Type and ultrastructure of Didymocystis wedli and Koellikerioides intestinalis (Digenea,Didymozoidae) cysts in captive Atlantic bluefin tuna (Thunnus thynnus Linnaeus, 1758)

Tissue encapsulation, one of the most common tissue reactions to invading parasites, is the hallmark sign of didymozoid (Digenea, Didymozoidae) infections in fish. Investigated were the types of intermediate filaments and ultrastructure of the connective tissue capsule elicited by the presence of didymozoids in the gills and intestine of Atlantic bluefin tuna (Thunnus thynnus Linnaeus, 1758). The evaluation was done performing TEM microscopy of two tissue‐embedded didymozoid species, along with monoclonal antibodies labeling (anti‐fish collagen type I, anti‐human cytokeratin, anti‐vimentin antibodies). Ultrastructure of Didymocystis wedli (Ariola, 1902) (prevalence = 61.75%, abundance = 28.91) encapsulated in gill filaments and Koellikerioides intestinalis ( Yamaguti, 1970 ) (prevalence = 54.65%, abundance = 10.96) in the intestinal submucosa showed that the thin parasitic hindbody tegumentum was directly embedded in layers of connective tissue bands. Only a few cellular elements (lymphocytes, fibroblasts and fibrocytes) infiltrated the connective tissue capsule, which differed between the two didymozoid species in thickness, not in the type of filaments expressed. Cysts showed positive reaction to extracellular collagen as well as appearing positive for the cytoskeletal intermediate filaments vimentin and cytokeratin.     

The distribution of connective tissue content in swimming muscle of cod (Gadus morbua)

The amount and distribution of connective tissue in the swimming muscle of cod (Gadus morhua) have been determined. By biochemical analysis, the collagen was found to make up 1,5 % of the total protein content. Measured by light microscopical morphometric analysis, the areal fraction of the connective tissue elements was found to be 3,0 % of the total muscle area. The areal fraction of myocommatal connective tissue mentioned above was found to be 2.3 %. The thickness of the endomysial sheath was calculated by morphometry based on electron microscopy, and was found to be 0.30 and 0.16 μ for red and white fibres, respectively. The areal fraction of the endomysial sheaths was 2.3 % in red muscle and 0.5% in white muscle. The endomysial sheaths make up 25% of the total connective tissue in the swimming muscles. These sheaths influence the binding properties of fish muscle products.     

Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle

Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.     

Disposition of arsenobetaine in two marine fish species following administration of a single oral dose of [14C]arsenobetaine

The distribution and excretion of arsenobetaine in fish were investigated using whole body autoradiography and liquid scintillation counting. A single dose of synthesised [(14)C]arsenobetaine was orally administered to Atlantic salmon, Salmo salar L., and Atlantic cod, Gadus morhua L. Arsenobetaine was distributed to most organs within both species. Nevertheless, there were species differences in tissue distribution and excretory pattern. The highest level of arsenobetaine in Atlantic salmon was present in muscle tissue, while high levels of arsenobetaine were found in both muscle and liver (including gall bladder) from Atlantic cod. The results suggest that the major route of excretion was via urine, which seemed to be more important in Atlantic cod than in Atlantic salmon. Elimination of arsenobetaine via bile appeared to be negligible in both species.     

Sulfated glycosaminoglycans in the extracellular matrix of muscle tissue in Atlantic cod (Gadus morhua) and Spotted wolffish (Anarhichas minor)

Two species of commercially important cold water fish were investigated for content of sulfated glycosaminoglycans (GAGs) in muscle tissue by use of in vivo 35S-sulfate labeling combined with different digestions (papain, chondroitinase ABC, keratanase and nitrous acid treatment), DEAE chromatography, SDS-PAGE and histology techniques. The species investigated in this study have different gaping properties. The non-gaping species, spotted wolffish (Anarhichas minor), contained 3-4 times more 35S-sulfated anionic components than the gaping species, Atlantic cod (Gadus morhua). The higher level of sulfation in wolffish was supported by light microscopy studies using Alcian blue staining with different concentrations of MgCl2 as critical electrolyte. Furthermore, the muscular connective tissue in the non-gaping species was dominated by chondroitin sulfate (CS)/dermatan sulfate (DS), whereas the gaping species was more dominated by heparan sulfate (HS). Moreover, structural differences were observed in the junctions between the myofibers, which were more pronounced in the wolffish. The histological studies revealed that the basement membrane area was rich in acidic mucopolysaccharides in both species.     

