Studies Of Isoenzymes And Karyotype Of The Tonguefish Cynoglossus Semilaevic Gunther

The tonguefish C. semilaevis (order Pleuronectiformes, family Cynoglossidae) is a rare and high-quality fish, and has recently emerged as an increasingly farmed species in northern China. Presently, the tonguefish resources have been depleted to be in severe danger partly due to overfishing, maritime pollution and its special particularities of reproduction (significant differences of abundance and body sizes between the females and males). Its chromosome number has been identified as 42. However, isozymes and chromosome banding trials are still lacking. In addition, the tonguefish shows a typical sexual dimorphism, with females being larger at first maturity and growing to a larger size than males. Production of all-female fish via genetic manipulation is of both theoretical interest and economical importance, and thus we were encouraged to undertake a detailed study of the isozymes, chromosome karyotype and banding patterns in C. semilaevis. Here we report an improved trypsin method for the isozymes, G-banding, C-banding and nucleolar organizer regions (NORs) of the chromosomes from the renal tissue cells of the tonguefish C. semilaevis, and describe the patterns and numbering of four isozymes and the chromosome G–bands, C-bands and NORs. The results of the project will lay a theoretic foundation not only for the protection of the rare resources but also for the seedling project of seafarming and sea ranching.1. IsoenzymesA total of 16 tonguefish including 8 male and 8 female Cynoglossus semilaevis with body weight of 67-375 g and body length of 11-41 cm were procured from Yellow Sea Fisheries Research Institute at Qingdao, China. All the fish were 1-year-old. Four isozymes, i. e. malic enzyme (ME), malate dehydrogenase (MDH), acid phosphatase (ACP) and esterase(EST), in seven tissues or organs: kidney, spleen, liver, gonad, heart, brain and muscle of tonguefish C. semilaevis, were analyzed using vertical polyacrylamide gel electrophoresis (PAGE) and biochemical staining. ME was present in all tissues examined. In female kidney and male brain, two bands (ME-1 and ME-2) were detected. ME-1 and ME-3 were present in male liver. There were four bands in female liver. In other tissues ME showed a single band (ME-1). ME-1 was highly active in both female and male liver.In all tissues except the female kidney, male liver and male brain, MDH showed two bands (MDH-2 and MDH-3). MDH-3 was detected only in the female kidney and male liver tissues. In male brain tissues, MDH showed only a single band (MDH-2).EST-3 was present in all the tissues. EST-1 was detected only in female and male liver, and EST-2 was present only in male spleen. EST-4 was present in female and male kidney, female and male liver and male brain tissues. EST-5 was detected in male liver and spermary. Just like the ME and MDH enzyme, the electrophoretic patterns of the EST showed sex-related differences between male and female C. semilaevis but not showed inter-individual difference in the same sex.ACP was active in all the tissues examined, but it was more complex then the other three isozymes. There were 16 bands of ACP in 7 tissues of female and male C. semilaevis. ACP-1, ACP-2, ACP-3 were detected in female liver, female spleen and male liver tissue respectively. ACP-4 was widely present in female and male kidney, heart, muscle and male spleen, spermary and brain tissues, but the active was different. ACP-5, ACP-6, ACP-7 and ACP-8 were present in female liver, spleen, ovary and brain tissues respectively with highly active. In female liver, brain and male liver tissues ACP-9 was detected with highly active. ACP-10 was showed in female and male brain, muscle, kidney and male spermary tissues. ACP-11 was only found in female and male liver. ACP-12 was showed in female and male spleen and male ovary tissues with lowly active. ACP-13 was detected in female and male heart. ACP-14 was present only in male kidney tissue. In female and male muscle, female kidney and male spermary ACP-15 was showed. ACP-16 was present in female and male brain and male kidney tissues with highly active. The electrophoretic patterns of acid phosphatase showed not only sex-related differences between male and female C. semilaevis but also differences between different tissues in the same individual.In summary, judging from electrophoretic patterns of four isozymes in C. semilaevis, it was found that (1) ME, MDH, ACP and EST were detected in all tissues, (2) the electrophoretic patterns of four isozymes showed sex-related differences between male and female C. semilaevis; (3) the electrophoretic patterns of ME, MDH, ACP and EST did not show inter-individual difference in the same sex. (4) ACP enzyme systems examined was tissue-specific. The electrophoretic patterns of ACP showed obvious differences between different tissues in the same individual C. semilaevis.Isozymes have been found to be the products of expression of genes. The sex-related differences between male and female C. semilaevis and differences between different tissues in the same individual of isozymic phenotypes of four isozymes in C. semilaevis may be due to controlling of sex-linkage gene, hormone or different physiological state. It is possible that further study may help to resolve this situation.2. Karyotype and banding studyChromosome karyotype and banding study remain lacking in Cynoglossus semilaevis heretofore. This paper reports the karyotype, G-band patterns, C-band pattern and nucleolar organizer regions of tonguefish Cynoglossus semilaevis. The metaphase chromosome preparations of the fries has been got from their fin by hot air drying methods, while the metaphase chromosomes of one year old fish which has physiologically sex differentiation has been got from their renal tissues by the method of PHA and colchicines injection. The karyotypes were examined. The result showed that there are 42 acrocentric chromosomes in diploid and their karyotype formulae is 2n = 42t, there existed heterotypic sex chromosome which belongs to ZW/ZZ type. The sex ratio in artificial bred stock is nearly 1:1. The 21st pair of chromosomes from females was heteromorphic, while the same pair chromosomes from males were homomorphic. The first description and numbering of the karyotyped bands in metaphase chromosomes were reported.G-banding method stains positively 68.1% of the chromosome surface in C. semilaevis, and a total of 171 G-bands (89 positive, 74 negative and 8 variable) has been recorded in the 20 pairs of autosomes and a pair of sex-chromosomes Z and W. The interstitial regions in all chromosomes including the pair of sex chromosomes had positive C-bands, and some chromosomes showed C-bands in centromeric or distal ends. The C-banding heretochromatin occupies 30.03% of the total chromosome surface in C. semilaevis, which is similar to that of amphibians such as Mixophyes fasciolatus (30.2%) and M. schevilli (20.7%), but is rather lower than that of cephalochordate Branchiostoma belcheri (54.3%). Silver staining revealed a single pair of nucleolar organizer regions (NORs) located in the telomeric regions of the Chromosome No. 2. The association of NORs with heterochromatin observed in vertebrates also occurs in C. semilaevis as the telomeric regions of the chromosome No. 2 are always stained positively with C-banding.