LI Si-Fa, ZHAO Yan FAN Wu-Jiang CAI Wan-Qi XU Ying-Fang

(1. Key Laboratory of Aquatic Genetic Resources and Utilization Certificated by the Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China; 2. Chinese Academy of Fishery Science, Yangtze River Fisheries Research Institute, Jingzho 434000, China)

Possible genetic reproductive isolation between two tilapiine genera and species:Oreochromis niloticusandSarotherodonmelanotheron

LI Si-Fa1,*, ZHAO Yan1, FAN Wu-Jiang1, CAI Wan-Qi1, XU Ying-Fang2

(1. Key Laboratory of Aquatic Genetic Resources and Utilization Certificated by the Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China; 2. Chinese Academy of Fishery Science, Yangtze River Fisheries Research Institute, Jingzho 434000, China)

Successful crossbreeding betweenOreochromis niloticusandSarotherodon melanotheronto produce a commercial hybrid has been difficult. The karyotypes and isoenzyme of these two species and their reciprocal hybrids (O. niloticus♀ ×S.melanotheron♂, S.melanotheron♀ ×O. niloticus ♂,the last not included in the isoenzyme study) were investigated via metaphase chromosomes obtained from head kidney cells and electropherogram of lactate dehydrogenase (LDH) isoenzymes from the liver, kidney, white muscle, heart, and eye balls. The diploid chromosome number (2n=44) and the fundamental number (NF=50) of the four tilapia genotypes were the same. However, the karyotype ofO. niloticushad three pairs of sub-metacentric (sm), twelve pairs of sub-telocentric (st), and seven pairs of telocentric (t) chromosomes, whileS.melanotheronhad one pair of metacentric (m), two pairs of sm, 12 pairs of st, and seven pairs of t chromosomes. The reciprocal hybrids both showed a mixed karyotype range between their parents: 0.5 pair of m, 2.5 pairs of sm, 12 pairs of st, and seven pairs of t chromosomes. In view of the electropherogram of isozymes, only the LDH of the kidney showed significant clear bands, with five bands inO.niloticus, three bands inS.melanotheron,and duplicated six bands in the hybrids. The bands varied depending on their activities and mobilities. We considered that the differences in karyotype and isoenzyme were related to the genetic mechanism for post-mating isolation, and provided some additional basic genetic background of their taxonomy.

Tilapiine;Oreochromis niloticus;Sarotherodon melanotheron;Karyotype; Isoenzyme; Reproductive isolation; Taxonomy

Belonging to the order Perciformes, family Cichlidae, Tilapiini is a highly diverse tribe with more than 70 species (Trewavas, 1983), commonly called tilapia. Tilapia is a generic term used to designate a group of commercially important Cichlid fish, which consists of three genera:Oreochromis(maternal mouthbreeder), Sarotherodon(paternal mouthbreeder) andTilapia(substrate breeder).Tilapia classification relies on differences in morphology and breeding behavior. In aquaculture, interspecific hybridization within the genus (such asO. niloticus×O. aureusfor the purpose of producing all male fish andO. niloticus×O. mossambicusfor the purpose of producing red tilapia) is commonly performed.

Culture of tilapia in brackish and salt waters remains poorly developed because few species possess both rapid growth and high adaptability to salinity. Since 1999, we have used fast growingO. niloticusand high salt toleratingS. melanotheronfor a crossbreeding program to produce a new variety of tilapia possessing both high salt tolerance and good growth rate (Li et al, 2008a, b; Liu et al, 2009). However, natural hybridization betweenO. niloticusandS. melanotheronproved impossible and the artificial reciprocal hybridization was unpredictable and unsuccessful, with only several hundred F1individuals produced from over 100 batches of artificial hybridization each year. Despite this, the F1fish were fertile and produced hundreds of F2off-spring, which were similar to those of F1and had ideal aquaculture characteristics such as: (1) better salt tolerance――can stand 15‰?25‰ salinity with good survival and growth, (2) reasonable grow rate―over 500 g in 5?6 months in south China, and (3) better testy than tilapia cultured in freshwater. However, the major obstacle for commercialization of this hybrid was the bottleneck of F1 production. In addition to ecological habits, we attempted to understand their mechanisms of reproductive isolation, including karyotype and isoenzyme point of view.

