虹鳉透明突变的遗传特征及其组织学观察

摘要：采用杂交方法对虹鳉（Poecilia reticulate）透明性状的遗传规律进行了研究。从F1自交和回交后代的表型分析,该性状由1对等位基因控制,呈隐性遗传,其遗传特征符合孟德尔基因分离定律。采用体视镜观察比较不同表型虹鳉体表色素细胞的差异,并应用石蜡切片和电镜技术对不同表型鱼的皮肤、腹膜等结构进行研究,结果显...     

Preference for Vertical-striped Backgrounds by the Oyanirami Coreoperca kawamebari,a Freshwater Serranid

The oyanirami Coreoperca kawamebari displays cross-stripes on its body when afraid; this pattern is also used as a signal to conspecifics. The fish preferred to rest at vertically- rather than horizontally-striped sites, and transparent stripes were more attractive than opaque ones, but no inclination was shown for any particular stripe widths. It is hypothesized that the cross-stripes first evolved as a pattern furthering crypticity among the vertical aquatic habitat plants, secondarily as a signal to conspecifics.     

Induction of mutations in males of the fish Oryzias latipes at a specific locus after gamma-irradiation

We have studied frequencies of mutations induced at the b locus of the fish, Medaka Oryzias latipes, after gamma-irradiation. Homozygotes for the b locus have colorless melanophores whose phenotypic expression can be distinguished from that of the wild type. An advantage of the use of oviparous fish for detection of skin color mutations is that the mutant phenotype can be confirmed as early as 1.5 days after fertilization because of the transparent egg membrane of the embryo. Wild-type (B/B) male fish were exposed to 4.75 or 9.5 Gy of 137Cs gamma-rays at a dose rate of 0.95 Gy/min and then mated with the female testers (b/b). A total of 77,761 F1 offspring were examined for mutation and other abnormalities. In the control, we had 1 mutant among 22,068 offspring, resulting in a mutation rate of 4.53 X 10(-5)/locus/gamete. However, this mutant embryo died before hatching. Therefore, in an attempt to present specific-locus mutation frequencies in the fish, the frequencies of color mutants that survived more than 4 days after hatching were used as frequencies of viable mutants; (number of viable color mutants)/(number of hatched fry that survived more than 4 days after hatching). In the 4.75 Gy-irradiated group the viable mutant frequencies were 45.0 X 10(-5), 69.7 X 10(-5) and 0/locus/gamete, while exposure to 9.5 Gy resulted in mutation rates of 217 X 10(-5), 130 X 10(-5) and 8.06 X 10(-5), respectively, for sperm, spermatids and spermatogonia. In comparison with viable color mutant frequencies those of the total color mutants, which include such mutants as ones that died before hatching (defined as number of total color mutants/number of fertilized eggs minus number of early deaths), were considerably higher. For sperm, spermatids, and spermatogonia after exposure to 4.75 Gy, the frequencies were 1180 X 10(-5), 629 X 10(-5) and 9.90 X 10(-5)/locus/gamete, respectively, and in 9.5-Gy-irradiated fish, the frequencies were 1940 X 10(-5), 953 X 10(-5) and 55.5 X 10(-5). Although our data are incomplete, the present results were compared with mutation induction in mice. We concluded that the frequencies of viable color mutants in the fish can be compared with those in mice.     

肉白水晶彩鲫和红鲫杂交的遗传学

通过肉白水晶彩鲫(Carassius auratustransparent colored var.)和红鲫(C.a.red var.),以及后代不同杂交组合的体色遗传学研究,结果表明,肉白水晶彩鲫和红鲫杂交、肉白水晶彩鲫自交,子代全部为水晶彩鲫型;红白水晶彩鲫自交,子代表现为水晶彩鲫型和红鲫型;红鲫自交,子代全部为红鲫型。初步推断肉白水晶彩鲫是体表鸟粪素缺失型的纯合体(-/-),红鲫是体表鸟粪素完全型的纯合体( / ),红白水晶彩鲫是杂合体( /-)。鸟粪素缺失型相对完全型是显性,但它们的杂交遗传不完全符合一对等位基因的孟德尔分离规律。对照经典遗传学中金鱼的透明和正常遗传实验,发现透明鱼与肉白水晶彩鲫相似,五花鱼与红白水晶彩鲫相似,正常鱼与红鲫相似。二者的主要区别是经典遗传学依据体表将水晶彩鲫分为透明鱼和五花鱼,本研究依据体色将水晶彩鲫分为肉白色、红白色和其他色,由于分类角度的不同,一些观点和结论就产生了差异。     

