The Morphology and Periodic Structures of the Otolith of the Chinook Salmon (Oncorhynchus tshawytscha), and Temperature-dependent Variation in Otolith Microscopic Growth Increment

Acra Zoologica (Stockholm). Vol. 72. No. 3. pp. 159-179. 1991 Printed in Great Britain

OO0 1-727219 1 $3.00+ .IN Pergamon Press plc

0 1991 The Royal Swedish Academy of Sciences

The Morphology and Periodic Structures of the Otolith of the Chinook Salmon (Oncorhynchus tshawytscha), and Temperature-dependent Variation in Otolith Microscopic Growth Increment Width R. W. Gauldie Hawaii Institute of Geophysics, School of Ocean, Earth Sciences and Technology, University of Hawaii, 2525 Correa Road, Honolulu, HI 96822, U.S.A.

(Acccpied for publicarion 12 October 1990)

Abstract Gauldie, R. W. 1991. The morphology and periodic structures of the otolith of the chinook salmon (Oncorhynchus Ishawytscha). and temperature-dependent variation in otolith microscopic growth increment width.-Acfa Zoologica (Stockholm) 72: 159-179.

The otolith (sagitta) of the chinook salmon (Oncorhynchus ishawyrscha) has a variable external crystalline morphology which is related to differences in the growth rate of crystals in different parts of the otolith. The internal crystal structure of the otolith is complex with different mineral phases in different growth fields of the otolith and a well-defined series of microscopic growth increments in both the dorsal and ‘the ventral parts (orientation in situ) of the otolith. The period of the microscopic growth increments was shown. by both a rearing experiment and interpolation from fish of known age, to be daily. By rearing sibling chinook salmon at different temperatures (while still maintaining them on the same diet) it was shown that the width of the daily growth increment varies with temperature, but neither multi- nor sub-daily increments appear in the area of regular. daily growth incrementation. lnclusions of the vaterite morph of calcium carbonate crystallizing in the botryoidal habit occur in the otherwise aragonitic otolith. Regularly spaced check rings in the sulcul part of the otolith are homologous with those occurring in the dorsal and ventral growth axis and are. on average. separated by about 28 microincrements.

R. W. Gauldie, Oceanic Biology, Hawaii Insrifute of Geophysics, 2525 Correa Road, Honolulu, HI 96822, U.S.A.

Introduction

Teleosts generally have three otoliths in each of the pair of endolymphatic sacs associated with the inner ear. Oto- iiths are composed of crystalline calcium carbonate, usually in the form of twinned aragonite (Gauldie & Nelson 1988). Twinning occurs when a mirror-image crystal is produced at one of the crystalline faces. In otolith aragonite it is the (110) face of the crystallographic plane. Twinning results in much faster growth of the crystal as well as simultaneous growth in two directions of what is, structurally, the same crystal. Otoliths are not as readily subject to metabolic reorganization as are bones, resorption occurring only under unusual circumstances (Mugiya & Uchimura 1989). Therefore, the pattern of ultrastructural changes laid down in the otolith in response to conditions that affect the otolith chemistry are retained over the life of the fish. Otoliths consequently provide in their ultrastructure a record of events in the life of the fish. As a result of this recording capacity, the patterns of otolith ultrastructure have been widely used to estimate the age of fish in preference to information in the other hard parts of fish, such as scales and bones (Bagenal 1974). Unfortunately, in spite of their wide usage in esti- mating fish age, the physiology of otoliths, especially that related to their deposition, is not well known. Ignorance of the physiology of otolith deposition has been a major

problem in the validation of age estimation techniques in many fish species. In the absence of physiological information, the validation of age estimation techniques has come to rely heavily on statistical correlation rather than on an understanding of cause and effect in the patterns of the ultrastructure of otoliths. To a large extent it was this lack of physiological information that led to the disquiet expressed by Beamish and McFarlane (1983) over the validity of ages estimated from otolith ultrastructure.

Unfortunately, the physiology of otoliths is complicated by the experimental difficulties of recording information from the endolymphatic sac of live fish, and the technical difficulties imposed by the growth of the otolith being controlled by chemical gradients in an extracellular fluid space. Although we are thus denied a ‘karyotic biominera- lization window’ into the otolith growth process (as described by Simkiss 1984), we have ready access to all of the growth processes of the otolith that have been recorded in the ultrastructure of the otolith itself. How- ever, in order to use the information recorded in the otolith it is necessary to assess the significance of both the external and internal ultrastructural patterns in otoliths whose age can be estimated with confidence.

The age of salmonids in general, and chinook salmon Oncorhynchus tshawytscha in particular, can be accurately determined to the annual level of precision because of

159

160 R. W. Gauldie

the general habit of salmonids of returning to their natal stream, and the development of techniques for tagging juvenile salmon. Salmon species present an almost unique experimental situation in which the age of wild fish can be measured directly and not by estimation from otoliths and scales.

Ageing procedures have been developed for many salmonids using scales (Bilton & Ludwig 1966; Mottley 1929). Ageing procedures using otoliths have been developed for sockeye and chum (Kim & Roberson 1968; Bilton & Jenkinson 1968) and chinook salmon (Flain 1982). For sockeye, chum, and chinook salmon the otolith ageing technique is based on a series of opaque and hyaline zones.

Opaque and hyaline zones in the oyster toadfish Opsanw tau appear to be caused by seasonal variation in the width of daily growth increments (Radtke et al. 1985). Daily growth increments have been described for sockeye salmon with evidence that their width is related to size change in the fish (Volk et al. 1983; Wilson & Larkin 1982). Daily growth increments have been described for chinook salmon, but with the qualification that under certain circumstances more than one increment may be deposited every 24 h period depending on the entrainment pattern of die1 rhythms (Neilson & Geen 1982; Neilson et al. 198%). In other species, from a wide number of fish families, increment deposition in the otolith appears to be daily in fish provided that fish are growing at normal physiological rates under normal circadian rhythms (Campana & Neilson 1985; Jones 1985). Physiological evidence supports daily increment deposition in fish otoliths as a function of endogenous die1 rhythms in calcium mobilization (Mugiya 1987) and of neuropeptide secretion (Gauldie & Nelson 1988).

The chinook salmon (Oncorhynchus tshawyrscha) otolith presents many advantages for the study of growth increments. Chinook salmon have a long history of hus- bandry so that fish can be reared with great reliability making the species experimentally tractable. Because the chinook salmon, in common with other salmonids, lives in a fluctuating environment (particularly temperature) it provides the opportunity for a large amount of possible temperature (and correlated growth)-dependent variation in individual fish to be recorded in the crystalline structure of the otolith. Thus, the chinook salmon otolith might be expected to provide a suitable experimental vehicle to test the range of variation in daily growth increment morphology that is likely to be found in teleost otoliths.

One of the disadvantages of the chinook salmon otolith is that its internal architecture (opaque zones, check rings, microincrements, etc. and their relations to the kind and spatial distribution of crystals) is quite complex and must be taken into account when decoding signals stored in patterns of the ultrastructure of the otolith. This paper describes the architecture of the chinook salmon otolith and establishes those regions of the otolith within which regular patterns of growth increments occur in both juvenile and adult fish. The effect of temperature on the variation of regular patterns of growth increments is described. The architecture of the chinook otolith is described in detail reflecting both the desirability of know- ing more about the organization of the otoliths of a species

which can be aged with great accuracy, as well as the limited amount of information concerning the ultrastructure of otoliths which is presently available in the literature (e.g. Degens et al. 1969; Dunkelberger et al. 1980).

Because the age of the chinook salmon can be accurately determined from annual opaque and hyaline zones in the otolith, it is possible to validate daily rings from annual rings. It is, therefore, possible to examine the morphology of chinook salmon otoliths in the light of their known periodicities, rather than assume periodicities based on preconceived patterns in the morphology of the otolith. In this study direct validation of daily increments was also made from juveniles which were reared from eggs. In addition, the period of the regular series of check rings which appear in the sulcus of the chinook otolith was determined. The periodicity of such check rings is of considerable importance to the theory of ageing fish from otoliths.

Materials and Methods

Two groups of fish were used in the investigation of otolith ultrastructure, a juvenile group and an adult group. The juvenile fish used in the experiment were drawn from a single pair of chinook salmon (Oncorhynchus rshawyrcha) from wild stocks of the Ouinsam River on Vancouver Island. British Columbia, Canada. Genetic uniformity was approximated by restricting the experiment to the offspring of a single pair mating. The fertilization of eggs took place on 27 October 1978. All the fish were subsequently reared together and fed to repletion for the duration of the entire experiment on the same batch of Oregon Moist Pellet. Mortality past the emergent fry stage was less than 0.5%. On 20 August 1979. a sample of 500 fish was randomly sorted into five groups of 100 fish each in separate tanks, which were then gradually acclimated over 5 days to five different temperature regimes.

