Inhibitory effect of 2,4-dibromophenol and 2,4,6-tribromophenol on larval survival and metamorphosis of the sea urchin <Emphasis Type="Italic">Strongylocentrotus nudus</Emphasis>

ABSTRACT:   As a possible factor leading to the low recruitment level of sea urchins in kelp forests, the inhibitory effect of 2,4-dibromophenol (DBP) and 2,4,6-tribromophenol (TBP) released from the large perennial brown algae Ecklonia kurome and Eisenia bicyclis on survival and metamorphosis of eight-armed larvae of the sea urchin Strongylocentrotus nudus was examined. The percentage of larvae that underwent metamorphosis in filtered sea water after 1 h exposure to one-half dilution of saturated dibromomethane solution (∼60 ppm) as a chemical inducer reached approximately 100% after 1 h, while that in filtered sea water containing 1 ppm TBP was reduced to 73%. This was further reduced to less than 40% in the presence of 10 and 20 ppm TBP after 2 h. In filtered sea water containing 1 and 10 ppm DBP, the proportion of metamorphosed larvae was reduced markedly to 43 and 5% after 2 h, respectively. All larvae exposed to 50 ppm TBP and to 20 and 50 ppm DBP died after 1 h. These findings suggest that DBP is more toxic than TBP for sea urchin larvae, strongly inhibiting their metamorphosis.     

Relationship between soak time and number of enmeshed animals in experimentally lost gill nets

In order to determine the effects of ghost fishing by lost gill nets, the relationship was examined between soak time and number of enmeshed animals in experimentally lost gill nets by using diving observations. Two experimental gill nets were set at 13 m depth in Tateyama Bay, Chiba Prefecture, Japan for approximately 200 days. One gill net was deployed in a small trough surrounded by artificial reefs, and the other was deployed on an adjacent open sandy bed. Twelve species of crustaceans, six species of gastropods, and five species of bony fish were enmeshed in the experimental gill nets. The number of enmeshed animals in the artificial reef gill net was substantially larger than that in the sandy bed gill net. The number of enmeshed animals in the experimental gill nets increased rapidly within one month after deployment, and then declined gradually showing fluctuations caused by the decrease in newly enmeshed animals, and the drop off from gill nets caused by the decomposition of dead animals. The decrease in the number of enmeshed animals was expressed by logarithmic equations, and based on these equations, the duration of capture function for the lost gill nets was calculated to be 284–561 days in the artificial reef gill net and 200 days in the sandy bed gill net. The duration of capture function for the lost gill nets for non-commercial by-catch species such as small crustaceans and gastropods was longer than for commercial species such as Japanese spiny lobster and bony fish.     

Universal PCR primers for ribosomal protein gene introns of fish

Human ribosomal protein (RP) gene sequences with respect to intron/exon structures and corresponding cDNA or genomic data of fish species were obtained from the GenBank database. Based on conserved exon sequences, 128 primer pairs for 41 genes were designed for exon-primed intron-crossing (EPIC) polymerase chain reaction (PCR). In reference to the draft genome sequences of the Pacific bluefin tuna (Thunnus orientalis), 12 primer pairs expected to amplify introns of the bluefin tuna with lengths of 500–1000 bp were selected and applied to six distantly related fish species belonging to the Orders Clupeiformes, Tetraodontiformes, Pleuronectiformes, Perciformes, Scorpaeniformes, and Anguilliformes. PCR amplification was observed for at least four species in each primer pair, and all fragments were larger than those expected for intronless amplification. Single fragment amplification was observed for at least seven primer pairs per species. Fragment sizes of the bluefin tuna for nine primer pairs corresponded to those expected from the genomic data. Thus, our primer pairs are potentially applicable to a wide variety of fish species and serve as an initial step for isolating single-copy nuclear DNA sequences.     

Androgen-induced pseudo-hermaphroditic phenotypes in female <Emphasis Type="Italic">Brevimyrus niger</Emphasis> Günther 1866 (Teleostei,Mormyridae)

This paper explores the plasticity of sexually dimorphic characters in subadult female Brevimyrus niger, an African weakly electric mormyrid species. Thirty-five fish were exposed in a staggered fashion (five fish a week) to aromatizable 17α-methyltestosterone over a period of 7 weeks; 18 fish served as untreated controls. 17α-MT induced precocious vitellogenesis that mirrored the natural maturational process during seasonal ovarian recrudescence. At the same time, 17α-MT exposure resulted in complete masculinization of the females’ anal fin support structure normally observed during rainy season in adult males. We discuss possible hormonal mechanisms acting along the brain-pituitary-gonad axis that would explain the occurrence of precocious vitellogenesis and the male-typical transformation of the female’s anal fin ray bases. Our findings are relevant to commercial aquaculture as the use of 17α-MT in fish hatcheries can pose serious environmental issues.     

Artificially induced tetraploid masu salmon have the ability to form primordial germ cells

Diploid gametes generated with tetraploid animals are a stepping stone to improving chromosome manipulation techniques. However, artificially induced tetraploid individuals generally die soon after hatching. Diploid gametes could be induced by in vivo cultures of tetraploid primordial germ cells (PGCs) through germ-line chimera. In the present study, characteristics of PGCs were studied in inviable tetraploid masu salmon, Oncorhynchus masou. Histological observation of tetraploid embryos revealed that the same or smaller numbers of PGCs were observed and they migrate into the genital ridges as did diploid PGCs during gonadogenesis. By whole-mount in situ hybridization using vasa messenger RNA (mRNA), 4–35 vasa-positive signals were detected in a pair of genital ridges of tetraploids. By cytological observation of genital ridge cell suspensions, several large round cells were observed, some of which extended pseudopodia. They also contained large nuclei and round granules in their cytoplasm, characteristics of PGCs. As the results suggest that inviable artificial tetraploids have PGCs, we expect to achieve diploid gamete production through surrogate propagation and tetraploid fish production.     

