Growth variability of juvenile soles Solea solea and Solea senegalensis, and comparison with RNA : DNA ratios in the Tagus estuary, Portugal

Growth variability and condition of juvenile soles Solea solea and Solea senegalensis , were assessed through RNA : DNA estimates and compared to absolute growth rates. Higher mean cohort RNA : DNA ratios were observed for cohort I at the beginning of estuarine occurrence for both species (4·42 and 4·87, for S. solea and S. senegalensis respectively). Despite different estuarine colonization habits, no significant differences were observed between RNA : DNA monthly variation for both sole species within the same year ( P  > 0·05 for 2003 and 2004). Juvenile S. senegalensis showed significant differences between RNA : DNA ratios obtained for the two nursery areas ( P  < 0·001). The decrease of seasonal growth rates with fish age was similar to seasonal variation of mean RNA : DNA values. Thus the RNA : DNA pattern of juvenile S. solea and S. senegalensis reflected growth and estuarine colonization patterns.     

Seasonal distribution and duration of the planktonic stage of Dover sole, Solea solea, larvae in the Bay of Biscay: an hypothesis

The possibility of deriving a prediction about the effect of seasonal variations in the duration of the planktonic larval phase on the dispersal of larval Dover sole was investigated. During six cruises, from February to May 1992, the distribution of sole larvae was studied along a 100-km transect, from the offshore spawning grounds to the coastal nurseries of the Bay of Biscay (France). Samples ( n = 189) were collected with a suprabenthic sampler, and vertical profiles of water temperature and salinity were recorded simultaneously. Counts of otolith increments of larval stage 4b (onset of metamorphosis) were used to estimate the duration of the planktonic life. Duration of the larval phase decreases by about 15 days (37%) with water temperature increase (between 8° in February and 11.2° C in May). Sole larvae occur from the coastal area to 100 km offshore. Within the same cruise, no difference in the duration of the planktonic life was observed between the larvae caught in the onshore and the offshore area. In spite of seasonal differences in abundance, the extent and the shape of the larval distributions during the period of study suggest that the seasonal variations in the duration of the planktonic life did not affect the larval distribution.     

Interaction between exopolysaccharide and oxygenation levels on habitat selection in the sole Solea solea (L.)

A habitat selection experiment was conducted to examine the behavioural response of sole Solea solea to a combination of sediment quality (exopolysaccharides, EPS-free, i.e. 0 mg l⁻¹ and EPS-rich, i.e. 4 mg l⁻¹) and water oxygenation level (100 and 35% air saturation). The distribution of sole was influenced differently by the type of substratum depending on the water oxygenation level. In normoxia, sole settled preferentially on sand whereas under hypoxic conditions, sole settled preferentially on the muddy substratum. In order to explain these apparently counter-intuitive observations, it is proposed that, via cutaneous respiration, young sole are able to take advantage of the large quantities of oxygen produced by microphytobenthic organisms present in the upper few millimetres of muddy substratum.     

Population genetic structure of turbot (Scophthalmus maximus L., 1758) in the Black Sea

Turbot, Scophthalmus maximus, is a commercially important demersal flatfish species distributed throughout the Black Sea. Several studies performed locally with a limited number of specimens using both mitochondrial DNA (mtDNA) and microsatellite markers evidenced notable genetic variation among populations. However, comprehensive population genetic studies are required to help management of the species in the Black Sea. In the present study eight microsatellite loci were used to resolve the population structure of 414 turbot samples collected from 12 sites across the Black Sea. Moreover, two mtDNA genes, COI and Cyt-b, were used for taxonomic identification. Microsatellite markers of Smax-04 and B12-I GT14 were excluded from analysis due to scoring issues. Data analysis was performed with the remaining six loci. Loci were highly polymorphic (average of 17.8 alleles per locus), indicating high genetic variability. Locus 3/20CA17, with high null allele frequency (>30%), significantly deviated from HW equilibrium. Pairwise comparison of the F_ST index showed significant differences between most of the surveyed sampling sites (P < 0.01). Cluster analysis evidenced the presence of three genetic groups among sampling sites. Significant genetic differentiation between Northern (Sea of Azov and Crimea) and Southern (Turkish Black Sea Coast) Black Sea sampling sites were detected. The Mantel test supported an isolation by distance model of population structure. These findings are vital for long-term sustainable management of the species and development of conservation programs. Moreover, generated mtDNA sequences would be useful for the establishment of a database for S. maximus.     