For the duration of the experiment, temperatures were maintained as closely as possible to 8". 10". 12". 14O and 16" Celsius. However. the initial acclimation period (of up to 5 days for the highest temperature regimes) led to a lower mean temperature and increased standard error. The consequent mean daily temperatures for each of the five treatment groups over the 43-day period were as follows: 63°C (control). 7.97 (nominal 8°C). 9.98 (nominal 10°C). 11.70 (nominal ITC), 13.94 (nominal 14°C) and 15.98 (nominal 16°C). Records of daily temperatures were kept throughout the experiment. as average temperature per day (Fig. I ) . The average degrees per day over the entire life of the fish for each temperature treatment group were 8.31 (control). 8.40 (nominal 8°C). 832 (nominal IOT), 8.61 (nominal 12°C). 8.74 (nominal 14°C) and 8.85 (nominal 16°C). Temperature treatment groups are identified by their average degrees per day values throughout the text. After 43 days all of the fish were anaesthetized. weighed, measured, frozen in liquid nitrogen, and subsequently stored at -20°C. The fish in the temperature control group were sacrificed on 3 October 1979. their sex determined and their otoliths removed. Their total age at this date was 348 days. The control group were sacrificed 2 days later bringing their total age to 350 days.

Fourteen fish died in the lowest temperature tanks during the 43-day temperature experiment. Examination of these fish failed to reveal any parasites or infectious agents. At the end of the experiment a further sample of 100 fish was taken randomly from the original population which had been maintained at 8.30"C. They were anaesthetized, weighed, measured, and frozen in liquid nitrogen and subsequently stored at -20°C.

During the period from emergence to the initiation of the 43-day temperature experiments. the water temperatures of the tank in which the fish were reared varied from a low of 7°C to a high of 13.5"C. Between 219 and 285 days water supplies were changed owing to low water levels resulting in a period of higher, and more variable daily temperatures. As a result, two sets of temperature data are available: the day-to-day variation over the entire life of the fish. and the 43-day temperature experiment itself.

When otoliths were removed it was evident that two morphological types of otolith were present. an aragonitic. the normal otolith, and a vateritic type. Aragonite and vaterite are two crystalline forms of cal-

Otoliths of the Chinook Salmon 161

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Fig. I. Daily water temperatures during the rearing experiment: the five treatment temperatures and the control are indicated by the nominal treatment temperatures. Day zero is the date of spawning. In the period 719-285 days water temperatures fluctuated and are marked in with solid lines. Temperatures over the experimental period are shown as alternate /I& and durk dois. The temperature change marked E came at 205 days counted back from the experimental otoliths.

cium carbonate. Upon removal. right and left otoliths wcre kept separate. The results reported here are for aragonite otoliths. Some of the vaterite data have been reported elsewhere (Gauldie 1986).

The adult fish used in the experiment came from a sample of adult chinook salmon otoliths from wild stocks of the Ouinsam River, Van- couver Island. and from wild stocks of New Zealand chinook salmon from the Raikaia River. [New Zealand chinook were introduced from North America at the turn of the century (McDowell 1978).] The otoliths of some New Zealand chinook salmon otoliths were stained with fluorescein which enhances the opaque and hyaline zones of the otolith (pen. commun., M. Flain. Fisheries Research Centre).

Scanning electron microscopy was carried out using a Philips 505 SEM after the specimens were sputter coated with gold. SEM specimens were mounted on aluminium stubs using either epoxy resin or double-sided sticky tape and silver conducting paint.

Specimens for optical analysis were mounted on glass slides with epoxy resin and then ground and polished at the appropriate plane using a Struers’ Pedemax petrographic grinder. Once in the appropriate plane the epoxy resin containing the otolith was removed from the slide by soaking in 70% alcohol for 2 h and then easing off the slide using a single blade razor. The ground otolith and epoxy resin support were then re-embedded on a glass slide, ground plane downwards, and then reground to form a thin (10-15 r m ) section in the horizontal plane.

Etching of otolith sections was performed with 0.1 M ethylene diam- ino tetracetic acid (EDTA). p H 2.5. Acetate peels were made in the conventional way after etching the polished surface of otolith sections with EDTA. Acetate peels and thin sections were photographed using a Wild M W photomicroscope.

Measurements of otolith axes, o r distances between opaque and

.

hyaline zones and other features of the otolith were made with a microscope micrometer.

Crystalline morph was tested by X-ray diffraction. Samples were lightly crushed in an agate mortar and pestle and examined with a Philips PW 1279/PW 1710. Errors attached to the proportions of the different crystalline morphs were ? 5%.

Plotting of the graphs of daily growth increments was carried out using a LOWESS program (Cleveland 1979) modified by Ian West of the Fisheries Research Centre. Wellington. New Zealand. The LOWESS program is a method of local weighting to smooth data to reveal underly- ing trends. The LOWESS method has the advantage of allowing the amcunt of information lost in the smoothing to be adjusted by changing the window size. i.e. the number of consecutive observations used to establish the running mean. The loss of information from smoothing data in this study was about 5%. which serves to emphasize the underly- ing pattern of peaks and troughs in microincrement widths.

The terminology associated with check rings has become less precise as knowledge of otolith structure has developed. In the results section, check rings are referred to without qualification as structures within a particular context. Qualification of the term check ring will be introduced in the discussion. In general, the terminology of otoliths has been developed in response to the needs of taxonomists and to meet the demands of fish age estimation procedures. The taxonomic nomenclature used here to describe the external features of the otolith is from Pannella (1980). However. as with other taxonomic nomenclatures. Pannella’s usage is based on outmoded ideas. For example. colliculum generally refers to a point of attachment of a nerve, muscle or ligament and we now know that there is no direct attachment to the otolith (Dunkelberger cf el. 1980). The internal structures o f the otolith have

162 R. W. Gauldie

often been assigned specific names even though there is no ultrastructural basis to distinguish between such structures (Gauldie 1988~). Conse- quently I have modified the standard (Pannella) nomenclature to use, where possible, ordinary, descriptive English words rather than to intro- duce new terms. The nomenclature for aragonite crystals used by Car- riker et af. (1980) has been followed wherever possible. Calcium carbonate occurs in four morphs: aragonite, calcite, vaterite and calcium carbonate monohydrate (Carlstrom 1%3). Within these morphs calcium carbonate crystals occur in different, although stable and often characteristic forms referred to as their crystal habit. For example, calcite can occur in a cuboidal or rhombohedra1 habit, and vaterite occurs in a number of different habits the most common of which, the botryoidal habit, occurs in salmonid otoliths (Gauldie 1986).

The possibility of multiple microscopic growth increments (i.e. more than one per day) caused by optical artifacts was further tested by using an extinction method to test for valid daily microscopic growth increments. The extinction method takes advantage of the refractive properties of otoliths which cause apparent multiple microscopic growth increments to appear when the gaussian focus of the microscope is altered (Gauldie & Nelson 1988). Thick sections result in a switch from light to dark diffraction patterns at the point of gaussian focus which can result in the appearance of an extra dark (or light) zone. Multiple dark zones caused by gaussian focusing problems become less apparent as the section thickness approaches zero. Focusing at the edge of the wedge-shaped section resulted in the microscopic growth increment being either in or out of focus without apparent multiple microscopic growth increments. Eight sections were examined in this way. Micro- scopic growth increments present some problems in definition. At the light microscopy level they are defined as a light, mineral-rich, zone, followed by a much narrower, dark, mineral-deplete zone (Gauldie & Nelson 1988). At the SEM level the definition is made in terms of the relative etch-sensitivity of the mineral-rich and mineral-deplete zones. Although it is a somewhat clumsy usage, microscopic growth increments require validation of their deposition periodicity before they can be identified as daily microscopic growth increments.

Results

This study is divided into three sections: (1) scanning electron microscopy of the topography of the chinook otolith; (2) the internal architecture of the otolith and the relationship between microscopic growth increments, check rings and opaque and hyaline zones; and (3) the relation between microscopic growth increment width and temperature.

