Gills Of Teleost Fish Research Articles

Gill-associated ammonia oxidizers are widespread in teleost fish.

Recent advances in sequencing methods have greatly expanded the knowledge of teleost-associated microorganisms. While fish-gut microbiomes are comparatively well studied, less attention has gone toward other, external organ-microbiome associations. Gills are particularly interesting to investigate due to their functions in gas exchange, osmoregulation, and nitrogen excretion. We recently discovered a branchial symbiosis between nitrogen-cycling bacteria and teleosts (zebrafish and carp), in which ammonia-oxidizing Nitrosomonas and denitrifying bacteria together convert toxic ammonia excreted by the fish into harmless dinitrogen (N2) gas. This symbiosis can function as a "natural biofilter" in fish gills and can potentially occur in all ammonotelic fish species, but it remains unknown how widespread this symbiosis is. In this study, we analyzed all publicly available gill microbiome data sets and checked for the presence of Nitrosomonas. We discovered that more than half of the described fish gill microbiomes contain 16S rRNA gene sequences of ammonia-oxidizing bacteria (AOB). The presence of gill-specific AOB was shown in both wild and aquacultured fish, as well as in marine and freshwater fish species. Based on these findings, we propose that ammonia oxidizers are widespread in teleost fish gills. These gill-associated AOB can significantly affect fish nitrogen excretion, and the widespread nature of this association suggests that the gill-associated AOB can have similar impacts on more fish species. Future research should address the contribution of these microorganisms to fish nitrogen metabolism and the fundamental characteristics of this novel symbiosis.IMPORTANCERecent advances in sequencing have increased our knowledge of teleost-associated microbiota, but the gill microbiome has received comparatively little attention. We recently discovered a consortium of nitrogen-cycling bacteria in the gills of common carp and zebrafish, which are able to convert (toxic) ammonia into harmless dinitrogen gas. These microorganisms thus function as a natural nitrogen biofilter. We analyzed all available gill microbiome data sets to determine how widespread gill-associated ammonia-oxidizing bacteria (AOB) are. More than half of the data sets contained AOB, representing both aquacultured and wild fish from freshwater and marine habitats. In total, 182 amplicon sequencing variants were obtained, of which 115 were found specifically in the gills and not the environmental microbiomes. As gill-associated AOB are apparently widespread in teleost fish, it is important to study their impact on host nitrogen excretion and the potential to reduce ammonia accumulation in (recirculating) aquaculture of relevant fish species.

Trace metals in the teleost fish gill: biological roles, uptake regulation, and detoxification mechanisms.

In fish, the gill plays a vital role in regulating the absorption of trace metals and is also highly susceptible to metal toxicity. Trace metals such as iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn) are involved in various catalytic activities and molecular binding within the gill, thereby supporting a range of physiological processes in this organ. While beneficial at normal levels, these metals can become toxic when present in excess. Conversely, nonessential metals like cadmium (Cd) and lead (Pb) can gain entry into gill cells through similar metal transport pathways, potentially interfering with various cellular processes. The transepithelial transport of these metals across the gill epithelium is governed by a variety of metal transport and metal binding proteins. These include the Cu transporter 1 (CTR1), divalent metal transporter 1 (DMT1), and members of the Zrt-/Irt-like protein (ZIP) and zinc transport (ZnT) families. Additionally, some of these metals can compete with major ions (e.g., calcium, sodium) for absorption sites in the gill. This complex crosstalk suggests an interdependent mechanism that balances metal uptake to meet physiological needs while preventing excessive accumulation. In this article, I review the roles of trace metals in proteins/enzymes that support the different functions in the gill of teleost fish. I also discuss current understanding of the pathways involved in regulating the branchial uptake of metals and their influence on ionic regulation, and the potential detoxification mechanisms in the gill. Finally, I summarize knowledge gaps and potential areas for further investigation.

Genetic characterisation of four Lamproglena spp. (Copepoda, Lernaeidae) from Africa and the first mitochondrial data.