Interactive effect of ammonia and crowding stress on ion-regulation and expression of immune-related genes in juvenile turbot (Scophthalmus maximus)

The present study was to investigate the interactive effect of ammonia and crowding stress on ion-regulation and expression of immune-related genes in juvenile turbot (Scophthalmus maximus). The fish were exposed to four total ammonia nitrogen (0, 5, 20, 40 mg/L TAN) and two stocking density. After 96 h of exposure, blood, gill, and liver samples were collected to measure biochemical parameters and mRNA levels of immune-related genes. The results showed that co-exposure to high TAN (20 and 40 mg/L) and high density significantly increased plasma sodium (Na⁺), gill Na⁺/K⁺-ATPase activity and mRNA levels. Following individual and combined exposure to high TAN and high density, the heat shock protein 70 (HSP 70), HSP 90, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) genes expression were obviously higher than their control. Conversely, the lysozyme (LZM) and hepcidin mRNA levels were down-regulated in liver of fish exposed to high TAN alone and combination of high TAN-density. Moreover, glutathione S-transferase (GST) mRNA levels significantly increased in treatments with individual high TAN and high density, but decreased in high TAN-density co-exposed fish. Overall, ion homeostasis and immune status were adversely influenced in high exposed turbot under high density.     

Coupling of heart rate and locomotor activity in sole, Solea solea (L.), and bass, Dicentrarchus labrax (L.), in their natural environment by using ultrasonic telemetry

Field measurements of distance moved and heart rate in sole, Solea solea (L.) and bass, Dicentrarchus labrax (L.) using ultrasonic telemetry revealed two different strategies. Heart rate in the sole increases during activity, accurately reflecting fluctuation in metabolic rate and so can be used as a measure of metabolic rate in the field. In contrast, the relatively stable value of the heart rate in bass during the whole tracking period whatever the activity level suggests that in this species heart rate in the field cannot be associated with metabolic rate determination.     

New microsatellite markers in turbot (Scophthalmus maximus) derived from an enriched genomic library and sequence databases

Scophthalmus maximus is an important commercially aquaculture fish species. We tackle the search for new microsatellites using two different approaches: an enriched partial genomic library and a screening of all turbot DNA sequences deposited in GenBank. Out of 15 genomic library derived loci, five gave working primer pairs, with expected heterozygosities (H_E) ranging from 0.13 to 0.91. Out of seven loci derived from database sequences, two amplified successfully with an H_E ranging from 0.56 to 0.63. The average allele number of these microsatellites was 5.7 per locus with a range between two and 15.     

Structural peculiarities of the tubercles in the skin of the turbot,Scophthalmus maximus (L., 1758) (Osteichthyes,Pleuronectiformes, Scophthalmidae)

The structure of the bony tubercles of the turbot, Scophthalmus maximus (L., 1758), was examined using ground sections, microradiography, SEM, and TEM. The tubercles are small, isolated, mineralized conical plates randomly distributed in the eyed side of the body. They are composed of three layers: the outer limiting layer, the external layer, and the basal plate, which make up the thin and flat elasmoid scales of Teleostei. The main difference between regular elasmoid scales and bony tubercles lies in the organization and the growth of the basal plate. Indeed, the conical shape of the tubercle is the result of a prominent thickening of the central part of the basal plate where the collagen matrix is organized in a complicated three-dimensional network. Densely packed thick collagen fibrils form superimposed plies organized in a plywood-like structure that resembles that of the elasmoid scales but it is criss-crossed by numerous vertical sheets of thin collagen fibrils. The tubercles originate from thin and flat plates located in the skin of larvae and juveniles, whose structure is that of regular-developing elasmoid scales. Thus, the tubercles of Scophthalmus maximus could be considered as modified elasmoid scales rather than bony structures. They might be the result of specific arrangements related to the general trend of reduction of the dermal skeleton in the teleostean lineage.     

