Aquacult Int (2008) 16:203–214
DOI 10.1007/s10499-007-9138-6
Gynogenesis and sex determination in large-scale loach
Paramisgurnus dabryanus (Sauvage)
Cuihong You Æ Xiaomu Yu Æ Deqing Tan Æ Jingou Tong
Received: 8 March 2007 / Accepted: 5 October 2007 / Published online: 8 November 2007
Ó Springer Science+Business Media B.V. 2007
Abstract Gynogenesis was induced using heterologous sperms in large-scale loach,
Paramisgurnus dabryanus (Sauvage), in which a ZW/ZZ sex determination was previously
proposed. Three microsatellite loci were used to monitor exclusive maternal inheritance of
gynogenetic progenies. The results showed that high percentages of meiogynogens were
produced at 4 min post-fertilization and mitogynogens were produced at 18 min postfertilization by heat shocks, while meiotic gynogenesis was induced by cold shocks within
a wide period and high heterozygosity was even observed in gynogens produced at 24 min
post-fertilization. The sex ratios of the F1 progenies in three gynogenetic families were
significantly deviated from 1:1 expectation with a female bias in two families and a male
bias in one family (P \ 0.05), and the other four gynogenetic families showed approximate
1:1 sex ratios. Moreover, the self-mating between gynogenetic F1 progenies and mating
between gynogenetic F1 progenies and normal individuals produced all-female progenies
or identical proportions of females and males. The data of sex ratios generally confirmed
that the sex determination in large-scale loach was determined by the putative ZW/ZZ
system, and the possible reasons causing the biased sex ratios are discussed.
Keywords Large-scale loach (Paramisgurnus dabryanus) Temperature shock Gynogenesis Sex ratio Sex determination
Introduction
In fish, it is rarely possible to characterize sex determination cytogenetically due to the low
occurrence of heteromorphic sex chromosomes (Devlin and Nagahama 2002). As small
food fish, loaches have been caught and consumed over thousands of years in China, and
C. You X. Yu D. Tan J. Tong (&)
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology,
The Chinese Academy of Sciences, Wuhan 430072, China
e-mail: jgtong@ihb.ac.cn
C. You
Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
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cultivation and farming of loaches in pond or cages have been encouraged over the past
two decades. Because female loaches usually grow faster than males, the development of
all-female loaches in aquaculture should increase production and bring more profits to
farmers. Large-scale loach, Paramisgurnus dabryanus (Sauvage) (Cobitidae, Cypriniformes), is endemic to mainland China and Taiwan. This loach species was proposed to
exhibit female heterogamete (ZW/ZZ) sex determination (Chang and Yu 1997). Sox genes
were also investigated in this species (Chang et al. 2000a, b). However, this assertion of a
possible chromosomal mechanism for sex determination in large-scale loach remains to be
supported by other evidence.
Gynogenesis is a mode of reproduction whereby offspring inherit only female chromosomes. It involves two steps: (1) elimination of paternal chromosomes, i.e., activation of
eggs with irradiated homologous or heterologous sperms, and (2) restoration of diploidy by
a physical or chemical shock, retaining the second polar body (meiotic gynogenesis) or
suppression of the first mitotic cleavage (mitotic gynogenesis). The former produces
homozygous offspring at a locus only if no recombination occurred, and the latter results in
fully homozygous offspring (Onozato 1984). Gynogenesis has been widely used in the
studies of fish genetics and breeding such as establishment of inbred lines, production of
all-female fish, and the investigation of sex determination (Rougeot et al. 2005; Gomelsky
2003; Felip et al. 2001). By inducing gynogenesis and scoring sex ratios of the progenies,
some fishes were determined to exhibit XX/XY sex determination, e.g., channel catfish
(Ictalurus punctatus), common carp (Cyprinus carpio), silver carp (Hypophthalmichthys
molitrix), and pond loach (Misgurnus anguillcaudatus) (Devlin and Nagahama 2002;
Nomura et al. 1998; Morishima et al. 2001), while others, e.g., white sturgeon (Acipenser
transmontanus) and beluga sturgeon (Huso huso), were believed to exhibit ZW/ZZ sex
determination (Van Eenennaam et al. 1999; Omoto et al. 2005).
