Journal of Fish Biology (2002) 61, 347359

doi:10.1006/jfbi.2002.2032, available online at http://www.idealibrary.com on

Growth, gonadal development and sex ratios of

meiogynogenetic diploid sea bass
A. F, F. P, M. C S. Z*
Consejo Superior de Investigaciones Cientficas (CSIC), Instituto de Acuicultura de
Torre la Sal, 12595 Ribera de Cabanes, Castellon, Spain

(Received 15 June 2001, Accepted 30 May 2002)

Gynogenesis showed little eect on general physiology and gonadal development in sea bass
Dicentrarchus labrax. Meiogynogenetic fish showed well-developed gonads indicating low
occurrence of developmental imbalances even after gynogenesis induction in this species. In
addition, the proportion of sexes of meiogynogenetic sea bass was similar to the diploid
controls in two independent trials, which did not deviate significantly from a 1 : 1 male : female
sex ratio. Even considering some environmental influence on sex dierentiation, as has been
previously demonstrated, the fact that the proportion of sexes was similar between gynogenetic
and control diploids essentially eliminates the possibility that in the sea bass the females are the
homogametic sex. Although the mechanism of sex determination of this species still remains
unknown, even after gynogenesis induction, the genetic mechanism of the ZW/ZZ type could
probably operate in the sea bass.
 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved.

Key words: Dicentrarchus labrax; growth; gonadal development; meiogynogenesis; sea bass;
sex determination; sex ratio.

INTRODUCTION
Fishes exhibit all sex determining mechanisms known in vertebrates (Hunter &
Donaldson, 1983). These include: (a) gonadal sex is controlled by sex chromo-
somes, including two monofactorial systems (XX/XY or ZW/ZZ) and six
systems having multiple sex chromosomes (Tave, 1993); (b) the polygenic system,
in which sex is also controlled by a number of autosomal genes (Tave, 1993); (c)
environmental sex determination, particularly temperature-dependent (Guiguen
et al., 1999). The sex chromosomes of some species are morphologically distinct,
but most fish species lack recognizable heterochromosomes (Tave, 1993).
Cytological techniques based on dierential banding are often not suitable to
elucidate the sex determination mechanism in fishes (Cano et al., 1996), and thus
other strategies are used. These include sex-reversal studies (Piferrer, 2001),
chromosomal manipulation (Felip et al., 2001), or analysis of sex-linked DNA
sequences (Devlin et al., 1991; Nakayama et al., 1994; Coughlan et al., 1999).
In many species, chromosomal manipulation based on the induction of
gynogenesis (i.e. exclusive maternal inheritance with no genetic contribution of
paternal chromosomes) has resulted in all-female progenies, proving that the
*Author to whom correspondence should be addressed. Tel.: +34 964 319500; fax: +34 964 319509;
email: zanuy@iats.csic.es
Present address: Institut de Cie`ncies del Mar, CSIC, Passeig Martim, 37-49, 08003 Barcelona, Spain.
347
00221112/02/080347+13 $35.00/0  2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved.
348 . .

