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Distribution of the salmonid Hpa 1 family in the salmonid species demonstrated by in vitro runoff transcription assay of total genomic DNA: a procedure to estimate repetitive frequency and sequence divergence of a certain repetitive family with a few known sequences.

An in vitro runoff transcription assay of total genomic DNA was developed. As an example of use of this assay, analysis of a highly repetitive sequence in the cherry salmon (Oncorhynchus masou) is described. Total genomic DNA of the cherry salmon was completely digested with Hpa 1, whose site is known to be in the tRNA-unrelated region of the cherry salmon Hpa 1 family. On transcription of the digested DNA in a HeLa cell extract, a discrete-sized RNA of about 100 nucleotides, constituting 70% of the transcripts, was produced, whereas on transcription of the undigested total DNA, only smeared RNA was obtained. In a fingerprint, the oligonucleotides of the discrete transcript from the digested total DNA were very distinct and exactly corresponded to those of a transcript from an Hpa 1 digest of a cloned DNA, but with few extra oligonucleotides. These results showed that the cherry salmon Hpa 1 family constitutes a major repetitive family in the genome of the cherry salmon. For determination of the distribution of the salmonid Hpa 1 family in other salmonid species, the same analysis was applied to DNAs from the chum salmon (Onchorhynchus keta), brown trout (Salmo trutta), Japanese common charr (Salvelinus leucomaenis pluvius), and Japanese huchen (Hucho perryi). The results showed that the salmonid Hpa 1 family is widespread in the genomes of salmonid species. A method and equations are also presented for estimating the relationship between the ratio of a given repetitive family to all the Pol III genes and its average sequence divergence by calculating the molar ratio of the runoff transcript to all the in vitro Pol III transcripts.

Animals

Exploring the Effect of Whole-Genome Duplication on Salmonid LincRNA Repertoire.

Long intergenic non-coding RNAs (lincRNAs) are key epigenetic regulators of genome function, yet their evolutionary dynamics following whole-genome duplication (WGD) events remain poorly understood. Salmonids, which underwent a lineage-specific autotetraploidization (salmonid-specific WGD, ~88-100 million years ago), provide an excellent model to investigate the retention, divergence, and functional potential of recently duplicated non-coding elements. LincRNA repertoires were compared across five genome-annotated salmonids (Oncorhynchus tshawytscha, O. kisutch, O. mykiss, Salmo salar, and S. trutta) and their closest non-duplicated relative, northern pike (Esox lucius). LincRNAs represented ~5-7% of annotated genes in all salmonids except S. salar (18%). Sequence conservation was low relative to coding genes, with only 11-68 highly similar (e-value < 1 &#xd7; 10-30; similarity > 70% and alignments > 100 nucleotides) putative orthologues shared between salmonids and northern pike, and 161-338 among salmonids alone. Synteny conservation was modest in lincRNAs, with lower conservation in putative orthologues (8-16%) compared to putative ohnologues (8-33%). Secondary structure conservation was associated with sequence similarity (&#x3c1; = -0.45; p = 2.2 &#xd7; 10-16), and the association was stronger among WGD ohnologues than orthologues. In S. salar and O. mykiss, lincRNA putative ohnologues showed weaker expression correlations than coding genes, suggesting widespread regulatory divergence, possibly through neo- and subfunctionalisation. Conserved salmonid lincRNAs showed enriched predicted interactions with miRNAs involved in tumour suppression, brain, bone, and muscle development (e.g., miR-455, miR-365, miR124, miR-133a, miR-140, and miR-9), a finding supported by limited transcriptomic data. Although salmonid WGD expanded lincRNA repertoires, lincRNAs have undergone rapid sequence and transcriptional divergence, with limited conservation across species based on sequence similarity, chromosomal position, synteny, and secondary structure. A subset of conserved lincRNAs retains structural features and regulatory signatures consistent with roles as miRNA sponges in brain, skeletal, and muscle development and tumour suppression, potentially acting within conserved regulatory networks. These findings provide new insights into lincRNA evolution following genome duplication and highlight the need for experimental validation of their regulatory functions.

