Continuous and discontinuous growth in the otolith of Macruronus novaezelandiae (Merlucciidae: Teleostei)

JOURNAL OF MORPHOLOGY 216:271-294 (1993)

Continuous and Discontinuous Growth in the Otolith of Ma cruronus novaezelandia e (Me r I u cc i id ae : Te I eos te i)

R.W. GAULDIE Hawaii Institute OfGeophysics, School of Ocean and Earth Science and Technology, University of Hawaii, Honolulu, Hawaii 96822

ABSTRACT Viewed by transmitted light, the lapillus and asterjcus otoliths Macruronus novaezelandiae (Merlucciidae) contain radial bands of similar width to the distances between steps on the surface of the otolith. The sagitta otolith has a multi-prismatic structure and shows differences in ultrastructure between its dorsal and ventral growth axes, as well as its sulcal (inward-facing) and anti-sulcal (outward-facing) parts. The ultrastructure of the sagitta shows that not all of the optical and etched checks in the central growth axis represent true discontinuities in the crystal growth of the otolith; they are the result of refraction around narrow optically active refractive bands. Microincrement growth along the dorsal prisms of the otolith from the primordium to the growing edge shows discontinuities in crystal growth at the boundary of the primordium and at the intersection of prisms. Parts of the ventral edge of the otolith show discontinuous crystal growth apparently caused by the physical growth restriction at the point at which the otolith is supported by the skull. Both the anti-sulcal and sulcal parts of the otolith often show discontinuities in the crystal structure alongside zones of continuous microincrement deposition, or evidence of continuous crystal growth, documenting simultaneous continuous and discontinuous growth in closely juxtaposed parts of the same otolith. u 1993 Wiley-Liss, Inc.

Inspection of the fish literature shows that an enormous amount of effort (and associated cost) has been expended in the collection ofotoliths and the interpretation of the different kinds of information thought to be contained in the morphology, chemistry, and ultrastructure of otoliths. The information contained in the otolith as morphological variation has been used in fish taxonomy (Gaem- ers, '84; Nolf, '85). The chemical information contained in the otolith in the form of elemen- tal ratios (Radtke, '89) has been used in life history studies (Gauldie et al., '921, and calcium carbonate mineralogy has been used as a probe for otolith deposition mechanisms (Gauldie, '92). However, the most widely ex- ploited source of information in the otoliths of fishes has been the discontinuous optical patterns in otolith ultrastructure. These patterns give rise to opaque and hyaline zones, check rings (both in ground and in break-and- burn sections; Christensen, '641, and daily microincrements (Campana and Nielson, '85). Although the overall size and shape of the otolith provide the conventional limits to mor-

phological studies of otoliths, the patterns of marks within otoliths can also be regarded as part of the otolith morphology (Dunkelber- ger et al., '80; Gauldie, '91; Zhang, '92). In pursuit of the morphological context of the internal patterns of marks in otoliths it is useful to begin by considering the back- ground about the interpretation of such marks. Thus far, the frequency and patterns of such optical structures have been used almost exclusively to estimate the age of individual fish (e.g., Beamish, '79).

In using any kind of mark in an otolith (more specifically the patterns of such marks) for age estimation, researchers often assume that a change in the growth rate of the fish has caused a simultaneous change in the growth rate of the otolith. For example, opaque and hyaline zones are often interpreted as an annual occurrence caused by a winter period of slow, or nil, somatic growth (e.g., Pinhorn, '69). However, there is an alternative assumption that otolith growth is independent of the growth of the fish, that the otolith acts as a clock, regularly deposit-

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272 R.W. GAULDlE

ing a microincrement every day (Campana and Nielson, '85). Somewhere between these two alternatives there will be situations in which there are temporary periods of uncoupling of otolith and somatic growth rates in response to stress (e.g., temperature, Moose- gaard et al., '88) from which otoliths recover their clock-like record of time.

Microscopic growth increments deposited daily in fish otoliths are evidence of a clock- like mechanism of deposition (Pannella, '71). Daily increments also appear in the hard parts of organisms from almost every phy- lum (Neville, '67). The daily pattern of microscopic growth increments in otoliths may be driven by at least two diurnal endogenous processes, calcium mobilisation (Mugiya, '87) and peptide secretion (Gauldie and Nelson, '88; Zhang, '92). Microscopic growth increments are laid down in otoliths over most of the life of at least some fishes (Laurs et al., '85; Ralston and Williams, '88a,b; Pulfrich and Griffiths, '88; Smith and Kostlan, '91). Although some fish respond to stress by de- positing more or less than one microscopic growth increment per day in their otoliths (Laroche et al., '82; Geffen, '831, other species have otolith microincrement deposition processes that are resistant to physiological stress (Mugiya and Muramatsu, '82; Cam- pana, '83). The most common stress that fish encounter over most of their lives is probably temperature change, and some fish respond with change of width of the increments (Wil- son and Larkin, '82; Radtke et al., '85; Gauldie, '91). Thus, the cycle of deposition in some species is perturbed in response to changing temperatures (Neilson and Geen, '82, '85). The clock-like deposition of microscopic growth increments is also apparent from our observation that the pattern of microincrement deposition is not correlated to the pattern of crystal deposition (Gauldie and Nelson, '90a).

The macroscopic structures of the otolith (opaque and hyaline zones, and checks) are unlike the daily microincrements in that they often lack a regular pattern of deposition. This irregularity forces separate age estimation conventions: one for periodic (e.g., annual) and one for aperiodic (e.g., stress or spawning related) structures (Keir, '60). Bro- ken and burned hemi-sections of otoliths have long been used to age fish on the basis of the annual periodicities of structures referred to as opaque and hyaline zones that appear when such hemi-sections of otoliths are ob-

served at low magnification under oil (Chris- tensen, '64). However, in other species with otoliths that show such opaque and hyaline zones at low magnification under oil, the zones proved to be refractive effects, not zonal changes in matrix density (Gauldie, '88a,b). Similar refractive effects in whole otoliths can also arise whenever whole otoliths are viewed at low magnification by both transmitted and reflected light (Gauldie, '88b; Davies et al., '88). However, thin sections of otoliths do show clear optical checks, or appearances of a discontinuity, when observed by transmitted light.

The presence of both daily microincrements and aperiodic macroincrements, such as optical checks, in the same otolith intro- duces a paradox. Their presence implies that there are two apparently inimicable processes at work simultaneously in the ontog- eny of the same otolith: 1) a continuous, daily, microincrement deposition that is resistant to metabolic stress in a t least some fish, particularly in adult fish, and 2) a discontinuous check ring deposition that is sensitive to metabolic stress in a t least some fish, particularly in adult fish. The resolution of this paradox is not simply a matter of choosing either microscopic growth increment ages, or annual check ring ages. Both microincrements and annual checks are part of the morphology of the otolith and are therefore unavoidably related to each other. The paradox of continuous and discontinuous growth has an impact on fisheries science. The fundamental problem underlying its resolution lies as much in understanding the morphology of the otolith as in the adjudication of the con- flicting evidence offered in support of various age estimation procedures.

Macruronus nouaezelandiae has a suitable otolith within which to resolve the paradox of continuous microincremental growth and discontinuous macroincremental checks. The sagitta of M. novaezelandiae has at least three kinds of optical structures, the patterns of which have been used to age fish, each yield- ing a different estimate. Opaque and hyaline zones in horizontal sections have been interpreted to give maximum ages of 9+ years (Kuo and Tanaka, '84). Optical check rings in the sulcal part of the otolith have been interpreted to give maximum ages of 25+ years (Kenchington and Augustine, '88). Microin- crements of the same width as what are regarded as daily increments in other species also occur (Gauldie and Nelson, '881, with

GROWTH IN THE OTOLITH OF MACRURONlJS NOVAEZELANDIAE 273

the average width implying maximum ages of less than 8years. The sagitta ofM. nouazelan- diae is typical of the Merlucciidae and represents a basic prismatic otolith structure common to most of the gadoids and to many other groups of fishes (Nolf, '85); this suggests that the observations made from the otolith of M. nouaezelandiae will be relevant to many other species. This paper describes structure and organization of the otoliths of M. nouaezelandiae in detail sufficient to estab- lish whether the morphological relationships between checks and microincrements can ex- plain the significance of simultaneous continuous and discontinuous growth.

MATERIALS AND METHODS

Endolymphatic sacs were dissected out of whole heads of the New Zealand hoki, Macru- ronus novaezelandiae (Merlucciidae: Teleos- tei) caught at sea and fixed in 10% neutral (pH 7.0) buffered formalin [see Dale ('76) for a description of the anatomy of the teleost otolith]. They were dissected within 2 weeks of fixation. Whole heads fixed in 10% neutral buffered formalin showed a slow drift to acid conditions (pH 6.8) after 6 months of storage. The otoliths used in this study were unaffected by acid storage conditions. Whole endolyrnphatic sacs, and dissected sacs with reflected otoliths, were photographed by transmitted light using a WILD photomicro- scope.

The maculae of sagittae were dissected out, dehydrated by alcohol series, and critical point dried, gold coated, and photographed mounted on aluminum pin stubs using a Philips 505 scanning electron microscope (SEM). Some sections of hoki otoliths were etched with 0.1 M (pH 2.0) ethylene diamino tetracetic acid (EDTA) before being coated with gold and photographed with the Philips 505 SEM.

Otoliths were also collected at sea and stored dry in paper envelopes. Clean, dry- stored otoliths were mounted whole on aluminum pin stubs, gold coated, and photographed using a Philips 505 SEM. Otoliths broken by thumb pressure were prepared and photographed in an identical way. Otoliths were split lengthwise by heating the whole otolith in a spirit lampflame. Most otoliths heated in this way will split spontaneously to varying degrees of completion.

Otoliths were washed in distilled water, and the clean, dry otoliths were ground and polished to the appropriate planes of observation (see Results) using a Struers petro-

graphic grinder and then mounted on glass slides with epoxy resin and re-ground to give optical sections of 20-30 pm thickness. Opti- cal photographs were made with a Zeiss photo microscope. Acetate peels of etched otolith sections were made with 2 pm acetate film using acetone as a solvent.

