The morphology and chemistry of the scales of the orange roughy and the smooth and spiky oreo dories

TISSUE AND CELL, 199123 (5) 677-708 0 1991 Longman Group UK Ltd.

R. W. GAULDIE*, G. COOTEt, I. F. WEST+ and K. P. MULLIGAN*

THE MORPHOLOGY AND CHEMISTRY OF THE SCALES OF THE ORANGE ROUGHY AND THE SMOOTH AND SPIKY OREO DORIES Keywords:

Scales, orange roughy. oreo dories, proton, carbon, oxygen

deuteron

microprobe,

calcium, fluorine,

ABSTRACT. Proton and deuteron microprobes were used to establish the two-, and threedimensional patterns of calcium, fluorine, carbon and oxygen variation in the scales of the orange roughy (Hoplostethus atlanticus), smooth 0x0 dory (J’seudocyttus macularus) and spiky Oreo dory (Neocyttus rhomboid&)

major part of the effort in the orange roughy fishery in 1977, stocks of orange roughy have steadily declined (Robertson, 1987) in the face of conservative (about 10% of estimated biomass) catch regimes (Robertson and Grimes, 1983) to the point where the virgin biomass has been reduced by 60-70% (Smith et al., 1991). The reasons for the decline in abundance may be related to the unusual environment of the orange roughy which has such characteristics as to indicate that orange roughy populations are stronger k-selected and are therefore very sensitive to even moderate fishing pressure (Gauldie et al., 1989). However, any kind of population dynamics theory to explain declines in orange roughy abundance hinge around establishing a satisfactory estimation of fish age. Four species of Oreo dories have been described from New Zealand waters (Ayling and Cox, 1982). The smooth Oreo also occurs in Australian, South African and South American waters (Last et al., 1983) and ranges in depth between 650 and 1200m (McMillan, 1985). All three species present problems in age estimation. Age estimates in orange roughy based on interpretations of different otolith structures result in maximum ages of about 20 years using microincrement widths, (Gauldie et al., 1989) to about 100 years using opaque zones in whole otoliths (Mace et al., 1990). The otolith of the Oreo dories has a

Introduction

The orange roughy Hoplostethus atlanticus (Trachichthyidae), and the Oreo dories, the smooth Oreo Pseudocyttus maculatus and the spiky Oreo Neocyttus rhomboidalis (Oreosomatidae), are species of the moderate deep water, 300m to about 11OOm. All three species are fished commercially in New Zealand waters. The orange roughy is one of five species of the family Trachichthyidae (related to the Beryciformes) that occur in New Zealand waters. Orange roughy have a world-wide distribution at depths from 450-1800m (Paulin, 1979; Merrett and Wheeler, 1983). Orange roughy occur sporadically in relatively low densities over much of the New Zealand continental shelf (Clark and King, 1986a, b), but in their spawning season they aggregate into very large and dense schools that once may have consisted of millions of tons (Robertson et al., 1982) providing a valuable fishery in both Australian and New Zealand waters. Since the inception of the * Hawaii Institute of Geophysics, University of Hawaii. 2525 Correa Road, Honolulu. HI 96822, USA. t Institute of Nuclear Sciences, Department of Scientific and Industrial Research, Private Bag, Gracefield, Lower Hutt, New Zealand, $ MAFFish, P.O. Box 297, Wellington, New Zealand. Received 26 November Revised 7 April 1991.

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complex crystalline structure that makes age estimation difficult (Davies et al., 1989), and no age estimates have been published. A possible alternative for age estimation is in the scales of all three species. However, the scales of all three species are usually small and show little optical structure that would lend any enthusiasm to using them in age estimation. Nonetheless, recent studies of the very high levels of fluorine in fish scales, coupled with the apparent temperaturedependent annual periodicity of fluorine variation (Gauldie et al., 1990) suggest a reexamination of the morphology and chemistry of the scales of the average roughy, smooth Oreo and spiky Oreo. Materials and Methods

The orange roughy grows to 40+ cm (standard length) which is large for a deep water fish and has a deep, laterally compressed shape. Nine orange roughy taken from a commercial catch off the Chatham Rise were sampled for scales around the lateral line dorsal to the pectoral fin. Four males (29,32, 33, 36cm SL) and five females (26, 30, 31, 33,35,36 cm SL) were sampled from catches made on the Chatham Rise, east of New Zealand. Scales were stored dry in paper packets. Ten samples of scales were taken for smooth and spiky Oreo respectively from the mid-body location and stored dry in paper bags. Scales were photographed optically with a WILD photomicroscope both with, and without, polarising filters. Scales were mounted on pin-stubs with epoxy resin and scanned using both a proton and deuteron beam following the methods described by Coote et al. (1982). Scales that were scanned, plus a sample of unscanned scales mounted on stubs, were dried, sputter coated with gold and photographed with a Philips 505 SEM. Some of the scales were scanned with a single trace across the areas of interest.

ET AL.

