Neil Hazon

PRIMARY SEQUENCE, TISSUE SPECIFICITY AND EXPRESSION OF THE NA+, K+-ATPASE ALPHA 1 SUBUNIT IN THE EUROPEAN EEL (ANGUILLA ANGUILLA)

The entire cDNA nucleotide sequence of the Na+,K(+)-ATPase alpha 1 isoform was cloned from the gills of the European eel (Anguilla anguilla) by a PCR based method. The amino acid sequence translated from the sequence shared 89.4 and 85.6% homology respectively with previously published Na+,K(+)-ATPase alpha subunit sequences from elasmobranch (Torpedo californica) and teleost (Catostomus commersoni) fish. The size of Na+,K(+)-ATPase alpha 1 mRNA transcripts in eel tissues was demonstrated to be 3.5 kb, except in the ovary where a 3.7 kb transcript existed. Na+,K(+)-ATPase alpha 1 mRNA was present at some level in all tissues investigated with the exception of cardiac and skeletal muscle where no Na+,K(+)-ATPase alpha 1 mRNA was detectable. The level of branchial Na+,K(+)-ATPase alpha 1 mRNA increased after the adaptation of freshwater eels to normal or double concentration seawater.

Somatostatin-related and glucagon-related peptides with unusual structural features from the European eel (Anguilla anguilla)

Peptides derived from prosomatostatins I and II and from two distinct proglucagons have been isolated from the pancreas of a teleost fish, the European eel (Anguilla anguilla). The product of prosomatostatin I processing, somatostatin-14, is identical to mammalian somatostatin-14. A 25-amino-acid-residue peptide (Ser-Val-Asp-Asn-Gln5-Gln-Gly-Arg-Glu-Arg10-Lys-Ala-Gly-Cys- Lys15-Asn-Phe-Tyr- Trp-Lys20-Gly-Pro-Thr-Ser-Cys25) is derived from prosomatostatin II. Compared with the corresponding peptides from other teleost fish, the eel somatostatin-25 contains the unusual substitution Pro for Phe at position 22. This peptide was also isolated in a form containing a hydroxylsyl residue at position 20. A 29-amino-acid-residue eel glucagon contains four substitutions relative to human glucagon Asn for Ser8, Glu for Asp15, Thr for Ser16, and Ser for Thr29). In common with mammalian and avian glucagons but unlike most other fish glucagons, the eel peptide possesses a glutamine residue at position 3. A peptide derived from a second proglucagon comprises 36 amino acid residues. A 7-residue C-terminal extension to the glucagon sequence shows structural similarity to the corresponding extension in ratfish (Hydrolagus colliei) glucagon and mammalian oxyntomodulin.

Isolation of high-molecular-weight C-type natriuretic peptide from the heart of a cartilaginous fish (European dogfish, Scyliorhinus canicula)

A high‐molecular‐weight form of C‐type natriuretic peptide (CNP) was isolated from both cardiac atria and ventrieles of European dogfish, Scyliorhinus canicula, and its primary structure was determined. The peptide consists of 115 amino acid residues, in which the C‐terminal 22 residues show high homology to CNPs identified to date. This is the first direct evidence for the presence of natriuretic peptide in the cartilaginous fish, and for the presence of CNP in an organ other than the brain.

Drinking behaviour in sea water and fresh water teleosts, the role of the renin-angiotensin system

Basal drinking rate and responses to administered angiotensin were examined in 12 species of fish. The responses of representative euryhaline, stenohaline marine and fresh water species to pharmacological manipulation of endogenous renin-angiotensin system (RAS) activity were also investigated.Basal drinking rates were consistently low in stenohaline and euryhaline fresh water fish, and all species examined showed an increased imbibition in response to administered angiotensin. Marine fish drank large volumes of water, rates varying considerably between species, with euryhaline species exhibiting lower rates than stenohaline groups. The extremely high drinking rates observed in the sea scorpion were associated with a high plasma osmolality. With the exception of the sea scorpion, all other species examined in sea water showed a further rise in drinking in response to exogenous angiotensin.Although the freshwater stenohaline carp showed a dipsogenic response to angiotensin, it was apparently unable to evoke this response when fish were acclimated to brackish water. The high drinking rates of both euryhaline and stenohaline fish held in sea water appeared dependent upon an activated endogenous RAS, and were lowered following inhibition of Al to All conversion by Captopril. Drinking was further stimulated in these marine species following stimulation of endogenous RAS activity by the administration of the hypotensive agent Papaverine. The study endorses a role for the RAS in the control of adaptive drinking in euryhaline and stenohaline marine teleosts.

