Species compositions and ecology of the riverine ichthyofaunas in two Sulawesian islands in the biodiversity hotspot of Wallacea.

This account of the riverine ichthyofaunas from the islands of Buton and Kabaena, off south-eastern mainland Sulawesi, represents the first detailed quantitative checklist and ecological study of the riverine fish faunas in the biological hotspot of Wallacea. The results are based on analysis of samples collected by electrofishing at a wide range of sites from July to September in both 2001 and 2002. While the fauna was diverse, with the 2179 fishes caught comprising 64 species representing 43 genera and 22 families, the catches were dominated by the Gobiidae (26 species and 25% by numbers), Eleotridae (seven species and 27% by numbers), Zenarchopteridae (three species and 22% by numbers) and Anguillidae (two species and 12% by numbers). The most abundant species were the eleotrids Eleotris aff. fusca-melanosoma and Ophieleotris aff. aporos, the anguillid Anguilla celebesensis, the zenarchopterids Nomorhamphus sp. and Nomorhamphus ebrardtii and the gobiids Sicyopterus sp. and Glossogobius aff. celebius-kokius. The introduced catfish Clarias batrachus was moderately abundant at a few sites. Cluster analysis, allied with the similarity profiles routine SIMPROF, identified seven discrete groups, which represented samples from sites entirely or predominantly in either Buton (five clusters) or Kabaena (two clusters). Species composition was related to geographical location, distance from river mouth, per cent contribution of sand and silt, altitude and water temperature. The samples from the two islands contained only one species definitively endemic to Sulawesi, i.e. N. ebrardtii and another presumably so, i.e. Nomorhamphus sp., contrasting starkly with the 57 species that are endemic to Sulawesi and, most notably, its large central and deep lake systems on the mainland. This accounts for the ichthyofaunas of these two islands, as well as those of rivers in northern mainland Sulawesi and Flores, being more similar to each other than to those of the central mainland lake systems. This implies that the major adaptive radiation of freshwater fishes in Sulawesi occurred in those lacustrine environments rather than in rivers.

Fish and macro-crustacean communities and their dynamics in the Severn Estuary.

The species of fish and macro-crustacean living within the Severn Estuary are reviewed. The fish community is notably species rich and exceeds 100 species in total for the estuary. Standardised long-term sampling at Hinkley Point in Bridgwater Bay gives a total complement of 83 for a single locality and this number is increasing by about one new species every two years. Most of these new species are moving in from centres of population lying to the south of the estuary. Almost all species of fish and macro-crustacean living within the estuary undertake regular migrations so that they tend to move seasonally in waves up and down the estuary. For fish, both species richness and the total abundance reach a maximum in late summer and autumn. The timing of this peak varies between the upper and lower estuary. This seasonal maximum is primarily caused by the arrival of the new recruits which use the estuary as a nursery. In contrast, crustaceans tend to be at their most diverse and abundant in early to mid summer. Using a 30-year time series of fish and crustacean abundance collected at Hinkley Point it is shown that major changes in the structure of the community are now underway and there are considerable recent changes in the abundance. However, some abundant species, including sand goby, Pomatoschistus spp., whiting, Merlangius merlangus and sprat, Sprattus sprattus, the three most abundant species in the estuary, have shown no long-term trend. At present, approximately 20% of the fish and macro-crustaceans observed in Bridgwater Bay are undergoing rapid, typically exponential, change in abundance. For a numerically abundant, diverse, fauna composed of approximately 90 species such levels of change are unexpected and suggest that the system is presently far from equilibrium. In some cases, the observed changes can be related to recent warming and the North Atlantic Oscillation. The overall increase in fish abundance observed may reflect a general improvement in water quality and a reduction in other anthropogenic impacts such as mortality in cooling-water intakes. The potential impacts of tidal power generation in the Severn Estuary are reviewed. There is considerable potential for any major installation to impact the fish and crustacean populations as they migrate and also alter the nature of the habitat resulting in changes in community composition. A particular difficulty in predicting the future impact of harnessing tidal energy is that the present community is already changing rapidly. The ability of fish and crustaceans to pass through the turbines unharmed will be a key issue in an assessment of the impact of tidal power generation.

To what extent are hepatic concentrations of heavy metals in Anguilla anguilla at a site in a contaminated estuary related to body size and age and reflected in the metallothionein concentrations?

