Swimbladder Tissue Research Articles

Deiodinase inhibition impairs the formation of the three posterior swim bladder tissue layers during early embryonic development in zebrafish

Thyroid hormone system disruption (THSD) negatively affects multiple developmental processes and organs. In fish, inhibition of deiodinases, which are enzymes crucial for (in)activating thyroid hormones (THs), leads to impaired swim bladder inflation. Until now, the underlying mechanism has remained largely unknown. Therefore, the objective of this study was to identify the process during swim bladder development that is impacted by deiodinase inhibition. Zebrafish embryos were exposed to 6 mg/L iopanoic acid (IOP), a model deiodinase inhibitor, during 8 different exposure windows (0–60, 60–120, 24–48, 48–72, 72–96, 96–120, 72–120 and 0–120 h post fertilization (hpf)). Exposure windows were chosen based on the three stages of swim bladder development: budding (24–48 hpf), pre-inflation, i.e., the formation of the swim bladder tissue layers (48–72 hpf), and inflation phase (72–120 hpf). Exposures prior to 72 hpf, during either the budding or pre-inflation phase (or both), impaired swim bladder inflation, while exposure during the inflation phase did not. Based on our results, we hypothesize that DIO inhibition before 72 hpf leads to a local decrease in T3 levels in the developing swim bladder. Gene transcript analysis showed that these TH level alterations disturb both Wnt and hedgehog signaling, known to be essential for swim bladder formation, eventually resulting in impaired development of the swim bladder tissue layers. Improper development of the swim bladder impairs swim bladder inflation, leading to reduced swimming performance. This study demonstrates that deiodinase inhibition impacts processes underlying the formation of the swim bladder and not the inflation process, suggesting that these processes primarily rely on maternal rather than endogenously synthetized THs since TH measurements showed that THs were not endogenously synthetized during the sensitive period.

Inhibition of the largemouth bass virus replication by piperine demonstrates potential application in aquaculture

Largemouth bass virus (LMBV) is a systemic viral pathogen that can cause high mortality rates in cultivated largemouth bass. However, no treatment is currently approved. Therapeutic strategies against LMBV infection are urgently needed. In this study, we investigated the antiviral activity of piperine against LMBV in vitro and in vivo. In vitro antiviral activity assay showed that 210.28 μM piperine significantly decreased LMBV major capsid protein (MCP) gene expression in epithelioma papulosum cyprinid (EPC) cells by a maximum inhibitory rate of >95%. Piperine treatment inhibited LMBV replication in a dose-dependent manner, with the half-maximal activity (IC50 ) of 34.61 μM. Moreover, piperine significantly decreased the viral titers and cytopathic effects (CPE), contributing to the protection of infected cells. With regard to the steps of piperine affecting the life cycle of viruses, piperine had a direct inactivating effect on LMBV. During the virus adsorption phase, piperine prevented the adsorption of LMBV to EPC cells. Furthermore, piperine played an antiviral role mainly in the later stages of viral infection (4-8h). To further evaluate the antiviral activity of piperine against LMBV in vivo, largemouth bass as a model organism was carried out in relevant experiments. Intraperitoneal injection of piperine (25 mg/kg) effectively improved the survival rate of LMBV-infected largemouth bass by 20%. In addition, RT-qPCR results of viral replication in liver, spleen, kidney, gill and swim bladder tissues showed that piperine significantly inhibited LMBV replication in vivo, thus protecting largemouth bass from LMBV-induced death. Together, our results suggested that piperine is a therapeutic and preventative agent against LMBV infection.

Aquaporin expression and cholesterol content in eel swimbladder tissue.

