Green light stimulates somatic growth in the barfin flounder Verasper moseri

We examined the effects of different light wavelengths-blue, green, and red-on the somatic growth of the barfin flounder Verasper moseri, a flatfish. The light sources used were fluorescent lamps and a combination of daylight and fluorescent lamps that produced ambient light. These light sources were filtered using blue, green, or red filters. During the experiments, the fish were reared in indoor tanks with running seawater of natural temperature and fed with commercial pellets twice daily until satiety. The tanks were white in color. Fish were exposed to constant light emitted from the fluorescent lamps (9:15, light:dark; 08:00-17:00, light) for 14 weeks from October or September to January or to ambient light with a 14-week natural photoperiod from September to December. The wavelengths that were filtered from the fluorescent lamp light modified the growth of the fish, i.e., fish reared under green or blue light exhibited a greater total length (TL; P<0.01) and body weight (BW; P<0.01) than those reared under red light. In contrast, in the case of fish exposed to filtered ambient light, fish reared under green light exhibited a greater TL (P<0.01) and BW (P<0.01) than fish exposed to other wavelengths-blue-, red-, and nonfiltered ambient light. Our results indicate that flounder growth was modified by certain wavelengths, namely, green and red light, which had growth-stimulating and growth-inhibiting effects, respectively.     

Development of polymorphic microsatellite markers from barfin flounder (Verasper moseri) and their cross-species amplification

Barfin flounder (Verasper moseri) is a rare fish species in the world. Here, we reported 10 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of barfin flounder (Verasper moseri). The number of alleles, observed, and expected heterozygosity per locus in a test population ranged from 2 to 6, from 0.3333 to 1.0000, and from 0.4866 to 0.7774, respectively. One locus significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these microsatellite loci in additional five fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in Verasper moseri. Gui-Dong Miao and Chang-Wei Shao Contributed equally to this work.     

Sequence and organization of the complete mitochondrial genomes of spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri).

In this work, the mitochondrial genomes for spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri) were completely sequenced. The entire mitochondrial genome sequences of the spotted halibut and barfin flounder were 17,273 and 17,588 bp in length, respectively. The organization of the two mitochondrial genomes was similar to those reported from other fish mitochondrial genomes containing 37 genes (2 rRNAs, 22 tRNAs and 13 protein-coding genes) and two non-coding regions (control region (CR) and WANCY region). In the CR, the termination associated sequence (ETAS), six central conserved block (CSB-A,B,C,D,E,F), three conserved sequence blocks (CSB1-3) and a region of 61-bp tandem repeat cluster at the end of CSB-3 were identified by similarity comparison with fishes and other vertebrates. The tandem repeat sequences show polymorphism among the different individuals of the two species. The complete mitochondrial genomes of spotted halibut and barfin flounder should be useful for evolutionary studies of flatfishes and other vertebrate species.     

Isolation and characterization of 31 polymorphic microsatellite markers in barfin flounder (Verasper moseri) and the cross-species amplification in spotted halibut (Verasper variegatus)

The construction of a genetic linkage map and evaluation of population genetic diversity both require large numbers of polymorphic molecular markers. In the present study, we report 31 polymorphic microsatellite markers, of which 28 were isolated from two repeat-enriched libraries constructed from genomic DNA, and three were detected in two genes (MCH-R1 and MCH-R2) of barfin flounder (Verasper moseri). A total of 94 alleles were detected with an average of 3.0 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to six, from 0.30 to 1.00 and from 0.33 to 0.78, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0016) and no significant linkage disequilibrum between pairs of loci was found. Cross-species amplification of these markers was evaluated using the closely related species spotted halibut (Verasper variegatus). This study will potentially be useful for stock management, constructing of a genetic linkage map, mapping economically important quantitative trait loci (QTL), and studying the population genetic diversity of barfin flounder (Verasper moseri).     