The conventional usage in otolith nomen- clature is that checks are non-annual optical (and etched) discontinuities in the otolith that are interpreted as spawning checks, or simply referred to as accessory checks (Hick- ling, '33; Keir, '60). In practice, it has proved difficult to recognise the difference between annual marks and either spawning or accessory checks, even in otoliths for which there are well-established ageing conventions (Woodhead, '68). Some species contain otoliths with very clear checks (particularly those in the sulcal side of the otolith) and these checks are interpreted as annual marks (Dan- nevig, '56; Beamish, '79). In other cases, otoliths have equally clear checks in the sulcal side of the otolith, but it has been shown that they deposit more than one such check each year (Irie, '60; Ikenouye, '69; Lee et al., '82). Staining studies (Dannevig, '56; Weide- man-Smith, '68) suggest that these well- defined sulcal checks represent protein layers accumulated during phases of slow, or no, mineral growth. Other studies question such a simple interpretation of staining studies (Gauldie et al., '901, although checks clearly represent zones that are both optically active and sensitive to etching by virtue of their lowered mineral density. However in many instances checks are thought to be growth discontinuities caused by metabolic or stress effects (Deelder, '81; Campana, '83).

The language used to describe such checks has become rather cumbersome. For example, the term annulus (which, in English, means circumferential band or zone) was appropriate to describe the opaque zones used in older ageing conventions. However, with the use of very narrow "zones" that are optically indistinguishable from checks, the convention has come to refer to each annual check as an annulus [even though annual checks often cannot be distinguished from non-annual by any internal ultrastructure (Gauldie, '88b)l. The result is that the zone between such checks would be an annulus between two annuli. Hence, I would prefer to drop the term annulus altogether. Not only is it open to misapplication, but it carries the connotation that an annual check can be

274 R.W. GAULDlE

independently identified by some inherent quality, which is as yet untrue, except on the basis of an a priori pattern in the mind of the observer. I t is ironic that the unwillingness to use the term “check ring,” going back to at least Keir (’601, has resulted in the use of the term annulus, as a latinization whose mean- ing has become almost exactly the same as the English term “check ring.” In this paper, checks are explored in sufficient structural detail so that arbitrary, prior definitions may be avoided as far as possible. Checks are therefore characterized in the text by terms such as optical checks and etched checks. The definitions are established in context.

Otoliths in this study are referred to as they are oriented in the laboratory, lying flat on a surface, sulcus side down. This avoids the cumbersome description necessary to ac- commodate orientation in situ. The anatomy of the gadoid inner ear is described by Dale (1976) and provides a clear review of both otolith orientation in situ and the anatomy of the nervous end-organs associated with each of the three otoliths in the inner ear. Crystal terminology follows Carriker et al. (’83). Where appropriate, ordinary English words have been used to describe otolith features, with the approximately equivalent older terminology from (Pannella, ’80) referred to in parentheses and italics.

The mineralogy of the otoliths was deter- mined using Raman spectra as described by Sharma (’89). The Raman technique provides a powerful and convenient tool for estab- lishing mineralogies without the preparation needed for x-ray diffraction studies. Raman spectra arise from the same lattice organization that generate x-ray diffraction spectra (Bischoff et al., ’85). For calcium carbonates, Raman spectra consist of a dominant spectral line that identifies the mineral as a calcium carbonate and higher harmonics that identify such crystalline morphs of the calcium carbonates as aragonite and calcite (Ur- mos et al., ’91).

RESULTS Terminology

The results are organised to reflect the growth of the otolith. The first section deals with the gross morphology and the crystalline structure of the surfaces of whole otoliths. The second level of growth of most otoliths is the organization of prismatic growth from the nucleus, and the restriction of prismatic growth. The third part deals with microincrements and the various checks

(optical and etched) that develop within the anti-sulcal growth axis of the prism. The fourth part deals with the microincrements and various checks found in the sulcal side of the otolith that underlies the main growth axis of the prism.

Gross morphology of otoliths The otoliths of the Macruronus novaezelan-

diae lie in three compartments of the endolymphatic sac, filled by a single reservoir of endolymphatic fluid. The largest otolith (sa- gittal occupies most of the endolymphatic sac, the middle-sized otolith (astericus) is housed in a diverticulum off the sac, and the smallest otolith (lapillus) lies in the atrium of the semi-circular canals. Each of the three otoliths of M. novaezelandiae has a distinctly different appearance when viewed by transmitted light (Fig. la). All three otoliths are formed of aragonite, unlike those of other species in which the lapillus has been described as vaterite (Lowenstam and Weiner, ’89; p. 196). Macula and otoconia

Reflection of the sagitta to one side dis- closes the macula underlying the sagitta and the massive innervation of the macula (Fig. lb). The macula extends along the length of the otolith, forming a bilobed structure with a constriction below, and close to, the location of the nucleus of the otolith. The macula of the sagitta has large numbers of associated otoconia embedded (Fig. 2a) in the otic membrane (Dunkelberger et al., ’80). The otoconia are aragonitic spherules (Fig. 2b) similar to those associated with otoliths in a number of teleost fish species (Dale, ’76; Gauldie et al., ’86). A sample of five whole endolymphatic sacs stored in formalin was inadvert- antly thawed and refrozen a number of times over a period of about 10 weeks. Examination of these endolymphatic sacs showed that whereas the otolith and macula had been replaced by patches of efflorescent orthorhombic aragonite laths (Fig. 2c). These structures were much larger ( - 10x1 than otoconia. Orthorhombic monoclinic crystals are the basic crystallographic type of the mineral series that includes aragonite (Bloss, ’71) but because of the extensive twinning in biomin- erals, orthorhombic aragonite is rarely observed in otoliths. Lapillus

An SEM picture of the smallest otolith (lapillus) shows a moderately coarse crystal-

GROWTH IN THE OTOLITH OF MACRUEONUS NOVAEZELANDIAE 275

Fig. 1. Macruronus nuvaezelandiae. a: After removal and cleaning, the three otoliths of Macruronus nouaezelandiae each have a distinctive appearance. The sagitta (S) shows a series of opaque and hyaline zones. The astericus (A) shows more radial opaque and hyaline zones (arrow) than the sagitta. A few indistinct opaque and hyaline zones occur along the ventral edge (arrow) of the lapillus (L). b: When the sagitta is reflected, the outline of the macula (M) conforms to the sulcus (S) shape and has a large nerve trunk (upper arrow) and extcnsive innervation (lower arrow). The bars are 1 mm to scale.

line surface with nodules on the surface and prismatic structure at the edge of the otolith (Fig. 3a). The surface of the lapillus consists of a series of steps partly obscured by the crystalline nodules on the surface. The average distance between the steps on the surface of the lapillus near its edge (Fig. 3a) is about 180 pm, similar to the width (174 pm) of the rings at the ventral edge of the lapillus observed by transmitted light as shown in Fig- ure la.

Astericus The astericus has the shape of a flattened

disc (Fig. 3b) with restricted growth on the in situ ventral margin that is closest to the entrance of the diverticulum in which the astericus lies. A sulcus-like groove below its

Fig. 2. Macruronus nouaezelandiae. a: Spherical otoconia (arrows) lie embedded in the otic membrane. The bar is 0.1 mm. b: Otoconia are of the crenellated spherule type. The bar is 10 pm to scale. c: Refrozen maculae show efflorescent crystalline aggregations growing out of the otic mombrane (left) with aragonitc crystals in the form of orthorhombic laths. The bar is 5 prn to scale.

nucleus (Fig. 3b) divides the astericus into unequal sized sections. The upper surface (external face) shows radial crystals growing from the nucleus of the astericus; they are crossed by a number of circular structures (Fig. 3b) resulting from steps in the radial crystal growth (Fig. 3c). The lower surface (internal face) is smoother than the upper surface and broken into prisms (Fig. 3d). The average width of those zones in the astericus that are opaque to transmitted light (Fig. la) is 126 pm and the average distance between steps OD the surface of the astericus in Figure 3c is 195 pm.

Sagitta The prismatic structure of the sagitta is

clearly evident in SEM pictures (Fig. 4a). At higher magnifications the upper, anti-sulcal surface of the sagitta (external face) consists of small crystals (Fig. 4b) that account for its smooth appearance. The upper surface of the

276 R.W. GAULDIE

Fig. 3. Macruronus nouaezelandiae. a: The lapillus of Macruronus nouaezelandiae has a coarse crystalline surface the ventral edge (E) of which shows a tendency to develop a scalloped appearance typical of an underlying prismatic structure. The surface of the lapillus has con- centric steps (arrows). Much of the surface of thc lapillus is covered with nodules (curved arrow). The bar is 1 mm. b: The astericus appears as a flattened disc with a medial suture (S, S ) dividing it into unequal sized sections. Crys-

sagitta typically shows weakly developed radial sculpturing on the surface of the otolith (Fig. 4a). However, the radial sculpturing observed on the surface is separated by widths similar to those of the opaque and hyaline zones seen by transmitted light in Figure la .

SEM pictures of the lower sulcal side (internal face) of the sagitta (Fig. 4c) show the sulcus as a bilobed flattened groove. The dorsal edge of the sulcal side of the otolith, particularly at the prismatic margins, has a smooth surface and shows a number of narrow, stepped, radiating surface bands (Fig. 4d). The surface banding in Figure 4d is seen to be caused by changes from a coarser to a finer crystalline surface structure (Fig. 4e).

The sulcus has the most complex crystal structure of the sagitta. The shape of the sulcus mimics that of the macula (Fig. Ib).

tals radiate from the central nucleus and are crossed by circular structures (arrows). The bar is 1 mm. c: The upper surface of the astericus shows a stepped appearance (arrows). Between steps a further less distinct series of radiating structures appears (open arrow within box) that is less clear at higher magnifications (right). The bar is 300 km. d: The lower surface of the astericus has a prismatic structure loading to a characteristically scalloped edge (El. The bar is 100 krn.

