Formation and oral administration of alginate microspheres loaded with pDNA coding for lymphocystis disease virus (LCDV) to Japanese flounder

Oral delivery of plasmid DNA (pDNA) is a desirable approach for fish immunization in intensive culture. However, its effectiveness is limited because of possible degradation of pDNA in the fish's digestive system. In this report, alginate microspheres loaded with pDNA coding for fish lymphocystis disease virus (LCDV) and green fluorescent protein were prepared with a modified oil containing water (W/O) emulsification method. Yield, loading percent and encapsulation efficiency of alginate microspheres were 90.5%, 1.8% and 92.7%, respectively. The alginate microspheres had diameters of less than 10 microm, and their shape was spherical. As compared to sodium alginate, a remarkable increase of DNA-phosphodiester and DNA-phosphomonoester bonds was observed for alginate microspheres loaded with pDNA by Fourier transform infrared (FTIR) spectroscopic analysis. Agarose gel electrophoresis showed a little supercoiled pDNA was transformed to open circular and linear pDNA during encapsulation. The cumulative release of pDNA in alginate microspheres was or=0.3) for anti-LCDV antibody from week 3 to week 16 for fish orally vaccinated with alginate microspheres loaded with pDNA, in comparison with fish orally vaccinated with naked pDNA. Our results display that alginate microspheres obtained by W/O emulsification are promising carriers for oral delivery of pDNA. This encapsulation technique has the potential for DNA vaccine delivery applications due to its ease of operation, low cost and significant immune effect.     

An investigation on pro-opiomelanocortin and processed peptides from the teleost fish Prochilodus platensis

Acid extracts of carefully dissected proadenohypophysis (PA) and metaadenohypophysis (MA) of the teleost Prochilodus platensis were subjected to chromatography in Sephadex G-50 after which several pro-opiomelanocortin (POMC) peptides were detected by means of three heterologous RIA systems: alpha-MSH, ACTH and beta-endorphin. Parallelism among extracts displacement curves ranged from 26% to 95% of those of the standard curves for the different systems employed. In PA chromatograms, peaks of ACTH immunoreactivity (IR) were detected at the positions of 30 kilodalton (K), 20K, 9K, a large 4.5K peak and 2K. Only one peak of beta-endorphin IR was detected at 30K. In MA chromatograms, ACTH IR detected similar peaks as in PA runs, but 4.5K peak was much smaller, whereas a large 2K peak roughly coincided with all alpha-MSH detected in the chromatograms. beta-Endorphin IR was detected mainly as a large peak coinciding with synthetic beta-endorphin in MA runs. Bioactivity was detected in both PA and MA 4.5K ACTH peaks, whereas little activity could be demonstrated associated with the 30K, 20K and 9K ACTH IR peaks. Prochilodus PAs and MAs were incubated with tritiated aminoacids and the extracts immunoprecipitated with ACTH, beta-endorphin and N-terminal POMC (N-POMC) antisera. The dissociated complexes were run in SDS polyacrylamide slab gel electrophoresis. The tritiated bands detected confirmed the results obtained with Sephadex chromatography. N-POMC immunoprecipitated peptides were located at 28K, 18K and 9K positions. The first two probably accounted for POMC and the N-POMC/ACTH intermediate respectively; the third corresponded to the mammalian 1-76N-POMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature adaptation and the contractile properties of live muscle fibres from teleost fish

