Seasonal variation of sound production in the Lusitanian toadfish Halobatrachus didactylus

Seasonal variation of sound production, which includes boatwhistles, grunts, croaks and double croaks, was studied in the Lusitanian toadfish Halobatrachus didactylus . Boatwhistles were emitted during the mating season in contrast with the other sound types, which were emitted all year round.     

Variability in the mating calls of the Lusitanian toadfish Halobatrachus didactylus: cues for potential individual recognition

The mating sounds (boatwhistles) of nesting batrachoidid Halobatrachus didactylus males were recorded in the Tagus Estuary from piers. Thirteen males with 16 boatwhistles per fish were analysed for 20 acoustic features. All variables showed larger between‐male than within‐male variation and differed significantly among individuals. Discriminant function analyses (DFA) considering seven of these variables assigned 90–100% of boatwhistles to the correct individual, depending on the number of males and number of sounds per male included in the model. The acoustic features that consistently best discriminated individuals were the dominant frequency of the middle tonal segment of the boatwhistle (P₂) and dominant frequency modulation, followed by P₂ pulse period, amplitude modulation and sound duration. These results suggest the possibility of individual recognition based on acoustic cues.     

Immunochemical studies of histone H5 from Halobatrachus didactylus.

1. Histone H5 from Halobatrachus didactylus was isolated by using perchloric acid (PCA) extraction of fish liver nuclei and trichloroacetic acid (TCA) precipitation. 2. A polyclonal antiserum was generated by immunizing rabbits with the antigen purified from SDS-PAGE. 3. By immunofluorescence the serum stains erythrocyte nuclei from H. didactylus but it does not react with mammalian cells. 4. By Western blotting, the anti-H5 antibody reacts with the isolated antigen at high titers. 5. Digestion of histone H5 with pepsin and cyanogen bromide suggests that the epitopes are located in the globular and C-terminal region of the H5 molecule excluding the N-terminal.     

Histochemical characterization of glycoconjugates in the epithelium of the extrapulmonary airways of several vertebrates

Summary The glycoconjugates of the extrapulmonary airways of 11 tetrapode vertebrates have been characterized by means of both conventional and lectin histochemistry. Abundant sialosulphomucins were detected in the secretory cells and periciliary layer of turtles, snakes, birds and mammals while only sialomucins were observed in amphibians. Neutral and traces of acidic mucins were detected in the secretory cells of lizards. The secretory cells of the amphibian airways were reactive to Con-A, DBA and WGA. No -l-fucose residues reactive with UEA-I or LTA were detected in amphibians. The goblet cells of the turtles were stained by DBA, SBA and WGA. Secretory cells of snakes and lizards reacted with Con-A and WGA. The mucous goblet cells of the birds were reactive to Con-A, LTA and WGA. In the chicken, they also showed affinity for PNA and SBA. The ciliated cells ofthe avian species studied were stained by Con-A and WGA. Mammalian goblet cells were reactive to Con-A, UEA-I and WGA. In the rat, affinity for DBA and SBA was also observed. The present results reveal the existence of marked differences in the sugar residues of the glycoconjugates of the extrapulmonary airways of tetrapode vertebrates. Only sialic acid residues appear to be constant constituents of the glycoconjugates of the airways of all species studied.     

Metazoan parasites as biological indicators of population structure of Halobatrachus didactylus on the Portuguese coast

A total of 366 Halobatrachus didactylus (Bloch & Schneider 1801) individuals were obtained between September and November 2000 from commercial fishing vessels at eight localities along the Portuguese coast and analysed for metazoan parasite infections. Three species of parasites were found: Hysterothylacium aduncum Rudolphi 1802; Progrillotia dasyatidis Beveridge et al. 2004, which constitutes a new host record for the Lusitanian toadfish; and Nerocila orbignyi Guérin‐Méneville 1829. Prevalence and mean abundance were determined for the total fish population in each sample. Significant differences in the number of parasitized fish among the samples analysed indicate differences in the H. didactylus populations along the Portuguese coast. The cluster analysis performed to prevalence data pointed out two major clusters: one composed of mainly estuarine samples and one that grouped samples from coastal areas; this is in agreement with the results obtained by morphological and genetic analysis performed for the same samples.     

Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and (TTAGGG)n by one-color and double-color FISH in the toadfish Halobatrachus didactylus (Teleostei: Batrachoididae)

The karyotype of Halobatrachus didactylus presents 46 chromosomes, composed of eight metacentric, 18 submetacentric, four subtelocentric, and 16 acrocentric chromosomes. The results of FISH showed that the major ribosomal genes were located in the terminal position of the short arm of a large submetacentric chromosome. They also showed a high variation in the hybridization signals. The products of amplification of 5S rDNA produced bands of about 420 pb. The PCR labeled products showed hybridization signals in the subcentromeric position of the long arm of a submetacentric chromosome of medium size. Double-color FISH indicated that the two ribosomal families are not co-located since they hybridizated in different chromosomal pairs. Telomeres of all the chromosomes hybridized with the (TTAGGG) _n probe. The GATA probe displayed a strong signal in the long arm of a submetacentric chromosome of medium size, in the subcentromeric position. The double-color FISH showed that the microsatellite GATA and the 5S rDNA gene are located in different chromosomal pairs. The majority presence of GATA probes in one pair of chromosomes is unusual and considering its distribution through different taxa it could be due to evolutionary mechanisms of heterochromatine accumulation, leading to the formation of differentiated sex chromosomes.     

Calculation of volumes and systolic indices of heart ventricle from Halobatrachus didactylus: echocardiographic noninvasive method

The purpose of this work is to calculate end-systolic and end-diastolic volumes of Halobatrachus didactylus ventricles, from two-dimensional (2D) echocardiographic images, comparing four different linear methods, and to derive systolic indices of ventricular function-fractional shortening, ejection fraction, stroke volume, and cardiac output independent of the Fick principle. Echocardiography provided high resolution images of cardiac structures and allowed accurate linear measurements. The Simpson algorithm proved to be the best method of calculating ventricle volumes. As a corollary, ventricular mass can be derived from echocardiographic volume data. This noninvasive method promises wide utilization in experimental comparative cardiovascular biology.     

Unusual glycoconjugates in the oesophagus of a tilapine polyhybrid

The aim of this work is to elucidate the glycoconjugate composition of the secretory products of the oesophageal mucous cells in a tilapine polyhybrid. Lectin histochemistry gave evidence of the presence of β-galactose, α-N-acetylgalactosamine and sialic acid residues in the terminal position. The majority of sialic acid belongs to short side chains; a few sialic acid residues are acetylated at the C₇ and/or C₈ and/or C₉ level. The heterogeneity of the carbohydrate chains may mask potential receptor sites for micro-organisms and hamper the formation of multiple bonds.     

Histochemical study of glycoconjugates in the nasal mucosa of the rat and guinea pig

Summary A histochemical study was carried out on the glycoconjugates of the nasal mucosa of rat and guinea pig using conventional techniques and peroxidase-labelled lectins. Both the respiratory mucosa and neuroepithelium were studied. Sulphate and sialic acid groups were found in the mucous layer of the neuroepithelia, Bowman's glands and goblet cells. In contrast, the nasal glands did not possess these groups, and only a few showed neutral mucins. Carbohydrate residues were more numerous in the acini of the Jacobson glands. Thus, the nasal glands in the rat and guinea pig are probably of a serous type because of the scarcity of carbohydrate residues.     