Leakiness of the swimbladder wall of teleost fishes must be prevented to avoid diffusional loss of gases out of the swimbladder. Guanine incrustation as well as high concentrations of cholesterol in swimbladder membranes in midwater and deep‐sea fish has been connected to a reduced gas permeability of the swimbladder wall. On the contrary, the swimbladder is filled by diffusion of gases, mainly oxygen and CO2, from the blood and the gas gland cells into the swimbladder lumen. In swimbladder tissue of the zebrafish and the Japanese eel, aquaporin mRNA has been detected, and the aquaporin protein has been considered important for the diffusion of water, which may accidentally be gulped by physostome fish when taking an air breath. In the present study, the expression of two aquaporin 1 genes (Aqp1aa and Aqp1ab) in the swimbladder tissue of the European eel, a functional physoclist fish, was assessed using immunohistochemistry, and the expression of both genes was detected in endothelial cells of swimbladder capillaries as well as in basolateral membranes of gas gland cells. In addition, Aqp1ab was present in apical membranes of swimbladder gas gland cells. The authors also found high concentrations of cholesterol in these membranes, which were several fold higher than in muscle tissue membranes. In yellow eels the cholesterol concentration exceeded the concentration detected in silver eel swimbladder membranes. The authors suggest that aquaporin 1 in swimbladder gas gland cells and endothelial cells facilitates CO2 diffusion into the blood, enhancing the switch‐on of the Root effect, which is essential for the secretion of oxygen into the swimbladder. It may also facilitate CO2 diffusion into the swimbladder lumen along the partial gradient established by CO2 production in gas gland cells. Cholesterol has been shown to reduce the gas permeability of membranes and thus could contribute to the gas tightness of swimbladder membranes, which is essential to avoid diffusional loss of gas out of the swimbladder.

A novel zebrafish model to emulate lung injury by folate deficiency-induced swim bladder defectiveness and protease/antiprotease expression imbalance

Lung injury is one of the pathological hallmarks of most respiratory tract diseases including asthma, acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). It involves progressive pulmonary tissue damages which are usually irreversible and incurable. Therefore, strategies to facilitate drug development against lung injury are needed. Here, we characterized the zebrafish folate-deficiency (FD) transgenic line that lacks a fully-developed swim bladder. Whole-mount in-situ hybridization revealed comparable distribution patterns of swim bladder tissue markers between wild-type and FD larvae, suggesting a proper development of swim bladder in early embryonic stages. Unexpectedly, neutrophils infiltration was not observed in the defective swim bladder. Microarray analysis revealed a significant increase and decrease of the transcripts for cathepsin L and a cystatin B (CSTB)-like (zCSTB-like) proteins, respectively, in FD larvae. The distribution of cathepsin L and the zCSTB-like transcripts was spatio-temporally specific in developing wild-type embryos and, in appropriate measure, correlated with their potential roles in maintaining swim bladder integrity. Supplementing with 5-formyltetrahydrofolate successfully prevented the swim bladder anomaly and the imbalanced expression of cathepsin L and the zCSTB-like protein induced by folate deficiency. Injecting the purified recombinant zebrafish zCSTB-like protein alleviated FD-induced swim bladder anomaly. We concluded that the imbalanced expression of cathepsin L and the zCSTB-like protein contributed to the swim bladder malformation induced by FD and suggested the potential application of this transgenic line to model the lung injury and ECM remodeling associated with protease/protease inhibitor imbalance.

Transcript levels of members of the SLC2 and SLC5 families of glucose transport proteins in eel swimbladder tissue: the influence of silvering and the influence of a nematode infection

The rate of glucose metabolism has been shown to be correlated to glucose uptake in swimbladder gas gland cells. Therefore, it is assumed that in the European eel silvering, i.e., the preparation of the eel for the spawning migration to the Sargasso Sea, coincides with an enhanced capacity for glucose uptake. To test this hypothesis expression of all known glucose transport proteins has been assessed at the transcript level in yellow and in silver eels, and we also included Anguillicola crassus infected swimbladders. Glucose uptake by rete mirabile endothelial cells could be crucial for the countercurrent exchange capacity of the rete. Therefore, this tissue was also included in our analysis. The results revealed expression of ten different members of the slc2 family of glucose transporters, of four slc5 family members, and of kiaa1919 in gas gland tissue. Glucose transporters of the slc2 family were expressed at very high level, and slc2a1b made up about 80% of all slc2 family members, irrespective of the developmental state or the infection status of the eel. Overall, the slc5 family contributed to only about 8% of all detected glucose transport transcripts in gas gland tissue, and the slc2 family to more than 85%. In rete capillaries, the contribution of sodium-dependent glucose transporters was significantly higher, leaving only 66% for the slc2 family of glucose transporters. Neither silvering nor the infection status had a significant effect on the expression of glucose transporters in swimbladder gas gland tissue, suggesting that glucose metabolism of eel gas gland cells may not be related to transcriptional changes of glucose transport proteins.

Anguillicola crassus infection affects mRNA expression levels in gas gland tissue of European yellow and silver eel.

