Development of an enzyme-linked immunosorbent assay (ELISA) for vitellogenin measurement in greenback flounder Rhombosolea tapirina

Vitellogenin (Vtg) was purified from the plasma of 17-estradiol (E₂)-injected male greenback flounder,Rhombosolea tapirina. The molecular weight of the native Vtg was estimated by gel filtration as 540 kD. SDS-PAGE and Western blotting analyses indicated that this protein consisted of three bands with molecular weights of 155, 104, 79 kD, respectively. A polyclonal antibody against the highest molecular weight band of putative Vtg was generated in sheep and an indirect antibody-capture competitive enzyme-linked immunosorbent assay (ELISA) was developed. The assay was validatedfor plasma Vtg measurement in greenback flounder. Serial dilutions of plasma from vitellogenic females parallelled the standard Vtg curve, whereas no cross-reaction was observed with the plasma of males in the ELISA. The Vtg ELISA was used to assess the induction of Vtg by E₂ in vivo in males. The induction of Vtg in greenback flounder showed a time- and dose-dependent response as in other species. In E₂-treated fish, detectable levels of Vtg were first found at 48 h, and reached a peak at 96 h post-injection. Plasma levels of Vtg increased as the E2 dose increased with a threshold of 0.1 mg kg^–1.     

Effects of dbcAMP on steroidogenesis in isolated ovarian follicles of Atlantic salmon

In vitro estradiol-17β (E₂) and testosterone (T) production were measured in vitellogenic follicles of Atlantic salmon in response to treatment with human chorionic gonadotropin (hCG) and 2-0-dibutyryladenosine 3′,5′-cyclic monophosphate (dbcAMP). DbcAMP stimulated T production but had no effect on E₂, whereas hCG treatment stimulated E₂ production but T production was low. Elevated intracellular [cAMP] may be part of the normal mechanism for inhibition of aromatase activity at the end of vitellogenesis.     

Effects of (D-Ala6, Pro9N ethylamide) — LHRH on plasma levels of gonadotropin, 17α,20β-dihydroxy-4-pregnen-3-one and testosterone in male goldeye (hiodon alosoides Rafineque)

Male goldeye were treated with (D-Ala⁶, Pro⁹-N ethylamide) — LHRH (LHRH-A) in saline or a silastic pellet (100 µg.kg^–1 body weight) and changes in plasma gonadotropin (GtH), 17,20-dihydroxy-4-pregnen-3-one (17,20P) and testosterone (T) levels were determined by radioimmunoassay. LHRH-A increased plasma GtH levels, with the response to LHRH-A in saline being of much greater magnitude and duration than the response to silastic pellet implants. Seasonal differences were found in the response to LHRH-A. Spermiated fish were the most responsive, recrudescing fish the least, and regressed fish displayed an intermediate response. Plasma 17,20P levels were elevated in response to LHRHA in fish of all sexual stages although the magnitude of the increase was not related to the magnitude of the increase in GtH levels. Treatment with LHRH-A also resulted in a transitory increase in plasma T levels. The endocrine control of GtH and steroid secretion in goldeye is discussed in relation to studies in cyprinids and salmonids. Address for reprint requests: Department of Zoology, University of Alberta, Edmonton, Alberta T6G 2E9 Canada.     

Capacity of fatty acid profiles and substrate utilization patterns to describe differences in soil microbial communities associated with increased salinity or alkalinity at three locations in South Australia

 Fatty acid methyl ester (FAME) profiles, together with Biolog substrate utilization patterns, were used in conjunction with measurements of other soil chemical and microbiological properties to describe differences in soil microbial communities induced by increased salinity and alkalinity in grass/legume pastures at three sites in SE South Australia. Total ester-linked FAMEs (EL-FAMEs) and phospholipid-linked FAMEs (PL-FAMEs), were also compared for their ability to detect differences between the soil microbial communities. The level of salinity and alkalinity in affected areas of the pastures showed seasonal variation, being greater in summer than in winter. At the time of sampling for the chemical and microbiological measurements (winter) only the affected soil at site 1 was significantly saline. The affected soils at all three sites had lower organic C and total N concentrations than the corresponding non-affected soils. At site 1 microbial biomass, CO₂-C respiration and the rate of cellulose decomposition was also lower in the affected soil compared to the non-affected soil. Biomarker fatty acids present in both the EL- and PL-FAME profiles indicated a lower ratio of fungal to bacterial fatty acids in the saline affected soil at site 1. Analysis of Biolog substrate utilization patterns indicated that the bacterial community in the affected soil at site 1 utilized fewer carbon substrates and had lower functional diversity than the corresponding community in the non-affected soil. In contrast, increased alkalinity, of major importance at sites 2 and 3, had no effect on microbial biomass, the rate of cellulose decomposition or functional diversity but was associated with significant differences in the relative amounts of several fatty acids in the PL-FAME profiles indicative of a shift towards a bacterial dominated community. Despite differences in the number and relative amounts of fatty acids detected, principal component analysis of the EL- and PL-FAME profiles were equally capable of separating the affected and non-affected soils at all three sites. Redundancy analysis of the FAME data showed that organic C, microbial biomass, electrical conductivity and bicarbonate-extractable P were significantly correlated with variation in the EL-FAME profiles, whereas pH, electrical conductivity, NH₄-N, CO₂-C respiration and the microbial quotient were significantly correlated with variation in the PL-FAME profiles. Redundancy analysis of the Biolog data indicated that cation exchange capacity and bicarbonate-extractable K were significantly correlated with the variation in Biolog substrate utilization patterns. Received: 8 March 2000     

