Ž .Aquaculture 192 2001 171–186www.elsevier.nlrlocateraqua-online

The formation of tetraploid stocks of red cruciancarp=common carp hybrids as an effect of

interspecific hybridization

Shaojun Liu a,), Yun Liu a, Gongjiang Zhou a, Xuanjie Zhang a,Chen Luo a, Hao Feng a, Xiaoxiao He a, Guihua Zhu b, Hui Yang b

a Life Science College, Hunan Normal UniÕersity, Changsha 410081, Hunan, People’s Republic of Chinab Donghu Fish Farm, Xiangying 414600, Hunan, People’s Republic of China

Received 30 October 1999; received in revised form 27 April 2000; accepted 3 June 2000

Abstract

Ž . Ž . ŽF –F hybrids of red crucian carp Carassius auratus red var. Y =common carp Cyprinus3 8. Ž .carpio L. ? were found to be allotetraploids by examining the numbers and karyotypes of

chromosomes, the DNA content of red blood cells, and the mean nuclear erythrocyte volumes.The chromosome numbers of F –F were 4ns200. The chromosome numbers of two types of3 8

Ž . Žtriploids produced by mating F –F ? , respectively, with diploid Japanese crucian carp C.3 8. Ž . Ž .auratus cuvieri T. et S. Y and Xingguo red carp Y were 3ns150. The mean DNA content

of F and the triploid are, respectively, two times and 1.5 times of that of diploid Japanese crucian3

carp. F hybrids had mean erythrocyte nuclear volumes about two times larger than diploid3

Japanese crucian carp. The hybrids F –F were fertile. It is easy for F –F to produce their3 8 3 8

tetraploid offspring. The triploids were sterile. Based on the analysis of karyotype and theintermediate appearance with two pairs of small bases of barbels, F –F hybrids were proved to be3 8

allotetraploids with two chromosome sets of red crucian carp and two chromosome sets ofcommon carp. It is concluded that the females and males of F hybrids were able to produce,2

respectively, diploid eggs and diploid spermatozoa and they fertilized to form tetraploid fish.q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Carassius auratus red var.; Cyprinus carpio; Hybridization; Tetraploidy; Stock; Triploidy

) Corresponding author. Tel.: q86-731-8872552; fax: q0086-731-8883310.Ž .E-mail address: lsj@hunnu.edu.cn S. Liu .

0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 00 00451-8

( )S. Liu et al.rAquaculture 192 2001 171–186172

1. Introduction

Intergenetic hybridization can result in genome-level alterations including the occur-rence of triploid hybrids, for example, F hybrids of Ctenopharyngodon idella=1

Ž .Hypophthalmichthys nobilis Marian and Kraszai, 1978; Beck et al., 1984 and F1Ž .hybrids of carp with Hemiculter eigenmanni Vasil’ev et al., 1975 . Study on polyploidy

Ž .in crucian carp=common carp was previously reported by Cherfas et al. 1994 . In thepresent study, the results of long-term investigation on fertile F –F tetraploids resulting3 8

from F hybrids of red crucian carp=common carp are presented. It is the first example2

of production of tetraploid fish by successive generations of hybridization without anyexperimental treatment. The first aim for creating tetraploids is the production oftriploids. Secondly, it is possible that the fertile tetraploids will develop into a newspecies with 200 chromosomes.

2. Materials and methods

2.1. General scheme of the production of hybrid progeny

Red crucian carp used in the present study is a diploid bisexual form. Red crucianŽ . Ž .carp Carassius auratus red var. and common carp Cyprinus carpio L. are the

members of different genera, so the crossing between them is considered as distantŽ . Ž .crossing. In 1978–1983, red crucian carp Y and common carp ? had been

artificially hybridized to produce a viable diploid hybrid, and 4.7% male F hybrids and1Ž .44.3% female F hybrids were found to be fertile Liu and Zhou, 1986 . The fertility of1

the F hybrids of this crossing is an exception to the general case of sterility in1

interspecific hybrids.F hybrids were produced by artificial fertilization in 1984. F hybrids were also2 2

produced in 1993 and 1998. In 1985 and 2000, F hybrids were produced by artificially3

fertilizing F ova with semen of F males. In 1986, F were produced by artificial2 2 4

fertilization from F . Because of breeding space limitation, F were artificially produced3 5

only in 1989. F , F , F , F and F were produced, respectively, in 1993, 1997, 1998,6 7 8 9 10Ž . Ž .1999, 2000 data for F and F not presented . Since 1986, the F –F hybrids ? were9 10 3 8