The orientation of the otolith is described in terms of sulcul and anti-sulcul side, i.e. ‘down’ and ‘up’, respect- ively, when the otolith is laid flat sulcus side down on the laboratory bench. Because all of the information in this paper refers to isolated otoliths, it seems unnecessarily cumbersome to continue to refer to the orientation found in situ. In addition, the standard usage of ‘inward face’ for the sulcul side, and ‘outward face’ for the anti-sulcul side applies only to a limited range of fish. However, the otolith is divided into two parts by the sulcus that are referred to as dorsal and ventral as they would be in situ. Canadian and New Zealand chinook salmon used in this study did not show any significant differences, but sample sizes were small and this study is concerned with the patterns of growth rather than regional variations in otolith morphology that might arise from different growth rates within otoliths.

Scanning electron microscopy of chinook otolith topography

The endolymphatic sac of the chinook salmon contains three otoliths, a large sagitta and two smaller otoliths,

the lapillus and asteriscus. The disposition of the three otoliths has the same organization (with minor species- specific variations) to that of the cod described in detail by Dale (1976) to which the reader is referred for a general description of the labyrinthine organs of teleosts.

The topography of the otolith comprises three sectors, the anti-sulcul surface, the sulcul surface and the sulcus itself.

The anti-sulcul surface. At low magnification the upper (anti-sulcul) surface of the chinook salmon otolith had a coarse appearance often with secondary deposits of calcium carbonate laid down after the deposition of the main body of the otolith (Fig. 2A). The sulcus groove lies under the otolith in this orientation, dividing the otolith into a dorsal and ventral segment. Both segments are of similar size in juveniles, but in adults the ventral segment is very much larger than the dorsal segment.

At higher magnifications there were differences in surface texture between the dorsal and ventral segments of the otolith (dorsal and ventral are in reference to the sulcus as is shown in Fig. 2A). The upper surface of the ventral segment had a coarse surface with a stepped, or scale-like appearance. The width of the steps or scales varied with a mean of 8 p m (15 otoliths). The steps or scales were always stepped down towards the edge of the otolith, i.e. the direction of growth (Figs 2B, C).

The upper surface of the dorsal segment had a finer texture (Fig. 2D) with no indications of the stepped surface. One of the hemispherical secondary growth nodules lying on the upper surface of the otolith can be seen in Fig. 2D.

The sulcus surface. The lower sulcus surface of the chinook salmon otolith had a complex structure. The lower surface of the whole otolith can be seen in Fig. 3A. The sulcus divides the otolith into a smaller dorsal region and a larger ventral region (Fig. 3A). The three main features of the otolith, sulcus, dorsal segment and ventral segment have distinctive features which are best described separ- ately.

The large, ventral lower surface showed a faint super- ficial pattern of zones about 330 p m wide (Fig. 3A). At low magnification the surface appeared relatively smooth with a pitted appearance which became coarser towards the edge of the otolith but was actually very smooth right at the edge of the otolith (Figs 3B, C). The pits on the ventral surface (Fig. 3B) were about 60 pm wide. The edge of the otolith often had the appearance of a lip separated by a shallow groove from the main body of the otolith (Figs 3B, C). The shallow groove appeared to have a coarser crystalline structure than either the main body of the otolith or the leading edge.

At the edge of the sulcus itself the pitted lower surface changed into a more coarsely textured surface (Fig. 3D) on which there were small nodules with the appearance of blunted ends of hexagons (Fig. 3E). Hollows appeared irregularly on the ventral surface (Fig. 3E). At the bottom of these hollows there were lath-like crystals (Fig. 3F) similar to those which have been found growing at the leading edges of the principal growth axes of otoliths in other species (Gauldie 1987).

Otolith of the Chinook Salmon 163

[email protected]. The otolith pictured is from an adult fish and is lying on the sulcus side, anti-sulcus side uppermost. The anterior (A! is to the right. The long posterior ( P ) has a projecting horn to the left of the picture. The sulcus groove is underneath (curved arrows) and divides the otolith in a narrow dorsal (D) sector and a much wider ventral (V) sector. Many nodules (arrow) can be seen on the upper surface. Scale bar 1 mm.-E. Successively higher magnifications of the upper surface of the ventral segment show a stepped or scale-like surface layer. Scale bar ZOll p m . 4 . The stepped or scale-like upper ventral surface varies in the width of the step or scale and the coarseness of its composite crystals. Scale bar 50 pm.--D. Higher magnifications of the upper surface of the dorsal segment show a finer textured surface than the ventral segment without steps or scales. One of the secondary growth nodules [arrow in (A)] can he seen in more detail (arrow) to have a spherical shape. Scale bar 100 km.

Some chinook otoliths showed more complex patterns of crystals associated with the pitted structure on the lower ventral surface. The organization of the pits was similar among (Figs 4A-C), but with differences between individuals. Generally the ‘floor’ of the pits had a smooth crystal structure. The ‘walls’ of the pits were built up of parallel rows of crystals, but the rows varied between individuals from nodule-like arrangements of crystals (Figs 4A, C) to well-organized rows (Fig. 4B). In some parts of the otolith well-organized rows occurred without pits (Fig. 4E). In all cases the crystals were invested with what appears to be an organic film.

The dorsal lower surface had a different structure from that of the ventral lower surface. The edge of the sulcus had a fine-grained crystal structure (Fig. 5A) which, between the sulcus and the edge of the otolith, developed an intersecting habit with individual crystals running in one direction and the blocks formed by those crystals running in another (Fig. 5B). In some places those blocks of crystals progressively developed into hexagonal prisms

(Fig. 5A) arranged as a series of zones or steps. At the edge of the otolith a smooth crystal habit appeared as in Figs 3B, C.

The sulcus. In the majority of wild chinook salmon the sulcus was a deep groove with a smooth interior surface (Fig. 6A). The crystals of the otolith grew parallel to the sides of the groove (Fig. 6A) except for a layer at the very edge of the sulcus which grew inwards towards the sulcus itself (Fig. 6A). The layer at the edge of the sulcus can be followed even when crystal deposition within the sulcus has obscured the original apex of the sulcus (Fig. 6A). The apex of the sulcus (the part closest to the nucleus) appeared to be gradually filled in with crystalline material. Detail of the crystals of the wall of the otolith showed groups of intersecting layers (Fig. 6B) whose appearance is similar to that of the crystals associated with the pits on the lower ventral surface (Figs 4A-D). However, a significant proportion (about 10%) of wild chinook salmon showed crystalline deposits in the sulcul

164 R. W. Gauldie

Figs 3A-F.-A. The sulcus clearly divides the otolith into a smaller dorsal (D) and a larger ventral ( V ) segment. A faint pattern of zones can be seen (urrows) on the ventral surface.-B. At low magnification the relatively smooth ventral surface can he seen t o have pits (urrow) in the central region and an irregular surface (open urrow) running along the ventral edge of the sulcus. An X x expansion of the edge of the otolith shows the irregular surface becoming quite smooth at the growing edge o f the otolith. The growing edge o f the otolith can he seen to he growing as a lip along the ventral surface with a shallow groove (G) running between the edge and the body of the already deposited main hody of the otolith. Scale bar 100 pm.-C'. Higher magnification of the shallow groove behind the leading edge (and the X x expansion of the right-hand frame) shows the coarseness of the crystalline material in the groove between the lip and the main hody o f the otolith. Scale bar SO pm.-D. The ventral surface shows hollows (arrows) and a groove running along the edge o f the sulcus (urrow). Scale bar I00 pm.-€. Higher magnification of the groove (D above) shows an irregular surface of small nodules. some approximating the hexagonal shape of free aragonite crystals. Scale bar 50 pm.-F. Holes or hollows in the ventral surface often show projecting lath-like crystals. Scale har I00 pm.


Figs 4A-D.-A-C. The consistent pattern of the pitted ventral lower surface can be seen hy comparing these three different otoliths. Generally the organization of pits is similar with a smooth 'floor'. but the 'walls' vary in arrangement from nodules to rows of crystals. Scale bars (A. B) 200 pn; (C) IOU pm.-D. Some areas of the ventral lower surface show structures which appear to be aggregations of crystal rows without pits but invested with an organic film. Scale bar 20() pm.

figs SA. I?.-A. The edge of the sulcus (E) has a fine-grained crystal structure which grows towards the cdgc o f the otolith. resulting in a stepped appearance (open urrows) and aggregating in places into hexagons (urrow:~) . Scale bar 2(M) pm.-B. Between the sulcus and the edge of the otolith the crystals develop an intersecting structure with hlocks of crystals running in one direction and crystals within blocks (rtrrons) running in another. Scale bar 200 pm.

