鲫鲤杂种一代(F1)自交二代(F2)的受精细胞学研究

荷包红鲫(♀)×湘江野鲤(♂)杂交产生的杂种一代(F     

Morphology of unfertilized mature and fertilized developing marine pelagic eggs in four types of multiple spawning flounders

Starry flounder Platichthys stellatus, spotted halibut Verasper variegates, turbot Scophthalmus maximus, and Japanese flounder Paralichthys olivaceus are four commercially cultivated multiple spawning flounders that spawn pelagic eggs. Through appropriate light and scanning electron microscope processing, the shape and surface structures (such as micropyle, pores, pore density, and paten) of unfertilized mature and fertilized developing eggs of the four species were observed and measured. First, individual or intraspecific comparisons of the surface structures of eggs at different developmental stages were made. Second, interspecific differences among the four species at the same developmental stage of unfertilized mature eggs were statistically computed and analyzed through one-way analysis of variance and hierarchical cluster analysis. Eggs of the same species collected at different stages of development tend to be different in morphology. Smoothing of the convoluted egg envelope surface and closure of the micropyle to serve as a final step of the polyspermy-preventing reaction are common after fertilization. Based on detailed morphology of micropyle of just-mature fertilizable eggs, turbot, starry flounder, and Japanese flounder each have a micropyle with a long canal but no distinct micropylar vestibule, type III of Riehl and Götting (Arch Hydrobiol 74:393–402, 1974). In contrast, spotted halibut has a micropyle with a distinct flat micropylar vestibule and a long canal, type II. Envelope surface microstructures, especially those in the micropyle region, are useful characters for egg identification among the four species. Cluster analysis using selected egg characters indicated the highest similarity between turbot and Japanese flounder and that starry flounder is obviously more similar to turbot and Japanese flounder than to spotted halibut.     

Hybridization and F1 backcrosses between two closely related tropical species of sea urchins (genus Echinometra) in Okinawa

Summary

Four species of sea urchins of the genus Echinometra, designated species A, B, C, and D, occur along the coast of Okinawa; they are distinguished by color pattern and other characteristics. The two most distinct species, Echinometra sp. A (Ea) with white-tipped spines and Echinometra sp. D (Ed) with nearly black spines (possibly E. oblonga), were examined for potential hybridization through a series of fertilization experiments and rearing of the resulting hybrids. Fertilization was reciprocally asymmetrical; Ed ova were readily fertilized by sperm of Ea, but less than 20% of the ova of Ea were fertilized by sperm of Ed. Nevertheless, hybrids resulting from crosses in both directions developed normally through larval and juvenile stages to produce sexually mature adults. Larvae of Ed♀×Ea♂ and Ed♂×Ed♀ were larger than those of Ea♂ × Ed♀ and Ea♂ × Ea♀, reflecting the larger eggs of Ed. However, Ea♂×Ea♀ juveniles and later stages, up to maturity, were consistently larger than those of equivalent ages of the other crosses; largest to smallest were Ea♂×Ea♀, Ed♂×Ea♀, Ea♂ × Ed♀, and Ed♂×Ed♀, respectively. Ova and sperm of the hybrids were viable and fertilizable in all combination of crosses, although fertilization of those of Ed♂×Ea♀ hybrids was consistently higher than those of Ea♂ × Ed♀ hybrids. These experiments represent the first reported successful production of hybrid sea urchins with viable gametes. They indicate that reproductive isolation is achieved by prezygotic isolating mechanisms in these two species. Of these mechanisms, gametic incompatibility is probably only partly involved, if at all, and differences in spawning times, habitat segregation, or other factors appear to be important for maintaining reproductive isolation in these closely related species.     

Reproductive isolating mechanisms contributing to asymmetric hybridization in Killifishes (Fundulus spp.)