Ž . Ž . Ž .mated with Japanese crucian carp C. auratus Cuvieri Y and Xingguo red carp YŽ . Ž .a variety of carp to produce, respectively, triple crucian carp T and triple common1

Ž .carp T hybrids.2

2.2. Methods of ploidy determination

To determine ploidy, chromosome counts were done on kidney tissue and culturedlymphocytes of common carp, red crucian carp, Japanese crucian carp, F –F hybrids,1 8

Ž .and the two types of triple hybrids T and T . The kidney tissue was grinded in 0.9%1 2

NaCl. For lymphocyte culture, the leukocytes were isolated from whole blood collectedfrom the caudal vein by centrifugation. The separated leukocytes were cultured in 10 ml

Ž . Ž . ŽRPMI 1640 medium Sigma at 268C supplemented with concanavalin A 10 mg

( )S. Liu et al.rAquaculture 192 2001 171–186 173

. Ž .mlr1 for stimulation of mitosis. Two hours prior to harvest, colchicine 0.5 mg mlr1was used to arrest the chromosomes at metaphase. For both the kidney cells andlymphocytes, hypotonic treatment was accomplished with 0.075 M KCl at 378C for 40

Ž .min, followed by fixation in 3:1 methanol–acetic acid three changes . Cells weredropped on cold, wet slides, and stained for 30–60 min in 4% Giemsa in pH 7.0phosphate buffer. Preparations were examined under oil at a magnification of 330= .Good-quality metaphase spreads were photographed and used for analysis of karyotypes.Length of entire chromosomes, long and short arms were measured. Chromosomes were

Ž .classified based on their long-arm to short-arm ratios according to Levan et al. 1964 :values of 1.0–1.7 were classified as metacentric; 1.7–3.0 as submetacentric; 3.1–7.0 assubtelocentric, and )7.1 as telocentric.

To measure the DNA content of red blood cells of F –F hybrids and T and2 4 1

Japanese crucian carp, 1–2 ml blood samples were collected from the caudal vein of theabove fish into syringes containing about 200–400 units of sodium heparin. The bloodsamples were fixed in the 3:1 methanol–acetic acid solution for 20 min, and then treated

Ž .with Nuclei Extraction Partec . Samples were then filtered, and stained with DAPIŽ .DNA Staining Solution Parc . The DNA content of each sample was measured using a

Ž .flow cytometer Cell Counter Analysiser, Parc .Ž .Nuclear micromeasurements of red blood cells RBC were also used to ascertain

ploidy. A 1–2 ml blood sample was drawn from F hybrid, T and Japanese crucian3 1

carp. Blood smears were fixed in ethanol and stained with Giemsa solution. To measurenuclear diameter, 20 erythrocytes from each fish were observed under oil at a 330= ,

Ž . 2using an ocular micrometer. Nuclear volume was calculated by 4r3 Pab , where awas the major semi-axis and b was the minor semi-axis of a perfect ellipsoid. Volumemay have been slightly over-estimated due to cell flattening.

2.3. The size measurements of gametes and embryonic deÕelopment

In the reproductive season, the semen stripped out from F , F , F , common carp and2 3 8Ž .scattered mirror carp Cyp. carpio haematopterus , was, respectively, fixed in 4%

formaldehyde–glutaraldehyde for studies under scanning electron microscope. In eachsample of semen of F , F , common carp and scattered mirror carp, 50 spermatozoa3 8

were measured. In the sample of semen of F , 80 spermatozoa were measured. The2Ž . 3volume of spermatozoon was calculated by 4r3 Pa , where a was the semi-diameter.