166 R. W . Cauldie

Figs 6A-D.-A. Cross-section o f the sulcus shows a narrow. deep, groove with smooth sides and vault (the part closest to the nucleus) in the majority of chinook salmon. The crystals of the otolith grow parallel to the walls o f the sulcus (closed urrows). hut at the very edge o f the sulcus there is a layer of crystals growing at right angles to the sulcus wall (open a r r o w ) . The upper part of the vault of the sulcus has heen filled with a secondary crystal deposit. Scale bar 200 pm.-B. Intersecting crystals grow close to the vault (expanded seccriou). hut at the dorsal lower surface hexagons develop (arrow). Scale bar 200 @m.--C. Botryoidal masses o f vaterite (urrow) occur in some chinook salmon otoliths. Scale bar 400 pm.-D. The vaterite masses lying in the sulcus have a zoned appearance which persists across apparent discontinuities (arrow) in the crystal mass. Scale bar 20 pm.

groove (Fig. 6C). These crystals had the rounded appearance of vaterite, a form of calcium carbonate with a characteristic botryoidal crystal form which occurs in chinook salmon (Gauldie 1986). The botryoidal masses had a zoned appearance with about a 10 Fm interval between zones (Fig. 6D).

The internal architecture and principal growth axis of microscopic growth increments

The internal architecture of the otolith can be conveniently described in two stages. The first stage is the description of the three-dimensional organization of the otolith that can be developed from the examination of broken sections of otoliths. The second stage is the description of microscopic growth increments which can be developed by grinding and etching otoliths in the horizontal and vertical (visd-vis the orientation of the sulcus) planes.

Three-dimensional organization of the chinook otolith. Sections broken at right angles to the sulcus showed that there was a growth axis running along the upper (i.e. the anti-sulcus or outward facing) edge of the otolith in both the ventral and dorsal segment (Figs 7A, B). Detail of the growth axis (Fig. 7B) showed the same pattern of epitaxial crystal growth described for other species (Gauldie 1988a). This pattern consisted of a growth axis beneath the surface of the otolith of crystals growing from the nucleus to the edge of the otolith. Crystals curved away from this axis towards the sulcus side of the otolith until they were more or less growing at right angles to the original growth axis, as well as to the upper surface (Fig. 7B). Crystalline discontinuities occurred as a boundary between the principal growth axis and both the upper and lower surfaces of the otolith, although otherwise the crystals appeared to be growing continuously out of the principal growth axis (Fig. 7B). The crystals of the principal growth axis grew towards the edge of the otolith before they curved away to lie orthogonal to the principal


growth axis (Fig. 7B). Some chinook salmon otoliths showed another, more

pronounced crystalline discontinuity, ‘spalling’ planes (Fig. 7C), with the same smooth, fine-grained appearance (Fig. 7D) observed in the otolith of the orange roughy (Gauldie 1987). (The term ‘spalling’ was introduced as an English word describing the effect of these structures in breaking the otolith into smaller pieces using thumb pressure.) The continuity of crystals in the. principal growth axis with those in the ventral sulcus side of the otolith allowed a measure of the different growth rates of what is essentially the same crystal occurring in both the principal growth axis and the sulcul part of the otolith. Measurements made in the thickest part of the otolith in Fig. 7A showed that the crystal twin which originates in the principal growth axis and continues to the edge of the otolith was about 2.25 times the length of the twin which grew to the sulcul side. Measurements in a number of

otoliths showed that this difference in growth rate of crystal twins ranged from 2 to 3 times.

Internal organization of microscopic growth increments, check rings and opaquelhyaline zones. Both horizontal and vertical sections of otolith showed microscopic growth increments as well as check rings. Horizontal cross-sections of otoliths showed information that was not simply associated with differences in growth rate of twinned aragonite crystals growing in the principal growth axis and sulcus parts of the otolith. There was other information about growth in the otolith which was revealed by the pattern of various zones and check rings, particularly in the anterior and posterior extremes of the otolith.

The horizontal cross-section of the otolith in Fig. 8 was from a typical aragonite otolith from the control group (the fish held at 8.31”C) at the age of 348 days from

Figs 7A-D.-A. Broken sections show a well-developed growth axis (urrows) running beneath the upper (anti-sulcus) surface (U) in both the dorsal (D) and ventral ( V ) part of the otolith. Crystals grow orthogonally (open urrows) to the growth axis in both the upper (U) and lower ( L ) parts of the otolith. A rounded vaterite crystalline inclusion (arrow) can be seen growing in the apex of the sulcus. The distance from the point marked with an open circle on the ventral principal growth axis to the edge of the otolith is about 2.25 times the distance measured from the same point to the edge of the sulcus. Scale bar 0.5 mm.-B. Detail of the growth axis shows epitaxial growth towards both the upper (U) and the lower ( L ) surfaces of the otolith as well as along the growth axis itself. The enlarged section shows the same crystal branching into both the upper and lower segments. but being interrupted by a fine crystalline anomaly before reaching the upper surface (arrows). and a more coarse crystalline anomaly (arrows) helow the principal growth axis in the lower section. Scale bar 200 pn.--C. Broken surface from the dorsal growth axis of the otolith from wild chinook salmon show the same appearance of spalling planes (arrows) observed in other species. Scale bar IN pm.- D. Detail of a spalling plane shows its smooth. fine-grained surface (crtn’ed urrow). Although the spalling planes interrupt the crystal. crystal p w t h appears to continue (arrow) from one side of the break to the other. Scale bar 20 pn.

168 R. W. Cauldie

Fig. 8. The typical horizontal section of an otolith is divided by the sulcus (which lies beneath the plane of this section and is shown by dorred lines) into a dorsal (D) and ventral ( V ) segment. The dorsal and ventral segments show the development of a system of sutures (S) which divide the otolith into the typical prism pattern found in many species. The anterior ( A ) and posterior ( P ) ends of the ventral segment (and to a lesser extent the dorsal segment) which run along the sulcul groove develop rostrums (H). The ventral segment is divided by sutures (arrows) into a semi-hemispherical segment centred on the nucleus (N) within which regular daily growth increments occur.

fertilization. The otolith had been ground down horizon- tally from the upper (anti-sulcus) side to the level of the nucleus. The sulcus lying underneath the plane of the grind has been shown in outline dividing the otolith into dorsal and ventral segments. The dorsal and ventral segments showed signs of the prism pattern which appears more strongly in the adult chinook salmon. The prism pattern is common to many otoliths from different species and is equivalent to the process of prismatic crystal growth in molluscs (Wilbur & Saleuddin 1983).

The anterior and posterior ends of the ventral segment showed the first stages of the projections (anterior and posterior rostrum) that grow at each end of the sulcus in the adult fish. The sutures or prismatic boundaries indicated by arrows in Fig. 8 conveniently mark the section of the ventral segment within which daily growth increments appear most clearly.

The internal architecture of the horizontal cross-section of the chinook salmon otolith can be conveniently treated as having five regions: the anterior and posterior rostrums

Figs YA. B.-A. The complex nested cones (arrows) in the dorsal anterior rostrum can be seen in an SEM of the etched surface. Scale bar 200 pm.-B. Light microscope sections show microincrements (small arrows) between the checks (large arrows) that define the nested cones.


following the sulcus, the dorsal segment, the nucleus and the ventral segment with daily growth increments.

The anterior and posterior rostrums. An SEM picture of the etched surface of otoliths from the control group of salmon showed the development of a complex series of check rings in the posterior and anterior rostrums associated with the sulcus (Fig. 9A). Using a light microscope, higher magnification showed microscopic growth increments between a complicated arrangement of check rings (Fig. 9B). This area of complex check ring deposition was directly above the sulcus.

The ventral segment. Acetate peels of the etched surface of the ventral segment of the control group showed a number of features which, although they showed some variation in detail, were none the less consistently present in otoliths from individual fish.

The prism pattern described in Fig. 8 becomes more pronounced as the otolith increases in size, and although there are growth axes in both the dorsal and ventral segments (Fig. 7A), it is evident that in adult chinook salmon otoliths the ventral growth axis is longer than that of the dorsal segment and has a more pronounced curvature. The curvature of the growth axes raises difficulties in cutting a section in the horizontal plane which will pass through all of the microscopic growth increments lying along the growth axes. Horizontal sections containing all of the microscopic growth ring sequence in the ventral segment usually show incomplete ring sequences in the dorsal segment. Useful information from horizontal sections is thus limited to certain parts of the otolith, the nucleus, the central part, the edge, etc.

The nucleus. The nucleus of the otolith of hatchery- reared fish often shows abnormalities, particularly multiple nuclei. Multiple nuclei ranging from a string of separate nuclei, to a multi-centred single nucleus have been described from different species (Rosenberg & Lough 1977; Tanaka et al. 1981; Geffen 1983), including chinook (Neilson et al. 19856).

