Journal of Fish Biology (1999) 55, 916930

Article No. jfbi.1999.1049, available online at http://www.idealibrary.com on

Sex ratios in ospring of sex-reversed sea bass and the

relationship between growth and phenotypic sex
dierentiation
M. B *, M. C*, S. Z* F. P
*Instituto de Acuicultura de Torre de la Sal, Consejo Superior de Investigaciones
Cientficas (C.S.I.C.), 12595 Torre de la Sal, Castellon, Spain and
Institut de Cie`ncies del Mar, C.S.I.C., Passeig Joan de Borbo s/n, 08039 Barcelona,
Spain

(Received 20 February 1999, Accepted 24 June 1999)

Groups of sexually undierentiated sea bass Dicentrarchus labrax were fed with the androgen
17
-methyltestosterone (MT) during sex dierentiation. MT treatment increased males from
793% in the controls (the usual 3 : 1 male : female sex ratio of cultured sea bass) to 1000%,
implying that in the treated groups one out of each five resulting males was a masculinized
female (neomale). Thirteen males from the MT treated groups were taken as the parental
generation and their sperm used to individually fertilize a pool of eggs from unrelated females.
The probability of having at least one neomale was 95% and most probably two or three of the
males used were neomales. The ospring from each family were reared separately under the
same environmental conditions. Samples were taken at 11 and 15 months of age, during and
after sex dierentiation, respectively. Results showed that females predominated among the
larger fish whereas males and undierentiated fish predominated among the smaller ones.
Intersexes exhibited an intermediate size. All fish with a body length smaller than 12 cm were
undierentiated. These results suggest that sex dierentiation is more dependent on length than
on age. At 15 months, sex ratios were male-biased in all families, except one (females ranged
from 5 to 50%) and only two families had sex ratios not significantly dierent from 1 : 1,
suggesting that the mechanism of sex determination in the sea bass is not of a XX/XY or
ZW/ZZ type since no family exhibited a female-biased progeny, as would be expected from
both types. Results support the hypothesis that factors other than genetic, i.e., environmental,
may act epigenetically on the sex determination mechanisms of sea bass, as has been
demonstrated in other fishes.  1999 The Fisheries Society of the British Isles

Key words: sex determination; sex dierentiation; sex-related growth; environmental eects;
fish, Dicentrarchus labrax.

INTRODUCTION
In contrast to birds and mammals, fish, amphibians and reptiles show a high
degree of plasticity in sex determination and sex dierentiation (Francis, 1992).
Fish, in particular, exhibit all types of sexuality known in vertebrates: gono-
chorism, hermaphroditism and unisexuality, and their sex determining mech-
anisms are varied and sometimes complex. Thus, in contrast to the single
mechanism of mammals (female homogamety XX/XY) and birds (male
homogamety ZZ/ZW), both XX/XY and ZZ/ZW systems are found in fish,
Present address: Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill
AB25 2ZD, Aberdeen, Scotland, U.K.
Author to whom correspondence should be addressed. Tel.: +34 964 319500; fax: +34 964 319509;
email: zanuy@iats.csic.es
916
00221112/99/110916+15 $30.00/0  1999 The Fisheries Society of the British Isles
 917