Although the macula is flat, its internal surface has many contours, pits, and ridges (Fig. 4c). The crystals of the sulcus are not signifi- cantly larger than those outside of the sulcus (average 3.9 pm, range 0.5-10 pm; versus average 4.05, range 1-8 km, respectively) but show a tendency to grow as isolated crystals often with evident traces of the approximately 64" twinning angles typical of aragonite (Fig. 5a). The edge of the sulcus itself has a complex crystalline structure, often with stepped arrays of crystals (Fig. 5b).

The prismatic structure ofthe sagitta The sagitta of Macruronus novaezelandiae

grows as a series of discrete prisms. Many species of fish have prismatic otoliths (Nolf, '85) but are not usually described in such terms in the fish otolith ageing literature.

GROWTH IN THE OTOLITH OF MACRLJRONUS NOVAEZELANDIAE 277

Fig. 4. Macruronus nouaezelandiae. a: The sagitta of Macruronus nouaezelandiae viewed from the anti-sulcal surface had a highly developed prismatic structure, of which the longest prisms (arrow) lead from the nucleus (N) to the dorsal edge (D) of the otolith. There are more prisms in the posterior (PI section of the otolith than in its anterior (A) part. Some surface sculpturing (open arrows) appears at the posterior and anterior ends. Note the groove (curved arrows) running along the ventral (V) edge of the otolith. ?'hc bar is 1 mm. b: The surface of the sagitta of Mucrwonus nouaezelandiae is composed of fine crystals. The right sidc (RS) frame shows them to be growing at different angles in the two prisms that meet to form the groove in the left side frame (LS). The RS frame i s 8 x the LS frame and the bar IS 50 pm. c: The sulcus

(S) of the sagitta appears as a flattened groove with a constriction at its centre. The prismatic structure (P) of the otolith appears in both the dorsal (D) and ventral (V) parts of the otolith, Both the dorsal (D) and vcntral (V) sulcal surfaces show stepped radiating bands (arrows). The ventral (V) surface also shows a groove running along (white arrow) the ventral edge. The bar i s 1 mm. d: The dorsal outer edge of tho sulcal surface of the hoki sagitta shows both a wider, stepped series of radiating bands (arrows) and narrower grooves (open arrows) towards the sulcus itself. The bar is 1 mm. e: The stepped, radiating structure (arrow, LS) at the dorsal edge of the sulcal surface is associated with a change (open arrows, RS) in the coarseness of the crystals of the otolith. Thc bar is 0.5 mm.

Many mollusc shells are also characterized by prismatic growth (see Wilbur and Saleud- din, '83: p. 268, for a molluscan analogue) that is caused by the persistence of separate bundles of calcium carbonate crystals grow-

ing from a single focus. The prisms of the sagitta appears to be added sequentially on the anterior and posterior ends of the otolith and grow in both the dorsal and ventral directions (Fig. 4a). The stepwise accretion of

278 R.W. GAULDIE

edge of the otolith to have a larger number of prisms than the dorsal. Prismatic growth at the ventral edge closest to the nucleus is apparently restricted (Fig. 4a) and forms a flattened groove slightly back from the ventral edge.

Splitting of the sagitta lengthwise by heating shows the prismatic structure of the otolith as well as the branching of ventral prisms and the development of checks along the ventral side of the otolith (Fig. 6a). The development of checks in the ventral part of the otolith of Macruronus nouaezelandiae shows the effects of growth restriction from contact with the floor of the otic cleft as described for other species (Gauldie and Nelson, '90b; Smith and Kostlan, '91). Dorsal prisms of split sections also show the sequential insertions of successive prisms along the anterior- posterior axis of the otolith (Fig. 6b).

Fig. 5. Macruronus nouaezelandiae. a: The sulcus has a coarse crystal texture with a tendency towards large single crystals oriented to each other at about the 64" twinning angle of aragonite and surface discontinuities (short arrow) are common. The bar is 1 brn. b The crystal structure of the edge of the sulcus is very coarse with individual crystals oriented vertically to thc macula and showing a stepped or discontinuous growth surface (arrows). The bar is 0.1 mm.

prisms means that each additional prism will be younger than the previous prism. The only parts of the otolith that can reflect all the life history of the fish are the oldest dorso-ventral prisms close to the nucleus.

The longest single prisms run from the nucleus to the dorsal edge of the sagitta (Fig. 4a). For the otolith in Figure 4a, there are 17 complete dorsal prisms from the nucleus to the posterior end, each of which runs from a more-or-less central axis to the dorsal edge (Fig. 4a). From the nucleus to the anterior end of the otolith, there are seven to eight complete prisms each of which runs from the more-or-less central axis to the dorsal edge in

Fig. 6. Macruronus nouaezelandiae. a: Asagitta split bv heating shows the orismatic structure twical of the

the otolith (Fig. 4a). In both the post&or and anterior directions the prisms become progressively ~ r n d e r (Fig. 4a). The branching of individual prisms causes the ventral

ventral (v', part of thebtolith with branchiliprisms (P) and pressure checks (arrows). The bar is 1 mm. b: The dorsal side of the split sagitta shows uninterrupted prisms inserted along the cdge (E) anterior posterior (A, P) axis. The bar is 1 mm.

GROWTH IN THE OTOIJTH OF MACRURONUS NOVAEZELANDIAE 279

Horizontal sections of the sa,gitta Whenever observed by transmitted light,

horizontal sections of the sagitta of Macruro- nus novaezelandiae show a succession of opaque and hyaline zones of differing widths organized along prisms (Fig. 7a). Some of the central part of the otolith of the specimen in Fig=ure 7a was partly obscured by the optically active material (dark by transmitted light) that is characteristic of the central part of the otolith. The part of the otolith from which the optically active material has been removed by grinding (Fig. 7a), one sees the

Fig. I. Macruronms nouaezelandiae. a: A horizontal optical section of the anterior part of the sagitta shows a large number of minor opaque zoncs, particularly tn- wards the anterior (A) end of the otolith. Prisms (P) can be seen running from the nucleus (N) towards both the ventral (V) and the dorsal (D) edge that has bccn lost in the grinding processes. The plane of grind has retained the ventral iV) edge that contains the major opaque zones or checks (arrows) which continue around from the anterior section. The central part of the otolith is obscured by highly light refractive material. The bar is 1 mm. b An etched horizontal section of the sagitta shows the primordium (PO) of the otolith and the prism boundaries by differential etching (arrow). Prisms decrease in

most central of the succession of major opaque zones that range from the nucleus to the edge of the otolith. The horizontal section of the otolith in Figure 7a shows the prismatic organization of both the dorsal and ventral parts of the otolith. Complete prisms run from the center of the otolith to both the dorsal and ventral edge (Fig. 7a).

The central part of the otolith is partly obscured in the optical section shown in Fig- ure 7a but an equivalent SEM of an etched horizontal section shows the large prirnor- dium characteristic of the otolith of Macruro-

size towards the anterior but their points of insertion appear to lie along the central axis. The bar is 1 mm. e: The micrnincrement structure of the primordium (PO) radiating from the nucleus (white arrow) is out of phave with tho increments of the dorsal (D) and ventral (V) prisms (PI the boundaries of which match apices of the primordium and their origin appears to be at the most concave part of the primordium boundary. The bar is 200 p m ~ d An etched horizontal section of the sagitta showed a clear boundary of etch-sensitive material between the primordium (PO) and the prism (PSI that continued between prisms. Detail of the point of intersection of the primordium and prism boundaries (right) showed that the crystals stopped growing. The bar is 10 km.

280 R.W. GAULDIE

nus novaezelandiae (Fig. 7b), as well as the difference in etch sensitivity between the material of the prisms and the prism boundaries. Prisms become smaller, and more curved, towards the anterior end of the otolith (Fig. 7a) as they decrease in size from the long, straight prisms near the primordium. The point of origin of the prisms becomes progressively more difficult to find as they proceed towards the anterior of the otolith, but their points of origin (or insertion) appear to lie along the central axis of the otolith directly over the macula (Fig. 7b). It is evident that sections along the nucleus to anterior axis cannot contain as much of the growth history of the otolith as the nucleus to dorsal axis in spite of covering a much greater length of otolith.

A horizontal optical section of a second otolith (Fig. 7c) a t higher magnification shows the nucleus within the primordium and the relationship between the primordium and the prismatic structure of the otolith. The boundaries of both dorsal and ventral prisms are located at the apices of the primordial polygon, implying that the prism radiation (the point of origin of the prisms) is a t the point of maximum convexity on the primordium polygon. Microincrements are deposited within the primordium, growing radially from a single nucleus. No multiple nuclei appear in 23 different primordia. The primordium has a polygonal shape whose axes vary in length, but microincrement counts along the longest axis of the primordium (not necessarily the same axis in each section) range from 54 to 103 with a mean of 82 for a sample of five otolith primordia. The primordium has a distinct boundary that is not simply a more strongly emphasized microincrement. This is evident from the discontinuous phase relationships of microincrements within the primordium compared to those of microincrements outside the primordium (Fig. 7c). Prisms also show distinct boundaries in optical sections (Fig. 7a). An SEM of the boundaries (and junction) of three prisms taken from an EDTA etched specimen shows that crystalline growth had stopped (Fig. 7d). Crys- tals from neighboring prisms do not merge into each other but show hexagonal termina- tion apices indicating normal crystal growth up to the point of cessation (Fig. 7d).

Microincrements i n horizontal sections Microincrement studies have been con-

fined to the longest dorsal prism because that is the prism that potentially contains more of

the life-history of the fish than any other part of the otolith. The growth axis in which microincrements are deposited lies in the anti- sulcal part of the prism. Checks also occur in this part of the prism, but the checks used in the ageing convention of Kenchington and Augustine ('88) are contained in the sulcal part of the prism, which is dealt with below. However, horizontal sections that reveal any part of the microincrement structure must be of necessity confined to the anti-sulcal growth axis.

Horizontal sections of the growth axis of the anti-sulcal part of the otolith show a progression of microincrements from the edge of the primordium to the edge of the otolith whenever the otolith is sectioned in the horizontal plane. Microincrements in the earliest part of the prismatic growth prove to be difficult to resolve. A typical image of microincrements out of 83 horizontal optical sections (or acetate peels of sections) aimed at the earliest part of the prism is shown in Figure 8a. The microincrement-like structure within the primordium is out of phase with, and much more clearly defined than, microincrements in the earliest part of prism growth. Microincrements in the prism progressively decrease in width along the prism but increase in clarity until they have the appearance of conventional microincrements (vide Campana and Neilson, '85). SEM pictures of the part of the prism containing low-resolution microincrements show them to be 5 to 10 km wide with crystals oriented at an angle to the plane of the section (Fig. 8b). Wide microincrements have been reported at the central part of prisms of otoliths of other species (Nishimura and Yamada, '88).

