Copyright C Munksgaard 2001

Ecology of Freshwater Fish 2001: 10: 122126

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ISSN 0906-6691

Short communication

Validation of the otoliths for age

determination in Barbus sclateri (Gnth.)
Escot C, Granado-Lorencio C. Validation of the otoliths for age determination in Barbus sclateri (Gnth.).
Ecology of Freshwater Fish 2001: 10: 122126. C Munksgaard, 2001
Abstract Otoliths were used to explore the timing of annulus formation
in Barbus sclateri. Lapillus exhibited opaque and translucent zones
whose combination was formed once a year. Opaque zones appeared
formed from July and January, and the translucent ones from February to
June. We describe a procedure to assess age by using the number of annulii, the condition of the otoliths edge and the capture date. The use
of otoliths for age determination in this cyprinid endemic to the Iberian
Peninsula provides a useful alternative to the traditional methods of scale
reading.

C. Escot1, C. Granado-Lorencio2
1

Aquatic Ecology Station, EMASESA, Sevilla,

Department of Plant Biology and Ecology,
Faculty of Biology, University of Sevilla, Spain
2

Key words: otolith; age determination; Barbus

sclateri
C. Granado-Lorencio, Department of Plant
Biology and Ecology, Faculty of Biology,
University of Sevilla, Apdo. 1095, Sevilla
E-41080, Spain; e-mail: granado/cica.es
Accepted for publication January 5, 2001

Un resumen en espanol se incluye detras del texto principal de este artculo.

Introduction

Material and methods

Studies on the age and growth of cyprinids endemic to the Iberian Peninsula have focused on the
interpretation of fish vertebrae and scales. When
the first vertebrae is used, only specimens up to 8
years have been reported due to imprecise determination of rings in older individuals (Lucena & Camacho 1978; Lucena et al. 1979). Moreover, Herrera (1991) and Rodrguez-Ruiz (1992) reported
difficulties in determining the location of the first
ring in scales of older specimens due to the occurrence of false marks.
Overall, otoliths have been considered the
most accurate bone for age determination in
slow-growing species (Beamish & McFarlane
1987), but its use to age cyprinids in uncommon.
The objective of this study was therefore to validate otoliths for aging Barbus sclateri Gnther
1868, one of most abundant members of stream
fish assemblages in the southern Iberian Peninsula. In particular, we attempt to detect annual
formations in otoliths to meet the assumptions
established by Francis et al. (1992) and Francis
(1995): 1) the existence of periodical marks in
the selected structure; 2) the ability to identify
these marks correctly (including discrimination
from false marks); and 3) the ability to convert
the number of marks into age.

Otoliths of 340 specimens of B. sclateri (60315

mm, standard length) were collected monthly with
electrofishing during a year in Rio Guadalete
(southwestern Spain) and in Rio Bocaleones, a
major tributary. In contrast to the former stream,
the latter is fed by springs and exhibits a rather
homogeneous stream flow and thermal regime
throughout the year (Prenda 1993). The recent
construction of a dam upstream the mouth of Rio
Bocaleones into Rio Guadalete prevented mixing
of the two fish populations during the last 3 years.
Otoliths were obtained from fresh fish just after
collection or from individuals frozen for a maximum of 2 hours after collection. The otoliths were
cleaned in a 10% sodium hypochloride solution
(bleach), and rinsed with distilled water. Then they
were immersed in 95% ethanol and stored dry.
Based on previous assessments of these otoliths
(Escot & Granado-Lorencio, 1998), the right-side
lapillus was placed into water and observed with a
microscope at 40 using reflected light on a black
background. It was not necessary to clear them
because these otoliths had clearly defined opaque
zones alternating with translucent zones, and the
operation of reflected light made the opaque zone
appear white and luminous, and the translucent
zone appeared dark.