Females of species of Lamproglena von Nordmann, 1832 are parasitic on the gills of teleost fishes and the 38 nominal species are based on mainly morphological data. Only four of these species have been genetically characterised and no mitochondrial data are available for the genus. The present study aimed to provide representative ribosomal DNA (rDNA) data for two additional species of Lamproglena from Africa: Lamproglena clariae Fryer, 1956 and Lamproglena hoi Dippenaar, Luus-Powell et Roux, 2001, alongside mitochondrial DNA (mtDNA) for these and two other African species, Lamproglena hemprichii von Nordmann, 1832 and Lamproglena monodi Capart, 1944. The four species were collected from Clariidae, Cyprinidae, Alestidae and Cichlidae, respectively. Representative 18S rDNA and 28S rDNA data were obtained for L. clariae and L. hoi, while cox1 mtDNA was obtained for all four species. The respective haplotypes supported the distinctness of all species using all three gene regions investigated. Interestingly, species appeared to be grouped more by geographical origin than host family, with L. hoi more closely related to other African species than to Asian species also collected from cyprinid hosts. Even though the results presented here greatly add to the molecular data available for Lamproglena, there are still 32 (>80%) species for which no genetic data are available. The interpretation of the results presented here is thus preliminary and much more data are required before the phylogeny of this genus, and other members of the family, such as Lernaea Linnaeus, 1758, can be studied appropriately.

Gill Transcriptomic Responses to Toxin-producing Alga Prymnesium parvum in Rainbow Trout.

The gill of teleost fish is a multifunctional organ involved in many physiological processes, including protection of the mucosal gill surface against pathogens and other environmental antigens by the gill-associated lymphoid tissue (GIALT). Climate change associated phenomena, such as increasing frequency and magnitude of harmful algal blooms (HABs) put extra strain on gill function, contributing to enhanced fish mortality and fish kills. However, the molecular basis of the HAB-induced gill injury remains largely unknown due to the lack of high-throughput transcriptomic studies performed on teleost fish in laboratory conditions. We used juvenile rainbow trout (Oncorhynchus mykiss) to investigate the transcriptomic responses of the gill tissue to two (high and low) sublethal densities of the toxin-producing alga Prymnesium parvum, in relation to non-exposed control fish. The exposure time to P. parvum (4–5 h) was sufficient to identify three different phenotypic responses among the exposed fish, enabling us to focus on the common gill transcriptomic responses to P. parvum that were independent of dose and phenotype. The inspection of common differentially expressed genes (DEGs), canonical pathways, upstream regulators and downstream effects pointed towards P. parvum-induced inflammatory response and gill inflammation driven by alterations of Acute Phase Response Signalling, IL-6 Signalling, IL-10 Signalling, Role of PKR in Interferon Induction and Antiviral Response, IL-8 Signalling and IL-17 Signalling pathways. While we could not determine if the inferred gill inflammation was progressing or resolving, our study clearly suggests that P. parvum blooms may contribute to the serious gill disorders in fish. By providing insights into the gill transcriptomic responses to toxin-producing P. parvum in teleost fish, our research opens new avenues for investigating how to monitor and mitigate toxicity of HABs before they become lethal.

Prolactin Influences Different Aspects of Fish Biology

Prolactin (PRL) is a 198 amino acid long polypeptide hormone, structurally similar to growth hormone, secreted by adenohypophysis in the vertebrates. The full-length amino acid sequence of PRL has been determined from a variety of teleost and non-teleost fish. Teleost PRL genes are shorter than mammalian. PRL exists in two forms, namely, PRL-1 commonly found in all vertebrates and PRL-2 found in several non-mammalian vertebrates including fish. This hormone binds to transmembrane Prolactin receptor (PRLR) which is a member of the class 1 cytokines receptor superfamily. We know that PRL has over 300 different functions in vertebrates. In fish, PRL plays an important role in migration, osmoregulation, parental behaviour, reproduction and development along with other hormones. Recent studies show prolactin has a role in cell proliferation in the gills of teleost fish. This review highlights diversified role of PRL in fish which may serve as avenues for further researches in near future.