Karyotypes are chromosome complements of related individuals (species) and the number of chromosomes is often used as an indicator of relationship among species and to determine hybridization potential between species. The application of electrophoretic methods to the taxonomic study of various fish groups has revealed that electropherograms of muscle, serum and hemoglobin tissue proteins are species specific (Thompson, 1960; Nyman, 1965; Li, 1998), and therefore are a valuable tool for the analysis of phylogenetic relationships between different groups within a family (Tsuyuki & Roberts, 1965; Li & Cai, 1995). Additionally, some isoenzymes have strong specificity in activity of a substrate, and genetic information can be easily obtained by using the electropherogram of isoenzymes such as lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and esterase (EST).

We conducted a karyotype and isoenzyme study of these two tilapia genera and their hybrid to: (1) understand the genetic mechanism of the hybridization barrier between the two species, and (2) provide some genetic basis for their taxonomy.

1 Materials and Methods

1.1 Karyotype study

1.1.1 Sample collection

We collectedO. niloticus, S. melanotheronand their reciprocal hybrids (about 100 g of body weight) from the State Zhongji Tilapia Farm, Hebei Province. Ten fish of each genotype were used for the study. Fish were maintained for one week in aerated aquaria (1m×1.5 m) with constant temperature of 30 ℃. Each specimen was injected intra-peritoneally with freshly prepared phytohemagglutinin (PHA) at 10 μg/g body weight, then returned to the aquarium for 16?18 h, and later injected with 0.01% of colchicine solution of pure colchicine at a dosage with 2 μg/g body weight.

1.1.2 Cell harvest

Three hrs after injection with colchicine, fish were euthanized by cutting the anterior cardinal aorta and vein. The anterior head kidney was taken out and pulverized with isotonic solution of NaCl. Cells drifting away from the tissue were moved to 0.075 mol/KCl solution at 37 ℃for 30 min for low osmotic pressure treatment and then centrifuged (8 min, 1 000 r/min). After removing the supernatant, the samples were fixed in fresh Carnoy’s solution (methanol:acetic acid=3:1) for 15 min, and then centrifuged (8 min, 1 000 r/min).

1.1.3 Spreading of cells and staining

The centrifuged cell suspension was spread by Pasteur pipette on slides washed by 95% ethanol and then swirled in distilled water. The slides were air dried on flame and 24 h later stained with 3% Giemsa in Sorensen’s buffer with pH of 6-8 for 40 min. The slides were rinsed in distilled water, air dried and mounted.

1.1.4 Examination of the karyotype

Spread chromosomes in metaphase were observed using an Olympus compound microscope and 50 fields from each specimen under x400 and x1 000 (oil immersion) magnifications, respectively, were photographed by an automatic camera fixed on the microscope. The number and total length of chromosomes were evaluated, and the chromosomes were arranged in pairs from the longest to the shortest. The centromeric position was determined according to the classification of Levan et al (1964).

1.2 Isoenzyme study

1.2.1 Specimens collection and preservation

LiveO. niloticus, S. melanotheronand their hybrids (about 300-600 g of body weight) were collected from the ponds at the State Zhongji Tilapia Farm, Huanghua city, Hebei province. Ten fish of each genotype were used for the study. The liver, kidney, white muscle, heart, and eye ball were taken after releasing blood by cutting the anterior cardinal aorta and vein. Tissues were put into small marked bags and preserved in liquid nitrogen, then transferred to a –80 ℃ refrigerator upon arrival to the laboratory.

1.2.2 Electrophoresis

Following National Standard GB/T 18654.13 (2008), electrophoresis was run at the DYY-12 electrophoresis set (Beijing Liuyi Company) with 7.0% polyacrylamide vertical continuous swimming, 300 V and 30 s for prepare-swimming and pre-swimming before and after adding the samples. Formal swimming lasted 5 h at 200 V. After staining, gel banding patterns were densitometrically analyzed and interpreted to quantify genetic variability among the tilapia genotypes. Lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and esterase (EST) were examined.