This paper reports optimal conditions to induce meiotic and mitotic gynogenesis in largescale loach by ultraviolet (UV) irradiation with temperature shock. The heterologous sperm
of red crucian carp (Carassius auratus) were used to ensure that any survival offspring are
truly gynogens but not the result of normal fertilization of homologous sperms as is possibly
caused due to the incomplete irradiation (Varadaraj 1990) or the photoreactivation of UVirradiated sperm (Ijiri and Egami 1980). The sexes of the gynogenetic progenies were also
identified. The major objective of these studies was to provide further genetic information to
understand the mechanism of sex determination in large-scale loach.
Materials and methods
Animals
Mature adults of large-scale loach, with a body weight between 30 and 44 g, were collected from local fish markets. Three batches of wild populations of large-scale loach were
also sampled from ponds and rice fields near Wuhan as natural controls. Red crucian carp
were obtained from the Fish Experiment Station of the Institute of Hydrobiology.
UV irradiation and gametes collection
Semen from red crucian carp were diluted 1:100 with cold Hanks’ solution and irradiated
for 5 min in the dark with a 15 W ultraviolet lamp, as described by Wu et al. (1981).
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Large-scale loaches were induced to spawn by a single intraperitoneal injection of human
chorionic gonadotropin (HCG) with a dose of 6-8 IU g-1 body weight for females and
2 IU g-1 body weight for males. A total of 20 female loaches and 15 male loaches were
used. Eggs were stripped into Petri dishes and wrapped with towel, together with irradiated
sperms, temporally stored at about 4°C and then in an ice box (about 10–12°C) for
warming shortly before fertilization and shocks.
Heat and cold shock experiments
The first cleavage of the large-scale loach, which occurred at about 30 min post-fertilization (p.f.) at 20°C, had been determined by self-mating in pilot studies. Both cold and
heat shocks were applied to induce gynogenesis. All the experiments involved the following four types of groups: (1) a diploid control group P1 (untreated eggs and loach
sperms), (2) a haploid control group N (untreated eggs and UV-irradiated crucian carp
sperms), (3) a hybrid control group P2 (untreated eggs and normal crucian carp sperms),
and (4) treated groups for the duplication of haploid genomes with different shocks.
For cold shocks, two experiments were designed. In the first one, cold shocks for 30 min
durations were initiated at 1–32 min p.f. with 2 min intervals. In the second one, cold
shocks began 6 min p.f. and lasted for 25, 30, 35, and 40 min, respectively. Three
experiments were used to investigate the effect of heat shocks. The first one was triggered
at 2–34 min p.f. with 2 min intervals and then at 40°C for 1 min. The second was initiated
at 4 min p.f. and shocked at 38–42°C with 0.5°C or 1°C intervals for 1 min. The third was
initiated at 4 min p.f. and then shocked at 40°C for 0.5–3 min with 0.5 min interval,
respectively. Cold and heat shocks were separately performed in an ice-water bath (0–4°C)
and thermostatic apparatus (LKB). All experiments were repeated two to three times,
except for the experiments on the activation times for the heat and cold shocks, in which
five and eight replicates were used, respectively. For each replicate a different female was
used.
The percentages of embryos surviving to gastrula, yolk absorption, and feeding stage
relative to the initial number of eggs in each group were determined as the fertilization
rate, the hatchery rate, and the survival rate, respectively. The larvae of the hybrid control
P2 could not survive and died before or just at hatching. All data were standardized to the
relative percentage (i.e., percentage survival of the embryos of the treated groups and
haploid control groups were expressed relative to the diploid control P1 from the same
female) to reduce the maternal effect among experiments, as described by Volckaert et al.
(1994). Consequently the fertilization rate of P1 controls was always 100%, although the
actual fertilization rate could not reach 100%. In a few cases, when the fertilization rate of
the P1 control was less than that of the treatments, the highest fertilization rate of the
treatments was set at 100%. All the relative data were arcsin-transformed and tested for
significance (P \ 0.05) by one-way analysis of variance (ANOVA).
