Sex determination by ultrasonography prior to artificial breeding of the pufferfish Arothron manilensis (Tetraodontiformes, Tetraodontidae).

Artificial breeding was induced in the pufferfish Arothron manilensis following ultrasonographic sex determination. Hormonal treatment of mature male and female specimens followed the collection (and measurement) of fully developed eggs by cannulation. Fertilized eggs (0.85 ± 0.02 mm diameter) were spherical, demersal and individually adhesive. Hatching occurred 5 days after fertilization, larvae being 2.23 ± 0.15 mm in total length and 2.08 ± 0.14 mm in notochord length. The larvae had all died within 14 days of hatching. To improve artificial breeding techniques for A. manilensis, it is necessary to determine more appropriate timing for hormone injection, as well as feeding nutrient-enhanced SS type Brachionus sp. to newly hatched larvae.

Tandem spawning and development in the African freshwater pufferfish Tetraodon schoutedeni.

To advance breeding techniques for the African freshwater pufferfish Tetraodon schoutedeni and observe tandem spawning closely, we monitored the reproduction of captive individuals. Eight spawning sessions (stable water temperature 24-25°C; daily light period 07:00-19:00) occurred between May 2016 and November 2017. After 65-150 min of tandem swimming (the male biting and clinging to the female's abdomen), 3-50 spherical, weakly adhesive eggs were spawned, being scattered onto the sandy substrate or water plants. The removal of cohabitants (potentially eating spawned eggs) and provision of small initial food items, such as small-type Brachionus spp., for larval fish were essential for successful breeding.

Crawler, a novel Tc1/mariner-type transposable element in Aspergillus oryzae transposes under stress conditions.

A novel active transposable element, designated Crawler, has been isolated from an industrial strain (OSI1013) of Aspergillus oryzae as an insertion sequence within the niaD gene encoding nitrate reductase. It is 1290bp in length with imperfect terminal inverted repeats of 28bp and is flanked by 2bp (TA) target site duplications. It contains an open reading frame with no introns that encodes a putative transposase (AotA) of 357 amino acid residues, which is highly homologous to the transposase existing in impala, a member of Tc1/mariner superfamily class II DNA transposon from Fusarium oxysporum. Southern blot analysis revealed that the OSI1013 strain has multiple copies (at least 16) of the element in the genome. Transcription of Crawler occurred under standard growth conditions, and was up-regulated in the presence of CuSO(4) or by heat shock at 42 degrees C. Moreover, transposition events of Crawler induced by various stress treatments were observed by transposon trapping, in which crnA and niaD genes were used as targets for insertion of the element. The excision analysis of Crawler inserted within promoter regions of the crnA gene revealed that CuSO(4) stress and heat shock treatment for conidia were most effective on its excision/transposition, and that acidic environment, oxidative stress, and UV irradiation also slightly induced transposition. To our knowledge, this is the first study reporting the observation of active transpositions of a resident class II transposon under various stress conditions in filamentous fungi.

Cloning of a novel tyrosinase-encoding gene (melB) from Aspergillus oryzae and its overexpression in solid-state culture (Rice Koji).

We have cloned a novel tyrosinase-encoding gene (melB) specifically expressed in solid-state culture of Aspergillus oryzae. A tyrosinase-encoding gene (melO) from A. oryzae was already cloned and the protein structures of its catalytic and copper binding domains were investigated. However, our recent results revealed that the melO gene was highly expressed in submerged culture but not in solid-state culture. Because tyrosinase activity was also detected in solid-state culture, we assumed that another tyrosinase gene other than melO is expressed in solid-state culture. Another tyrosinase gene was screened using the expressed sequence tag (EST) library. One redundant cDNA clone homologous with the tyrosinase gene was found in the collection of wheat bran culture. Northern blot analysis revealed that the gene corresponding to the cDNA clone was specifically expressed in solid-state culture (koji making), but not in submerged culture. Molecular cloning showed that the gene carried six exons interrupted by five introns and had an open reading frame encoding 616 amino acid residues. This gene was designated as melB. The deduced amino acid sequence of the gene had weak homology (24%-33%) with MelO and other fungal tyrosinases but the sequences of the copper binding domains were highly conserved. When the melB gene was expressed under the control of the glaB promoter in solid-state culture, tyrosinase activity was markedly enhanced and the culture mass was browned with the melanization by MelB tyrosinase. These results indicated that the melB gene encodes a novel tyrosinase associated with melanization in solid-state culture.

Isolation and characterization of a novel gene encoding alpha-L-arabinofuranosidase from Aspergillus oryzae.

We cloned and characterized a novel gene (abfA) encoding alpha-L-arabinofuranosidase (alpha-L-AFase) from Aspergillus oryzae. One clone homologous to the alpha-L-AFase gene of Thermotoga maritima was found in an expressed sequence tag (EST) library of A. oryzae and a corresponding gene was isolated. Molecular analysis showed that the abfA gene carried six exons interrupted by five introns and had an open reading frame encoding 481 amino acid residues. The amino acid sequence similarity at active sites to the alpha-L-AFases from other organisms indicated that the alpha-L-AFase encoded by abfA was classified as a family 51 glycoside hydrolase. When the abfA was overexpressed in the homologous hyperexpression system of A. oryzae, a large amount of alpha-L-AFase was produced as intracellular protein. The apparent molecular mass of the purified enzyme was estimated to be 228,000 by gel filtration and that of its subunit as 55,000 by SDS-PAGE, suggesting that the enzyme is a tetramer. The enzyme hydrolyzed p-nitrophenyl-alpha-L-arabinofuranoside but not other p-nitrophenyl glycosides. These results demonstrated that the abfA gene encodes a functional alpha-L-AFase.

A novel amine oxidase-encoding gene from Aspergillus oryzae.

We cloned a novel gene (aoxA) encoding amine oxidase (AOX) from Aspergillus oryzae. One cDNA clone showing extreme homology to the AOX-encoding genes was found in an expressed sequence tag (EST) library of A. oryzae. Molecular analysis revealed that the aoxA carried four exons interrupted by three introns and had an open reading frame encoding 672 amino acid residues. The deduced amino acid sequence showed about 83.5% identity to the Aspergillus niger AO-I. The strictly conserved residues for co-factor and copper binding in copper/quinine-containing AOXs were also preserved at Tyr 405, His 456, His 458 and His 617 in the cDNA sequence. When the aoxA was overexpressed in the homologous hyperexpression system of A. oryzae, AOX activity in the transformant was enhanced 75-fold. An apparent molecular weight of 159,000 by gel filtration and a subunit molecular weight of 75,000 by SDS-PAGE of the purified enzyme were estimated, suggesting that the enzyme molecule is a homo-dimer similar to other copper/quinine-containing AOXs. The A. oryzae AOXA preferentially oxidized aliphatic monoamines of C2-C6 rather than aromatic amines or diamines. From these results, the aoxA gene product obtained by homologous hyperexpression system of A. oryzae is undoubtedly a functional AOX.