The sizes of eggs produced respectively by F , F , Japanese crucian carp and red crucian2 8

carp were measured. In each sample of eggs of F , Japanese crucian carp and red8

crucian carp, 50 eggs were measured. In the samples of eggs of F , 220 eggs were2

measured.The embryonic development of F , F T and diploid red crucian carp, at water3 4, 1

temperature of 20–238C, was observed under a microscope.

2.4. ObserÕation of fertility and growth rate of hybrids

All fish samples were raised for 1 year or more than 1 year. Thirty one-year-oldF –F and 37 1-year-old T were dissected. The gonads were removed and fixed in3 8 1

( )S. Liu et al.rAquaculture 192 2001 171–186174

Bouin’s solution for preparation of tissue sections. Parafin-embedded sections were cutand stained with hematoxylin and eosin. In the reproductive season, the 1-year-old orolder T and T fish were examined to verify their fertility. The growth rate of T and T1 2 1 2

hybrids, Japanese crucian carp and common carp were compared under the sameconditions.

3. Results

3.1. The chromosome number and karyotypes

The chromosome numbers were counted from metaphases of all samples. The redŽ . Ž .crucian carp, common carp, and F –F were diploids 2ns100 Table 1; Fig. 1A,B,C .1 2

Ž . Ž .F –F hybrids ,were tetraploids 4ns200 Table 1; Figs. 1D and 2A,B . T and T3 8 1 2Ž . Ž .were triploids 3ns150 Table 1; Fig. 2C .

Based on the analysis of karyotype, F and F hybrids had the same karyotype as1 2Ž .their parents — red crucian and common carp, containing 22 metacentric m , 34

Ž . Ž . Ž .submetacentric sm , 22 acrocentric subtelocentric, st and 22 telocentric t chromo-Ž . Ž .somes Fig. 1A,B,C . F –F hybrids had the same karyotype formula Fig. 1D :3 8

44mq68smq44stq44t. Although red crucian carp and common carp had the samekaryotype formula, a difference existed between their longest submetacentic chromo-somes. Table 2 shows that the ratio of long-arm to short-arm of the longest submetacen-

Ž .tic chromosome RLS in red crucian carp, common carp, F –F and F The RLS in red1 3 8.

Table 1Ž . Ž .Distribution of chromosome numbers for common carp CC , red crucian carp RC , Japanese crucian carp

Ž .JC , F –F hybrids, T and T in samples of 50 cells per fish type1 8 1 2

Fish type Chromosome numbers

90–95 95–100 100–145 145–150 150–190 190–200 200–210

CC 5 42 3RC 3 41 6JC 2 43 3F 2 46 21

F 6 42 22

F 1 45 43

F 5 44 14

F 3 40 75

F 6 42 26

F 4 45 17

F 6 39 58

T 2 43 51

T 3 40 72

( )S. Liu et al.rAquaculture 192 2001 171–186 175

Ž . Ž . Ž . Ž .Fig. 1. Karyogram of common carp A , red crucian carp B , F hybrid C and F hybrid D . The longest2 8Ž . Ž . Ž .submetacentric chromosomes sm are marked 1 and magnified E . Bars3 mm.

Ž .crucian carp is lower than that in common carp Fig. 1A,B,E . In diploid F , the two2Ž .members of the largest submetacentric chromosome pair sm differ in their RLS. One,1

whose ratio of long-arm to short-arm is lower, fits and was apparently inherited from thered crucian carp. The other, whose RLS is higher, fits and was apparently inherited from

Ž Ž .the common carp Table 2; Fig. 1C,E . Among the four longest submetaccentricŽ .chromosomes sm of tetraploid F and F , two chromosomes with a lower ratio of1 3 8

( )S. Liu et al.rAquaculture 192 2001 171–186176

Ž . Ž . Ž . Ž .Fig. 2. Metaphase chromosome spreads of F A , F B 4Ns200 , and T hybrid 3Ns150 . Bars3 mm.3 8 1

long-arm to short-arm come from the red crucian carp, while the other two chromo-Ž .somes, with a higher RLS, come from common carp Table 2; Fig. 1D,E .