Using Illumina sequencing, we investigated transcriptional changes caused by the nematode Anguillicola crassus within yellow and silver eels by comparing swimbladder samples of uninfected yellow with infected yellow eels, and uninfected silver with infected silver eels, respectively. In yellow eel gas gland, the infection caused a modification of steady state mRNA levels of 1675 genes, most of them being upregulated. Functional annotation analysis based on GO terms was used to categorize identified genes with regard to swimbladder metabolism or response to the infection. In yellow eels, the most prominent category was ‘immune response’, including various inflammatory components, complement proteins, and immunoglobulins. The elevated expression of several glucose and monocarboxylate transporters indicated an attempt to maintain the level of glucose metabolism, even in due to the infection thickened swimbladder tissue. In silver eel swimbladder tissue, on the contrary, the mRNA levels of only 291 genes were affected. Genes in the categories ‘glucose metabolism’ and ‘ROS metabolism’ barely responded to the infection and even the reaction of the immune system was much less pronounced compared to infected yellow eels. However, in the category ‘extracellular matrix’, the mRNA levels of several mucin genes were strongly elevated, suggesting increased mucus production as a defense reaction against the parasite. The present study revealed a strong reaction to an Anguillicola crassus infection on mRNA expression levels in swimbladder tissue of yellow eels, whereas in silver eels the changes ware almost negligible. A possible explanation for this difference is that the silvering process requires so much energy that there is not much scope to cope with the additional challenge of a nematode infection. Another possible explanation could be that gas-secreting activity of the silver eel swimbladder was largely reduced, which could coincide with a reduced responsiveness to other challenges, like a nematode infection.

Anguillicola crassus Infection Significantly Affects the Silvering Related Modifications in Steady State mRNA Levels in Gas Gland Tissue of the European Eel.

Using Illumina sequencing, transcriptional changes occurring during silvering in swimbladder tissue of the European eel have been analyzed by comparison of yellow and silver eel tissue samples. Functional annotation analysis based on GO terms revealed significant expression changes in a number of genes related to the extracellular matrix, important for the control of gas permeability of the swimbladder, and to reactive oxygen species (ROS) defense, important to cope with ROS generated under hyperbaric oxygen partial pressures. Focusing on swimbladder tissue metabolism, levels of several mRNA species encoding glucose transport proteins were several-fold higher in silver eels, while enzymes of the glycolytic pathway were not affected. The significantly higher steady state level of a transcript encoding for membrane bound carbonic anhydrase, however, suggested that CO2 production in the pentose phosphate shunt and diffusion of CO2 was of particular importance in silver eel swimbladder. In addition, the mRNA level of a large number of genes related to immune response and to sexual maturation was significantly modified in the silver eel swimbladder. The modification of several processes related to protein metabolism and transport, cell cycle, and apoptosis suggested that these changes in swimbladder metabolism and permeability were achieved by increasing cell turn-over. The impact of an infection of the swimbladder with the nematode Anguillicola crassus has been assessed by comparing these expression changes with expression changes observed between uninfected yellow eel swimbladder tissue and infected silver eel swimbladder tissue. In contrast to uninfected silver eel swimbladder tissue, in infected tissue the mRNA level of several glycolytic enzymes was significantly elevated, and with respect to extracellular matrix, several mucin genes were many-fold higher in their mRNA level. Modification of many immune related genes and of the functional categories “response to DNA damage stimulus” and “cellular response to stress” illustrated the damaging effect of the nematode infection. This study has identified a range of cellular processes in the swimbladder of silver eels that appear to be altered by nematode infection. These altered cellular processes could contribute to detrimental changes in swimbladder function that, in turn, may lead to impairment of spawning migration.

Anguillicola crassus impairs the silvering-related enhancements of the ROS defense capacity in swimbladder tissue of the European eel (Anguilla anguilla).