Summary The contractile properties of swimming muscles have been investigated in marine teleosts from Antarctic (Trematomus lepidorhinus, Pseudochaenichthys georgianus), temperate (Pollachius virens, Limanda limanda, Agonis cataphractus, Callionymus lyra), and tropical (Abudefduf abdominalis, Thalassoma duperreyi) latitudes. Small bundles of fast twitch fibres were isolated from anterior myotomes and/or the pectoral fin adductor profundis muscle (m. add. p). Live fibre preparations were viable for several days at in vivo temperatures, but became progressively inexcitable at higher or lower temperatures. The stimulation frequency required to produce fused isometric tetani increased from 50 Hz in Antarctic species at 0°C to around 400 Hz in tropical species at 25°C. Maximum isometric tension (P_o) was produced at the normal body temperature (NBT) of each species (Antarctic, 0–2°C; North Sea and Atlantic, 8–10°C; Indo-West Pacific, 23–25°C). P₀ values at physiological temperatures (200–300 kN·m^–2) were similar for Antarctic, temperate, and tropical species. A temperature induced tension hysteresis was observed in muscle fibres from some species. Exposure to <0°C in Antarctic and <2°C in temperate fish resulted in the temporary depression of tension over the whole experimental range, an effect reversed by incubation at higher temperatures. At normal body temperatures the half-times for activation and relaxation of twitch and tetanic tension increased in the order Antarctic>temperate>tropical species. Relaxation was generally much slower at temperatures <10°C in fibres from tropical than temperate fish. Q₁₀ values for these parameters at NBTs were 1.3 2.1 for tropical species, 1.7–2.6 for temperate species, and 1.6–3.5 for Antarctic species. The forcevelocity (P-V) relationship was studied in selected species using iso-velocity releases and the data below 0.8 P₀ iteratively fitted to Hill's equation. The P-V relation at NBT was found to be significantly less curved in Antarctic than temperate species. The unloaded contraction velocity (V_max) of fibres was positively correlated with NBT increasing from about 1 muscle fibre length·s^–;1 in an Antarctic fish (Trematomus lepidorhinus) at 1°C to around 16 muscle fibre lengths·s^–1 in a tropical species (Thalassoma duperreyi) at 24°C. It is concluded that although muscle contraction in Antarctic fish shows adaptations for low temperature function, the degree of compensation achieved in shortening speed and twitch kinetics is relatively modest.Abbreviations ET environmental temperature - m. add. p major adductor profundis - m. add. s. major adductor superficialis - NBT normal body temperature - P ₀ maximum isometric tension - P-V force velocity - SR sarcoplasmic reticulum - T 1/2 a half activation time - T 1/2 r half relaxation time - V _max unloaded contraction     

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake (Merluccius capensis) and tilapia (Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp7, Leu8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp7, Leu8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp7, Leu8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln8-GnRH) and Trp7, Leu8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln8-GnRH and Trp7, Leu8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp7, Leu8-GnRH, while tilapia brain contains both Trp7, Leu8-GnRH and Gln8-GnRH. Additional GnRH molecular forms were detected in both species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species

AIMS: The development and evaluation of a protocol based on polymerase chain reaction (PCR) and nucleic acid hybridization techniques for the specific detection of lymphocystis disease virus (LCDV) in several marine fish species. METHODS AND RESULTS: The pair of primers for PCR, OBL3 and OBL4, was designed based on published nucleotide sequence (LCDV-1) and amplifies a fragment within the major capsid protein. The sensitivity was evaluated using DNA from purified viral particles, as well as from cells inoculated with several viral concentrations. The PCR combined with slot blot was the most sensitive methodology, detecting 2.5 ng of viral DNA. Using this methodology LCDV was detected at 5 days postinoculation from SAF-1 cells initially inoculated with 10(-5) TCID(50) ml(-1). The combination of PCR with membrane hybridization has also been proved to be adequate to detect LCDV from apparently healthy carriers by means of caudal fin sample analysis. This asymptomatic infection was also demonstrated by classical virological methods (cell culture and immunoblot). CONCLUSIONS: The protocol described in this study allows the specific detection of LCDV, both in cell cultures and in fin homogenates from asymptomatic fish. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of asymptomatic carriers by a rapid molecular method using caudal fin sampling, which does not imply animal killing, could be an important tool to control epizootics caused by LCDV, as fish could be analysed before their introduction and/or mobilization in farm facilities.     

Stannius corpuscles and plasma calcium levels during the reproductive cycle in the cichlid teleost fish,Oreochromis mossambicus

Summary The corpuscles of Stannius (CS) of the cichlid Oreochromis mossambicus (formerly Sarotherodon mossambicus) were studied in relation to sexual maturation and plasma calcium levels. After sexual maturation, the CS are enlarged in female fish, because of an increase in size and number of the type-1 cells. During the ovarian cycle, the size of the CS increases in parallel with the growth of the ovaries. Concurrently, plasma total calcium increases markedly until spawning. This increase is mainly accounted for by calcium bound to proteins (vitellogenins), but the ultrafiltrable calcium fraction is also slightly higher than in males. Ovariectomy is followed by a reduction in the size of the CS, mainly a result of involution of the type-1 cells, and by a reduction in plasma calcium to levels typical for males. Gonadectomy in males does not affect size or ultrastructure of the CS, or plasma calcium levels. Since the type-1 cells of the CS are the presumptive source of a hypocalcemic hormone, we conclude that activation of the CS during the female reproductive cycle is a response to elevated calcium levels that accompany ovarian maturation. We suggest that the CS respond in particular to the elevated ultrafiltrable or ionic calcium levels.     