( )S. Liu et al.rAquaculture 192 2001 171–186 177

Table 2Ž .The ratio of long-arm to short-arm of the longest submetacentric chromosomes RLS in different fish types

Ž .20 longest submetacentric chromosomes were measured in each sample

Fish type RLS

Red crucian carp 1.989"0.059Common carp 2.557"0.045

a bF 1.923"0.111 ; 2.443"0.1401a bF 1.671"0.090 ; 2.481"0.0942a bF 1.728"0.122 ; 2.292"0.2953a bF 1.673"0.128 ; 2.527"0.3728

a The chromosome that comes from red crucian carp.b The chromosome that comes from common carp.

3.2. The DNA content

Ž .The DNA content of Japanese crucian carp 2ns100 was used as the standard. Theresults of the distribution of DNA content of all samples are shown in Table 3 and Fig.3A,B,C,D. Peak No. 1 in each pattern refers to the majority of cells analyzed. Thesmaller peak No.2 refers to two fused nuclei of erythrocytes. The ratios of mean channelnumber of the first peaks of F , T and F to Japanese crucian carp are, respectively,2 1 3

0.93, 1.50 and 2.03, indicating that F is diploid, T is triploid and F is tetraploid.2 1 3

3.3. The mean erythrocyte nuclear Õolume measurements for F , T and Japanese3 1

crucian carp

The results of the measurements of the mean erythrocyte nuclear volume for F , T3 1

and Japanese crucian carp are shown in Table 4. The mean erythrocyte nuclear volumeratio of F hybrid to diploid Japanese crucian carp was not significantly different3Ž .P)0.05 from the 2:1 ratio. The mean erythrocyte nuclear ratio of T to Japanese1

Ž .crucian carp was not significantly different P)0.05 from the1.5:1 ratio.

Table 3Ž .The distribution of DNA content in Japanese crucian carp JC , F –F , and T2 4 1

Fish type Mean DNA content Ratio

Observed Expected

JC 48.51aŽ .F 45.03 F rJC 0.93 12 2aŽ .T 72.55 T rJC 1.50 1.51 1aŽ .F 98.68 F rJC 2.03 23 3

a Ž .The observed ratio was not significantly different P )0.05 from the expected ratio.

()

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Ž . Ž . Ž .Fig. 3. Cytometric histogram of DNA fluorescence for diploid Japanese crucian carp A , F hybrid B , T hybrid and F hybrid D .2 1 3

( )S. Liu et al.rAquaculture 192 2001 171–186 179

Table 4Ž .Mean erythrocyte nuclear volume measurements for F , T and Japanese crucian carp JC . For each sample,3 1

30 erythrocyte nuclear volumes were measured

Fish Type Major axis Minor axis Volume Volume ratio3Ž . Ž . Ž .mm mm mm

Observed Expected

JC 6.37"0.56 3.37"0.56 36.92"8.86aŽ .T 7.47"0.69 3.71"0.45 54.38"12.90 T rJC 1.47 1.51 1aŽ .F 9.71"1.38 3.81"0.63 73.87"17.64 F rJC 2.00 23 3

a Ž .The observed ratio was not significantly different P )0.05 from the expected ratio.

3.4. The sizes of gametes and hatching rate of hybrids

The results of measurements of the mean volume of spermatozoa of common carp,scatter mirror common carp, F , F and F are shown in Table 5. The volume of2 3 8

Ž .spermatozoa of the common carp Table 5; Fig. 4A almost equal to that of the scatteredŽ . Ž .mirror carp, which is also diploid fish 2ns100 Li, 1998 , indicating that both of

them produced the similar haploid spermatozoa. The volume of spermatozoa of F and3Ž .F Table 5; Fig. 4B is two times larger than that of the common carp, indicating that F8 3

and F produced diploid spermatozoa. Five types of spermatozoa existed in the8Ž . Ž .water-like semen of F , containing 48.8% diploid Fig. 4C , 40% haploid Fig. 4C and2

a few tetraploid, octoploid and aneuploid spermatozoa whose volume was obviouslysmaller than that of the haploid volume produced by the common carp.