Animals

Evolution of the salmonid mitochondrial control region.

To explore the evolutionary nature of the salmonid mitochondrial DNA (mtDNA) control region (D-loop) and its utility for inferring phylogenies, the entire region was sequenced from all eight species of anadromous Pacific salmon, genus Oncorhynchus; the Atlantic salmon, Salmo salar; and the Arctic grayling, Thymallus arcticus. A comparison of aligned sequences demonstrates that the generally conserved sequence elements that have been previously reported for other vertebrates are maintained in these primitive teleost fishes. Results reveal a significantly nonrandom distribution of nucleotide substitutions, insertions, and deletions that suggests that portions of the salmonid D-loop may be under differential selective constraints and that most of the control region of these fishes may evolve at a rate similar to that of the remainder of their mtDNA genomes. Maximum likelihood and Fitch parsimony analyses of 9 kb of aligned salmonid sequence data give evolutionary trees of identical topology. These results are consistent with previous molecular studies of a limited number of salmonid taxa and with more comprehensive, classical analyses of salmonid evolution. Predictions from these data, based on a molecular clock assumption for the mtDNA control region, are also consistent with fossil evidence that suggests that species of Oncorhynchus could be as old as the Middle Pliocene and would have thus given rise to the extant Pacific salmon prior to about 5 or 6 million years ago.

Animals

Shaping and reshaping of salmonid genomes by amplification of tRNA-derived retroposons during evolution.

Three families of tRNA-derived repeated retroposons in the genomes of salmonid species have been isolated and characterized. These three families differ in sequence, but all are derived from a tRNA(Lys) or from a tRNA species structurally related to tRNA(Lys). The salmon Sma I family is present in the genomes of two species of the genus Oncorhynchus but not in other species, including five other species of the same genus. The charr Fok I family is present only in four species and subspecies of the genus Salvelinus. The third family, the salmonid Hpa I family, appears to be present in all salmonid species but is not present in species that are not members of the Salmonidae. Thus, the genome of proto-Salmonidae was originally shaped by amplification and dispersion of the salmonid Hpa I family and then reshaped by amplification of the Sma I and Fok I families in the more recently evolved species of salmon and charr, respectively. We speculate that amplification and dispersion of retroposons may have played a role in salmonid speciation.

Animals

Species-specific amplification of tRNA-derived short interspersed repetitive elements (SINEs) by retroposition: a process of parasitization of entire genomes during the evolution of salmonids.

Fourteen members of the Hpa I subfamilies of tRNA-derived SINEs in particular salmonid species were isolated from genomic libraries of chum salmon, kokanee, coho salmon, masu salmon, and steelhead. Alignment of the sequences of these 14 members, together with those of 4 members already published, 3 of which were previously demonstrated to have been amplified specifically in certain lineages, revealed the presence of five subfamilies with particular diagnostic nucleotides. The amplification of members of the same subfamily in different salmonid lineages and the amplification of members of different subfamilies in the same salmonid lineage suggest that multiple dispersed loci were responsible for amplification or, alternatively, that SINEs were transmitted horizontally between species. These two possibilities are not mutually exclusive. Our results also indicate that the Hpa I SINEs in salmonids behave like parasites. The amplification of these SINEs is ongoing and continues to shape the evolution of salmonid genomes.

Animals

Enterocytozoon salmonis n. sp.: an intranuclear microsporidium from salmonid fish.