2 Results

2.1 Karyotype

The arm ratio and type of chromosome sets ofO. niloticus, S. melanotheronand the hybrids are shown in Tab. 1. The metaphase spread of the chromosomes of the four investigated tilapia genotypes is shown in Fig.1. Chromosomes size ranged from 2.1 to 8.0 μm. We found 78%, 83%, and 79% of metaphases studied contained 44 chromosomes for each of the three groups, respectively, therefore the diploid chromosome number for all was 2n=44. The karyotype ofO. niloticushad three pairs of sub-metacentric (sm), 12 pairs of sub-telocentric (st), and seven pairs of telocentric (t) chromosomes, and a fundamental number (NF) of 50. The karyotype ofS. melanotheronhad one pair of metacentric (m), two pairs of sm, 12 pairs of st, and seven pairs of t chromosomes, andNFof 50. However, the karyotype of the reciprocal hybrids was the same: 0.5 pairs of m, 2.5 pairs of sm, 12 pairs of st, seven pairs of t, andNFof 50 (Fig. 2)

2.2 Isoenzymes

Only the LDH from the kidney showed clear bands (Fig. 3). There were five bands (LDH1, LDH2, LDH3, LDH4and LDH5) inO. niloticus,three bands (LDH1, LDH2and LDH3) inS. melanotheron, and six bands (LDH1, LDH2, LDH3, LDH4, LDH5and LDH6) in the hybrid. All LDH isoenzymes were at the anodal side of the gel, and were numbered from the anodal side, with the most anodal being LDH1. Tab.2 lists the relative activity and relative mobility of the LDH bands in the kidneys ofO. niloticus, S. melanotheronand their hybrids. The densitometric patterns are shown in Fig. 3.

3 Discussion

Taxonomy is the theory and practice of naming, describing and classifying organisms. However, the taxonomic and evolutionary relationship among the Tilapiini species remains controversial. Trewavas (1983) reclassified the tilapiini into three genera:Oreochromis, SarotherodonandDanakiliaand suggested that the first two mouth brooding genera could have arisen as one or possibly two splits from the ancestral line, withSarotherodonbeing conservative andOreochromismore progressive. Unfortunately, there is a lack of genetic information to support this classification. Sodsuk and McAndrew (1991) analyzed 44 enzyme loci of 15 species from the three genera and concluded that despite the minor rearrangements within the major classes, Trewavas’s (1983) classification not only clearly described the biological characteristics of the species within each genus, but also reflected the evolution of this group. Previous research has clarified that the LDH isoenzyme of fish is coded by two genes on different loci, which is common among the various groups of vertebrates, and its electrophoretic pattern shows marked differences among fish species (Markert & Faulhaber,1965). This isoenzyme pattern has also used as evidence for the hypothesis of the evolution of vertebras from fish to mammals and is realized mainly by gene duplication (Ohnoet al, 1968).

Tab. 1 Arm ratio and type of the chromosomes set of Oreochromis niloticus, Sarotherodon melanotheron and their reciprocal hybrids

Fig. 1 Metaphase chromosome of the Cichlid fish

BothO. niloticus and S. melanotheronare the most representative species in the generaOreochromisandSarotherodon. They occupy different ecosystems in Africa, withS. melanotheroncovering a natural geographical area of the brackish estuaries and lagoons along the cost of Zaire to Senegal and O.niloticusliving in fresh waters from Senegal to Gambia, Volta to Chad, Nile, Ethiopia, and Baringo (Trewavas, 1983). Due to geographical isolation, no natural hybrid has been reported between these two species. In addition, well known differences in breeding habits exist between the two species, e.g.,O. niloticusis a maternal mouthbreeder andS. melanotheronis paternal mouthbreeder, which provides a reasonable ecological and physiological explanation for their reproductive isolation.