In a process called silvering, European eels prepare for their long-distance migration from European freshwater systems to the Sargasso Sea for reproduction. During this journey, eels perform extended diel vertical migrations, and the concomitant changes in hydrostatic pressure significantly affect the swimbladder, functioning as a buoyancy organ. As the swimbladder is primarily filled with oxygen, the tissue has to cope with extreme hyperoxic conditions, which typically are accompanied by the generation of reactive oxygen species (ROS) and oxidative stress. In addition, since the introduction of the parasitic nematode Anguillicola crassus in the early 1980s, swimbladder function of most of the European eels is impaired by the infection with this parasite. However, the exact pathways to detoxify ROS and how these pathways are affected by silvering or the infection are still unknown. In swimbladder and muscle tissue from uninfected and infected yellow, and from uninfected and infected silver eels, we measured the level of lipid peroxidation, which increases with ROS stress. To assess the capacity of the ROS defense systems, we analyzed the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR), and determined the concentration of the antioxidant glutathione (GSH + GSSG). In swimbladder tissue, we found increased concentrations of GSH + GSSG as well as higher activities of SOD, GPx and GR, suggesting that SOD and the glutathione cycle are important for ROS detoxification. Comparing swimbladder tissue of uninfected yellow with uninfected silver eels, the concentration of GSH + GSSG and the activity of SOD were higher after silvering, corresponding with lower levels of lipid peroxidation. Whereas in yellow eels the infection with A. crassus had no effect, in silver eels the capacity to cope with ROS was significantly impaired. In muscle tissue, silvering or the infection only affected the activity of SOD but in exactly the same way as in swimbladder tissue.Electronic supplementary materialThe online version of this article (doi:10.1007/s00360-016-0994-0) contains supplementary material, which is available to authorized users.

Improved ROS defense in the swimbladder of a facultative air-breathing erythrinid fish, jeju, compared to a non-air-breathing close relative, traira.

The jeju Hoplerythrinus unitaeniatus and the traira Hoplias malabaricus are two closely related erythrinid fish, both possessing a two-chambered physostomous swimbladder. In the jeju the anterior section of the posterior bladder is highly vascularized and the swimbladder is used for aerial respiration; the traira, in turn, is a water-breather that uses the swimbladder as a buoyancy organ and not for aerial oxygen uptake. Observation of the breathing behavior under different levels of water oxygenation revealed that the traira started aquatic surface respiration only under severe hypoxic conditions and did not breathe air. In the jeju air-breathing behavior was observed under normoxic conditions, and the frequency of air-breathing was significantly increased under hypoxic conditions. Unexpectedly, even under hyperoxic conditions (30 mg O2 L−1) the jeju continued to take air breaths, and compared with normoxic conditions the frequency was not reduced. Because the frequently air-exposed swimbladder tissue faces higher oxygen partial pressures than normally experienced by other fish tissues, it was hypothesized that in the facultative air-breathing jeju, swimbladder tissue would have a higher antioxidative capacity than the swimbladder tissue of the water breathing traira. Measurement of total glutathione (GSSG/GSH) concentration in anterior and posterior swimbladder tissue revealed a higher concentration of this antioxidant in swimbladder tissue as compared to muscle tissue in the jeju. Furthermore, the GSSG/GSH concentration in jeju tissues was significantly higher than in traira tissues. Similarly, activities of enzymes involved in the breakdown of reactive oxygen species were significantly higher in the jeju swimbladder as compared to the traira swimbladder. The results show that the jeju, using the swimbladder as an additional breathing organ, has an enhanced antioxidative capacity in the swimbladder as compared to the traira, using the swimbladder only as a buoyancy organ.

The effects of venting and decompression on Yellow Tang (Zebrasoma flavescens) in the marine ornamental aquarium fish trade.

Each year, over 45 countries export 30 million fish from coral reefs as part of the global marine ornamental aquarium trade. This catch volume is partly influenced by collection methods that cause mortality. Barotrauma in fish resulting from forced ascent from depth can contribute to post-collection mortality. However, implementing decompression stops during ascent can prevent barotrauma. Conversely, venting (puncturing the swim bladder to release expanded internal gas) following ascent can mitigate some signs of barotrauma like positive buoyancy. Here, we evaluate how decompression and venting affect stress and mortality in the Yellow Tang (Zebrasoma flavescens). We examined the effects of three ascent treatments, each with decompression stops of varying frequency and duration, coupled with or without venting, on sublethal effects and mortality using histology and serum cortisol measurements. In fish subjected to ascent without decompression stops or venting, a mean post-collection mortality of 6.2% occurred within 24 h of capture. Common collection methods in the fishery, ascent without decompression stops coupled with venting, or one long decompression stop coupled with venting, resulted in no mortality. Histopathologic examination of heart, liver, head kidney, and swim bladder tissues in fish 0d and 21d post-collection revealed no significant barotrauma- or venting-related lesions in any treatment group. Ascent without decompression stops resulted in significantly higher serum cortisol than ascent with many stops, while venting alone did not affect cortisol. Future work should examine links in the supply chain following collection to determine if further handling and transport stressors affect survivorship and sublethal effects.

Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland)

Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm2 tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies.

Characterization of apoptosis induced by grouper iridovirus in two newly established cell lines from barramundi, Lates calcarifer (Bloch)

Two new cell lines have been established from the muscle and swim bladder tissues of barramundi, Lates calcarifer, and designated as BM (barramundi muscle) and BSB (barramundi swimbladder), respectively. The cells multiplied well at 28 degrees C in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum, and have been continuously subcultured more than 100 times to date. Morphologically, BM cells were mostly fibroblastic, whereas BSB were mostly epithelial. Both cell lines were susceptible to grouper iridovirus (GIV) and displayed characteristics of apoptosis after viral infection. The induction of apoptosis was further assayed in GIV-infected BM and BSB cells by various methods. The inhibition of cell growth by GIV was demonstrated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Morphological observations revealed typical apoptotic features in the infected cells, including cell shrinkage and rounding, chromosome condensation and formation of apoptotic body-like vesicles. Chromosome fragmentation was detected by DNA laddering and TUNEL assays. Finally, the appearance of phosphotidylserine on the outer leaflet of apoptotic cell membranes was confirmed by annexin V staining. This is the first report of apoptosis induced by GIV in fish cells.

Temporal and spatial expression of RNases from zebrafish ( Danio rerio)

We have recently isolated and characterized three zebrafish ( Danio rerio) RNases, ZF-RNase-1,-2 and -3, endowed with diverse bioactivities, including microbicidal and angiogenic activities. In the present study we have analyzed their temporal and spatial gene expression profiles . Our results indicate that the three ZF-RNase genes have a differential expression pattern, with ZF-RNase-1 having the most unique and dynamic expression profile. This is characterized by expression in embryonic stages and later on, in larvae, juvenile and adult organisms. In contrast, ZF-RNase-2 and -3 are only expressed either in juvenile or adult organisms. Moreover, analysis of spatial expression of ZF-RNase-1, -2 and -3 detected the three different transcripts in liver, heart, gut and swim bladder tissues. Interestingly, ZF-RNase-1 was the only gene expressed in the brain of embryonic and adult organisms. Collectively, the results suggest that the three ZF-RNases may have potential distinct functional role(s) in zebrafish either during embryonic development and/or later on, in juvenile as well as in adult organisms. Indeed, taking advantage of zebrafish as an excellent viable model to study gene function, this study opens the way to an investigation of the in vivo role(s) of ZF-RNase-1 during embryonic development, as well as, during organogenesis.

Migratory response of European eel (Anguilla anguilla) phagocytes to the eel swimbladder nematode Anguillicola crassus

The migratory response of peripheral blood granulocytes and monocytes from the European eel Anguilla anguilla (L.) to infective larvae of the swimbladder nematode Anguillicola crassus Kuwahara, Niimi and Hagaki, 1974 was examined by means of light microscopical histology and with an in vitro assay using a modified Boyden chamber. Histological examination of experimentally infected eels revealed that, already 8 days postinfection, an infiltration of inflammatory cells around L3 of A. crassus in the swimbladder tissue can occur. In the Boyden chamber, in presence of infective larvae of A. crassus (L3), neutrophil granulocytes and monocytes showed a higher migration activity than in the absence of L3. In conclusion, infection of European eels with A. crassus leads to an activation of the defence cells resulting in an increased migration activity compared to uninfected eels.

Establishment, characterization, and viral susceptibility of two cell lines derived from goldfish Carassius auratus muscle and swim bladder

Goldfish Carassius auratus are common aquarium fish and have a significant economic and research value, having considerable worth to fisheries as a baitfish and the ability to adapt to a range of habitats. Two cell lines were established from goldfish muscle and swim bladder tissue, in order to create a biological monitoring tool for viral diseases. Cell lines were optimally maintained at 30 degrees C in Leibovitz-15 medium supplemented with 20% fetal bovine serum. Propagation of goldfish cells was serum dependent, with a low plating efficiency (>16%). Karyotyping analysis indicated that both cell lines remained diploid, with a mean chromosomal count of 104. Results of viral challenge assays revealed that both cell lines shared similar patterns of viral susceptibility and production to infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, snakehead rhabdovirus, and spring viremia carp virus. Both cell lines demonstrated a higher sensitivity and significantly larger viral production than control brown bullhead cells for channel catfish virus. These newly established cell lines will be used as a diagnostic tool for viral diseases in this fish species and also for the isolation and study of goldfish viruses in the future.