When species hybridize, one F1 hybrid cross type often predominates. Such asymmetry can arise from differences in a variety of reproductive barriers, but the relative roles and concordance of pre-mating, post-mating prezygotic, and post-zygotic barriers in producing these biases in natural animal populations have not been widely investigated. Here, we study a population of predominantly F1 hybrids between two killifish species (Fundulus heteroclitus and F. diaphanus) in which >95% of F1 hybrids have F. diaphanus mothers and F. heteroclitus fathers (D♀ × H♂). To determine why F. heteroclitus × F. diaphanus F1 hybrids (H♀ × D♂) are so rare, we tested for asymmetry in pre-mating reproductive barriers (female preference and male aggression) at a common salinity (10 ppt) and post-mating, pre-zygotic (fertilization success) and post-zygotic (embryonic development time and hatching success) reproductive barriers at a range of ecologically relevant salinities (0, 5, 10, and 15 ppt). We found that F. heteroclitus females preferred conspecific males, whereas F. diaphanus females did not, matching the observed cross bias in the wild. Naturally rare H♀ × D♂ crosses also had lower fertilization success than all other cross types, and a lower hatching success than the prevalent D♀ × H♂ crosses at the salinity found in the hybrid zone centre (10 ppt). Furthermore, the naturally predominant D♀ × H♂ crosses had a higher hatching success than F. diaphanus crosses at 10 ppt, which may further increase their relative abundance. The present study suggests that a combination of incomplete mating, post-mating pre-zygotic and post-zygotic reproductive isolating mechanisms act in concert to produce hybrid asymmetry in this system.     

散鳞镜鲤与兴国红鲤、龙州镜鲤的杂种优势以及鳞被、体色的遗传

本研究希望通过鲤鱼品种间杂交出现的杂种优势来提高鲤鱼的产量以及在杂种的后代中选择具有优良的经济性状的类型培育出新的鲤鱼品种。两组杂交的杂种一代具有明显的杂种优势,可以直接应用于渔业生产上并可望获得增产效果。根据对鳞被和体色的遗传特性的研究结果断定散鳞镜鲤与兴国红鲤的杂种二代中出现的红色镜鲤的鳞被和体色是由纯合型隐性基因控制的,可以预期它的后代不再出现分离而可能成为一个稳定的新品种。研究了不同品种及其杂种的血清蛋白聚丙烯酰胺盘状电泳图谱,希望借此作为一种选种的工具。       

Effects of long-term starvation on hepatocyte ultrastructure of olive flounder Paralichthys olivaceus

The effects of nutritional conditions on alterations in condition factor, liver-somatic index, and hepatocyte ultrastructure in the olive flounder Paralichthys olivaceus were examined. Twelve weeks of starvation significantly decreased the condition factor and the liver-somatic index in the olive flounder. Hepatocytes underwent marked ultrastructural changes in response to 12 weeks of starvation. Compared to those of the initial control and fed group, the prominent features characterizing the hepatocytes of the starved group were: reduction in cell and nucleus size; apparent loss of nucleoli; condensation of chromatin; loss of stored glycogen; reduction of endoplasmic reticulum profile; increase in the number of electron-dense bodies containing large amounts of iron; and increased mitochondrial size. Results suggest that the histological changes caused by ultrastructural alterations in the hepatocytes can be used as alternative indicators to identify starvation in cultured P. olivaceus.     

Experimental hybridization among Oryzias species. II. Karyogamy and abnormality of chromosome separation in the cleavage of interspecific hybrids between Oryzias latipes and O. javanicus

In interspecific hybridization between Oryzias latipes and O. javanicus, all hybrid embryos failed to develop and died before hatching. Cytological examination of fertilization and early development was performed to discover the cause of lethal development. When O. latipes eggs were inseminated by sperm of O. javanicus, the cortical reaction was induced normally. Chromosomal material in the fertilized eggs was visualized using the DNA-specific fluorochrome Hoechst. The spermatozoon was capable of penetrating into the egg cytoplasm through the micropyle, and the sperm nucleus transformed to the male pronucleus. The female pronucleus that formed after extrusion of the second polar body migrated towards the male pronucleus. The female and the male pronuclei underwent DNA synthesis and encountered each other in the center of the blastodisc, fused with one another and formed a zygote nucleus before breakdown of the nuclear envelope. Metaphase chromosomes with electron dense chromatin regions were abnormally divided into each blastomere in cleavage. The abnormally separating chromatin masses were also labeled by BrdU. The abnormal separation resulting in partial loss of fragmented chromatin might be a cause of abortive development in the interspecific hybrids between O. latipes and O. javanicus.     