We explored how hepatic [metal]s in Anguilla anguilla at a contaminated estuarine site are influenced by body size, age and season, and the extent that [Cu], [Cd] and [Zn]s are reflected in [metallothionein (MT)]s. Although each [metal] and [MT] increased significantly with length, weight and age, those biotic variables explained <10% of the variation in each [metal] and only 11-16% for [MT]. Seasonal changes in [Cu] and [Cd] were paralleled by [MT]. The variation in [MT] explained by Cu (42%) was greater than by Zn (16%) and Cd (13%), and seasonally lay between 43 and 69% for those metals collectively. Thus, hepatic [MT] in eels is closely correlated with hepatic [heavy metal]s. However, the great variability among [MT]s for eels of similar sizes and ages, which reflects marked variability in hepatic [heavy metal]s, means that this variable reflects imprecisely the contamination level at a particular site.

Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in European eels Anguilla anguilla from United Kingdom estuaries.

A total of 94 European eels (Anguilla anguilla) were collected from five estuaries in the UK. The deconjugated metabolites of polycyclic aromatic hydrocarbons (PAHs) in the bile of the eels were separated using HPLC. Six PAH metabolites were identified: 1-hydroxy (1-OH) metabolites of phenanthrene, pyrene and chrysene; and the 1-OH, 3-OH and 7,8 dihydrodiol metabolites of benzo[a]pyrene (BaP). The mean concentration of the six metabolites was greatest in eels from the Tyne (49 microM) followed by the Wear (33 microM), Tees (19 microM), Thames (4 microM) and Severn (2 microM) estuaries. Although 1-OH pyrene was always the dominant compound, there were significant differences (P<0.05) between sites and between estuaries for some metabolites. Normalising the molar concentration of the bile metabolites to the bile biliverdin absorbance reduced sample variation. When the metabolites identified were each expressed as a percentage of the total detected, the metabolite profile was characteristic for each estuary.

Correlative changes in metabolism and DNA damage in turbot (Scophthalmus maximus) exposed to benzo[a]pyrene.

Juvenile turbot (Scophthalmus maximus) were injected intraperitoneally with either corn oil or 5 mg/kg benzo[a]pyrene (BaP) dissolved in corn oil and sampled I and 3 days after injection. After 1 day, no elevation of 7-ethoxyresorufin O-deethylase (EROD) activity was observed, however bile metabolites (BaP-7,8 dihydrodiol representing 70% of the total metabolites) and a single hepatic DNA adduct spot (0.47 adducts/10(8) nucleotides) identified by 32P-postlabelling were formed. No BaP metabolites or DNA adducts were observed in either control or carrier control fish. Fish sampled after 3 days reported 5-fold higher (P < 0.05) levels of EROD activity, a shift in the bile metabolite profile towards BaP phenol formation (1OH and 30H BaP comprising up to 60% of total metabolites detected) and the formation of two adduct spots (0.86 and 0.71 adducts/10(8) nucleotides). These results show that BaP can be metabolised and form hydrophobic DNA adducts in turbot without EROD elevation. Following EROD elevation, a shift in the profile of both BaP metabolites and BaP metabolite-DNA interactions occurs indicative of other oxidative processes.

Bile metabolites of polycyclic aromatic hydrocarbons in three species of fish from the Severn Estuary.

Six metabolites of polycyclic aromatic hydrocarbons (PAHs) were identified and quantified from the bile of 31 common eels (Anguilla anguilla), 29 European flounders (Pleuronectes flesus), and 15 conger eels (Conger conger) collected from the Severn Estuary and Bristol Channel during 1997. The bile metabolites were deconjugated by enzymatic hydrolysis and separated by reverse-phase HPLC with fluorescence detection. The major metabolite present in all fish was 1-hydroxy pyrene (75-94% of all metabolites detected) with lower proportions of 1-hydroxy chrysene (2-15%) and 1-hydroxy phenanthrene (2-8%), and small amounts of three benzo[a]pyrene derivatives (<3%). Metabolite concentrations (normalized to biliverdin content) were significantly higher in common eels than in the other two species and tended to be higher in all species at the beginning of the year than at the end. The data confirm the importance of 1-hydroxy pyrene as the key PAH metabolite in fish bile and suggest that the common eel is an ideal species for monitoring PAHs in estuarine environments.

Assessing chemical toxicity with the bioluminescent photobacterium (Vibrio fischeri): a comparison of three commercial systems.