The Origin and Evolution of the Surfactant System in Fish: Insights into the Evolution of Lungs and Swim Bladders

Several times throughout their radiation fish have evolved either lungs or swim bladders as gas-holding structures. Lungs and swim bladders have different ontogenetic origins and can be used either for buoyancy or as an accessory respiratory organ. Therefore, the presence of air-filled bladders or lungs in different groups of fishes is an example of convergent evolution. We propose that air breathing could not occur without the presence of a surfactant system and suggest that this system may have originated in epithelial cells lining the pharynx. Here we present new data on the surfactant system in swim bladders of three teleost fish (the air-breathing pirarucu Arapaima gigas and tarpon Megalops cyprinoides and the non-air-breathing New Zealand snapper Pagrus auratus). We determined the presence of surfactant using biochemical, biophysical, and morphological analyses and determined homology using immunohistochemical analysis of the surfactant proteins (SPs). We relate the presence and structure of the surfactant system to those previously described in the swim bladders of another teleost, the goldfish, and those of the air-breathing organs of the other members of the Osteichthyes, the more primitive air-breathing Actinopterygii and the Sarcopterygii. Snapper and tarpon swim bladders are lined with squamous and cuboidal epithelial cells, respectively, containing membrane-bound lamellar bodies. Phosphatidylcholine dominates the phospholipid (PL) profile of lavage material from all fish analyzed to date. The presence of the characteristic surfactant lipids in pirarucu and tarpon, lamellar bodies in tarpon and snapper, SP-B in tarpon and pirarucu lavage, and SPs (A, B, and D) in swim bladder tissue of the tarpon provide strong evidence that the surfactant system of teleosts is homologous with that of other fish and of tetrapods. This study is the first demonstration of the presence of SP-D in the air-breathing organs of nonmammalian species and SP-B in actinopterygian fishes. The extremely high cholesterol/disaturated PL and cholesterol/PL ratios of surfactant extracted from tarpon and pirarucu bladders and the poor surface activity of tarpon surfactant are characteristics of the surfactant system in other fishes. Despite the paraphyletic phylogeny of the Osteichthyes, their surfactant is uniform in composition and may represent the vertebrate protosurfactant.

Development of the swimbladder in the European eel ( Anguilla anguilla).

The swimbladder of the adult eel, Anguilla anguilla, with its bipolar countercurrent system, the rete mirabile, is a widely used model for swimbladder function, but very little is known about the development of this swimbladder. Our histological studies on the developing swimbladder revealed that during metamorphosis the swimbladder becomes present as a dorsal outgrowth of the esophagus. It is filled with surfactant, and gas was not detected in the swimbladder. In the young glass-eel, the epithelial (gas gland) cells of the swimbladder are columnar, but do not yet have the typical basolateral labyrinth established in adult animals. Few blood vessels are found in the swimbladder tissue, and the submucosa is present as a thick layer of connective tissue, giving a large diffusion distance between blood vessel and swimbladder lumen. Within the next 2 or 3 months of development, gas gland cells develop their typical basolateral labyrinth, and the thickness of the submucosa is significantly reduced, resulting in a short diffusion distance between blood vessels and the swimbladder lumen. The first filling of the swimbladder with gas is observed while the gas gland cells are still in a poorly differentiated status and it appears unlikely that these cells can accomplish their typical role in gas deposition. The presence of small gas bubbles in the swimbladder as well as in the ductus pneumaticus at the time of initial swimbladder inflation suggests that the swimbladder is filled by air gulping or possibly by taking up gas bubbles from the water.

Swim bladder gas gland cells produce surfactant: in vivo and in culture.

