Partial nucleotide sequences, and routine typing by polymerase chain reaction-restriction fragment length polymorphism, of the brown trout (Salmo trutta) lactate dehydrogenase, LDH-C1*90 and *100 alleles

The cDNA nucleotide sequences of the lactate dehydrogenase alleles LDH-C1*90 and *100 of brown trout (Salmo trutta) were found to differ at position 308 where an A is present in the *100 allele but a G is present in the *90 allele. This base substitution results in an amino acid change from aspartic acid at position 82 in the LDH-C1 100 allozyme to a glycine in the 90 allozyme. Since aspartic acid has a net negative charge whilst glycine is uncharged, this is consistent with the electrophoretic observation that the LDH-C1 100 allozyme has a more anodal mobility relative to the LDH-C1 90 allozyme. Based on alignment of the cDNA sequence with the mouse genomic sequence, a local primer set was designed, incorporating the variable position, and was found to give very good amplification with brown trout genomic DNA. Sequencing of this fragment confirmed the difference in both homozygous and heterozygous individuals. Digestion of the polymerase chain reaction products with BslI, a restriction enzyme specific for the site difference, gave one, two and three fragments for the two homozygotes and the heterozygote, respectively, following electrophoretic separation. This provides a DNA-based means of routine screening of the highly informative LDH-C1* polymorphism in brown trout population genetic studies. Primer sets presented could be used to sequence cDNA of other LDH* genes of brown trout and other species.     

Histopathological alterations and parasite infection in fish: indicators of multiple stress factors

Within the scope of the multidisciplinaryresearch project Valimar (1995–1999), thepresent study emphasizes the use ofhistopathological investigations to evaluatethe effects of the different chemical impactof two small streams on the general health andcondition of the two sentinel fish species,brown trout (Salmo trutta f.fario) and loach (Barbatula barbatula).Parasitological investigations were alsoincluded to assess a possible relationshipbetween toxicant exposure, parasitic load, andthe occurence of histopathological organchanges. According to the multitiered approachof the Valimar project, fish from the field,individuals exposed to stream water undersemi-field conditions, and fish fromlaboratory experiments were investigated.Under semi-field conditions, thehistopathological responses in brown troutproved to reflect the different levels ofcontamination of the two small streams moreclearly than those detected in loach. Whencaptured from the field, both fish speciesrevealed, in most cases, a higher degree ofhistopathological alterations than thoseindividuals exposed at the respective siteunder semi-field conditions. Simulation of thetoxic load of the more polluted stream in alaboratory experiment did not result in thesame kind of tissue lesions in brown trout asin the field and semi-field investigations.Whereas results from the laboratoryexperiments could be specified astoxicant-induced, the histopathologicalalterations in fish from both semi-field andfield studies represent stress responses whichintegrate the effects of various abiotic andbiotic stress factors including both toxicantsand parasitic diseases.     

Chromosome evolution in the Salmonidae (Pisces): an update

The karyotypes of salmonid fishes including taxa in the three subfamilies Coregoninae, Thymallinae and Salmoninae are described. This review is an update of the (Hartley, 1987) review of the chromosomes of salmonid fishes. As described in the previous review, the karyotypes of salmonid fishes fall into two main categories based on chromosome numbers: the type A karyotypes have diploid numbers close to 80 with approximately 100 chromosome arms (2n = 80, NF = 100), and the type B karyotypes have diploid numbers close to 60 with approximately 100 chromosome arms (2n = 60, NF = 100). In this paper we have proposed additional sub categories based on variation in the number of chromosome arms: the A' type with NF = 110-120, the A" type with NF greater than 140, and the B' type with NF less than 80. Two modes of chromosome evolution are found in the salmonids: in the Coregoninae and the Salmoninae the chromosomes have evolved by centric fusions of the Robertsonian type decreasing chromosome numbers (2n) while retaining chromosome arm numbers (NF) close to that found in the hypothetical tetraploid ancestor so that most extant taxa have either type A or type B karyotypes. In the Thymallinae, the chromosomes have evolved by inversions so that chromosome arm numbers (NF) have increased but chromosome numbers (2n) close to the karyotype of the hypothetical tetraploid ancestor have been retained and all taxa have type A' karyotypes. Most of the taxa with type B karyotypes in the Coregoninae and Salmoninae are members of the genus Oncorhynchus, although at least one example of type B karyotypes is found in all of the other genera. These taxa either have an anadromous life history or are found in specialized lacustrine environments. Selection for increases or decreases in genetic recombination as proposed by Qumsiyeh, 1994 could have been involved in the evolution of chromosome number in salmonid fishes.     

Distribution and biology of Galaxias truttaceus (Galaxiidae) in south-western Australia, including first evidence of parasitism of fishes in Western Australia by Ligula intestinalis (Cestoda)