Cloning and expression of three aquaporin homologues from the European eel (Anguilla anguilla): effects of seawater acclimation and cortisol treatment on renal expression

Background information. The European eel (Anguilla anguilla) is able to osmoregulate over a wide range of environmental salinities from FW (freshwater) to hyperconcentrated SW (seawater). Successful acclimation is associated with strict regulation of ion and water transport pathways within key osmoregulatory epithelia to enable animals to survive the dehydrating or oedematous conditions. These observations suggested that homologues of the AQP (aquaporin) water channel family were expressed in the eel and that these proteins may contribute to the water transport and osmoregulation in all euryhaline teleosts.

The renin-angiotensin system and vascular and dipsogenic regulation in elasmobranchs

The role of a renin-angiotensin-like system (RAS) in the regulation of blood pressure and drinking has been investigated in the elasmobranch, Scyliorhinus canicula. Injection of exogenous angiotensin II produced, as expected, a vasopressor response, though injection of the converting enzyme inhibitor, Captopril, alone produced little change in resting blood pressure. Papaverine, a smooth muscle relaxant, reduced blood pressure which completely recovered within 30 min. A subsequent injection of Captopril produced a rapid vasodepressor response with no recovery over 2 hr. The low basal levels of drinking in dogfish were not altered by Captopril injection but angiotensin II-induced increased drinking and papaverine administration resulted in markedly stimulated water intake, which was inhibited by coadministration with Captopril. Captopril inhibition of the recovery in blood pressure and associated dipsogenic response following the papaverine-induced hypotension is consistent with the activation of a RAS-like system in the dogfish. This and other evidence supporting the presence of a RAS-like system in elasmobranchs are discussed in relation to other vertebrates.

Sex-biased investment in yolk androgens depends on female quality and laying order in zebra finches (Taeniopygia guttata)

The Trivers–Willard hypothesis predicts sex biases in parental investment according to parental condition. In addition, parents may need to sex bias their investment if there is an asymmetry between the sexes in offspring fitness under different conditions. For studying maternal differential investment, egg resources are ideal subjects because they are self contained and allocated unequivocally by the female. Recent studies show that yolk androgens can be beneficial to offspring, so here we test for sex-biased investment with maternal investment of yolk testosterone (T) in zebra finch (Taeniopygia guttata) eggs. From the Trivers–Willard hypothesis, we predicted females to invest more in male eggs in optimum circumstances (e.g. good-condition mother, early-laid egg), and more in female eggs under suboptimal conditions (e.g. poor-condition mother, late-laid egg). This latter prediction is also because in this species there is a female nestling disadvantage in poor conditions and we expected mothers to help compensate for this in female eggs. Indeed, we found more yolk T in female than male eggs. Moreover, in accordance with our predictions, yolk T in male eggs increased with maternal quality relative to female eggs, and decreased with laying order relative to female eggs. This supports our predictions for the different needs and value of male and female offspring in zebra finches. Our results support the idea that females may use yolk androgens as a tool to adaptively manipulate the inequalities between different nestlings.

The effects of dietary sodium loading on the activity and expression of Na, K-ATPase in the rectal gland of the European Dogfish (Scyliorhinus canicula)