Others were scanned with up to 50 parallel traces, the data from which was displayed as three dimensional plots of elemental concentration. Analysis of the three dimensional data was undertaken using the LOWESS techniques (Cleveland, 1979) available through the ‘S’ statistical package. The LOWESS technique uses a running mean method to reveal the underlying functional relationships between variables without the need to fit the data to an a priori parametric equation. In this paper we are interested in the rise and fall in the fluorine content of the scale as a possible seasonal indicator of the validity of what are otherwise very weakly defined annual marks. Scale terminology refers to annual marks as annuli, which makes the object of our interest the annulus between annuli. To avoid these linguistic contortions we refer to annual marks as such, and where appropriate the inter-annual zone, following the nomenclature of Gauldie et al. (1990). ReSXdts 1. Orange rougby scales

la. Scale morphology The orange roughy has small irregularly shaped scales (Figs 1,2) except for the scales along the lateral ‘line (Fig. 3). The scales along the belly ridge are almost in the form of scutes and were not sampled for this study. Typically scales ranged in size from about 1 mm in diameter to up to 5 mm across the long axis. Scales from the lateral line were much larger, up to 20mm, with a characteristic winged appearance with a complex central structure with lacunae presumably connected to the lateral line system. The part of the scale protruding from the skin had a spiny structure arranged in rows (Fig. 1). Optical pictures showed possible annual marks in the section of the scale that was buried in the tissue of the skin (Fig. 1). However, although the annual marks were

Fig. 1. The spiny part of the orange roughy scale is located in the external (E) part of the scale. Scale marks similar to ammal marks (arrows) are weakly defined and occur in the internal (1) part of the scale. x 17. Fig. 2. Polarising filters have little or no effect on the legibility of the annual marks in the scales above. X 17.

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poorly defined and could not be seen in all of the scales taken from the same fish, similar marks were visible in the smaller as well as in the larger scales (Fig. 1). Crossed polarising filters made the annual marks less visible (Fig. 2). A lateral line scale from the same individual showed a much clearer pattern of marks (Fig. 3) on the lateral edges of the scale. SEM pictures of scales showed possible annual marks (Fig. 4), but they require subjective interpretation being in the form of variations in widths between circuli, rather than checks (e.g. Kelley, 1988). The sample of scales in Figures 1 and 2 were more-or-less juxtaposed in the fish. There is a wider variation in scale width (the range is about 89% of the mean) within the sample, than in the depth of the scale (the range is about 36% of the mean) particularly in the smooth part of the scale that is within the dermis. The numbers of spines were also more closely related to the width rather than the depth of the scales. Although there was evidently variation in growth between scales, the greatest variability was in the width of the scale. The proportion of erosion, or replacement, scales, was very low, about 1 scale in 50. Even these scales showed circuli patterns that were disordered rather than eroded (Fig. 5). Scales that were deliberately broken showed an internal structure of protein fibres oriented at about 90” to each other with an

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amorphous mineral layer on the outer surface and a smaller amount of mineral infiltrating the scale layers (Fig. 6). The external surface of the scale was mineralised between circuli (Fig. 7). The mineralised surface was composed of rounded crystals, aggregating into strands of beaded crystals that were between O-5 and 1 pm in diameter and up to 5 pm in length (Fig. 7). Detail of the circuli showed a smooth-surfaced toothed structure at the edge of the circuli. (They were probably damaged in handling leading to the cracks and broken teeth in Fig. 8), but further back from the circuli itself the surface showed rounded crystals about 1 pm in diameter (Fig. 8). One scale showed what may have been genuine erosion in the form of an etch-pit at the focus of the scale (Fig. 9). The crystals within the etchpit have the same granular appearance of crystals elsewhere in the scale with diameters between 0.5 and 1 pm. An SEM of a scale mounted endodermal side up showed a number of marks similar to annual marks (Fig. 10). The undersurface of the scale showed a difference in structure between the external and internal parts of the scale. The internal part of the scale had many concentric bands that appear to coalesce into a much smaller number of bands on the external part of the scale (Fig. 10). The undersurface of the scale showed the alignment of proteins very clearly on a weakly mineralised surface (Fig. 11). Protein

Fig. 3. Lateral line scales of orange roughly have a winged appearance with annual type marks visible (arrows) along the lateral sides. The central part of the scale has an overlaid appearance with a complex structure of spines and lacunae. x 11.5. Fig. 4. An SEM picture of an orange roughy scale shows the spinose external part of the scale and marks (arrows) consisting of variations in the widths of circuli that may be equivPlent to annual marks in the part of the scale buried in the dermis. x24. Fig. 5. An SEM picture of an erosion-type orange roughy scale showed a disordering of circuli patterns [inset] rather than erosion of the scale. LHS at x 13. Fig. 6. A broken orange roughy scale showed [inset] protein layers oriented at 90” to each other. An amorphous mineral layer (M) coats the external part of the scale with a small amount of mineralisation (arrows) between protein layers. The surface crystal in the form of small granules. LHS at x88. Fig. 7. The part of the scale buried in the dermis has an amorphous mineral surface (enlarged in inset) that is composed of rounded crystals sometimes beaded together (arrow). LHS is x420. Fig. 8. Circuli of the orange roughy scale have a smooth mineralised surface at the toothed edge, but show small rounded crystals on the rest of the surface (arrows). x6400.