Ploidy levels were determined by chromosome preparation at the gastrula stage
according to Yu et al. (1980) with slight modifications. Putative diploid gynogens, together
with three groups of P1 controls, were reared to maturity in indoor tanks at room temperature (19–29°C).
Fish sexing was based on morphological characters, as described by Zhao and Wu
(2002), and on gonad dissection. Comparisons of the observed sex ratios against the
expected ratio (1:1) were performed using the chi-square test (Zar 1974). Self-matings
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Table 1 Cross-species amplification in large-scale loach P. dabryanus (P. dab.) using microsatellites from
pond loach M. anguillicaudatus (M. ang.)
Locus
GenBank accession no. Annealing temperature (°C)
No. of alleles
Allele size (base pair)
M. ang.
P. dab.
M. ang. P. dab. M. ang.
P. dab.
Mac15 AB060177
60
56
6
9
142–168
124–184
Mac37 AB060181
60
60
5
11
79–101
80–112
Mac50 AB060182
60
52
5
4
88–94
84–110
between F1 gynogenetic progenies, and matings between F1 gynogenetic progenies and
normal individuals, were carried out to investigate the sex ratios of the second generations.
Genetic identification using microsatellite DNA markers
Fifteen pairs of microsatellite primers, originating from pond loach (Morishima et al.
2001), were used to monitor maternal inheritance in the present study. Preliminary crossspecies amplifications showed that genotypic segregations for ten of these microsatellite
loci were in good agreement with the expected Mendelian mode in two full-sib families of
large-scale loach, from which three loci, namely Mac15, Mac37, and Mac50, were finally
chosen because of their high polymorphism (Table 1). Ten progenies were sampled from
each shock and control group for microsatellite identification. Female loaches with heterozygous genotypes were chosen to measure the recombination rates between
microsatellite loci and the centromeres.
Genomic DNA was extracted from fin tissues or fry according to the traditional phenolchloroform method. Polymerase chain reaction (PCR) was performed in a 12.5 ll reaction
mixture, which contained 1 9 buffer (with 1.5 mM Mg2+ ), 0.4 U Taq polymerase,
100 lM dNTP, 2.5 pmol primers, and 50–100 ng template DNA, under the following
conditions: an initial denaturation for 5 min at 94°C, followed by 37 cycles of denaturation
at 94°C for 40 s, annealing at 52–60°C for 35 s and extension at 72°C for 35 s, and a final
extension at 72°C for 10 min. PCR products were separated by 8% polyacrylamide gels,
and fragments were visualized by ethidium bromide staining and UV transillumination
(Syngene GeneGenius).
Results
Optimal conditions for gynogenesis
There was no significant difference in fertilization rate between different groups
(P [ 0.05), while a significant difference was observed in hatchery rate and survival rate
between the shock groups and control groups (P \ 0.05). In cold shocks, three peaks of
survival rates (15.5%, 6.67%, and 1.80%) were observed at 6, 12, and 24 min p.f.,
respectively (Fig. 1). One of the females produced the highest yield of 107 gynogens at
6 min p.f. with a 23.42% actual survival rate (data not shown). When duration-dependent
survival rates in cold shocks were explored, no significant difference was observed among
the different shock groups for 25, 30, 35, and 40 min, respectively (P [ 0.05, Fig. 2). For
heat shocks, two peaks were observed at 4 min and 18 min p.f., with survival rates of
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Fig. 1 Effect of different activation times of cold shocks on gynogenesis in large-scale loach. Cold shocks
were applied at 0–4°C for 30 min. P1, diploid control; N, haploid control
9.69% and 5.62%, respectively (Fig. 3). The highest yields of gynogens were produced
from the same female with 104 and 45 putative gynogenetic diploids at 4 min and 18 min
p.f., respectively (data not shown). Moreover, shock temperature and shock duration were
tightly linked in heat shocks. For instance, when shocks were applied at 40°C, the optimum
duration was 0.5 min (Fig. 4), and when shocks were applied for 1 min, 39°C was the
optimum shock temperature (Fig. 5). Additionally, a few spontaneous diploids were
occasionally found in the haploid controls with mean survival rates of no more than 1%.