One hundred and twenty otoliths from the hatchery- bred group were ground down to thin sections with the horizontal plane passing through the nucleus. Of these, only one showed a centre with a single nucleus and radiating microscopic growth increments (Fig. 10).

The nucleus of the chinook salmon otolith consisted of a spherical body (a circle in section) around which a few faintly outlined microscopic growth increments grew before encountering a structure which showed up as a dark check ring in optical photographs of thin sections (Fig. lo), or as a deep groove in etched otoliths. This first check ring must have been the boundary of the ‘primordium’ of the otolith. Beyond this first check there was a regular series of microscopic growth increments. All of the other otoliths examined showed multi-nucleated centres. Multi-nucleated centres apparently perturb the normal deposition of crystalline mineral in the nuclear region resulting in a loss of legibility of microscopic growth increments near the nucleus.

Fig. 10. A thin section of a juvenile chinook salmon otolith shows a nucleus (A‘). consisting of a spherical structure bounded by a check ring (arrows). A series of very fine lines (shown as bars) lie between the core of the nucleus and the first check ring ( P and arrows) which marks the boundary of the nucleus. Scale bar 20 pm.

The dorsal segment. Because of the disruptive effects of multiple nuclei at the centre of the otolith, the microscopic growth increments in the dorsal segment of the otolith were best described from the outside inwards towards the nucleus.

None the less readable sequences of microscopic growth increments occurred at the centre of the otolith near the nucleus (Fig. 10) and progressed through the central part of the otolith (Fig. 11A) to the edge. The width of the microscopic growth increment changed in those parts of the otolith that correspond to the period temperature change which started at day 219 (Fig. 11A). Microscopic growth increments still occurred, but in sequences sometimes interspersed with crystal discontinuities in the otolith (Fig. 11B).

Check rings in the salmon otolith [and in other species, Gauldie (1988u)], have different appearances by SEM and optical sections. For example, in Fig. 9A SEM pictures showed after etching what could reasonably be described as a check ring (within the wide range of the usage of the term). However, optical sections of the same part of the otoliths (Fig. 9B) showed that in their unetched form, check rings appear as wider, and optically more dense, i.e. more refractive, growth increments. A similar effect has been noted between unetched and etched check rings in the otolith of Curanx georgiunus (Gauldie & Radtke 1990). A number of distinct check rings appeared in different otoliths consistently at about the same number of days after spawning as the microscopic growth increment count.

The first check ring occurs at 34 days from the edge of the otolith (i.e. the rings being counted from the edge towards the multi-nucleated centre). inspection of the temperature graph (Fig. 1) shows a temperature pulse at 34 days prior to the end of the experiment.

The second check ring came at 45 days from the edge.

170 R. W. Gauldie

Figs IIA, B.-A. Readable daily growth increments occur through the centre of the otolith with more-or-less uniform width until the growth increments corresponding to the start (day 219: arrow) of the temperature treatment experiment is reached. Scale bar 20 Fm.--B. Readable sequences of daily growth increments occur in parts of the otolith which are not on the principal growth axis: but in these parts of the otolith the sequence may be disrupted by crystalline discontinuities.

For the control group, this check came on the day on which the control group was sub-sampled for the fish that went into the temperature treatment experiments. Inspection of Fig. 1 shows a temperature pulse at the 45- day period as well, so that the 45-day check ring may be due to either temperature, or disturbance, or both.

Between days 68 and 134 from the edge there was a long series of temperature fluctuations. Although the beginning and end of this period was marked by check rings, there was a period in the otolith corresponding to days 68-94 in which wide microscopic growth increments were deposited and another period corresponding to days 94-134 in which microscopic growth increment widths were narrower.

At day 205 counted back from the edge there was a check ring corresponding to a temperature change (marked ‘E’ in Fig. 1).

At day 267 counted back from the edge there was a check ring (F). The disruptive effect of multiple nuclei makes it difficult to count back exactly 83 days from this check ring to the nucleus, but extrapolation of microscopic growth increment widths from the region of the F check ring indicates that day 350 counted from the edge would lie near the most central nucleation site. Feeding was noted on day 100. There was a fainter check ring at about 100 days from the nucleus which may mark first feeding, but this ‘feeding’ check varies in intensity among individuals.

The generality of the relations between check ring location and temperature was demonstrated by counting back from the edge to check rings B, C, D, E and F in five otoliths. The results are shown in Table 1.

The variation in counts was low and may have been due either to counting errors or variation in microscopic growth increment deposition. However, it must be noted that these five otoliths counted were taken from the five best preparations. Other preparations showed clear sequences of rings within which ring counts were stable, for example, the sequence C to D was often found even if check ring B was obscured and within the sequence C

Table I ~~ ~

Check ring

B C D E F ~.

Otolith I 45 68 I34 205 267 Otolith 2 44 M 135 206 267

47 69 134 207 268 Otolith 3 Otolith 4 43 68 I34 20s 267 Otolith 5 44 67 135 206 268

to D there were about 66 rings corresponding to the appropriate number of days. Similarly, check ring B could be located at about 45 days from the edge when all other check rings were obscured. Thus it may be possible, but unlikely, that multiple microscopic growth increments were laid down in some of the otoliths from this experiment.

Some of the check rings that were visible in sections of otoliths (Fig. 12A) and acetate peels could be seen by SEM to have resulted from protrusions of material left after the EDTA etching process (Fig. 12A). The.nature of the protruded material was difficult to judge from the SEM pictures. In some cases the material appeared as if it were a deposit on the surface of the otolith (Fig. 12B); but in other cases had the appearance of etch resistant crystalline ridges (Fig. 12C). Etch resistant material sometimes appeared running parallel to the direction of growth of the crystals of the otolith (Fig. 12A). In the parts of the otoliths that contained the groups of wider microscopic growth increments corresponding to the periods of heightened temperature, EDTA etching often left a series of ridges (Fig. 12C).

A number of triangle-shaped areas (Fig. 13A) sometimes appeared at the ventral edge of juvenile otoliths in optical sections. EDTA etching revealed a series of protrusions at the edges of the triangles when photographed with SEM (Fig. 13B) which were similar to the


figs 12A-C.-A. EDTA etching reveals narrow bands of etch resistant material (open arrows) corresponding to the growth increments seen optically in thin sections. Some of the etch resistant material runs parallel to the direction of crystalline growth (arrow). Scale bar 3 pm.- B. Some sections show etch resistant material lying as a deposit (arrows) on the surface. Scale bar 10 pm.--C. EDTA etching of the sequences of wider daily growth increments corresponding to periods of heightened temperature leaves a series (arrows) of etch resistant ridges. Scale bar 25 pm.

check ring type material seen in other parts of the otolith. Occasionally, vaterite inclusions occur in the otolith itself (Gauldie 1986), not just in the sulcus. SEM studies of etched sections with vaterite inclusions (Fig. 14) showed clear terminal boundaries of etch resistant material that formed a distinct raised protrusion similar to those of the

triangle-shaped areas and the higher temperature microincrements. Otoliths were set whole in epoxy resin and then ground down to the plane of interest making it unlikely that the etch resistant protrusions were contaminants, e.g. glue.

Adult wild New Zealand chinook otoliths have ventral and dorsal growth axes in which microscopic growth increments occurred. Chinook salmon have well-validated annual, opaque zones (Flain 1982). Therefore the number of microscopic growth increment counts between annual zones could be used to establish the period of the microincrements. To avoid the problems of curvature of the principal growth axis in the horizontal plane, vertical sections were made through the nucleus of the otolith aligned along the centres of prismatic growth (Fig. 15A).

At low magnifications, vertical sections have other features which provide useful background to the microscopic growth increment issue. Growth axes below the dorsal and ventral upper surfaces could be clearly seen growing away from the nucleus (Fig. 15B). Opaque and hyaline zones corresponding to those observed in the whole otolith could be seen in section (Fig. 15B). In addition, well- defined check rings could be seen lying between and among the opaque and hyaline zones (Fig. 15B). At higher magnifications the opaque and hyaline zones in the sulcul side of the otolith could be seen as optically dense regions (Fig. 15C), and microscopic growth increments could be seen between those check rings. The check rings appeared to be simply more strongly emphasized (i.e. more optically refractive) microscopic growth increments (Fig. 15C), and, although there was some uncertainty owing to the variation in optical empha- sis common to all microscopic growth increments, there were 13 (range 1&15) apparent check rings between opaque zone and opaque zone in both the growth axis, and between the homologous checkings in the sulcul side of the otolith (Fig. 15B).