Transparent adult zebrafish as a tool for in vivo transplantation analysis

The zebrafish is a useful model for understanding normal and cancer stem cells, but analysis has been limited to embryogenesis due to the opacity of the adult fish. To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells. In a hematopoiesis radiation recovery assay, transplantation of GFP-labeled marrow cells allowed for striking in vivo visual assessment of engraftment from 2 hr-5 weeks posttransplant. Using FACS analysis, both transparent and wild-type fish had equal engraftment, but this could only be visualized in the transparent recipient. In a tumor engraftment model, transplantation of RAS-melanoma cells allowed for visualization of tumor engraftment, proliferation, and distant metastases in as little as 5 days, which is not seen in wild-type recipients until 3 to 4 weeks. This transparent adult zebrafish serves as the ideal combination of both sensitivity and resolution for in vivo stem cell analyses.     

Water temperature and fish growth: otoliths predict growth patterns of a marine fish in a changing climate

Ecological modeling shows that even small, gradual changes in body size in a fish population can have large effects on natural mortality, biomass, and catch. However, efforts to model the impact of climate change on fish growth have been hampered by a lack of long‐term (multidecadal) data needed to understand the effects of temperature on growth rates in natural environments. We used a combination of dendrochronology techniques and additive mixed‐effects modeling to examine the sensitivity of growth in a long‐lived (up to 70 years), endemic marine fish, the western blue groper (Achoerodus gouldii), to changes in water temperature. A multi‐decadal biochronology (1952–2003) of growth was constructed from the otoliths of 56 fish collected off the southwestern coast of Western Australia, and we tested for correlations between the mean index chronology and a range of potential environmental drivers. The chronology was significantly correlated with sea surface temperature in the region, but common variance among individuals was low. This suggests that this species has been relatively insensitive to past variations in climate. Growth increment and age data were also used in an additive mixed model to predict otolith growth and body size later this century. Although growth was relatively insensitive to changes in temperature, the model results suggested that a fish aged 20 in 2099 would have an otolith about 10% larger and a body size about 5% larger than a fish aged 20 in 1977. Our study shows that species or populations regarded as relatively insensitive to climate change could still undergo significant changes in growth rate and body size that are likely to have important effects on the productivity and yield of fisheries.     

Identification of the causative gene of a transparent phenotype of juvenile red sea bream Pagrus major

Deformities in cultured fish species may be genetic, and identifying causative genes is essential to expand production and maintain farmed animal welfare. We previously reported a genetic deformity in juvenile red sea bream, designated a transparent phenotype. To identify its causative gene, we conducted genome-wide linkage analysis and identified two single nucleotide polymorphisms (SNP) located on LG23 directly linked to the transparent phenotype. The scaffold on which the two SNPs were located contained two candidate genes, duox and duoxa, which are related to thyroid hormone synthesis. Four missense mutations were found in duox and one in duoxa, with that in duoxa showing perfect association with the transparent phenotype. The mutation of duoxa was suggested to affect the transmembrane structure and thyroid-related traits, including an enlarged thyroid gland and immature erythrocytes, and lower thyroxine (T₄) concentrations were observed in the transparent phenotype. The transparent phenotype was rescued by T₄ immersion. Loss-of-function of duoxa by CRISPR–Cas9 induced the transparent phenotype in zebrafish. Evidence suggests that the transparent phenotype of juvenile red sea bream is caused by the missense mutation of duoxa and that this mutation disrupts thyroid hormone synthesis. The newly identified missense mutation will contribute to effective selective breeding of red sea bream to purge the causative gene of the undesirable phenotype and improve seed production of red sea bream as well as provide basic information of the mechanisms of thyroid hormones and its related diseases in fish and humans.Subject terms: Agricultural genetics, Animal breeding     

