Morales Nin 2000

Review of the growth regulation processes of

otolith daily increment formation

Beatriz Morales-Nin*

Instituto MediterraÂneo de Estudios Avanzados (CSIC/UIB), Miguel, Marques 21, 07190 Esporles, Mallorca, Spain

Abstract

Otolith growth is a complex phenomenon integrating various factors that can be considered either as endogenous or

exogenous, although they are always regulated by the physiology of the ®sh. Both types of factors may operate upon the

anabolism and catabolism of the ®sh, and these processes are re¯ected in the rhythmic deposition of the two main constituents

of the otolith: the organic matrix and the aragonite layers. Both components re¯ect the endogenous process in their periodicity,

and the exogenous process in the amount of material laid down in the otolith, resulting in how and where the increments are

formed.

At the endogenous level, several morphological and functional aspects are relevant. The main one is the role of the otolith

as a mechano-receptor in the inner ear. Thus, inner-ear anatomy and function regulate otolith growth and morphology, which

in turn determine the daily growth rates. Environmental conditions, transmitted through the physiology of ®sh, affect the

otolith growth rate (increment width) but increment periodicity may be disrupted only in extreme cases of physiological stress.

The current state of the art is reviewed and the otolith growth paradigm is summarized. Relevant subjects not yet studied are

pointed out for future research. # 2000 Elsevier Science B.V. All rights reserved.

Keywords: Fish; Biomineralization; Otolith growth

1. Introduction

Otolith formation involves rhythmic variations in

the deposition and size of organic matrix ®bres and

carbonate crystals, resulting in the formation of

macroscopic translucent and opaque rings and micro-

scopic zonations (growth increments). For these

structures to be of use in age estimation, they must

be regulated by an endogenous rhythm linked to a

periodic environmental cycle or synchronized to

periodic events.

Translucent and opaque rings were considered as

time signals and ®rst used to determine age of ®sh in

the 19th century (Reibisch, 1899). The presence in

otoliths of structures with a periodicity lower than

seasonal was ®rst described in hake otoliths by Hick-

ling (1933), while Pannella (1971) was the ®rst to

describe the presence of increments formed with daily

periodicity, as inferred by counting the number of

increments and contrasting them with the age esti-

mated by the number of macroscopic zonations. He

also showed how small variations in increment width

Fisheries Research 46 (2000) 53±67

* Tel.: �34-97-161-1721; fax: �34-97-161-1761.

E-mail address: ieabmnqps.uib.es (B. Morales-Nin)

0165-7836/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 5 - 7 8 3 6 ( 0 0 ) 0 0 1 3 3 - 8

and appearance resulted in the formation of macro-

structures.

The general pattern of microincremental growth

found in many plants and organisms has been proved

to be daily for many species and habitats, and is a

general phenomenon thought to occur in most ®sh

(Campana and Neilson, 1985; Gauldie, 1991). Only

the most severe stress seems to alter the daily nature of

the increments (Mugiya and Uchimura, 1989). How-

ever, without a knowledge of the nature of the endo-

genous regulator it is not possible to predict how

environmental conditions are liable to in¯uence incre-

ment periodicity.

The complex physiology of otolith growth and

formation has been less studied, although several

authors have analysed the otolith constituents

(CarlstroÈm, 1963; Degens et al., 1969; Mugiya,

1974; Mugiya and Takahashi, 1985; Dunkelberger

et al., 1980; Gauldie, 1993; Morales-Nin, 1986a,b;

Gauldie and Xhie, 1995). The ®rst otolith growth

model in ®sh based on the chemistry of the endolymph

has appeared recently (Romanek and Gauldie, 1996).

This evolution from description to modelling using

different approaches is very promising and should

include in the future the interactions between the

organic matrix and the aragonite, the 3D otolith

growth (Bailey et al., 1995), and the sacculus bio-

chemistry.

In this contribution, the complex otolith growth is

reviewed and described by considering both endogen-

ous factors, such as the otolith's function in the inner

ear of ®sh, and exogenous factors, such as light, water

temperature, or food intake. Finally, the implications

of otolith growth are discussed and future relevant

questions are highlighted.

2. Endogenous otolith growth regulation

Otoliths act as mechano-electrical sound and dis-

placement transducers that convert shear forces into

electrical impulses by distorting the hair cells of the

nervous endorgan or macula (Fig. 1).

Fish otoliths are composed of calcium carbonate

and a keratin-like protein (Degens et al., 1969) laid

down following the endogenous diel rhythms in cal-

cium metabolism (Mugiya, 1987) and of neuropeptide

secretion at the inner ear (Gauldie and Nelson, 1988).