Fig. 2 Karyograms of the Cichlid fish

Fig. 3 Electropherograms and densitometric patterns of lactate dehydrogenase (LDH) in kidney of the Cichlid fish

Tab. 2 Relative activity and mobility of bands of lactate dehydrogenase (LDH) in kidney of Oreochromis niloticus, Sarotherodon melanotheron and their hybrid (O. niloticus × S. melanotheron)

Some tilapia species are appropriate for both intensive and extensive pisciculture due to positive characteristics such as fast growth, tolerance for poor water quality, and wide range of feed habits. However, their uncontrolled and prolific breeding at a small size in mixed sex culture is a constraint on their efficient production. The interspecific hybrids ofO. niloticus♀×O. Aureus ♂produced a high male ratio, which resolves the over reproduction problem in tilapia culture.

The culture of tilapia in brackish and salt waters remains poorly developed because few species have both rapid growth and ability to withstand marked variations in salinity. For example,O. niloticusis characterized by a high growth rate but low salt tolerance, whileS. melanotheronis characterized by high salt tolerance but poor growth rate. We expected that the hybridization between these two species could produce a hybrid sharing both good salt tolerance and growth. In our study, hybridization experiments betweenO. niloticusandS. melanotheronwere conducted in two national tilapia seed farms located in south and north China, respectively. Firstly, no natural matings occurred in the ponds (under semi-natural conditions). Secondly, it was difficult to produce hybrids through artificial fertilization, with only a few offspring (F1) produced by reciprocal practice (Li et al, 2008b). Thirdly, successful fertilization during artificial fertilization, even if eggs and sperm met, was rare or absent, possibly due to a lack of effective interaction between the sperm and eggs. Finally, even if fertilization occurred, most embryos failed to develop properly. The above issues are in contrast to the well known phenomenon that interspecific and intraspecific crossbreeding is rather common in Tilapiini, both in the wild and in hatcheries. Many closely related species produce fully viable and fertile F1and backcross progeny in semi-natural conditions and are commercially utilized. For example,O. niloticus×O. mossambicusandO. niloticus×O. aureus, which belong to the same genus, have of same chromosome number (2n=44). Harvey et al (2002a) reported that differences in chromosome number does not prevent the production of inter-specific hybrids betweenO. niloticus(2n=44) andO. karongae(2n=38),although the experiment was only at the laboratory scale with very small number and makes no mention of whether the hybrids were sterile or not.

A great deal has been written about reproductive isolation mechanisms, which can be categorized into spatial and ecological (geographical) isolation and genetic isolation approaches. The geographical barrier plays a significant role in pre-mating (spatial, seasonal, habitat, and behavioral), while the genetic barrier plays an inherent role in post-mating (structure, gametic incompatibility, hybrid non-viability or weakness, and hybrid sterility, Merrell, 1981). Previous research has shown strong evidence related to geographical barriers, but little regarding genetic barriers.

It was difficult to determine generic level differences in karyotype and isoenzyme betweenO. niloticusandS. melanotheronin the present study. We recognized that these differences might not be enough to conclude that they were responsible for the obstacles of hybridization, but we believe that in addition to the differences in natural distribution and breeding behavior that influence pre-mating isolation, the differences in karyotype and isoenzymes may relate to the genetic mechanism for post-mating isolation.

Our results suggest thatO. niloticus and S. melanotheronhave a conservative but different karyotypic structure. Their diploid numbers were both 2n=44, which is in agreement with previous research (Li, 1998; Harvey et al, 2002b). The most interesting finding was the significant difference in the composition of m and sm chromosomes between the two species, specificallyO. niloticushad three pairs of sm andS. melanotheronhad one pair of m and two pairs of sm. However, the number of st and t in the two species were the same, and the hybrids were of mixed m and sm composition. Harvey et al (2002a) reported thatS. melanotheronhas 2 m + 15 sm/st + 5t and Sofy et al (2008) reported thatO. niloticushas 1sm + 13st + 7t, which differ from our present findings. These differences may reflect the difficulties in obtaining high quality metaphase chromosome preparations and in accurately measuring chromosome arm lengths.