Expression of the tyrosinase-encoding gene in a colorless melanophore mutant of the medaka fish, Oryzias latipes

In the medaka fish Oryzias latipes many mutants for body colors have been isolated. Among them, a colorless melanophore mutant b, carrying b alleles homozygously, has pigmented black eyes but orange-colored skin with amelanotic melanophores, suggesting the presence of a tissue-specific mechanism of melanin formation. To cast light on the molecular basis of the mechanism, we have cloned cDNAs for tyrosinase (Tyr), a key enzyme in melanin biosynthesis, from the wild-type (wt) fish. DNA sequence analysis revealed that all clones encode a protein of 540 amino acids, having five potential glycosylation sites and two copper-binding sites that are characteristic features of Tyr. Genomic DNA blot analysis disclosed that the Tyr gene is present as a single copy in the fish genome. Using a cDNA clone as a probe, RNA blot analysis was carried out. In the wt, the 2.2-kb Tyr mRNA was expressed in eyes and skin but not in liver, corresponding to tissue-specific melanin formation. In the b mutant, contrary to expectation, the mRNA was detected not only in eyes but also in amelanotic skin. Therefore, pigmentation of the skin controlled by b is not directly related to expression of the Tyr gene.     

Infectious gastroenteritis caused by Vibrio harveyi (V. carchariae) in cultured red drum, Sciaenops ocellatus

Summary An outbreak of serious mortality among the cultured red drum Sciaenops ocellatus (L.) characterized by a swollen intestine containing transparent yellow fluid (ascites and gastroenteritis) occurred in July 2000 in Taiwan. A motile strain Rd 0700 was isolated from head kidney and/or the intestinal yellow fluid on tryptone soya agar (TSA) supplemented with 2% (w/v) NaCl and/or thiosulfate citrate bile salt (TCBS) sucrose agar plates. Applying biochemical characteristics, this strain was characterized and identified as Vibrio harveyi (V. carchariae). The bacteria could be re‐isolated from kidney, liver, and the transparent yellow fluid of swollen intestine of fish after bacterial challenge. The LD₅₀ values of the organism and its extracellular products (ECP) were 2.9×10⁷ colony forming units (CFU) and 3.85 μg protein g^?1 fish body weight, respectively. All moribund/dead fish exhibited gastroenteritis except those killed within 12 h. This is a first report showing that intraperitoneal (i.p.) injection of the ECP from V. carchariae is lethal to red drum and can reproduce gastroenteritis in the fish.     

The ADP-ribosylating toxin, AexT, from Aeromonas salmonicida subsp. salmonicida is translocated via a type III secretion pathway

AexT is an extracellular ADP ribosyltransferase produced by the fish pathogen Aeromonas salmonicida subsp. salmonicida. The protein is secreted by the bacterium via a recently identified type III secretion system. In this study, we have identified a further 12 open reading frames that possess high homology to genes encoding both structural and regulatory components of the Yersinia type III secretion apparatus. Using marker replacement mutagenesis of aopB, the A. salmonicida subsp. salmonicida homologue of yopB in Yersinia, we demonstrate that the bacterium translocates the AexT toxin directly into the cytosol of cultured fish cells via this type III secretion pathway. An acrV mutant of A. salmonicida subsp. salmonicida displays a calcium-blind phenotype, expressing and secreting significant amounts of AexT even in the presence of CaCl2 concentrations as high as 10 mM. This acrV mutant is also unable to translocate AexT into the cytosol of fish cells, indicating AcrV is involved in the translocation process. Inactivation of either the aopB or acrV gene in A. salmonicida subsp. salmonicida (resulting in an inability to translocate AexT) is accompanied by a loss of cytotoxicity that can be restored by trans complementation. Finally, we present data indicating that preincubation of the wild-type bacteria with antibodies directed against recombinant AcrV-His protein provides fish cells protection against the toxic effects of the bacterium.     