Histochemical differentiation of glycoconjugates occurring in the tilapine intestine

The glycoconjugates secreted by the anterior and posterior intestinal segments of Tilapia spp. were characterized by means of both conventional histochemical procedures (PAS, AB, AB-PAS, LID, HID) and a battery of 12 horseradish peroxidase labelled lectins. Some sections were treated with glycolytic enzymes and KOH before conventional and lectin stainings. The large majority of the mucopolysaccharides secreted by the goblet cells of the anterior segment are carboxylated while only a few carry sulphate groups. The mucopolysaccharides in the anterior intestine contained chondroitin, heparin and chondroitinsulphates which can provide protection for intestinal mucosae. DBA lectin staining demonstrated the presence of some endocrine cells in the anterior segment.     

Histochemical analysis of glycoconjugates in the domestic cat testis

The localization and characterization of oligosaccharide sequences in the cat testis was investigated using 12 lectins in combination with the beta-elimination reaction, N-Glycosidase F and sialidase digestion. Leydig cells expressed O-linked glycans with terminal alphaGalNAc (HPA reactivity) and N-glycans with terminal/internal alphaMan (Con A affinity). The basement membrane showed terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,3GalNAc, alpha/betaGalNAc, and GlcNAc (SNA, PNA, HPA, SBA, GSA II reactivity) in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc (RCA120 staining) and alphaMan in N-linked oligosaccharides; in addition, terminal Neu5acalpha2,3Galbeta1,4GlcNac, Forssman pentasaccharide, alphaGal, alphaL-Fuc and internal GlcNAc (MAL II, DBA, GSA I-B4, UEA I, KOH-sialidase-WGA affinity) formed both O- and N-linked oligosaccharides. The Sertoli cells cytoplasm contained terminal Neu5Ac-Galbeta1,4GlcNAc, Neu5Ac-betaGalNAc as well as internal GlcNAc in O-linked glycans, alphaMan in N-linked glycoproteins and terminal Neu5Acalpha2,6Gal/ GalNAc in both O- and N-linked oligosaccharides. Spermatogonia exhibited cytoplasmic N-linked glycoproteins with alphaMan residues. The spermatocytes cytoplasm expressed terminal Neu5Acalpha2,3Galbeta1,4 GlcNAc and Galbeta1,3GalNAc in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-linked glycoconjugates. The Golgi region showed terminal Neu5Acalpha2,3Galbeta1,4GlcNac, Galbeta1,4GlcNAc, Forssman pentasaccharide, and alphaGalNAc in O-linked oligosaccharides, alphaMan and terminal betaGal in N-linked oligosaccharides. The acrosomes of Golgi-phase spermatids expressed terminal Galbeta1,3GalNAc, Galbeta1,4GlcNAc, Forssmann pentasaccharide, alpha/betaGalNAc, alphaGal and internal GlcNAc in O-linked oligosaccharides, terminal alpha/betaGalNAc, alphaGal and terminal/internal alphaMan in N-linked glycoproteins. The acrosomes of cap-phase spermatids lacked internal Forssman pentasaccharide and alphaGal, while having increased alpha/betaGalNAc. The acrosomes of elongated spermatids did not show terminal Galbeta1,3GalNAc, displayed terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-glycans and Neu5Ac-Galbeta1,3GalNAc in O-linked oligosaccharides.     

Secretory glycoconjugates in the epithelium of the goat prostate

Summary Secretory glycoconjugates in the epithelium of the goat prostate were investigated with various electron microscopical histochemical methods that involved periodic acid—thiocarbohydrazide—silver proteinate, colloidal gold-labelled lectin and related procedures. The three types of cells in the epithelium previously differentiated with the light microscope were substantiated: mucus-producing cells, protein-secreting cells and cells intermediate between both types. These three cell types contained varying amounts of neutral glycoconjugates; the histochemical nature of these carbohydrates was determined with particular emphasis upon the histophysiological functions of the accessory sex glands.     