Microsatellite and karyologic analysis
Maternal inheritance in gynogenesis was confirmed using three microsatellite loci: Mac15,
Mac37, and Mac50. For example, at the locus Mac15, two genotypes of large-scale loach
haploids and hybrid diploids were observed in the hybrid control (Fig. 6a), and no paternal
DNA was observed to transmit to the gynogenetic progenies in all the treated groups and
the haploid controls (Fig. 6b–g). The karyotypes showed that diploids of the large-scale
loach had 48 chromosomes and the hybrid embryos between large-scale loach and crucian
carp had 74 chromosomes.
Spontaneous diploids in the haploid controls were found to have high heterozygosity,
with only one out of ten progenies from a female being homozygous at three microsatellite
loci (e.g., Mac15 in Fig. 6b). High heterozygosity was also observed in diploid progenies
produced by cold shocks at 6, 12, and 24 min p.f., respectively (Fig. 6c–e). For example,
70%, 20%, and 40% heterozygosity were separately observed at Mac15, Mac37, and
Mac50 at 24 min p.f. On the contrary, in heat shocks, all homozygous diploids were
produced at 18 min p.f., while 60% of heterozygous genotypes were observed at 4 min p.f.
at Mac15 (Fig. 6g,f). Microsatellite data suggested that meiotic gynogenesis was induced
at 4 min p.f. and mitotic gynogenesis was induced at 18 min p.f. by heat shocks.
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Fig. 2 Effect of different durations of cold shocks on gynogenesis in large-scale loach. All eggs were
triggered at 6 min post-fertilization. P1, diploid control; N, haploid control
Fig. 3 Effect of different activation times of heat shocks on gynogenesis in large-scale loach. All eggs were
exposed to 40°C for 1 min. P1, diploid control; N, haploid control
Fig. 4 Effect of different durations of heat shocks on gynogenesis in large-scale loach. All eggs were
triggered at 4 min post-fertilization, and then exposed to the 40°C treatment. P1, diploid control; N, haploid
control
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209
Fig. 5 Effect of different shock temperatures of heat shocks on gynogenesis in large-scale loach. All eggs
were triggered at 4 min post-fertilization and then shocked for 1 min. P1, diploid control; N, haploid control
Among three microsatellite loci, a high marker-centromere recombination rate ([50%) in
meiotic gynogenesis was found at the locus Mac15. This microsatellite locus may be a
candidate molecular marker to identify meiogynogens or mitogynogens in large-scale loach.
Sex ratios in progeny of gynogenesis
After 7–11 months of rearing at room temperatures (19–29°C), a total of 135 F1 gynogenetic progenies from seven females survived, all originating from cold shocks. The sex
ratios in the progenies from three females deviated significantly from the expected 1:1 ratio
(P \ 0.05), with two biased towards females (families 2 and 3) and one biased towards
males (family 1); the other four gynogenetic families showed an approximate 1:1 sex ratio
(P [ 0.05) (Table 2). In the three P1 controls, two groups were significantly female-biased
and one male-biased (P \ 0.05), although approximate 1:1 sex ratios were observed in
three batches of nature control (P [ 0.05) (Table 2). The sex ratios of gynogenetic F2
progeny are shown in Table 3. Those females (putative ZW) from gynogenetic families 1
or 2 that mated with its sib male (putative ZZ) or with a normal male (putative ZZ)
produced identical proportions of females and males, and all females were produced when
gynogenetic males (putative ZZ) from family 1 mated with gynogenetic females (putative
WW) from family 2 or with a normal female (putative WW) (P \ 0.05). These sex ratio
data, to some extent, support the putative ZW/ZZ sex determination in large-scale loach.