Reproduction of the summer flounder, Paralichthys dentatus (L.)

The reproduction of summer flounder, Paralichthys dentatus (L.), occurring in the Middle Atlantic Bight was studied from 1974–1979. Males dominated the length interval between 21–35 cm t.l. and were essentially absent in samples >55 cm t.l. Females were more abundant in all length intervals >45 cm t.l. Length at maturity ranged from 24–27 cm t.l. for males and from 30–33 cm t.l. for females which coincided with length at age two. Ovarian egg diamerter frequencies indicated summer flounders are serial spawners and the trend in mean and maximum maturity indexes (% ovary weight of total fish weight) indicated spawning began in September and continued through February in 1975 and probably into March in 1976. Annual condition cycle of males peaked in September and was lowest in April while females' cycle reached a maximum in October which coincided with peak spawning time. Fecundity was related to length, weight and ovary weight for 1974–1977 and the length-fecundity equation F = 0.0007975 L ^3.402 was found to be the best predictive relationship. The overall reproductive strategy of summer flounder was discussed and apparently tends to maximize reproductive potential through an extended spawning season, early maturation, high fecundity, serial spawning and extensive spawning migrations.     

Embryonic and larval staging of summer flounder (Paralichthys dentatus)

Early development of flatfishes such as the summer flounder Paralichthys dentatus (Pleuronectiformes) has not been extensively documented, largely because of a dearth of material; however, the recent expansion of flatfish aquaculture has made embryos of P. dentatus readily available for developmental studies. We divide development of P. dentatus embryos and larvae into two main periods, pre- and posthatching, and assign stages within each of those primary divisions. Stages from fertilization to hatching loosely follow the general teleost staging scheme suggested by Shardo ([1995] J Morphol 225:125-167); stages from hatching through metamorphosis are aligned with the series used for Japanese flounder, P. olivaceus (Minami [1982] Nippon Suisan Gakkaishi 48:1581-1588; Fukuhara [1986] Nippon Suisan Gakkaishi 52:81-91). Although length, width, and age may serve as approximate indicators of developmental progression in summer flounder, these characteristics are too variable to form the sole basis of a staging table. Therefore, we define stages by morphological criteria drawn from the development of the jaw apparatus and digestive system, eye migration, and notochord tip flexion. Examination of these morphological features in hatched larvae allows accurate and consistent assessment of developmental stage despite variation in timing and size. The staging scheme for flounder embryonic and larval development presented here should facilitate both experimental and comparative research on summer flounder and other flatfish species.     

Molecular cloning, expression, and functional analysis of caspase-10 from Japanese flounder Paralichthys olivaceus

We isolated and sequenced caspase-10 cDNA and gene from Japanese flounder, Paralichthys olivaceus. The Japanese flounder (JF)-caspase-10 cDNA consisted of 2282 bp and encoded 495 amino acid residues. The characteristic death effector domains (DEDs) of caspases were observed in JF-caspase-10 as well as the three aspartic acid residues (D-186, -382 and -392), which are potential cleavage sites for the large and small subunit structures. The amino acid residue (His-325) and pentapeptide (QACQG), which are involved in catalytic activity, were absolutely conserved in Japanese flounder-caspase-10. JF-caspase-10 gene has a length of 6.6 kb and consists of 11 exons and 10 introns similar to that of human. The strong expression of JF-caspase-10 mRNA was detected in the gills, peripheral blood leukocytes, spleen and posterior kidney, while the weak expression was observed in the head kidney, heart, intestine, skin and stomach. The over-expression analysis of JF-caspase-10 in Japanese flounder cell line HINAE was shown to induce apoptosis 24h post-transfection using TUNEL assay.     