Electron microscopical examination of gas gland cells of the physostome European eel (Anguilla anguilla) and of the physoclist perch (Perca fluviatilis) revealed the presence of significant numbers of lamellar bodies, which are known to be involved in surfactant secretion. In the perch, in which the gas gland is a compact structure and gas gland cells are connected to the swim bladder lumen via small canals, lamellar bodies were also found in flattened cells forming the swim bladder epithelium. Flat epithelial cells are absent in the eel swim bladder, in which the whole epithelium consists of cuboidal gas gland cells. In both species, Western blot analysis using specific antibodies to human surfactant protein A (SP-A) showed a cross-reaction with swim bladder tissue homogenate proteins of approximately 65 kDa and in the eel occasionally of approximately 120 kDa, probably representing SP-A-like proteins in a dimeric and a tetrameric state. An additional band was observed at approximately 45 kDa. Western blots using antibodies to rat SP-D again resulted in a single band at approximately 45 kDa in both species, suggesting that there might be a cross-reaction of the antibody to human SP-A with an SP-D-like protein of the swim bladder tissue. To localize the surfactant protein, eel gas gland cells were cultured on permeable supports. Under these conditions, the gas gland cells regain their characteristic polarity. Electron microscopy confirmed the presence of lamellar bodies in cultured cells, and occasionally, exocytotic events were observed. Immunohistochemical staining using an antibody to human SP-A demonstrated the presence of surfactant protein only in luminal membranes and in adjacent lateral membranes. Only occasionally, evidence was found for the presence of surfactant protein in lamellar bodies.

Infection of the glass-eel swimbladder with the nematode Anguillicola crassus.

The ability of the nematode Anguillicola crassus to infect eel larvae (glass-eel stage) was tested. The results show that glass-eels fed on infected copepods, the natural intermediate host of the nematode, can be infected. Light microscopical examination of the infected developing swimbladder tissue revealed that the infection results in a significant thickening of the connective tissue. The basolateral labyrinth of gas gland cells is very much reduced in infected swimbladders, and the distance of gas gland cells to blood capillaries is enlarged. Critical swimming speed, defined as the speed where the larvae were no longer able to swim against the current, was similar in infected and uninfected animals. At intermediate speeds (about 60-80% of critical swimming speed) infected eels showed a slightly higher swimming activity than control animals. Resting oxygen consumption, measured as an index of metabolic activity, within the first 2 months of infection was higher in control animals, which may be due to a reduced rate of activity in infected glass-eels. By 4-5 months after the infection, however, it was significantly higher in infected animals. This may indicate that at this stage a higher activity of the animals is required to compensate for the increase in body density, but swimming performance of infected and non-infected glass-eels was not significantly different. Oxygen consumption during swimming activity, measured in a swim tunnel at 50% of maximal swimming speed, also was not affected. The results thus show that even glass-eels can be infected with A. crassus, and this probably contributes to the rapid spread of the nematode in Europe. While aerobic metabolism during swimming activity is not affected at this stage of infection, the swimbladder tissue shows severe histological changes, which most likely will impair swimbladder function.

Vasodilation of swimbladder vessels in the european eel (Anguilla anguilla) induced by vasoactive intestinal polypeptide, nitric oxide, adenosine and protons

The effects of &bgr;-adrenergic stimulation, vasoactive intestinal polypeptide (VIP), adenosine, the nitric oxide (NO)-releasing agent sodium nitroprusside and of metabolic end-products of gas gland cell metabolism on swimbladder blood flow were investigated using saline- or blood-perfused swimbladder preparations of the freshwater European eel Anguilla anguilla. While &bgr;-adrenergic vasodilation was not detectable, a bolus injection of adenosine (100 microl, 10(-)7 mol l-1) and application of VIP (10(-)7 mol kg-1) caused a significant decrease in perfusion pressure in saline-perfused swimbladder preparations. Immunohistochemical analysis revealed the presence of VIP-immunoreactive nerve fibres in the swimbladder artery and in the swimbladder vein (seawater-adapted eels were used for immunohistochemical studies). Application of sodium nitroprusside also elicited a small, but significant, decrease in perfusion pressure in saline-perfused swimbladder preparations, while preincubation of swimbladder tissue with N(&ohgr;)nitro-l-arginine, a non-selective inhibitor of nitric oxide synthase, significantly enhanced the flow-induced increase in perfusion pressure. Lactate, the major metabolic end-product of gas gland cell metabolism, had no effect on perfusion pressure. In contrast, an increase in proton concentration in both saline- and blood-perfused preparations induced a vasodilation, as indicated by a significant decrease in perfusion pressure. The results demonstrate that VIP, NO, adenosine and protons may induce a vasodilation of swimbladder blood vessels. None of these effects, however, compares in time span with the previously described immediate, short-lasting vasodilation of swimbladder vessels elicited by pulse stimulation of the vagus nerve.