Molecular cloning and expression of caspase-3 in the protandrous cinnamon clownfish, Amphiprion melanopus, during sex change

The caspase-3 appears to be a key protease in the apoptotic pathway. We identified caspase-3 complementary DNAs from the ovaries of the protandrous cinnamon clownfish (Amphiprion melanopus), and investigated its mRNA and proteins, and activity levels during the sex change (I, mature male; II, male at 90 days after removing of the female; and III, mature female). The nucleotide sequence of the caspase-3 cDNA was 969 base pairs in length with open reading frames encoding peptides of 282 amino acids. The caspase-3 mRNA and protein, and activity levels in stages of the mature gonad are higher than those of the development gonad stage. To understand the effect of gonadotropin-releasing hormone (GnRH) on gonad apoptosis, we examined expression of genes caspase-3 mRNA and activity level in immature cinnamon clownfish gonads after GnRH analogue (GnRHa). The findings support the hypothesis that caspase-3 expression is associated with both testicular and ovarian development, and suggests that it may play a role in the control of ovarian development in cinnamon clownfish. Also, we demonstrate that GnRH agonists stimulate caspase-3 production which can in turn stimulate apoptosis. The present study provides a framework for better understanding of the role of caspase-3 during sex change processes in fish.     

Isolation of Sulfated Anticoagulant Compound from Fermented Red Seaweed Grateloupia filicina

The red alga Grateloupia filicina was fermented with 0.15 g/mL sucrose at 25 C, and anticoagulant activity was measured biweekly up to the 10th wk by activated partial thromboplastin time (APTT). A sulfated polysaccharide compound having anticoagulant activity was purified from the sixth wk of fermentation by fast protein liquid chromatography using diethylaminoethyl cellulose followed by a sepharose‐4B column. The concentration of the purified sulfated anticoagulant polysaccharide was 136.65 μg/mL, and the sulfate concentration of the purified compound was 26.33 μg/mL. The presence of just a single spot on agarose gel electrophoresis confirmed that the compound was purified, and polyacrylamide gel electrophoresis showed that the purified compound was of high molecular mass. The purified sulfated anticoagulant polysaccharide consisted of galactose, glucose, and mannose in an approximate molar ratio of 0.95 : 0.01 : 0.03. Both purified anticoagulant and commercial heparin showed >1000 sec APTT activity at 136.65 μg/mL. As the technique of fermentation is inexpensive, the purified anticoagulant compound could have significant potential in the medical and pharmaceutical industry.     

Effects of LED light spectra on the growth of the yellowtail clownfish <Emphasis Type="Italic">Amphiprion clarkii</Emphasis>

Growth hormone (GH) is an essential polypeptide required for the normal growth and development of vertebrates. We have studied the effects of light-emitting diodes (LEDs) emitting different spectra (red, green, and blue) on the GH of yellowtail clownfish Amphiprion clarkii. Full-length GH cDNA from the pituitary of the yellowtail clownfish was first cloned and then the expression of GH mRNA under different light spectra was measured. GH mRNA expression was significantly higher under green and blue light than under red light spectra. These results indicate that in yellowtail clownfish, short-wavelength LED enhances growth more than long-wavelength LED, and that LED lights are more effective for enhancing growth than white fluorescent bulbs. Injection of melatonin resulted in significantly higher expression levels of GH mRNA compared to the control. We therefore conclude that green and blue light enhance GH levels and that melatonin plays a role in modulating growth of the yellowtail clownfish.     

Quantitative investigation of Enteromyxum leei (Myxozoa: Myxosporea) infection and relative condition factor in cultured olive flounder Paralichthys olivaceus (Temminck and Schlegel)

Enteromyxum leei has been reported to cause emaciation disease in various fish species. To determine the effect of parasite intensity on cultured olive flounder Paralichthys olivaceus, we investigated the relationship between the relative condition factor (rCF = CF/standard CF × 100) and parasite load with quantitative polymerase chain reaction (qPCR) and the challenge test. A total of 57 cultured olive flounders were obtained from 11 fish farms and divided into five groups based on their rCF. We investigated the parasite intensity in the posterior intestine of the fish. The parasite load was closely matched to severe loss of body weight. In addition, olive flounders were inoculated either orally or anally with intestinal scrapings of infected fish or phosphate‐buffered saline. The fish were reared at natural water temperature and transferred to different tanks, and the water temperature was adjusted to 20°C after 6 weeks of inoculation. When the water temperature was increased to 20°C, the rCF decreased in the experimentally infected group. The results demonstrated that qPCR can be utilized to determine the relative abundance of E. leei in olive flounders and water temperature is an important factor to track the progress of the emaciation disease.     

Morphological and molecular characterization of Pseudocohnilembus longisetus Thompson, 1965 from farmed black rockfish Sebastes schlegelii in Korea

The morphology, infraciliature, silverline system, and the small subunit ribosomal RNA (SSU rRNA) of the little-known marine scuticociliate Pseudocohnilembus longisetusThompson, 1965 from the diseased black rockfish Sebastes schlegelii in Korea were studied. This scuticociliate possessed the typical characteristics of the genus Pseudocohnilembus, but could be discriminated from Pseudocohnilembus hargisi, and Pseudocohnilembus persalinus in terms of the body size, shape, the number of somatic kineties and kinetids in somatic kinety 1, and the number/position of contractile vacuole pores. The SSU rRNA gene of P. longisetus was sequenced in order to gain a better understanding of appropriate phylogenetic classification. The SSU rRNA was 1754 bp and the sequence was deposited in GenBank under accession number FJ899594. The SSU rRNA gene sequences of P. longisetus had an identity of 98.1%, 96.8% and 95.3% with P. hargisi, P. persalinus, and Pseudocohnilembus marinus SSU rRNA sequences, respectively. Our population of P. longisetus belonged to the genus Pseudocohnilembus and was in an isolated position based on the SSU rRNA gene tree, which was consistent with the conclusions based on the morphological studies. However, further investigation is required to determine the pathogenicity of this species.