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Fig. 13. The raw calcium counts from the first orange roughy scale showed a clear peak in the centre at around the point (asterisk) nearest to the focus (F) of the scale and minor peaks (arrow) of varying intensities. The scale refers to each point number of the scan.

fibres can be seen running parallel northsouth (Fig. 11) with a few fibres of the next layer growing at about 90” to the first layer. Small mineral granules on this surface had diameters less than, or equal to about 1 ,um. lb. Scale chemistry: proton microprobe

The results below are organised by each scale so that each section is denoted by a scale number of OSl, 0S2, etc. Back-calculated length-at-age of the scales are shown in Figure 47. OSl. The first orange roughy scale was scanned longitudinally (Fig. 12) from left to right at 20 ,um intervals. The break along the microprobe scan line of this and following

scales was due to drying in preparation for scanning electron microscopy. The calcium and fluorine traces are shown separately as raw data in Figures 13, 14 and as smoothed data in Figure 15. The smoothed data in Figure 15 showed a good match between peaks in the calcium and fluorine records. Fluorine counts between peaks and troughs in the smoothed data ranged from about lO50% of the trough values. The fluorine count at the focus was about 4 times higher than in other parts of the scale. Calcium counts at the focus were also higher than in the rest of the scale. The range between peaks and troughs in the calcium counts was between about lo-30%. The fluorine to calcium ratio

Fig. 9. An etch pit appeared at the focus of one orange roughy scale rcvcaling crystals (arrow) that were greater in number in the internal (I). than in the external of the scale. x3600. Fig. 10. An orange roughy scale mounted concentric marks (arrows). X30.

endodermal

side uppermost

granular (E). part

showed a number

of

Fig. 11. Detail of the endodermal surface of the scale [inset] showed the protein structure with a weak mineralisation (small arrows) and the beginnings (large arrow) of the next. orthogonal protein layers. LHS is X88. Fig. 12. An SEM of the first orange roughy scale showed the proton microprobe scan running from left to right. The location of the troughs in the calcium scan of Figure 15 below are shown by arrows and scale textures by asterisks. x41.

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Fig. 14. The raw fluorine counts from the first orange roughy scale showed a clear peak at the focus of the scale and minor peaks. The scale refers to each point number of the scan.

in most of the scale was about 0.44, except at the focus where the ratio was about 0.23. The smoothing programme (LOWESS) was set to remove about 5% of the apparent noise. The smoothed data showed a peak in both calcium and fluorine counts near the focus of the scale. The average distance between circuli was 40 pm so that the trend

of peaks in the smoothed data cannot be related to the circuli. However there were 76 circuli countable in the scale and there were 82 peaks in the raw calcium data (counting shoulders as peaks), which given the difficulty of reading circuli near the focus may indicate that a local mineral concentration at each circulus shows up as a peak.

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Fig. 15. Smoothed calcium (smooth line) and fluorine data (dotted line) from the first orange roughy scale showed a clear series of peaks and troughs, particularly in the calcium record.

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Point number Fig. 17. The smoothed calcium (solid line) and fluorine (dotted line) traces run from the spiny external side to the left of the traces, to the smooth internal side to the right with the crossover point marked by an arrow. Possible troughs in the calcium trace on the internal side are marked with arrows, and peaks on the external side with asterisks.

The annual mark on a scale is made visible by the circuli being closer together because the scale is growing more slowly in the (presumably) winter season. Slower growth will also result in less mineral and lower fluorine levels (Gauldie et al., 1990). Therefore the troughs in the calcium record should correspond to the annual marks (annuli) usually observed in scales. The scale in Figure 12 has been marked with arrows along the proton scan at the equivalent points to troughs in the trace. Many of the arrows lie close to scale features (marked with asterisks) that could be readily interpreted as annual marks. If the system of peaks and troughs could be regarded as annual, then the fish that this scale came from, a 35 cm male, could be regarded as 6-7 years old (OS1 in Fig. 47). OS2. A second scale (Fig. 16) was scanned along the caudal axis that includes the spiny

part of the scale that provides the external spines from which the orange roughy takes its name. The calcium and fluorine scans are shown after 5% smoothing in Figure 17. The spiny section of the scale corresponds to the left-hand side of Figure 17. There are 6 rows of spines on the scale and there are 12 peaks, including shoulders, in the calcium and fluorine traces equivalent to that part of the scale. There are five possible troughs in the calcium and fluorine traces in the part of the scale embedded in tissue. These are marked in Figure 17 and their equivalent locations are shown by arrows in Figure 16. Some of the arrows in Figure 16 correspond to structures (marked by asterisks) that would correspond to annual marks in other scales. The fluorine content of the scales was higher in the spiny part of the scale, with counts of up to 1600 compared to counts of up to 500

Fig. 16. The second orange roughy scale was scanned from the point (E) on the external side of the scale through the focus to the edge (I) of the external part of the scale. Troughs in the calcium record (Fig. 17) are shown as arrows and scale features as asterisks. x36. Fig. 18. The third orange roughy scale was scanned across the longitudinal axis. The relative positions of the troughs in the calcium and fluorine trace (Fig. 19, below) are marked by arrows and structures in the scale that could be interpreted as annual marks are indicated by asterisks. x26.