Ground and polished sections of the otolith generally show regular sequences of microincrements (Fig. 8c). When etched with EDTA, the regular sequences of microincrements appear as stacks of small crystals oriented more-or-less orthogonal to the plane of the microincrement (Fig. 8d). The stacks of small crystals are separated by gaps caused by the removal of EDTA-etch-sensitive material. The nature of the EDTA sensitive material is difficult to test directly, but electron backscatter measurements of polished, unetched otolith surfaces of otoliths show changes in electron-absorbing and electron-reflecting material (Fig. 8e). Protein is less dense than calcium carbonate so that the dark, electron- absorbing (and therefore less dense) bands may be due to protein. The banding shows

GROWTH IN THE OTOLITH OF MACRURONUS NOVMZELANIK4E 281

Fig. 8. Macruronus nouaezelandiae. a: The primordium of the sagitta can be seen in an acetate peel of an etched optical horizontal section as a polygonal structure with a series of rings radiating from the nucleus (N). Within the primordiuin microincrements are more readily visible than those outside. On the outside of the primordium on the dorsal (D) side there was typically a region of wide increments (white arrows). The bar is 50 km. b: SEM pictures ofthe wider microincrements of the earliest part of the prism showed that the part of the microin- cremcnt that usually contains the mineral component of the microincrement (arrowed) has widened because of the angle of orientation to the plane of the section but retains a similar relation to the width of the narrow protein component (open arrow). The bar is 10 p.m. c:

major and minor cycles of apparent protein density (Fig. 8e). Examination of the etched stacks of small crystals (Fig. 8d) shows traces of disruption between the more stongly etched spaces, which may correspond to the minor cycles of protein density. The differential etching effect is very important as it provides a chemical probe that can be used to define a microincrement.

Etching with EDTA also shows changes in the degree to which the otolith of Macruro- nus novaezelandiae is etch-sensitive, provid- ing further chemical information on the morphology of the otolith. The differences in

Horizontal optical sections of the sagitta shows sequences of microincrements characterized by regular periodic changes in the optical density of the organic moiety (arrows) of the microincrement. The bar is 20 p.m. d EDTA etched horizontal section of the mgitta shows the microincrement series to have been composed of stacks of small crystals separated by gaps left by removal of etch sensitive organic material. In places the stacked crystals show traces of etch disruption that may reflect sub-daily organic deposits. e: Electron backscatter measurements of the polished surface of horizontal sections of the sagitta showed a progression of dark bands of varying intensity (arrows) corresponding to the electron absorbing protein component of the microincrement. The bar is 10 p.m.

etch-sensitivity result in varying degrees of removal of otolith material (Fig. 9a). Exami- nation of the otolith shows that the most heavily etched areas are those that have the narrowest microincrements (Fig. 9b). Etch- ing shows a consistent pattern of stacks of crystals perpendicular to the parallel zones of etch-sensitive material (Fig. 9b). Etching sometimes reveals structures that are so de- pleted in mineral content (or so rich in etch- sensitive material) that they can be regarded as either narrow, and mineral depauperate, microincrements or extremely wide exam- ples of the etch-sensitive band of microincre-

282 R.W. GAULDIE

Fig. 9. Macruronus nouaezelandiae. a: SEM pictures of the etched surface of horizontal sections of the sagitta showed that there was a great deal of variation in etch sensitivity across the surface of the hoki otolith. The bar IS 100 km. b: Higher magnifications show that the most heavily etched areas have the narrowest rnicroincrements (arrow) and the least etched areas had the widest microincrements (open arrow). The most heavily etched areas also have higher protein band to mineral band ratios than the least etched areas. In some parts of the least etched areas the etch-sensitive band is barely visible (curved arrow). The bar is 10 bm. c: Prolonged etching ( > 5 min) results in deep clefts at the etch sensitive areas (arrow) as well as loss of resolution of microincrements (open arrow). The bar is 100 pm.

ments (Fig. 9b). Prolonged etching results in both a general loss of resolution of the microincrement structure, and the deep clefts that form at prism boundaries and at the parts of the otolith that have the most narrow microincrements (Fig. 9c). Prolonged etching rein- forces the observation that both microincrements and etched checks can be artefacts of an etching process that removes an unknown amount of an unknown material.

Microincrements defined by etching form a more-or-less continuous series of layers along the central growth axis of the anti-sulcal part of the dorsal prism. Acetate peels have the advantage of avoiding refraction effects a t the edges and boundaries of sections of otolith material. An acetate peel of an etched otolith shows continuous microincrement deposition to the growing edge of the longest dorsal prism (Fig. 10a). The more narrow, central parts of the dorsal prism show constriction in the deposition of microincrement layers apparently imposed by the process of prismatic growth. This constriction effect results in apparent fusion of microincrements in the more radial parts of the prism while discrete layering persists in the central part of the prism (Fig. lob).

Microincrement layers are not always in phase in the adjoining parts of different prisms. For example, a section of three con- joined prisms shows some clearly in-phase microincrements, whereas other microincrement patterns of different prisms appear to be independent of each other (Fig. 1Oc).

Vertical sections of the sagitta General

In addition to division into successive prisms, the otolith of Macruronus nouaezelandiae is divided into anti-sulcal and sulcal parts as are the otoliths of other teleosts (Gauldie, '88b; '91). There are, consequently, two sources of information about the periodic structures of the otolith. The first source is information from the growth axis in the anti-sulcal part of the otolith (ageing studies of Kuo and Tanaka, '84). The second source provides apparently different information about age in vertical sections of the sulcal part of the otolith (Kenchington and August- ine, '88). This division is followed in the ensuing description of the morphology of the otolith.

Vertical sections of the otolith of Macruro- nus nouaezelandiae indicate periodic structures in both the anti-sulcal and sulcal parts of the otolith. The morphological interrela-

GROWI'H IN THE OTOLITH OF MACRURONUS NOVAEZEIANDIAE 283

Fig. 10. Mucruronus nouaezelandiae. a: Microincre- ment deposition can be followed right out to the edge of the otolith. The bar is 10 km. b: Acetate peels of the microincrement structure of the prisms of the sagitta show regular layering in the central ( C , C) part of the

tionship between these two parts is therefore an important part of the explanation of the apparent paradox of continuous and discontinuous growth of otoliths.

Division of the vertical section into anti-sulcal and sulcal parts

Vertical sections of otoliths of Macruronus nouaezehndiae observed by transmitted light show a number of consistent features. Crys- talline anomaly divides the otolith into sulcal and anti-sulcal parts (Fig. I la) . The anti- sulcal part is wide compared with that of other species (Gauldie, '88a,b, '91) and has a layered appearance with both microincrements and optical checks (Fig. l l a ) . The structure of etch-defined and optically observed microincrements from the central part of the anti-sulcal growth axis of the otolith is similar to that of the previously described horizontal sections, but in passing into the sulcal segment, some of these anti-sulcal rni- croincrernents are lost by fusion (Fig. l lb ) . The process of fusion also results in the layers of checks of the anti-sulcal segment (Fig. l lb ) . Vertical sections also show a different

prism, with radial fusion of microincrements (arrows). The bar is 10 bm. c: Microincrements at prism boundaries may be in phase (arrow) for only part of the sequence. The bar is 10 km.

view of the nucleus in the anti-sulcal part of the otolith a t the center of a flattened primordium with radiating microincrements (Fig. l lc) .

Comparison of the anti-sulcal and sulcal parts of the sagitta

The growth axis in the upper anti-sulcal segment of the otolith equivalent to those described for horizontal sections contains a variety of structures including opaque and hyaline zones, checks, and microincrements. It is evident from Figures l la and b that many structures in the anti-sulcal part of the otolith grow out of the anti-sulcal part of the otolith into the sulcal part of the otolith. To allow comparison of these structures, a vertical section has been photographed by transmitted light and also mounted on a pinstub and etched (Figs. 12a,b). This allows comparison of the features observed by light microscopy with the ultrastructural features observed by SEM. The upper, anti-sulcal section of the otolith observed by transmitted light shows a large number of wide opaque zones and narrow zones, or optical checks (Fig.

284 R.W. GAULDIE

Fig. 11. Mucruronus nouaezelundiue. a: A vertical section of a sagitta shows a line of division (open arrow) between the upper, anti-sulcal (A) part of the otolith and the lower, sulcal6) part of the otolith which runs across the otolith from the ventral (V) to the dorsal (D) edge. Both the anti-sulcal and sulcal parts of the otolith contain check ring-like structures (arrows). The bar i s 1 mm. b Microincrements (arrow) occur in vertical sections and

apparently fuse (open arrow) to form the check-like structures that pass into the sulcal part of the otolith. The bar is 20 pm. c: Vertical optical sections of the sagitta show the primordium as a flattened structure overlaying the sulcul side of the otolith ( S ) with distinct microincrements coming away from the nucleua (N). The bar is 100 pm.

12a). Scanning electron microscopy of the same section shows that only some of those optical checks correspond to etched checks in the otolith (Fig. IZb), and that only a few of the etched check rings penetrate into either the anti-sulcal or sulcal part of the otolith. Most etched checks are confined to the central part of the anti-sulcal growth axis. The opaque boundary between the central growth axis and the sulcal part of the otolith (Fig. 12a) does not correspond to an observable boundary in the crystalline structure between the sulcal and anti-sulcal parts of the broken otolith (Fig. 12b).

Comparison of Figures 12a and b shows that few of the identifiable opaque zones of the sulcal part of the otolith correspond to etched checks, The ratio for the otolith in Figure 12a, b is about ten optical zones to one

etched check. In addition, the distances between checks is greatest in the central part of the anti-sulcal growth axis, decreasing as the checks (both optical and etched) approach or pass into both the sulcal and anti-sulcal parts of the otolith. However, the frequency of optical and etched checks (number per mm) increases towards the dorsal edge (i.e., the growing edge) of the otolith, and there is a parallel decrease in the progressive narrowing of inter-check distances between checks as they approach the sulcus surface b., the lower surface) of the otolith.