female was the homogametic sex. Even in species with a XX/XY system,
however, various proportions of gynogenetic males have sometimes been
recorded (Felip et al., 2001). Hence, in zebra fish Danio rerio (Hamilton)
and common carp Cyprinus carpio L., gynogenetics not only showed a high
variability in the percentage of sexes but a proportion of males was also obtained
when they were mated with homogametic females (Komen & Richter, 1983;
Thorgaard, 1983). These results suggest the presence of a minor female sex gene
located on a sex chromosome or autosome, or that environmental factors may
be influencing the sex determination mechanism (Komen & Richter, 1983;
Thorgaard, 1983). Understanding the way sex is determined is important
because this knowledge can be useful in the production of monosex populations
to improve the production of cultured species, for the conservation of aquatic
biological diversity and for the control of populations (Piferrer, 2001).
The sea bass Dicentrarchus labrax (L.) is a well established species in Europe
for both basic and applied research. Despite the fact that several experimental
approaches have been used, the sex determination mechanism has not yet been
elucidated. Past studies have included: cytological characterization of the
chromosomes using dierent staining techniques (Cataudella et al., 1978;
Arefyev, 1989; Vitturi et al., 1990; Sola et al., 1993; Cano et al., 1996); progeny
testing of sex-reversed fish, i.e. obtaining all-male populations by androgen
treatment and the use of their sperm to fertilize normal females (Blazquez et al.,
1999); development of DNA probes as markers of sex-specific DNA sequences
using restriction fragment length polymorphisms (RFLPs) and subtractive
hybridization (Martnez et al., 1999). An alternative approach is the identifi-
cation of the sex ratios in gynogenetics. Preliminary studies conducted on the
induction of gynogenesis in the sea bass have reported in some instances the
percentage of females obtained (4282%) (Colombo et al., 1997). The assess-
ment of the true gynogenetic nature of the fish used and sexed was not done in
these studies. The present study on the sexuality of gynogenetic sea bass has
been carried out after fulfilling two essential conditions: (1) using an optimized
method for the induction of meiogynogenesis in this species, as described
previously (Felip et al., 1999a) and (2) confirming, by AFLP (amplified fragment
length polymorphism) markers, the exclusive maternal genomic contribution in
induced meiogynogenetics and thus establishing their true gynogenetic origin
(Felip et al., 2000). Besides assessing the sex ratios of gynogenetic sea bass
obtained in two independent trials, this study also examined the growth, gonadal
development and some key body indices of these fish.

MATERIAL AND METHODS

PRODUCTION OF MEIOGYNOGENETIC DIPLOIDS AND PARENTAL
TESTING
During the reproductive season (JanuaryFebruary), sea bass female broodstock
were intraovarically cannulated to determine the stage of oocyte development. After
hormonal treatment to induce final oocyte maturation and ovulation in females, only
those producing eggs of good quality were used (Felip et al., 1999a). Two male and two
female adult sea bass were chosen for this experiment. Gametes were obtained by
stripping. The milt of each male (M) was diluted with an appropriate extender (Billard,
1984) and used without or after UV irradiation to fertilize the eggs of one female (F).
 349

Two families (G1, G2) originating from each dierent male-female pair cross (G1, M1-F1
and G2, M2-F2) were produced. In turn, each family was divided into two groups: (1) one
served as the diploid controls and (2) in the other gynogenesis was induced. Each diploid
and meiogynogenetic sea bass group from each family was reared in duplicate tanks and
maintained under natural conditions of photoperiod and temperature [eight tanks in
total=two families with two experimental treatments per family (diploids and
gynogenetic diploids) and two replicates per experiemental treatment].
Meiogynogenesis was induced by genetically inactivating diluted sperm with UV-light
at 35 00040 000 erg mm
2 before fertilization (Felip et al., 1999a). Diploidy was
restored by applying a cold shock of 0 C for 10 min starting 5 min after fertilization
(Felip et al., 1997). The assessment of the gynogenetic origin was done by AFLP
fingerprinting, confirming the induction of 95% of true meiogynogenetic sea bass in both
families (5% of fish were nongynogenetic diploid individuals among gynogenetic fish), as
previously reported (Felip et al., 2000).

GROWTH PERFORMANCE
Fish were fed according to standard procedures, including rotifers and Artemia as live
food organisms during larval rearing with a prey density of 110 ml
1 (on days 415).
On day 16, larvae were fed with Artemia nauplii enriched with essential nutritive
components (Super Selco, Artemia Systems N.V., Belgium). Fry were weaned progres-
sively on c. day 60 with inert feed of the appropriate size (Inve Aquaculture N.V.,
Baasrode, Belgium and Ewos S.A., Madrid, Spain). All fish were hand fed one to four
times a day and maintained in 200 l tanks provided with slow-flowing, filtered,
UV-sterilized and aerated sea water in a closed system. At 45 months of age, fish were
transferred to 500 l circular fibreglass tanks. Each tank was provided with a seawater
inlet at the top and one to four oxygen inlets at the bottom to aerate the sea water.
Between 5 to 24 months of age, fish were periodically weighed (nearest 001 g) and fork
length (LF) measured (nearest mm) to determine growth performance. The number of
diploids and meiogynogenetics was 4042 fish per replicate per treatment in G1 and 5259
fish per replicate per treatment in G2.