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Fig. 19. The smoothed calcium (solid line) and fluorme (dotted line) traces show a series of peaks and troughs, with peaks near the focus (arrow).

in the internal part of the scale. The trough to peak variation in calcium in the spiny part of the scale ranged from 3-5 times, and from a few percent up to 3 times in the internal part of the scale. There was generally a good match between peaks in the calcium and fluorine traces with fluorine counts varying

350q

from about 2550% of calcium counts. The point corresponding to the focus (arrow in Fig. 17) has high calcium and fluorine counts, but it is evident from Figure 16 that the more mineralised external part of the scale appears to have overgrown the focus of the scale. If the five troughs in the scale corresponded to

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Fig. 20. The smoothed calcium (solid line) and fluorine (dotted line) traces for the fourth orange roughy scale showed a series of peaks and troughs running from the spiny external, left hand side to the smooth, internal right hand side. The external side stops at the focus which is shown by an arrow.

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Fig. 21. The raw calcium data for the first of the multiple scanned orange roughy scale showed higher levels on the external (E) side than on the internal (I) side. Some circular elements in the distribution of the raw data are visible (dark line). The maximum value of calcium counts was 453.

annual marks then this fish, a 29cm male, would have been five years old (OS2 in Fig. 47). 0S3. A third scale was scanned across the longitudinal axis of the scale close to the focus (Fig. 18). The calcium and fluorine patterns showed a well defined series of peaks and troughs (Fig. 19). There was a good correspondence between peaks and troughs in calcium and fluorine. However, the proportion of the fluorine to calcium varied from both sides of the scale, ranging from about 12% to about 23%. Peak to trough variation

was very large, up to 1200 times, in both fluorine and calcium. Both calcium and fluorine levels were higher at the focus. The relative positions of all the troughs are shown by arrows along the path of the trace in Figure 18. Structures that would correspond to annual marks in scales are marked by dots in Figure 18. If the pattern of peaks and troughs in this otolith could be interpreted as annual, then the fish from which the scale was taken, a 36 cm male, was between 7 and 8 years old (OS3 in Fig. 47). OS4. A fourth scale was scanned for cal-

Fig. 22. The raw fluorine data showed higher levels on the external (E) side than on the internal (I) side, with highest levels at the centre of the scale at a point (asterisk) corresponding to the focus. Maximum fluoride count was 208.

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Fig. 23. The smoothed calcium data showed that some of the circular elements of the scale could be seen (open arrows) as well as linear elements (closed arrows). Maximum fluorine count was 324. Counts were higher on the external (E) side, and at the focus (F).

cium and fluorine along the caudal axis (Fig. 20) and yielded a series of 6 peaks and troughs on the external part of the scale and four troughs on the endodermal side of the scale (Fig. 20). There was good correspondence in both calcium and fluorine traces. The highest values for calcium and fluorine were at the focus. Except at the focus, fluorine levels were about 25% of calcium levels, with peak to trough variation between 10 and 50%. If these troughs cor-

respond to annual marks this fish (a 26 cm female) would be 5-6 years old (OS4 in Fig. 47). Multiple scans. Three orange roughy scales were scanned by 50 parallel lines of 100 scan points each 50 pm apart. The raw calcium data for one of these scans is shown in Figure 21 and the raw fluorine data is shown in Fig. 22. The fluorine data showed a clear maximum at a point corresponding to the focus. Maximum fluorine counts (208) were

Fig. 24. The smoothed fluorine data a stronger external/internal difference in fluorine levels than the corresponding calcium data but otherwise less structure, although a series of peaks can be seen along the lateral axis marked by two arrows. Maximum fluorine count was 107. Counts were higher on the external (E) side, and at the focus (E).

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Fig. 25. Smoothed calcium data from the second scale showed a pronounced linear effect resulting in a succession of peaks and troughs running orthogonal to the internal (1) side of the scale. Maximum calcium count was 477.

about half of the maximum (453) calcium counts. After smoothing, more of the under lying structure of the data becomes evident. The smoothed data for calcium and fluorine is shown in Figures 23, 24 respectively. It is evident that there are higher calcium and fluorine levels towards the external side than on the endodermal side of the scale. The raw data showed that although the information is very dense on the calcium surface generated by the trace, there is evidence of concentric calcium deposition (lines in Fig. 21). However, the smoothed data (Fig. 23) showed in addition to the concentric

calcium a very strong linear trend towards parallel deposition of both calcium and fluorine along the head-to-tail axis of the fish (i.e. the external to internal axis of the scale). Concentric deposition is more evident in the smoothed fluorine data (Fig. 24), but even there, there are strong parallel orientations in fluorine peaks and troughs. Both calcium and fluorine smoothed plots showed a maximum count at the centre of the scale corresponding to the focus (Figs 23, 24). There are a series of peaks and troughs towards the lateral edges of the scale (Figs 23, 24). If these troughs correspond to the

Fig. 26. Smoothed fluorine data from the second scale showed more emphasised linear effect than in the calcium data, with ridges of higher fluorine counts running orthogonal to the internal (I) side of the scale. Maximum fluorine count was 488.