The otolith is in contact with a sensory epithelium

(macula) that is usually attached to the sulcus of the

otolith (Fig. 2) (Dunkelberger et al., 1980; Fay and

Popper, 1980; Platt and Popper, 1981). In some spe-

cies, otoconia appear on the membranous protein

between the sagitta and the haircell bundles in the

macular area (Dale, 1976). Their possible role as

sound transducers or as a source of calcium for sagittal

Fig. 1. Drawing of a left Merluccius spp. inner ear showing the three otic chambers (L: lagena, U: Utriculus, S: sacculus), otoliths (as:

asteriscus, lp: lapillus, sg: sagitta) and the sensory maculae ( ml: macula lagena, mu: macula utriculi, ms: macula sacculi). The semi-circular

channels (Sc) and their ampulla (am) are also included. The innervation to the macula sacculi is indicated by a thicker arrow. Drawn by A.

Lombarte (CSIC).

54 B. Morales-Nin / Fisheries Research 46 (2000) 53±67

growth is unknown. The calcium carbonate is in the

form of twinned aragonite, although abnormal crystal-

line otoliths are composed of calcite (Morales-Nin,

1985) or vaterite (Gauldie, 1986). Twinning is a

complex process (Bloss, 1971) which stabilizes crystal

polymorphisms and increases the growth rate of the

crystal (Smith, 1974; Davey et al., 1993).

The labyrinth of ®sh is involved in the maintenance

of equilibrium and has nervous cells sensitive to

pressure, movement, and sound vibrations (Lowen-

stein, 1971). The membranous labyrinth of ®sh con-

sists of three connecting epithelial chambers, each

containing one otolith (Fig. 1). These chambers also

communicate with the three semi-circular canals. The

lumen of the entire system is ®lled with endolymph,

which is similar to plasma (Enger, 1964). Fish endo-

lymph has a high sodium concentration (110±140 nM;

Enger, 1964), although the saccular endolymph in ®sh

is more alkaline than plasma (Mugiya and Takahashi,

1985). In teleosts, a saccular potential of about

�10 mV has been reported (Enger, 1964). This sug-

gests that in all cases, energy-dependent mechanisms

maintain the K� concentration of the endolymph.

Another feature of ®sh saccular endolymph is its high

anhydride carbonate content (Mugiya and Takahashi,

1985). Biomineralization by formation of calcium

carbonate in the otolith shifts the carbonic acid equi-

librium towards acid values (Cameron, 1990). The

observation that endolymph ¯uid is more alkaline than

plasma suggests that H� ions are pumped out by the

epithelium and that this ion is not in electrochemical

equilibrium. Thus, at least for K� and H�, energy-

dependent mechanisms appear to maintain gradients

between the plasma and endolymph (Mayer-Gostan

et al., 1997).

The endolymphatic and otic sac ¯uids are bicarbo-

nate buffered, but teleosts are not good pH regulators,

so that increased H� ion concentrations resulting from

activity (lactic acid load) are soon transported into the

perilymph and endolymph. Temperature also controls

H� ion availability in bicarbonate-buffered systems.

The pH of the endolymph and temperature are there-

fore an important control of otolith growth (Romanek

and Gauldie, 1996).

The otolith is precipitated from the ¯uid of the

endolymphatic sac of the inner ear, with the chemical

composition of the ¯uid being tightly determined by

the function of the otolith itself (Gauldie et al., 1995).

In order for calcium carbonate to form, calcium as

Ca2� must be available at the site of nucleation.

Calcium ions are a necessary counterpart to the uni-

valent cations in the neuromuscular excitation pro-

cess. The sensitivity of the whole neuromuscular

system depends on the concentration of Ca2�, since

an increase in concentration inhibits sensitivity and a

decrease stimulates it. Calcium reaches the endo-

lymph primarily from the blood plasma (Kalish,

1989, 1991; Wright et al., 1992). The concentration

of Ca2� in the sacculus ¯uid is below the point of

super-saturation and there are changes in the move-

ment of calcium ions during the process of otolith

growth (Mugiya, 1966, 1987). Seasonal variation in

free Ca2� ions ranges from 65.4% of total calcium

levels during fast growth to 79.1% during slow growth

(Mugiya, 1966), which probably represents the range

over which Ca2� can vary without physiological dys-

function of the neural mechanisms of the macula

(Gauldie and Nelson, 1990). In addition, neurosecre-

tory activity in the macula has a daily cycle related to

the deposition of daily microincrements (Gauldie and

Nelson, 1988).