Weaning requirements of larval mulloway,Argyrosomus japonicus

Mulloway (Argyrosomus japonicus) is an emerging aquaculture species in Australia, but there is a need to improve the production technology and lower costs, including those associated with larval rearing and live feeds. Three experiments were conducted to determine appropriate weaning strategies from live feeds, rotifers (Brachionus plicatilis) and Artemia, to cheaper formulated pellet diets. Experiment 1 examined the effects of feeding Artemia at different levels [0%, 50% or 100% ration of Artemia fed from 18 days after hatching (dah); based on current hatchery protocols] and a pellet diet from two larval ages (14 or 23 dah). In addition, rotifers were supplied to larvae in all treatments for the duration of the experiment (14–29 dah), at which time all larvae were successfully weaned onto the pellet diet. No significant (P>0.05) differences existed between the growth of fish fed a 50% and 100% ration of Artemia; however, fish fed a 0% ration of Artemia had significantly (P<0.05) reduced growth. The time of pellet introduction had no significant (P>0.05) effects on the growth of larvae. Experiments 2 and 3 were designed to determine the size [total length (TL), mm] at which mulloway larvae selected Artemia equally or in preference to rotifers, and pellet (400 μm) equally or in preference to Artemia respectively. Each day, larvae were transferred from a holding tank to experimental vessels and provided with rotifers (2 mL^?1), Artemia (2 mL^?1) or a combination of rotifers (1 mL^?1) and Artemia (1 mL^?1) (Experiment 2), and Artemia (2 mL^?1), a pellet diet or a combination of Artemia (1 mL^?1) and a pellet diet that was broadcast every 15 min (Experiment 3). After 1 h, a sub‐sample of larvae was randomly selected from each replicate vessel (n=5) and the gut contents were examined under a light microscope. Mulloway larvae began selecting Artemia equally to rotifers at 5.2 ± 0.5 mm TL and selected pellets equally to Artemia at 10.6 ± 1.8 mm TL. Our results have led to the establishment of weaning protocols for larval mulloway, which optimize larval growth while reducing feed cost by minimizing the amount of Artemia used during production.     

The effect of stocking density and repeated handling on the growth of juvenile mulloway, Argyrosomus japonicus (Temminck & Schlegel 1843)

The effect of stocking density on the growth of mulloway, Argyrosomus japonicus, was tested with 17 g fish stocked at 4.08, 8.16, or 16.32 kg m⁻³ in 50 l aquaria. Weight checks were carried out every 2 weeks to track performance. Each density treatment was also compared to a nonhandled control group to establish if handling during weight checks influenced the growth of mulloway. Mulloway performed poorly at the lowest density and, under the current experiment conditions, growth did not appear to be negatively affected by regular handling.     

Impact of a change in tillage and crop residue management practice on soil chemical and microbiological properties in a cereal-producing red duplex soil in NSW, Australia