Much of the information provided by checks lies in their perceived patterns. The patterns of what appear to be the same checks growing continuously from anti-sulcal to sulcal part differ between the anti-sulcal growth axis and the sulcal part of the otolith. I t is

GROWTH IN THE OTOLITH OF MACRURONUS N O V A E Z E W D I A E 285

in the central growth axes and the inter- check distances in the sulcal part for the otolith of homologous pairs of checks. Mea- surements of ten pairs of homologous checks from each of five otoliths yield a range of correlations from r = 0.21 to r = 0.62, with a mean of r = 0.39.

Etching shows that there are underlying boundary zones in the crystalline structure of the otolith that do not necessarily correspond to the boundary zones caused by changes in the refractive index of otolith sections viewed by transmitted light. The boundary zones revealed by etching are clearly the most likely structures to represent genuine discontinuities in the crystalline growth of the otolith. Detailed analysis of the crystal structure of the otolith reveals four kinds of discontinuities: etched checks, spalling lines, sulcal checks, and disordered crystal checks. Etched checks

Fig. 12. Macruronus nouaezelandiae. a An SEM of the etched surface of the vertical sagitta section shows a series of etched checks proceeding along the principal growth axis (two are shown by vertical arrows). Some of these checks continue into the upper, anti-sulcal (A) part of the sagitta (long arrow) with a progressive decrease in the distance between checks towards the centre of the otolith. Checks appeared in the sulcal (S) part of the otolith (horizontal arrows) with a different appearance to those in the rest of the otolith, but could be seen to be related to, and continue from, etched checks in the central growth axis. The check rings in the sulcal part of the otolith are more parallcl as they proceed out of the central growth axis. A line of demarcation (open arrows) persists between the sulcal and anti-sulcal sections but has the appearance of a change in crystal texture, rather than a check. (The curved arrows identify the same cracks as in the panel b, helow). The bar is 1 mm. b An optical view of a vertical sagitta section in (a) above shows a line of demarcation (open arrows) between the anti- sulcal (A) and sulcal (S) parts of the otolith. Many checks occur in the principal growth axis of the anti-sulcal section. Vertical arrows had the checks corresponding to the etched chock rings in panel (A). Checks can be seen in the sulcal section (short arrows); these become progressively more narrow and more evenly spaced towards the centre of the otolith. The curved arrows identify the same crack marks as in a. The bar is 1 mm.

evident that the otolith of Macruronus novaezelandiae yields different patterns for homologous checks from the anti-sulcal growth axis and from the sulcal part of the otolith. The difference in patterns of homologous checks can be demonstrated by testing the correIation between the inter-check distances

In order to reduce distortion due to the etching process, the otolith section is lightly etched for 10 seconds as shown in Figure 13a. The etched checks of the anti-sulcal growth axis show varying degrees of penetration into the sulcal and anti-sulcal parts of the otolith, with a tendency simply to fade into the general crystal structure of the otolith. The etched check that was selected for this study has been marked in Figure 13a by four arrows each indicating a point of examination. A series of SEM pictures starts from the first arrow in the sulcal side of the otolith.

At the first point of examination, the point a t which the etched check ring faded into the crystal structure is shown on the left side of Figure 13b; the traces of the crystalline discontinuity fade from the right side. This kind of fading is important in understanding otolith structure. It occurs in otoliths of other species (Gauldie, '91) and represents a point at which both continuous and discontinuous growth occurs in closely juxtaposed crystals that are separated by only a few nanometers within the fluid from which they precipitate.

At the second point of examination, the etched check has widened into a shallow groove. Within this groove several minor etched lines appear, although the major etched band can still be followed (Fig. 13c). The major etched line is narrow, about 0.1 pm, and in places continuous crystal growth crosses the line (Fig. 13c). I t is difficult to perceive this kind of check as a true disconti-

286 R.W. GAULDIE

Fig. 13. Macruronus novaezelandiae. a: This etched vertical section of the sagitta shows a series of structural checks ofvaryingwidth passing down the principalgrowth axis of the otolith and penetrating into the anti-sulcal (A) and sulcal (S) parts of the otolith. A series of detailed observations were made along one of the checks at the points indicated by arrows starting from the large arrow on the left. The bar is 0.3 mm. b Higher magnification of the first point (large arrow in (a) above) at which the checks fade out into the sulcal part o f the otolith shows the progressive loss of detectable insertion of the check into the crystal structure of the otolith. The bar is 10 km. c: At the second point, the check disperses into a wide structure within which a main check can be seen (open arrow) as well as less pronounced bands (small arrows) that may correspond to rnicroincrements. Disruption of the crystal structure by the main check hand is incom-

nuity because some crystal material continues across the check. At the third point of observation, farther around the etched check, the groove becomes more narrow and has a single line of etch 0.1 to 0.3 km wide (Fig. 13d). At this point the etched check ring is orthogonal to the axes of crystal growth, with some indications that crystals on the nucleus side of the etch continue to grow on the other side of the check ring (Fig. 13d). However, other less-etched discontinuities in the crystal structure of the check ring in Figure 13d

plete in some places (curved arrows). The bar is 1 km. d: Higher magnification of the third point of the check shows a narrowing of the check within which the main check band can he seen (open arrow). Very narrow microincrements lying close to the center of the check (small arrows) progressively widen as well as change their angle of approach to the check ring itself. The bar is 1 km. e: The fourth point of the check shows that the check ring in the anti-sulcal part of the otolith appears as a deep cleft running through a more coarsely crystalline structure than that of the rest of the otolith. The illustration shows the point of fusion of two checks in the anti-sulcal section of the otolith. In spite of the obvious discontinuity of the check rings, crystalline bundles apparently maintain their continuity (arrows) across both checks. The bar is 10 Fm.

may be the basic lamellae of the otolith crystal (Gauldie et al., '92).

At the fourth point of observation, in the anti-sulcal part of the otolith above the nucleus, the check becomes much deeper and wider, and, in a manner paralleling the fusion of microincrements, merges with another etched check, Even in this most ex- treme form of the check, the crystals on one side of the check remain oriented perpendicular to those on the other side (Fig. 13e). Crystals on both sides of the merged etched

GROWTH IN THE OTOLITH OF MACR URONUS NOVAEZELANDIAE 287

Fig. 14. Macruronus nouuezelandiae. a: Detail of the dorsal tip nf the growth axis of the sagitta (box, left) shows the growth of the radial (R) crystal along the growth axis (arrow) with medial (MI twinned aragonite crystals growing to either side of the main growth axis. The bar is 0.5 mm. b: A broken section of a sagitta shows a number of spalling lines in both the central gruwth axis and anti-sulcal part (arrows) of the otolith. The bar is 1 mm. c: At higher magnifications spallinglines are charac- torised by a fine grained crystalline surface (open arrow).

check show indications of apparent continuity of individual crystals on either side of the check.

Broken sections of the otolith of Macruro- nus novaezelandiae (Fig. 14a) show that twinned crystals grew so that morphologi- cally they are the same crystal with a radial component growing along the anti-sulcal growth axis, and two medial components growing towards the sulcal and anti-sulcal surfaces, respectively. In effect, one part of the crystal (the radial, central growth axis component) grows much faster than the other parts (the medial, sulcal and anti-sulcal components) of the same crystal. It is this kind of growth that gives broken sections of the otolith the appearance of being virtually continuous crystals within each prism. Measure-

In some cases, the crystalline discontinuity implied by the spalling fails to penctrate the crystalline structure of the sagitta completely. The bar is 100 pm. d: A thin vertical section of hoki otolith has been lightly etched and broken by thumb pressure. Breaks against the crystal structure of the otolith appear at two of the more strongly etchcd checks (arrows), but not at another more strongly etched check (open arrow), a further break appears at a weakly etched check (curved arrow) that had a surface typical of spalling line. The bar is 0.1 mm.

ment of the distance of the medial crystal from nucleus to the sulcal edge, compared to measurement of the radial twin of that crystal, shows the sulcal medial crystal to average about 33% of the length of the radial twin. Correlation of the patterns of homologous checks rings between the anti-sulcal and sulcal parts of the otolith indicates only about 39%) of the variation in the sulcal check patterns could be explained by the variation in the anti-sulcal check pattern. The similar- ity between differences in crystal length and the decrease in variability between the anti- sulcal and sulcal parts of the otolith suggest that substantial differences in crystal growth rates between different parts of the otolith are responsible for much of the life history

288 R.W. GAULDIE

record of the anti-sulcal otolith part that is lost, or censored, in its sulcal part.

Spalling lines Breaking of some otoliths of Macruronus

novaezelandiae by thumb pressure gives rise to distinctive discontinuities, This appearance is similar to that found in the otoliths of other species (Gauldie et al., '91). Spalling lines are uncommon in some otoliths, but very common in others and occur along the anti-sulcal growth axis (Fig. 14b). These spalling lines are characterised by a fine-grained crystalline surface (Fig. 14c). The relationship between the spalling lines of the otolith and checks has been examined as follows. A vertical section of the otolith was lightly etched to show the location of checks and then broken by thumb pressure. The otolith section breaks along two of the more strongly etched lines without showing a smooth, fine- grained surface typical of a spalling line. How- ever, further along the section the otolith breaks at a weakly etched line and does show a surface typical of a spalling line (Fig. 14d).

Sulcal check rings Checks in the sulcal part of the otolith

have been used to estimate age in Macruro- nus novaezelandiae (Kenchington and Augus- tine, '88). The sulcal section of the etched otolith shows a series of parallel etched checks typical of this type of check in otoliths of other species. These sulcal etched checks correspond to some of the optical checks observed in the sulcal part of the otolith (see Figs. 12a,b). Their fine structure is quite different from that of checks in either the anti-sulcal growth axis, or in the anti-sulcal part of the otolith. The crystals in the sulcal part of the otolith are very large compared with those in either the central or anti-sulcal parts of the otolith, and show a strongly jointed crystal structure (Fig. 15a). Although sulcal checks apparently show some sections of clear structural discontinuity at the point of etching, some crystals grow continuously right through the etch-sensitive material of the discontinuity (Fig. 15b).