透明鲫—一个适合活体研究的鱼类模型

目的了解鲫成体透明的原因,探讨该性状的应用特性,为透明鲫作为水生实验动物材料系统开发提供基础。方法对透明鲫进行繁殖,并观察其后代性状,了解透明性状的遗传规律;体视镜观察透明鲫色素细胞的种类与分布,并与鲫比较;组织切片和压片确认微孢子虫对透明鲫的感染,并观察感染症状的变化。结果鲫的透明性状可以遗传,大部分后代表现为通体透明,心、肝、肾、肠、鳔、鳃、脊椎等组织器官肉眼清晰可见。与正常鲫比较,透明鲫的主要色素细胞为黄色素细胞,并未发现虹彩色素细胞,黑色素细胞的数量也大为减少。微孢子虫对鱼体的感染过程可直观观察,病原的扩散和空间分布能实时获得,具普通鱼类无法比拟的应用优势。结论虹彩色素细胞的缺失是鲫透明突变的结构基础。由于透明鲫内部器官可直接观测,无需依靠解剖或复杂仪器系统,在同一动物身上可能获得一系列的动态试验数据,或可作为模型材料广泛应用于生命科学不同领域。     

Mutation of cyp19a1b results in sterile males due to efferent duct obstruction in Nile tilapia

The involvement of estrogen in male fertility has been well established in mammals. However, less is known about the role of estrogen in fish male reproduction. Our recent study revealed that Cyp19a1a deficiency had no effect on fertility in male fish. In this study, expression of Cyp19a1b, but not Cyp19a1a, was detected by immunohistochemistry in Leydig cells of tilapia testes. cyp19a1b mutation resulted in a significant decrease in the concentration of 17β‐estradiol in serum and sterility in XY fish, as no offspring were obtained when crossed with control XX fish at 240 days after hatching (dah). No sperm was obtained from the mature mutants by in vitro extrusion. Further examination of the mutant gonads revealed excessive semen accumulation and testicular hypertrophy. Semen collected from the mutant testes during autopsy contained sperm with a normal morphology that showed no significant differences in motility, VCL, BCF, STR, or fertility compared with control sperm. Efferent ducts from the mutant testes, which had low‐convolution levels, fewer branches, and no blood vessels observed inside the walls, were significantly smaller in size. qRT‐PCR analyses showed downregulated expression of ion exchange genes. There was increased apoptosis in the epithelial cells of the efferent ducts and other somatic cells of the testes as revealed by TUNEL staining, as well as upregulation of apoptosis gene expression in the mutants. At 360 dah, mutant fish showed testicular atrophy and efferent duct fibrosis. These results demonstrated that estrogen deficiency caused by Cyp19a1b mutation resulted in male sterility due to efferent duct obstruction.     

萍乡肉红鲫的性腺发育研究

萍乡肉红鲫(Pingxiang red-transparent crucian carp,Carassius auratus L.)是在江西省萍乡地区分布的天然三倍体鲫突变体经人工选育后获得的遗传性状基本稳定的后代,具有两性生殖和雌核生殖两种生殖方式.研究以F5代萍乡肉红鲫为材料,自孵化后每满1个月开始取性腺,观察了其卵巢1周年性成熟和精巢的发育过程,结果表明萍乡肉红鲫的性腺为1年成熟类型.卵巢发育进程町以分为6个时期,卵母细胞发育相应可分为6个时相.统计了卵巢成熟系数周年变化,体重为95 g左右的雌性萍乡肉红鲫,其成熟卵巢的成熟系数约为(11.73±2.8)%,成熟的卵母细胞内充满卵黄,相对怀卵量为(3018±310)粒/g.萍乡肉红鲫精巢属于小叶型,在精小叶中可观察到不同发育阶段的生殖细胞.由精原细胞分裂而来的仞级精母细胞经分裂增殖,产生次级精母细胞并最终发育成为精子.萍乡肉红鲫的精巢发育程序与普通鲫鱼和鲤鱼相似,卵巢和精巢的发育过程基本同步,孵化后50日龄内性腺分化不明显,到70日龄左右开始出现雌雄分化,3月龄发育为第1期,4-5月龄发育为第2期,6-7月龄发育至第3期,7-10月龄可见第4期卵巢,1年即可成熟产卵,精巢可排出精液.结果表明,该鲫鱼突变体的性腺发育与普通二倍体鲤(鲫)鱼的性腺发育方式类似.     