The otic ¯uid is regulated by ATP-driven membra-

nous pumps that regulate the biochemistry of both the

endolymphatic ¯uid and the surrounding perilympha-

tic ¯uid (Rauch, 1963). A calcium-binding protein

may not be involved in the Ca transport into the

Fig. 2. Scanning electron microphotograph of the rostral area of

the macula in Merluccius capensis showing the kinocilia. Scale bar

5 mm. Photo by A. Lombarte (CSIC).

B. Morales-Nin / Fisheries Research 46 (2000) 53±67 55

endolymph; a paracellular transport of Ca across the

macula has been demonstrated (Kalish, 1991).

Protein is released from the cells of the macula and

taken up onto the otolith (Watabe et al., 1982; Gauldie

and Nelson, 1988; Zhang, 1992). The distribution of

matrix material appears to occur in two phases. The

®rst phase is in association with the twinning plane of

the basic aragonite crystal (Gauldie and Xhie, 1995).

The second phase forms a dense band of ®bres corre-

sponding in size and orientation to the narrow dis-

continuous unit of the daily microincrement (Fig. 3)

(Dunkelberger et al., 1980; Morales-Nin, 1986b). This

phase may re¯ect the diel change in protein concen-

tration in the macula (Gauldie and Nelson, 1988) that

results in the daily insertion of a matrix signal into the

otolith structure. The otolith protein acts as a signi®-

cant component of the mechanism controlling the

shape of the otolith (Degens et al., 1969; Dunkelberger

et al., 1980; Mugiya, 1987; Gauldie, 1991, 1993;

Zhang, 1992).

Otolith calci®cation is limited by the number of

nucleation sites provided by the matrix (Crenshaw,

1982; Mann et al., 1989), as well as physico-chemical

conditions at the otolith surface. Therefore, the rate of

matrix production by the matrix-producing cells of the

sacculus (Saitoh and Yamada, 1989; Wright, 1991)

will ultimately determine the rate of otolith calci®ca-

tion. Further, the soluble matrix of salmon otoliths

contains a glycoprotein capable of inhibiting calci®-

cation in vitro (Wright, 1991). Consequently, the

matrix is involved in the periodic deposition of

mineral and matrix-rich layers. This role should be

investigated in the future.

The otolith matrix is composed of a collagen-type

protein (Degens et al., 1969). The matrix is more

dense in the early development phase, and its ami-

noacidic composition changes in relation to age (Mor-

ales-Nin, 1986a,b). Although the matrix protein has a

high af®nity for calcium carbonate, it does not seem to

affect the growth rates of the aragonite (Gauldie,

1990). However, the presence of more-alkaline amino

acids in young-of-the-year hake and sea bass was

related to a more dense crystalline structure in the

nuclear area (Morales-Nin 1986a,b).

Phase differences in the secretion rate of calcium

and protein result in the daily microincrements

(Fig. 4). These are composed of two units: an incre-

mental unit (L-unit, from its lighter aspect under the

light microscope), rich in aragonite acicular micro-

crystals in a three-dimensional organic matrix; and a

discontinuous unit (D-unit, from its darker aspect

under the light microscope), in which the organic

®bres predominate, thereby forming a discontinuity

(Fig. 3) (Dunkelberger et al., 1980; Morales-Nin,

1987b; Mugiya, 1987; inter alia). The precipitation

Fig. 3. Scanning electron microphotography showing the organic

matrix of a demineralized sagittal section from a Dicentrarchus

labrax sagittal otolith. The thick fibers forming layers (arrows)

correspond to the D-units of the daily growth increment. Scale bar

3 mm.

Fig. 4. Light microscope microphotography of the daily growth

increments (arrows) in a sagittal section of a Merluccius merluccius

otolith. Note the rhythmical growth patterns (line marks) resulting

from small changes in increment width and translucency. Scale bar

100 mm.


of material from the sacculus ¯uid is controlled by

changes in the pH (Wright et al., 1992) under hormo-

nal control (Mugiya, 1986, 1990; Mugiya and

Yoshida, 1995).

The mechanism of otolith growth is very unusual

amongst biomineral tissues, in that, most of the oto-

liths does not grow in contact with any cellular tissue.

Otoliths are joined by a protein matrix (the otic

membrane) to the cells of the macula along the sulcal

groove of the otolith (Dunkelberger et al., 1980). The

cells of the macula are the source of both the calcium

ions and the proteins that constitute the otolith

(Mugiya, 1965, 1966, 1987; Gauldie and Nelson,

1988). However, recent studies have shown that the

saccular tissue has ionocites in a dense mesh network

around, but separated from, the macula and also in a

second area opposite the macula, with smaller, less

dense patches (Mayer-Gostan et al., 1997). These

results suggest that not all the otolith crystallisation

is regulated by the macula as the only source of both

calcium ions and proteins, although, its modulatory

role is probably predominant. Thus, otolith crystal-

lisation is produced, except in the area close to the

macula, out of the ¯uids in the endolymphatic sac,

which are alkaline bicarbonate buffers with free cal-

cium ion levels of �10 mM (Mugiya, 1966).