The results of measurements of the sizes of the eggs of F , F , Japanese crucian carp2 8

and red crucian carp are shown in Table 6. The mean diameter of the Japanese cruciancarp is almost equal to that of the red crucian carp, indicating that the two kinds of

Table 5Ž . Ž .Mean volume measurements for the spermatozoa of common carp CC , scattered mirror carp SC , F , F and2 3

F8

Fish type Number of Mean diameter Volume Volume ratio3Ž . Ž .sperm mm mm

Observed Expected

CC 50 1.90"0.21 3.61"0.26aŽ .SC 50 1.91"0.19 3.68"0.53 SCrCC 1.02 1aŽ .F 50 2.40"0.02 7.25"0.18 F rCC 2.00 23 3aŽ .F 50 2.39"0.08 7.15"0.69 F rCC 1.98 28 8

aŽ .F 39 2.42"0.17 7.45"0.65 F rCC 2.06 22a 2aaŽ .F 32 1.92"0.15 3.70"0.31 F rCC 1.03 12b 2baŽ .F 2 3.05"0.10 14.86"1.03 F rCC 4.11 42c 2caŽ .F 2 3.80"0.07 28.60"0.16 F rCC 7.92 82d 2d

Ž .F 5 1.38"0.13 1.40"0.42 F rCC 0.392e 2e

a Ž .The observed ratio was not significantly different P )0.05 from the expected ratio.

( )S. Liu et al.rAquaculture 192 2001 171–186180

Ž .Fig. 4. A–B Scanning electron micrographs of the spermatozoa in the semen stripped-out, respectively, fromŽ . Ž . Ž .the common carp A and the F hybrid B . Bars3 mm. C Scanning electron micrograph of the8

spermatozoa in the water-like semen stripped-out from a 1-year-old F male, showing the diploid spermato-2Ž . Ž . Ž .zoon the lower arrow and the haploid spermatozoon the upper arrow . Bars1.9 mm. D Light microscopy

Ž .of the eggs produced by F female, showing three size classes of eggs, the large size eggs L , the middle size2Ž . Ž .eggs M and the small size eggs S . Bars0.2 cm.

diploid fish have the similar eggs in size. The mean diameter of the diploid eggsproduced by F is obviously larger than that of Japanese crucian carp. Three size groups8

Ž .of eggs Fig. 4D existed among the eggs produced by F . The eggs with the diameter of2

0.17 cm were similar to the eggs of F in size, indicating they were diploid eggs. During8

reproductive season in 2000, after the artificial fertilization of the eggs and semen of F ,2

among 2500 fertilized eggs with the diameter of 0.17 cm, about 500 embryos were ableto develop into the hatching stage, but most of the larvae were dead past hatching, only16 fish survived. The eggs with the diameter of 0.13 cm were similar to the eggs of the

( )S. Liu et al.rAquaculture 192 2001 171–186 181

Table 6Ž . Ž .Mean size measurements for the eggs of F , F , Japanese crucian carp JC and red crucian carp RC2 8

Ž .Fish type Number of eggs Mean diameter cm

JC 50 0.14"0.01RC 50 0.13"0.01F 50 0.17"0.018

F 110 0.20"0.012L

F 79 0.17"0.012M

F 31 0.13"0.012S

red crucian carp in size, indicating that they were haploid eggs. Among 1400 fertilizedeggs with the diameter of 0.13 cm, about 150 embryos were able to develop into thehatching stage, but most of the larvae were also dead past hatching, only two fishsurvived. All fertilized eggs with the diameter of 2.0 cm were dead before the stage ofgastrula, indicating that those eggs were aneuploid.

The hatching rate of F and T and red crucian carp were, respectively, 51%, 62.5%4 1

and 73%. Compared with the diploid red crucian carp, the F –F hybrids had higher3 8

abnormalities during the hatching period.