Integration of Transcriptome, Gross Morphology and Histopathology in the Gill of Sea Farmed Atlantic Salmon (Salmo salar): Lessons From Multi-Site Sampling.

The gill of teleost fish is a multifunctional organ involved in many physiological processes such as gas exchange, osmotic and ionic regulation, acid-base balance and excretion of nitrogenous waste. Due to its extensive interface with the environment, the gill plays a key role as a primary mucosal defense tissue against pathogens, as manifested by the presence of the gill-associated lymphoid tissue (GIALT). In recent years, the prevalence of multifactorial gill pathologies has increased significantly, causing substantial losses in Atlantic salmon aquaculture. The transition from healthy to unhealthy gill phenotypes and the progression of multifactorial gill pathologies, such as proliferative gill disease (PGD), proliferative gill inflammation (PGI) and complex gill disorder (CGD), are commonly characterized by epithelial hyperplasia, lamellar fusion and inflammation. Routine monitoring for PGD relies on visual inspection and non-invasive scoring of the gill tissue (gross morphology), coupled with histopathological examination of gill sections. To explore the underlying molecular events that are associated with the progression of PGD, we sampled Atlantic salmon from three different marine production sites in Scotland and examined the gill tissue at three different levels of organization: gross morphology with the use of PGD scores (macroscopic examination), whole transcriptome (gene expression by RNA-seq) and histopathology (microscopic examination). Our results strongly suggested that the changes in PGD scores of the gill tissue were not associated with the changes in gene expression or histopathology. In contrast, integration of the gill RNA-seq data with the gill histopathology enabled us to identify common gene expression patterns associated with multifactorial gill disease, independently from the origin of samples. We demonstrated that the gene expression patterns associated with multifactorial gill disease were dominated by two processes: a range of immune responses driven by pro-inflammatory cytokines and the events associated with tissue damage and repair, driven by caspases and angiogenin.

A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD)

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.

Approximation of immune response abilities in salmonid gills using the RTgill-W1 cell line as an in vitro model

Teleost fish gills represent a considerable exchange surface with the external environment. Our group has previously presented evidence regarding the expression and detection of regulatory and effector molecules of the immune response system in salmonid gills. This observation is supported by increased macrophage infiltration in this organ. However, it is probable that gill epithelial tissue also possesses immunomodulatory abilities. The RTgill-W1 cell line presents an epithelial morphology and forms tight monolayer sheets, thereby representing a key model for understanding how fish gills are able to identify the presence of microorganisms and trigger the immune response. The aim of this study was to determine if the epithelial cells of salmonid gills are able to detect pathogen associated molecular patterns (PAMPS) through intracellular receptors, using the activation of intracellular signaling intermediaries to secrete immunomodulatory cytokines. The RTgill-W1 line expressed TNFα, IL-1β, and type 1 IFN in response to stimulation with LPS and Poly I:C. Knockdown of the NOD-like receptor (NLRC5) with siRNA (siNLRC5) decreased the expression of IFN-α/β in RTgill-W1 cells treated with Poly I:C. In turn, knockdown of the TRIM protein (siTRIM3) in cells treated with LPS resulted in the downregulation of TNFα and IL-1β. Additionally, the cell line responded to rIFN-γ stimulation by upregulating TRIM8, a facilitator of the JAK/STAT pathway. These data provide interesting evidence regarding the immune function of the gill epithelium. This studied was financed through Proyecto FONDECYT N° 1140797

Phosphorylation increases the catalytic activity of rainbow trout gill cytosolic carbonic anhydrase.