Microscopic growth increments occurred as a regular series in both dorsal and ventral segments. The chinook otolith is relatively large (compared with cod Gadus morhua that have similar ages for most of their size range) for its age, particularly in the ventral segment, so that the microscopic growth increments in most of the otolith are also relatively large and hence easily legible in optical preparations (Fig. 16A). The dorsal segment was much smaller than the ventral segment and was, furthermore, recurved at the edge which led to a narrowing of the microscope growth increments and a consequent decrease in legibility. There were about 365 microscopic growth increments that could be regarded as equivalent parts of successive opaque and hyaline zones in the ventral growth axis in a sample of five otoliths. In the dorsal growth axis of the same five otoliths there were about 365 microscopic growth increments between what could be regarded as equivalent parts of successive opaque and hyaline zones, although the width of the microscopic growth increment was less in the dorsal than in the ventral growth axis. The opaque zones in both dorsal and ventral sections were quite wide making it difficult to locate exactly the beginning and end of year (see Figs 15A, B). More precise location of year endings may have shown some variation around the observed 365 days.

172 R. W. Gauldie

Figs 13A, B.-A. Triangle-shaped invasive areas (arrows) sometimes appear in optical sections at the ventral edge of juvenile otoliths-B. Etch resistant material appears in SEM sections as protrusions (arrows) at the edges of invasive areas. Scale bar 19 pm.

rings had changed. As a result, measurements of 10 random pairs of distances between homologous check rings occurring in both the principal growth axis and the sulcus part of the otolith from 10 otoliths were not correlated, ? = 0.2.

The relationship between the microscopic growth increment width and temperature

Fig. 14. SEM studies of etched wavefronts associated with botryoidal vaterite inclusions show terminal boundaries of etch resistant material (arrows). Scale bar 40 p n .

For one otolith the widths of the daily increments were measured along the ventral growth axis so as to include three successive annual opaque zones. The variation in ring width has been plotted in Fig. 17.

Check rings occurred in both the dorsal and ventral growth axes in a way which indicated that they marked a shift in the direction of the growth of the crystals of the otolith (Fig. 16B). The check rings in the growth axes were therefore homologous with those in the sulcul part of the otolith.

The homology of check rings in the growth axes and the sulcul part of the otolith allowed a particular check ring to be traced from the principal growth axis through the sulcul section to the sulcus itself (Fig. 15B). However, the microscopic growth increments of the principal growth axis merge as they pass out of the principal growth axis into the sulcul part of the otolith (Fig. 16B). Thus the check rings observed in the sulcul part of the otolith were homologous with those observed in the principal growth axis, except that the pattern of distances between check

Information about the effects of temperature on microscopic growth increment deposition in different individuals was found in the pattern of microscopic growth increments deposited in the 43-day controlled temperature experiment. This experiment confirmed the temperature effect on microscopic growth increment width, which was shown by taking an average of microscopic growth increment counts from the final 43 days of otolith and plotting the average against the average temperature over that period. The results are shown in Fig. 18 for a random sub-sample of otoliths from different temperatures. There was a linear relationship between average microscopic growth increment width and temperature with a correlation coefficient r = 0.76, i.e. about 58% of the variance in microscopic growth increment width is due to temperature. The relationship between average microscopic growth increment width and temperature is given by Width = 1.19 + 0.085 Temperature. The low value of the intercept (1.19) may not have any biological significance. The variance of the slope (0.085) is quite high, 0.19, resulting in a coefficient of variation of 20.9%. However, the change in average microscopic growth increment width due to temperature was much lower than the change in body size during the 43-day controlled temperature experiment. The average body size in the highest temperature group was 160 g, an increase of 300% over the average body size in the lowest temperature group. The average microscopic growth increment width in the lowest temperature group was 1.70 Fm and the average microscopic growth increment width in the highest temperature group was 2.5 p.m, an increase of 41%.


Fig. 15A. Whole adult chinook salmon show opaque zones. and prisms (open arrows) when observed by transmitted light. The marks on the otolith indicate the dorsal (D) and ventral (V) axes along which the otolith was cut. The otolith is shown from the upper surface with anterior ( A ) and posterior ( P ) ends marked. Scale bar 1.3 pm.

-

Fig. I5B. Sections of adult chinook salmon when observed by transmitted light show opaque zones (0) which carry through from the growth axes to the sulcus, as well as check rings (arrows) lying between the opaque and hyaline zones of the sulcus. The dorsal (D) and ventral (V) growth axes are characterized by opaque (by transmitted light) material with regular check rings (crtrved arrows) which are homologous to some of the check rings in the sulcus part of the otolith. Scale bar 0 .S mm.

174 R. W. Gauldie

Fig. 15C. At higher magnifications opaque zones (0) can be seen as optically dense regions. Check rings (open arrows) appear to be simply more emphasized (i.e. more refractive) microscopic growth increments between which microscopic growth increments (arrows) appear.

Discussion

SEM studies showed that there was a marked dissimilarity between the crystalline topographies of the upper and lower surfaces of the otolith. The upper surface had a stepped or scale-like appearance with nodules of secondary growth crystals. The lower surface was flatter, without the radiating sculpture, but showed a range of crystal habits that were generally coarse at the edge of the otolith and at the sulcus, but smoother in between. The significance of the range of crystal habits, the various nodules and crystalline textures (in Figs 3 and 4) can be explained as differences of growth rates between crystals in various parts of the otolith resulting in finer or coarser crystals in various parts of the otolith depending on whether they are growing more, or less, rapidly (de Jong 1959). The significance of pits observed in Fig. 4 are unknown. They are too large to be associated with the kinocilia of the macula that are almost two orders of magnitude smaller. However, the surface sculpturing introduces banding patterns similar in appearance to opaque and hyaline zones caused by refractive changes in both transmitted and reflected light examination of whole otoliths. -The refractive changes are caused by steps in the surface of the otolith (Davies el al. 1988; Gauldie 19886), that is, by virtue of its crystal calcium carbonate composition, strongly optically refractive. A further problem in interpretation arises from the relation between the stepped surface structure and some of the internal check rings (Gauldie 19886).

Fig. 16A. Microscopic growth increments (arrows) in the ventral growth axis of vertical sections of the adult otolith. Microscopic growth increments merge (open a r r o w ) as they pass out of the growth axis into the sulcus part of the otolith. The sulcus part of the otolith (S) contains many structures similar to microscope growth increments which persist in spite of the merging process. Variation in optical density of the microscopic growth increments in the sulcus part of the otolith results in the persistence of opaque zones (0). Scale bar IOU b m .

The 330 pm wide zones in Fig. 3A cannot be explained. There are about nine faintly discernible zones in Fig. 3A, which means that they cannot be annual zones because chinook salmon do not grow to that age. The average width of microscopic growth increments in the segment containing the zones is about 2.5 pm, which gives them a probable period of about 130 days. There is no evidence in the literature to support a significant


Fig. i6B. Crystals in the growth axis of the chinook salmon otolith (open arrows) indicate a shift in the direction (arrows) of otolith growth occurring at check rings ( C ) at both the anti-sulcus ( A ) and sulcus (S) pans of the otolith.

biological cycle in salmonids with an approximately 130- day cycle. Other zones, or scale-like stepped structures were observed on the upper ventral surface with an average width of 8 pm. The process of otolith growth is not well known, but there are good reasons to expect that otolith growth is controlled to give otoliths a certain size and shape. Controlled growth would imply changes in the direction of crystal growth at fairly short intervals which could be expected to translate into a stepped, or zoned, structure. This would also account for the zoned appearance of the pits and hollows of the crystal masses appear- ing in the sulcus (Figs 8D, E).

The dorsal and ventral parts of the otolith are of a similar size in the juvenile otolith, but the ventral segment becomes much larger (by about 4 times) in the adult. The steps, or scales, on the ventral surface are about four times the width of the daily growth increments in the dorsal part of the otolith and may be equivalent, but appropriately wider, growth increments to daily growth increments elsewhere.

However, it can be seen that the lower surface is smoother and more homogeneous than the upper surface. This would be consistent with the idea of otolith growth between the sulcus and the growing tip being controlled by the macula (Gauldie & Nelson 1988), while the upper

0

F 1

2 12

200 400

I

I I :

600 800 1000

number of micminaements

Fig. 17. Daily growth increment is plotted against distance along the ventral growth axis which included three successive annual opaque zones (pecked lines). and 13 peaks in daily growth ring width (arrowed) between the major peaks of the annual zones.