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Fig. 27. The third scale was laterally expanded with respect to the internal (I) side of the scale resulting in a complex pattern of ridges in calcium counts running from the ccntrc to the edge of the scale along the lateral axes (arrows). Maximum calcium count was 197.

annual marks then the fish from which this scale was taken (a 31 cm female) would be about 5 years old. A second set of smoothed calcium and fluorine scans are shown in Figures 2.5,26 for

a scale taken from a 29 cm male. The parallel effects are very pronounced in this scale, but along the lateral sides of the scale there appear to be a series of four to five troughs from the focus to the edge of the scale.

Fig. 28. The smoothed fluorine data from the third scale showed a very similar topography to the smoothed calcium data with apparently better resolution of the sequence of peaks and troughs in the lateral (L) part of the scale. Maximum fluorine count was 88.

Fig. 29. The pattern of the microprobe scans for carbon and oxygen can bc seen in the smaller of the orange roughy scales that were scanned by the dcutcron beam. Fig. 34. The smooth Oreo scale is almost circular with the focus (F) located closer to the edge of the otolith. The scale shows a well developed series of annual marks or annuli (arrows). x37.

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Fig. 30. The patternof carbon distribution for the smaller orange roughy scale was coarsely defined, but showed lower levels towards the centre (arrow) and higher levels towards the external (E) side. Maximum carbon count was 2562.

A third set of smoothed calcium and fluorine scans are shown in Figures 27.28 for a scale taken from a 36 cm female. This particular scale was laterally expanded, but not as extreme as with the lateral line scales. From the focus of the scale to the lateral edge there were apparently between 8 and 9 troughs that may correspond to annual marks. Ic. Scale chemistry: deuteron microprobe

The deuteron beam of the microprobe provides a heavier particle, and consequently higher energy is available to develop a signal from lighter elements such as carbon and

oxygen. Carbon and oxygen traces were taken from two scales that were scanned with 20 parallel beams. The smaller of the two scales (Fig. 29) was about 5 cm square resulting in the 20 scans being about 350 pm apart, yielding a coarse picture of the otolith chemistry. The three dimensional reconstructions of the carbon and oxygen scans are shown as raw data in Figures 30, 31 respectively. The larger of the two scales was 15.9 mm by 12.7mm giving an even coarser resolutions from 20 x 20 scans. The three dimensional reconstructions of the carbon and oxygen scans are shown as raw data in Figures 32, 33.

Fig. 31. The pattern of oxygen distribution for the smaller orange roughy scale was coarsely defined but showed higher levels at the centre and lower levels at the edges. The external side is shown as E. Maximum oxygen count was 896.

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Fig. 32. The second, larger orange roughy scale showed a decrease in carbon towards the centre with increased levels towards the edges. The external (E) side is toward the viewer. Maximum oxygen count was 921.

2. Oreo dory scales 2a. Scale morphology

of the smooth Oreo

The smooth Oreo has very small, easily dislodged scales. Although they are almost circular, the focus of the scale is displaced to one side of the scale (Fig. 34). The smooth Oreo scale showed a well-defined series of annual marks (annuli) that result from the interruptions in the normally concentric pattern of the scale circuli (Fig. 35). Most of the smooth Oreo scale lies within the dermis so that the part used in ageing forms the largest part of the scale. Smooth Oreo scales are very thin (=20 pm) with a typical cross-ply protein structure and mineralised outer surface (Fig. 36). The circuli appear as more heavily mineralised components of the outer surface (Fig. 36). The broken section of the smooth Oreo scale revealed granular crystals within

the mineralised part of the scale (Fig. 36). Crystals ranged in diameter from 0.5-l pm within the mineral layer, about 2 pm in diameter on the surface (Fig. 36). 2b. Scale chemistry of the smooth Oreo

Two scales from smooth Oreo were scanned along the head-to-tail axis. SMOl. The first scale was scanned at 200 points at 17ym intervals (Fig. 37). There were 17 circuli along the path of the scan at approximately 36 pm average intervals. The smoothed calcium and fluorine traces are shown in Figure 38. The relative amount of fluorine increases towards the external part of the scale. Unlike the orange roughy scale, the focus has lower levels of both calcium and fluorine than other parts of the scale. The ratio of fluorine to calcium in the smooth Oreo scale was about 10% with peak to trough

Fig. 33. Oxygen levels in the second, larger orange roughy scale showed higher values towards the centre than at the edges of the scale. The external (E) side is toward the viewer. Maximum oxygen count was 921.

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Point number Fig. 38. The smoothed calcium (solid line) and fluorine (dotted

line)

data showed

a series of

peaks and troughs running from the interior edge of the scale (LHS) through the focus whose

relative position is marked with an arrow. Troughs in the calcium scan are marked with asterisks.

variation of about 5%. There were approximately 11 troughs in the calcium and fluorine records (Fig. 38) whose equivalent locations are marked on the trace in Figure 37 with arrows. There are structures in the scale (marked by dots) equivalent to the annual marks found in other scales, most of which correspond with the arrows on the trace. If the troughs therefore represent annual events, the fish from which this scale was taken (a 36 cm male) would have been ll12 years old (SMOl in Fig. 47). SM02. The second scale was scanned at 400 points each 9 pm apart (Fig. 39). There were approximately 82 circuli along the scan path at approximately 43pm average intervals. There were approximately 10 troughs in the calcium and fluorine traces shown Figure 40. The scan did not pass through the focus. Overall, fluorine levels were about 12% of calcium levels, with peak to trough variation of about 25%. Fluorine and calcium

