The role of aquaporin 3 in teleost fish

The aquaporin isoform, AQP3 has now been identified in a number of different teleost fish species, with additional DNA sequence information on AQP3 genes in further fish species available in genome databases. In zebrafish (Danio rerio), the AQP3 gene is present as two duplicate isoforms resulting from a teleostean fish genome-wide duplication. A further splicoform/isoform has also been identified in rainbow trout (Oncorhynchus mykiss). The identification of these AQP3 isoforms in other fish species is consequently explored. The role of AQP3 in physiological/osmoregulatory processes, in various teleost organs is then described. In teleost gill, AQP3 is expressed in 'chloride' cells, and in some species, in other epithelial cell types, where it may have a number of different functions including the prevention of dehydration. In eel esophagus, immunohistochemistry shows that AQP3 is expressed in surface epithelial cells in the anterior esophagus, but in mucus cells within the epithelium of the posterior esophagus. In eel intestine, AQP3 is found in macrophage-like cells and probably plays no part in osmoregulatory processes. In the rectum, as in the posterior esophagus AQP3 is expressed in mucus cells. In eel kidney, AQP3 is expressed in a subset of renal tubules, and localizes to the apical pole of tubule cells. There is no apparent change in the location or protein abundance of renal AQP3 following the acclimation of eels from freshwater to seawater.     

Genome evolution and biodiversity in teleost fish

Teleost fish, which roughly make up half of the extant vertebrate species, exhibit an amazing level of biodiversity affecting their morphology, ecology and behaviour as well as many other aspects of their biology. This huge variability makes fish extremely attractive for the study of many biological questions, particularly of those related to evolution. New insights gained from different teleost species and sequencing projects have recently revealed several peculiar features of fish genomes that might have played a role in fish evolution and speciation. There is now substantial evidence that a round of tetraploidization/rediploidization has taken place during the early evolution of the ray-finned fish lineage, and that hundreds of duplicate pairs generated by this event have been maintained over hundreds of millions of years of evolution. Differential loss or subfunction partitioning of such gene duplicates might have been involved in the generation of fish variability. In contrast to mammalian genomes, teleost genomes also contain multiple families of active transposable elements, which might have played a role in speciation by affecting hybrid sterility and viability. Finally, the amazing diversity of sex determination systems and the plasticity of sex chromosomes observed in teleost might have been involved in both pre- and postmating reproductive isolation. Comparison of data generated by current and future genome projects as well as complementary studies in other species will allow one to approach the molecular and evolutionary mechanisms underlying genome diversity in fish, and will certainly significantly contribute to our understanding of gene evolution and function in humans and other vertebrates.     

Identification of protein kinase C (PKC) isoforms in teleostean, amphibian and avian pigment cells

The beta isoform of protein kinase C (PKC) has been described as the main isoform involved in the stimulation of melanogenesis in mammalian skin melanocytes. Little is known about PKC isoforms in non-mammalian pigment cells. In neopterigian fish (holostei and teleostei), PKC is associated with pigment granule aggregation within the pigment cells (skin lightening), whereas in elasmobranchs and tetrapods, the activation of PKC leads to pigment granule dispersion (skin darkening). In an attempt to a better understanding of this distinct functional behavior upon PKC activation, we decided to investigate the PKC isoforms expressed in pigment cell lines of teleost fish, amphibians and birds, using RT-PCR followed by cloning and sequencing. Our results demonstrate the presence of messenger RNA (mRNA) for the following PKC isoforms: beta 1, lambda and iota in GEM-81 cells (Carassius auratus erythrophoroma), beta 1, beta 2 and zeta in Xenopus laevis (amphibian) melanophores; beta 1 and lambda in Gallus gallus (chicken) primary melanocytes. Beta 1 PKC seems to be conserved throughout phylogeny, but the diversity of the other isoforms in the different groups may account for the functional differences after PKC activation, which are observed between teleost and tetrapod pigment cells.     