3.5. The appearance, color and fertility

Common carp has two pairs of barbels, one longer pair being located on either side ofthe lower jaw, and the other shorter pair being located on either side of the upper jaw,whereas red crucian carp has no barbel. F –F also had two pairs of barbels. However,1 2

the lengths of the longer and shorter barbels in F –F were, respectively, shorter than1 2

those in common carp. In F –F , only two pairs of small bases of barbells existed,3 8

which are hardly seen by the naked eye. Compared with the barbels in F –F , the length1 2

of barbels in F –F had drastically decreased. T has only one pair of very short barbels3 8 1

on either side of the lower jaw. T had two pairs of barbels, which were similar to those2

of the common carp. The color of F –F , like that of common carp, was gray. The shape1 8

of F –F was intermediate to red crucian carp and common carp. F –F had the similar1 2 3 8

shape also intermediate to red crucian carp and common carp, but a little different fromthat of F –F . F –F grew slower than F –F . With the above features, it was quite easy1 2 3 8 1 2

to identify F –F hybrids from other diploids and triploids.3 8

F were easily produced. However, it was somewhat difficult to produce F . The2 3

semen of fertile F was water-like, in which there were fewer spermatozoa compared2

with the semen produced by the common carp. The number of fertile F hybrids was2

few. During the reproductive season, the white semen and mature ova could be easilystripped out respectively from males and females of F –F and they were able to3 8

fertilize to produce the viable offspring. Under light microscope, many mature ova thatwere full of yolk were observed in the ovary of F hybrid at maturity. Numerous sperm3

were found in the lobules of the testis of F hybrid. Under scanning electron microscope,3Ž .numerous spermatozoa Fig. 4B were observed in the semen produced by F male.8

( )S. Liu et al.rAquaculture 192 2001 171–186182

Table 7Gonad structure in 37 1-year-old triploid crucian carp

Gonad structural type Number of fish Percentage

Testis 17 45.94Ovary-like 12 32.43Fat tissue 8 21.62

We could not strip out semen or mature ova from 1-year-old or older T and T1 2

during the reproductive season, and no fish was produced by T or T . Three structural1 2Ž .types of gonad were observed among 37 1-year-old T Table 7 . The first were testes1

that consisted of many lobules filled with many spermatids, but no mature spermatozoabeing observed. The second were ovary-like gonads that contained many small oogo-nium-like cells and a few larger primary oocytes, but no mature ova being observed. Thethird was fat tissue found in the gonad location, neither ovary nor testis being observed.Detailed description of gonadal structures of T and T will be presented elsewhere.1 2

The growth rate of T was 20% faster than Japanese crucian carp. The growth rate of1

T was 30% faster than common carp.2

4. Discussion

Counting the chromosome number is a direct and accurate method for determiningthe ploidy level of the samples. Use of flow cytometry to examine the DNA content ofsamples is a rapid, simple and accurate method. The DNA content of some fish, such as

Ž . Žtriploid brook trout Dube et al., 1991 , triploid and tetraploid common carp Cherfas et.al., 1993 , was determined by this method. The erythrocyte nuclear measurement is also

Ža simple and useful method to determine the ploidy Wolters et al., 1982a; Benfey and.Sutterlin, 1984; Varadaraj and Pandian, 1990; Cherfas et al., 1991, 1993 . However, this

method is not very accurate. In the present study, all three methods were applied and alldata obtained were in agreement and showed the following ploidy of progeny: diploidyof F –F hybrids, tetraploidy of F –F hybrids, triploidy of T and T .1 2 3 8 1 2

The difference of the values of the RLS in red crucian carp, common carp, andF –F , F , F and the difference of the length of barbels in common carp and F –F1 2 3 8 1 8

provided genetic evidence indicating that F –F were hybrids and not the outcome of1 8

gynogenesis or androgenesis. That is to say, F –F were allodiploids and F –F were1 2 3 8Ž .allotetraploids amphidiploids with two chromosome sets of red crucian carp and two

chromosome sets of common carp.How were the allotetraploids produced? Like the F , F was also a hybrid with one1 2

set of chromosome of red crucian carp and one set of chromosome of common carpŽ .Fig. 1C . Both the males and females of the F hybrids were able to produced different2