Molecular cloning of multiple chitinase genes in Japanese flounder, Paralichthys olivaceus

Three different chitinase genes (fChi1, fChi2 and fChi3) were identified from Japanese flounder, Paralichthys olivaceus. The deduced amino-acid sequences of flounder chitinases revealed a typical chitinase structure containing a catalytic glyco-18 domain, a hinge region and a chitin binding domain type 2. The fChi1 and fChi2 mRNAs were predominantly expressed in the gastric glands of stomach. In contrast, expression of fChi3 was found in spleen, pancreas, stomach, intestine, liver, kidney and gonads of adult flounder by RT-PCR. The expression level of fChi3 in the adult tissues was below the detection limit of in situ hybridization (ISH) analysis; however, ISH signals were detected in the liver of flounder larvae. These results suggest that fChi1 and fChi2 are acidic chitinases that digest dietary chitin and that fChi3 probably is a macrophage specific chitinase (chitotriosidase) for biodefense and has an important unknown role in the liver during larval stages.     

Cryopreservation of flounder (Paralichthys olivaceus) embryos by vitrification

Conventional cryopreservation of complex teleost embryos has been unsuccessful, possibly because their large size (1-7 mm diameter), multi-compartmental structure and low water permeability lead to intracellular ice formation and chilling injury. To overcome these obstacles, we have developed a vitrification procedure for cryopreservation of flounder (Paralichthys olivaceus) embryos. In initial toxicity tests, propylene glycol (PG) and methanol (MeOH) were less toxic to embryos than dimethylformamide (DMF) or dimethyl sulfoxide (Me2SO), whereas ethylene glycol (EG) and glycerol (Gly) were toxic to all tested embryos. Embryos between four-somite and tail bud stages were more tolerant to vitrifying solutions than embryos in other developmental stages. Four vitrifying solutions (FVS1-FVS4) were prepared by combining a basic saline solution (BS2) and cryoprotectants PG and MeOH in different proportions (FVS1: 67, 20 and 13%; FVS2: 60, 24 and 16%; FVS3: 55, 27 and 18%; FVS4: 50, 30 and 20% of BS2, PG and MeOH, respectively). Their impact on flounder embryos was then compared. FVS1 produced the highest survival rate; whereas deformation rate was highest for FVS4. Five-step equilibration of embryos in FVS2 resulted in higher survival rates than equilibration in 4, 3, 2 or 1 steps. Flounder embryos varying from the 14-somite to the pre-hatching stage were cryopreserved in the four vitrifying solutions in liquid nitrogen for 1-7 h. From eight experiments, 20 viable thawed embryos were recovered from 292 cryopreserved embryos. Fourteen larvae with normal morphology hatched successfully from the 20 surviving frozen-thawed embryos from five experiments. Embryos at the tail bud stage exhibited greater tolerance to vitrification than embryos at other stages. These results establish that cryopreservation of flounder embryos by vitrification is possible. The technology has many potential applications in teleost germplasm resource conservation.     

主成分分析在猪肌肉组织化学特性方面的应用

选择3个亲本猪种(莆田黑猪、大白猪和杜洛克猪)和2个杂种猪大莆(大白猪♂×莆田黑猪♀)和杜莆(杜洛克♂×莆田黑猪♀)共150头猪作为试验材料,在相同饲养管理条件下饲养,体重达90kg时屠宰,取背最长肌肉样,测定了肌肉纤维形态、数量和肌肉组织化学成分,并对与肉质有关的12个性状进行了主成分分析和聚类分析.结果表明:不同性状之间呈现不同程度的相关,其中以肌纤维面积与均径的相关性最高.12个性状可简化为2个主成分因子,即肌肉纤维因子和肌肉营养因子,且这2个主成分因子可保留87.132%的信息量,可基本反映原指标提供的信息量.同时,综合评定结果显示,在5个猪种中以杜莆猪肉质最好.     

