Trevor Anderson

A newly developed ELISA showing the effect of environmental stress on levels of hsp86 in Cherax quadricarinatus and Penaeus monodon

The induction of hsps by stress in Cherax quadricarinatus and Penaeus monodon was investigated using SDS-PAGE, Western blotting and ELISA techniques. Western blotting showed the presence of an immuno-reactive protein to mouse alpha-human hsp70 IgG1 monoclonal antibody at a mass of 86 kDa (hsp86) in pleopod samples but was not sensitive enough to detect differences in response to stress. An enzyme-linked immunosorbent assay (ELISA) was developed using this antibody for the detection of hsp86 in the pleopods of C. quadricarinatus and P. monodon. Using this assay, significantly higher levels of hsp86 were detected in hyperthermally stressed C. quadricarinatus (21 to 70 g) and P. monodon (14 to 32 g) and hypoosmotically stressed P. monodon (14 to 32 g). Male C. quadricarinatus and P. monodon were thermally stressed with an increase in temperature from 24 to 33 degrees C for a period of 2 h then a recovery period of 6 h. SDS-PAGE gels of thermally stressed C. quadricarinatus and P. monodon samples revealed an increase in protein band intensity at 97 kDa (C. quadricarinatus) and 43 and 35 kDa in P. monodon. A 25 kDa mass protein was induced in C. quadricarinatus when thermally stressed. P. monodon were osmotically stressed with a decrease from 31 to 15 ppt for 2 h with a recovery of 6 h. SDS-PAGE gels revealed increased intensity of bands at 35 and 43 kDa and a 100 kDa band was induced demonstrating a wide range response of protein profile to stress in these species. SDS-PAGE gels of both species investigated also revealed an apparent reduction in band intensity of the haemocyanin subunits in stressed samples. The ELISA described here constitutes the first quantitative assay for the detection of a hsp in crustaceans and the following investigations are believed to be the first to describe the response of hsps to stress in C. quadricarinatus and P. monodon. In doing so, they provide a sound basis for future studies of the role of hsps in physiological functions in commercially cultured crustaceans.

Apparent digestibility of tuna oil for common carp, Cyprinus carpio — effect of inclusion level and adaptation time

Abstract The effects of inclusion level and adaptation time on the apparent digestibility of tuna oil for common carp were determined by incorporation into a casein-based reference diet at 10% and 15% inclusion. Chromium oxide (1%) was used as an external indicator and faeces were collected via siphon. Analyses were performed on faeces pooled over 10 consecutive days (Days 8 to 17) and unpooled daily faecal samples. Apparent digestibility coefficients (ADCs) determined from pooled faeces indicated an effect of inclusion level on the digestibility of tuna oil with a significant decrease in digestibility from 10% inclusion (83.1 ± 2.4%) to 15% inclusion (59.8 ± 1.5). Digestibility of crude energy from tuna oil decreased significantly from 76.6 ± 3.9% at 10% inclusion to 38.0 ± 2.1% at 15% inclusion. The inclusion of tuna oil did not affect dietary protein digestibility. Analysis of unpooled daily faecal samples indicated a prolonged period of adaptation for common carp fed the 15% inclusion diet, with digestibility of tuna oil at 15% inclusion increasing markedly over the duration of the trial. Daily variability in digestibility was also observed. These results emphasise that ADCs for tuna oil should be determined over the range of inclusion levels to be used and that it is important to ensure the complete adaptation of the experimental animals to diets prior to the pooling of faeces for analysis.

Sex change strategy and the aromatase genes

Abstract5′flanking regions of CYP19A1/A2 genes are reported for three sex changing fish.

Effect of rearing water temperature on protandrous sex inversion in cultured Asian Seabass (Lates calcarifer)

Asian Seabass, Lates calcarifer (Bloch, 1790), is a protandrous species cultured for Aquaculture. The cultured Asian Seabass in Australia exhibits precocious sex inversion before 2years of age. This phenomenon highly affects on maintaining a proper broodstock in a hatchery. The effect of temperature on sex inversion inducement in Asian Seabass was thus investigated at five different temperature regimes experienced in Australia. Asian Seabass (14months) grown in fresh water under natural temperature in a commercial farm in Queensland were transported to the research facility at James Cook University, Australia and held in fresh water at 28°C until acclimatized to the experimental conditions. Fish were acclimated to the experimental conditions (30ppt salinity) over the first and final week (22°C, 25°C, 28°C, 31°C and 34°C) of one month acclimatizing period. Fish were fed daily with a commercial pellet (50% protein, 18MJkg(-1)) to satiety. Blood, brain and gonad collected before transfer to the experimental temperature regime in the final week of acclimatization and at the end of the experiment were analysed. Plasma sex steroids level and aromatase activity of brain and gonad were also measured. There was an increase in plasma estradiol levels with increasing temperature from 25°C while no significant difference was observed among all treatment temperatures except at 25°C. However, fish held at 22°C showed higher estradiol level than at 25°C and 28°C. Significantly higher (p<0.05) plasma testosterone levels were detected in fish held at 31°C and 34°C while a reducing trend was observed towards lower temperature regimes. Fish held at 22°C had significantly lower plasma testosterone than all others as well those sampled at the beginning. The plasma 11-ketoTestosterone was at non-detectable levels in all experimental temperatures as shown at the beginning. The average aromatase activity in brain was highest at 28°C among all temperatures, but no significant differences (p>0.05) observed. The Average aromatase activity in gonad was highest at 31°C followed by at 34°C and 28°C. No or very low level of gonad aromatase activity recorded in fish sacrificed prior to treatment. The aromatase activity was greater in brain than in gonad suggesting that the aromatase produced in the brain yet to transfer to the gonad or brain is the first place to response for culture environmental temperature. It is concluded that plasma sex steroids levels and aromatase activity in Asian Seabass have positive response to increasing temperature in culture facilities.