sometimes even in species of the same genus (Solari, 1994). In addition,

polygenic systems have been reported (Kallman, 1984). Several studies have also
shown that the genetic sex can be altered or changed by environmental or social
factors (Francis, 1992; Bla zquez et al., 1998a). In addition, most karyological
studies reveal no morphologically distinguishable sex chromosomes in fish
(Francis, 1992; Solari, 1994), which further complicates the elucidation of their
genetic sex.
However, the use of indirect approaches has clarified the sex determination
mechanism of several fish (Solari, 1994). These include studies of the inheritance
of sex-linked colour genes (Aida, 1921; Winge & Ditlevsen, 1947) and breeding
experimentally sex-reversed fish with normal fish and studying the sex ratios of
their progenies (Hunter & Donaldson, 1983). Thus, female homogamety (XX)
was demonstrated in the chinook salmon Oncorhynchus tshawytscha (Walbaum)
(Hunter et al., 1983), the catfish Ictalurus punctatus (Rafinesque) (Davies et al.,
1990), the Siamese fighting fish Betta splendens Regan (Kavumpurath &
Pandian, 1992), the guppy Poecilia reticulata (Peters) (Kavumpurath & Pandian,
1993), and the tilapia Oreochromis niloticus L. (Jalabert et al., 1974). Likewise,
female heterogamety (ZW) was demonstrated in another tilapia Oreochromis
aureus Steindachner (Hopkins et al., 1979) and in a poecilid Poecilia sphenops
Cuvier & Valenciennes (George & Pandian, 1995).
The European sea bass Dicentrarchus labrax L., is a gonochoristic teleost, and
the subject of intense research. It has no morphologically distinct sex chromo-
somes (Arefyev, 1989; Vitturi et al., 1990; Sola et al., 1993) although a recent
study suggests that males and females could dier in the amount and pattern of
distribution of heterochromatin (Cano et al., 1996). In addition, no sex-specific
DNA sequences have been detected to date (Martnez et al., 1999). Although the
sex determination mechanism of this species has not yet been identified, its
elucidation would be very useful for both fundamental and applied research. In
addition, sea bass gonads remain sexually undierentiated during most of the
first year of life and it is not known why the sex ratios of stocks born in captivity
are consistently skewed in favour of males (Carrillo et al., 1995). This suggests
that environmental factors may influence its sex dierentiation. In this regard,
we have shown recently that a low rearing water temperature (15 C) early in life
during sex dierentiation not only results in delayed growth, but also in an
all-male population. In contrast, a temperature higher than usual (25 C) does
not aect the 3 : 1 male : female sex ratios usually found in cultured populations
(Bla zquez et al., 1988b). However, whether this eect of temperature is direct or
mediated through growth is not known at present.
To take advantage of the ability to masculinize sexually undierentiated sea
bass by androgen treatment (Bla zquez et al., 1995), experimentally masculinized
sea bass were bred with normal females. The sex ratios of the resulting progenies
were then studied in an attempt to understand the sex determination mechanism
of this species.

MATERIALS AND METHODS

ORIGIN OF FISH
Parental males were obtained by androgen treatment of sexually undierentiated
juveniles (Bla zquez et al., 1995). Briefly, four groups (n=60 fish per group) of sea bass
918 . .

(224004 g; mean...) were reared in 500-l aquaria. Two groups were fed a diet
containing 17
-methyltestosterone (MT) at 10 mg kg
1 from day 126 to day 226 after
fertilization, which comprises the critical period of sex dierentiation. The other two
groups were fed an untreated diet and served as the controls. At 20 months of age, some
of the fish (n=30 per group) were sampled at random and the sex determined
histologically. MT treatment increased the proportion of males from 793% in the
controls (i.e., the usual 3 : 1 male : female sex ratio of reared sea bass) to 1000%
(Bla zquez et al., 1995).

PROGENY TEST
At 3 years, 13 males from the treated groups and two unrelated females were taken
randomly as the parental (P) generation. These fish were induced to spawn as described
previously (Carrillo et al., 1995). Eggs of both females were obtained by abdominal
massage, pooled, mixed gently and divided into 13 batches of 70 ml each. Each batch
of eggs was fertilized individually with the sperm from one of the 13 males, resulting in
13 dierent families (F1, . . ., F13). Eggs were fertilized and incubated following standard
procedures in our laboratory. After hatching, larvae were transported to the Station of
Aquaculture Sepia International (Gravelines, France) in isotherm containers filled with
sea water and oxygen (1 : 1 vol : vol). Very little or no mortality was observed as a
consequence of transport. Each family was reared separately in 600-l aquaria at 225 C
and fed a standard diet for sea bass.

SAMPLING AND HISTOLOGICAL PROCEDURES

Fish were sampled at two previously determined ages, during and after sex dierenti-
ation (Bla zquez et al., 1995). To determine the relationship between size and sex
dierentiation, fish from each family were measured at 11 months under anaesthesia with
01 g l
1 of MS-222 (Sigma Chemical Co., St Louis, MO, U.S.A.). A size distribution
histogram was made with lengths divided into six intervals. Half of the fish in each
interval were selected randomly to avoid any bias towards one size and thus possibly one
sex (Bla zquez et al., 1998b). In total about half (n=3250) of the fish from each family
were killed at 11 months of age during sex dierentiation (January). The remaining half
was returned to the tanks for further rearing and killed (n=2846) once sex dierentiation
was completed at 15 months of age. At each sampling time, fish were measured (fork
length; precision 01 cm), weighed (precision 001 g) and their gonads removed, embedded
in paran, cut at 7
m, stained with Cleveland Wolfes (Herlant, 1960) and examined
with a light microscope. This staining procedure allowed accurate visualization of
connective tissue making it ideal to examine sexually undierentiated gonads, as they
contain large areas of this tissue. From each fish, at least five cross-sections covering
dierent portions of the gonad were examined to determine their phenotypic sex.