The developmental stages of a recently described microsporidian from the nucleus of hematopoietic cells of salmonid fish were found to be unique among the Microsporida. All observed stages, including meronts, sporonts, and spores were in direct contact with the host cell nucleus (principally hematopoietic cells) of chinook salmon (Oncorhynchus tshawytscha). There is no parasitophorous vacuole and sporogony does not involve formation of a pansporoblastic membrane as with other members of the suborder Apansporoblastina. The extrusion apparatus differentiates prior to division of sporogonial plasmodia. The spores are ovoid (1 x 2 microns) and uninucleate, and possess a coiled polar tube with 8-12 turns. Developmental stages of the salmonid microsporidian are similar to those described for Enterocytozoon bieneusi as found in the intestinal mucosa of human AIDS patients. However, the intranuclear development, different cell types, and host infected clearly separate the salmonid and human parasites. Accordingly, the intranuclear parasite of salmonids is given the name Enterocytozoon salmonis n. sp. within the suborder Apansporoblastina.

Animals

Loss of duplicate gene expression in salmonids: evidence for a null allele polymorphism at the duplicate aspartate aminotransferase loci in brook trout (Salvelinus fontinalis).

Unusual phenotypic distributions at the muscle-specific, duplicate aspartate aminotransferase (AAT) loci were found in wild populations of brook trout (Salvelinus fontinalis), a species of the tetraploid-derivative Salmonidae. Analysis of these phenotypic distributions ruled out disparate gene frequencies, nonrandom association between the two loci, and inbreeding as possible explanations; however, models incorporating a null allele fit the data. Inheritance data from hatchery populations of brook trout also indicated a null allele polymorphism. This proposed AAT null allele, along with other null allele polymorphisms in salmonids, is evidence that loss of duplicate gene expression is still occurring. In contrast, there is no such evidence of ongoing loss of duplicate gene expression in the Catostomidae, another tetraploid-derivative lineage. We interpret this and other differences between salmonids an catostomids as reflecting an autotetraploid origin for salmonids and an allotetraploid origin for catostomids. The significance of these findings is also considered with respect to current models of the rate of loss of duplicate gene expression in tetraploid-derivative organisms.

Alleles

Genetic basis of creatine kinase isozymes in skeletal muscle of salmonid fishes.

The genetic basis of isozyme phenotypes of creatine kinase (CK) from extracts of skeletal muscle of salmonids has been resolved through breeding data including double heterozygous crosses and backcrosses of rainbow trout (Salmo gairdneri), and backcrosses of coho salmon (Oncorhynchus kisutch). The two-three-, or four-banded phenotypes of homozygous individuals and all heterozygous and hybrid phenotypes of ten salmonid species are readily explained by the following model: (1) there are no detectable heterodimers either between allelic products at a single locus or between loci: (2) each allele is represented electrophoretically by two bands, presumably a reflection of stable posttranslational modification of a single polypeptide unit; (3) CK of salmonid muscle is encoded by two loci--CK-1 and CK-2. The distance separating the paired bands reflecting each allele provides a basis for two groupings--a broad-spaced group (including all species of Oncorhynchus tested excepting O. masou) and a narrow-spaced group (including all species of Salmo tested and O. masou). The relationships among species suggested by the relative mobilities and spacings of these CK bands are consistent with taxonomic schemes inferred from morphological, cytogenetic, and other isozymic data.

Animals

Palmitate-binding, serum albumin-like proteins in salmonids.

There has been considerable controversy over the existence of serum albumin in fish. One of the physiological functions of albumin is to bind free fatty acids. This characteristic was used to screen the plasma of seven species of salmonids. Each species contains a protein fraction that (i) binds palmitate, (ii) has a molecular mass similar to that of human serum albumin, and (iii) is one of the most rapidly migrating proteins when salmonid plasma is subjected to anodal polyacrylamide gel electrophoresis. We conclude therefore, that salmonids have serum albumins that are homologous to the serum albumin of higher vertebrates.

Animals

The development and validation of a radioimmunoassay to measure plasma ACTH levels in salmonid fishes.