Cytoplasmic carbonic anhydrase (CAc) in the gill of teleost fish contributes to ionic regulation and acid–base balance by catalyzing the reversible reaction of CO2 and water, CO2 + H2O ↔ H(+) + HCO3(-). Regulation of CAc abundance and activity therefore is expected to fine-tune responses to ionic or acid–base challenges. The present study investigated the potential for gill CAc of rainbow trout, Oncorhynchus mykiss (tCAc), to undergo reversible phosphorylation. The activity of tCAc was approximately doubled by phosphorylation achieved through in vitro stimulation of endogenous protein kinases; kinase stimulation doubled phospho-threonine content from that observed in tCAc isolated under conditions where both kinases and protein phosphatases were inhibited. In vitro incubation to preferentially stimulate specific kinases implicated protein kinase G (PKG) in mediating the increase in tCAc activity. The kinetic parameters of turnover number (k cat) and substrate affinity (K m) were similarly affected by stimulation of either kinase or phosphatase action. However, phosphorylation via kinase stimulation significantly increased the efficiency of tCAc (V max /K m), and this factor may have contributed to the elevation of tCAc activity. In addition, phosphorylation of tCAc by kinase stimulation significantly increased the inhibition constant (K i) for acetazolamide. These results demonstrate that tCAc is subject to reversible phosphorylation; future work should focus on identifying the physiological situation(s) in which phosphorylation of trout branchial CAc occurs.

Hydrodynamic resistance and flow patterns in the gills of a tilapine fish

The gills of teleost fishes are often discussed as an archetypal counter-current exchange system, capable of supporting the relatively high metabolic rates of some fishes despite the low oxygen solubility of water. Despite an appreciation for the physiology of exchange at the gills, many questions remain regarding the hydrodynamical basis of ventilation in teleost fishes. In this study, the hydrodynamic resistance and flow fields around the isolated gills of a tilapia, Oreochromis mossambicus, were measured as a function of the applied pressure head. At ventilatory pressures typical of a fish at rest, the hydrodynamic resistance of the gills was nearly constant, the flow was laminar, shunting of water around the gills was essentially absent, and the distribution of water flow was relatively uniform. However, at the higher pressures typical of an active or stressed fish, some of these qualities were lost. In particular, at elevated pressures there was a decrease in the hydrodynamic resistance of the gills and substantial shunting of water around the gills. These effects suggest mechanical limits to maximum aerobic performance during activity or under adverse environmental conditions.

Implication du gène Fhit dans la régulation de l’invasion tumorale

This short review traces the history of in vitro experimental methods that have been used to help elucidate the ion transport mechanisms of teleost fish gills. It begins with an isolated gill preparation published by Denis Bellamy in 1961 and progresses through many different approaches and concludes with current techniques. Among them are perfused gill arches, primary cultures of gill epithelia, isolated opercular skin preparations, whole embryos in vitro, the yolk-ball technique, dissociated gill epithelial cells, vibrating microprobe and scanning ion-selective microelectrodes; currently all are combined with molecular biological techniques. Each new approach brought new findings but is subject to certain limitations and each has contributed significantly to this important subfield of comparative physiology.

Stanniocalcin Has Deep Evolutionary Roots in Eukaryotes

Vertebrates have a large glycoprotein hormone, stanniocalcin, which originally was shown to inhibit calcium uptake from the environment in teleost fish gills. Later, humans, other mammals, and teleost fish were shown to have two forms of stanniocalcin (STC1 and STC2) that were widely distributed in many tissues. STC1 is associated with calcium and phosphate homeostasis and STC2 with phosphate, but their receptors and signaling pathways have not been elucidated. We undertook a phylogenetic investigation of stanniocalcin beyond the vertebrates using a combination of BLAST and HMMER homology searches in protein, genomic, and expressed sequence tag databases. We identified novel STC homologs in a diverse array of multicellular and unicellular organisms. Within the eukaryotes, almost all major taxonomic groups except plants and algae have STC homologs, although some groups like echinoderms and arthropods lack STC genes. The critical structural feature for recognition of stanniocalcins was the conserved pattern of ten cysteines, even though the amino acid sequence identity was low. Signal peptides in STC sequences suggest they are secreted from the cell of synthesis. The role of glycosylation signals and additional cysteines is not yet clear, although the 11th cysteine, if present, has been shown to form homodimers in some vertebrates. We predict that large secreted stanniocalcin homologs appeared in evolution as early as single-celled eukaryotes. Stanniocalcin's tertiary structure with five disulfide bonds and its primary structure with modest amino acid conservation currently lack an established receptor-signaling system, although we suggest possible alternatives.