STATISTICAL ANALYSIS
To calculate the probability of choosing x neomales, P(x), when n=13, males from the
MT treated groups were taken randomly as the parental generation, the following
formula was used:

(Zar, 1996), for values of x from 0 to 13, where p=021 (female proportion), and q=079
(male proportion) in the control groups (i.e., p+q=1). Since in a binomial distribution
xx=1 P(x)=1 [i.e., P(0)+P(1)+. . .+P(n)=1], the probability of selecting at least 1
neomale (113 neomales) was calculated as: 13 x=1 P(x)=1
P(0) i.e., 1 minus the
probability of selecting none (Zar, 1996).
Likelihood ratio tests were performed to compare the observed sex ratios at 15 months
of age (sexual dierentiation complete) in each of the 13 families against the male : female
sex ratios of 3 : 1 (usually found in cultured sea bass), 2 : 1 (sometimes observed) and 1 : 1
 919

0.30

0.25

Probability, P(x) 0.20

0.15

0.10

0.05

0.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Number of neomales, x
F. 1. The theoretical probabilities for selecting a determinate number of neomales (013) when 13 fish
were taken randomly from a stock of sea bass treated with the androgen 17
-methyltestosterone,
which increased the proportion of males from 79 to 100%.

(rarely observed) (Robin & Brusle , 1983; Bla zquez et al., 1995). As intersex gonads
consisted mainly of testicular tissue, intersexes were pooled with males for analysis. This
was supported further by a significant negative correlation (r=
076; P=0003) between
the percentage of intersexes and males but not between intersexes and females (r=015;
P=0626). For those families that could be fitted to more than one sex ratio (3 : 1, 2 : 1
or 1 : 1), the P value from the likelihood ratio tests was used to determine which of the sex
ratios they adjust to best (Zar, 1996). For growth-related sex dierentiation, data from
all fish were pooled, regardless of family of origin, and classified by phenotypic sex.
One-way ANOVA was used to detect sex-related growth dierences. Normality was
ensured after logarithmic transformation of data, when required, and Bartletts test used
to verify homogeneity of variances (Zar, 1996). Multiple comparisons between means
were made with Tukeys multiple range test. In all cases, dierences were accepted as
significant when P<005.

RESULTS
PROBABILITY OF SELECTING A NEOMALE IN THE PARENTAL (P)
GENERATION
The 13 males used for the progeny test came from a group consisting of 100%
males as a result of masculinization by MT treatment. The untreated controls
had 79% males, indicating that 21% of males in the treated groups were fish that
would otherwise have developed into females if not treated with androgen.
These sex-reversed females were phenotypic males (neomales). The probability
of selecting a determinate number of neomales (one to 13) when 13 fish were
randomly taken from the MT-treated groups as the P generation for the progeny
test was between two and three neomales (Fig. 1). The probability of having no
neomale P (0) in the parental generation was calculated as follows:

then the probability of selecting at least one neomale was 1
P(0)=0954, i.e.,
95% (Fig. 1). Together this means that, with this experimental design, most
920 . .

probably two or three males were neomales and that, in any case, the probability
of having at least one neomale in the progeny test was 95%.

SEX DIFFERENTIATION
Histological examination of cross-sections of gonads during sex dierentiation
revealed the presence of dierent phenotypes (Fig. 2): (1) sexually undierenti-
ated fish, with filiform gonads made of fibrous connective tissue with some
scattered germinal cells [Fig. 2(a)]; (2) dierentiating males, with larger gonads
still containing an important amount of connective tissue, but the germ cells
starting to form the testicular lobules [Fig. 2(b)]; (3) males, with well formed
testes with testicular lobules containing both the germinal (spermatogonia) and
the somatic cells [Fig. 2(c)]; (4) intersex fish, which were essentially males with
testes containing some scattered previtellogenic oocytes intermingled among the
testicular lobules [Fig. 2(d)]; and (5) females, with ovaries arranged in lamellae,
containing previtellogenic oocytes [Fig. 2(e)].
At 11 months of age, all females had completed the process of sex dierenti-
ation since their ovaries were filled with previtellogenic oocytes. In contrast,
some males were still in the process of sex dierentiation since their testes were
not completely filled with lobules. Thus, sex dierentiation in the sea bass
finished earlier in females than in males.