A radioimmunoassay (RIA) capable of determining blood ACTH levels in salmonid fishes was developed and validated. The RIA used an antibody raised against mammalian ACTH, iodinated human ACTH as tracer, and human 1-39 ACTH as standard. Incubation of the standard or unknown with antibody for 3 days before addition of as little high-specific activity tracer as practicable (1500 cpm; equivalent to 5 pg ACTH) produced a very sensitive RIA; the operating range was 5 to 200 pg ACTH/ml. Extracts of both pars distalis and neurointermediate lobe of the pituitary glands from a range of salmonid species diluted parallel to the ACTH standard in the RIA. There was always considerably more ACTH-immunoreactivity (ACTH-IR) in the pars distalis extracts than in the neurointermediate lobe. Generally plasmas also diluted parallel to the ACTH standard, with the exception only of the plasma from sexually mature female salmonids, which diluted very non-parallel to the standard, leading to unrealistically low estimates of the ACTH-IR level. The use of heparin as an anticoagulant during collection of samples caused problems when these plasmas were immunoassayed; instead EDTA was found to be a suitable anticoagulant. When the ACTH-IR was extracted from a pool of plasma obtained from acutely stressed salmon and chromatographed on a column of BioGel P6, followed by subsequent ACTH RIA of the fractions, only a single sharp peak of ACTH-IR was detected, which eluted in the position of authentic 1-39 ACTH. The plasma ACTH-IR level in unstressed fish was low, and near the detection limit of the RIA. An acute stress, produced by crowding and confinement for 30 min, increased ACTH-IR approximately 10-fold, and plasma cortisol levels 50-fold, but the plasma alpha-MSH level was not affected. Dexamethasone-treated fish did not respond to this stressor with any increase in either ACTH or cortisol levels.

Adrenocorticotropic Hormone

Different cellular distributions of two somatostatins in brain and pancreas of salmonids, and their associations with insulin- and glucagon-secreting cells.

Invariant somatostatin-14 (SST-14) and somatostatin-25 (SST-25), isolated from coho salmon pancreas (Plisetskaya et al., 1986a) are likely coded by two distinct somatostatin genes. The present study was undertaken to investigate whether these genes are expressed in the same or in different cell types in the pancreatic islets and in the brain of two salmonids: rainbow trout and coho salmon. Antibodies generated against SST-14, mammalian (m) SST-28(1-14), salmon (s) SST-25, salmon insulin, and salmon glucagon were used as immunocytochemical probes. Two distinct cell types containing SSTs were revealed in the pancreas of both salmonid species: one cell type immunoreactive to both SST-14 and mSST-28(1-14) and the other cell type immunoreactive only to sSST-25. The SST-14/mSST-28(1-14)-positive cells were limited to the more central parts of the islets, in apposition to the insulin-positive cells: sSST-25-positive cells were located more peripherally and were associated topographically with the glucagon-positive cells. In contrast to the pancreas, neurons in the neurohypophysis and hypothalamus of the rainbow trout and coho salmon contained only SST-14-like and mSST-28(1-14)-like immunoreactivities, while immunoreactivity to sSST-25 was completely absent. These results suggest that differentiation in the pancreas and brain of salmonid fishes results in cell types in which SST genes are separately expressed. The close topographical association of sSST-25 with glucagon cells, and of SST-14 with insulin cells, in the pancreatic islets implies yet unknown functional regulatory relationships that require detailed study.

Animals

Linkage relationships reflecting ancestral tetraploidy in salmonid fish.