寄主型和迁飞型棉蚜的交配行为

棉蚜Aphis gossypii Glover种群存在寄主利用和迁飞能力上的明显分化,有棉花型和瓜型、迁飞型和滞留型之分。但是,棉花型与瓜型之间,以及迁飞型与滞留型之间是否能发生交配行为,尚无研究报道。本文在低温和短光照条件下分别诱导棉花型、瓜型、迁飞型和滞留型棉蚜的性蚜,并进行性蚜间的交配行为观察。结果表明,迁飞型和滞留型性蚜间可以发生交配行为,杂交时发生交配行为的个体比率与自交时无显著差异,但是杂交时雄蚜寻找配偶所花的时间要显著长于自交时,并且迁飞型雌蚜(M♀)与滞留型雄蚜(S♂)杂交时的交配持续时间也显著长于自交时。迁飞型和滞留型棉蚜同型交配容易完成,棉蚜的迁飞型和滞留型已在交配行为上产生了一定的分化。棉花型和瓜型棉蚜的性蚜间可以发生交配行为,并且两品系在正交和反交时雄蚜的寻偶时间与交配持续时间均无显著差异。     

紫花含笑（♀）与灰岩含笑（♂）及其杂种F1代叶表皮微形态和叶结构的比较观察

通过比较观察紫花含笑（♀）和灰岩含笑（♂）及其杂种F1代叶表皮微形态和叶结构发现,紫花含笑和灰岩含笑在叶表皮微形态及叶解剖结构方面有很大差异。杂种F1代个体间在叶表皮微形态及叶解剖结构方面变异很大,为连续的数量遗传;其中57．7％以上的杂种F1代气孔密度高于父母本。杂种F1代矮化型植株叶片气孔密度较小,推测其抗寒性较强,可以通过进一步杂交改良,获得矮化型的盆栽含笑新品种。观察结果可为深入探讨含笑属种间杂种F1代的遗传变异,并从这些杂种F1代中选育观赏新品种积累科学资料。     

Experimental horizontal transmission of viral hemorrhagic septicemia virus (VHSV) in Japanese flounder Paralichthys olivaceus

Infection by viral hemorrhagic septicemia virus (VHSV) has recently occurred among wild and farmed Japanese flounder Paralichthys olivaceus in Japan. In the present study, horizontal transmission of VHSV among Japanese flounder was experimentally demonstrated by immersion challenge. Exposure to a flounder isolate (Obama25) of VHSV revealed a dose-response, with higher mortality (81 and 70%) at the 2 higher exposure levels (6.0 and 4.0 log10 TCID50 ml(-1)). In a second experiment, high titers of VHSV were expressed from moribund and dead flounder based on virus detection in holding-tank waters 2 to 3 d prior to death of the fish and 1 d after death. The virus could not be detected in tank waters 2 d after death. Finally, a third cohabitation experiment in small tanks demonstrated horizontal transmission of VHSV from experimentally infected to uninfected fish.     

Larval morphometry of the Japanese flounder,Paralichthys olivaceus

The larval development of the Japanese flounder,Paralichthys olivaceus, was surveyed using two types of morphometric analyses, modified allometry and polar coordinate analysis by principal component analysis (PCA). In the former, centroid size was used as a growth index instead of total length (TL), such enabling the determination of more detailed changes in each character than ordinary allometry based upon TL. Polar coordinate analysis disclosed two remarkable inflexions during the larval development ofP. olivaceus. Postlarvae ofP. olivaceus were found to undergo four developmental phases. From the point of view of metamorphosis, the phases were named drifting larva, premetamorphic larva, metamorphic larva and postmetamorphic larva, respectively. These phases were also tested by other characters related to flounder metamorphosis.