SEX RATIOS OF F1, . . ., F13

At 11 months, all but one family (F3) contained sexually undierentiated fish.
These ranged from 0 to 30%, whereas males (both dierentiated and dierenti-
ating) ranged from 13 to 74%; intersex from 0 to 45% (only F1 had no intersex
fish), and females from 6 to 36% (Table I). At 15 months of age, all fish had
completed sex dierentiation, as shown by the absence of undierentiated fish
and dierentiating males. Females ranged from 5 to 50%, intersex fish from 0 to
42% and males from 28 to 85% (Table II). Likelihood ratio tests showed that at
this age, and with the sample size used, 12 out of the 13 families could be fitted
to (i.e., did not significantly dier from) a 3 : 1 male : female sex ratio. Eleven of
these families could also be fitted to a 2 : 1 sex ratio and one of them (F10) to a
1 : 1 sex ratio. However, and based on the P values from the likelihood ratio
tests, eight of these families (i.e. F1, F4, F5, F6, F7, F8, F11 and F12) supported a
3 : 1 male : female sex ratio best and three of them (F3, F10 and F13) a 2 : 1
male : female sex ratio. The remaining family (F9) could only be fitted to a 1 : 1
sex ratio (Fig. 3).

GROWTH
At 11 months of age, when some fish were still sexually undierentiated,
females were the largest (180023 cm; 798323 g). Intersexes were inter-
mediate (166020 cm; 631246 g) between females and dierentiated males
(158012 cm; 512129 g) and dierentiating males (145011 cm;
385105 g), whereas undierentiated fish were the smallest (141014 cm;
362117 g) (Fig. 4). Unlike females, there were no males >21 cm and >150 g.
Likewise, there were no females <135 cm and all fish <12 cm were sexually
undierentiated. Statistical dierences (ANOVA; P<005) in length and weight
were found between all phenotypes except between dierentiating males and
 921

F. 2. Sea bass gonads at 11 months of age. (a) Undierentiated gonad. Germ cells were located
randomly along the entire length of the gonad. (b) Dierentiating testis. Germ cells
(spermatogonia) were grouped in small cysts that later gave rise to the testicular tubules. Note the
fibrous-connective tissue located in the centre of the gonad. (c) Testis. Spermatogonia were
present in the testicular tubules, which were well formed. (d) Intersex gonad. Note the presence of
previtellogenic oocytes (arrowheads) embedded in the testis. (e) Ovary. Previtellogenic oocytes
located in the ovarian lamellae. Bar=100
m.
922 . .

T I. Sex proportions in each sea bass family resulting from the progeny test at 11
months of age

Sex (%) F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13

Female 18 23 26 8 12 6 12 23 325 25 36 11 26
Intersex 0 45 15 27 19 10 26 28 125 18 19 42 14
Male 16 13 44 23 41 48 26 23 175 34 19 36 40
Male* 36 0 15 15 12 26 12 13 15 7 17 0 10
Undier. 30 19 0 27 16 10 24 13 225 16 9 11 10

F1 . . . F13, Family 1 . . . Family 13.

Male*, dierentiating males; Undier., undierentiated fish.

T II. Sex proportions in each sea bass family resulting from the progeny test at 15
months of age

Sex (%) F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13

Female 27 5 33 15 15 15 15 23 50 38 22 26 30
Intersex 4 21 22 20 0 0 4 19 10 16 24 9 42
Male 69 74 45 65 85 81 58 40 46 54 65 28

F1 . . . F13, Family 1 . . . Family 13.

undierentiated fish (Fig. 4). These sex-related dierences were maintained at

15 months, when all the fish were sexually dierentiated (Fig. 5). Females were
still the largest fish (224021 cm; 1473460 g), intersexes were intermediate
(216026 cm; 1266499 g) and males were the smallest (211014 cm;
1218255 g). All females had a body length >18 cm and no males >300 g
were observed. In length, females and intersexes were not statistically dierent
from each other with males dierent from females but not from intersexes. In
weight, both intersexes and males were significantly smaller than females
(ANOVA; P<005) (Fig. 5).