The inhibition of light emitted by the bioluminescent bacterium, Vibrio fischeri, is the basis for several toxicity bioassays. The inhibitory effects of 81 chemicals, after 5 min contact time, were studied at eight concentrations using reagents from three commercial assay systems (ToxAlert 10, Microtox and LUMIStox). Solubility in water was the limiting factor in determining the selection of chemicals for study. The effective nominal concentrations (EC) resulting in 20, 50 and 80% inhibition were determined using Ln dose/Ln gamma plots and the results obtained for each system were compared by linear regression. The chemical concentrations producing 10-90% inhibition extended over 9 orders of magnitude and ranged from a minimum of 0.001 ppm to a maximum of 1,000,000 ppm. The toxicity of many chemicals was apparently related to their pH in solution and at high chemical concentrations, to osmotic imbalance. The fact that the same operator tested the same solutions simultaneously on three different systems reduced sources of error and variability and improved the consistency and reliability of the results. Only five compounds gave EC 50s that varied more than three-fold between assays. These data provide comparisons of toxicity that have not been previously available and demonstrate that, when used under standardised conditions, these bioluminescence-based toxicity assays produce very similar results.

Hepatic metallothionein as a biomaker for metal contamination: age effects and seasonal variation in European flounders (Pleuronectes flesus) from the Severn Estuary and Bristol Channel.

Hepatic concentrations of metallothionein [MT] and three metals (Cu, Zn, Cd) were determined in 242 European flounders (Pleuronectes flesus) collected from power stations at Oldbury-upon-Severn and Hinkley Point, located in Severn Estuary and Bristol Channel, UK, respectively, between March 1996 and February 1998. A model involving three-factor analysis of variance (ANOVA) was used to examine variation in MT and metal concentrations with respect to season, year and site; with age-class included as a covariate in the analysis. Hepatic concentrations of MT and Cd (and to some degree, Cu, but not Zn) increased significantly with age. The model explained 38, 25, 17 and 26% of the variation in MT, Cu, Zn and Cd, respectively, with significant effects due to season, and to a lesser extent, to year. Site was only a significant factor for Cd which was higher in fish from Hinkley. Correlation between the individual concentration of MT and each metal alone, or in combination, was poor, and explained only an additional 3.0% of the residual variation in MT, most of which was attributable to Cu (2.7%). Compared to other industrialised estuaries, Cd concentrations were high (>20 micro g-1 in some individuals). The study emphasises the importance of seasonal variation and other factors in biomonitoring programmes and highlights the limitations of using [MT] as a biomarker for metal contamination in flounders from the Severn Estuary.

The distribution of melanin-concentrating hormone in the lamprey brain.

In addition to its novel, colour-regulating hormonal role in teleosts, the melanin-concentrating hormone (MCH) serves as a neuromodulatory peptide in all vertebrate brains. In gnathostome vertebrates, it is produced in several neuronal cell groups in the hypothalamus. The present work examines the organisation of the MCH system in the brain of lampreys, which separated from gnathostome vertebrates at an early stage in evolution. In all three lamprey genera examined-Petromyzon, Lampetra, and Geotria spp.-MCH perikarya were found in one major anatomical site, the periventricular dorsal hypothalamic nucleus of the posterior hypothalamus. Axons from these cell bodies projected medially into the ventricular cavity, and laterally to the neuropile of the lateral hypothalamus. From here, they extended anteriorly and posteriorly to the fore- and hindbrain. Other fibres extended dorsomedially to the habenular nucleus. In Lampetra, but not in Petromyzon, MCH fibres were seen in the pituitary neurohypophysis, most prominantly above the proximal pars distalis. The hypothalamic region in which the MCH perikarya are found forms part of the paraventricular organ (PVO), which is rich in monoamines and other neuropeptides. The association of MCH neurones with the PVO, which occurs also in many other nonmammalian vertebrates, may reflect the primary location of the MCH system. These MCH neurones were present in ammocoetes, postmetamorphic juveniles, and adults. They were more heavily granulated in adults than in young lampreys but showed no marked change in secretory appearance associated with metamorphosis or experimental osmotic challenge to indicate a role in feeding or osmoregulation. In sexually maturing Lampetra fluviatilis, however, a second group of small MCH neurones became detectable in the telencephalon, suggesting a potential role in reproduction and/or behaviour.

Fish fauna of the Severn Estuary. Are there long-term changes in abundance and species composition and are the recruitment patterns of the main marine species correlated?