Disordered crystal checks Occasionally (about 1 in 20) the etched

otoliths show an unusually complex set of sulcal checks, the structure of which may offer some insight into the processes of check deposition. This kind of sulcal check occurs in otoliths that have a well-defined boundary

Fig. 15. Mucruronus novaezelandiae. a: Aragonite crystals in the sulcal side of the sagitta are very large compared to crystals in the other parts of the otolith. Crystalline jointing occurs in the crystals of the sulcus (white arrow) as well as checks (arrows) through which crystals pass with little or no change in orientation or boundaries. The bar is 0.1 mm. b: Higher magnification of sulcal check rings shows crystal growth continuing through the checks (arrows). The bar is 10 km.

between the anti-sulcal growth axis and the sulcal part of the otolith (Fig. 16a). Checks in the anti-sulcal part of the otolith are very well defined in both the anti-sulcal and sulcal part of the otolith, but characteristically only penetrate about halfway into the main growth axis. The general shape of these checks appears to reflect the shape which the edge of the otolith would have had at the time when those check were being deposited. In some checks (Fig. 16b) microincrements continue to grow past the check ring, but the check appears to deflect the angle of growth of the otolith, and the orientation of both crystals and microincrements. In another case (Fig. 16~1, although microincrements continue to be deposited (below the central structure and out of sight in this picture) there is a pronounced check. Although there are signs of diminishing microincrement width, the

GROWTH IN THE OTOLITH OF MACRURONUS NOVAEZELANDIAE 289

check itself appears to be formed of relatively disorganised small crystals, rather than very closely spaced microincrements. Both kinds of checks show that both continuous and discontinuous growth can occur simultaneously in the same part of the otolith. The microincrements leading up to this check (Fig. 16b) show the unusual feature of the protein component of the microincrement being wider than the mineral component.

DISCUSSION Hierarchy of discontinuities

The otoliths of Macruronus novaezelandiae provide a hierarchy of both real and apparent discontinuities in the morphology from the gross anatomical level, to the most minute level available from scanning electron microscopy. To facilitate discussion, this hierarchy conveniently can be divided into sections.

Whole otoliths The first and most obvious discontinuity in

otolith growth is provided by the differences in size of the three otoliths of the endolymphatic sac. Although bathed in the same endolymphatic fluid, the three otoliths clearly grow at very different rates. These differences irn- ply an otolith-specific growth control mechanism. A recent study by Zhang ('92) supports in part the Gauldie and Nelson ( '88) hypoth- esis of otolith growth control by the macula. The basic theme of chemical modulation of otolith growth, as shown in discontinuities between whole otoliths, reappears in one form or another throughout the entire hierarchy of otolith chemistry.

All three otoliths-lapillus, astericus, and sagitta-of Macruronus nouaezelandiae show

Fig. 16. Macruronus nouaezelandiae. a: An etched section of the sagitta shows a well-defined boundary between the upper anti-sulcal surface (A) and the lower sdcal ( S ) side of' the otolith. Well-defined checks (filled arrow) in the anti-sulcal (A) section show a change in structure (open arrow) as they pass through the bound- my into the sulcal scction of the otolith. The bar is 1 mm. b: Checks (filled arrow) interrupt the growth of some parts of tho otolith although microincrement deposition (open arrow) continues around the check. The bar is 100 km. c: Checks (filled arrow) consisting of small crystals cause a marked discontinuity in the microincrement deposition pattern. Microincrements leading up to the check show a larger protein (etch sensitive) than mineral (open arrow) moiety. The bar is 100 pm.

a regular banding pattern of opaque and hyaline zones when viewed whole by transmitted light. Examination of whole otoliths shows that all three otoliths had steps on their surfaces of approximately the same width as the banding patterns viewed by transmitted light. I conclude, therefore, that the opaque zones are caused by refraction at these steps. Similar apparent opaque and hyaline zones caused by surface sculpturing have been reported for other species (Gauldie, '88a; Dav- ies et al., '88). The cause of surface step formation is unknown, but it takes the form of a decrease in crystal size on the otolith surface.

A further discontinuity in growth appears as differences in structure between the small

290 R.W. GAULDIE

crystals on the anti-sulcal surface and the much larger crystals on the sulcal surface. These differences in size imply two different chemical environments between the endolymphatic fluid and the fluids near the macula. The existence of such an otolith-specific chemical gradient between endolymphatic fluid and macula is implied by the large differences between otoliths.

Discontinuous crystal growth Discontinuities caused by cessation of crys-

talline growth occurred in the otoliths of Macruronus nouaezelandiae. Ontogenetically, the first of the growth cessations is the primordium boundary. The primordium of the sagitta typically grows from a single nucleus and lays down between 50 to 120 microincrements before a distinct check in growth appears. This check defines the boundary of the polygonal primordium. Growth stops at this boundary, and then restarts in the form of prisms growing from the boundary of the polygonal primordium. The evidence for stop- ping and restarting growth is that the crystals of the primordium are out of phase with the crystals of the prisms. There is no indication of crystal growth in the primordium continuing into the prismatic region of the otolith. There are no signs of smaller crystals associated with the primordial boundary, but it may be possible that the deeper etch associated with this boundary i s caused by the relatively higher surface-to-volume area that would be expected from very small crystals whose growth has been attenuated by the organic material associated with the primordial boundary.

Prismatic structure of the otolith The prisms of the otolith of Macruronus

novaezelandiae provide a third-order discontinuity in otolith growth. It is evident from the prismatic structure of the sagitta that only the oldest prism of the otolith can carry all of the life history of the fish in one continuous crystalline sequence. Periodic structures, such as opaque zones, can be followed around the otolith because successive zones must of necessity encircle the otolith, but the youngest prism will contain only the newest zone, and only the oldest prism will contain all the zones. The confluence of prisms provides another example of cessation of crystal growth. Removal of the etch-sensitive material between prisms leads to crystals with hexagonal apices, as would be expected from

crystals that have stopped growing, rather than have been occluded by organic material. In addition, there i s no evident alignment of crystals across the inter-prism boundary.

Prisms appear to grow consecutively by insertion along the transverse anterior posterior axis of the otolith. This stepwise accretion is probably the most discontinuous form of growth that is possible to achieve in an otolith. I t implies a succession of new nucleation points, each growing into a discrete prism. The prismatic structure of otoliths has received scant attention in the literature, yet it is evident that the mechanism of prism formation has to be understood as a prerequi- site to understanding the physiology of the growth of this kind of otolith. Microincrements within prisms

The pattern of microincrement growth within the prism, immediately from the nucleation site of the prism, is difficult to resolve because of the poor legibility of the increments involved. Ultrastructural features near the nucleus of otoliths are often difficult to identify and sometimes have had to be in- ferred in otoliths of other species (Campana et al., '87). In part, this may be because the microincrements near the primordial boundary of the otolith are atypically wide, as is true in some other species (Nishimura and Yamada, '88). Increase of the mineral-to- protein ratio by an increase in the amount of mineral deposited in any one day may dilute the etch-sensitive materia1 enough to make the microincrement more difficult to define by etching. In addition, the high angle to the plane of the crystals in horizontal sections in this part of the otolith complicates resolution of the microincrement structure.

Otolith microincrements are typically the smallest ultrastructural features regularly examined in otolith studies (Campana and Neil- son, ,851, although the crystalline lamellae of some otoliths are sometimes of a similar order of magnitude (Gauldie et al., '92). Al- though this study of microincrements was restricted to the sagitta, the lapillus ofMacru- ronm novaezelandiae has also been shown to have daily microincrement deposition until at least 55 days post-first feeding (Thresher et al., '89).

The microincrements observed in horizontal sections of prisms of the sagitta show a tendency to fuse towards the prism boundary; this parallels the fusion of microincrements in the vertical plane. The effect of horizontal and vertical fusion requires analy-

GROWTH IN THE OTOLITH OF MACRURONUS NOVAEZELANUIAE 291

sis of microincrement number and width to be carried out, if possible, in the most central part of the growth axis of the prism. Fusion sometimes occurs simultaneously in contiguous prisms so that fused microincrements may meet at prism boundaries. It is difficult to avoid the conclusion that fusion is respond- ing to some general signals, or conditions of crystallisation, that are affecting the whole otolith. Fusion also results in an increase in etch sensitivity, so that fusion contributes to the patterns of etched checks observed at lower magnifications. Because fusion typically takes place at the edges of the prisms it may represent the specific slowing-down of the growth of a particular crystal face. This kind of face-specific inhibition has been sug- gested as the fundamental mechanism of shape control in biogenic calcium carbonates (Weisbuch et al., '91).

Within the central part of the growth axes of the prisms of the sagitta, microincrements vary considerably in width. Decreasing microincrement width in etched sections is always associated with a generally greater degree of etch. Microincrement width decreases as the microincrement series approaches checks pro- duced by etching. It is therefore possible that such etched checks represent decrease in growth rate rather than growth cessation and consist of closely spaced narrow microincrements. Very light etching lends support to this interpretation of some optical checks representing slowed, rather than stopped, growth.

Microincrements have separate mineral and organic phases. Etching the organic material results in parallel stacks of oriented crystals. I t is difficult to see whether the organic phase of the microincrement is a mixture of organic material and very small crystal material or is completely organic material with no crystal component. There is no indication of very small crystallisation indicating renucleation at the leading edge of the mineral phase of the microincrement, but if small crystals had been present they would have been etched rapidly. Check-like discontinuities

Broken sections of otoliths of Macruronus novazelandiae show spalling lines that are discontinuities characterized by very small crystals that result in a fine-grained fracture surface quite unlike the terminating screw dislocations characteristic of broken aragonites. Etching and fracture studies show that a t least some of the etched checks correspond

to spalling lines. Some of the otolith crystals on either side of spalling lines show apparent alignment across the check, but the fine structure of spalling lines strongly suggests a renucleation episode indicating a cessation and restarting of otolith growth. One hoki otolith shows an atypical pattern of checks in the main growth axis. One of these checks shows a cluster of ataxic crystals inserted between and among sequences of regular otolith growth. Ataxic crystal inserts have been observed in the otoliths of oreosomatids (Dav- ies et al., '88). Such inserts may indicate local cessation of growth, followed by a secondary in-filling of crystals. Repair or damage by secondary in-filling in molluscan aragonites i s characterised by ataxic crystal deposition (Watabe, '81). However, microincrements continue to grow past the insertion of ataxic crystals, showing simultaneous continuous and discontinuous growth virtually side-by- side in the same otolith.