Little is known of the physiological regulatory

mechanisms controlling increment formation. How-

ever, a hormonal involvement has been implicated,

since hypophysectomy has been found to cause a

reduction in otolith growth (Mugiya, 1990) or otolith

demineralisation (Simmons, 1971), and otolith miner-

alisation in hypophysectomised ®sh can be restored by

injection of pituitary extract (Simmons, 1971). Wright

et al. (1992) suggested that as plasma calcium con-

centration is regulated by hyper- and hypo-calcemic

hormones, diel changes in the plasma concentration of

these hormones may be indirectly responsible for the

periodic decline in otolith calci®cation. Moreover,

Wright et al. (1992) induced hypocalcemia in Salmo

salar parr and showed a short-term net loss of calcium

from the otolith. Carbonate crystallisation in molluscs

involves neural control (Zylstra et al., 1978). Neural

control of calcium concentration in the sacculus is a

physiological explanation of the direct tracking of

seasonal and daily total calcium levels of the blood

plasma by the endolymph (Mugiya and Yoshida,

1995).

3. Otolith shape and microstructure

Otoliths are mechano-electrical transducers in the

inner ear of ®sh (Fay, 1980; Schuijf, 1981) acting as

statoliths through the mediation of the sensorial

macula and kinocilia (Popper, 1976; Lombarte and

Popper, 1994). The particular sound frequencies to

which the otolith responds, as a transducer, depends on

the shape of the otolith (Gauldie, 1988). This implies

that otolith shape, and a certain proportion between

the otolith shape and the sensory area, have to be

maintained throughout the life span (Lombarte and

Popper, 1994). Otolith shape is often complex and is

species-speci®c (Nolf, 1985) and genus-speci®c (Gae-

mers, 1984). Lombarte and CastelloÂn (1991) showed

how the otolith shape is regulated by the species and to

a lesser degree by environmental factors. Otolith

shape might be controlled by several factors, such

as the shape of the otic capsule and the cranium, and

the checks and discontinuities in otolith growth con-

trolled by the macula (Gauldie, 1988; Lombarte and

Morales-Nin, 1995). The relationships between the

shapes of the macula, sulcus acusticus, and otolith, and

their relationships to environmental conditions have

been recently studied in Merluccius (Torres et al.,

2000). The otolith shape is controlled by the position

and relative size of the seasonal translucent rings, as

shown in Merluccius, where the species can be deter-

mined by the translucent ring's radius, with better

results when more rings are considered (Torres, 1997).

Otolith formation starts with a primordium, which

is generally the ®rst calci®ed tissue in the embryo. The

nucleus is formed when the ®rst discontinuous unit

(Dunkelberger et al., 1980) is laid down. This gen-

erally corresponds to hatching, ®rst feeding, or start of

activity (Brothers and McFarland, 1981; Morales-Nin,

1992), although some species with long embryonic

periods may start forming increments before hatching.

The nucleus is usually circular, but it can be elongated

as in gobiids (Iglesias et al., 1997) or multiple as in

salmonids (Geffen, 1983; Neilson et al., 1985; Gaul-

die, 1991).

The ®rst increments may be homogeneous in thick-

ness if the ®sh has yolk sac reserves, or they may

decrease in thickness until the ®sh starts exogenous

feeding. In the otoliths of many ®sh species, additional

planes of growth are formed, from which a new series

of increments appears to emanate. These accessory


growth centres or secondary primordia (Fig. 5) are

frequent in species that undergo a marked habitat

change at the transition from the larval to juvenile

stage. Accessory growth centres are common in ¯at-

®sh. For example, Toole et al. (1993) demonstrated in

Dover sole that the formation of the ®rst accessory

centre corresponded to the initiation of metamorpho-

sis, while the last was formed months later when the

left eye traversed the mid-dorsal ridge. The accessory

centres formation also has been related to the transi-

tion from larva to juvenile in Merluccius merluccius

(Morales-Nin and Aldebert, 1997) and Ammodytes

marinus (Wright, 1993). The prisms of aragonite

develop from these centres; additional prisms formed

later have the effect of keeping the shape properties

(Fig. 6). The prismatic otoliths occur in a wide range

of species, but are particularly common in gadoids

(Gaemers, 1984).