The effect of a change of tillage and crop residue management practice on the chemical and microbiological properties of a cereal-producing red duplex soil was investigated by superimposing each of three management practices (CC: conventional cultivation, stubble burnt, crop conventionally sown; DD: direct-drilling, stubble retained, no cultivation, crop direct-drilled; SI: stubble incorporated with a single cultivation, crop conventionally sown), for a 3-year period on plots previously managed with each of the same three practices for 14 years. A change from DD to CC or SI practice resulted in a significant decline, in the top 0-5 cm of soil, in organic C, total N, electrical conductivity, NH₄-N, NO₃-N, soil moisture holding capacity, microbial biomass and CO₂ respiration as well as a decline in the microbial quotient (the ratio of microbial biomass C to organic C; P <0.05). In contrast, a change from SI to DD or CC practice or a change from CC to DD or SI practice had only negligible impact on soil chemical properties (P >0.05). However, there was a significant increase in microbial biomass and the microbial quotient in the top 0-5 cm of soil following the change from CC to DD or SI practice and with the change from SI to DD practice (P <0.05). Analysis of ester-linked fatty acid methyl esters (EL-FAMEs) extracted from the 0- to 5-cm and 5- to 10-cm layers of the soils of the various treatments detected changes in the FAME profiles following a change in tillage practice. A change from DD practice to SI or CC practice was associated with a significant decline in the ratio of fungal to bacterial fatty acids in the 0- to 5-cm soil (P <0.05). The results show that a change in tillage practice, particularly the cultivation of a previously minimum-tilled (direct-drilled) soil, will result in significant changes in soil chemical and microbiological properties within a 3-year period. They also show that soil microbiological properties are sensitive indicators of a change in tillage practice.     

Cold and dark or warm and light: variations on the theme of environmental control of reproduction

Seasonality is a common feature of teleost reproduction and is most marked at higher latitudes. Seasonal cycles are principally entrained by photoperiod and temperature but a range of other variables including lunar phase and social interaction may also operate. Photoperiod information is transduced through the light inhibition of pineal melatonin synthesis but the mechanism by which this regulates the reproductive endocrine system is not clear. There is less information on the specific effects of temperature but these are likely to involve effects on expression and activity of regulatory proteins. Environmental regulation of high latitude species appears to operate across a hierarchy of variables with photoperiod then temperature as primary drivers, whereas in tropical habitats, the hierarchy may be inverted.     

Effects of slow release gonadotropin releasing hormone analog on milt characteristics and plasma levels of gonadal steroids in greenback flounder, Rhombosolea tapirina

Spermiated male greenback flounder (Rhombosolea tapirina) were implanted with gonadotropin releasing hormone analog (GnRHa) pellets at different dosages to examine their effects on milt characteristics and plasma levels of testosterone (T), 11-ketotestosterone (11KT) and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP). Milt and blood samples were collected for up to 28 or 42 days post-implantation (p.i.) in two separate experiments using fish that were slightly or moderately spermiated, respectively. In both experiments, fish treated with GnRHa pellets showed a consistent and significant increase in milt volume relative to controls, and the increase of milt volume was more rapid than the increase in numbers of spermatozoa. Spermatocrit (Sct) was significantly lower in GnRHa-treated fish than in controls. Plasma levels of T and 11KT were elevated at 7 and 14 days p.i. in slightly spermiated fish treated with GnRHa and elevated plasma T and 11KT levels were accompanied by increased milt volume early in the spermiation period. In contrast, there was no significant difference in plasma T levels between GnRHa-treated and control fish in fish that were moderately spermiated at the time of implant. Treatment with GnRHa tended to result in lower plasma levels of 11KT and higher levels of 17,20βP relative to controls, and there was a positive relationship between the elevation of plasma 17,20βP and the increase in milt volume in response to implantation of GnRHa pellet. Slow release GnRHa administration had no effect on sperm activity or pH and osmolality of seminal plasma in either experiment.     

The dynamics of in vitro 17 β-estradiol secretion by isolated ovarian follicles of the rainbow trout (Oncorhynchus mykiss)

This study investigated the effects of human chorionic gonadotropin (hCG), 17-hydroxyprogesterone (17P) and testosterone (T) on the in vitro production of 17-estradiol (E₂) by isolated ovarian follicles of the rainbow trout (Oncorhynchus mykiss). 17P at 100 ng ml^-1, and hCG at 100 IU ml^-1 stimulated E₂ production relative to controls, whereas lower doses were ineffective. T was the most effective in stimulating E₂ production, followed by 17P and hCG respectively. The timecourse of E₂ production was investigated for both static culture, and incubations with media replacement, with follicles being exposed to hormone treatment for 30 min, 1 or 3 h, or constantly. E₂ production was observed after 30 min, 3 and 3-6 h in response to T, 17P and hCG respectively. Under static culture, E₂ levels reached maximal levels in 6 h. Longer incubations resulted in further metabolism of E₂ to E₂-glucuronide, which resulted in the blurring of treatment effects after 18 h. Incubations with media replacement resulted in higher E₂ production than in static culture. The results indicate that a 6 h incubation period is sufficient to produce significant increases in E₂ production in response to hCG, 17P and T, and that incubations longer than 12 h result in losses E₂ from the incubation media. These findings have implications for the validity of using static cultures to examine the effects of hormone treatment on the activity of steroid converting enzymes in vitro.