Fig. 35. The annual marks result from,changes on the surface of the scale. x 150.

levels increased towards the internal edge of the scale. There was generally a good match between the pattern of fluorine and calcium. The maximum fluorine count was about 25% of the maximum calcium count. The equivalent locations of the calcium and fluorine troughs are shown with arrows along the trace in Figure 18a. There are structures in the scale (marked by dots) equivalent to the annual marks (or annuli) found in other scales, most of which correspond to the arrows on the trace. If the troughs therefore represent annual events, the fish from which this scale was taken (a 32 cm female) would have been 8 years old (SM02 in Fig. 47). Multiple scans. A single smooth Oreo scale was scanned by 50 parallel scans of 100 observation points. Three dimensional smoothed plots of calcium and fluorine for the smooth Oreo scale shown in Figures 41, 42. The smooth Oreo scale is unusual in that calcium and fluorine levels are low at the centre of

in both the spacing and structure of circuli

Fig. 36. Broken scales showed a mineral&d upper layer that included mineralised circuli (C) and a thin protein layer consisting of protein layers oriented at 90” (arrows). x2550.

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Fig. 40. The calcium (solid line) and fluorine (dotted line) data showed a series of peaks and troughs running from the interior (LHS) of the scale to the exterior (RHS). Troughs in the calcium trace are marked with asterisks.

Fig. 41. The smoothed calcium data from a multiple scanned smooth Oreo scale showed generally lower counts in the central part of the scale. but little in the way of periodic structures, although with increased counts at the internal (I) edge. The maximum calcium count was 192.

Fig. 37. The first smooth Oreo scale was scanned from the interior edge (I) through the focus (F). Points equivalent to troughs in the calcium/fluorine traces (below) are shown as arrows. Marks in the scale that could be interpreted as annual marks (or annuli) are shown as asterisks. x35. Fig. 39. The second smooth Oreo scale was scanned from the interior edge (I) to one side of the focus (F). Points equivalent to troughs in the calcium/fluorine traces (below) are shown as arrows. Marks in the scale that could be interpreted as annual marks (or annuli) are shown as asterisks. ~35.

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Fig. 42. The smoothed fluorine data from a multiple scanned smooth ore” SC& showed a marked central depression in counts, but little in the way of periodic structures. Counts wcrc higher at both the external (E) and internal (I) edges. The maximum fluorine count was 53.

2d. Scale chemistry of the spiky Oreo

in Figure 45. There are structures in the scale (marked by asterisks) equivalent to the annual marks (or annuli) found in other scales, most of which correspond to the arrows on the trace. Two large arrows in Figure 45 mark the two troughs on the external side of the focus. In the calcium and fluorine traces in Figure 46 they are marked with arrows. Fluorine and calcium levels corresponding to that at the focus would have similar values to the second trough in Figure 46. Fluorine levels varied between 5 and 10% of calcium levels. If the troughs represent annual events, the fish from which this scale was taken (a 27 cm male) would have been at least 9 years old. Part of the internal edge of the scale was obscured by resin so that this fish may have been older than 9 years.

One scale from the spiky Oreo was scanned along the head to tail axis at 400 points at about 6 ,um intervals (Fig. 4.5). There were 83 circuli along the path of the scan at approximately 30pm intervals. The smoothed calcium and fluorine traces are shown in Figure 46. There were 9 troughs in the calcium fluorine scans (Fig. 46) whose equivalent positions are marked on the trace

3. Back-calculation of growth from scales Back-calculated lengths-at-age are shown for four orange roughy scales and two smooth Oreo scales in Figure 47. Back calculation were made from the widths between apparent annual marks seen on the scales that correspond to troughs in the calcium/fluorine traces. Part of the internal edge of the spiky

the scale, rising at the edges. Although the calcium and fluorine patterns are highly structured there is little indication of the kind of periodic structures seen in other three dimensional maps of scales. 2c. Scale morphology of the spiky Oreo The scale of the spiky Oreo is set deeply in the tissue with a narrow protruding rim of spines (Fig. 43). The spiky Oreo scale shows a number of surface structures that have the appearance of annual marks observed in the scales of other fishes (Fig. 43). The annual marks have a characteristic pattern where they enter the evidently heavily mineralised spiked external part of the scale (Fig. 44).

Fig. 43. The scale of the spiky oreo shows the focus (F) displaced towards the external, mineralised, spiky edge. Periodic structures (arrows) similar in appearance to the annual marks found in other scales appear on the surface. x52. Fig. 44. Grooves (arrows) appear in the mineraked external edge of the otolith that may indicate the anomalies in the circuli that result in allual-type marks in the scale. x200.

