Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, <Emphasis Type="Italic">Notemigonus crysoleucas</Emphasis>

Baitfish such as golden shiners are subjected to stress during harvesting, grading, and transport. Their small size makes it difficult to measure the stress response with the biological indicator cortisol using conventional assay methods for plasma. This paper examines the development and validation of methods for whole-body cortisol extraction from individual baitfish. Three types of extracts were tested: (1) an ethyl ether unaltered extract (UA); (2) an extract reconstituted in phosphate buffered saline (PBS); (3) an extract that had been increased in volume by the addition of food-grade vegetable oil (VO). These extracts were evaluated using validation tests with radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). The UA extract produced inadequate volumes of extract for multiple assays and could not be used for the determination of cortisol in a single fish. The PBS reconstitution method failed the precision recovery of serial dilutions (62.3%), linearity (R ²: 0.7864), and parallelism validation tests. The VO volume-boosting method passed all validation tests [intra-assay coefficent of variation (%CV): 16.3 for ELISA and 5.9 for RIA; inter-assay %CV: 10.3; spiked recovery: 102.0%; dilution recovery: 93.0%; linearity R ²: 0.9435; log of serial dilutions was parallel] and provided enough extract for multiple assays from an individual baitfish. Based on these results, we conclude that the VO volume-boosting method presents a means for determining cortisol from individual baitfish using either RIA or ELISA assays.     

Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

The sea cucumber (Stichopus japonicus) is able to undergo autolysis in response to a variety of environmental and mechanical cues. Within the framework of a long-term study of this phenomenon we have purified a protease from the body wall of the sea cucumber by means of ion-exchange chromatography with DE-52 cellulose and gel filtration chromatography with Sephadex G-100. The final enzyme preparation was nearly homogeneous on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 35.5 kDa. The purified enzyme exhibited a maximum activity for the hydrolysis of casein at pH 7.0 and 50°C and a remarkable stability at pH 4.0–7.0 and 40–60°C. Based on the inhibition and activation profiles obtained with numerous specific protease inhibitors and an activator, the protease purified from the body wall of the sea cucumber was defined to be a cysteine-like protease.     

A comparative account of the structure of the growth hormone encoding gene and genetic interrelationship in six species of the genus <Emphasis Type="Italic">Labeo</Emphasis>

The growth hormone (GH) gene isolated and cloned from various Labeo species (L. rohita, L. calbasu, L. fimbriatus, L. gonius, L. bata, and L. kontius) is shown to contain a single copy in the haploid genome, with an overall size of ∼2.5 kb. The GH gene in all the Labeo species studied has five exons and four introns of various sizes with the exon/intron boundary sequence of GT/AG. The length variation of the GH gene between the species is found to be due to length variation in the form of several deletions in the third intron. The length of individual exons is the same in all the species with an open reading frame (ORF) of 630 bp (210 amino acids) except in L. rohita, which has a 9 bp deletion in the fourth exon, resulting in a shorter GH of 621 bp (207 amino acids). The similarity in the nucleotide and amino acid sequences between the different Labeo species is greater than 97%, in spite of eight amino acids being altered in the GH protein of Labeo that reside outside the conserved domain sequence required for its function. Nucleotide substitutions are seen in the form of 20 transitions and three transversions in the ORF of the GH gene. Both types of transitions (A–G; T–C) and only one type of transversion (A–C) are detected in the GH gene. Codon preference in GH gene shows a strong preference for G and C in the wobble position of the codons. Genetic interrelationships determined between Labeo and other species of fishes using nucleotide sequence of GH cDNA supports the overall teleost classification of Nelson (Fishes of the World. Wiley, New York, 1984) with separate clades for Ostariophysi, Protacanthopterygii, and Acanthopterygii. Besides, the unweighted pair group method with arithmetic means (UPGMA) analysis clearly distinguishes between the species having five exons and four introns in the GH gene from the species having six exons and five introns in the same gene. The Labeo species analyzed in the present study could be clustered into two groups using the maximum-parsimony method on the intron sequences data of the GH gene.     

Purification of anaphase promoting complex/cyclosome from goldfish oocytes

Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin-specific ubiquitinating system, including anaphase-promoting complex/cyclosome (APC/C) is thought to be responsible for cyclin B destruction. To learn more about the molecular mechanism of cyclin B degradation, a molecular study of the ubiquitinating system in goldfish has been undertaken. For biochemical preparation of APC/C, we first conducted the cloning, sequencing and expression analysis of goldfish, Carassius auratus, cdc27 that encodes a subunit of APC/C from goldfish ovary. The deduced amino acid sequence is highly homologous to cdc27 from other species. Then recombinant goldfish Cdc27CT (C-terminal half of Cdc27) was expressed in Escherichia coli, and an antibody was raised against purified recombinant protein. Polyclonal antiserum cross-reactive with Cdc27 was obtained. By the assay using the antibody, APC/C was purified by column chromatographs.     