8 8 10 12 14 1%

Treatment temperature ("C)

Fig. 18. Average widths of increments from within the part of the otolith corresponding to the temperature treatment experiment are plotted against treatment temperatures.

surface could be subject to uncontrolled precipitation out of the endolymphatic fluid. However, there are differences in crystal texture in various parts of the lower surface indicating some variability in the control of crystal growth. For example, pits similar to those in Fig. 3B appear also on the lower ventral surface of the otolith of Fundulus heteroclitus (Dunkelberger er al. 1980). The pits .

176 R. W. Gauldie

are about 60 pm wide and their role is unknown. Their presence, along with that of the hollows on the lower surface (Figs 3D, F) and the appearance of coarse crystals (Fig. 3E) at the edge of the sulcus indicate local variations in crystal growth rate on the ventral lower surface. In addition, a common quality of all the features of the lower surface is that the crystals grow at right angles to the macula, but with a gradually decreasing angle until they reach the edge of the otolith where the crystals are in the same plane as those on the upper surface.

The pits on the lower ventral surface (Figs 4A-C) and their associated crystal types (Figs 4D, E) draw attention to a new problem in otolith growth. The ventral lower surface is clearly under some kind of constraint or control which causes it to grow in a characteristic fashion. The macula, which contains the most obvious cellular organization of the endolymphatic sac, is confined to the sulcus. If there is a protein-based control mechanism for the arrangement of pits (which seems the most likely option) then the protein must either diffuse between the otolith and the sac, or must be produced by hitherto undiscov- ered cells associated with the sac. The arrangement of pits and intervening crystals shown in Figs 4A-C may provide a vital clue to one of the most puzzling features of otoliths, the incomplete penetration of the otolith by check rings.

The chinook salmon otolith when ground down into a single plane (particularly the horizontal plane) shows a number of clear check rings which apparently impede the growth of the otolith (examples are in Figs 9 and 10). Continued grinding down of the otolith in that plane often causes check rings to disappear and reappear in successive grinding planes. Similar effects have been observed in the snapper otolith (Gauldie 1988~). The implication is that some, at least, of the check rings in the otolith are discontinuous. The pits in Figs 4A-C show that, in one plane, the crystalline growth of the otolith is discontinuous in patches. Check rings, however, are laid down at the growing edge of the otolith. It is possible that the grooves at the edge of the otolith (Fig. 3B) are a generalized form of the same crystalline effect as the pits resulting in check rings at the growing edge of the otolith which range from small patches of discontinuities to virtually complete discontinuities that could be properly referred to as check rings.

Check rings in this context refer to optical or SEM discontinuities either before or after etching, i.e. they refer to an identifiable structure within the architecture of the otolith. The older use of the term check ring referred to a mark in the annuli of scales or otoliths that did not fall into the appropriate pattern of annual marks (usually in the form of opaque and hyaline zones) con- sidered appropriate for the species (Hickling 1933). Some check rings in the chinook otolith may also have their origins in the structural discontinuities which appear to be associated with spalling lines, but in optical sections it is not possible to distinguish one structural type of check ring from another. Consequently reference to the term check ring can be read only in context.

The lower dorsal surface shows a quite different crystalline habit to that of the lower ventral surface. Apart from the edge of the sulcus the crystals appear to be growing

towards the edge of the otolith forming into intersecting blocks, sometimes aggregating into hexagons before assuming a smooth appearance at the edge of the otolith. The different crystal habits of the dorsal and ventral lower processes indicate different rates of growth, different directions of growth, and hence different growth control processes, presumably different proteins.

The sulcus of the otolith presents, by its very nature, the part of the otolith in which crystalline growth has been most severely inhibited. One of the features of the sulcus is that the crystals lining the wall of the sulcus are at right angles to the macula surface. However, behind the layer of vertical crystals, the normal growth of the medial twin crystal continues, that is, parallel to the wall of the sulcus (Fig. 6A). There is evidently some constraint, or some signal that must be associated with the kinocilia-bearing cells of the macula, which lies within the sulcus, which serves to orient aragonite crystals at right angles to the source of the signal. If this supposition were correct, then such a signal, diffusing away from the macula, would be responsible for the re-orientation of the medial twin of the radial crystals of the central growth axis, thereby determining the shape of the otolith. Further weight is added to this supposition by similarity of the crystals lining the sulcus and those associated with pits on the lower ventral surface. That signals of some kind originate in the sulcus can be argued from the presence of vaterite in the sulcus itself.

Vaterite deposition (instead of aragonite) is due to the presence of glutamic acid or aspartic acid (Kitano & Hood 1965) either in solution or as residues on the matrix protein. In the chinook salmon otolith the availability of such amino acids may be partly controlled by temperature and partly under genetic control (Gauldie 1986). The presence of vaterite in the sulcus of otherwise aragonitic otoliths indicates a source of glutamic acid or aspartic acid in the macula, a source which fluctuates regularly to give a layered appearance to the vaterite deposits in the sulcus. If the source were not in the macula, vaterite would be deposited at the edges, not the sulcus, of the otolith.

The crystalline topography of the chinook salmon otolith shows that it is a complex structure consisting of different sections composed of crystals with different habits. The implications are that there are different processes occurring in the mineralization of the upper and lower surfaces, as well as the dorsal and ventral segments, and within the sulcus itself, processes which are characterized by apparent differences in growth rate between the upper (faster growing, finer texture) and lower (slower growing, coarser texture) growth axes. Further differences in mineral morph also occur, resulting in vaterite crystals growing in the sulcus of the otolith. One can conclude from the qualitative data presented here on otolith morphology that there is variation in growth rate in different parts of the chinook salmon otolith which is related to the processes controlling size and shape of the otolith which results in variation in crystal size, and hence otolith appearance. The chinook salmon otolith appears to be a veritable mosaic of crystalline structures each storing a time-dependent coded message about the growth history of various sectors of the otolith.

Otolith of the Chinook Salmon 177

The pattern of mineralization supplies the information that the biologist uses to recognize time-dependent structures (e.g. annual opaque and hyaline zones, annual check rings and daily microincrements). Therefore, the complex mineralization patterns of the chinook salmon otolith require the biologist to assess which growth axes of the otolith have the appropriate crystalline structure, and the continuity of growth, necessary for the resolution of the pattern of interest before the search for such patterns and the study of their periodicities, can begin. In the chinook salmon, examination of the internal architecture reveals two growth axes, dorsal and ventral, along which continuous daily deposition of crystalline material occurs. The choice of axis depends on the size of the fish because the dorsal axis is longer in older fish. In addition, in other species it has been shown (Gauldie & Nelson 1990) that larger otoliths may rest on the bottom of the otic cleft resulting in interruption of growth of the otolith. The dorsal growth axis is free of this effect and must therefore be the axis of choice in ageing chinook salmon.

Broken sections of wholly aragonitic otoliths revealed epitaxial growth (Degens 1976) along axes in both the dorsal and ventral segments. These axes run from the nucleus more or less orthogonally to the plane of the sulcus, slightly below the upper surface of the otolith. The qualitative differences in growth rate of otolith crystals, indicated by their crystalline morphology, is supported by direct measurements of the twinned epitaxial crystal which occurs in both the principal growth axes and sulcul part of the otolith. The crystals of the principal growth axis grow 2-3 times faster than their twins in the sulcul part of the otolith. Ground sections showed that other parts of the otolith (particularly the rostrums of the otolith associated with the sulcus) have a complex pattern of growth-complex in the sense that the pattern gives the appearance of folded structures within which continuous linear epitaxial growth was unlikely to have occurred. One of the difficulties associated with ageing otoliths from daily growth increments is the possibility that growth increment deposition may become intermittent, particularly in older fish (Ralston & Miyamoto 1983). The folded check ring structure in the chinook salmon otolith indicates that it is possible for the growth increment deposition in certain parts of the otolith to be interrupted, but also that it is clear from the architecture of the otolith when such interruption has occurred.

Other kinds of growth interruptions also occur in the chinook otolith. The sutures between prisms are obviously boundaries along which growth does not occur. However, etching shows ridges of edge-resistant material in the axes in which growth does occur which is similar in appearance to the resistant material located in the sutures. One can conclude from such observations that it is as difficult to establish an ultrastructural criterion to indicate that otolith growth has stopped, as it is to find an ultrastructural criterion to separate annual check rings from accessory check rings in other species (Gauldie 1988~).