Mechanosensitive signalling in fish gill and other ion transporting epithelia

Epithelia involved in vectorial salt transport respond to apical and basolateral changes in osmotic activity by moderating the transmural solute transport rate simultaneously with underlying volume regulatory mechanisms involved in regulatory volume increase (RVI) and decrease (RVD). This review examines rapid osmotic responses in salt secreting epithelia of marine and euryhaline teleost fish, with inclusion of recent results from other ion transporting epithelia that also respond rapidly to osmotic shock. Mitochondrion-rich chloride secreting cells of marine teleost fish gills and skin, when exposed to hypertonic shock, activate NaCl secretion via phosphorylation of Na(+), K(+), 2Cl(-) cotransporter (NKCC1) in the basolateral membrane and activation of anion channels in the apical membrane. Conversely, NaCl secretion is inhibited when chloride secreting cells are swollen osmotically. Mammalian airway epithelial cells also possess NKCC1 basally and apical anion channels [Cystic Fibrosis Transmembrane conductance Regulator (CFTR)]; with hypotonic shock, this epithelium releases ATP and NaCl secretion is stimulated via purinergic receptors, while hypertonic shock inhibits Na(+) uptake. In the eye, the ciliary epithelium activates Cl(-) channels in response to hypotonic shock as RVD, an effect that modulates transepithelial fluid transport rates. In the renal A6 cell line, K(+) and Cl(-) effluxes activate during RVD and RVI Na(+) transepithelial absorption. A common theme in these systems is ATP release in hypotonic shock with subsequent RVD-effective mechanisms such as NKCC1 inhibition and K(+) and Cl(-) efflux, but there are different effects of osmotic changes on transepithelial transport, apparently depending on the role of the epithelial system.

Expression of Ol-foxi3 and Na +/K +-ATPase in ionocytes during the development of euryhaline medaka ( Oryzias latipes) embryos

Osmoregulation is a vital function that is essential to all vertebrates. Ionocytes are epithelial cells responsible for this function and have been extensively studied in adult teleost fish gills. The euryhaline medaka ( Oryzias latipes) has recently emerged as an investigative model because of its ability to acclimatize easily to water presenting various salinities. However, no studies to date have focused on the development of ionocytes in medaka embryos. We first analyzed the distribution of ionocytes in the skin and gills during development, using a specific marker of differentiated ionocytes (the Na +/K +-ATPase pump, or NKA). Strikingly, we were able to identify two ionocyte domains on the yolk surface ectoderm, that we named the Vitellin Zone (VZ) and the Lateral Zone (LZ). In zebrafish, ionocyte differentiation has been shown to be controlled by two forkhead-box genes, foxi3a and foxi3b. We cloned the medaka foxi3 ortholog which appeared to be highly similar to foxi3b. Whole-mount in situ hybridizations performed on medaka embryos revealed that Ol-foxi3 is expressed in differentiated ionocytes of the pharyngeal endoderm, the branchial arches and the yolk epidermis, as well as in epibranchial placode territories. We further focused on the expression patterns of the yolk epidermis and compared the expression of Ol-foxi3 with that of the non-neural progenitor marker p63. We evidenced that Ol-foxi3 is expressed in progenitor cells which are first of all located uniformly in the VZ and then transitorily clustered in the LZ. Taken together, these data contribute to a clearer understanding of osmoregulatory tissue ontogenesis in euryhaline fish.