122

Otoliths reading in Barbus sclateri

Results and discussion

Opaque and translucent zones in the whole lapillus

were clearly visible (Fig. 1). In several otoliths, the
translucent zones were cleared to detect the main
axis that became diffused when getting nearer the
superior or inferior part of the otolith. Translucent
zones were observed in the edge of the otoliths
throughout the year (Fig. 2a), but the proportion
of otoliths with a translucent zone showed an unimodal annual cycle. Over 80% of the otoliths
showed a marginal translucent zone in April and
May. More than 80% of the otoliths from Rio Guadalete exhibited a translucent edge in April, and
those from Rio Bocaleones exhibited a translucent
edge between March and June (Fig. 2b, c).

Fig. 1. A view of the whole lapillus of B. sclateri under magnifying glass, submerged into water on a black background with
reflected light. The larger otolith above is from an 11 year old
female, 260 mm total length, captured in Rio Bocaleones in
November (bar1 mm). The smaller otolith below is from a 6
year old male, 92 mm total length, also captured Rio Bocaleones in January (bar0,5 mm). The black arrows indicate each
annulus

The distal portion of each translucent zone was

considered the annullus. The term annullus is associated with the translucent zone that is formed
once a year in calcificed structures of fishes growing in temperate climates (Casselman 1987). The
areas of complete growth for each year were represented by the combination of one opaque zone and
a subsequent translucent zone. The number of annuli of the whole lapillus was counted along the
longest antero-posterior axis on its dorsal face
(anti-sulcal).
We applied the marginal increase analysis
method (Baillon 1992) to validate age. This technique encompasses qualitative and quantitative examination of the otolith edge over the growth
period. The percentage of the otolith with opaque
or translucent zones on the edge was estimated
(Ross & Stevens 1995; Thompson & Beckam 1995;
Yosef & Casselman 1995); the width of the two
most external zones of growth were measured
(each opaque zone of the last annulus), and the
relative width of the marginal increment (MI) was
calculated as the ratio of the width of the actual
growth zone to the width of the previous years
growth zone (Maceina & Betsill 1987; Gooley
1992; see also Kalish et al. 1995 for the glossary
of terms).

Fig. 2. Plot of percentage of otoliths with translucent edges

(bars) and of the mean marginal increments (dark squares, with
standard errors) during the study period. A. Individuals of B.
sclateri from both streams pooled. B. Individuals from Rio Guadalete and C. Individuals from Rio Bocaleones

123

Escot & Granado-Lorencio

Table 1. Analysis of the variance (anova) for the effects of capture time
(month) and place (river) on the marginal increment (MI) of the otoliths of B.
sclateri during the study period. The Bonferroni comparisons were used to
determine differences between adjacent means.
Source of
variation

Sum of
squares

df

Mean
squares

Month
Place
Month*Place
Error

3.76
0.09
0.81
16.50

10
1
10
289

0.38
0.09
0.08
0.06

6.58 0.0001
1.56
0.212
1.41
0.174

FebMarAprMayJunJulAugSepOctNovDec

Fig. 3. Monthly variations of the mean marginal increment for

the age-groups indicated in (a) Rios Guadalete and (b) Bocaleones (b)

The time of capture influenced significantly influenced the amount of opaque material at the
edge of the otolith (ANOVA, P0.0001), but no
environmental influence was significant (Table 1)
(Fig. 2a, b, c). There were three differential stages
of otolith growth throughout the year. The otolith
growth appeared at a minimum between February
to May and increased significantly between June
and September.
Given that there was a clear pattern in the
opaque zones growth for each river dataset, we
further tested whether the same pattern occurred
in all age-classes. We selected age-groups so that
we could compare the greatest number of ages and
yet have an adequate sample size. These were 24,
124