Ž . Ž .sizes of spermatozoa Table 5; Fig. 4C and eggs Table 6; Fig. 4D , respectively.Among the eggs produced by F female, the eggs with the diameter of 0.17 cm similar2

to the diploid eggs in size produced by F , were considered to be diploid eggs. Among8

( )S. Liu et al.rAquaculture 192 2001 171–186 183

the spermatozoa stripped out from F male, the spermatozoa with the diameter of 2.422

mm similar to the diploid spermatozoa in size produced by F were considered to be8Ž .diploid sperm. The rate of diploid sperm was higher 48.8% than that of any other types

of sperm. So, it is concluded that the formation of tetraploids in F hybrids was due to3

the fertilization of the diploid eggs and diploid spermatozoa produced by F . The2

gonadal structure of F of red crucian carp=common carp was previously described by2Ž .Zhong 1994 . In the ovary of fertile F spermatogonium-like cells existed near mature2,

ova, and in the testis of fertile F , a few ova existed near the lobules filled with mature2

sperm, indicating that the formation of gametes of F was not abnormal, providing the2

occasion to produce abnormal gametes including diploid gametes. The feature ofproducing diploid gametes in F was apparently maintained through further generations2

Ž .of hybridization F –F . Both the fertile male and female tetraploid F were found and3 8 3

they were mated to produce the F tetraploid hybrids. The tetraploid hybrids could be4

inherited from one generation to another.The larger eggs with the diameter of 0.2 cm were not able to produce living fish,

Ž .being considered as aneuploid eggs. Cherfas et al. 1994 reported that the aneuploideggs could not produce living fish. Based on the variation of gametes in size producedby F , in theory, it is possible that diploid, triploid, hexoploid and other ploid level fish2

also exist among F hybrids although until now only tetraploid fish were found among3

F hybrids. The research work is expected to be continued in the future.3Ž .Cherfas et al. 1994 found that the male F hybrids of crucian carp, C. gibelio=1

Ž .common carp Cyp. carpio produced unreduced eggs. They reported that ADiploid eggsand aneuploid eggs differed in size, diploid eggs being distinctly larger. Only largerdiploid eggs are capable of normal development after fertilization, while small eggs arenot.B Those larger eggs that fertilized with the normal spermatozoa produced triploidfish. However, F males were completely sterile. The sex reversal method was used to1

Ž .produce F males with XX chromosomes. Inverted F male XX produced allote-1 1Ž . Ž .traploids by mating with F female XX , which produced unreduced eggs XX .1

Ž .However, in theory, only the female allotetraploids XXXX could be produced. In thepresent study, the eggs with the diameter of 0.17 cm were similar to the larger diploid

Ž .eggs described by Cherfas et al. 1994 and they were also considered to be diploideggs. In the present study, only few smaller eggs with the diameter of 0.13 cm were ableto develop into living fish. We conclude that most of the smaller fertilized eggs couldnot survive past hatching due to their aneuploidy. We propose that the reason of theformation of tetraploids in the present study is due to a mechanism similar to that

Ž .described by Cherfas et al. 1994 , except that in the present study both F male and2

female, which produced the tetraploids, were fertile, and both the diploid spermatozoaand diploid eggs were found respectively in the water-like semen of F males and2

among the eggs of F females.2Ž .Cherfas et al. 1994 thought that the occurrence of unreduced eggs is due to the

endoreduplication of some germ cells in early oogenesis, which resulted in doubling ofchromosome number. This process was followed by two subsequent meiotic divisionsŽ .as in the case of regular meiosis . We think that this mechanism is reasonable and it canwell explain the formation of the diploid eggs produced by F females, and diploid,2

tetraploid and optoploid spermatozoa produced by F males The tetraploid and opto-2 .

( )S. Liu et al.rAquaculture 192 2001 171–186184

ploid sperm can be produced if the endoreduplication of some germ cells in earlyspermatogenesis occurred two times or four times in succession, followed by twosubsequent meiotic divisions.