Duplication of phospholipase C-delta gene family in fish genomes

Fishes possess more genes than other vertebrates, possibly because of a genome duplication event during the evolution of the teleost (ray-finned) fish lineage. To further explore this idea, we cloned five genes encoding phosphoinositide-specific phospholipase C-delta (PLC-delta), designated respectively PoPLC-deltas, from olive flounder (Paralichthys olivaceus), and we performed phylogenetic analysis and sequence comparison to compare our putative gene products (PoPLC-deltas) with the sequences of known human PLC isoforms. The deduced amino acid sequences shared high sequence identity with human PLC-delta1, -delta3, and -delta4 isozymes and exhibited similar primary structures. In phylogenetic analysis of PoPLC-deltas with PLC-deltas of five teleost fishes (zebrafish, stickleback, medaka, Tetraodon, and Takifugu), three tetrapods (human, chicken, and frog), and two tunicates (sea squirt and pacific sea squirt), whose putative sequences of PLC-delta are available in Ensembl genome browser, the result also indicated that the two paralogous genes corresponding to each PLC-delta isoform originated from fish-specific genome duplication prior to the divergence of teleost fish. Our analyses suggest that an ancestral PLC-delta gene underwent three rounds of genome duplication during the evolution of vertebrates, leading to the six genes of three PLC-delta isoforms in teleost fish.     

Comparative analysis of teleost fish genomes reveals preservation of different ancient clock duplicates in different fishes

Clock (Circadian locomotor output cycle kaput) was the first vertebrate circadian clock gene identified in a mouse forward genetics mutagenesis screen. It encodes a bHLH-PAS protein that is highly conserved throughout evolution. Tetrapods also have the second Clock gene, Clock2 or Npas2 (Neuronal PAS domain protein 2). Conversely, the fruit fly, an invertebrate, has only one clock gene. Interrogation of the five teleost fish genome databases revealed that the zebrafish and the Japanese pufferfish (fugu) each have three clock genes, whereas the green spotted pufferfish (tetraodon), the Japanese medaka fish and the three-spine stickleback each have two clock genes. Phylogenetic and splice site analyses indicated that zebrafish and fugu each have two clock1 genes, clock1a and clock1b and one clock2; tetraodon also have clock1a and clock1b but do not have clock2; and medaka and stickleback each have clock1b and one clock2. Genome neighborhood analysis further showed that clock1a/clock1b in zebrafish, fugu and tetraodon is an ancient duplicate. While the dN/dS ratios of these three fish clock duplicates are all <1, indicating that purifying selection has acted upon them; the Tajima relative rate test showed that all three fish clock duplicates have asymmetric evolutionary rates, implicating that one of these duplicates have been under positive selection or relaxed functional constraint. These results support the view that teleost fish clock genes were generated from an ancient genome-wide duplication, and differential gene loss after the duplication resulted in retention of different ancient duplicates in different teleost fishes, which could have contributed to the evolution of the distinct fish circadian clock mechanisms.     

The Physiology and Evolution of Urea Transport in Fishes

This review summarizes what is currently known about urea transporters in fishes in the context of their physiology and evolution within the vertebrates. The existence of urea transporters has been investigated in red blood cells and hepatocytes of fish as well as in renal and branchial cells. Little is known about urea transport in red blood cells and hepatocytes, in fact, urea transporters are not believed to be present in the erythrocytes of elasmobranchs nor in teleost fish. What little physiological evidence there is for urea transport across fish hepatocytes is not supported by molecular evidence and could be explained by other transporters. In contrast, early findings on elasmobranch renal urea transporters were the impetus for research in other organisms. Urea transport in both the elasmobranch kidney and gill functions to retain urea within the animal against a massive concentration gradient with the environment. Information on branchial and renal urea transporters in teleost fish is recent in comparison but in teleosts urea transporters appear to function for excretion and not retention as in elasmobranchs. The presence of urea transporters in fish that produce a copious amount of urea, such as elasmobranchs and ureotelic teleosts, is reasonable. However, the existence of urea transporters in ammoniotelic fish is curious and could likely be due to their ability to manufacture urea early in life as a means to avoid ammonia toxicity. It is believed that the facilitated diffusion urea transporter (UT) gene family has undergone major evolutionary changes, likely in association with the role of urea transport in the evolution of terrestriality in the vertebrates.     

