shifts in ecosystem structure and function in the northwest Atlantic. In the Georges Bank-southern New England region, commercially important species have declined, and the ecosystem shifted to one dominated by economically undesirable species such as skates and dogfish. Aggregated abundance indices indicate a large increase of small and medium-sized elasmobranchs in the early 1980s following the decline of many commercial species. It has been hypothesized that ecological interactions such as competition and predation within the Georges Bank region were responsible for and are maintaining the "elasmobranch outburst" at the heart of the observed ecosystem shift. We offer an alternative hypothesis invoking population connectivity among winter skate populations such that the observed abundance increase is a result of migratory dynamics, perhaps with the Scotian Shelf (i.e., it is an open population). Here we critically evaluate the survey data for winter skate, the species principally responsible for the increase in total skate abundance during the 1980s on Georges Bank, to assess support for both hypotheses. We show that time series from different surveys within the Georges Bank region exhibit low coherence, indicating that a widespread population increase was not consistently shown by all surveys. Further, we argue that observed length-frequency data for Georges Bank indicate biologically unrealistic population fluctuations if the population is closed. Neither finding supports the elasmobranch outburst hypothesis. In contrast, survey time series for Georges Bank and the Scotian Shelf are negatively correlated, in support of the population connectivity hypothesis. Further, we argue that understanding the mechanisms of ecosystem state changes and population connectivity are needed to make inferences about both the causes and appropriate management responses to large-scale system change.

First record of the sicklefin devilray Mobula tarapacana (Myliobatiformes: Mobulidae) from Australian waters.

The sicklefin devilray Mobula tarapacana (Philippi, 1892) is one of the largest and least known mobulids (Compagno & Last 1999; Couturier et al. 2012). Collections, fisheries bycatch data and visual records suggest it is primarily oceanic, with a circumglobal tropical and warm temperate distribution (Notarbartolo-di-Sciara 1987; Compagno & Last 1999; Gadig & Sampaio 2002; White et al. 2006a, b; Weir et al. 2012; Sobral 2013; Tomita et al. 2013). In the Pacific Ocean it has been recorded from Chile, Gulf of California, Japan, Taiwan, South China Sea and Indonesia (Compagno & Last 1999; White et al. 2006b; Couturier et al. 2012; Tomita et al. 2013).

Contrasting Responses to Harvesting and Environmental Drivers of Fast and Slow Life History Species.

According to their main life history traits, organisms can be arranged in a continuum from fast (species with small body size, short lifespan and high fecundity) to slow (species with opposite characteristics). Life history determines the responses of organisms to natural and anthropogenic factors, as slow species are expected to be more sensitive than fast species to perturbations. Owing to their contrasting traits, cephalopods and elasmobranchs are typical examples of fast and slow strategies, respectively. We investigated the responses of these two contrasting strategies to fishing exploitation and environmental conditions (temperature, productivity and depth) using generalized additive models. Our results confirmed the foreseen contrasting responses of cephalopods and elasmobranchs to natural (environment) and anthropogenic (harvesting) influences. Even though a priori foreseen, we did expect neither the clear-cut differential responses between groups nor the homogeneous sensitivity to the same factors within the two taxonomic groups. Apart from depth, which affected both groups equally, cephalopods and elasmobranchs were exclusively affected by environmental conditions and fishing exploitation, respectively. Owing to its short, annual cycle, cephalopods do not have overlapping generations and consequently lack the buffering effects conferred by different age classes observed in multi-aged species such as elasmobranchs. We suggest that cephalopods are sensitive to short-term perturbations, such as seasonal environmental changes, because they lack this buffering effect but they are in turn not influenced by continuous, long-term moderate disturbances such as fishing because of its high population growth and turnover. The contrary would apply to elasmobranchs, whose multi-aged population structure would buffer the seasonal environmental effects, but they would display strong responses to uninterrupted harvesting due to its low population resilience. Besides providing empirical evidence to the theoretically predicted contrasting responses of cephalopods and elasmobranchs to disturbances, our results are useful for the sustainable exploitation of these resources.