Other structures formed are discontinuities called

checks, and bands of thin increments (Fig. 6) that

create an abrupt change in the appearance of incre-

mental structures associated with the shift from a

pelagic to a benthic habitat (Victor, 1982; Brothers

et al., 1983). Otolith growth rates also may change at

transitions between life history stages (Morales-Nin,

1980; Campana, 1989; Karakiri and von Western-

hagen, 1989). These checks have been interpreted

as markers of life history events such as hatching,

metamorphosis, environmental stress, or habitat tran-

sitions (Campana, 1984; Neilson et al., 1985; Karakiri

et al., 1989; Gartner, 1991; Geffen, 1996; Modin et al.,

1996; inter alia). Checks are lacking in species that do

not undergo marked changes during their life span,

such as the neotenic pelagic goby Aphia minuta

(Iglesias et al., 1997).

Several authors have noted a general decrease of

increment width with age. This trend might be related

to a change of metabolism with age resulting in a

lower tissue pH (Love, 1980), which might affect

otolith growth rate (Gauldie and Nelson, 1990). The

catabolic phase of metabolism in ®shes is charac-

terised by low tissue pH due to the hydrolysis of

proteins. Conversely, peptide condensation requires

higher pH levels, so the anabolic phase would typi-

cally have alkaline or neutral pH levels. Check rings

appeared in Caranx georgianus otoliths at metabolic

stages in which the pH was lowered (Gauldie and

Radtke, 1990).

4. Exogenous otolith growth regulation

Many characters that are related to environmental

properties are expressions of a genetic property that

may or may not manifest itself. Thus, regulation of

phenotype is mainly dependent on the genotype and is

Fig. 5. Accessory growth centres (arrows) on: (a) a scanning electron micrograph of a sagittal etched section of an otolith of Coelorhynchus

coelorhynchus. Scale bar 60 mm. (b) A light microscopy micrograph of a sagittal section from an otolith of Helicolenus dactylopterus. Scale

bar 100 mm.


related to species characteristics as well as to indivi-

dual variability. Campana and Neilson (1985) pro-

posed that the periodicity of the increment formation

is under an endogenous control and is entrained to

photoperiod, although other factors, such as tempera-

ture ¯uctuations and feeding frequency, could mask

the circadian rhythmicity and result in the formation of

sub-daily increments. Evidence in support of an endo-

Fig. 6. Crystal discontinuities: (a) Coelorhynchus coelorhynchus discontinuities on the aragonite prisms in a transversal section. (b) Minor

discontinuities (arrows) in the incremental growth and between prisms (double arrow) of a Sparus aurata otolith. (c) Checks as major

discontinuities on the crystalline structure of a Merluccius merluccius otolith. (d) Crystal discontinuities between contiguous prisms and the

incremental growth (e) Merluccius angustimanus transversal section showing the differential otolith growth in the sulcal side (arrow) and anti-

sulcal side. (a, c, d: scanning electron micrographs. The lower panel is 600�, the upper panel shows 3000� the area marked by a square in the

lower panel; b: light microscopy 400�; e: light microscopy 30�.


genous regulation of increment formation has come

from several species and experimental conditions. The

daily rhythm of the increment formation continued in

®sh held under constant light (Campana, 1984) or

darkness (Radtke and Dean, 1982), or in absence of

cyclical variations in major environmental factors

(Wright et al., 1992).

Tanaka et al. (1981) demonstrated that in Tilapia

nilotica the order of formation of the continuous and

discontinuous units was dependent on photoperiod, as

a reversal of the light±dark cycle was found to induce a

reversal in the order of the two increments. Using 45

Ca and 1H-labelled glutamate, the association of

photoperiod and the diurnal rhythmicity was proved

in both calci®cation and organic matrix formation, the

two cycles being in antiphase (Mugiya et al., 1981;

Mugiya, 1987). Wright et al. (1992), in experiments

with Salmo salar and 45 Ca, showed that the otolith

calci®cation was entrained to the light±dark cycles,

with calcium accumulation onto otoliths declining at

night and resuming at dawn. This cycle coincided with

a diel decline in calcium plasma concentration.

A decrease in light might affect both ®sh physiology

and the ability of visual predators to obtain food. In

this connection, a laboratory experiment induced the

formation of translucent zones in Lepomis macro-

chirus otoliths by varying photoperiod and restricting

food intake (Schramm, 1989).