DISCUSSION
The present study constitutes the first attempt at identifying the mechanism of
sex determination in the sea bass by breeding sex-reversed fish with normal fish.
This was feasible due to the possibility of obtaining all-male populations
by androgen treatment (Bla zquez et al., 1995). Nevertheless, two types of
diculties are usually encountered with this strategy. First, experiments are
time-consuming since, for example, 23 years of rearing are required in the sea
bass to obtain sucient sperm from the masculinized females. Thereafter, at
least an additional year is needed before the phenotypic sex of the progeny can
be assessed unambiguously. Second, significant space facilities are required since
progenies of individual crosses need to be reared separately. Thus, in designing
this type of experiment, a compromise needs to be reached in terms of time and
 923

5
3:1

4
F4
Number of families

0.43
2:1

3
F5
0.43 1:1

2
F6 F8 F1 F13
0.31 0.80 0.82 0.66

1
F2 F7 F11 F12 F3 F10 F9
0.06 0.31 0.80 0.90 1.00 0.63 1.00

0
0 10 20 30 40 50
Females (%)
F. 3. Sex ratios of the dierent families at 15 months of age after the completion of sex dierentiation.
Numbers in the squares represent the sex ratios (males : females) to which the progenies of each
family could be fitted. Numbers inside the bars represent the P value from the likelihood ratio test
for each of the families. Intervals are: (049), (5099), . . ., (550599).

space allocation. Furthermore, in species with a 1 : 1 male : female sex ratio, the
chance of selecting a neomale for the progeny test is 1 in 2 but in this study, with
21% females in the untreated groups, the chances were essentially 1 in 5. For this
reason we had to use 13 crosses (thus 13 aquaria) to have a 95% probability of
having at least one neomale in the P generation and most probably between two
and three.
No sex ratios indicative of any known chromosomal sex determination
mechanism were found in any of the 13 progenies examined. A polygenic sex
determination mechanism in the sea bass seems unlikely because it would result
in a high variation of the sex ratios among successive progenies originating from
the same progenitors (Kallman, 1984), something that is not observed. It is
interesting to note that not all the families exhibited similar sex ratios between
the two sampling times, as in families F2 and F11. In these families, the
percentages of females were 23 and 36 at 11 months, and 5 and 22 at 15 months,
respectively. This suggests either that the number of females decreased, i.e. that
some females dedierentiated , which we find unlikely, or that these values are
perhaps distorted by the amount of intersex and/or undierentiated fish present
in the sample at 11 months. Similarly, the proportion of intersex fish in the
dierent families was not consistent between the two sampling times (i.e. F13 had
14% and 42% intersexes at 11 and 15 months, respectively). Although sampling
errors cannot be completely ruled out, they seem very unlikely as several gonadal
924 . .

100
a
75 18.0 0.23 Females 79.8 3.23a
n = 98
50

25

75 16.6 0.20b Intersexes 63.1 2.46b

n = 84
50

25

0
Frequency (%)

75 15.8 0.12c Males 51.2 1.29c

n = 151
50

25

0
14.5 0.11d Males* 38.5 1.05d
75 n = 57
50

25

0
14.1 0.14d Undifferentiated 36.2 1.17d
75 n = 69

50

25

0
10 15 20 25 0 50 100 150 200 250
Length (cm) Weight (g)
F. 4. Frequency histogram of length and weight in 11 month old sea bass according to phenotypic sex.
The sex and the number of fish (n) are represented in the central part of the figure. Mean weight
and length... are represented on top of each histogram. For each variable (length and
weight), dierent superscripts indicate statistical dierences between sexes (ANOVA; P<005).
Males*, Dierentiating males.

cross-sections were observed from each fish when assessing the gonadal sex, to
ensure that intratesticular oocytes would not be missed if present. The presence
of males with intratesticular oocytes has already been described in cultured sea
bass (sometimes up to 34%; Brusle & Roblin, 1984), being attributed to a
possible juvenile intersexuality. In fact, Dicentrarchus labrax L. was traditionally
part of the family Serranidae but was the only gonochoristic species in a family
otherwise consisting entirely of hermaphrodites. This species was later included
in the family Moronidae (gonochoristic) to overcome this problem (Tortonese,
 925

50
22.4 0.21a Females 147.3 4.60a
40 n = 117
30

20

10
0
ab
40 21.6 0.26 Intersexes 126.6 4.99b
Frequency (%)

n = 64
30

20

10

40 21.1 0.14b Males 121.8 2.55b

n = 263
30

20

10

0
15 20 25 30 75 150 225 300 375
Length (cm) Weight (g)
F. 5. Frequency histogram of length and weight in 15 month old sea bass according to phenotypic sex.
The sex and the number of fish (n) are represented in the central part of the figure. Mean weight
and length... are represented on top of each histogram. For each variable (length and
weight), dierent superscripts indicate statistical dierences between sexes (ANOVA; P<005).

