Morphology of Diplomonad Flagellates: Spironucleus torosa N. Sp. from Atlantic Cod Gadus morhua L., and Haddock Melanogrammus aeglefinus (L.) and Hexamita salmonis Moore from Brook Trout Salvelinus fontinalis (Mitchill)

ABSTRACT. A diplomonad flagellate, Spironucleus torosa n. sp. is described from Atlantic Cod Gadus morhua and haddock Melanogrammus aeglefinus . This is believed to be the 1st confirmed report of Spironucleus from a marine fish. Organisms swimming in the rectal lumen were broadly pyriform to elongate, and measured 10.5–18.6 μm long and 3.2–13.3 μm wide; other elongate organisms were attached to the rectal epithelium, via apical extensions appearing continuous with the microvilli. The posterior end of the body was extended into a caudal projection, on either side of which was a posteriolateral ring-shaped protrusion or torus, with a recurrent flagellum emerging from its centre. A symmetrical system of microtubules and lamellae, forming a "V" in protargol impregnated specimens, supported the flanges of the body surrounding the tori, the tori themselves and the caudal projection. Supranuclear microtubules were an inverted V to U shape in transverse section, and an electron dense band accompanied the cytostomes. Lightly staining homogenous cytoplasm was usually present in the anterior part of the body, the remainder being highly vacuolated with numerous dark granules. In swimming organisms, rough endoplasmic reticulum (RER) was present around the nuclei and cytostomes, and bacteria were occasionally seen in the cytoplasm. In "attached" organisms, RER was reduced, and bacteria were absent. Hexamita salmonis Moore from Salvelinus fontinalis was studied by light and scanning electron microscopy for comparison; its cytoplasm was not highly vacuolated. The two recurrent flagella emerged close together from the blunt posterior end of the body.     

Gape morphology of cod Gadus morhua L., haddock Melanogrammus aeglefinus (L.) and whiting Merlangius merlangus (L.) through metamorphosis from larvae to juveniles in the western Irish Sea

Variations in standard length ( L _S), gape size ( S _G) and jaw length ( L _J) were studied in larval and juvenile gadoids (cod Gadus morhua , haddock Melanogrammus aeglefinus and whiting Merlangius merlangus ) from 4 to 70 mm. The increase in S _G and L _J was not linear with respect to L _S. The relationship was best described by segmented regression lines in all three species, with an inflection point at c . 10·5 mm. The S _G and L _J increased more rapidly in relation to larval L _S for individuals smaller than this inflection point size. The rates of increase slowed significantly post-inflection, an effect more noticeable in S _G data compared to L _J data. In each case, the inflection point fell in the intermediate period of development between the larval and juvenile stages, which could be considered as metamorphosis. Published equations that have been used to predict S _G from L _J lead to the overestimation of gape. New relationships are presented, which may be used to predict S _G from measurements of either L _S or upper jaw length in cod, haddock and whiting.     

Species‐specific TaqMan probes for simultaneous identification of (Gadus morhua L.), haddock (Melanogrammus aeglefinus L.) and whiting (Merlangius merlangus L.).

We report the development of a multiplex, single tube (TaqMan) PCR assay for the identification of three commercially important gadoid species, the cod (Gadus morhua L.), the haddock (Melanogrammus aeglefinus L.) and the whiting (Merlangius merlangus L.). The assay unambiguously identifies known adult tissue samples, and ethanol‐preserved eggs from all three species with greater than 98% accuracy, providing a powerful tool for the development of catch independent stock assessment methods, mapping of spawning sites and the detection of commercial fraud in the fishing and food production industries.     

The carbohydrate moiety of serum IgM from Atlantic cod (Gadus morhua L.)

The carbohydrate moiety of cod serum IgM was analysed using oligosaccharide sequencing techniques. The carbohydrate moiety constituted about 10% of the molecular weight of cod IgM, was associated with the constant region of the heavy chains (Fc), and was composed of N-linked complex type oligosaccharides. Considerable heterogeneity was observed. Sixteen different glycan structures were identified, over 60% were sialylated and 40% contained core fucose. The carbohydrate moiety of cod IgM was shown to provide protection against protease digestion, and partial deglycosylation abolished the antigen binding property of natural cod anti-TNP-BSA antibody.     

Isolation and identification of Viral Haemorrhagic Septicaemia (VHS) viruses from cod Gadus morhua with the ulcus syndrome and from haddock Melanogrammus aeglefinus having skin haemorrhages in the North Sea

During fish disease surveys for marine rhabdoviruses in 1993 and 1995, the cod ulcus syndrome was seen widely in all ages of cod, especially the 2 to 5+ year classes. Viral Haemorrhagic Septicaemia Virus (VHSV) was isolated from a small proportion of the lesion-positive fish and these isolates were identified by immunofluorescence or ELISA. A serendipitous observation of dermal petechiae on haddock was made. VHSV was isolated from this lesion for the first time indicating a new host species for VHSV.     