Ž . Ž .Myers 1986 used two species of tilapia, Orechromis mossambicus Peters and O.Ž .niloticus L. , and one of their hybrids, O. mossambicus=O. niloticus to produce auto

Ž . Ž .intraspecific and allotetraploids interspecific by means of combination of pressureand cold treatments. Results indicated that allotetraploids were markedly more viablethan autotetraploids. In the present study, from F to F alloetraploids, it is easy to3 8

produce tetraploid offspring and the allotetraploid offspring are viable. Although F –F3 8

allotetraploids had a higher abnormality rate than diploid crucian carp during theŽ .embryonal development, they still keep a high enough hatching rate 51% .

In theory, allotetraploids and autotetraploids should be fertile. However, among theŽ .allotetraploids artificially produced by crossing Japanese crucian carp Y with red

Ž .crucian carp ? by means of heat shock, the allotetraploid males were found to beŽ .fertile while the allotetraploid females were not found Chen et al., 1997 . In the present

study, allotetraploids occurred spontaneously and the males and females were all foundto be fertile. All F –F allotetraploids kept the same shape and color. They can be stably3 8

inherited from one generation to another. Today, we have produced about 50,000 F –F3 8

allotetraploids. They have formed a stable fertile allotetraploid population. F and F9 10

hybrids were also produced, respectively, last year and this year.The main practical interest in tetraploids is for producing triploids. Triploids are

expected to be sterile, since homologous chromosomes cannot pair properly during themeiotic development of gonads and the random separation of trivalent chromosome at

Žanaphase results in the formation of aneuploid gametes. In some triploid fish Gervai et.al., 1980; Wolters et al., 1982b , both the females and males were found to be sterile. In

Žother triploid fish Lincoln and Scott, 1984; Allen et al., 1986; Benfey et al., 1986;.Hussain et al., 1996 , the females were found to be completely sterile and a few of

abnormal spermatozoa were found in the testis of the males. However, the eggsfertilized with such spermatozoa were unable to develop into a living fish, so this kind

Žof triploid males were still considered to be sterile Benfey et al., 1986; Hussian et al.,. Ž1996 . In the present study, T and T were proved to be triploids. All triploids T and1 2 1

.T were found to be sterile, no offspring being produced by T or T . The three types2 1 2

of structures found in the gonad location of 37 1-year-old T showed that T is sterile.1 1

The descriptions about the gonadal structures of triploid fish have been reported in manyŽ . Žfish, such as Nile Tilapia Hussian et al., 1996 , channel catfish Wolters, et al.,

.1982a,b . However, the report that only fat tissue exists in the gonad location of someŽ .triploid fish is rare. Hulata et al. 1980 reported that reduced or absent gonads with

Žaccumulation fatty mass in the dorsal part of abdomen cavity substituting the missing.gonads existed in the F hybrids of Chinese common carp=European common carp,1

offspring of F =Chinese common carp and offspring of F =European common carp.1 1Ž .The two types of triploids T and T have showed several advantages in large-scale1 2

production, such as sterility and faster growth. The growth rate of T was also measured1Ž .by Zhou et al. 1999 . In that report, the growth rate of T was 21.8% faster than1

Japanese crucian carp and 70.8% faster than Pengze crucian carp. Among those threeŽ .kinds of fish, the survival rate of T 92.3% was higher than that of Penze crucian carp1

( )S. Liu et al.rAquaculture 192 2001 171–186 185

Ž . Ž .90.6% and that of Japanese crucian carp 81.3% . Production of triploids by crossingtetraploids with normal diploids is a simpler, more economical and accurate method. Inthe present study, the most important thing is that a fertile allotetraploid population was

Ž .obtained. With the allotetraploid population, triploid fish T and T have been1 2

produced on a large scale in China.

Acknowledgements

We thank the numerous people who helped in various stages of this project,particularly Zhaogui Cai, Zhanzhou Yao, Xiangyang Zhong, Biqian Liu and Fang Hu,for their assistance in the laboratory and field. The research was funded by ChineseScience and Technology Ministry, and Hunan Science and Technology Department.

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