Three GnRH systems in the brain and pituitary of a pleuronectiform fish,the barfin flounder Verasper moseri

To clarify the possible function of gonadotropin-releasing hormone (GnRH) in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri, the distribution of three forms of GnRH in various areas of the brain was examined by radioimmunoassay, and the localization of GnRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary was determined by immunocytochemistry. The dominant form in the pituitary was seabream GnRH (sbGnRH), levels of which were much higher than those of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In contrast, sbGnRH levels were extremely low in all other brain areas examined. Levels of sGnRH and cGnRH-II were high in the anterior and posterior part of the brain, respectively. sbGnRH-ir cell bodies were located in the preoptic area, whereas sbGnRH-ir fibers were localized mainly in the preoptic area-hypothalamus-pituitary and formed a distinctive bundle of axons projecting to the pituitary. sGnRH-ir cell bodies were located in the ventromedial part of the rostral olfactory bulbs and in the terminal nerve ganglion (the transitional area between the olfactory bulb and the telencephalon). cGnRH-II-ir cell bodies were localized to the midbrain tegmentum. sGnRH-ir and cGnRH-II-ir fibers were observed throughout the brain except in the pituitary gland. These results indicate that sbGnRH is responsible for the neural control of the reproductive endocrinology of the barfin flounder (hypothalamo-hypophysial system), and that sGnRH and cGnRH-II function as neurotransmitters or neuromodulators in the brain.     

Isolation and characterization of microsatellite DNA markers in the rare species barfin flounder (Verasper moseri) and its closely related species spotted halibut (V. variegatus)

Barfin flounder and spotted halibut have been selected in Japan as target species for stock enhancement due to the fact that the number of individuals of these species in the wild has been decreasing in the last three decades. Barfin flounder is now considered to be a rare species as its situation is critical. The first microsatellite DNA markers for barfin flounder and spotted halibut are described in this study. Cross‐amplification of barfin flounder markers was examined in spotted halibut species and vice versa.     

Identification of two forms of vitellogenin-derived phosvitin and elucidation of their fate and roles during oocyte maturation in the barfin flounder, Verasper moseri

A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phosphatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.     

Two forms of vitellogenin, yielding two distinct lipovitellins, play different roles during oocyte maturation and early development of barfin flounder, Verasper moseri, a marine teleost that spawns pelagic eggs.

Two forms of vitellogenin (Vg), Vg A and Vg B, were identified in serum from estrogen-treated barfin flounder (Verasper moseri). Structural changes of lipovitellins (Lvs) derived from the two Vgs were examined during vitellogenesis and oocyte maturation. Two Lvs, vLv A and vLv B, were identified electrophoretically and immunologically in postvitellogenic oocytes. Each appeared to be composed of distinct heavy chains (vLvH A, M(r) 107,000, and vLvH B, M(r) 94,000) and light chains (vLvL A, M(r) 30,000, and vLvL B, M(r) 28,000) when analyzed by SDS-PAGE. Results from N-terminal amino acid sequencing and Western blotting using antisera to vLvH A and vLvH B verified that there are two Vg polypeptides in serum from estrogen-treated fish, Vg A (M(r) 168,000) and Vg B (M(r) 175,000), which give rise to vLvH A-vLvL A and vLvH B-vLvL B, respectively. N-terminal sequencing revealed two sequences for both phosvitin and beta'-component, supporting the concept of duality for all three classes of Vg-derived yolk proteins. During oocyte maturation, native dimeric vLv B was dissociated into a native M(r) 170,000 monomer (oLv B). Meanwhile, vLv A was extensively cleaved including complete degradation of vLvH A into free amino acids. We propose that the quantitative ratio of vLv A to vLv B in postvitellogenic oocytes regulates the buoyancy of the spawned pelagic eggs by controlling availability of free amino acids which function as osmotic effectors during oocyte hydration. The vLv A/vLv B ratio likely also controls the proportional availability of different types of nutrients, free amino acids versus Lv, for use during embryonic development.     

Molecular Cloning of Growth Hormone Complementary DNA in Barfin Flounder (Verasper moseri)

The olive flounder (family Paralichthidae; Paralichthys olivaceus) growth hormone (ofGH) appears to be the most derived among known growth hormones, with the deletion of 14 consecutive amino acids in the carboxy-terminal region. To ascertain if this deletion is common to all flounders, growth hormone complementary DNA of the barfin flounder (bfGH) (family Pleuronectidae; Verasper moseri) has been cloned. It was amplified by polymerase chain reaction using single-strand cDNA from the pituitary gland. Excluding the poly(A) tail, the bfGH cDNA is 919 nucleotides long and contains a 609-bp open reading frame encoding a putative signal peptide of 17 amino acids and a mature protein of 186 amino acids. Northern blot analysis detected 1.0 kb of bfGH messenger RNA in the pituitary gland, which is a reasonable value considering the poly(A) tail. The deduced amino acid sequence of bfGH has 78% identity with the sequence of ofGH. A major difference is the presence of a 14 amino acid segment (140–153) in bfGH, as in other growth hormones, suggesting that this deletion in the olive flounder occurred after the divergence of the Pleuronectoidae. Received May 7, 1999; accepted July 13, 1999.     