cDNA fragments of both the alpha- and beta-subunits of the Na, K-ATPase and a cDNA fragment of the secretory form of Na-K-Cl cotransporter from the European dogfish (Scyliorhinus canicula) were amplified and cloned using degenerate primers in RT-PCR. These clones were used along with a sCFTR cDNA from the related dogfish shark, Squalus acanthias to characterise the expression of mRNAs for these ion transporters in the dogfish rectal gland subsequent to an acute feeding episode. Following a single feeding event where starved dogfish were fed squid portions (20 g squid/kg fish), there was a delayed and transient 40-fold increase in the activity of Na, K-ATPase in crude rectal gland homogenates. Increases in enzyme activity were apparent 3 h after the feeding event and peaked at 9 h before returning to control values within 24 h. These increases in activity were accompanied by small and transient decreases in plasma sodium and chloride concentrations lasting up to 3 days. Significant increases in the expression of mRNAs for alpha- and beta-subunits of the Na, K-ATPase, the Na-K-Cl cotransporter and CFTR chloride channel were detected but not until 1-2 days after the feeding event. It is concluded that the transient increase in Na, K-ATPase activity is not attributable to increases in the abundance of alpha- and beta-subunit mRNAs but must be associated with some, as yet unknown, post-transcriptional activation mechanism.

Genetic differentiation of populations of the copepod sea louse Lepeophtheirus salmonis (Krøyer) ectoparasitic on wild and farmed salmonids around the coasts of Scotland: Evidence from RAPD markers

Abstract Sea trout are the sea-going migratory form of the freshwater brown trout ( Salmo trutta L.) and since 1989 there have been marked declines in their stocks on the west coasts both of Scotland and Ireland. Various factors have been attributed as possible causal agents in these stock declines, including fresh water acidification, overfishing, climatic fluctuations, habitat degradation and sea lice parasitic burdens. The putative impact of infestations of sea trout by the ectoparasitic copepod sea louse, Lepeophtheirus salmonis (Kroyer), has featured prominently in the controversy, especially with regard to the role of inshore commercial salmon farms as a possible source of infestation of wild salmonids by sea lice. This study focused on the population genetics of L. salmonis around the coasts of Scotland: We sampled fish from wild and cultured stocks and included salmon ( Salmo salar L.), rainbow trout ( Oncorhynchus mykiss Walbaum) and sea trout as host species. Analyses of allozyme variation of sea lice were confined to data for two polymorphic loci ( Fum, Got-2 ) and conformed to our initial expectation — that the inclusion of a planktonic larval phase in the life cycle of the copepod, in addition to the high mobility of the host fish, would enhance gene flow and preclude genetic differentiation of L. salmonis populations as a result of random drift alone. DNA polymorphism was quantified by means of PCR and RAPD analysis. Six primers were screened for 16 samples (from wild and farmed salmon, wild sea trout and farmed rainbow trout) — including the east, north and west coasts of Scotland — and the data analyzed by AMOVA (Analysis of Molecular Variance). In contrast to the allozyme results, the RAPD analysis showed striking patterns of genetic differentiation around the coasts of Scotland. The overall pattern was one of genetic homogeneity of L. salmonis populations sampled from wild salmon and sea trout. All of the L. salmonis samples taken from farmed salmon and rainbow trout did, however, show highly significant levels of genetic differentiation, both between wild and farmed salmonids and among the various farms themselves. Evidence of high levels of small-scale spatial or temporal heterogeneity of RAPD marker band frequencies was shown for the one farm from which repeat samples (July and November, 1995) were analysed. Samples of sea lice taken from west coast wild sea trout subjected to RAPD analysis also revealed the occurrence of putative “farm markers” in some individual parasites, indicating that they had possibly originated from salmon farms.

Primary sequence, tissue specificity and mRNA expression of the Na+,K+ -ATPase ?1 subunit in the European eel (Anguilla anguilla)

The entire amino acid coding sequence of the Na+,K+-ATPase β1 isoform was cloned from the gill of the European eel (Anguilla anguilla) by a PCR based method. The amino acid sequence translated from the nucleotide sequence shared 61.4 and 56.2% homology respectively with previously published Na+,K+-ATPase β1 isoform sequences from the clawed toad (Xenopus laevis) and the ray (Torpedo californica) an elasmobranch fish. The size of the Na+,K+-ATPase β1 mRNA transcript in eel tissues was demonstrated to be 2.35 Kb. Detectable levels of Na+,K+-ATPase β1 mRNA were found at some level in all tissues except liver and cardiac muscle. The level of branchial Na+,K+-ATPase β1 mRNA was observed to increase after the adaptation of fresh water eels to normal or double concentration sea water.