Effect of calcium ion on the thermal denaturation of subfragment-1 and rod regions of squid myosin upon the heating of myofibrils

Thermal inactivation of Ca²⁺ ATPase of squid myofibrils was significantly suppressed in the presence of Ca²⁺. Monomeric myosin content decreased much faster than Ca²⁺ ATPase inactivation in Ca medium, which was well explained by fast rod denaturation. In contrast, rod denaturation was slower than S-1 in EDTA medium. The decrease in monomeric myosin content was explained by faster S-1 denaturation. Comparing the S-1 and rod denaturation rates at a fixed temperature, it was concluded that S-1 denaturation was suppressed by Ca²⁺ whereas the rod denaturation was not. An unfolding experiment with isolated myosin rod confirmed that there was no stabilizing effect of Ca²⁺ on myosin rod. It was concluded that significant stabilization of the S-1 portion by Ca²⁺ generated the apparently different myosin denaturation patterns in the two media.     

Growth and reproduction of the lion's paw scallop Nodipecten subnodosus in a suspended culture system at Guerrero Negro lagoon,Baja California Sur,Mexico

The lion's paw scallop, Nodipecten subnodosus (Sowerby) has considerable aquacultural potential due to its fast growth and large adductor muscle. Prior investigations throughout northwestern Mexico's littoral have reported highly variable growth rates; furthermore, no studies are available of the environment on growth and gametogenesis in this species under culture conditions. This investigation assesses the effect of food availability and temperature on the growth and gametogenesis of N. subnodosus in a suspended culture system at Guerrero Negro lagoon, Mexico. After 1 year of cultivation, N. subnodosus reached 69.13 mm in shell height (SH) (0.196 mm day^?1, 14 months old). Two significant growth spurts were observed: over the two first months of culture (August and September 2001, mean growth rate 0.4 mm day^?1) and in September 2002 (0.3 mm day^?1), both related to high temperatures and chlorophyll a concentrations. The onset of gametogenesis occurred in April 2002, with an increase in temperature (10‐month‐old scallops, 54.5 mm SH). The first spawning occurred in October and November (86.2 and 93 mm SH), with peak temperatures. These results, together with the analysis of previous reports, indicate that N. subnodosus has a higher preference for temperate areas; therefore, the Guerrero Negro lagoon appears to be a suitable site to culture this species.     

Relative potencies of natural estrogens on vitellogenin and choriogenin levels in the Indian freshwater spotted snakehead, <Emphasis Type="Italic">Channa punctata</Emphasis>: in vivo and in vitro studies

The relative efficacies of three natural estrogens viz., estrone (E₁), estradiol-17β (E₂) and estriol (E₃) to induce synthesis of vitellogenin (Vg) and choriogenin (Chg) were assessed in primary hepatocyte cultures of the Indian freshwater spotted snakehead, Channa punctata. Hepatocytes were isolated from the spotted snakehead liver by a non-enzymatic protocol. Optimum culture conditions were standardized for ensuring their viability and functioning. Isolated hepatocytes were cultured for 48 h for monolayer formation and then exposed to various concentrations (0.001–10 μM) of the three estrogens. Competitive homologous ELISAs, developed and validated for spotted snakehead Vg and Chg were employed to determine the amounts of these two proteins secreted into the culture medium after 48 h of incubation. The results reveal that although all the three estrogens were effective in inducing the production of Vg and Chg in a dose-dependent manner, there were differences in their relative potencies. Of three estrogens, E₁ was the least potent and could induce synthesis of Vg and Chg only at a minimum concentration of 0.5 μM; whereas significant levels of both the proteins were quantified in culture medium by exposing the hepatocytes to E₂ or E₃ even at a concentration of 0.001 μM. All three estrogens were effective in inducing synthesis of Vg and Chg in vivo also. These results suggest the possibility of employing the above in vitro experimental design to monitor the presence of estrogens/estrogen-like chemicals in natural waters, which could interfere with the estrogen receptor system of fish. This study further points to the possibility of using Chg, in addition to Vg, as a parameter for screening various chemicals for their estrogenic activity.     

Microsatellite marker isolation and cultured strain identification in <Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>

Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.     

Cryopreservation effects on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier 1818)

The effects of straws volume, cryoprotectants and thawing temperatures were evaluated on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier), an important Colombian fish species. Sexually mature fish were induced to ovulation or spermiation with a carp pituitary extract. A pool of suitable sperm samples was diluted in glucose, egg yolk, dimethyl sulphoxide (DMSO‐10%), methanol (MET‐10%) or ethylene glycol (ETG‐5%) and packed in 0.5, 2.5 or 5.0 mL straws and frozen in nitrogen vapour. The thawing process was performed in a 35 or an 80 °C water bath. The fertility was evaluated after 6 h post fertilization. The highest motility percentage (33 ± 3%) was observed with sperm cryopreserved with DMSO, packed in 5 mL straws and thawed at 35 °C. The treatments with DMSO and MET packed in 0.5 and 5.0 mL straws and thawed at 35 °C showed the highest fertility (higher than 71%) and the lowest fertility was obtained with MET‐2.5 mL (9 ± 5%). In all the treatments, a significant decrease in the sperm quality was observed at 80 °C. Sperm cryopreserved with DMSO‐10% or MET‐10%, packed in 2.5 or 5.0 mL straws are suitable to achieve acceptable fertilization and to fertilize high amounts of eggs.