Influence of DNA isolation from historical otoliths on nuclear-mitochondrial marker amplification and age determination in an overexploited fish, the common sole (Solea solea L.)

Historical otolith collections are crucial in assessing the evolutionary consequences of natural and anthropogenic changes on the demography and connectivity of commercially important fish species. Hence, it is important to define optimal protocols for purifying DNA from such valuable information sources while avoiding any damage to the physical structure of the otolith. Before being able to conclude on the harmlessness of a method, it is important to validate protocols on different kinds of otoliths by testing purification methodologies under standardized conditions. Here we compare the effect of two DNA extraction methods on the success in identifying the age in an overexploited marine fish, the common sole (Solea solea L.). To ensure optimal future population genetic and demographic analyses, we assessed DNA quantity and tested the DNA quality by investigating the amplification success of a mitochondrial and nuclear marker. Our results show that the choice of the DNA extraction method had a significant effect on the success of using these otoliths in age and growth analyses. Standard commercial and published protocols resulted in a severe damaging of the otolith structure, hampering accurate preparation and analyses of the morphological structures of the otoliths. Shortening the lysis time and lowering the EDTA (ethylene diamine tetraacetic acid) and SDS (sodium dodecylsulphate) concentration turned out to be beneficial for the stability of otolith structure, while maintaining an overall high DNA quality measured through polymerase chain reaction amplification success. We therefore recommend that care should be taken when choosing the extraction method for a molecular study on archived samples, in order to enable the maximal use of information embedded in historical material.     

Composition of the seminal Fluid and ultrastructure of the spermatozoon of turbot (Scophthalmus maximus)

Mean values of the composition of the seminal fluid of 30 turbots were: osmotic pressure =306 mosml⁻¹, pH =7–31, total protein =8.8mg ml¹, Na⁺=133.0 mmoll¹, K⁺=3.80mmoll⁻¹, Cl⁻=129mmol l⁻¹. The rounded nucleus, the reduced middle piece and the typical '9 + 2' structure of the flagellum mean that the spermatozoon of turbot can be considered to be of a primitive type.     

Gene diversity analysis in natural populations and cultured stocks of turbot (Scophthalmus maximus L.)

The genetic variability of eight fish-farm and three natural populations of turbot was studied by electrophoretic analysis of 35 enzymatic loci. The results showed low genetic variability in natural populations of turbot ( H _T = 0·029 ± 0·013) in comparison with other flatfish species. Great genetic similarity was revealed among the natural populations studied, which indicates high rates of gene flow in this species. The hatchery stocks showed less genetic variation than the wild populations analysed, which suggests genetic drift phenomena involved in the foundation and management of broodstocks. In addition, the heterozygosity differences detected among the hatchery stocks analysed are correlated with inverted levels of fluctuating asymmetry, which supports the existence of inbreeding depression phenomena in turbot culture.     

Histological changes in starved turbot larvae (Scophthalmus maximus) quantified by digital image analysis

In turbot larvae, Scophthalmus maximus , deprived of food for 24 h there was a significant increase in the specific surface of the epithelium and the corresponding microvillous border of the foregut accompanied by slight cellular degeneration. Following 48 h starvation Sarvae showed severe tissue degeneration in the foregut mucosa, progressing to extensive mucosal desquamation and cellular sloughing. Intracellular vacuolation of the epithelium and loss of microvilli was also extensive and the ability of the gut to absorb food must be severely impaired, with further starvation probably resulting in larval death. There was no evidence of parasitic infection in any of the larvae sampled and all observed alterations are attributable to food deprivation.     