Fish were collected from the intake screens of the Oldbury Power Station in the Severn Estuary in each week between early July 1972 and late June 1977 and at least twice monthly between early January 1996 and late June 1999. The annual catches, after adjustment to a common sampling effort, demonstrate that the abundance of fish at Oldbury was far greater in the 1990s than 1970s, mainly due to marked increases in the numbers of certain marine species, such as sand goby, whiting, bass, thin-lipped grey mullet, herring, sprat and Norway pout. These increases may reflect the great improvement that occurred in the water quality of the Severn Estuary between these decades. The only species that declined markedly in abundance was poor cod. Modest declines in flounder and River lamprey paralleled those occurring elsewhere in the UK. The species composition in the two decades also differed, reflecting changes not only in the relative abundances of the various marine estuarine-opportunistic species, which dominated the ichthyofauna, but also in those of the suite of less abundant species in the estuary. The cyclical changes undergone each year by the species composition of the fish fauna of the Severn Estuary reflect sequential intra-annual changes in the relative abundances of species representing each of the marine, diadromous and freshwater categories. New approaches have been developed to test whether or not large sets of correlations between patterns of recruitment amongst abundant marine species (internal correlations), and between those patterns and salinity and water temperature within the estuary (cross-correlations), were significant. The correlation profile analyses found no evidence that the annual recruitment strengths of these species were either intercorrelated, or correlated with either one or a combination of both of the above environmental variables. Yet, the timings of the recruitment of these species into the estuary were intercorrelated, i.e. a slightly earlier or later than normal immigration by one species in a given year was paralleled by the same trend in other species. However, this association in recruitment times could be linked neither to salinity nor water temperature within the estuary, nor to a combination of these two variables. These results indicate that, while the factors that influence the annual recruitment strengths of the juveniles of different marine species vary, inter-annual differences in the phasing of events that regulate spawning times and/or larval dispersal influence, in the same direction, the times when marine species are recruited into the estuary.

Towards the development of molecularly imprinted polymer based screen-printed sensors for metabolites of PAHs.

This paper describes the fabrication of a sensor for 1-hydroxypyrene (1-OHP) based on a screen-printed carbon electrode (SPCE) modified with a molecularly imprinted polymer (MIP); 1-OHP was chosen as a model metabolite of polyaromatic hydrocarbons (PAHs). It was shown that 1-OHP could be readily oxidised at a plain SPCE and the electrochemical mechanism was found to involve an ECE (electron transfer-chemical reaction-electron transfer) process. The MIP for 1-OHP was prepared using only divinylbenzene (DVB) and styrene as monomers and the binding was only based on hydrophobic interactions. Batch binding studies revealed that optimum uptake of 1-OHP by the MIP occurred from solutions containing 35% water in methanol. Selectivity of the binding sites in the MIP was examined by performing uptake studies in the same solution containing either phenol or 1-naphthol; the specific binding of 1-OHP was twenty times greater than the former and five times greater than the latter. Preliminary calibration studies were performed with the MIP-SPCE using a two-step approach; accumulation was carried out in 35% water in methanol followed by measurement in 50% methanol-0.025 mol dm(-3) phosphate buffer pH 12. This two-step non-competitive affinity assay gave encouraging results and indicated potential for use in pollution studies.

Supernatants from leucocytes treated with melanin-concentrating hormone (MCH) and alpha-melanocyte stimulating hormone (alpha-MSH) have a stimulatory effect on rainbow trout (Oncorhynchus mykiss) phagocytes in vitro.

Melanin-concentrating hormone (MCH) and alpha-melanocyte stimulating hormone (alpha-MSH) are widespread vertebrate neuropeptides. In teleost fish the peptides are involved in the hormonal control of skin pigmentation, but they have also been shown to modulate corticosteroid secretion in both fish and mammals. alpha-MSH has additional potent anti-inflammatory actions in mammals and both peptides stimulate leucocyte phagocytosis in rainbow trout in vitro. The effects of these peptides on phagocytosis and the release of immunomodulatory factors by rainbow trout head kidney leucocytes were investigated in vitro. Neither MCH nor alpha-MSH had any effect on the adherence of phagocytes to glass slides or the activity of isolated phagocytes. When added to mixed leucocyte suspensions, however, MCH (50 and 100nM) and alpha-MSH (1 and 10nM) significantly increased the percentage of cells undergoing phagocytosis and MCH (50nM), but not alpha-MSH, stimulated the phagocytic index. In subsequent experiments, isolated phagocytes were exposed to supernatants derived from mixed leucocyte suspensions exposed to MCH (50 and 100nM) and alpha-MSH (1 and 10nM). Supernatants from leucocytes exposed to all doses of the peptides significantly increased the percentage phagocytosis and those from cells stimulated with MCH (100nM) and alpha-MSH (1 and 10nM) increased the phagocytic index of the phagocytes. The results suggest that cells other than phagocytes are required for MCH and alpha-MSH to exert their stimulatory effects on trout phagocytic cells through the release of one or more macrophage-activating factors.