GONADAL DEVELOPMENT, SEX RATIOS AND BODY INDICES

Fish were sampled at two previously determined ages, when the histological sex
dierentiation was being completed, at 1011 months of age, and later, at 2324 months
of age (Blazquez, 1996). The proportion of sexes and gonadal development was
determined in these two samples. When possible, the sex was determined macro-
scopically, and then verified on a fish by histological analysis. Histological procedures
were carried out according to conventional techniques using the Cleveland Wolfes
methodology for staining (Herlant, 1960). Gonadal cell stages were classified according
to previous morphological studies of the gametogenesis in female (Mayer et al., 1988) and
male (Rodrguez et al., 2001) sea bass. The gonado-somatic (IG), hepato-somatic (IH)
and fat visceral index (IVF) (gonad, liver and fat mass, respectively, expressed as
percentages of total body mass), were measured when fish were 2324 months old.

STATISTICAL ANALYSIS
Data are expressed as mean... from two replicates for diploid and
meiogynogenetic fish in the G1 and in the G2 families. Growth performance was analysed
at each sampling date by using a two-way ANOVA test and a Barletts test for the
homogeneity of variances. Percentages of IG, IH and IVF were arc sine transformed
before statistical analysis. A t-test was used to compare the body indices between
diploids and meiogynogenetics. Since no significant dierences were found between
males and females, no sex distinctions were considered for these analysis. A
2 test was
used, however, to determine significant dierences in sex ratios between diploid and
meiogynogenetic sea bass. Significant dierences were accepted when P<005.
350 . .

(a) (b)
400 35

30
Group 1
300
25

20
200
15

10
100
5
Mass (g)

LS (cm)
0 0
(c) (d)
400 35

30
Group 2
300 25

20
200
15

10
100
5

0
0 5 10 15 20 25 0 5 10 15 20 25
Age (months)
F. 1. Changes in body mass (a) (c) and fork length (b) (d) of diploid () and meiogynogenetic () sea
bass derived from two independent crosses from 5 to 24 months. Mean... from two replicates
per ploidy and group. No significant dierences were observed (P>005).

RESULTS
Previously, Felip et al. (1999a) have shown that, under laboratory conditions,
the mean survival of normal meiogynogenetic larvae at hatching was 35% of the
diploid control group. Accurate calculation of survival was not possible in the
present study due to the large number of eggs required for these experiments
(175250 ml of eggs; 1 ml contains c. 750 eggs). Nevertheless, the expected lower
survival of meiogynogenetic larvae in the G1 and G2 families was noted, which
decreased significantly immediately after hatching in comparison to their respec-
tive diploid controls. Diploid and meiogynogenetic fish completed the phases of
larval rearing and weaning succesfully and as soon as larvae were weaned,
survival of meiogynogenetic and diploid control sea bass was essentially similar
throughout this experiment.
No significant dierences in growth between diploid and meiogynogenetic
sea bass were observed during the first 2 years of age both in G1 [Fig. 1(a), (b)]
and in G2 [Fig. 1(c), (d)]. At 24 months of age, the diploid controls in the G1
were 361791764 g and 29405 cm while meiogynogenetics of the same
progeny were slightly smaller (345651667 g and 28405 cm), although
statistical analysis showed no significant dierences (P>005). In the G2 progeny
at the same age, meiogynogenetics were 214222038 g and 23408 cm
in comparison to diploids of 267552101 g and 25708 cm (P>005).
 351

T I. Gonado-somatic, hepato-somatic and visceral fat indices in two groups of diploid

(control) (Ctrl) and meiogynogenetic diploid (Mgy) sea bass at 2324 months of age

Group n IG IH IVF P

Ctrl (G1) 26 171028 225010 241014 P>005

Mgy (G1) 26 244052 244011 279018
Ctrl (G2) 19 169025 213016 256029 P>005
Mgy (G2) 18 117027 193012 264026