Fifteen classical linkage groups were identified in two salmonid species (Salmo trutta and Salmo gairdneri) and three fertile, interspecific hybrids (S. gairdneri X Salmo clarki, Salvelinus fontinalis X Salvelinus namaycush and S. fontinalis X Salvelinus alpinus) by backcrossing multiply heterozygous individuals. These linkage relationships of electrophoretically detected, protein coding loci were highly conserved among species. The loci encoding the enzymes appeared to be randomly distributed among the salmonid chromosomes. Recombination frequencies were generally greater in females than in males. In males, certain linkage groups were pseudolinked with other linkage groups, presumably because of facultative multivalent pairing and directed disjunction of chromosomes. Five such pseudolinkage groups were identified and they also appeared to be common among species and hybrids. Duplicate loci were never classically linked with each other, although some exhibited pseudolinkage and some showed evidence of exchanging alleles. Gene-centromere recombination frequencies estimated from genotypic distributions of gynogenetic offspring were consistent with map locations inferred from female intergenic recombination frequencies. These linkage relationships support the contention that all extant salmonids arose from a common tetraploid progenitor and that this progenitor may have been a segmental allotetraploid.

Animals

Fish infected with Sphaerospora spp. Thélohan (Myxosporea) from waters enzootic for proliferative kidney disease of salmonids.

Sphaerospores were found among three species of fish examined from waters known to be enzootic for proliferative kidney disease (PKD) of salmonids. They were detected in the renal tubules of both hatchery-reared rainbow trout (Salmo gairdneri) exposed to the infectious stage of PKD and in chubs (Gila bicolor) in the headwaters of a hatchery where PKD is enzootic. Sticklebacks (Gasterosteus aculeatus) collected near net pens where Pacific salmon had experienced a PKD epizootic were also found to harbor sphaerospores in the lumen of the kidney tubules. The latter two host species contained developmental stages of a myxosporidan in the blood and in the lumen of the kidney tubules which are similar to those of PKX, the causative agent of PKD in salmonid fish. The sphaerospores observed in the rainbow trout are the first to be observed in this species. The similarity to previously observed developmental stages, rarity, and presence of these sphaerospores in salmonid fish from a hatchery where PKD is enzootic suggest that they are the most mature stage of the PKX myxosporidan yet observed.

Animals

Transfer, expression, and inheritance of salmonid growth hormone genes in channel catfish, Ictalurus punctatus, and effects on performance traits.

We examined expression and inheritance of salmonid growth hormone genes RSVLTR-rtGH1 cDNA and RSVLTR-csGH cDNA, transferred to channel catfish (Ictalurus punctatus) by microinjection. One to 9 copies of the foreign DNA were inserted in either head-to-tail tandem array at single insertion sites or single copies at multiple insertion sites. All P1 transgenic catfish evaluated produced salmonid growth hormone regardless of the construct. Five P1 x P1 matings were accomplished. The spawning rate and fertility of these P1 transgenics in artificial spawning conditions were comparable to those of normal channel catfish. In two of three years, 100% spawning and 100% hatch were obtained. Percent transgenic progeny observed in the five matings were 20, 52, 7, 47, and 0%, which was lower (P < 0.001, chi 2) than the 75% inheritance expected assuming the P1 brood stock had at least one copy of the foreign gene integrated and were not mosaics in the germ line. At least 7 of 10 P1 were mosaics, and a minimum of 2 of 10 P1 did not possess the salmonid growth hormone genes in their germ line. P1 transgenics grew at the same rate as their nontransgenic full siblings, which is not surprising because the P1 were mosaics. F1 transgenic progeny in two families possessing RSVLTR-csGH cDNA grew 26% faster, to 40 to 50 gm, than their nontransgenic full siblings when evaluated communally. One F1 progeny group produced by RSVLTR-rtGH1 cDNA x RSVLTR-csGH cDNA mating and one F1 progeny group (parents either RSVLTR-rtGH1 cDNA or RSVLTR-csGH cDNA) grew at the same rate as normal full siblings when grown communally to 25 gm and 60 mg, respectively. In families where F1 progeny grew faster than controls, the range in body weight and coefficient of variation for the transgenic full siblings were less than those for controls. In families where F1 progeny grew at the same rate as controls, range in body weight and coefficient of variation were similar for transgenic and normal individuals. The percent deformities observed in P1 transgenics (13.6%) was higher (P < 0.05) than in microinjected P1 nontransgenics (5.1%). Percent deformities in transgenics and control F1 channel catfish was not different (p > 0.05; 0.5 and 2.8%, respectively).