Histochemical study of glycoconjugates in the epididymis of the hamster (Mesocricetus auratus)

Summary The glycoconjugates of hamster epididymis were investigated with conventional and lectin histochemistry. A zone of the caput epididymis, with particular histochemical characteristics, has been differentiated. β-Elimination in combination with lectins was used to establish the presence and distribution of N- and O-linked glycoconjugates. The epithelium, spermatozoa and the intertubular matrix were rich in glycoconjugates. The Golgi apparatus and stereocilia of the principal cells were intensely positive with HPA, PNA and SBA lectins. β-limination indicated that these cells contained abundant O-linked glycoconjugates. Apical and clear cells presented a common lectin affinity; their reactivities towards WGA and UEA-I were very positive. These cells probably contain abundant N-glycoconjugates. The spermatozoa were stained by periodic acid-Schiff (PAS) and by all the lectins (especially in the acrosome), except by those with an affinity for α-l-fucosyl residues; the most intense reaction was found with HPA, WGA, PNA and SBA. Changes in the sperm lectin binding along the ductus were observed: sperm flagellum abruptly acquired WGA and PNA labelling from the posterior caput, and HPA reactivity was negative only in the zone between the caput and the corpus.     

Oxidative stress in toadfish (Halobactrachus didactylus) cardiac muscle. Acute exposure to vanadate oligomers

Vanadate solutions as ‘metavanadate’ (containing ortho and metavanadate species) and ‘decavanadate’ (containing manly decameric species) (5 mM; 1 mg/kg) were injected intraperitoneously in Halobatrachus didactylus (toadfish), in order to evaluate the contribution of decameric vanadate species to vanadium (V) intoxication on the cardiac tissue. Following short-term exposure (1 and 7 days), different changes on antioxidant enzyme activities—superoxide dismutase (SOD), catalase (CAT), selenium-glutathione peroxidase (Se-GPx), total glutathione peroxidase (GPx), lipid peroxidation and subcellular vanadium distribution were observed in mitochondrial and cytosolic fractions of heart ventricle toadfish. After 1 day of vanadium intoxication, SOD, CAT and Se-GPx activities were decreased up to 25%, by both vanadate solutions, except mitochondrial CAT activity that increased (+23%) upon decavanadate administration. After 7 days of exposure, decavanadate versus metavanadate solutions promoted different effects mainly on cytosolic CAT activity (−56% versus −5%), mitochondrial CAT activity (−10% versus +10%) and total GPx activity (+1% versus −35%), whereas lipid peroxidation products were significantly increased (+82%) upon 500 μM decavanadate intoxication. Accumulation of vanadium in total (0.137±0.011 μg/g) and mitochondrial (0.022±0.001 μg/g) fractions was observed upon 7 days of metavanadate exposure, whereas for decavanadate, the concentration of vanadium increased in cytosolic (0.020±0.005 μg/g) and mitochondrial (0.021±0.009 μg/g) fractions. It is concluded that decameric vanadate species are responsible for a strong increase on lipid peroxidation and a decrease in cytosolic catalase activity thus contributing to oxidative stress responses upon vanadate intoxication, in the toadfish heart.     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, alpha-D-mannose and alpha-D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; alpha-D-mannose and alpha-D-glucose residues and terminal sialic acid-galactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

Summary In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, -D-mannose and -D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; -D-mannose and -D-glucose residues and terminal sialic acidgalactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

Histochemical analysis of glycoconjugates in the eccrine glands of the raccoon digital pads

The distribution and selectivity of complex carbohydrates in the eccrine glands of the digital pads in the North American raccoon (Procyon lotor) were studied using light and electron microscopic histochemical methods, particularly lectin histochemistry. In the eccrine glands, the dark cells exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine and N-acetyl-neuraminic acid); the clear cells contained numerous glycogen particles and showed a distinct reaction of alpha-L-fucose. The presence of complex carbohydrates with various terminal sugars was evident in the excretory duct cells. In addition, beta-D-galactose and N-acetyl-neuraminic acid residues were mainly observed in the luminal secretion. The glycoconjugates produced by the eccrine glands of the raccoon digital pads may protect the epidermis against physical damage or microbial contamination. In this way, the normal functioning of the sensory apparatus of the foot pads is ensured.