摄食水平和投喂频率对大菱鲆幼鱼生长及生化成分的影响

以大菱鲆幼鱼(7.56±0.03 g)为试验对象,在水温17～19℃下,设计了4个摄食水平梯度(1.0％、1.5％、2.0％和饱食)和3个投喂频率梯度(1、2和3次·d-1),探讨了不同摄食水平和投喂频率对其生长及生化成分的影响.结果表明:随着摄食水平的升高,大菱鲆幼鱼的终末体重、相对增重率和摄食率显著升高(P＜0.05);特定生长率随摄食水平的增加呈线性升高;湿重、干重、蛋白质和能量转化效率出现先升高后降低的趋势;摄食水平对鱼体的体成分和组间表观消化率无显著影响(P＞0.05);不同投喂频率下,终末体重、相对增重率、特定生长率及转化效率呈先升高再降低的趋势,在2次·d-1时取得最大值;投喂频率对鱼体的体成分和组间表观消化率无显著影响(P＞O.05).     

Growth and gonadal development of gynogenetic diploid Scophthalmus maximus

The survival, growth, gonadal development and sex ratios of gynogenetic diploid turbot Scophthalmus maximus between 9 and 36 months of age were investigated. Gynogenesis was induced by activation of freshly collected eggs with diluted (1 : 10 with Ringer) and UV‐irradiated (30000 erg mm⁻²) sperm, followed by a cold shock at −1 to 0° C for 25 min, starting at 6·5 min after fertilization. The exclusive maternal inheritance of the resulting gynogenetic diploids was confirmed in all individuals used in the experiment by microsatellite markers. Mean total length and mass throughout the experiment was higher in the controls than in the gynogenetics. The gonadal development in gynogenetic males proceeded normally, and in gynogenetic females it was delayed during the first sexual maturation but was normal during the second one. The sex ratio was 1 male (M) : 1 female (F) in the controls, as expected, but 1 M : 3 F in the gynogenetics. In a second, independent experiment, carried out with fish originating from different broodstock, however, the sex ratio was 1 M : 1 F in the controls and 0 M : 1 F ( i.e . 100% females) in the gynogenetics. Together, these results suggested that the main sex‐determining genetic component in turbot fits well to female homogamety, in accordance with data from other flatfishes. These results indicate that it will be possible to obtain near or all‐female turbot stocks through induced gynogenesis or by crosses involving hormonally sex‐reversed fish.     

养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析

2004年夏季山东海阳一带养殖大菱鲆(Scophthalmus maximusL.)暴发溃疡病。症状主要表现为体表病灶部位出血、肌肉溃烂、眼球凸出等,解剖可见鳃丝贫血,肝脏充血,肾脏和胆囊肿大,肠壁充血并呈透明状,从出现发病症状起,大约一周后开始出现死亡。从病鱼体表溃疡部位及内脏分离出优势菌并命名为H1,经人工感染证实H1即为引起本次养殖大菱鲆体表溃疡症的病原菌。对病原菌进行鉴定揭示该菌革兰氏染色阴性,菌体呈短杆状,极生单鞭毛。综合该菌在形态、生理生化、API20NE与API20E自动鉴定结果、16S rDNA同源性等方面的特性,确认H1为哈维氏弧菌(Vibrio harveyi),该菌对呋喃妥因、菌必治等多种抗生素敏感。哈维氏弧菌是海水养殖鱼类的常见致病菌,但作为养殖大菱鲆的病原菌属首次报道。     

大菱鲆白化相关基因(DEN-1、DEN-2)的分离鉴定及特征分析

应用mRNA差异显示技术,对比研究正常与白化大菱鲆有眼侧皮肤组织,克隆差异表达基因,经测序,RT-PCR验证,差异表达基因片段DEN-1(GenBank登录号:DQ886390)与DEN-2(GenBank登录号:DQ886391)均在白化大菱鲆有眼侧皮肤组织中下调表达;通过BLAST检索发现,DEN-1与GenBank中的斑马鱼和牛的类似尿苷二磷酸-葡萄糖:糖蛋白葡糖基转移酶(UGGT)基因、与挪威鼠类似尿苷二磷酸-葡萄糖:神经酰氨葡糖基转移酶1(Ugcgl1)基因有较高同源性;DEN-2与原鸡、斑马鱼、人、挪威鼠、家鼠和狗的眼缺乏同源框4(eya4)基因的同源性均较高。采用相对定量RT-PCR对正常鱼有眼侧皮肤(N1)、白化鱼有眼侧皮肤(A1)、正常鱼无眼侧皮肤(N2)、白化鱼无眼侧皮肤(A2)进行表达谱分析,以内参β-actin基因分别对DEN-1和DEN-2表达量进行标准化的光密度扫描显示:DEN-1和DEN-2在4种皮肤组织中有相似的表达模式,即表达量:N1>N2≈A2>A1。电子延伸将DEN-2延伸为497bp的片段,该延伸片段与多种动物的eya4基因在核酸和蛋白水平均有很高同源性。本研究对DEN-1和DEN-2的不同组织器官和不同发育阶段的电子表达谱分析,将对其进一步研究提供有价值参考。     