The Yangtze River dolphin or baiji (Lipotes vexillifer): population status and conservation issues in the Yangtze River,China

• 1. Baiji were sighted 17 times during three recent simultaneous multi‐vessel surveys in the Yangtze River, China (November 4–10, 1997; December 4–9, 1998; October 31–November 5, 1999). There were 11 sightings in 1997 (consisting of 17 animals), five in 1998 (seven animals), and two in 1999 (four animals). It was concluded that 13 individuals could be considered as a minimum number of the baiji currently in the Yangtze River.
• 2. An annual rate of population decrease was roughly estimated as 10%. From the body sizes observed, the proportions of old, adult and immature individuals were approximately estimated at 57, 26, and 17% respectively.
• 3. Baiji showed a significant attraction to confluences and sand bars with large eddies. The present distribution range of the baiji is less than 1400 km in length in the Yangtze main river. Distances between the two nearest groups of baiji appear to be increasing.
• 4. Two typical sightings are described, in which surfacing and movements of baiji were recorded. Baiji were often found swimming together with finless porpoise. In the surveys they occurred in the same group in 63% of occurrences. Interactions between baiji and finless porpoise are described and discussed.
• 5. Human activities are the main threats to the baiji. Illegal electrical fishing accounted for 40% of known mortalities during the 1990s. Engineering explosions for maintaining navigation channels have become another main cause of baiji deaths. The last hope of saving the species may be to translocate the remaining baiji into a semi‐captive reserve, known as the ‘Baiji Semi‐natural Reserve’.

Copyright © 2003 John Wiley & Sons, Ltd.     

Observations on growth, sexual maturity and spawning performance of pond-reared Penaeus merguiensis

This 12‐month preliminary study investigated the development of sexual characters, primary sexual maturity, ovarian maturity and spawning performance of pond‐reared Penaeus merguiensis in relation to culture conditions in south‐east Queensland, Australia. Post‐larvae of P. merguiensis were produced and cultured in two 60‐m³ tanks during the first 14 weeks. Before winter, they were harvested and stocked in three different overwintering facilities: a 200‐m² covered pond, two 60‐m³ outdoor tanks and a 15‐m³ indoor, recirculated tank at a stocking density of 10 individuals m^?2. The development of sexual characters was found to be similar to that reported previously in wild P. merguiensis. Males matured at younger ages and smaller sizes than females. Overall, the average size at primary sexual maturity of pond‐reared P. merguiensis was 23.1 mm carapace length for males (possessed spermatophores) and 29.3 mm for females (being impregnated). Water temperature and the availability of natural food strongly influenced prawn growth, maturity rate and their subsequent spawning performance. Growth, maturity rate and spawning performance of prawns in the covered pond were significantly higher than in the other overwintering facilities. Prawns started mating at 6–7 months, reached full ovarian maturation and spawned as early as about 8 (peaked at 9–11) months from hatching, producing high fecundity and viable larvae. There was a strong relationship (P < 0.001) between prawn size and fecundity. The results of this study suggest a potential for using pond‐reared broodstock P. merguiensis for hatchery production and for domestication or selective breeding programmes.     

Reducing the potential for processing contaminant formation in cereal products

Processing contaminants may be defined as substances that are produced in a food when it is cooked or processed, are not present or are present at much lower concentrations in the raw, unprocessed food, and are undesirable either because they have an adverse effect on product quality or because they are potentially harmful. The presence of very low levels of processing contaminants in common foods is becoming an increasingly important issue for the food industry, as developments in analytical techniques and equipment bring foods under closer and closer scrutiny. This review considers the formation of lipid oxidation products, hydrogenation of polyunsaturated fatty acids to prevent lipid oxidation and the associated risk of trans fatty acid formation. The formation of acrylamide in the Maillard reaction is described, as well as the genetic and agronomic approaches being taken to reduce the acrylamide-forming potential of cereal grain. The multiple routes for the formation of furan and associated chemicals, including hydroxymethylfurfuryl, are also described. The evolving regulatory and public perception situations for these processing contaminants and their implications for the cereal supply chain are discussed, emphasising the need for cereal breeders to engage with the contaminants issue.     

Plant growth and survival of five introduced and two native/naturalized perennial grass genotypes exposed to two defoliation managements in arid Argentina

The field performance of the native Pappophorum vaginatum, the naturalized Eragrostis curvula and various cultivars of the introduced Achnatherum hymenoides and Leymus cinereus was evaluated as potential forage resources in rangelands of arid Argentina during the warm seasons of 2007/2008 and 2008/2009. Plants of these grass species, obtained from seeds, were transplanted to the field in 2006, when they were 1 year old. During the study years, there were two defoliation managements: plants of all study genotypes either remained undefoliated (controls) or were defoliated twice a year during spring at 5 cm stubble height. Despite tiller number being lower (P < 0·05) on defoliated than on undefoliated plants, and total leaf length per unit basal area being similar (P > 0·05) between defoliation managements by mid‐spring, there were no differences (P > 0·05) in dry weight production between defoliated and undefoliated plants in all genotypes at the end of the second growing season. Plants of one or more of the introduced genotypes showed a similar (P > 0·05) or greater (P < 0·05), but not lower, tiller number per plant and per square centimetre, daughter tiller production, total leaf length and dry weight production per unit basal area than the native species at the end of the first and/or second growing seasons. These morphological variables were similar (P > 0·05) or greater (P < 0·05) in the native than in the naturalized genotype. Plant survival, however, was lower (P < 0·05, overall mean = 20%) in the introduced than in the native (>70%) or naturalized (>39%) genotypes at the end of the first or second growing seasons.