Nevertheless, although body mass and LS increased gradually in both progenies,

growth performance in the G1 was greater than in the G2 under the same natural
conditions of photoperiod and temperature after 2 years. At 2324 months of
age, there were no significant dierences in IG, IH and IVF between diploids and
meiogynogenetics regardless of the family considered (P>005) (Table I).
The gonads of the control and meiogynogenetic sea bass at 1011 months of
age in the G1 and G2 families could be divided into four categories: undier-
entiated, intersexes, males and females (Table II). The undierentiated gonads
consisted mainly of connective tissue and scattered germ cells [Fig. 2(a)]. Those
gonads exhibiting testicular lobules filled with spermatogonia and two or more
scattered intratesticular oocytes per cross section were classified as intersexes
[Fig. 2(b)]. Gonads composed of testicular lobules with spermatogonia, includ-
ing mitotic proliferation and a complete absence of oocytes were classified as
testes and were of a similar appearance in diploid control groups [Fig. 2(c)] and
meiogynogenetic diploid groups (Fig. 2(d)]. The ovaries of females were
characterized by the presence of oogonia, chromatin nucleolus oocytes and
oocytes at the early perinucleolus stage arranged in lamellae projecting into a
central lumen both in diploid controls [Fig. 2(e)] as well as in meiogynogenetics
[Fig. 2(f)].
At 2324 months of age, intersexes were not observed in any group, however,
fish with undeveloped gonads were observed (sexual dierentiation in the sea
bass is completed at c. 1011 months of age, older fish whose gonads showed
unrecognizable male or female characteristics were considered as undeveloped).
Thus in G1, 8% of controls and 154% of meiogynogenetics had undeveloped
gonads. In G2, one meiogynogenetic fish (55%) had undeveloped gonads
whereas the control fish were sexually dierentiated (Table II). On the other
hand, well-developed testes were filled with mature spermatozoa in both controls
[Fig. 3(a)] and meiogynogenetics [Fig. 3(b)]. The ovaries of diploid control fish
[Fig. 3(c)] contained many immature nonvitellogenic oocytes with scattered
early vitellogenic oocytes. Furthermore, some oocytes showed an advanced
vitellogenic stage with numerous lipid vesicles and yolk granules. Ovarian
development in meiogynogenetic females [Fig. 3(d)] was similar to diploids,
although one out of 12 females in the G1 exhibited small and thread-like ovaries,
with oogonia and some oocytes in the early meiotic stage.
Since females complete sexually dierentiation earlier (c. 9 months) than males
(c. 11 months) in this species (Blazquez, 1996), fish with undierentiated gonads
in the first sample (1011-month-old fish) were considered as putative males. On
T II. Phenotypical categories of histological cross-sections of gonads in two groups of diploid control (Ctrl) and meiogynogenetic (Mgy)
sea bass at 1011 and 2324 months of age. Numbers of fish (percentage) are given for each category and sex ratios

Age Undierentiated Undeveloped Intersexes Males Females

Group Fish1 Sex-ratio2
2 P
(months) n n n n n

Ctrl (G1) 11 39 6 (154) 0 2 (51) 11 (282) 20 (513) 1:1 017 >005

Mgy (G1) 33 2 (61) 0 2 (61) 12 (364) 17 (515) 1:1
Ctrl (G1) 23 26 0 2 (8) 0 12 (46) 12 (46) 1:1 017 >005
Mgy (G1) 26 0 4 (154) 0 10 (385) 12 (461) 1:1
Ctrl (G2) 10 58 23 (397) 0 0 9 (155) 26 (448) 1:1 131 >005
Mgy (G2) 59 25 (424) 0 1 (17) 10 (169) 23 (39) 1:1
Ctrl (G2) 24 19 0 0 0 9 (474) 10 (526) 1:1 119 >005
Mgy (G2) 18 0 1 (55) 0 9 (50) 8 (445) 1:1

1
Number of sacrificed fish. 2The sex ratio is computed considering undierentiated and intersex fish as males at 1011 months of age and males and females with
well-developed gonads at 2324 months of age. Fish with undeveloped gonads were not considered as sex could not be determined.
 353

F. 2. Photomicrographs of gonads from diploid and meiogynogenetic sea bass at 1011 months of age.
(a) Undierentiated gonad, (b) intersex, (c) testis of a diploid male, (d) testis of a meiogynogenetic
male, (e) ovary of a diploid female and (f) ovary of a meiogynogenetic female. Bar 50
m.

the other hand, intersexes were considered as males, since their gonads were
composed mostly of testicular tissue (Blazquez et al., 1999). Accordingly, the
proportion of males was calculated computing undierentiated and intersex fish
as males. Hence, the sex ratio in the G1 and in the G2 at 1011 months of age
was 1 : 1 male : female for both control and meiogynogenetic fish (Table II).
Similar results were observed at 2324 months of age (Table II).