Effects of calcium and phosphate on the corpuscles of Stannius of the teleost fish,Oreochromis mossambicus

Summary Removal of the corpuscles of Stannius (CS) in Oreochromis mossambicus leads to hypercalcemia and hypophosphatemia. The effects on CS size and ultrastructure of different calcium and phosphate concentrations of the ambient water and of the food were investigated. A six-fold increase of the calcium concentration of the water leads to a four-fold increase in CS volume; this is mainly caused by an increase in the size and number of the type-1 cells. The effect of external calcium is most probably mediated by the calcium concentration of the blood plasma. Plasma ionic calcium may be the relevant factor. Changes in the calcium concentration of the food had no effect on the CS. Similarly, hyperphosphatemia or hypophosphatemia induced by high phosphate concentrations of the water or the food, or by a phosphate-deficient diet, had no noticeable effect on the CS. The results support the hypothesis that the type-1 cells produce the hypocalcemic factor of the CS. There is no evidence for the production by the CS of an endocrine factor involved in the control of phosphate metabolism.     

Comparative physiology and molecular analysis of carbonic anhydrase from the red blood cells of teleost fish

This study investigates the early evolution of vertebrate red blood cell (rbc) carbonic anhydrase (CA) by examining the physiological and molecular properties of rbc CA in teleost fish. When representatives of four different families of teleosts were compared, it was found that differences in overall rbc CA activity were due to different concentrations of CA, rather than differences in the enzymes kinetic properties. Additional molecular analysis of CA from the rbcs of rainbow trout provided further evidence that critical elements of the enzyme, such as the active site, have been highly conserved during vertebrate evolution. The active site of the trout CA differed from that of gar rbc CA at only two amino acid positions. The rainbow trout rbc CA sequence also showed high sequence homology with CA sequences from other fish tissues, and fits into an emerging group of fish CAs that are basal to mammalian CA I, II and III. Northern blot analysis of the tissue expression of the sequenced CA indicated that it is primarily found in the rbcs, but high amounts of cytosolic CA activity were also found in the gill, suggesting the presence of other cytosolic CA isozymes in this species.Abbreviations Az acetazolamide - CA carbonic anhydrase - MP maximum parsimony - NJ neighbour joining - RACE rapid amplification of cDNA ends - rbc red blood cellCommunicated by L.C.-H. Wang     

Fine structure and metabolism of multiply innervated fast muscle fibres in teleost fish

Summary Both the fast and slow muscle fibres of advanced teleost fish are multiply innervated. The fraction of slow-fibre volume occupied by mitochondria is 31.3%, 25.5% and 24.6%, respectively, for the myotomal muscles of brook trout (Salvelinus fontinalis), crucian carp (Carassius carassius), and plaice (Pleuronectes platessa), respectively. The corresponding figures for the fast muscles of these species are 9.3%, 4.6% and 2.0%, respectively. Cytochrome-oxidase and citrate-synthetase activities in the fast muscles of 9 species of teleost range from 0.20–0.93 moles substrate utilised, g wet weight muscle^-1 min^-1 (at 15° C) or around 4–17% of that of the corresponding slow fibres. Ultrastructural analyses reveal a marked heterogeneity within the fast-fibre population. For example, the fraction of fibres with <1% or >10% mitochondria is 0,4,42% and 36, 12 and 0%, respectively, for trout, carp and plaice. In general, small fibres (<500 m²) have the highest and large fibres (>1,500 m²) the lowest mitochondrial densities. The complexity of mitochondrial cristae is reduced in fast compared to slow fibres.Hexokinase activities range from 0.4–2.5 in slow and from 0.08–0.7 moles, g wet weight^-1 min^-1 in fast muscles, indicating a wide variation in their capacity for aerobic glucose utilisation. Phosphofructokinase activities are 1.2 to 3.6 times higher in fast than slow muscles indicating a greater glycolytic potential. Lactate dehydrogenase activities are not correlated with either the predicted anaerobic scopes for activity or the anoxic tolerances of the species studied. The results indicate a considerable variation in the aerobic capacities and principal fuels supporting activity among the fast muscles of different species. Brook trout and crucian carp are known to recruit fast fibres at low swimming speeds. For these species the aerobic potential of the fast muscle is probably sufficient to meet the energy requirements of slow swimming.     