Peptide transport and animal growth: the fish paradigm

Protein digestion products are transported from the intestinal lumen into the enterocyte both in the form of free amino acids (AAs), by a large variety of brush border membrane AA transporters, and in the form of di/tripeptides, by a single brush border membrane transporter known as PEPtide Transporter 1 (PEPT1). Recent data indicate that, at least in teleost fish, PEPT1 plays a significant role in animal growth by operating, at the gastrointestinal level, as part of an integrated response network to food availability that directly supports body weight. Notably, PEPT1 responds to both fasting and refeeding and is involved in a phenomenon known as compensatory growth (a phase of accelerated growth when food levels are restored after a period of growth depression). In particular, PEPT1 expression decreases during fasting and increases during refeeding, which is the opposite of what observed so far in mammals and birds. These findings in teleost fish document, to our knowledge, for the first time in a vertebrate model, a direct correlation between the expression of an intestinal transporter, such as PEPT1, primarily involved in the uptake of dietary protein degradation products and animal growth.     

Osmoregulation and epithelial water transport: lessons from the intestine of marine teleost fish

For teleost fish living in seawater, drinking the surrounding medium is necessary to avoid dehydration. This is a key component of their osmoregulatory strategy presenting the challenge of excreting excess salts while achieving a net retention of water. The intestine has an established role in osmoregulation, and its ability to effectively absorb fluid is crucial to compensating for water losses to the hyperosmotic environment. Despite this, the potential for the teleost intestine to serve as a comparative model for detailed, integrative experimental studies on epithelial water transport has so far gone largely untapped. The following review aims to present an assessment of the teleost intestine as a fluid-transporting epithelium. Beginning with a brief overview of marine teleost osmoregulation, emphasis shifts to the processing of ingested seawater by the gastrointestinal tract and the characteristics of intestinal ion and fluid transport. Particular attention is given to acid–base transfers by the intestine, specifically bicarbonate secretion, which creates the distinctly alkaline gut fluids responsible for the formation of solid calcium carbonate precipitates. The respective contributions of these unique features to intestinal fluid absorption, alongside other recognised ion transport processes, are then subsequently considered within the wider context of the classic physiological problem of epithelial water transport.     

Environmental and nutritional regulation of expression and function of two peptide transporter (PepT1) isoforms in a euryhaline teleost

Expression and function of the oligopeptide transporter PepT1 in response to changes in environmental salinity have received little study despite the important role that dipeptides play in piscine nutrition. We cloned and sequenced two novel full-length cDNAs that encode Fundulus heteroclitus PepT1-type oligopeptide transporters, and examined their expression and functional properties in freshwater- and seawater-acclimated fish and in response to fasting and re-feeding. Phylogenetic analysis of vertebrate SLC15A1 sequences confirms the presence of two PepT1 isoforms, named SLC15A1a and SLC15A1b, in fish. Similar to other vertebrate SLC15A1s, these isoforms have 12 transmembrane domains, and amino acids essential for PepT1 function are conserved. Expression analysis revealed novel environment-specific expression of the SLC15A1 isoforms in F. heteroclitus, with only SLC15A1b expressed in seawater-acclimated fish, and both isoforms expressed in freshwater-acclimated fish. Fasting and re-feeding induced changes in the expression of SLC15A1a and SLC15A1b mRNA. Short-term fasting resulted in up-regulation of PepT1 mRNA levels, while prolonged fasting resulted in down-regulation. The resumption of feeding resulted in up-regulation of PepT1 above pre-fasted levels. Experiments using the in vitro gut sac technique suggest that the PepT1 isoforms differ in functional characteristics. An increased luminal pH resulted in decreased intestinal dipeptide transport in freshwater-acclimated fish but suggested an increased dipeptide transport in seawater-acclimated fish. Overall, this is the first evidence of multiple isoforms of PepT1 in fish whose expression is environmentally dependent and results in functional differences in intestinal dipeptide transport.     