Discussion
Gynogenesis was successfully induced in large-scale loach by cold and heat shocks using
heterologous sperms of crucian carp in the present study. Shocks can retain the second
polar body by inhibiting spindle formation (meiotic gynogenesis) or can suppress the first
cleavage division by inhibiting cytokinesis (mitotic gynogenesis) (Pandian and Koteeswaran 1998). A successful application of shocks to induce meiotic or mitotic gynogenesis
relies on several factors, e.g., shock magnitude, shock duration, and activation time. In
pond loach, which was very close to large-scale loach in morphology, physiology, and
taxonomy (Li et al. 1983), meiotic gynogenesis at 5 min p.f. and mitotic gynogenesis at
17 min p.f. could be induced by heat shocks at 25°C (Morishima et al. 2001). Similar
activating times were observed in the large-scale loach (4 min and 18 min p.f.) in this
study.
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Fig. 6 Exclusive maternal inheritance of gynogenetic progenies in large-scale loach at the microsatellite
Mac15. Samples in the figure included the hybrid control P2 (a), the haploid control N (b), three batches of
gynogens produced at 6 (c), 12 (d), and 24 min (e) post-fertilization by cold shocks, and two batches of
gynogens produced at 4 (f) and 18 min (g) post-fertilization by heat shocks, respectively. M: pBR322/MspI
size marker; Lane 1, maternal (egg) donor in the gynogenesis; lane 2, paternal (sperm) donor of red crucian
carp in the gynogenesis; lanes 3–12, ten gynogenetic progenies from the different treatments
Microsatellites, with codominance, high polymorphism, and random distribution along
the genome, have been widely used in the confirmation of the exclusively maternal
transmission and in the estimation of recombination rates in gynogens (e.g., Galbusera
et al. 2000; Morishima et al. 2001). The genetic patterns of the three microsatellite loci in
the present study clearly showed that most gynogens produced by cold shocks were still
meiotic heterozygotes even at a very late stage post-fertilization. High heterozygosity in
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211
Table 2 Sex ratios of F1 progeny in the gynogenetic families, the positive diploid control (P1), and the
nature control in large-scale loach (P. dabryanus)
No.
Age
(month)
Total no.
of fish
No. of
females
No. of
males
P1 control
1
11
37
12
25
32.43
3.89*
2
9
10
9
1
90
4.90*
3
7
18
16
2
88.89
9.39*
Mean
Gynogenetic induction
70.44
1
11
23
6
17
17.39
4.35*
2
11
36
34
2
94.44
26.69*
3
7
21
17
4
80.95
6.86*
4
7
22
16
6
72.73
3.68
5
9
11
7
4
63.64
0.36
6
9
7
3
4
42.86
0
7
11
15
4
11
26.67
2.4
Mean
Natural control
% females
v2 (1:1)
Treatment
56.95
1
77
45
32
58.44
1.87
2
46
26
20
56.52
0.54
3
54
30
24
55.56
0.46
Mean
56.84
*P \ 0.05
Table 3 Sex ratios of F2 progenies from two gynogenetic families in P. dabryanus
Gynogenetic
family
Mating group
Total no.
of F2 progeny
No. of F2
females
No. of F2
males
% females
v2 (1:1)
1
F1$1 9 #3
18
9
9
50
0
F1$1 9 F1#1
14
7
7
50
0
$4 9 F1#2
7
7
0
100
5.14*
F1$2 9 #4
11
5
6
45.45
0
F1$3 9 F1#a1
17
17
0
100
15.06*
2
a
This male was from family 1
*P \ 0.05
spontaneous gynogenetic diploids or meiogynogens could be explained by high recombination levels or meiotic nondisjunction of all or some chromosomes (Galbusera et al.
2000; Tanck et al. 2001; Bertotto et al. 2005). Previous investigations demonstrated that
cold shocks could be successfully initiated within a wide period in tilapias, common carp
and ornamental carp (Don and Avtalion 1988; Linhart et al. 1995; Cherfas et al. 1994). A
maturation delay effect blocking the second meiotic division instead of the first zygotic
cleavage (Libertini et al. 2002) may explain the meiogynes produced by a late cold shock
in large-scale loach. Alternatively, it could also be possible that cold shocks caused the
resorption of the second polar body of the ‘‘fertilized’’ eggs even at the time of the first
cleavage, resulting in gynogens with heterozygosity (meiogynogens). In this study, we
further proved that cold shocks could not induce mitotic gynogenesis as effectively as heat
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shocks in large-scale loach, as shown in other studies (Nam et al. 2004; Pandian and
Koteeswaran 1998).