Cathepsin D from Atlantic cod (Gadus morhua L.) liver. Isolation and comparative studies

The isolated cathepsin D-like enzyme from Atlantic cod (Gadus morhua L.) liver was shown to be a monomer with a molecular mass of approximately 40 kDa. It was inhibited by Pepstatin A and had an optimum for degradation of haemoglobin at pH 3.0. The purified enzyme had lower temperature stability than bovine cathepsin D. Antibodies raised against the purified enzyme and against two C-terminal peptides of cod cathepsin D recognized a 40 kDa protein in immunoblotting of the samples from the purification process. Both antisera showed cross reactivity with a similar sized protein in liver from cod, saithe (Pollachius virens L.), Atlantic herring (Clupea harengus L.) and Atlantic salmon (Salmo salar L.). A protein of same size was detected in wolffish (Anarhichas lupus L.) liver with the antibody directed against the purified enzyme. This antibody also recognized the native enzyme and detected the presence of cathepsin D in muscle of cod, saithe, herring and salmon. These antibodies may be useful in understanding the mechanisms of post mortem muscle degradation in fish by comparing immunohistochemical localization and enzyme activity, in particular in cod with different rate of muscle degradation. They may also be used for comparing muscle degradation in different fish species.     

Differences in the susceptibility of brown trout, Salmo trutta L., and American brook trout, Salvelinus fontinalis (Mitchill), to infestation by the peritrich ciliate, Scyphidia sp.

A hitherto undescribed species of Scyphidia has been shown to infest the skin of brown trout. However, it is incapable of colonizing the skin of American brook trout, even when these fish are co-cultivated with heavily infested brown trout. Possible mechanisms accounting for this host/parasite specificity are discussed.     

Respiratory burst activity of Atlantic cod (Gadus morhua L.) blood phagocytes differs markedly from that of rainbow trout

In the present study we investigated the respiratory burst (RB) activity of Atlantic cod (Gadus morhua L.) blood phagocytes and we evaluated how the RB activity of cod blood cells differ from that of trout. The RB activities were measured directly from highly diluted whole blood as luminol-amplified chemiluminescence (CL) under various conditions. Studies regarding the blood dilutions for cod whole blood chemiluminescence measurements (WBCL) revealed that at a final blood dilution of 1.5 microl ml(-1) or less the CL response was strictly proportional to the number of phagocytes. This range of blood dilution did not markedly differ from that of trout. However, the opsonisation capacity of cod plasma was markedly poorer. The RB activity of phagocytes was most active at 15 degrees C when heterologous cod serum was used as a source of opsonin, whereas at final blood dilution of 8.0 microl ml(-1) (when homologous cod plasma was at a higher concentration) the highest RB activity was observed at 10 degrees C. Aeromonas salmonicida strain MT004 (As MT004) induced higher RB activity than the two known pathogens for cod, atypical A. salmonicida and Vibrio anguillarum. Cod blood phagocytes were more responsive to plastic surfaces and the adhesion response of phagocytes was partly inhibited but did not totally vanish even at a final gelatin concentration of 0.4%. Moreover, cod serum enhanced the adherence of phagocytes and cod blood phagocytes also showed slow spontaneous degranulation. Finally, within the tested anticoagulants (heparin, Na-citrate, EDTA) heparin treated blood phagocytes generated the highest RB activity.     

Twenty-three novel microsatellite markers developed from Atlantic cod Gadus morhua L. expressed sequence tags

Twenty-three novel polymorphic microsatellite markers were developed from c. 2000 expressed sequence tags of Atlantic cod Gadus morhua L. Gene identity was determined at 12 loci, confirming the associated microsatellites as type I markers. These microsatellite markers provide useful tools for studies of population genetics and reproductive ecology and for constructing linkage maps of G. morhua .     

Site fidelity of Atlantic cod Gadus morhua L. as deduced from telemetry and stable isotope studies

A study was performed to test the hypothesis that Atlantic cod Gadus morhua captured within fjords do not migrate across the sill into outer fjord segments or coastal waters. An 18 month long telemetry experiment, corroborated by recapture patterns, showed that 40% of the tagged fish crossed the sill. The majority of the fish that crossed the sill, however, returned after short excursions of only a few days. Many fish remained stationary at or near the release location, and migrations up the fjord into brackish water appeared more frequent than migration out of the fjord. The stable isotope analyses showed significant differences between the inner and the outer fjord segments, and this was seen for both adults and juveniles. This suggests that the Atlantic cod in the two areas have different diets and probably utilize different foraging ranges. Thus, both the isotope studies and the telemetry experiment suggest limited cross-sill migration and rather high site fidelity. Marketing restrictions only apply to fish caught in the fjord proper, not the comparatively clean outer fjord system beyond the sill. Based on this study of migratory tendencies, it is concluded that contamination levels in Atlantic cod in both the Frierfjord proper and the outer fjords probably result from local exposure and appear little affected by inter-fjord migration.     