The control of drinking in elasmobranch fish with special reference to the renin-angiotensin system

In summary, it is evident that teleost and elasmobranch fish respond to extra-cellular dehydration by increasing drinking rate mediated by an increase in circulating levels of ANG II. However, although the primary stimulus for drinking may be the same, clearly the mechanisms involved in regulating ion and water balance are entirely different. In order to maintain ion and water balance in the face of cellular and extra-cellular dehydration, the integration and hormonal control of renal and extra-renal function in elasmobranchs has developed in a very different manner to that described for teleost fish.     

Pervasive indels and their evolutionary dynamics after the fish-specific genome duplication

Insertions and deletions (indels) in protein-coding genes are important sources of genetic variation. Their role in creating new proteins may be especially important after gene duplication. However, little is known about how indels affect the divergence of duplicate genes. We here study thousands of duplicate genes in five fish (teleost) species with completely sequenced genomes. The ancestor of these species has been subject to a fish-specific genome duplication (FSGD) event that occurred approximately 350 Ma. We find that duplicate genes contain at least 25% more indels than single-copy genes. These indels accumulated preferentially in the first 40 my after the FSGD. A lack of widespread asymmetric indel accumulation indicates that both members of a duplicate gene pair typically experience relaxed selection. Strikingly, we observe a 30-80% excess of deletions over insertions that is consistent for indels of various lengths and across the five genomes. We also find that indels preferentially accumulate inside loop regions of protein secondary structure and in regions where amino acids are exposed to solvent. We show that duplicate genes with high indel density also show high DNA sequence divergence. Indel density, but not amino acid divergence, can explain a large proportion of the tertiary structure divergence between proteins encoded by duplicate genes. Our observations are consistent across all five fish species. Taken together, they suggest a general pattern of duplicate gene evolution in which indels are important driving forces of evolutionary change.     

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.     

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.     

Identification of novel tropomyosin 1 genes of pufferfish (Fugu rubripes) on genomic sequences and tissue distribution of their transcripts

Fugu genome database enabled us to identify two novel tropomyosin 1 (TPM1) genes through in silico data mining and isolation of their corresponding cDNAs in vivo. The duplicate TPM1 genes in Japanese pufferfish Fugu rubripes suggest that additional an ancient segmental duplication or whole genome duplication occurred in fish lineage, which, like many other reported Fugu genes, showed reduction in genomic size in comparison with their human homologue. Computer analysis predicted that the coiled-coil probabilities, that were thought to be the most major function of TPM, were the same between the two TPM1 isoforms. We confirmed that the tissue expression profiles of the two TPM1 genes differed from each other, which implied that changes in expression pattern could fix duplicated TPM1 genes although the two TPM1 isoforms appear to have similar function.     

Tetraodon genome analysis provides further evidence for whole-genome duplication in the ray-finned fish lineage