An immunological study of glycosaminoglycans in the connective tissue of bovine and cod skeletal muscle

The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl₂ (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.     

Immunohistochemical study of types I, III and IV collagen in diseased human gingiva of patients with rapidly progressive periodontitis: a light and electron microscopic study

The distribution of type I, III and IV collagens and their ultrastructural organization have been studied in diseased gingival connective tissue of patients with rapidly progressive periodontitis. This disease is characterized by acute destruction of the gingival collagenous components. The use of an immunofluorescent procedure has shown that the diseased connective tissue was made up of both type I and III collagens but that type III collagen was less resistant to acute inflammation. Ultrastructural immunolabelling, using the peroxidase procedure has shown that the large, dense bundles of type I collagen of PI, the main pattern of organization of the gingival connective tissue offered a better resistance to acute destruction than PII, a loose pattern of organization mainly composed of type III collagen. Type IV collagen was exclusively located in degraded lamina densa of basement membrane.     

Immunohistochemical analysis of the distribution of type VI collagen in the lingual mucosa of rats during the morphogenesis of filiform papillae

Iwasaki, S., Yoshizawa, H. and Aoyagi, H. 2012. Immunohistochemical analysis of the distribution of type VI collagen in the lingual mucosa of rats during the morphogenesis of filiform papillae. —Acta Zoologica (Stockholm) 93 : 80–87. We examined the distribution after immunostaining of immunofluorescence of type VI collagen, differential interference contrast (DIC) images, and images obtained using confocal laser‐scanning microscopy in transmission mode, after toluidine blue staining, during morphogenesis of the filiform papillae, keratinization of the lingual epithelium and myogenesis in the rat tongue on semi‐ultrathin sections of epoxy resin‐embedded samples. Immunoreactivity specific for type VI collagen was dispersed over a relatively wide range of connective tissue in the mesenchyme of fetuses on day 15 after conception (E15), at which time the lingual epithelium was composed of one or two layers of cuboidal cells and the lingual muscle was barely recognizable. Slight immunoreactivity specific for type VI collagen was scattered within the lamina propria in fetuses on E17 and on E19, and immunoreactivity was relatively distinct on the connective tissue around the lingual muscle during myogenesis. In fetuses on E19, the epithelium was already stratified squamous. At postnatal stages from P0 to P14, keratinization of the lingual epithelium advanced gradually as morphogenesis of the filiform papillae proceeded during postnatal development. In newborns on P0, myogenesis of the tongue was almost completed. The intensity of immunoreactivity specific for type VI collagen at postnatal stages was mainly restricted on the endomysium and perimysium around the lingual muscle, while scant immunoreactivity was evident in the connective tissue in the lamina propria. Immunoreactivity around the fully mature lingual muscle on P7 and P14 was weaker than that on E19 and P0. Thus, type VI collagen appeared in the connective tissue that surrounded the lingual muscles such as the endomysium and perimysium, in parallel with changes in extracellular components during myogenesis of the tongue.     

Molecular characterisation and expression analysis of interferon gamma in Atlantic cod (Gadus morhua)

Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. In this study, the cDNA and genomic sequences of Atlantic cod IFN-γ were cloned and found to encode a putative protein containing 194 amino acids with a 24 amino acid signal peptide sequence. The gene is composed of four exons and three introns similar to IFN-γ genes of other vertebrates. The cod IFN-γ showed only 14–29% amino acid identity with other fish IFN-γ and 9–17% identity with IFN-γ from higher vertebrates. However, cod IFN-γ possesses the typical IFN-γ motifs in the C-terminal end of the protein and displays an alpha-helix structure similar to mammalian IFN-γ. The promoter region contains a putative ISRE element indicating up-regulation by type I IFNs and dsRNA. Real time RT-PCR analysis confirmed that IFN-γ gene expression was up-regulated in organs of cod injected with the dsRNA polyinosinic:polycytidylic acid (poly I:C), which is a strong inducer of type I IFNs. Injection of cod with formalin-killed Vibrio anguillarum also increased IFN-γ expression in head kidney, but to a much lesser extent than poly I:C. The gene expression results thus indicate a role for IFN-γ in innate immune response against both virus and bacteria in Atlantic cod.     