A lagged effect of feeding upon both increment

width and the coupling of otolith and somatic growth

were observed by Secor et al. (1989). Also, otolith

growth rates were dependent on prey density in Clu-

pea harengus (Moksness et al., 1995) and Sparus

aurata (Morales-Nin et al., 1995). The effect of tem-

perature on the growth of the otolith has been much

studied and generally shows a positive relationship

between otolith growth and temperature (Campana

and Neilson, 1985). On the other hand, Salvelinus

alpinus otolith growth rate and somatic growth rate

respond differently to changing temperature (Mose-

gaard et al., 1988). Their results suggest that tempera-

ture continues to enhance otolith growth beyond the

point at which somatic growth is adversely affected. A

negative effect of temperature upon microincrement

width has also been observed (GutieÂrrez and Morales-

Nin, 1986; May and Jenkins, 1992; Ralston, 1995).

Laboratory and ®eld studies suggest that the effects

of episodic and chronic hypoxia on ®sh growth may be

highly dependent upon the degree and extent of oxy-

gen reduction, although understanding of the process

is limited due to the lack of understanding of the

numerous mechanisms through which oxygen reduc-

tion may affect the biology of ®shes (Hales and Able,

1995). Otolith growth is correlated to oxygen con-

sumption independent of growth rate (Wright, 1991).

Reduced otolith and somatic growth has been related

to hypoxia levels in Centropristis striata, with differ-

ent effects upon the coupling of otolith and somatic

growth depending on the oxygen level (Hales and

Able, 1995). Anaerobic stress may result in otolith

reabsorption (Mugiya and Uchimura, 1989).

A number of ®sh species, particularly anadromous

salmonids, but also marine species, show immediate

signs of disturbed physiology and metabolism at low

oxygen concentrations (Hales and Able, 1995). A low

dissolved oxygen level of 3 mg lÿ1 was critical for

herring larvae (Clupea harengus) (De Silva and Tytler,

1973). In smelt (Osmerus eperlanus) a direct correla-

tion between dissolved oxygen and width of the

microincrements was established, with a threshold

effect on otolith growth for oxygen concentrations

<4.5 mg lÿ1 (Sepulveda, 1994). Similar effects have

been described for daily increments in otoliths of dab

(Limanda limanda) under oxygen de®ciency condi-

tions (Karakiri and Temming, 1988). According to

Brett (1979), there is a critical concentration below

which growth rate declines at low oxygen levels.

Check formation in the otoliths of juvenile Pleur-

onectes platessa was related to ontogenic changes in

feeding and activity patterns related to tides (Geffen

and Nash, 1995). Similarly, rhythmic growth patterns

and checks in Merluccius capensis, M. paradoxus, and

Genypterus capensis were related to activity patterns

and different life strategies (Morales-Nin, 1987a).

5. Otolith growth modelling

Two main aspects determine otolith growth: the

microincrement periodicity and the microincrement

width.

Gauldie and Radtke (1990) have proposed two

mechanisms that may underline the formation of

microincrements: (a) obligatory microincrementation,

when the cycle of crystal and protein deposition that

forms a single microincrement is part of the diel


physiological cycle of the ®sh, and (b) facultative

microincrementation, when the cycle of crystal and

protein deposition that forms a single microincrement

is part of the metabolic effort of the ®sh. Thus, changes

in growth rate would cause changes in the period of the

microincrement, i.e., more or less than one increment

per day.

Obligatory microincrementation is part of the daily

and seasonal physiological cycles. Observations in the

®eld and in the laboratory have shown that micro-

increment width may change in response to tempera-

ture and diet, but the period of microincrement

deposition remains daily (Gauldie and Radtke, 1990).

The facultative microincrementation may occur

during the early life of some species, when the ®rst

increments are free-running and regulated by the

metabolic rate (Geffen, 1982, 1992; ReÂ et al., 1985;

Mosegaard et al., 1988; Maillet and Checkley, 1990).

By contrast, the ®rst increments of skipjack tuna

(Katsuwonus pelamis) otoliths are daily for a few days

after hatching (Radtke, 1983), but oxytetracycline

marked and recapture experiments have shown that

this can be interrupted or diminished for some late

juveniles and adults (Wild et al., 1995). This corre-

sponded to sub-optimal conditions for growth, due to a

lack of nourishment for this species, while for yellow-

®n tuna (Thunnus albacares) the increment formation

proved to be daily under conditions of better nutri-

tional status (Wild et al., 1995). The same process has

been described during early development, when

experimental stress caused sparse feeding and an

interval of non-daily increment formation in both tuna

species (Uchiyama and Strusaker, 1981). Indirect

evidence involving laboratory experiments with larvae

of other species also implicates an inadequate nutri-

tional level as a cause of non-daily increment forma-

tion (Taubert and Coble, 1977; Geffen, 1982; Jones

and Brothers, 1987). In salmonids it has also been

associated with changes in feeding intensity (Neilson

and Geen, 1985; Neilson et al., 1985; Wright et al.,

1990). In juvenile out migrating Auke Bay pink

salmon, Oncorhynchus gorbuscha, increment periodi-

city was most closely correlated with growth rate of

the otolith, and therefore the increment number did not

represent age (Volk et al., 1995). Facultative micro-

incrementation requires high levels of anabolic activ-

ity or some other sources of increased tissue pH

(Gauldie and Radtke, 1990).