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Fig. 45. A spiky oreo scale was scanned from the external (E) side to the internal (I) side, bypassing the focus. Points equivalent to troughs in the calcium/fluorine traces (below) are shown by arrows. Marks in the scale that could be interpreted as annual marks are shown as asterisks. x28.

d;

50

100

150

200

250

300

350

';b

400

Fig. 46. The smoothed calcium (solid line) and fluorine (dotted line) data showed a series of peaks and troughs running from the exterior edge (LHS) to the interior edge (RHS). The two troughs on the external side are shown by arrows the remainder by asterisks.

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Fig. 47. Back-calculated lengths-at-age are shown for orange roughy scales (0s 1, 2, 3, 4) and &moth Oreo scales. (SM6 1, 2). _

Oreo scale was obscured, and therefore length-at-age back calculations were not made. Discussion The basic structure of the scales of the orange roughy and the Oreo dories consists of alternating protein layers intersecting at about 90” and mineralised with granular crystals of calcium phosphate. This kind of organisation of protein and mineral occurs in most, if not all, teleosts (Lansing and Wright, 1976). Granular crystals have been described in other scales. Granules consisting of aggregated calcium phosphate crystals (described as spheritic mineralisation) have been reported in the scales of Carassius aurutus (Zylberberg and Nicolas, 1982) and Cyprinodon vaviegatus (Olson and Watabe, 1980) as ranging in size from 0.04 and O-2 pm in C. variegatus to about 0.14pm in C. aurutus. Granular crystals in the scales of the orange roughy and Oreo dories were larger, 0.5 and 1 pm, and l-2 pm respectively, but smaller than the granular crystals reported for Amia calva scales by Meunier (1981) that were up to 50 pm in diameter. Much larger crystals, up to 150 pm in diameter, identified as Mandl’s corpuscles also occurred in the scale of Amina calva (Meunier, 1981). Spheritic granules were described by Zylberberg and Nicolas (1982) as resulting from fusion of single crystals of calcium phosphate deposited by the mineralising front of the scale. Zylberberg and Nicolas (1982) present arguments that spheritic mineralisation occurs without a protein matrix, i.e. it is a

crystalline effect. If this is so, the size of the crystal ought to reflect both its own growth rate, and consequently, the growth rate of the scale. The similarity of granule size observed in the scales of the orange roughy and the Oreo dories are reflected in the similar size-at-age of individuals. The circuli of the scales of the three species were mineralised structures with a smootherless granular appearance similar to that of the scales. The differences in the average widths of circuli between the focus and the internal edge of the scale were not very great, 30-43 ,um. The development of circuli are ascribed Hemichoromis bimaculatus by Sire and Geraudie (1983) in which they argue that circuli arise when two superficial scleroblasts overlap over a short distance. Variation in the widths between circuli that give rise to annual-type marks in the scale might then be ascribed directly to seasonality in the metabolic activity of scleroblasts. The period of the cycles in fluorine and calcium were much longer than the periods of the circuli. In some scales the circuli may generate higher frequency peaks in calcium and fluorine but the underlying periodicity in calcium and fluorine was not related to the circuli. The spines of the orange roughy scales have higher levels of fluorine relative to calcium, as did the external part of the scale in all three species. In life the spines are invested with scleroblasts, otherwise they would not grow. Nonetheless, there is still an intimate relationship with the surrounding water from which the scale may only be separated by scleroblasts of much less than a micron thickness (Lanzing and Wright,

GAULDIE ET AL.

1976). Even with such intimate contact there must be some concentrating mechanism in the scleroblast that increases the level of fluorine in the scale by an enrichment factor of about lo5 with respect to sea-water (Carpenter, 1969). Orange roughy scale spines occur in rows whose existence may imply a pattern of growth that is scale-specific, and not necessarily directly related to body growth. The lateral line scales of the orange roughy showed a complex central structure presumably related to lateral line function that may be unrelated to other growth processes, which may be, nonetheless, expressed in the annual marks of the distal parts of the lateral scales. In addition, orange roughy scales showed more variation in width (dorsoventral) than they did along their caudal axis. There is a lot of individual variation in scale area, but scale length along the caudal axis is conserved. Orange roughy scales were characterised by weak, sometimes virtually non-existent, annual marks on the scales, but had welldeveloped periodicities in their fluorine and calcium levels as both single traces and in three dimensional plots. Calcium and fluorine variation holds promise of being useful in ageing orange roughy, but the problem remains of what causes the variation in fluorine and calcium. Other studies have shown a temperature component in fluorine composition of scales (Gauldie et al., 1990). However, at the depths at which orange roughy occur temperature variation is low, of the order of l-2°C. It is much more probable that fluorine and calcium variation is related to growth, rather than temperature related changes in mineral metabolism. Glycine uptake studies have shown that scales grow in response to any kind of growth affecting controls (Adelmann, 1987). The growth of even fishes at depth may be periodic representing seasonality of a variety of kinds (Tyler, 1987). The cyclical changes in calcium and fluorine are therefore presumably related to the seasonality of the growth process rather than temperature. Fluorine commonly occurs in biological calcium phosphates possibly as a replacement for the hydroxyl radical in hydroxy apatite (Posner et al., 1980). Fluorine levels in the orange roughy scales varied between 10 and 50% of the calcium count, averaging about