Immunolocalization of Na +/K +–ATPase and Na +/K +/2Cl − cotransporter in the tubular epithelia of sea snake salt glands

The sublingual salt gland is the primary site of salt excretion in sea snakes; however, little is known about the mechanisms mediating ion excretion. Na +/K +–ATPase (NKA) and Na +/K +/2Cl − cotransporter (NKCC) are two proteins known to regulate membrane potential and drive salt secretion in most vertebrate secretory cells. We hypothesized that NKA and NKCC would localize to the basolateral membranes of the principal cells comprising the tubular epithelia of sea snake salt glands. Although there is evidence of NKA activity in salt glands from several species of sea snake, the localization of NKA and NKCC and other potential ion transporters remains unstudied. Using histology and immunohistochemistry, we localized NKA and NKCC in salt glands from three species of laticaudine sea snake: Laticauda semifasciata, L. laticaudata, and L. colubrina. Antibody specificity was confirmed using Western blots. The compound tubular glands of all three species were found to be composed of serous secretory epithelia, and NKA and NKCC were abundant in the basolateral membranes. These results are consistent with the morphology of secretory epithelia found in the rectal salt glands of marine elasmobranchs, the nasal glands of marine birds and the gills of teleost fishes, suggesting a similar function in regulating ion secretion.

Comparative immunolocalization of Na+/K+‐ATPase and Na+/K+/2Cl− cotransporter in the kidneys of freshwater and marine snakes

In contrast with the marine sea snakes, watersnakes in the genus Nerodia are not known to have functional salt glands. Despite this physiological difference, at least two species of watersnake are known to inhabit marine environments; the remaining members of this genus occupy freshwater habitats. The mechanism by which marine watersnakes regulate ion balance without functional salt glands is unknown. In this study, we compared the cellular anatomy of kidneys from two species of watersnake, Nerodia fasciata (freshwater) and N. clarkii (marine), and one species of sea snake, Laticauda semifasciata (marine). Specifically, we examined the localization of Na+/K+‐ATPase (NKA) and Na+/K+/2Cl− cotransporter (NKCC), two proteins that are known to regulate membrane potential and drive salt secretion in all vertebrate secretory cells examined to date. Using histology and immunohistochemistry, we localized NKA and NKCC to the basolateral membranes of the distal tubules in all three species of snake. These results are consistent with the localization of these proteins in other secretory tissues such as the gills of teleost fishes and the salt glands of marine elasmobranchs, birds, and reptiles, and suggest a potential role for ion regulation at the level of the kidney. This research was funded by the National Geographic Society (8058‐06 to HBL) and the National Science Foundation (IOB‐0519579 to DHE).

Área mitocondrial relativa nos epitélios branquial e renal do baiacu estuarino tropical <i>Sphoeroides testudineus </i>após aclimatação a água do mar isosmótica

The gills of teleost fishes are responsible both for gas exchangein respiration and salt transport in osmoregulation (JOBLING, 1995; ZADUNAISKY, 1996; PERRY, 1997; VAN DER HEIJDEN et al., 1997; EVANS et al., 1999; EVANS et al., 2005). In marine teleosts the gill epithelium secretes salt mainly through chloride cells (JOBLING, 1995; ZADUNAISKY, 1996; PERRY, 1997; VAN DER HEIJDEN et al., 1997; FERNANDES et al., 1998; EVANS et al., 2005). These cells are typically located between secondary lamellae at their insertion in the gill filament (inter-lamellar region) or in the gill filament itself (LAURENT & DUNEL, 1980; PERRY, 1997; FERNANDES et al., 1998; EVANS et al., 2005). These cells are rounded, display abundant mitochondria, a tubular system of endomembranes, sub-apical vesicles, and extensive intercellular junctional complexes (LAURENT & DUNEL, 1980; JOBLING, 1995; ZADUNAISKY, 1996; PERRy, 1997; EVANS et al., 2005).