An introduction to the practical and ethical perspectives on the need to advance and standardize the intracoelomic surgical implantation of electronic tags in fish

The intracoelomic surgical implantation of electronic tags (including radio and acoustic telemetry transmitters, passive integrated transponders and archival biologgers) is frequently used for conducting studies on fish. Electronic tagging studies provide information on the spatial ecology, behavior and survival of fish in marine and freshwater systems. However, any surgical procedure, particularly one where a laparotomy is performed and the coelomic cavity is opened, has the potential to alter the survival, behavior or condition of the animal which can impair welfare and introduce bias. Given that management, regulatory and conservation decisions are based on the assumption that fish implanted with electronic tags have similar fates and behavior relative to untagged conspecifics, it is critical to ensure that best surgical practices are being used. Also, the current lack of standardized surgical procedures and reporting of specific methodological details precludes cross-study and cross-year analyses which would further progress the field of fisheries science. This compilation of papers seeks to identify the best practices for the entire intracoelomic tagging procedure including pre- and post-operative care, anesthesia, wound closure, and use of antibiotics. Although there is a particular focus on salmonid smolts given the large body of literature available on that group, other life-stages and species of fish are discussed where there is sufficient knowledge. Additional papers explore the role of the veterinarian in fish surgeries, the need for minimal standards in the training of fish surgeons, providing a call for more complete and transparent procedures, and identifying trends in procedures and research needs. Collectively, this body of knowledge should help to improve data quality (including comparability and repeatability), enhance management and conservation strategies, and maintain the welfare status of tagged fish.     

Shaping up: a geometric morphometric approach to assemblage ecomorphology

This study adopts an ecomorphological approach to test the utility of body shape as a predictor of niche relationships among a stream fish assemblage of the Tickfaw River (Lake Pontchartrain Basin) in southeastern Louisiana, U.S.A. To examine the potential influence of evolutionary constraints, analyses were performed with and without the influence of phylogeny. Fish assemblages were sampled throughout the year, and ecological data (habitat and tropic guild) and body shape (geometric morphometric) data were collected for each fish specimen. Multivariate analyses were performed to examine relationships and differences between body shape and ecological data. Results indicate that a relationship exists between body shape and trophic guild as well as flow regime, but no significant correlation between body shape and substratum was found. Body shape was a reliable indicator of position within assemblage niche space.     

Refuge use by fish as a function of body length-related metabolic expenditure and predation risks

Theory predicts that staying in a refuge has benefits in terms of predator avoidance and costs in terms of lost feeding opportunities. In this study, we investigated how the relative importance of these costs and benefits changes with increasing body length. This is of particular interest in animals such as fish, which show continuous growth throughout their lives. Our results suggest that larger fish are subject to lower predation risks and are less affected by food deprivation than small fish, with fish decreasing their responses to food-deprivation treatments more strongly with increasing body length than to predation treatments. This may explain our observation that large fish emerged later from a refuge than small ones and spent shorter times outside the refuge. The key role of differential responses to food deprivation was further illustrated by the finding that the relative weight loss of individual fish was strongly correlated with a reduction in hiding time even in the absence of body length differences. The importance of inter-individual differences in metabolic rates for the decision-making behaviour of animals is discussed.     

Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus)