Vibrio splendidus biotype 1 as a cause of mortalities in hatchery-reared larval turbot, Scophthalmus maximus (L.)

AIMS: To characterize bacteria associated with turbot larvae feeding on Artemia and identify pathogens causing mortalities in larvae. METHODS AND RESULTS: To identify bacteria associated with mortalities in larval turbot rearing, bacteria were isolated from homogenates of Artemia or from several batches of well-performing or poorly performing turbot larvae. Samples were plated onto marine agar and were characterized using biochemical tests and BIOLOG GN plates. Total culturable aerobic bacteria ranged from 1.9 x 10(5) to 1.8 x 10(6) CFU per larva and >96% of bacteria identified were vibrios. Almost all bacteria were haemolytic and clustered into two phenons represented by Vibrio alginolyticus and Vibrio splendidus. The bacterial flora of Artemia was almost entirely V. alginolyticus, whereas V. splendidus biotype 1 dominated the larval turbot gut flora (69/115 isolates in seven experiments) and formed four different groups based on BIOLOG GN reactions. Of 16 isolates tested for virulence towards turbot larvae, four of the 11 V. splendidus biotype 1 isolates were lethal and all belonged to the same group of V. splendidus biotype 1 isolates. CONCLUSIONS: In a commercial turbot hatchery, the microbial flora of the larval gut was dominated by V. splendidus biotype 1. Four of the 11 V. splendidus biotype 1 isolates caused mortalities in larval turbot and all belonged to one group of the biotype 1 strains identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of four isolates of V. splendidus that are pathogenic for turbot larvae from three separate batches of larval turbot will allow these to be compared with avirulent isolates to define how V. splendidus causes mortalities in larval turbot.     

盐度对大菱鲆幼鱼鳃Na -K -ATPase活力、血清离子浓度 和激素水平的影响

采用饲养试验方法,研究了平均体质量为(7.16±0.07)g的大菱鲆(Scophthalmus maximus)幼鱼分别在盐度12、18、24、30和36下饲养60 d后,其鳃Na+-K+-ATPase活力、血清离子浓度、生长激素(GH)、皮质醇激素(COR)、特定生长率(SGR)和饲料效率(FCE)的变化。结果表明:幼鱼鳃Na+-K+-ATPase活力、血清Na+浓度均随盐度的升高而上升,分别在3.48~8.30 U/mg和169.99~180.00 mmol/L之间,其中12盐度组最低,36盐度组最高。幼鱼血清中K+和Cl-浓度分别在2.20~3.47 mmol/L和136.67~142.00 mmol/L之间,各盐度组之间差异不显著。幼鱼血清中GH和COR浓度分别在0.41~1.66 ng/ml和35.33~76.41 ng/ml之间;其中GH在36盐度组最高,12盐度组最低,而COR在12盐度组最高,36盐度组最低。幼鱼SGR和FCE分别在(1.45~2.00)%/d和1.12%~1.38%之间,与盐度的相关性不显著,两者均为12盐度组最低。由此可见,盐度变化显著影响大菱鲆幼鱼鳃Na+-K+-ATPase活力、血清Na+浓度和激素含量。本研究对大菱鲆养殖生理生态条件分析具有重要参考意义,研究结果可为大菱鲆养殖的盐度选择提供理论依据。