Otolith growth in Caranx georgianus shows differ-

ent phases depending on the metabolism. During the

catabolic phase, the relationship between increment

width and body size was weak, while in the anabolic

phase of metabolism there was a stronger relationship.

Microincrement width in both phases was correlated

linearly with temperature (Gauldie and Radtke, 1990).

The patterns of daily growth of otoliths during early

development, in relation to environmental factors,

have been studied by time-series analysis of increment

width data (GutieÂrrez and Morales-Nin, 1986; Thor-

rold and Williams, 1989; Maillet and Checkley, 1991;

May and Jenkins, 1992; Ralston, 1995). These studies

deal in several ways the changes in otolith growth rate

due to ®sh size, which confounds the underlying

otolith growth. Thorrold and Williams (1989) asso-

ciated increment width data with speci®c calendar-

dates for ®ve daily larval cohorts but made no adjust-

ment for the effect of age on increment width when

looking for a temperature effect. Likewise, May and

Jenkins (1992) back-calculated a daily calendar-date

time series of somatic growth rate from increment

width data but made no adjustment for the effect of

different size compositions on different dates. GutieÂr-

rez and Morales-Nin (1986) divided the ®rst life

period into three stanzas of approximately equal

length and developed daily increment width time

series for each one. Maillet and Checkley (1991)

partitioned the ®rst 50 days of life into two growth

stanzas, which were analysed separately, but also

divided each observed otolith growth rate by its

expected value from a regression of speci®c otolith

growth rate on age. Ralston (1995) removed the effect

of specimen age on otolith growth rate by means of an

analysis of variance of the calendar-date effect. These

studies have used cross correlation functions and

transfer function models to model the time structure

between the dependent (otolith growth) and indepen-

dent (environmental factors) variables. However, Ral-

ston (1995) has shown a non-linear response of otolith

growth rate to two variables. Also, he points out that

the autoregressive multiple regression allows the inter-

actions between variables to be analysed without

assuming the steady-state linear response of the

dependent series assumed in the cross correlation

and transfer functions. The strong autoregressive nat-

ure of otolith growth causes inertia in the growth

processes that buffers the otolith from exogenous


in¯uences and induces a lag between otolith response

and environmental perturbations (GutieÂrrez and Mor-

ales-Nin, 1986; Maillet and Checkley, 1991; Ralston,

1995). The existence of lags of variable and unknown

length complicates the analysis of otolith growth and

the in¯uence of environmental factors upon it.

Many recent studies have demonstrated that the

relationship between otolith size and somatic size

depends upon growth rate (Reznick et al., 1989; Secor

and Dean, 1989, 1992; Casselman, 1990; Mugiya and

Tanaka, 1992). This dependency biases traditional

approaches to back-calculation (Campana, 1990) and

in ®sh of the same size results in larger otoliths in

slow-growing individuals. This phenomenon uncou-

ples somatic and otolith growth rates (Mosegaard et al.,

1988) and obscures the use of daily increment widths

as a measure of somatic growth rate. In general,

alterations in otolith growth rate do not scale directly

to somatic growth rate but instead provide a conser-

vative representation of growth variability.

Several papers have dealt with otolith growth based

on the chemical properties of the otic sacculus and of

the aragonite. The model of otolith growth proposed

by Gauldie and Nelson (1990) suggests that Ca2� ions

released at the macula cannot be immediately avail-

able for reaction into calcium carbonate. The avail-

ability of Ca2� increases from the macula to the otolith

apex where the growth is maximal, and declines along

the anti-sulcus surface. The otolith formation is facili-

tated by carbonic anhydrase, which increases the rate

of Ca(CO3)2 production. The sequence of reactions in

which carbonic anhydrase is involved can be summar-

ized as

CO2 � H2O�H2CO3�H�HCO3ÿ�2Hÿ � CO3

2ÿ

Although there are no data for ®sh, the levels of

carbonic anhydrase in molluscs change markedly with

age, resulting in continuous minerallization but with

growth rate changing in response to carbonic anhy-

drase activity, as well as other extrinsic factors such as

temperature (Gauldie and Nelson, 1990).