Comparison of behavioural development between Japanese flounder (Paralichthys olivaceus) and spotted halibut (Verasper variegatus) during early life stages

Behavioural development was compared between two flatfish species (Japanese flounder and spotted halibut) from hatching to settlement (juvenile stage) in order to speculate on the ecology of their early life stages and to provide fundamental knowledge for improving seedling production techniques for stock enhancement. Fish were cultured under identical rearing conditions (500‐L tank maintained at 17.8 ± 0.4°C, 34 ppt, 10L : 14D light regime and an initial stocking density of 20 larvae L^?1). Behavioural observations were conducted at about 4‐day intervals from hatching to the juvenile stage. Fish were sampled randomly from the rearing tank, and one fish was transferred into a 250‐ml observation container. Behaviour was video‐recorded for 5 min without food and for an additional 5 min with live feed (rotifer or Artemia). All behavioural data were sorted according to eight developmental stages and compared among developmental stages and between species. The average standard length of the spotted halibut was significantly greater than that of the Japanese flounder in all developmental stages, while the development of Japanese flounder was faster than that of the spotted halibut. For Japanese flounder, feeding, swimming and Ohm‐posture (typical shivering behaviour observed during early life stages in flatfishes) frequency were highest before metamorphosis (mean ± SD; 1.0 ± 2.0 attacks min^?1, 24.0 ± 9.6 actions min^?1, 1.1 ± 1.1 counts min^?1, respectively). Spotted halibut expressed feeding behaviour frequently from the beginning of metamorphosis (3.6 ± 5.2 attacks min^?1), had relatively low swimming activity during all developmental stages, and showed a peak of Ohm‐posture frequency during the flexion stage (2.6 ± 1.0 counts min^?1).     

Development of polymorphic microsatellite markers in barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) by the cross-species amplification

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two commercially important flatfish species in the Northeast Asia. In the present study, we reported polymorphic microsatellite markers in V. moseri and V. variegatus by the cross-species amplification of microsatellite primers developed previously in two other related marine fish species. A total of 244 polymorphic microsatellite markers were selected for cross-species amplification in V. moseri and V. variegatus, of which 182 markers deriving from Atlantic halibut (Hippoglossus hippoglossus) and 62 markers deriving from Japanese flounder (Paralichthys olivaceus). A sample of 10 individuals were detected. As a result, a total of 67 loci showed polymorphisms in V. moseri, and 62 loci showed polymorphisms in V. variegatus, with the observed number of alleles per locus ranging from two to five in V. moseri, and from two to seven in V. variegatus, respectively. This paper provided more candidate microsatellite markers which could be useful for construction of genetic linkage maps, evaluation of population genetic structure and stock management of V. moseri and V. variegatus.     

Genetic linkage maps of barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) based on AFLP and microsatellite markers

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two economically important marine fish species for aquaculture in China, Korea and Japan. Construction of genetic linkage maps is an interesting issue for molecular marker-assisted selection (MAS) and for better understanding the genome structure. In the present study, we constructed genetic linkage maps for both fish species using AFLP and microsatellite markers based on an interspecific F₁ hybrid family (female V. moseri and male V. variegatus). The female genetic map comprised 98 markers (58 AFLP markers and 40 microsatellite markers), distributing in 27 linkage groups, and spanning 637 cM with an average resolution of 8.9 cM. Whereas the male genetic map consisted of 86 markers (48 AFLP and 38 microsatellite markers) in 24 linkage groups, covering a length of 625 cM with an average marker spacing of 10 cM. The expected genome length was 1,128 cM in female and 1,115 cM in male, and the estimated coverage of genome was 56% for both genetic maps. Moreover, five microsatellite markers were observed to be common to both genetic maps. This is the first time to report the genetic linkage maps of V. moseri and V. variegatus that could serve as the basis for genetic improvement and selective breeding, candidate genes cloning, and genome structure research.     