58 and 916 years in Rio Guadalete, and 24, 5

8, 912 and 1318 years in Rio Bocaleones. As
shown in Fig. 3 those patterns were consistent
among age-groups for both rivers.
Overall, the results confirmed the presence of
one opaque zone followed by a translucent zone in
the Barbels lapillus, and that the combination of
both is formed once a year. A few false translucent
zones were apparent, but they were easily detectable because they did not occur in the areas above
and below the nucleus. False zones were similar to
those of the annuli, but they were found in only
one part of the structure, similar to the results obtained by Casselman (1987).
The formation of the annulus in calcified tissues
is complete when the translucent zone, which
forms once a year, is followed by opaque material
(Casselman 1987). This corresponded to the minimum marginal growth values detected for our barbel population. Since, besides marginal growth, we
included the periodical interpretation of the edges
appearance (opaque or translucent percentages),
we were able to determine the timing of the formation of the two zones: the opaque zone was formed
in both populations from July to January and the
translucent zone was formed from February to
June. Further observations on the reproduction of
B. sclateri in these streams suggest that reproduction extends from February to June (Lucena &
Camacho 1978; Herrera et al. 1988; Herrera 1991;
Rodrguez-Ruiz 1992).
Francis (1995) considered the interpretation of
the edge as a major factor in estimating age, yet it
is important to distinguish between the time of the
year when the zone is formed and the time when
it becomes visible. We found that the otoliths show
very fine translucent edges during the period when
opaque material in the edge most frequently appears. This event has being termed pseudo-hyaline
or pseudo-translucent edge (North 1988) and
would be the time in the otoliths growth when
there is no difference between the opaque and the
translucent zones. Also, Mina (1968) identified this
problem of visual identification of a zone: ...we
can only observe that a new zone has been laid,
when the change in the deposition of calcium has
been enough to produce a change in the optical
density of the material and therefore can be easily
detected... (see also Lecomte-Finiger 1992). This
phenomenon can be further complicated when
working with whole or sectioned otoliths where the
zones correspond to three-dimensional layers, in
contrast to fine sections in which the zones are
two-dimensional structures (Francis 1992).
From July to January (the time of greater frequency of opaque edges), our observations of translucent zones were probably misinterpreted, and this

Otoliths reading in Barbus sclateri

resulted from difficulty to distinguish between
translucent and the pseudo-translucent zones. Because during this period there were no apparent
growth in this pseudo-translucent edge, and there
was growth in the opaque zone, the formation of the
latter may have taken place before winter in Rio Guadalete and before the end of winter and the onset
of spring in Rio Bocaleones, and in the two rivers,
the translucent zone was formed before the summer.
Once the rings were counted (annulli) they were
converted into age. This last step is referred to by
Francis et al. (1992) as the interpretation of the
edge. This implies a decision on the moment (or
date) when the most external ring counted was
formed. The conversion of the number of rings
into age implies considerations on the relationship
between this date and two previous dates, i.e., time
of capture and birthdate (Francis 1995). For this
purpose, we took January 1 as the birth date for
the two fish stocks, which may not necessarily coincide with the date of natural birth (Willliams &
Bedford 1974). Consequently, the precise age of
each individual fish was determined taking the following criteria into account: 1) If the otolith had
an opaque edge, the age was equal to the number
of annulli, regardless of the date of capture. 2) If
the otolith had a translucent edge, estimating the
age depended on the date of capture as follows: if
fish came from the July to December, the age was
equal to the number of annulli with a posterior
opaque zone, and if the fish came from January to
June, age was equal to the number of annulli with
a posterior opaque zone plus one.
In conclusion, if the condition of the edge of the
otolith is taken into consideration, otolith reading
provides a useful alternative to other methods previously applied for age determination in B. slcateri
and probably in other cyprinids endemic to the Iberian Peninsula.
Resumen
1. Utilizamos otolitos para determinar el momento de la fomacion de anillos anuales en Barbus sclateri. Examenes microscopicos detallados pusieron de manifiesto la ocurrencia de zonas
opacas y traslucidas en el Lapillus cuya combinacion se produce
una vez al ano. Las zonas opacas aparecieron desde Julio hasta
Enero mientras que las translucidas lo hicieron desde Febrero
hasta Junio.
2. Describimos un procedimiento para evaluar la edad utilizando el numero de anillos, la condicion del lmite del otolito y la
fecha de captura. La utilizacion de otolitos para determinar la
edad de este ciprnido endemico de la Pennsula Iberica oferta
una alternativa a los metodos mas tradicionales basados en lectura de escamas.

Acknowledgements
This research was supported by the Department of Plant Biology and Ecology and by the Aquatic Ecology Station of Sevil-

la. We thank to all those who assisted during the successive

stages of this study, especially Dr. D. Rodriguez, Dr. L. Encina,
E. Mellado and Dr. J. Prenda.

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