DISCUSSION
This work is an attempt to identify the mechanism of sex determination in the
sea bass by gynogenesis induction. It is the first time that the proportion of sexes
354 . .

F. 3. Photomicrographs of testes from diploid (a) and meiogynogenetic (b) male sea bass at 2324
months of age exhibiting active spermatogenesis. Note the formation of primary spermatocytes
(ps), secondary spermatocytes (ss), spermatids (sp) and spermatozoa (sz). Ovary of diploid (c) and
meiogynogenetic (d) female sea bass at 2324 months of age, containing perinucleolar oocytes (po)
and vitellogenic oocytes (vo). Bar 50
m.
 355

is analysed in true gynogenetic sea bass. That is, in contrast to the early studies,
in this work the individuals sexed were previously demonstrated to have
exclusive maternal inheritance. This study shows unequivocally that in sea bass
the females are not the homogametic sex and on the other hand it reports
information concerning growth performance of gynogenetically produced fish,
which is available only for a few species.
Gynogenetic ospring of several fish species typically exhibit lower develop-
mental and survival rates than their corresponding diploid controls. This is
probably due to the expression of deleterious recessive alleles, i.e. inbreeding
eects plus a higher homozygosity when compared to diploids (Leary et al.,
1985; Don & Avtalion, 1988; Ihssen et al., 1990). Most studies on the artificial
induction of gynogenetic diploids, however, are terminated just after enough
time has passed for the elucidation of the genetic sex. Lower growth perform-
ance of gynogenetic diploids in comparison with diploid controls has been
reported in Misgurnus anguillicaudatus (Cantor) (Suzuki et al., 1985) and Salmo
salar L. (Johnstone & Stet, 1995). In Pagrus major Temminck & Schlegel
(Sugama et al., 1990), Clarias macrocephalus Gunther (Na-Nakorn, 1995) and
Gnathopogon caerulescens Sauvage (Fujioka, 1998) similar growth was obtained
between gynogenetics and controls. In this study, there were no significant
dierences between the diploid control and the meiogynogenetic sea bass
originating from the same breeders, but there were significant dierences
between the performance of progenies originating from dierent breeders. This
suggests that observed dierences in growth between the two progenies may be
due to genetic factors from individual females (Sugama et al., 1990; Fujioka,
1998), indicating that phenotypic growth is more dependent on maternal eects
than on eects of induction in the gynogenetics. On the other hand, despite the
fact that fish were produced under optimal treatment conditions and reared
under standard procedures, the number of viable fish was low. This is probably
due to the lower survival of marine fishes, with fragile gametes, with respect to
that of freshwater species with stronger gametes (Planas & Cunha, 1999).
In the present study, the body indices measured at 2 years of age showed
similar values between diploid control and meiogynogenetic sea bass. This
suggests that their higher inbreeding does not interfere with their performance,
showing a normal process of the accumulation of fat reserves for maturation as
well as an adequate function of the liver for the synthesis of vitellogenin. As far
as is known, no previous information existed on comparing body indices of
diploid and gynogenetic diploid fishes.
Regarding gonadal development, the presence of intersexes has been reported
previously in this species (Brusle & Roblin, 1984; Blazquez et al., 1999). The
number of fish with undierentiated gonads at 1011 months suggests that
although complete histological gonadal dierentiation in this species occurs at
c. 9 months in females and c. 11 months in males (Blazquez, 1996), maternal
eects and genetic factors as well as the size of the fish and environmental
conditions may be delaying this process, depending on the stock. Furthermore,
the presence of undeveloped gonads in 2324 month old diploid fish may indicate
alterations in the gonadal development under natural conditions.
Gonadal morphology of gynogenetic sea bass showed a normal development
of the gonads in this species. Gynogenetic female sea bass showed that they were
356 . .