The growth axes of the chinook salmon can be demonstrated in both the horizontal and vertical planes. In the juvenile chinook salmon the growth axis in the dorsal segment is most clearly demonstrated in the horizontal plane. The growth axis in the dorsal segment of the

juvenile chinook salmon shows a clear sequence of microscopic growth increments. Although there was some ambiguity associated with the number of growth increments laid down in the early development of the primordium of otolith, the discrepancy (9 out of an expected 348) is very low. It is therefore reasonable to assume that the microscopic growth increments are laid down every day. Further supporting evidence comes from the correspondence between days elapsed and temperature-dependent markers which appear in the otolith.

Vertical sections of the otoliths of adult chinook show that the opaque zones visible in the whole otolith persist in the sulcul part of the otolith. The opaque zones have been shown to be annual (i.e. winter) zones in the chinook, chum and sockeye otoliths so that the period of structures, particularly microscopic growth increments and check rings, can be calibrated from the opaque zones. By counting microscopic growth increments between successive opaque zones, it was shown that there were about 365 microscopic growth increments deposited each year, thereby confirming the results of the rearing experiment. In addition, it was shown that about 13 check rings in the sulcus of chinook salmon otoliths have a lunar period. The period of check rings is to some extent qualified since there is (at high magnification) subjectivity in defining a check ring in the sulcus part of the otolith. However, about 13 minor peaks in daily growth increment width between major annual peaks were observed in Fig. 15B.

The check rings of the principal growth axis can be followed into the sulcus part of the otolith. However, the pattern of homologous check rings changes between the principal growth axis and the sulcus part of the otolith. The correlation coefficient of the regression of the distance between check rings in the principal growth axis and the distance between homologous check rings in the sulcus part of the otolith is low, indicating that wide differences in interpretation of check ring patterns are possible depending on where in the otolith they are read, and what significance is attached to the pattern of check rings.

Part of the processes of pattern change in check ring disposition involves changes in the numbers of microscopic growth increments which appear to have merged together as they proceeded from the principal growth axis into the sulcus part of the otolith. The merging process may be due to the difference in growth rate between the twinned crystals growing in the principal growth axis and sulcus part of the otolith. Thus, the slower-growing sulcul part of the otolith loses faster-growing components (such as daily microincrements) but still records slower-growing components such as the lunar check rings. A similar effect has been observed in the orange roughy otolith (Gauldie 1990). Given the consistent lunar rhythms observed in both sulcul and growth axis check rings, and daily growth increment widths, one might expect similar effects among the check rings commonly observed in the sulcul side of the otoliths of many, if not all, species.

Multiple (i.e. non-daily) growth increments are reported in chinook salmon otoliths by Neilson & Geen (1982). The fish in this experiment were maintained at fixed 24-h periodicities cued by natural light and were not subject to physiological perturbations caused by changing

178 R. W. Gauldie

the periodicity of environmental patterns. It is not unreasonable to expect that the various physiological stresses associated with perturbed periodicities may perturb the normal processes of growth increment deposition. However, chinook salmon growth, unlike coho salmon growth, has been shown to be unaffected by arti- ficial photoperiod cycles (Clarke et al. 1981), which implies that the cueing mechanism for chinook salmon otolith increments is independent of the growth rate of both the fish and the otolith (whose size is strongly correlated to that of the fish). Studies of calcium mobilization (Mugiya 1987) and neuropeptide control (Gauldie & Nel- son 1988) of otolith deposition both imply a strong central nervous system component that can be disentrained by readjusting environmental cues, or by physiological stress. But the range of conditions experienced by fish in this experiment were not intended to subject the fish to physiological perturbation, and therefore the data reported here, consistent daily increments, can be regarded as representative of the otolith response in the absence of stress. Multiple rings due to optical persistence during changes in gaussian focus (Gauldie & Nelson 1988) can be tested simply by grinding preparations to the point of extinction. Such preparations do not show multiple rings in chinook otoliths.

The temperature fluctuations recorded over the life of the juvenile chinook salmon were correlated with the width of daily growth increments. About 58% of the variance in increment width of experimental fish could be accounted for statistically by temperature. Recalculation from the graphs of scale increments of different temperatures for the cod (Dannevig 1933) shows also that about half the variation in scale increment could be accounted for by temperature. The temperature treatment experiment coming at the end of the life of the juvenile salmon confirms the correlation between temperature and the width of the daily growth increment. However, if the increase in microincrement width were due to simple temperature effects on the chemical crystallization rate, one would expect that there would be at least a doubling of ring width with every 10°C rise in temperature, the well-known QlO rate (Netter 1969). However, the average increase in width from 6" to 16°C observed was about 50%. It is unlikely, therefore, that the change in daily microincrement width is a simple temperature effect. I t is more likely that the change in daily microincrement width is a biological effect tied to the increase in growth rate of the fish which occurred over the same temperature range. A similac relationship between otolith ring width and growth rate has been reported in other species of the genus Oncorhynchus (Wilson & Larkin 1982; Volk et al. 1983), and between scale incremental growth and fish growth rate in the cod (Dannevig 1933). However, the relative increase in width over the range of experimental temperatures was much less than the relative change in body weight of fish in the same experiment. The effects of temperature on the width of daily microincrements parallels the observation of Radtke et al. (1985) that opaque and hyaline zones arise from seasonal differences in the width of daily microincrements in the otolith. Chin- ook salmon fry were reared under cycling temperature regimes by Neilson & Geen (1985). They showed that

mean increment width 'reflected fish growth under a var- iety of water temperature and ration regimes' on the basis of statistical analysis of their temperature cycling data. Their data do not allow comparison with the direct effect of temperature presented here. Unfortunately the fry used in the Neilson & Geen (1985) experiment were subject to chlorine stress that caused significant mortali- ties (Neilson & Geen 1985; p. 93, paragraph 3) so that direct comparison of data may not be possible.

The chinook salmon otolith has a number of features of importance to the general problem of ageing fish from otoliths. Variation in crystal morph and habit shows that there are qualitative differences in the morphology of different parts of the chinook salmon otolith which can be explained as differences in growth rate in different parts of the otolith. Differences in growth rate lead to the principal growth axes being 2-2.5 times the width of the otolith between the anti-sulcul and sulcul surfaces of the otolith. Differences in growth rate lead to a slower deposition rate in the sulcul part of the otolith which results in a loss of faster transient information (such as daily microincrement deposition) but the retention of slower transient information (such as check rings) in the sulcul part of the otolith. The incomplete record of the sulcul part of the otolith could result in a misinterpretation of the nature and period of check rings if they were treated in isolation from the information stored in the principal growth axis.

The otolith of the chinook salmon can be used to test the relationship between the variation in morphology in different growth fields of otoliths, and the periodic structures generated by that variation in morphology-a relationship which illustrates the way in which variation in growth rates lead to the qualitative variation in crystal structure. The known age of salmon otoliths confirms that there is continuous growth in some axes of the otolith, but that growth information is censored by differential growth rates between different parts of the otolith. This kind of censorship imposed by morphological constraints limits the use of ageing techniques based on both microincrements and opaque and hyaline zones to continuous growth axes in the otolith, particularly the dorsal growth axis in otoliths of adult fish.

Acknowledgements

The experiments leading to this paper were made possible by the generous material and financial assistance provided by Canada Fisheries and Oceans. I am particularly indebted to Mr A . S. Wood, Dr R. Brett, Mrs Rose Curtis of the Pacific Biological Station. Nanaimo, and the technical staff at the Rosewall Creek Hatchery, Vancouver Island. Samples of New Zealand chinook otoliths were provided by Mr M. Flain and Dr Peter Todd of the Freshwater Fisheries Centre, Christ- church, New Zealand.

Scanning electron microscopy was carried out at the SEM unit. Vic- toria University of Wellington. Statistical and technical assistance was provided by Ian West and Ms Dianne Dunlop, K. P. Mulligan and R. Thomson. Some of the research leading to this paper was carried out at the MAFFish Fisheries Research Centre, P. 0. Box 297 Wellington, New Zealand. Photographs were printed by Alan Blacklock of MAFF- ish. The paper was completed in the Hawaii Institute of Geophysics, School of Ocean, Earth Sciences and Technology, University of Hawaii, Honolulu, with support from the NSF grants OCE-88-00686 and INT- 87-23084 made to Richard Radtke while the author was Visiting Ichthy-


ologist at the Hawaii Institute of Geophysics. SOEST contribution number 2461.

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Documents

The Morphology and Periodic Structures of the Otolith of the Chinook Salmon (Oncorhynchus tshawytscha), and Temperature-dependent Variation in Otolith Microscopic Growth Increment