Sex determination in fish is controlled by complex mechanisms. Sex chromosomes of
fishes develop from purely polygenic controls, to those with dominant sex-determination
factors mixed with autosomal controls, to highly evolved sex chromosomes with heterogametic (XY) males or heterogametic (ZW) females (Devlin and Nagahama 2002). Chang
and Yu (1997) proposed a female heterogamete mechanism (ZW/ZZ) for large-scale loach
based on C-banding results, in which a deeply stained band was located at the middle
region of the long arm of the putative female W chromosome. The results of this study
generally confirmed the previous conclusion by inducing gynogenesis and by scoring the
sex ratios of the gynogenetic F1 and F2 progenies.
All gynogenetic progenies of a male heterogamete (XY) fish would be females, while
gynogenetic progenies of a female heterogamete (ZW) fish may exhibit variable proportions of males, females and, sometimes, intersexes, due to the crossing over between the
centromere and the sex-determining locus during meiosis and the survival of the WW
individuals (Purdom 1993). The supposed sex-determining locus of large-scale loach might
be similar to that of the typical male-heterogametic sex determination in some fish, e.g.,
medaka (Matsuda et al. 2002). Gynogenetic F1 females would possess either a single (ZW)
or double (WW) sex-determining locus of the W chromosome and males (ZZ) would lack
the sex-determining locus. The unexpected sex ratios of gynogenetic F1 progenies in some
families (e.g., strong bias towards males in family 1 or to females in family 2, Table 2),
might be interpreted as the presence of a lethal locus on the W chromosome close to the
sex-determining locus and the high mortality of homogeneous recessive alleles, as suggested by Komen et al. (1991) in other fish. Supposing that the lethal locus is more distant
from the centromere than the sex-determining locus, then putative WW females with
heterozygous lethal loci would not die if a single cross-over occurred between them.
Furthermore, the two loci would have been both distant to the centromere because high
recombination rates between the sex-determining loci and the centromere were observed,
resulting in many unexpected ZW females in gynogenesis. Therefore, a female with ZW or
WW genotypes would produce totally different sex ratios and show a maternal effect.
To interpret the sex determination of large-scale loach by sexing gynogenetic progenies,
some environmental factors might also be considered. Increasing evidence has shown that
temperature, PH, salinity, population density, and some other factors, may influence sex
differentiation in some fish (Devlin and Nagahama 2002; Baroiller et al. 1999), although
the specific mechanism remains unknown. For example, an increase of male diploids in
gynogenetic groups was observed in pond loach (with possible XX/XY sex determination)
when they were reared at 25–30°C (Nomura et al. 1998). It is unclear whether a similar
effect of temperature on sex ratios exists in large-scale loach. As the progenies of both the
gynogenetic and control groups were reared at a wide range of room temperatures (19–
29°C) in the present study, such an effect cannot be excluded, although these details were
not explored in the present study. The proportion of females in the positive controls P1
(mean 70.44%) was higher than that in the nature controls (mean 56.84%), perhaps due to
the temperature effect or a biased distribution and/or recombination of sex-determining
factors of sex chromosome in artificial crosses, as shown in tilapia (Devlin and Nagahama
2002). Nevertheless, temperature effect, if it had, might have not been cumbersome for the
investigation of possible chromosomal mechanism for sex determination in large scaleloach in this study, considering the fact that the same rearing conditions were applied to all
the gynogenetic progenies among the different groups.
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In summary, the optimal conditions to induce meiotic and mitotic gynogenesis in largescale loach are described. The data for the sex ratios from gynogenetic F1 and F2 progenies
generally support the previously proposed ZW/ZZ sex determination. Factors that may
cause biased sex ratios are discussed. By applying a combined approach with possible
cytogenetical and molecular evidence, it is hoped to elucidate the mechanism for sex
determination in more fish species, which is informative for possible breeding programs
towards sex control.
Acknowledgements The authors would like to thank X. Liao and C. Wang for technical assistance. This
study was partially supported by grants from the NSFC (30370225 and 30271011) and the NSF of Hubei
Province (2003ABA119).
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