25%. The smooth Oreo scale had less fluorine, about 16% of calcium, and the spiky Oreo had less still, less than 10% of calcium. Samples sizes were small, but there appears to be a trend downwards across the three species. Variation between peaks and troughs of both fluorine and calcium varied among species. In the orange roughy scales peak to trough variation was as high as that described for Arripis Trutta and Chrysophrys auratus (Gauldie et al., 1990). The pattern of peaks and troughs in fluorine and calcium in all three species matches the pattern (even though it is weakly expressed) of annual-type marks in the scale. The deuteron probes showed a more-orless inverse relationship between oxygen and carbon that may reflect no more than the greater thickness of the mineralised layer towards the centre of the scale. The coarseness of the grid may have precluded the observation of periodic structures in the carbon/oxygen ratio. Studies of developing scales suggest that the mineralised layer may also reflect the degree of mucopolysaccharide concentration associated with the collagen fibres of the scale (Sire and Geraudie, 1983). In which case, carbon levels should also show periodic variation within the mineralised part of the scale. The scales of the smooth Oreo have well developed marks of the type usually regarded as annual in the scales of other fishes. Fluorine and calcium traces have similar periodicities suggesting that there are indeed periodic structures and are worth the effort of further investigation for age estimation. The scale of the smooth Oreo and spiky Oreo scale has a spiked edge and strongly mineralised surface in the part of the otolith protruding from the tissue. The marks corresponding to annual marks in other otoliths were very strongly emphasized in the strongly mineralised part of the scale. The good correspondence between presumed annual marks and cycles in fluorine and calcium suggest that the ‘annual’ marks are indeed annual in the scales of oreosomatids. One of the strongest differences between orange roughy and oreosomatid scale chemistry lies in the behaviour of fluorine and calcium at the focus of the scale. The focus of the orange roughy scale was marked by high counts of fluorine and calcium, although the fluorine to calcium ratio was lower than in

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other parts of the scale. Conversely, fluorine and calcium counts at the focus of the oreosomatid scale are lower than in other parts of the scale. The focus of the scale represents the juvenile part of the life history of scale and there are significant differences in the early life histories of the orange roughy and oreosomatids. The oreosomatids go through a metamorphosis from the post-hatching oreosoma phase to juveniles at some as yet undetermined time in their early life. The oreosoma stage is marked by horny protruberances (not scales) growing out of a papallose dermis. The oreosoma gradually metamorphoses into a juvenile with the small cycloid scales typical of the adult. The oreosoma stage occurs at adult depths (Myers, 1960) long after the pelagic eggs have hatched and the larva have descended from surface waters. Consequently the earliest part of the oreosomatid scale is developed (a) in colder water, and (b) after the fastest growth period in the early life of the fish. By contrast, orange roughy hatch in the upper water column and at even 3cm are well-formed juveniles of the adult. Therefore, orange roughy scales begin to develop as with other teleosts at the earliest part of post larval metamorphosis and are therefore laid down (a) in warmer water, and (b) during the fastest growth period in the life of the fish. Temperature effects may be indistinguishable from metabolic effects with our present technology, but the differences in fluorine and calcium behaviour at the focus of the scale show that chemical techniques are capable of detecting significant differences in life history between species. From a fisheries perspective, the fluorine and calcium variation in the scale disappears to offer a useful corroboration of age estimation from the marks in the scales of all three species that are generally regarded as annual marks (annuli) in scales. Growth curves based on back-calculation of widths between annual marks show faster growth rates than expected from orange roughy growth curves published by both Gauldie et al. (1989) and Mace et al. (1990).

The Lee effect usually results in smaller estimated size back-calculated from scales of older fish than the size actually observed (Gutreuter, 1987). However, occasionally the reverse Lee effect occurs (Deason and Hile, 1947) when mortalities of smaller, slower growing fish are higher than those of larger, faster growing fish. In the case of the orange roughy, there is a biomodality in length frequencies (Gauldie et al., 1989) that is consistent with higher mortality of smaller, slower growing fish. Growth curves have not been published for smooth Oreo to allow comparison, so that it must be borne in mind that, the back-calculated growth curves for smooth Oreo presented here may be biased by the Lee effect. The chemistry of scales is a new and important tool in the study of individual life histories (including age) of fishes. However the very high gradients between ambient fluorine and fluorine levels reported here and elsewhere is the scales of teleosts (Gauldie et al., 1990) and shark enamaloid and dentine (Legeros and Suga, 1980) points towards the critical role of scale scleoroblasts in scale chemistry. Much work has been done on the metabolic chemistry (Adelman, 1987) and natural history (Fishelson, 1984) of the cells that support the growth of teleost scales, and no doubt patterns in various scale elements will continue to be used, but the key to fully exploiting the information in the scale still lies in the physiology of scale scleroblasts. Acknowledgements

This paper was written while R. W. Gaudie was visiting Ichthyologist at the Hawaii Institute of Geophysics, School of Ocean, Earth Sciences and Technology, University of Hawaii. This research was supported partly by MAFFish, New Zealand, The Institute of Nuclear Sciences, New Zealand and by NSF Grants OCE 88-00686 and INT 87-23084 made to Richard Radtke. We are indebted to Mr. Peter McMillan of MAFFish for his assistance in obtaining samples. This is SOEST contribution no. 2444.

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