Cytoplasmic carbonic anhydrase isozymes in rainbow troutOncorhynchus mykiss: comparative physiology and molecular evolution

It is well established that the gills of teleost fish contain substantial levels of cytoplasmic carbonic anhydrase (CA), but it is unclear which CA isozyme(s) might be responsible for this activity. The objective of the current study was to determine if branchial CA activity in rainbow trout was the result of a general cytoplasmic CA isozyme, with kinetic properties, tissue distribution and physiological functions distinct from those of the red blood cell (rbc)-specific CA isozyme. Isolation and sequencing of a second trout cytoplasmic CA yielded a 780 bp coding region that was 76% identical with the trout rbc CA (TCAb), although the active sites differed by only 1 amino acid. Interestingly, phylogenetic analyses did not group these two isozymes closely together, suggesting that more fish species may have multiple cytoplasmic CA isozymes. In contrast to TCAb, the second cytoplasmic CA isozyme had a wide tissue distribution with high expression in the gills and brain, and lower expression in many tissues, including the red blood cells. Thus, unlike TCAb, the second isozyme lacks tissue specificity and may be expressed in the cytoplasm of all cells. For this reason, it is referred to hereafter as TCAc (trout cytoplasmic CA). The inhibitor properties of both cytoplasmic isozymes were similar (Ki acetazolamide 1.21+/-0.18 nmol l(-1) and 1.34+/-0.10 nmol l(-1) for TCAc and TCAb, respectively). However, the turnover of TCAb was over three times greater than that of TCAc (30.3+/-5.83 vs 8.90+/-1.95 e4 s(-1), respectively), indicating that the rbc-specific CA isoform was significantly faster than the general cytoplasmic isoform. Induction of anaemia revealed differential expression of the two isozymes in the red blood cell; whereas TCAc mRNA expression was unaffected, TCAb mRNA expression was significantly increased by 30- to 60-fold in anaemic trout.

Na(+), Cl(-), Ca(2+) and Zn(2+) transport by fish gills: retrospective review and prospective synthesis.

The secondary active Cl(-) secretion in seawater (SW) teleost fish gills and elasmobranch rectal gland involves basolateral Na(+),K(+)-ATPase and NKCC, apical membrane CFTR anion channels, and a paracellular Na(+)-selective conductance. In freshwater (FW) teleost gill, the mechanism of NaCl uptake is more controversial and involves apical V-type H(+)-ATPase linked to an apical Na(+) channel, apical Cl(-)-HCO-3 exchange and basolateral Na(+),K(+)-ATPase. Ca(2+) uptake (in FW and SW) is via Ca(2+) channels in the apical membrane and Ca(2+)-ATPase in the basolateral membrane. Mainly this transport occurs in mitochondria rich (MR) chloride cells, but there is a role for the pavement cells also. Future research will likely expand in two major directions, molded by methodology: first in physiological genomics of all the transporters, including their expression, trafficking, operation, and regulation at the molecular level, and second in biotelemetry to examine multivariable components in behavioral physiological ecology, thus widening the integration of physiology from the molecular to the environmental levels while deepening understanding at all levels.

A view of early vertebrate evolution inferred from the phylogeny of polystome parasites (Monogenea: Polystomatidae).

The Polystomatidae is the only family within the Monogenea to parasitize sarcopterygians such as the Australian lungfish Neoceratodus poisteri and freshwater tetrapods (lissamphibians and chelonians). We present a phylogeny based on partial 18S rDNA sequences of 26 species of Polystomatidae and three taxon from the infrasubclass Oligonchoinea (= Polyopisthocotylea) obtained from the gills of teleost fishes. The basal position of the polystome from lungfish within the Polystomatidae suggests that the family arose during the evolutionary transition between actinopterygians and sarcopterygians, ca. 425 million years (Myr) ago. The monophyly of the polystomatid lineages from chelonian and lissamphibian hosts, in addition to estimates of the divergence times, indicate that polystomatids from turtles radiated ca. 191 Myr ago, following a switch from an aquatic amniote presumed to be extinct to turtles, which diversified in the Upper Triassic. Within polystomatids from lissamphibians, we observe a polytomy of four lineages, namely caudatan, neobatrachian, pelobatid and pipid polystomatid lineages, which occurred ca. 246 Myr ago according to molecular divergence-time estimates. This suggests that the first polystomatids of amphibians originated during the evolution and diversification of lissamphibian orders and suborders ca. 250 Myr ago. Finally, we report a vicariance event between two major groups of neobatrachian polystomes, which is probably linked to the separation of South America from Africa ca. 100 Myr ago.