Ultrastructural studies on oogenesis in Symphyla

Summary The paired ovaries of Symphyla are sac-shaped. Their interior is filled with synchronously developing oocytes surrounded by a simple follicular epithelium. Previtellogenic oocytes have spherical or oval nuclei, the latter containing a large number of small nucleoli. Part of the nuclear surface is covered by a thick coat made up of a granular substance of medium electron density. The coat is penetrated by cisternae of the smooth endoplasmic reticulum. Micropinocytosis is the principal source of yolk in Symphyla; a small amount of yolk material arises within the mitochondrial cristae. Vitellogenic oocytes are characterized by a highly active rough endoplasmic reticulum synthesizing a material, probably lipoprotein in nature, which is incorporated into lipid droplets.Supported by Government Problem Grant II-1.3.13. The author is grateful to Dr. F. Kaczmarski of the Medical Academy, Kraków, for the use of EM facilities in his laboratory and to Dr. W. Hüther, Ruhr-Universität, Bochum, for the identification of the specimens     

Ultrastructural studies on oogenesis in symphyla

Summary In the oocytes of Hanseniella nivea, cortical granules are formed in the peripheral ooplasm during late stages of oogenesis. Single Golgi elements are involved in the process. Concurrent with the formation of cortical granules is the appearance of a chorion on the oocyte surface. Precursors of this envelope are most likely synthesized by follicle cells.     

Morphological and histochemical studies on oogenesis in Callosobruchus analis Fabr. (Bruchidae-Coleoptera)

The ovary in Callosobruchus analis consists of telotrophic ovarioles with the so called nurse cells confined to one chamber at the anterior end of the ovariole. There are three types of lipids in the ovary: (1) L₁ bodies that are present in the early oocytes, in the posterior prefollicular tissue and in the follicular epithelium and contain unsaturated phospholipids; (2) L₂ bodies that have a complete or incomplete sheath of phospholipids and a triglyceride core; (3) L₃ bodies that are formed of highly saturated triglycerides. Lipids are absent from the trophic tissue. In a mature oocyte the L₁ and L₂ bodies are cortical in distribution while the L₃ bodies are centrally located. The mitochondria contain lipoproteins with RNA. The yolk spheres are acid mucopolysaccharides and protein in nature. The precursors of the yolk spheres appear first in the cortical coplasm and are absent from the follicular epithelium or the trophic tissue. The nucleolus of the oocyte shows evidence of extrusions that are believed to pass into the ooplasm. There are no nutritive cords connecting the trophic tissue to the oocytes; nor is there any evidence of any histochemically demonstrable nutritive material being contributed to the oocyte by the trophic tissue. The circumstantial evidence points towards a contribution of the raw materials to the oocyte by the haemolymph either through or in between the follicular epithelium in some soluble form or as submicroscopic particles.     

Histological study of oogenesis pathology in smoky mutant mice

The various pathology of female mice reproductive system with the mutation smoky (smk) was observed. This recessive autosomal gene dilutes the black colour of hair to the light smoky. The cases with ovarian agenesis and dysgenesis of the mutants WR-smk/smk and B6-smk/smk without the germ cells or with the acute deficit was discovered. The oogenesis pathology of the mutants B6-smk/smk was revealed by the block of oocyte growth and their nucleus development in the growing nonatretic follicles leading to the female infertility. Endogene causes of the female hypogonadism and infertility associated with the mutation smoky are proposed.     

Changes in brain GnRH mRNA and pituitary GnRH peptide during testicular maturation in barfin flounder

The pleuronectid barfin flounder (Verasper moseri) expresses three forms of gonadotropin-releasing hormone (GnRH) in the brain. To clarify the physiological roles of the respective forms during testicular maturation, changes in brain GnRH mRNA levels and pituitary GnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. Fish hatched in April 2000. The gonadosomatic index remained low until October 2001 and then rapidly increased in January 2002. Fish continued to grow from hatching through testicular maturation. Fish spermiated in March 2002. The amount of seabream GnRH (sbGnRH) mRNA per brain significantly increased in January 2002 and remained at high levels in March 2002. The amounts of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) mRNA per brain did not show significant changes during the experimental periods. Pituitary sbGnRH peptide content significantly increased in March 2002. Pituitary sGnRH peptide and cGnRH-II peptide contents were extremely low compared to sbGnRH peptide levels and showed no significant changes during the experiment. These results indicate that sbGnRH is involved in the testicular maturation of barfin flounder.