Some observations upon the relationship of microhaematocrit values to haemoglobin concentrations and erythrocyte numbers in the carp Cyprinus carpio L. and brook trout Salvelinus fontinalis (Mitchill)*

Simultaneous determinations of microhaematocrit, erythrocyte numbers and haemoglobin concentrations have been obtained for samples of carp and trout of both sexes acclimated to various temperature regimes, and varying seasonal and nutritional states. Because haematological parameters have been used as indices of physiological condition in fishes correlational and multivariant regression analyses were carried out in an effort to determine whether valid correlations existed between the readily-determined parameter, microhaematocrit, and the other 2 variables. Results obtained indicate that microhaematocrit can be used as a general index of haematological status in the carp but not the brook trout.     

Eight new microsatellite markers in Atlantic cod (Gadus morhua L.) derived from an enriched genomic library

One hundred and fifty random clones from an enriched genomic library of Atlantic cod were sequenced. Primer pairs were designed for 15 microsatellites containing perfect di‐, tri‐, tetra‐ and hexanucleotide repeats and characterized in 96 unrelated fish. Eight markers were successfully amplified, with the number of alleles ranging from two to nine per locus and observed heterozygosity ranging from 0.341 to 0.977. Loci Gmo‐G13 and Gmo‐G14 had a significant excess of homozygotes. All loci conformed to the Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.     

Clostridium perfringens toxin types from wild-caught Atlantic cod (Gadus morhua L.), determined by PCR and ELISA

Ninety-five fecal samples from Atlantic cod (Gadus morhua L.), caught along the northern Norwegian coast, were examined bacteriologically for occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types and for genes encoding enterotoxin and the novel beta2 toxin for further subclassification. In addition, a commercial enzyme-linked immunosorbent assay (ELISA) kit for detection of C. perfringens alpha, beta, and epsilon toxin was used. Clostridium perfringens could be isolated in 37 fecal samples (38.9%) from cod. All isolates were C. perfringens toxin type A (alpha toxin positive) as determined by PCR and also ELISA. In addition, in isolates from two cod (2.1%) the gene encoding for beta2 toxin was found (A, beta2) by PCR. Genes encoding for beta, epsilon, and iota toxins and enterotoxin were not found. This is the first detection of C. perfringens alpha and beta2 toxin in cod and of beta2 toxin in fish in general. The origin of this bacterium in cod is discussed.     

Isolation and characterization of complement component C3 from Atlantic cod (Gadus morhua L.) and Atlantic halibut (Hippoglossus hippoglossus L.)

Complement component C3 was isolated from the plasma of cod (Gadus morhua L.) and halibut (Hippoglossus hippoglossus L.). Fast protein liquid chromatography (FPLC) techniques, involving ion exchange and gel filtration columns, were used. The purified proteins were analysed by SDS-PAGE which showed a two-chain structure, alpha- and beta-chains, as seen in higher vertebrates. Both proteins had intra-chain thioesters located within their alpha-chains and N-terminal amino acid sequencing confirmed their identity with reference to known C3 amino acid sequences from other species. Specific antibodies were prepared against cod and halibut C3 and tested in Western blotting on sera and purified C3. The proteolytic fragmentation of C3 was tested with trypsin, pepsin, papain and the extracellular product (ECP) from the bacterium Aeromonas salmonicida ssp. achromogenes (Asa). Both trypsin and papain were successful in cleaving C3 whereas pepsin and ECP had no effect. Carbohydrate moieties were detected in the alpha- and beta-chains of cod and halibut C3 and N-linked oligosaccharides were removed from the C3 with PNGase treatment, revealing a difference in C3 glycosylation between the two species.     