Tissue distribution, inhibition and activation of gelatinolytic activities in Atlantic cod (Gadus morhua)

Gelatinolytic activities in fish tissues with properties like matrix metalloproteinases (MMPs) have been paid little attention. However, they have been proposed to participate in post mortem degradation during storage and the disintegration of pericellular connective tissue during spawning. In this paper the distribution of gelatinolytic activities in liver, heart, muscle, gill, and male and female gonad of Atlantic cod (Gadus morhua) was studied by using gelatin SDS-PAGE, proteinase inhibitors, gelatin and lentil lectin Sepharose affinity chromatography. The amount of gelatin degrading enzymes varied from tissue to tissue. Most of the gelatin binding enzymes were found to be matrix metalloproteinases by adding galardin, a broad range MMP inhibitor, to the incubation buffer. A 72 kDa form of cod gelatin degrading enzyme had properties similar to human proMMP-2, as it could be activated by p-aminophenylmercuric acetate and trypsin. Like the human MMP-2 it did not bind to lentil lectin. An 83 kDa cod gelatin degrading enzyme had properties similar to the 92 kDa progelatinase B (proMMP-9). These properties were also similar to that of the 72 kDa form, except that the 83 kDa cod gelatinase was bound to lentil lectin, showing that it is a glycoprotein like MMP-9.     

Spatial distribution and orientation of dermatan sulfate in human medial collateral ligament

The proteoglycan decorin and its associated glycosaminoglycan (GAG), dermatan sulfate (DS), regulate collagen fibril formation, control fibril diameter, and have been suggested to contribute to the mechanical stability and material properties of connective tissues. The spatial distribution and orientation of DS within the tissue are relevant to these mechanical roles, but measurements of length and orientation from 2D transmission electron microscopy (TEM) are prone to errors from projection. The objectives of this study were to construct a 3D geometric model of DS GAGs and collagen fibrils, and to use the model to interpret TEM measurements of the spatial orientation and length of DS GAGs in the medial collateral ligament of the human knee. DS was distinguished from other sulfated GAGs by treating tissue with chondroitinase B, an enzyme that selectively degrades DS. An image processing pipeline was developed to analyze the TEM micrographs. The 3D model of collagen and GAGs quantified the projection error in the 2D TEM measurements. Model predictions of 3D GAG orientation were highly sensitive to the assumed GAG length distribution, with the baseline input distribution of 69+/-23 nm providing the best predictions of the angle measurements from TEM micrographs. The corresponding orientation distribution for DS GAGs was maximal at orientations orthogonal to the collagen fibrils, tapering to near zero with axial alignment. Sulfated GAGs that remained after chondroitinase B treatment were preferentially aligned along the collagen fibril. DS therefore appears more likely to bridge the interfibrillar gap than non-DS GAGs. In addition to providing quantitative data for DS GAG length and orientation in the human MCL, this study demonstrates how a 3D geometric model can be used to provide a priori information for interpretation of geometric measurements from 2D micrographs.     

Expression of the oligopeptide transporter, PepT1, in larval Atlantic cod (Gadus morhua)

The intestinal absorption of di- and tri-peptides generally occurs via the oligopeptide transporter, PepT1. This study evaluates the expression of PepT1 in larval Atlantic cod (Gadus morhua) during the three weeks following the onset of exogenous feeding. Larval Atlantic cod were fed either wild captured zooplankton or enriched rotifers. cDNA was prepared from whole cod larvae preceding first feeding and at 1000 each Tuesday and Thursday for the following three weeks. Spatial and temporal expression patterns of PepT1 mRNA were compared between fish consuming the two prey types using in situ hybridization and quantitative real-time PCR. Results indicated that PepT1 mRNA was expressed prior to the onset of exogenous feeding. In addition, PepT1 was expressed throughout the digestive system except the esophagus and sphincter regions. Expression slightly increased following first-feeding and continued to increase throughout the study for larvae feeding on both prey types. When comparing PepT1 expression in larvae larger than 0.15-mg dry mass with expression levels in larvae prior to feeding, no differences were detected for larvae fed rotifers, but the larvae fed zooplankton had significantly greater PepT1 expression at the larger size. In addition, PepT1 expression in the zooplankton fed larvae larger than 0.15-mg dry mass had significantly greater expression than rotifer fed larvae of a similar weight. Switching prey types did not affect PepT1 expression. These results indicate that Atlantic cod PepT1 expression was slightly different relative to dietary treatment during the three weeks following first-feeding. In addition, PepT1 may play an important role in the larval nutrition since it is widely expressed in the digestive tract.     