1986). However, from an evolutionary point of view, the sea bass is still very
close to a family of hermaphrodites.
If a XX/XY sex determination mechanism was operating in the sea bass, then
the progeny obtained from mating a neomale (XX) with a normal female (XX)
should have been all-female or at least mostly females, if it is assumed that some
genetic females may develop as males (see below). However, this was not the
case in any of the 13 families despite a 95% probability that at least one neomale
was among the 13 selected males for the progeny test. A previous study
performed by Cano et al. (1996) suggested that the likely type of mechanism of
sex determination in the sea bass was XX/XY. This was based on the dierent
pattern of distribution of heterochromatin in both homologues from chromo-
some pair 24. However, due to the small size of fish chromosomes this technique
is not as specific as it is for other vertebrates (Gold et al., 1990). Studies using
other approaches such as the induction of gynogenesis and the study of sex ratios
in gynogenetics have been inconclusive in ascribing a XX/XY sex determination
mechanism to the sea bass. Female proportions in the gynogenetic groups varied
from 30% to 80% (Colombo et al., 1996; Gorshkova et al., 1996; Felip, Carrillo,
Zanuy & Piferrer, unpublished data), but 100% was never achieved as would be
expected from a species with an XX/XY system. Together, these results support
the idea that an XX/XY system does not operate in the sea bass.
926 . .

Similarly, if a ZW/ZZ sex determination system was operating, the progeny

resulting from mating a neomale (ZW) with a normal female (ZW) should render
a 1 : 3 or 1 : 2 male : female sex ratio, depending on the viability of WW
individuals. However, a ZW/ZZ system is more likely to explain the results of
family F9 rather than the usual sex ratios of sea bass. Thus, males exhibiting
intratesticular oocytes could be genetic females whose phenotype had been
modified by environmental factors. Keeping this in mind, if F9 females and
intersexes are considered genetic females and grouped together, then the female
proportion increases to 60% and adjusts to a 1 : 3 sex ratio, the result that one
would expect from a ZW/ZZ system. In this regard, an unusual ZW/ZW /ZZ
system has been proposed for the cyprinid Scardinius erythrophthalmus L.
(Koehler et al., 1995). It was postulated that the ZW females could in fact be
sex-reversed ZZ males. Accordingly, genetic ZW females would dierentiate
into phenotypic males whereas ZW females would not undergo sex change.
Another possibility that cannot be ruled out is a dierential mortality towards
the females. If a high female mortality occurs during early development (i.e.
6555% of the females die), the results obtained from the progeny tests are as
expected from a ZW/ZZ system. Assuming that F9 is the sole sex-reversed male
family, which itself has the reasonable prior probability of 161%, the 1 : 3 ratio
of males to females at birth would become a 1 : 1 ratio after the death of some
females (5157%). The same would apply for the rest of the crosses, where no
neomale would theoretically be involved, resulting in 2631% females after death
that fits into a 3 : 1 or a 2 : 1 ratio of males to females, also consistent with the
same ZW/ZZ system.
Some studies aiming to identify the sex determination mechanism in other
species by selective breeding also had unexpected results. Such is the case of
Oncorhynchus tshawytscha (Hunter et al., 1983), for which some of the families
obtained from the progeny tests that should have rendered all-female popula-
tions contained variable male proportions. Similar results were reported for
Oreochromis niloticus (Calhoun & Shelton, 1983) and O. aureus (Hopkins et al.,
1979; Mair et al., 1987), in which a possible environmental eect on the sex ratios
was suggested to be responsible for the presence of some females in what should
have been an all-male population. The male-biased sex ratios found in most
families, and usually reported in cultured sea bass, could then be related to the
influence of environmental factors as is the case with reptiles (reviewed by Pieau
et al., 1994) and some fish (reviewed by Francis, 1992; Bla zquez et al., 1998a). In
this regard, it has been shown recently that a low water temperature (15 C)
induced an all-male population in the sea bass also resulting in delayed growth
(Bla zquez et al., 1998b). This led to the hypothesis that the eect of temperature
was not direct but mediated through growth. The present study shows a clear
relationship between phenotypic sex and growth. However, a dierent exper-
imental approach should be taken to determine a possible growth-mediated
eect of temperature on the final phenotypic sex in this species.
It has been proposed that in fish with a long sexually undierentiated period,
the length attained by individuals at a certain age seems to be the determinant for
sex dierentiation (Hunter & Donaldson, 1983). Sea bass exhibit a long sexually
undierentiated period comprising most of the first year of life (Roblin & Brusle ,
1983; Bla zquez et al., 1995). Our growth data suggest that the beginning and
 927