Ontogeny of feeding and respiration in larval Atlantic cod Gadus morhua (Teleostei,Gadiformes): I. Morphology

Cranial development in larval Atlantic cod Gadus morhua was studied throughout ontogeny using specimens treated by staining and clearing, scanning electron microscopy and histology. Newly hatched cod larvae have closed mouths, no operculii, five well-developed branchial arches, and transversii ventralis muscles. During the endogenous feeding (yolk-sac) stage, viscerocranial structures remain simple and nonarticulated. Six days after hatching at 5°C, articulation occurs between the quadrate/Meckel's cartilage and the hyomandibula/cranium. Integration of skeletal elements results in a functional jaw that facilitates the transition from endogenous to exogenous feeding. During later ontogenetic stages, the opercular apparatus and levator-operculi coupling develops, facilitating the transition of cutaneous to branchial respiration. Overall, feeding and respiratory needs are met by changes in form (including composition) and function during larval fish growth and are correlated with demands of energy acquisition essential to survival. © 1996 Wiley-Liss, Inc.     

Basic physico-chemical parameters of milt from sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss)

The aim of the study was to compare the physico‐chemical parameters of milt from sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss). Milt was collected by stripping and spermatozoa concentrations, were determined and compared with sperm motility and spermatocrit values along with seminal plasma indices (pH, osmolality, sodium, potassium, chlorine, calcium, magnesium, glucose and protein concentrations). The highest spermatozoa concentration of 22.3 ± 6.7 × 10⁹ ml^?1 was found in the sea trout milt, and was significantly different of those observed in brook trout (11.9 ± 3.3 × 10⁹ ml^?1) and rainbow trout (10.7 ± 4.4 × 10⁹ ml^?1). The values for pH and K⁺ did not differ significantly among species. The mean pH was 8.0 in the milt of each species and the K⁺ concentrations ranged from 24.8 ± 7.2 to 30.5 ± 7.6 mm L^?1. Considerable differences were determined for the Ca²⁺ ions concentrations. The highest value was found in sea trout (1.7 ± 0.3 mm L^?1), while in the rainbow trout it was 0.7 ± 0.5 and in the brook trout 0.4 ± 0.1 mm L^?1. The most pronounced differences were found in the glucose concentration cause of its unnaturally low concentration in rainbow trout of the mean value of 6.0 ± 15.2 mg L^?1. The mean value in sea trout and brook trout was 185.0 ± 172.4 and 231.2 ± 148.4 mg L^?1 respectively. For all species, protein mean values were below 1.3 g L^?1. The mean osmolality was between 230.6 ± 98.6 and 272.0 ± 26.4 mOsm kg^?1 in the species studied. No correlation was found between any components determined in milt and the spermatozoa motility (P > 0.05). The sperm concentration was positively correlated with the protein content in the milt of the three species studied, other less exhibited correlation was found.     

Effect of low and high temperatures on chymotrypsin from Atlantic cod (Gadus morhua L.); comparison with bovine alpha-chymotrypsin

1. Cod chymotrypsin displays higher enzyme activity compared to bovine alpha-chymotrypsin when assayed at low temperatures (3-15 degrees C). 2. Both enzymes are inactivated when incubated at temperatures between 60 and 70 degrees C. 3. When incubated at 99 degrees C the cod enzyme retains about 50% of the initial activity measured at room temperature. 4. Preincubation at boiling temperature renders the cod chymotrypsin active at 70 degrees C whereas the bovine enzyme is rapidly inactivated.     

Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin.

1. Two chymotrypsins with isoelectric points pI 6.2 and 5.8 were purified from the pyloric caeca of Atlantic cod using a phenyl-Sepharose column and chromatofocusing chromatography. The apparent molecular weight was 26,000 as judged by SDS-polyacrylamide gel electrophoresis and gel filtration. 2. The cod enzymes differed from bovine chymotrypsin in having a slightly higher molecular weight and more acidic pI points. N-terminal amino acid sequence analysis of cod chymotrypsin B showed considerable similarity with bovine chymotrypsin. 3. Heat stability and stability towards acidic pH were reduced in the cod enzymes. Generally, the cod and bovine chymotrypsins responded similarly to various protease inhibitors. However, the cod chymotrypsins were less sensitive to aprotinin inhibition but more sensitive towards soybean trypsin inhibitor and cysteine. 4. Kinetic properties were examined and the cod enzymes found to be more active towards both ester (N-benzoyl-tyrosine ethyl ester) and amide (N-benzoyl-tyrosine-p-nitroanilide) substrates. The observed differences in kinetic properties are indicative of an adaptive response towards the low temperature environment in which the cod lives.     

Use of hydrolysates from Atlantic cod (Gadus morhua L.) viscera as a complex nitrogen source for lactic acid bacteria

Hydrolysates of cod viscera were tested as an alternative to commonly used complex nitrogen sources (peptones and/or extracts) for the type strains of the lactic acid bacteria Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus sakei and Pediococcus pentosaceus. Comparative studies with MRS-like media containing different nitrogen sources showed that all the fish hydrolysates performed equally well or better than commercial extracts/peptones for all selected lactic acid bacteria.