The freshwater trout minnow, Galaxias truttaceus, is restricted to the small catchments of the Goodga and Kalgan Rivers in Western Australia. Its large geographic separation from populations in south-eastern Australia, and subsequent reproductive isolation and variation in the prevailing environmental conditions, has created marked differences in biology (and morphology) between the eastern and western populations of G. truttaceus. The biology (spawning period, longevity, growth rates, diet and parasitism) of G. truttaceus in the Goodga River is described and then compared with information on the biology of diadromous and landlocked populations in south-eastern Australia (i.e. Tasmania) (see Humphries 1989). In the Goodga River, ca. 34 and 8% of males and females, respectively, attain maturity at the end of their first year, while only four mature males and one mature female 0+ fish were found in the Tasmanian populations. Adults migrate upstream prior to spawning which peaks during April and May. Larvae, which hatch at ca. 6.5mm (cf. 7.5–9.0mm in Tasmania), move downstream into Moates Lake for a few months before re-entering the river. Of the 810 G. truttaceus collected, ca. 53, 34, 10, 2, 1, 0.2 and 0.1% belonged to the 0+, 1+, 2+, 3+, 4+, 5+ and 7+ age classes, respectively. In contrast, the Tasmanian populations have a much higher proportion of older fish. At the end of their first, second and third years, the males on average attain 60, 84 and 95mm total length (TL), respectively, whereas females attain 63, 89 and 103mm TL, respectively at those ages. Only one fish > 140mm TL was captured, which contrasts with the Tasmanian fish, where a substantial proportion are > 140mm TL. The diet of fish > 40mm TL consisted of between 65 and 96% terrestrial fauna (mainly coleopterans and hymenopterans) in the different seasons. Larval fish diets were largely comprised of copepods.The occurrence of the introduced cestode Ligula intestinalis in ca. 7% of G. truttaceus represents the first record of this parasite in Western Australia. It was found to cause gonadal retardation and gross morphological deformities, the latter of which possibly increases the risk of avian predation.     

Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout

Onehundred and forty-eight out of onehundred and fifty strains of Vibrio anguillarum isolated from vibriosis in Danish marine aquaculture produced bacterial communication signals, acylated homoserine lactones, eliciting a response in the Agrobacterium tumefaciens (pZLR4) monitoring system. One strain, a serotype O4, induced a strong response in the Chromobacterium violaceum (CV026) monitoring system. Profiles of AHLs determined by TLC separation revealed the presence of at least four AHLs and a compound similar to N-3-oxo-decanoyl homoserine lactone (3-oxo-C10-HSL) was present in all strains. The production rate of the presumed 3-oxo-C10-HSL followed the growth rate of V. anguillarum whereas the production rate of a small AHL (Rf value of 0.74) increased faster than the growth rate of V. anguillarum indicating autoinduction. AHLs were produced by all serotypes (O1 to O10) and by non-typable strains. During infection with V. anguillarum, AHLs could be extracted from liver, kidney and muscle of rainbow trout and AHLs were detected both in vitro and in vivo when cell numbers reached 10(7) per ml or gram. Preliminary investigations of interactions between AHLs and the fish immune system were carried out determining oxidative burst of fish macrophages exposed to 3-oxo-C10-HSL. No activation or suppression of the superoxide anion production in the head kidney macrophages was seen when treated with the AHL compound in concentrations of 1 nM-10 microM. Our data show that AHLs are produced by almost all V. anguillarum strains and that no clear pattern relating AHL production to disease or virulence appear.     

The functional characterisation of CK-1, a putative CC chemokine from rainbow trout (Oncorhynchus mykiss)

Recently a number of cytokine homologs have been cloned in teleost fish, including several that resemble chemokines, but to date few have been confirmed using functional assays. Chemokines are a family of cytokines that are able to induce chemotaxis in leucocytes. In this study CK-1, a rainbow trout chemokine, was functionally characterised. Recombinant CK-1 is able to attract rainbow trout peripheral blood leucocytes (PBL) in a micro-chemotaxis chamber. A greater number of PBLs migrated in response to CK-1 than to negative controls, either media alone or equivalent concentrations of beta2M, while comparable numbers migrated to the positive control, recombinant human C5a. The tissue distribution of CK-1 mRNA was also assessed by Northern blotting of RT-PCR and showed that expression is constitutive in the liver and gut, and is inducible by intraperitoneal injection of phytohemagglutinin in PBL and the head-kidney. Continuous cell lines generated from the gut and pituitary gland of the rainbow trout also express CK-1 message, whilst Southern analysis shows that CK-1 is a single copy gene. Finally, CK-1 shows the greatest amino acid similarity CCL20/LARC/Mip-3alpha as well as similar gene structure and expression pattern.     

A genetic analysis of the London strain of rainbow trout

The London strain of rainbow trout (Oncorhynchus mykiss) was created by interbreeding three other strains of rainbow trout and therefore was expected to have higher levels of genetic variation than other strains of rainbow trout. We examined 129 London strain rainbow trout from Indiana by allozyme electrophoresis to assess levels of genetic variation and to examine the relationship between the London strain and other hatchery strains. When using the same loci to compare with other hatchery strains the London strain showed levels of genetic variation within the range of other hatchery strains: mean heterozygosity of 0.053 (0.031-0.099), 1.27 (1.20-1.60) alleles per locus and 20.0% (20.0-40.0%) of the loci were polymorphic. The London strain is somewhat distinct from other hatchery strains (D=0.009-0.072), in part because of the high frequency of the sIDHP*40 allele.     

Construction and characterization of BAC libraries for three fish species; rainbow trout, carp and tilapia

Bacterial artificial chromosome (BAC) libraries are important tools for genomic research. We have constructed seven genomic BAC libraries from three fish species, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio) and tilapia (Oreochromis niloticus). The two rainbow trout BAC libraries have average insert sizes of 58 and 110 kb. The average size of inserts in the carp BAC library is 160 kb. The average insert sizes of the four tilapia BAC libraries are 65, 105, 145 and 194 kb, respectively. These libraries represent good coverage of each genome (2-64 x coverage). The libraries can be screened by conventional colony hybridization and provide a starting point for the construction of high-density filtres or polymerase chain reaction (PCR) screening approaches. These BAC libraries will facilitate the positional cloning of quantitative trait loci (QTLs) for a variety of economically important traits in these species.