morales nin 2000
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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.
56 B. Morales-Nin / Fisheries Research 46 (2000) 53±67
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
B. Morales-Nin / Fisheries Research 46 (2000) 53±67 57
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.
58 B. Morales-Nin / Fisheries Research 46 (2000) 53±67
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�.
B. Morales-Nin / Fisheries Research 46 (2000) 53±67 59
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
60 B. Morales-Nin / Fisheries Research 46 (2000) 53±67
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; Re 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
B. Morales-Nin / Fisheries Research 46 (2000) 53±67 61
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
62 B. Morales-Nin / Fisheries Research 46 (2000) 53±67
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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