Animals

[Epidemiology of infectious hematopoietic necrosis (IHN) of salmonid fish in France: study of the course of natural infection by combined use of viral and seroneutralization test and eradication attempts].

Infectious haematopoietic necrosis (IHN), a rhabdoviral infection of salmonid fish, was considered to be an exotic disease in Europe until it was recognized in France and Italy in 1987. In France, the existence of this new condition led the authorities in charge of animal health to order epidemiological studies to be undertaken. These studies were based upon virological, serological and experimental diagnostic methods and also encompassed disease eradication attempts. Studies were conducted at 7 fish farming sites, involved 1,545 salmonid fish, of which 848 were sacrificed, and represented 262 virological examinations and 1,782 serum neutralization tests. The presence of the IHN virus was detected in the 7 trout farm fish populations that were located in 5 regions, one of which was situated 600 km from the place where the first isolation of IHN virus was made. Moreover, 6 out of 7 rainbow trout populations reared in these farms also harboured viral haemorrhagic septicaemia virus (VHSV) often resulting in overt disease. Rainbow trout was the only salmonid fish species found infected with IHN. Overt infection, which was observed in fish ageing less than 2,200 degrees-days, always occurred at water temperatures below 14 degrees C, and the younger fish were more susceptible (mortality rate > or = 80%). Although the IHN virus is easily isolable from fish undergoing overt infection, it was hardly detectable in survivors until they were adults, at which stage the virus was shedded via sexual products which constituted suitable materials for virological examination and disease transmission assays. Survivors of overt and dormant IHN infection developed consistent immune response and special attention was paid to neutralizing antibodies (NAb) to IHN virus. The detection of such NAb in fish from infected farming sites or other NAb from presumably IHN-free sites, correlated fairly well with the presence and further detection of IHN virus among such fish populations. Our data provide arguments for considering the serological technique as a suitable means of completing fish health surveillance programmes for IHN. Although our results are in agreement with part of the existing knowledge on IHN, they differed in several points: rainbow trout was the only susceptible fish species; overt IHN was always recorded in juveniles and at water temperatures below 14 degrees C; IHN virus could not be recovered from the mucus of infected broodfish; IHN infection usually coexisted with VHS infection in same fish population; and serology was widely and successfully used for the diagnosis of IHN.

Animals

The role of hormones in the acquisition of sperm motility in salmonid fish.

In salmonid fish, spermatozoa taken from the testes are immotile, but acquire motility during their passage through the sperm duct. Using male masu salmon (Oncorhynchus masou), we found that gonadotropin-induced testicular production of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), the oocyte maturation-inducing hormone of salmonid fish, is responsible for the acquisition of sperm motility. However, neither testosterone (T) nor 11-ketotestosterone (11-KT), the two major androgens in teleost fish, were effective. We also present evidence that 17 alpha, 20 beta-DP action is mediated through an increase in sperm duct pH, which in turn increases the cAMP content of sperm allowing the acquisition of motility.

Animals

The fine structure of the epidermis of two species of salmonid fish, the Atlantic salmon (Salmo salar L.). and the brown trout (Salmo trutta L.)

The fine structure of epidermal mucous cells of two species of salmonid fish has been described. Mucous cells are, next to filament-containing cells, the most commonly encountered cells in fish epidermis. The development of the cells as they progress to the periphery has been characterised. They are initially difficult to distinguish from filament-containing cells: later, they can be recognised by the presence of much smooth-surfaced E.R. The mucigenesis and the subsequent secretion of mucus has been observed and it is essentially comparable to that which occurs in the mucous cells of the mammalian intestine. The mucous layer of the epidermal surface seems to mainly comprise of the products of these mucous cells and the "cuticle" seen in other species has not yet been observed in the salmonid species investigated here.

Animals