Two auxotrophic genes that play essential roles in bacterial cell wall biosynthesis--alanine racemase (alr) gene and aspartate semialdehyde dehydrogenase (asd) gene--knock-out Edwardsiella tarda (Δalr Δasd E. tarda) was generated by the allelic exchange method to develop a combined vaccine system. Green fluorescent protein (GFP) was used as a model foreign protein, and was expressed by transformation of the mutant E. tarda with antibiotic resistant gene-free plasmids harboring cassettes for GFP and asd expression (pG02-ASD-EtPR-GFP). In vitro growth of the mutant E. tarda was similar to wild-type E. tarda when D-alanine and diaminopimelic acid (DAP) were supplemented to growth medium. However, without d-alanine and/or DAP supplementation, the mutant showed very limited growth. The Δalr Δasd E. tarda transformed with pG02-ASD-EtPR-GFP showed a similar growth pattern of wild-type E. tarda when D-alanine was supplemented in the medium, and the expression of GFP could be observed even with naked eyes. The virulence of the auxotrophic mutant E. tarda was decreased, which was demonstrated by approximately 10? fold increase of LD?? dose compared to wild-type E. tarda. To assess vaccine potential of the present combined vaccine system, olive flounder (Paralichthys olivaceus) were immunized with the GFP expressing mutant E. tarda, and analyzed protection efficacy against E. tarda challenge and antibody titers against E. tarda and GFP. Groups of fish immunized with 10? CFU of the Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP showed no mortality, which was irrespective to boost immunization. The cumulative mortality rates of fish immunized with 10? or 10? CFU of the mutant bacteria were lowered by a boost immunization. Fish immunized with the mutant E. tarda at doses of 10?-10? CFU/fish showed significantly higher serum agglutination activities against formalin-killed E. tarda than PBS-injected control fish. Furthermore, fish immunized with 10?-10? CFU/fish of the mutant E. tarda showed significantly higher ELISA titer against GFP antigen than fish in other groups. These results indicate that the present double auxotrophic genes knock-out E. tarda coupled with a heterologous antigen expression has a great strategic potential to be used as combined vaccines against various fish diseases.     

FliC,a Flagellin Protein,Is Essential for the Growth and Virulence of Fish Pathogen Edwardsiella tarda

Edwardsiella tarda is a flagellated Gram-negative bacterium which causes edwardsiellosis in fish. FliC, as a flagellar filament structural protein, is hypothesized to be involved in the pathogenesis of infection. In this study, a fliC in-frame deletion mutant of a virulent isolate of E. tarda was constructed through double crossover allelic exchange by means of the suicide vector pRE112, and its virulence-associated phenotypes and pathogenicity were tested. It was found that the deletion of fliC significantly decreased the diameter of flagella filaments. In addition, the mutant showed reduced pathogenicity to fish by increasing the LD₅₀ value for 100-fold compared to the wild-type strain, as well as showed impaired bacterial growth, reduced motility, decreased biofilm formation and reduced levels of virulence-associated protein secretion involved in the type III secretion system (TTSS). The phenotypic characteristics of the fliC deletion mutant uncovered in this investigation suggest that fliC plays an essential role in normal flagellum function, bacterial growth, protein secretion by TTSS and bacterial virulence.     

Isolation of mutants of<Emphasis Type="Italic"> Vibrio anguillarum</Emphasis> defective in haeme utilisation and cloning of<Emphasis Type="Italic"> huvA</Emphasis>, a gene coding for an outer membrane protein involved in the use of haeme as iron source

The isolation of Vibrio anguillarum mutants lacking the ability to use haemin and haemoglobin as the only iron sources, as well as the identification of a gene involved in haeme utilisation are described. One of the isolated mutants defective in haeme utilisation lacked an iron-regulated outer membrane protein of 79-kDa. Although growth on haeme as iron source was completely abolished, the haemin and haemoglobin binding activities remained intact in the mutant, suggesting that the absent protein is not the only one involved in haeme binding. The wild-type phenotype in this mutant was restored by transformation with a cosmid clone (pML1) containing a 21-kb DNA fragment isolated from a gene library derived from the parental strain of V. anguillarum. Sequence analysis of pML1 subclones led to the finding of an ORF, huvA, that codes for a 79-kDa protein (HuvA) and whose sequence shows high identity with haeme receptors from Vibrio choleare (HutA) and Vibrio vulnificus (HupA). The sequence of huvA from the V. anguillarum haeme-utilisation mutant revealed a single mutation, leading to the synthesis of a truncated HuvA protein of 70 kDa. The parental strain and the cosmid-complemented mutant showed a higher degree of virulence for fish than the mutant strain in experimental infections in which fish were previously overloaded with haemin. This finding suggests that haeme uptake plays an important role in V. anguillarum multiplication in fish tissues when free haeme is available.