Distribution of connective tissue proteins in chick muscle spindles as revealed by monoclonal antibodies: a unique distribution of brachionectin/tenascin

The distribution of chick muscle spindles of eight connective tissue proteins (collagen types I, IV, V, and VI, laminin, heparan sulfate, fibronectin, and brachionectin/tenascin) was examined by immunofluorescent histochemistry. Intrafusal fibers were surrounded by layers of collagen type VI and fibronectin, and by an external lamina containing collagen type IV, laminin, and heparan sulfate. Most of these layers displayed a different pattern of staining at the sensory region of the equator than at the polar region. The crescent-like sheath that caps each intrafusal fiber and sensory terminal at the equator was strongly positive for collagen type I and weakly positive for collagen type V. The outer spindle capsule contained laminin, heparan sulfate, collagen types IV and VI, brachionectin/tenascin, fibronectin, and to a lesser degree also collagen types I and V. Brachionectin/tenascin had the narrowest distribution of any of the connective tissue macromolecules studied. It was found only in the outer capsule and in the coverings of blood vessels and nerves associated with the outer capsule.     

The effects of body mass and feeding on metabolic rate in small juvenile Atlantic cod

Apparent specific dynamic action (SDA) amplitude in young juvenile Atlantic cod Gadus morhua (1 to 8 g wet mass), fed a standardized meal and then exercised in a circular swimming respirometer at a constant swimming speed of 0·5 ± 0·3 body lengths s^-1, occurred within l h after feeding in all juveniles. SDA amplitude were 1·4 to 1·8 times higher in fed fish compared to unfed fish, and rates of oxygen consumption decreased as body mass increased. SDA duration had a tendency to decrease with increasing body mass and was shortest, at 6 h, in the smallest fish (1–1·5 g), but increased to 10–11 h in the largest fish. Apparent SDA in fed fish ( R _r) scaled with a mass exponent of 0·89, while maximum metabolic rate ( R _max) determined by chasing fish to exhaustion and then measuring oxygen consumption for 12 h, and unfed routine metabolic rate (R_r) scaled with a mass exponent of 0·79 and 0·76 respectively. Relative aerobic scope ( R _max– unfed R _r) did not appear to vary over the 1 to 8 g increase in wet mass. These results show that as body mass increased in young juvenile Atlantic cod: (1) apparent SDA ( R _f) increased more rapidly than R _max, and (2) apparent SDA took up >98% of the relative aerobic scope and that young Atlantic cod allocated most of the energy to growth, and left little for other metabolic activities.     

Age-Related Changes in Collagen and Glycosaminoglycans of Rat Gingiva1

Age-related changes in collagen and glycosaminoglycans (GAGs) of rat gingiva were studied biochemically and histologically. The components of isolated GAGs were identified as dermatan sulphate, hyaluronic acid, and heparan sulphate. In histological studies, hyaluronic acid was present in all regions of the gingiva, whereas dermatan sulphate and heparan sulphate were present only in gingival connective tissue. However, there was no significant difference in the relationship between aging, and the content or distribution of GAGs. On the other hand, histological findings showed that collagen fibers were markedly increased in number and the vascular composition was decreased with increasing age. In biochemical studies, the content of collagen, especially of insoluble collagen, was greatly increased with age, whereas collagen biosynthesis and collagenolytic activity were markedly decreased. In addition, lysyl oxidase activity was also significantly decreased with age. The results indicate that the rate of collagen turnover decreases and collagen fibers increase in stability in rat gingiva with increasing age. These observed age-related changes may affect the ability of gingiva to respond to local irritants.     

Detection of TIMP-2-like protein in Atlantic cod (Gadus morhua) muscle using two-dimensional real-time reverse zymography

Matrix metalloproteinases (MMPs) have been proposed to participate in postmortem degradation of fish muscle connective tissues during storage. In the extracellular matrix (ECM) of mammals, a group of specific tissue inhibitors of metalloproteinases (TIMPs) contributes in regulating the MMPs present. However, little information exists on the presence of TIMPs in fish. In this paper, the presence of TIMPs in the muscle of Atlantic cod (Gadus morhua) was investigated using gelatin affinity chromatography, real-time reverse zymography (RTRZ) and mass spectrometry (MS). Using RTRZ inhibitory action against cod muscle, proteinases binding to gelatin were detected in the muscle. The inhibitor had similar molecular weight (21 kDa) as a human recombinant TIMP-2 used as a reference sample. Because isoforms of TIMP-2 homologues with similar molecular weight have been suggested in fish, a two-dimensional RTRZ (2D RTRZ) method was designed. The new method showed the existence of only one form with inhibitory action against cod muscle proteinases. Finally, de novo sequencing of two peptides derived from the cod muscle inhibitor showed high homology to TIMP-2s both from human and other teleosts.