able to produce eggs with an excellent quality after ovarian stimulation by

hormonal treatment (unpubl. data). These results are in agreement with what
has been observed in other species, however, there are studies where alterations
in the gonadal development of gynogenetic fishes is reported as a consequence of
inbreeding eects or to the presence of deleterious recessive genes related with
reproduction (Piferrer et al., 1994; Fujioka, 1998). This has been observed in C.
carpio (Nagy et al., 1978; Komen et al., 1992), Oncorhynchus kisutch (Walbaum)
(Piferrer et al., 1994) and G. caerulescens (Fujioka, 1998). By contrast, eects of
inbreeding on gonad development of gynogenetic M. anguillicaudatus (Suzuki
et al., 1985) and C. macrocephalus (Na-Nakorn, 1995) were not extreme and
females had an apparently normal ovulation response after hormone injection.
In a XX/XY system, all-female progenies are expected when gynogenesis is
induced. Thus, neomales (masculinized females) mated with normal females
(XX) would be expected to produce all females. On the other hand, in a ZW/ZZ
system a higher proportion of females would be also expected, although the
viability of the WW super-female has been considered low (Yamazaki, 1983).
Induced gynogenetic diploids of many species are all or almost all females
(Felip et al., 2001). Even in species with a female homogametic system
(XX/XY), however, the presence of gynogenetic males have been recorded. The
proportions ranged from 51 to 100% (Felip et al., 2001).
The present results showed that the proportion of sexes in the diploid controls
was 1 : 1 (male : female). Although a high proportion of males (3 : 1) has been
usually observed in several hatcheries at dierent places (Colombo et al., 1997)
as well as under laboratory rearing conditions (Carrillo et al., 1995; Blazquez,
1996; Felip et al., 1999b), other sex ratios including 2 : 1, 1 : 1 or even 1 : 4 have
been reported in diploid control sea bass (Colombo et al., 1997). The proportion
of sexes in the meiogynogenetics was similar to the diploid controls in the two
progenies analysed (1 : 1) in this study. By contrast, the percentages of females
and males of meiogynogenetic sea bass observed in other experimental locations
varied considerably (range 4282%) (Colombo et al., 1997). The production of
a second generation of gynogenesis with potential viable WW females would
result in a monosex female production, providing evidence for female
heterogamety. Based on sex determination studies in fishes, temperature seems
to influence this mechanism (Guiguen et al., 1999). A study in the sea bass has
indicated that the rearing temperature aects sex ratio in this species (Blazquez
et al., 1998; Pavlidis et al., 2000). Thus, genetic factors in combination with
environmental factors may give an explanation to the unequal proportions of
sexes observed in this species.
This paper has evaluated the growth performance of gynogenetic sea bass as
well as the gonadal morphology of these fish. Taking into account the available
data plus data presented here, the situation regarding sex determination in the
sea bass can be summarized as follows: (1) some data suggest the existence of sex
chromosomes in this species (Cano et al., 1996); (2) there are also data indicating
that in this species temperature can aect the process of sex dierentiation
(Blazquez et al., 1998; Pavlidis et al., 2000); (3) in crosses involving sex-reversed
females with normal females no all-female stocks were obtained (Blazquez et al.,
1999); (4) gynogenetics had the same sex ratio than their corresponding controls
(this study). Together, the information available so far indicates that sea bass
 357

probably has a chromosomal system of sex determination that can be aected by

the environment. It is unlikely, however, that the system is of the XX/XY type.
Hence, ZW/ZZ sex determination mechanism is proposed although, further
studies are required to confirm the mechanism of sex determination in this
species.

The authors wish to thank J. Ramos for his help in the production of gynogenetic sea
bass. We especially thank C. Marin for rearing the fish and for his help in sampling
and P. Monfort for his help with histology. This research was supported by EC grant
No. AIR2-CT93-1543 and by CICYT grant No. AGP94-1321-EC to S.Z. A.F. was
supported by a grant from the Government of Valencia.

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