A whole-genome duplication in the ray-finned fish lineage has been supported by the analyses of the genome sequence of the Japanese pufferfish, Fugu rubripes. Recently, genome sequence of a second teleost fish, the freshwater pufferfish, Tetraodon nigroviridis, was completed. Comparisons of long-range synteny between the Tetraodon and human genomes provided additional evidence for the whole-genome duplication in the ray-finned fish lineage. In the present study, we conducted phylogenetic analysis of the Tetraodon and human proteins to identify ray-finned fish lineage-specific (‘fish-specific’) duplicate genes in the Tetraodon genome. Our analyses provide evidence for 1087 well defined fish-specific duplicate genes in Tetraodon. We also analyzed the Fugu proteome that was predicted in the recent Fugu genome assembly, and identified 346 duplicate genes in addition to the 425 duplicates previously identified. We estimated the ages of duplicate genes using the molecular clock. The ages of duplicate genes in the two pufferfishes independently support a large-scale gene duplication around 380–400 Myr ago. In addition, a burst of recent gene duplications was evident in the Tetraodon lineage. These findings provide further evidence for a whole-genome duplication early in the evolution of ray-finned fishes, and suggest that independent gene duplications have occurred recently in the Tetraodon lineage.     

Fugu genome analysis provides evidence for a whole-genome duplication early during the evolution of ray-finned fishes

With about 24,000 extant species, teleosts are the largest group of vertebrates. They constitute more than 99% of the ray-finned fishes (Actinopterygii) that diverged from the lobe-finned fish lineage (Sarcopterygii) about 450 MYA. Although the role of genome duplication in the evolution of vertebrates is now established, its role in structuring the teleost genomes has been controversial. At least two hypotheses have been proposed: a whole-genome duplication in an ancient ray-finned fish and independent gene duplications in different lineages. These hypotheses are, however, based on small data sets and lack adequate statistical and phylogenetic support. In this study, we have made a systematic comparison of the draft genome sequences of Fugu and humans to identify paralogous chromosomal regions ("paralogons") in the Fugu that arose in the ray-finned fish lineage ("fish-specific"). We identified duplicate genes in the Fugu by phylogenetic analyses of the Fugu, human, and invertebrate sequences. Our analyses provide evidence for 425 fish-specific duplicate genes in the Fugu and show that at least 6.6% of the genome is represented by fish-specific paralogons. We estimated the ages of Fugu duplicate genes and paralogons using the molecular clock. Remarkably, the ages of duplicate genes and paralogons are clustered, with a peak around 350 MYA. These data strongly suggest a whole-genome duplication event early during the evolution of ray-finned fishes, probably before the origin of teleosts.     

Short-term low-salinity tolerance by the longhorn sculpin, Myoxocephalus octodecimspinosus

The bottom-dwelling, longhorn sculpin, Myoxocephalus octodecimspinosus, is traditionally viewed as a stenohaline marine fish, but fishermen have described finding this sculpin in estuaries during high tide. Little is known about the salinity tolerance of the longhorn sculpin; thus, the purposes of these experiments were to explore the effects of low environmental salinity on ion transporter expression and distribution in the longhorn sculpin gill. Longhorn sculpin were acclimated to either 100% seawater (SW, sham), 20% SW, or 10% SW for 24 or 72 hr. Plasma osmolality, sodium, potassium, and chloride concentrations were not different between the 20 and 100% treatments; however, they were 20-25% lower with exposure to 10% SW at 24 and 72 hr. In the teleost gill, regulation of Na(+), K(+)-ATPase (NKA), Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), and the chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR) are necessary for ion homeostasis. We immunolocalized these proteins to the mitochondrion-rich cell of the gill and determined that acclimation to low salinity does not affect their localization. Also, there was not a downregulation of gill NKA, NKCC1, and CFTR mRNA or protein during acclimation to low salinities. Collectively, these results suggest that down to 20% SW longhorn sculpin are capable of completely regulating ion levels over a 72-hr period, whereas 10% SW exposure results in a significant loss of ions and no change in ion transporter density or localization in the gill. We conclude that longhorn sculpin can tolerate low-salinity environments for days but, because they cannot regulate ion transporter density, they are unable to tolerate low salinity for longer periods or enter freshwater (FW). The genus Myoxocephalus has three FW species, making this group an excellent model to test evolutionary and physiological mechanisms that allow teleosts to invade new low salinities successfully.