perhaps even the course of sex dierentiation in this species could be related
more to the attained length rather than the age, since at 11 months undierenti-
ated fish coexist with dierentiated fish, the former being always significantly
shorter in length than the latter. Moreover, females were always the biggest
individuals since they always comprised the upper classes of the distribution
either in length or weight in contrast to undierentiated fish, which exhibited the
smallest size. In this regard, a study performed by Francis & Barlow (1993) with
Cichlasoma citrinellum (Gu nther) showed that the attained length of the fish was
a determinant of the future sex of the individuals. Moreover, it has been shown
recently that the sex ratio in Paralichthys olivaceus (Temminck & Schlegel) can
be greatly aected by the existence of slower-growing fish within the population
(Tabata, 1995).
One aspect that deserves to be mentioned is the intermediate size of intersexes
as compared with males and females. This implies that a sea bass, for the sole
reason of having intratesticular oocytes, reaches a size between that of a male
and that of a female, albeit closer to that of the male. It would seem that the
presence of oocytes in the testes could not account for this phenomenon and thus
its significance is not apparent. However, it might be that sea bass genetic
females grow more than genetic males, independent of phenotypic sex.
Accordingly, intersexes would represent those genetic females that, for still
unknown reasons, develop like males (the intratesticular oocytes being a
reminiscent sign of their genetic sex) but without completely suppressing their
potential growth as females. This idea could be seen as an apparent contradic-
tion between grouping males and intersexes together when analysing the sex
ratios. However, it is strictly correct as the sex ratios were calculated on the basis
of the gonadal phenotype rather than the genotype of the individuals, and the
intersex gonads were essentially phenotypic testis with some intratesticular
oocytes. Nevertheless, the presence of intersexes reported in this study supports
and strengthens the hypothesis of a possible environmental factor modulating
sex dierentiation in this species.
In summary, the present study did not reveal the mechanism of sex determi-
nation of the sea bass since no sex ratios indicative of a particular system were
observed. However, it established the relationship between growth and sex
dierentiation, demonstrating that even before sex dierentiation is complete,
females predominate among the larger fish, while males predominate among the
smallest. A purely genetic basis of sex determination cannot be overruled despite
that, to the best of our knowledge, there is no known simple Mendelian
mechanism capable of giving a 3 : 1 male : female sex ratio generation after
generation. Although a dierential mortality towards the females cannot be
excluded, the absence of a genetic sex-linked marker makes it dicult to assess at
present. The possibility of environmental factors modulating a genetic system
should also be considered to explain male-biased sex ratios. However, trying
to separate intrinsic genetic factors from environmental ones is not easy.
Therefore, future experiments will need to follow the molecular approach, not
successful so far in the sea bass (Martnez et al., 1999), but fruitful in the
isolation of sex-specific DNA sequences and other genetic markers in birds
(Ogawa et al., 1997), reptiles (Ganesh et al., 1997), and some fish such as
Oncorhynchus tshawytscha (Devlin et al., 1991, 1994), Leporinus elongatus
928 . .

Valenciennes (Nakayama et al., 1994), the brown trout Salmo trutta L. (Prodo hl,
1994), the channel catfish Ictalurus punctatus (Liu et al., 1996), and the medaka
Oryzias latipes (Temminck & Schlegel) (Matsuda et al., 1997) to determine
further the possible role of the environment in the phenotypic sex.

We thank J. Monfort for technical assistance and Sepia International for providing the
facilities to rear the fish. M.B. was supported by a scholarship from the Generalitat
Valenciana. This work was supported by an EU grant AIR2-CT93-1543 and CICYT
grants AGF93-0127 and AGF94-1321-CE to S.Z.

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