The structure of the coeliac ganglion of a teleost fish Myoxocephalus scorpius

Summary In order to compare the structure of a teleost sympathetic ganglion with those of other vertebrates, light, fluorescence histochemical and electron microscopy were carried out on the coeliac ganglion of the scorpion fish, Myoxocephalus scorpius. In common with studies on other vertebrates, fluorescence histochemistry distinguished two cell types: a) principal neurones which exhibited low levels of specific catecholamine fluorescence and comprise the majority of neurones in the ganglia, and b) smaller intensely fluorescent cells, some of which had processes tens of micrometers long.With the electron microscope, the principal cells were seen to make axodendritic and axosomatic synapses with axons containing mainly 30 nm agranular vesicles at the synaptic site while in other vertebrates usually only one or other synaptic association is present.Both the somata and the processes of intensely fluorescent cells contain 300–600 nm diameter vesicles many of which have electron dense cores. These cells are also innervated by axons containing 30 nm agranular vesicles.     

Central and peripheral effects of ghrelin on energy balance, food intake and lipid metabolism in teleost fish

Ghrelin was first identified and characterized from rat stomach as an endogenous ligand for the growth hormone secretagogue receptor. Ghrelin and its receptor system are present not only in peripheral tissues such as stomach and intestine, but also in the central nervous system of mammals. Interestingly, administration of ghrelin induces an orexigenic effect and also modifies locomotor activity, suggesting its involvement in feeding control and the regulation of energy balance, in addition to the regulation of growth hormone release. Information about ghrelin in non-mammals, such as teleost fish, has also been increasing, and important data have been obtained. An understanding of the evolutionary background of the energy regulation system and the central and peripheral roles of ghrelin in teleost fish could provide indications as to their roles in mammals, particularly humans. In this review, we overview the central and peripheral effects of ghrelin on energy balance, locomotor activity, and lipid metabolism in teleost fish.     

Immunocytochemical investigation of forebrain control by somatostatin of the pituitary in the teleost Poecilia latipinna

Summary An extensive system of somatostatin-immunoreactive neurons has been localized in the forebrain and pituitary of the molly (Poecilia latipinna), using the unlabelled antibody immunocytochemical method.In the hypothalamus, reactive perikarya were scattered throughout the parvocellular divisions of the preoptic nucleus. These cells were smaller in size and more ventral in position than those which stained with antisera to the neurohypophysial hormones, vasotocin and isotocin. A few very small somatostatin-immunoreactive cells were observed in the tuberal region and in the nuclei of the lateral and posterior recesses — areas which were rich in somatostatin-immunoreactive fibres.Somatostatin cells were also found in a small area of the ventral thalamus, mainly in the dorsolateral nucleus. Some of these neurons were large and multipolar, and appeared to form tracts of fibres into the posterior hypothalamus. In the telencephalon there were a few stained cells in the ventral area, with a complex pattern of fibres occurring in parts of the dorsal area.Somatostatin-immunoreactivity was intense in the central and posterior neurohypophysis, and particularly in its finger-like projections into the proximal pars distalis, around groups of growth hormone cells. Examination of material from fishes under various experimental conditions provided evidence for the somatostatin fibres originating from the preoptic neurons being involved in the control of growth hormone secretion.     

Microstructure,mineral and mechanical properties of teleost intermuscular bones

There is an increasing interest in understanding teleost bone biomechanics in several scientific communities, for instance as interesting biomaterials with specific structure-function relationships. Intermuscular bones of teleost fish have previously been described to play a role in the mechanical force transmission between muscle and bone, but their biomechanical properties are not yet fully described. Here, we have investigated intermuscular bones (IBs) of the North Atlantic Herring with regard to their structure and micro-architecture, mineral-related properties, and micro-mechanical tensile properties. A total of 115 IBs from 18 fish were investigated. One cohort of IBs, containing 20 bones from 2 smaller fish and 23 bones of 3 larger fish, was used for mechanical testing, wide-angle X-ray scattering, and scanning electron microscopy. Another cohort, containing 36 bones from 7 smaller fish and 36 bones from 6 larger fish, was used for microCT. Results show some astonishing properties of the IBs: (i) IBs present higher ductility, lower Young's modulus but similar strength and TMD (Tissue Mineral Density) compared to mammalian bone, and (ii) IBs from small fish were 49% higher in Young’s modulus than fish bones from larger fish while their TMD was not statistically different and crystal length was 8% higher in large fish bones. Our results revealed that teleost IB presents a hybrid nature of soft and hard tissue that differs from other bone types, which might be associated with their evolution from mineralized tendons. This study provides new data regarding teleost fish bone biomechanical and micro-structural properties.     