Such an anhydride CO2-driven system has a number

of important consequences (Gauldie and Nelson,

1990): (1) The rate of otolith growth must generally

be controlled by metabolic rate. (2) Otolith deposition

must be continuous while the organism lives (except in

hibernation and aestivation), because the reaction is

driven by the organism's metabolism. (3) The level of

carbonic anhydrase may vary in different physiologi-

cal circumstances leading to different growth rates. (4)

As the system proposed is a chemical one, temperature

must be the main external factor controlling otolith

growth rate. The authors also proposed otoconial

stores of calcium in the otolithic membrane of the

maculae, with subsequent dissolution and precipitation

to the growing sagitta crystal surface at rates determined

by the pH gradient between the metabolic acidosis of

the respiring macula, and the carbonic-anhydrase-

driven alkalinity of the endolymphatic sac ¯uids.

Considering tissue accumulation as the accumula-

tion of chemical potential energy, Gauldie (1990)

proposed a model for the formation of calcium car-

bonate derived from the Gibbs±Helmholtz equation.

The chemical reaction is characterised in terms of the

rate of change in calcium. More recently, Romanek

and Gauldie (1996) have proposed a model based on

the precipitation kinetics of aragonite and the chem-

istry of ¯uids within the endolymphatic sac of ®sh.

The model includes the theory of the free-ion activity

coef®cients (Debye±HuÈckel coef®cient) to link the

actual concentration of an ion to its activity for the

dissolved ions present in the otic ¯uid. The precipita-

tion rate is related to the saturation state, which

depends on pH, temperature, and ¯uid chemistries.

The model uses increment width as a proxy for the rate

of aragonite precipitation. The results suggest that

endolymphatic ¯uid has a relatively simple chemistry

that lacks crystal growth inhibitors. When used in

conjunction with measurements of otolith size and

microincrement width, the model can be used to

constrain the age of ®sh and provide estimates for

the water temperature that ®sh experience in the

course of their lives.

However, the formation of biogenic calcium carbo-

nate in otoliths might act differently due to the action

of the organic matrix both as nucleation centres and as

inhibitor of calcium-carbonate crystallisation. Thus,

these models based in inorganic aragonite precipita-

tion, should be developed in the future to include the

organic matrix interaction.

6. Conclusion

One of the most important questions for ®sheries

managers and for ®sh biologists is the issue of ®sh age


determination. The relevance of this subject is clear

when considering the number of papers published that

re¯ect the research effort concentrated on ®sh age and

the relationships between environmental variables and

®sh growth. However, several main questions are still

unanswered: (1) What is the mechanism that relates

the growth marks in the otoliths with the age of the

®sh? (2) Is it possible to validate the proposed

mechanism with observed results? (3) How do phy-

logeny and stock affect the otolith increment patterns?

(4) How do the environmental and physiological

responses and processes affect check and zone

formation?

Although much progress has been made in recent

years in understanding otolith growth regulation, sev-

eral points still need further study. The main problem

is still the necessity to identify the signalling factors

regulating calcium transport and matrix production in

the sacculus. Experimentation at the sacculus level

may make it possible to identify the endogenous

regulator of otolith growth. Biogenic crystal growth

is still poorly understood, and the role of the organic

matrix in crystal growth and shape regulation should

be further investigated. Crystalline otoliths which

have abnormal organic components might provide

a useful tool to compare the role of the organic

matrix.

Otolith growth and its three-dimensional develop-

ment should be considered, because the internal struc-

ture of the otolith might be biased due to differential

development.

Some evidences indicate that at least two different

growth processes are at work in otoliths. Crystal

growth rate might re¯ect extrinsic environmental

effects, while checks in development re¯ect intrinsic

metabolic effects.

Another important point is the problems in the

statistical inferences from individuals to populations

and the need to evaluate the variation in increment

number at age or at length. Also, it is dangerous to

extrapolate from one life phase to an other, due to

differential otolith growth during the life span of a

given species. Thus, extrapolations beyond the range

of one study are unreliable and should be avoided.

Nevertheless, the current progress in the complex ®eld

of ®sh age determination provides a ®rm and promis-

ing foundation for future studies designed to address

the remaining unsolved questions.

Acknowledgements

My colleagues from the European Fish Ageing

Network (European Union FAIR PL 96.1304) and

specially Dr. P.J. Wright are thanked for their friendly

help in otolith studies. Dr. R.W. Gauldie is thanked for

many interesting hours spent talking about otoliths.

Dr. A. Lombarte for his revision of the text and for

Figs. 1 and 2. Dr. C. Rodgers revised the English text.

Ms. E. Batanero typed the manuscript. The SEM

micrographs were processed by J.M. FortunÅo.

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