Bythaelurus tenuicephalus n. sp., a new deep-water catshark (Carcharhiniformes, Scyliorhinidae) from the western Indian Ocean.

A new dwarf deep-water catshark, Bythaelurus tenuicephalus, is described based on one adult and one juvenile male specimen from off Tanzania and Mozambique in the western Indian Ocean. The new species differs from its congeners by its slender head and snout, which is only slightly bell-shaped in dorsoventral view without distinct lateral indention. All other Bythaelurus species have distinctly bell-shaped snouts with a strong lateral indention anterior to outer nostrils. Compared to its congeners in the western Indian Ocean, B. tenuicephalus n. sp. also has broader claspers in adult males (base width 2.1% TL vs. 1.5-1.8% TL). It further differs from B. clevai by attaining a smaller maximum size and having a color pattern of fewer and smaller blotches, larger oral papillae, a shorter snout, and broader claspers without knob-like apex and with a smaller envelope and a subtriangular (vs. subrectangular) exorhipidion. Compared to B. hispidus, the new species has a longer snout, a longer dorsal-caudal space, broader clasper without knob-like apex, and fewer vertebral centra. In contrast to B. lutarius, B. tenuicephalus attains a smaller maximum size and has a blotched (vs. largely plain) coloration, numerous (vs. lacking) oral papillae, shorter anterior nasal flaps, a longer caudal fin, a shorter pelvic anal space, and shorter and broader claspers.

Predictive habitat suitability models to aid conservation of elasmobranch diversity in the central Mediterranean Sea.

Commercial fisheries have dramatically impacted elasmobranch populations worldwide. With high capture and bycatch rates, the abundance of many species is rapidly declining and around a quarter of the world's sharks and rays are threatened with extinction. At a regional scale this negative trend has also been evidenced in the central Mediterranean Sea, where bottom-trawl fisheries have affected the biomass of certain rays (e.g. Raja clavata) and sharks (e.g. Mustelus spp.). Detailed knowledge of elasmobranch habitat requirements is essential for biodiversity conservation and fisheries management, but this is often hampered by a poor understanding of their spatial ecology. Habitat suitability models were used to investigate the habitat preference of nine elasmobranch species and their overall diversity (number of species) in relation to five environmental predictors (i.e. depth, sea surface temperature, surface salinity, slope and rugosity) in the central Mediterranean Sea. Results showed that depth, seafloor morphology and sea surface temperature were the main drivers for elasmobranch habitat suitability. Predictive distribution maps revealed different species-specific patterns of suitable habitat while high assemblage diversity was predicted in deeper offshore waters (400-800 m depth). This study helps to identify priority conservation areas and diversity hot-spots for rare and endangered elasmobranchs in the Mediterranean Sea.

Fine structural and cytochemical observations of dental epithelial cells during the enameloid formation stages in red stingrays Dasyatis akajei.

The fine structure and the localization of nonspecific acid phosphatase (ACPase), nonspecific alkaline phosphatase (ALPase), and calcium-dependent adenosine triphosphatase (Ca-ATPase) activities in the dental epithelial cells in tooth germs of Dasyatis akajei in the later stages of enameloid formation were investigated. Numerous invaginations of the distal cell membrane of the inner dental epithelial (IDE) cells were observed at the early stage of enameloid maturation. The invaginations contain many fine granular and filamentous substances; the lamina densa, which was thicker during the former stages, is obscure. Granules exhibiting defined ACPase activity were usually found in the IDE cells during the stages of enameloid mineralization and maturation. IDE cells are putatively involved in the removal of degenerated enameloid matrix during these stages. Marked ALPase activity was detected at the proximal and the lateral cell membranes of the IDE cells from the late stage of enameloid matrix formation to the early stage of enameloid maturation. Strong activity of Ca-ATPase was localized at the proximal and the lateral cell membranes of the IDE cells during the stages of enameloid mineralization and maturation. ALPase and Ca-ATPase activity is probably related to crystal formation in the enameloid and the removal of degenerated enameloid matrix from the enameloid.