Acknowledgements:We wish to express our gratitude to Professor Zhang K.J., Shanghai Ocean University, and Mr. Jing W. K, Tianjin Fish Farm, for their help with chromosome sample preparation, and to Professor Louis Bernatchez, Laval University, Canada, and Lin Junda, Florida Institute of Technology, USA, for assistance with English.

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尼羅羅非魚(yú)和薩羅羅非魚(yú)遺傳生殖隔離的初步證據(jù)

李思發(fā)1,*, 趙 巖1, 范武江1, 蔡完其1, 許映芳2

（1.上海海洋大學(xué) 農(nóng)業(yè)部水產(chǎn)種質(zhì)資源與利用重點(diǎn)開(kāi)放實(shí)驗(yàn)室,上海201306; 2.中國(guó)水產(chǎn)科學(xué)研究院長(zhǎng)江水產(chǎn)研究所,湖北 荊州434000）

羅非魚(yú)類（Tilapiini）含3個(gè)屬70余種, 種間和屬間頗易人工雜交, 但尼羅羅非魚(yú)（Oreochromis niloticus）和薩羅羅非魚(yú)（Sarotherodon melanotheron）人工雜交難度大, 產(chǎn)苗概率甚低, 要獲得數(shù)量足夠的可用于生產(chǎn)的雜交子代相當(dāng)困難。該文對(duì)這兩種魚(yú)及其正交（O. niloticus♀ ×S. melanotheron ♂）和反交（S. melanotheron♀ ×O. niloticus ♂）子代的頭腎細(xì)胞的核型進(jìn)行了比較。此外, 采用同工酶電泳方法檢測(cè)腎、肝、眼、肌肉、心中乳酸脫氫酶等4種同工酶的表型差異。4種遺傳型羅非魚(yú)具有相同的染色體二倍數(shù)（2n=44）和總臂數(shù)（NF=50）, 但各具不同的染色體類型, 尼羅羅非魚(yú)為3對(duì)近中著絲點(diǎn)染色體（sm）、12對(duì)近端著絲點(diǎn)染色體（st）和7對(duì)端著絲點(diǎn)染色體（t）; 薩羅羅非魚(yú)為1對(duì)中間著絲點(diǎn)染色體（m）、2對(duì) sm、12對(duì) st和7對(duì) t;正反雜交子代表現(xiàn)為介于雙親之間的混合類型, 為0.5對(duì)m、2.5對(duì)sm、12對(duì)st和7對(duì)t。在同工酶中, 僅見(jiàn)腎臟乳酸脫氫酶電泳結(jié)果有清晰差異, 尼羅羅非魚(yú)出現(xiàn)5條譜帶, 薩羅羅非魚(yú)3條, 而雜交子代6條, 且所有譜帶的遷移率和活性均表現(xiàn)出多態(tài)性。據(jù)此初步認(rèn)為, 核型和同工酶方面的差異可能是導(dǎo)致這兩種不同屬羅非魚(yú)生殖隔離的遺傳原因, 這些差異也可能為這兩種（屬）魚(yú)的分類學(xué)提供新的遺傳背景資料。

羅非魚(yú)類；尼羅羅非魚(yú)；薩羅羅非魚(yú)；核型；同工酶；生殖隔離；分類

2011-03-17；接受日期：2011-07-13

羅非魚(yú)行業(yè)技術(shù)體系(nycytx-48-3);公益性行業(yè)(農(nóng)業(yè))科研專項(xiàng)：羅非魚(yú)大規(guī)格魚(yú)種規(guī)?；嘤c生態(tài)養(yǎng)殖技術(shù)研究(nyhyzx07-044-01);國(guó)家科技支撐計(jì)劃專題：耐鹽羅非魚(yú)新品種選育(2006BAD01A1203)

Q959.483; Q951.3 ; Q959.483.09

A

0254-5853-(2011)05-0521-07

10.3724/SP.J.1141.2011.05521

date: 2011-03-17; Accepted date: 2011-07-13

*Corresponding author (通信作者), E-mail: sfli@shou.edu.cn

book=522,ebook=492