An important kind of discontinuity in otolith growth occurs in the ventral side of the otolith as a result of apparent growth occlusion by the weight of the otolith on the floor of the skull. This occlusion produces a series of severe, parallel checks running along the ventral edge of the otolith. Checks are visible in etched, optical and broken-and-burned hemisections, and similar checks have been described in other otoliths (Gauldie and Nel- son, '90b; Gauldie, '91; Smith and Kostlan, '91).

Vertical optical sections of the otolith show that the otolith of Macruronus novazelandiae is divided into sulcal and anti-sulcal parts by a boundary line that runs below the central growth axis. The same kind of boundary appears in the otoliths of the Hoplo- stethus atlanticus (Gauldie, ' 8 7 , '901, Chrysophrys auratus (Gauldie, '88b), and On- corhynchus tshawytscha (Gauldie, '91). Sur- prisingly, there is little or no evidence of such boundaries in broken sections of otoliths. The crystals in the sulcal part of the otolith are larger than those in the anti-sulcal part of the otolith. The checks in these large crystals, although prominent, show incomplete penetration of the crystal structure of otolith by the check and alignment of crystals on either side of the check. Alignment in the sulcal (and anti-sulcal) crystals across checks indicates that growth has not stopped, in the sense that there is no evidence of a renucleation necessary to re-initiate otolith growth. I t is difficult to believe that re-

292 R.W. GAULDIE

nucleation could produce such an orderly alignment of some crystals that apparently cross the check intact, whereas closely juxtaposed crystals clearly do not. An alternative explanation is that this kind of check is a chemical effect caused by low concentrations of contaminants. Characteristically, crystals precipitate out of contaminated solutions as the pure crystalline lattice form. As crystalli- zation continues, the relative concentration of contaminants rises to the saturation level, when they precipitate and cause disordered, but not discontinued, crystal growth. This effect is widejy used by organic chemists to purify contaminated materials and, inversely, in the zone refining process to purify crystals. Zones of denser inclusions are a common feature of the recrystallization of radiax- iaI fibrous calcite (Kendall, ’85); pseudo- discontinuities that are very similar in appearance to the sulcal “checks” occur in magnesium-calcite crystals as a result of su- persaturation gradients during precipitation (Lighty, ’85).

Some of the checks in the sulcal part of the otolith are formed by the fusion of microincrements passing out of the central growth axis. The fusion process results in only some of the information contained in the patterns of periodic structures in the central growth axis being transferred into the sulcal side of the otolith. Similar fusion has been described in the otolith of Hoplostethus atlanticus (Gauld- ie, ’90) and Oncorhynchus tshawytscha (Gauldie, ’91). The extent of the reduction of the information contained in the microincrement sequence of the anti-sulcal growth axis caused by fusion can be illustrated by compar- ing the patterns of widths between checks in the central growth axis with the pattern of widths between homologous checks in the sulcal side of the otolith. The low correlations observed, r = 0.39, indicate that the fusion process produces substantial reduction of the information contained in microincrement sequences between the central growth axis and of the information contained in the microincrement sequences in the sulcal side of the otolith. The mechanism underlying this effect may be connected to the basic twinning habit of otolith aragonite. Twinning results in the simultaneous growth of medial crystals along the anti-sulcal growth axis and radial crystals terminating a t the anti-sulcal and sulcal surfaces. Because of slower growth, the sulcal, (i.e., radial), crystals are much shorter than the medial crystals of the cen-

tral growth axis. Measurement along the nucleus-to-sulcal side compared to the nucleus- to-dorsal edge shows that the sulcal radial crystals average about 33% of the length of the medial crystals of the anti-sulcal growth axis. The slower growth of the sulcal crystals implied by this difference in length may account for the greater size of the sulcal crystals. However, it is difficult to avoid the conclusion that slower growth of the sulcal crystals results in a substantial loss of poten- tial life history information in the sulcal part of the otolith compared to the anti-sulcal part.

I t was evident from the comparison of optical and SEM sections that most (about 90%) of the opaque zones in both the growth axis and the sulcal section of the otolith ofMacru- ronus novaze1andia.e are optical effects, pre- sumably due to refraction and not to cessation of crystal growth.

CONCLUSIONS

Interactions between the structures of checks and microincrements show that the sagitta of Macruronus nooaezelandiae can be separated into a number of separate structural phases within which the apparent paradox of simultaneous continuous and discontinuous growth may occur. There are three kinds of discontinuities within which continuous growth occurs, but these discontinuities were out of phase with each other. Such discontinuities occur 1) between primordium and prism, 2) between contiguous prisms, and 3) between the anti-sulcal and sulcal parts of individual prisms. In addition, there are four kinds of pseudo-discontinuous growth in the otolith. Pseudo-discontinuities arise from 1) etched checks, 2) spalling lines, and 3) sulcal checks. Sulcal checks could be further divided into two types: a boundary check partially crossed by crystals and a check formed by insertions of ataxic crystals.

I suggest that the most parsimonious explanation of the continuity of the crystalline structure of the sagitta can be made as follows.

The gross morphology of the sagitta grows by accretion: First forms a primordium with a distinct terminating discontinuous boundary, followed by succession of discrete prisms. Within prisms, the primary growth process appears to be a continuous sequence of microincrements in the radial crystal twin of the anti-sulcal part of the otolith that partially fuse into checks that cross a fusion boundary into the sulcal part of the otolith. Pseudo-

GROWTH IN THE OTOLITH OF MACRIJRONUS NOVAEZELANDIAE 293

discontinuities occur in both the sulcal and anti-sulcal parts resulting from partial inter- ruption of the patterns of microincremental growth. In most cases the microincrements. grow past pseudo-discontinuities, thereby re- taining both the qualitative and quantitative properties of the microincrement sequence and giving rise to the apparent paradox of simultaneous continuous and discontinuous growth within the same otolith. True discontinuities occur along the ventral edge of the otolith and in the prismatic part of the otolith as spalling lines whose frequencies vary greatly between otoliths.

ACKNOWLEDGMENTS

I am indebted to Kevin Mulligan, Brooks Bays and Alan Blacklock for technical assis- tance. Electron microscopy was carried out at the electron microscope facility of Victoria University of Wellington. SOEST contribu- tion number 3174.

LITERATURE CITED Beamish, R.J. (1979) New information on the longevity

of the Pacific ocean perch (Sebastes alutus). J. Fish. Res. Board Can. 36t1395-1400.

Bischoff, W.D., S.K. Sharma, and F.T. MacKenzie (1985) Carbonate ion disorder in synthetic and biogenic mag- nesian calcites: A Raman spectral study. Am. Mineralo-

Bloss, F.D. (1971) Crystallography and Crystal Chemis- try. New York Holt, Rinehart and Wilson.

Campana, S.E. (1983) Feeding periodicity and the production of daily growth increments in otoliths of steelhead trout (Salno gairdneri) and starry flounder (Platich- thys stellatus). Can. J. Zool. 61:1591-1594.

Campana, S.E., and J.D. Nielson (1985) Microstructure of fish otoliths. Can. J. Fish. Aquat. Sci. 42t1014-1032.

Campana, S.E., J.A. Gagne, and J. Munro (1987) Otolith microstructure of larval herring (Clupea harengus): Image or reality? Can. J. Fish. Aquat. Sci. 44:1922- 1929.

Carriker, M.R., R.E. Palmer, and R.S. Prezant (1983) Functional ultramorphology of the divsoconch valves of the oyster Crussostrea uirginica. Proc. Natl. Shell- fish. Assoc. 74t829-836.

Christcnsen, J.M. (1964) Burning of otoliths, a technique of age determination of soles and other fish. J. Conseil.

Dale, T. (1976) The labyrinthine mechanoreceptor organs of the cod Gadus morhua L. (Teleostei: Gadidae). Norw. J. Zool. 24t8.5-125.

gist 70:581-589.