Capillary distribution and metabolic histochemistry of the lateral propulsive musculature of pelagic teleost fish

Metabolic and vascular adaptation of teleost lateral propulsive musculature to an active mode of life was investigated in four pelagic teleosts (mackerel, yellowtail scad, pilchard and Australian salmon). Histochemical profiles and capillarisation data of the red and white muscle were compared to those of less active demersal species. Pelagic white muscle stained positively for the aerobic enzymes succinate dehydrogenase and NADH diaphorase, and had both subsarcolemmal and intermyofibrillar mitochondria which corresponded to the loci of the histochemical stain. Subsarcolemmal mitochondria tended to be localised close to capillaries. In contrast, white muscle from demersal species was unstained for the same enzymes and was devoid of mitochondria. Red muscle of all species had abundant mitochondria and stained intensely for aerobic enzymes. Capillarisation was quantified by determining the percentage of fibres surrounded by a given number of peripheral capillaries, mean fibre diameter, mean number of peripheral capillaries, capillary: fibre ratio and sharing factor where appropriate. Red muscle of mackerel, Australian salmon, pilchard and scad are better vascularised than red muscle of the flathead having 153, 200, 242, 291 and 309 microns 2 of cross-sectional fibre area per peripheral capillary, respectively. White muscle of mackerel, pilchard and scad are better vascularised than white muscle of the Australian salmon and flathead having 2040, 3367, 4992, 9893 and 10,469 microns 2 of cross-sectional fibre area per peripheral capillary, respectively. Red muscle of Australian salmon had distinct regional variation. Deep red muscle was found to be more highly vascularised (4.2 peripheral capillaries per muscle fibre) than lateral red muscle (1.9 peripheral capillaries per muscle fibre). Red muscle of the other species was less heterogeneous. White muscle capillarisation was slightly variable in all species. It is concluded that the white muscle of the pelagic species studied is functionally and structurally adapted for sustained aerobic activity with relatively abundant mitochondria being preferentially situated close to the source of gas and metabolite exchange.     

A comparative study on the innervation and the vascularization of the bulbus arteriosus in teleost fish

Summary The structure of the bulbus arteriosus of a wide range of teleost fish is described with particular reference to the vascularization and innervation. The adventitia of the organ consists of blood vessels and large nerve bundles in a collagen matrix. The nerve bundles contain monoamines, and fluorescence studies show small terminal bundles penetrating the muscular media; this is confirmed by electron microscopy. The media consists of an extensive elastic tissue matrix with a spiral arrangement of smooth muscle cells joined end to end by desmosomes and presumed electrotonic junctions. The muscle cells are innervated only at the adventitia/media boundary and the significance of this innervation is discussed. It is proposed that there is a correlation between the degree of vascularization and innervation and the activity of a particular species offish.     

The distribution of capillaries in the somatic musculature of two vertebrate types with particular reference to teleost fish

The distribution of capillaries in teleost and rat striated muscles was investigated using a number of different methods. A new method for directly viewing capillaries was developed. Teleost white muscle has a capillary: fibre (C:F) ratio of between 0.2 and 0.3; and 0.6 to 1.0 peripheral capillaries per muscle fibre. 26-49% of fibres had no peripheral capillaries. Values for the rat gastrocnemius were 1.2, 2.6 and 4.8% respectively which compares well with literature values. Flathead red muscle had a C:F ratio of between 1.9 and 2.5; and between 5.3 and 6.6 peripheral capillaries per muscle fibre depending on the method used. Values for rat soleus were 1.8 and 4.1 respectively. Teleost pink fibres had an intermediate number of capillaries. Rat striated muscle, particularly the gastrocnemius, was found to be heterogeneous with respect to the distribution of capillaries. Flathead red muscle was homogeneous whilst teleost white muscle was only slightly variable. Flathead red muscle fibres are well suppled with subsarcolemmal mitochondria. These show a clumped distribution corresponding to the position of capillaries. In contrast teleost white fibres are almost totally devoid of these and all other mitochondria. No differences were observed in the vascularisation of either muscle type along the length of the fish. The results are discussed in relation to the division of labour between fibre types during swimming.