Modulation of the fish immune system by hormones.

Immune-neuroendocrine interactions in fish, as in mammals, have become a focus of considerable interest, with the modulation of immune responses by hormones receiving particular attention. Cortisol, growth hormone (GH), prolactin (PRL), reproductive hormones, melanin-concentrating hormone (MCH) and proopiomelanocortin (POMC)-derived peptides have all been shown to influence immune functions in a number of fish species. This review summarises the known effects of these hormones on the fish immune system, as well as the often complex interactions between different hormones. The possible implications for fish health, with respect to aquaculture and the changes in immunocompetence that take place during different stages in the fish life cycle are also discussed.

Catalytic properties of CYP1A isoforms in the liver of an agnathan (Lampetra fluviatilis) and two species of teleost (Pleuronectes flesus, Anguilla anguilla).

The catalytic activity of CYP1A isoforms and the effect of mammalian CYP1A-specific inhibitors in liver S9 fractions were studied in an agnathan (River lamprey, Lampetra fluviatilis, 30-33 cm) and in two species of teleost fish (European flounder, Pleuronectes flesus, 11-18 cm and common eel, Anguilla anguilla, 31-48 cm). Ethoxyresorufin O-deethylation (EROD), caffeine N-demethylation/C-oxidation and phenacetin O-deethylation (POD) activity increased 3-4-fold in flounders and 17-46-fold in eels, 5 days after fish were injected (i.p.) with 100 mg kg(-1) benzo(a)pyrene (B[a]P). In lampreys, basal EROD activity was very low and no increase in activity was observed following exposure to B[a]P. While the apparent Michaelis constant (K(m)) for each assay showed only small changes after B[a]P injection, maximum reaction velocity (V(max)) values increased by up to 19- and 84-fold for EROD activity, 4- and 35-fold for caffeine-related metabolism and 4- and 19-fold for POD activity in flounders and eels, respectively. The mammalian CYP1A2 inhibitor furafylline (50 microM-1 mM) reduced activity in the EROD, caffeine and POD assays to 65, 21 and 20% of control values in flounders and to 85, 10 and 5% of control values in eels, respectively. By contrast, low concentrations (0.025-0.050 microM) of the mammalian CYP1A1 inhibitor ellipticine completely abolished EROD activity, but had no effect (up to 1 mM) on caffeine metabolism or POD activity in either species. While the inhibitor studies strongly suggest that two separate enzymes are present in flounders and eels, the monophasic Michaelis-Menten kinetics obtained in all the assays imply that only a single CYP1A protein is present that has substrate and inhibitor specificities characteristic of both mammalian CYP1A1 and CYP1A2 isoforms.

In the trout, CRH and AVT synergize to stimulate ACTH release.

Anterior pituitaries of the rainbow trout (Oncorhynchus mykiss) were incubated with graded concentrations of arginine vasotocin (AVT) or synthetic rat corticotrophin-releasing hormone (rCRH-41), alone or in combination, and the ACTH secreted into the medium was measured by a sensitive cytochemical bioassay. The aim was to determine the relative potencies of the two secretogogues and whether, in this fish species, they act synergistically. Rat CRF-41 and AVT both produced concentration-dependent increases in ACTH release. The minimum effective concentration for both peptides was approximately 1 nM but, at higher concentrations, the efficacy of CRF-41 was greater than that of AVT. Clear evidence of synergy between the two peptides was obtained. The response of the trout thus falls in line with observations in mammals and contrasts with findings for the goldfish.

Neoplastic transformation of prostatic and urogenital epithelium by the polyoma virus middle T gene.

Male transgenic mice expressing the polyomavirus middle T (PyV-MT) gene exhibited growth and developmental abnormalities in prostatic and other urogenital epithelium. Expression of PyV-MT was directed to these tissues by a novel, androgen-inducible expression vector based on the rat C3(1) gene. Epithelial growth disturbances (hyperplasia, dysplasia, and invasive carcinoma) were observed in the ventral and dorsal prostate, coagulating gland, epididymis, and vas deferens. The abnormalities were characterized by histological disorganization, nuclear pleomorphism, increased mitoses, and abnormal DNA content. Transgene transcription was detected in affected tissues, indicating that the C3(1)-based vector targeted androgen-sensitive urogenital tissues, especially the prostate. These results demonstrated that expression of a gene, the protein of which is known to interact with cellular proteins involved in signal transduction, dramatically disrupted urogenital growth and development.