29t73-8 1.

~~~~ ~

Dannevig, E.H. (1956) Chemical composition of the zones in cod otoliths. J. Conseil. 21r156-159.

Davies, N.M., R.W. Gauldie, S.A. Crane, and R.K. Thomp- son (1988) Otolith ultrastructure of two ore0 species, Pseudocyttus maculatus, and Allocyttus sp. Fish. Bull., U.S. 86:499-515.

Deelder, C.L. (1981) On the age and growth of cultured eels, Anguilla anguilla (Linnaeus, 1758). Aquaculture 26:15-22.

Dunkelberger, D.G., J.M. Dean, and N. Watabe (1980) The ultrastructure of the otolith membrane and otolith in the juvenile mummichog, Fundulus heteroclitus. J. Morphol. 163t367-377.

Gaemers, P.A.M. i1984) Taxonomic position of the cichli- dae (Pisces, Perciformes) as demonstrated by the morphology of their otoliths. Neth. J. Zool. 34.566595.

Gauldie, R.W. (1987) The finc structure of check rings in the otolith of the New Zealand orange roughy (Hoplo- stethus atlanticus). N.Z. J. Mar. Freshw. Res. 21267- 274.

Gauldie, R.W. (1988a) The effect of surface sculpturing on the interpretation of opaque and hyaline zones in the orange roughy otolith. J . Appl. Ichthyol. 4t140- 146.

Gauldie, R.W. (198813) Similarities in fine structure of annual, and non-annual, check rings in the otolith of the New Zealand snapper (Chrysophrys auratus). N.Z. J. Mar. Freshw. Res. 22:273-278.

Gauldie, R.W. (1990) Phase differences between check ring locations in the orange roughy otolith. Can. J. Fish. Aquat. Sci. 47:760-765.

Gauldie, R.W. (1991) The morphology and periodic structures of the otolith of thc chinook salmon (Oncorhyn- chus tshawytscha), and temperature dependent variation in otolith microscope growth increment width. Acta. Zool., Stockh. 72:159-179.

Gauldie, R.W., and D.G.A. Nelson (1988) Aragonite twinning and neuroprotein secrction are the cause of daily growth rings in fish otoliths. Comp. Biochem. Physiol. 7 3 2 - 2 9 .

Gauldie, R.W., and D.G.A. Nelson (1990a) Interactions between crystal ultrastructure and microincrement layers in fish otoliths. Comp. Biochem. Physiol. 97r449- 459.

Gauldie, R.W., and D.G.A. Nelson i1990b) Otolith growth in fishes. Comp. Biochem. Physiol. 97t.119-135.

Gauldie, R.W., D. Dunlop, and J. Tse (1986) The simultaneous occurrence of otoconia and otoliths in four teleost species. N.Z. J. Mar. Freshw. Res. 20;93-99.

Gauldie, R.W., N.M. Davies, G. Coote, and I. Vickridge (1990) The relationship between organic material and check rings in fish otoliths. Comp. Biochem. Phyaid. 97t461-474.

Gauldie, R.W., G. Coote, K.P. Mulligan, I.F. West, and N.R. Merrett (1991) Otoliths of deep water fishcs: structure, chemistry and chemically-coded life histo- ries. Cornp. Biochem. Physiol. 100:1-32.

Gauldie, R.W., G. Coote, K.P. Mulligan, and I.F. West (1992) A chemical probe of the microstructural organis- ation of the fish otoliths. Comp. Biochem. Physiol. 102t533-545.

Geffen, A.J. (1983) The deposition of otolith rings in Atlantic Salmon, Salrno salur L., embryos. J. Fish. Biol. 23:467-474.

Hickling, C.F. (1933) The Natural History of the Hake. Part IV. Age-Determination and the Growth Rate. Fishery Invest., Lond., Ser. 2, 13(2), 120 pp.

Ikenouye, H. (1969) Age determination hy otolith of a Japanese alfonsin, B e y x splenderis, with special refcr- ence to growth. J. Tokyo Univ. Fisher. 55r91-98.

Irie, T. (1960) The growth of the fish otolith. J. Fac. Fish. Anim. Husb. Hiroshima Univ. 2203-230.

Keir, R.W. (1960) Answers to the questionnaire on age reading. ICNAF mimeographed document Serial No. 714 (D.C.21, document No. 4 , 6 7 pp.

Kenchington, T.J. and 0. Augustine (1988) Age and growth of blue grenadier, Macruronus nouaerelandiae (Hector), in south-eastern Australian waters. Aust. J. Mar. Freshw. Res. 38;625-646.

Kendall, A.C. (1985) Radiaxial fibrous calcite: A reap- Draisal. In N. Schneidermann and P.M. Harris (eds): karbonatc Cements. Spec. Publ. No. 36, SOC. Econ.Pal. Min. pp. 59-77.

294 R.W. GAULDIE

Kuo, C.-L., and S. Tanaka (1984) Time of ring formation of otolith and growth curve of hoki Macruronus nouaez- elancliae. Bull. Jap. SOC. Sci. Fish. 50t1627-1633.

Laurs, R.M., R. Nishimoto, and J.A. Wetherall (1985) Frequency of increment formation on sagittao of North Pacific albacore (Thunnus alalunga). Can. J . Fish. Aquat. Sci. 42:1552-1555.

Laroche, J.L., S.L. Richardson, andA.A. Rosenberg (1982) Age and growth of a pleuronectid, Parophrys uetulus, during the pelagic larval period in Oregon coastal waters. Fish. Bull. US . 80:93-104.

Lee, D.W., E.D. Prince, andM.E. Crow (1982) Interpreta- tion of growth bands on vertebrae and otoliths of Atlantic bluefin tuna, ‘I’hunmus thymus . In E.D. Prince and L.M. Pulos (cds): Proceedings of the International Workshop on Age Determination of Oceanic Pelagic Fishes: Tunas, Billfishes, and Sharks. NOAA Technical Report NMFS 8, pp. 61-70.

Lighty, R.G. (1985) Preservation of internal reef porosity and diagenetic sealing of submerged barier reef, south- east Florida Shelf. In N. Schneidermann and P.M. Harris (eds): Carbonate Cements. Spec. Publ. No. 36, SOC. Econ. Pal. Min. pp. 123-152.

Lowenstam, H.A., and S. Weiner (1989) On Biomineral- ization. New York and Oxford: Oxford University Press.

Moosegaard, H., H. Svedang, and K. Taberman (1988) Uncoupling of somatic and otolith growth rates in Arctic char (saluelinus alpinus) as an effect of differences in temperature response. Can. J. Fish. Aquat. Sci. 45:151&1524.

Mugiya, Y. (1987) Phase difference between calcification and organic matrix formation in the diurnal growth of otoliths in the rainbow trout, Salmo gairdneri. Fish. Bull., US . 85395-401.

Mugiya, Y., and J. Muramatsu (1982) Time-marking methods for scanning electron microscopy in goldfish otoliths. Bull. Jap. Soc. Sci. Fish. 48r1225-1232.

Neilson, J.D., and G.H. Geen (1982) Otoliths of chinook salmon (Olzcorhy??.chus tschawj&cha): Daily growth increments and factors influencing their production. Can. J. Fish. Aquat. Sci. 391340-1347.

Neilson, J.D., and G.H. Geen (1985) Effects of feeding regimes and die1 ternpcrature cycles on otolith increment formation in juvenile chinook salmon, Oncorh,yn- chus tshawylscha. Fish. Bull. U.S. 89r91-101.

Neville, A.C. (1967) Daily growth layers in animals and plants. Biol. Rev. 42t421-441.

Nishimura, A., and J. Yamada (1988) Geographical differences in early growth of wall eye pollack Tlzeragru chalcogramma, estimated by back-calculation of otolith daily increments. Mar. Biol. 97:459-465.

Nolf, D. (1985) Otolithi piscium. In H.P. Schultze (ed): Handbook of Paleoichthyology. Stuttgart: Gustav Fischer, vol. 10, pp. 1-145.

Pannella, G. (1971) Fish otoliths: Daily growth layers and periodical patterns. Science 173t1124-1127.

Pannella, G. (1980) Growth patterns in fish sagittae. In D.C. Rhoads and R.A. Lutz (eds): Skeletal Growth of Aquatic Organisms. New York: Plenum Press, pp. 519- 560.

Pinhorn, A.T. (1969) Fishery and biology of Atlantic cod (Gadus rnorhua) off the Southwest coast of Newfound- land. J. Fish. Res. Board Can. 26r3133-3164.

Pulfi-ich, A., and C.L. Griffiths (1988) Growth, sexual maturity and reproduction in the hottentot Pacbymet- opon blochii (Val.) S. Afr. J. Mar. Sci. 7:25-36.

Radtke, R.L. (1989) Strontium-calcium concentration ratios in fish otoliths as environmental indicators. Comp. Biochem. Physiol. 921189-193.

Radtke, R.L., M.L. Fine, and J. Bell (1985) Somatic and otolithic growth in the oyster toadfish (Opsanus tau L.). J. Exp. Mar. Biol. Ecol. 90.259-275.

Ralston, S., and H.A. Williams (1988a) Numerical integra- tion of daily growth increments: An efficient means of ageing tropical fishes for stock assessment. Fish. Bull., U S . 89;461472.

Ralston, S., and H.A. Williams (1988b) Depth Frequen- cies and Growth Characteristics of Deep Slope Fishes of the Marianas Archipelago. NOAA Technical Memo- randum NMFS-SWFC 113,47 pp.

Sharma, S.K. (1989) Applications of advanced Raman spectroscopic techniques in earth sciences. In H.S. Bist, J.R. Durig, and J.F. Sullivan (cds): Raman Spec- troscopy Sixty Years On: Vibrational Spectra and Struc- ture. Amsterdam, New York: Elsevier, Vol. 17B, pp. 513-568.

Smith, M.K., and E. Kostlan (1991) Estimates of age and growth of ehu (Etelis carbunculus) in four regions of the Pacific from the density of daily increments. Fish. Bull. U.S. 89r461-472.

Thresher, R.E., B.D. Bruce, D.M. Furlani, and J.S. Gunn (1989) Distribution, advection, and growth of larvae of the southern temperate gadoid, Macruronus nouaeze- 1andia.e (Teleostei: Merlucciidae), in Australian coastal waters. Fish. Bull. US. 87:29-48.

Urmos, J., S.K. Sharma, and F.T. MacKenzie (1991) Characterizations of some biogenic carbonates with Raman spectroscopy. Am. Mineralogist 76:641-646.

Watabe, N. (1981) Crystal growth of calcium carbonate in invertebrates. Prog. Cryst. Growth. Charact. 4;99- 147.

Weisbuch, I.L., L. Addadi, M. Lahar, and L. Leiserowitz (1 991) Molecular rccognition at crystal interfaces. Sci- ence 253;637-645.

Weidemann-Smith, S. (1968) Otolith age reading by means of surface structure examination. J. Conseil.

Wilbur, K.M., and A.S.M. Saleuddin (1983) Shell formation. In A.S.M. Saleuddin, K.M. Wilbur (eds): The Mollusca. Vol. 4, Physiology, Part 1. New York: Aca- demic Press.

Wilson, K.H., and P.A. Larkin (1982) Relationship between thickness of daily growth increments in sagittae and change in body weight of sockeye salmon (On- corhynchus nerlta) fry. Can. J. Fish. Aquat. Sci. 39; 1335-1339.

Woodhead, P.M.J. (1968) Seasonal changes in the calcium content of the blood of arctic cod. J. Mar. Biol. Ass. U.K. 48:81-91.

Zhang, Z. (1992) Relationship of saccular ultrastructure to otolith growth in the teleost Oreochromis niloticus. J . Morphol. 211r1-10.

32:270-277.

Documents

Continuous and discontinuous growth in the otolith of Macruronus novaezelandiae (Merlucciidae: Teleostei)