Expression of multiple insulin and insulin-like growth factor receptor genes in salmon gill cartilage

In mammals, one of the major actions of insulin-like growth factor I (IGF-I) is to increase skeletal growth by stimulating new cartilage formation. IGF-I stimulates chondrocytes in vitro to synthesize new cartilage matrix, measured by enhanced uptake of 35S-sulfate, but the addition of insulin does not produce a similar effect except when added at high concentrations. However, recent studies have shown that, in teleosts, both insulin and IGF-I are potent activators of 35S-sulfate uptake in gill cartilage. To further characterize the growth-promoting activities of these hormones in fish, we have used reverse transcriptase-linked PCR to analyze the expression of insulin receptor family genes in salmon gill cartilage. Partial cDNA sequences encoding the tyrosine kinase domains from six distinct members of the IR gene family were obtained, and sequence comparisons revealed that four of the cDNAs encoded amino acid sequences that were highly homologous to human IR whereas the encoded sequences from two of the cDNAs were more similar to the human type I IGF receptor (IGF-R). Furthermore, a comparative reverse transcriptase-linked PCR assay revealed that the four putative IR mRNAs expressed in toto in gill cartilage were 56% of that found in liver whereas the expressed amount of the two IGF-R mRNAs was 9-fold higher compared with liver. These results suggest that the chondrogenic actions of insulin and IGF-I in fish are mediated by the ligands binding to their cognate receptors. However, further studies will be required to characterize the binding properties and relative contribution of the individual IR and IGF-R genes.

Evolution and divergence of sodium channel genes in vertebrates

Invertebrate species possess one or two Na+ channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na+ channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na+ channel gene followed by tandem duplications of Na+ channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na+ channel genes in a teleost fish. Using an approach designed to clone all of the Na+ channel genes in a genome, we found six Na+ channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na+ channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na+ channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na+ channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na+ channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than {D[A(BC)]}.

Demonstration of the extrinsic coagulation pathway in teleostei: Identification of zebrafish coagulation factor VII

It is not known whether the mammalian mechanism of coagulation initiation is conserved in fish. Identification of factor VII is critical in providing evidence for such a mechanism. A cDNA was cloned from a zebrafish (teleost) library that predicted a protein with sequence similarity to human factor VII. Factor VII was shown to be present in zebrafish blood and liver by Western blot analysis and immunohistochemistry. Immunodepletion of factor VII from zebrafish plasma selectively inhibited thromboplastin-triggered thrombin generation. Heterologous expression of zebrafish factor VII demonstrated a secreted protein (50 kDa) that reconstituted thromboplastin-triggered thrombin generation in immunodepleted zebrafish plasma. These results suggest conservation of the extrinsic coagulation pathway between zebrafish and humans and add credence to the zebrafish as a model for mammalian hemostasis. The structure of zebrafish factor VIIa predicted by homology modeling was consistent with the overall three-dimensional structure of human factor VIIa. However, amino acid disparities were found in the epidermal growth factor-2/serine protease regions that are present in the human tissue factor–factor VIIa contact surface, suggesting a structural basis for the species specificity of this interaction. In addition, zebrafish factor VII demonstrates that the Gla-EGF-EGF-SP domain structure, which is common to coagulation factors VII, IX, X, and protein C, was present before the radiation of the teleosts from the tetrapods. Identification of zebrafish factor VII significantly narrows the evolutionary window for development of the vertebrate coagulation cascade and provides insight into the structural basis for species specificity in the tissue factor–factor VIIa interaction.

Intraembryonic hematopoietic cell migration during vertebrate development.

Vertebrate hematopoietic stem cells are derived from vental mesoderm, which is postulated to migrate to both extra- and intraembryonic positions during gastrula and neurula stages. Extraembryonic migration has previously been documented, but the origin and migration of intraembryonic hematopoietic cells have not been visualized. The zebrafish and most other teleosts do not form yolk sac blood islands during early embryogenesis, but instead hematopoiesis occurs solely in a dorsal location known as the intermediate cell mass (IM) or Oellacher. In this report, we have isolated cDNAs encoding zebrafish homologs of the hematopoietic transcription factors GATA-1 and GATA-2 and have used these markers to determine that the IM is formed from mesodermal cells in a posterior-lateral position on the yolk syncytial layer of the gastrula yolk sac. Surprisingly, cells of the IM then migrate anteriorly through most of the body length prior to the onset of active circulation and exit onto the yolk sac. These findings support a hypothesis in which the hematopoietic program of vertebrates is established by variations in homologous migration pathways of extra- and intraembryonic progenitors.

Expression cloning of a cDNA encoding a fish prolactin receptor.

By using an expression cloning strategy, we isolated a single positive clone encoding a tilapia prolactin (PRL) receptor. Tilapia PRL188 was used to screen a freshwater tilapia kidney expression library transfected in COS cells. The tilapia PRL receptor is a mature protein of 606 amino acids. The extracellular domain is devoid of the tandem repeat units present in birds and has two pairs of cysteine residues, a Trp-Ser-Xaa-Trp-Ser motif, and two potential N-glycosylation sites. The cytoplasmic domain contains 372 amino acids, including box 1, a sequence previously shown to be important for signal transduction in mammalian species. Thus, the general structure is similar to the long form of mammalian PRL receptors; however, amino acid comparisons reveal a rather low identity (approximately 37%). Northern blot analysis shows the existence of a single transcript in osmoregulatory tissues and reproductive organs. This localization is in agreement with known functions of PRL in teleosts.

Origin and diversity of the Sox transcription factor gene family: genome-wide analysis in Fugu rubripes

The SOX family of transcription factors are found throughout the animal kingdom and are important in a variety of developmental contexts. Genome analysis has identified 20 Sox genes in human and mouse, which can be subdivided into 8 groups, based on sequence comparison and intron-exon structure. Most of the SOX groups identified in mammals are represented by a single SOX sequence in invertebrate model organisms, suggesting a duplication and divergence mechanism has operated during vertebrate evolution. We have now analysed the Sox gene complement in the pufferfish, Fugu rubripes, in order to shed further light on the diversity and origins of the Sox gene family. Major differences were found between the Sox family in Fugu and those in humans and mice. In particular, Fugu does not have orthologues of Sry, Sox,15 and Sox30, which appear to be specific to mammals, while Sox19, found in Fugu and zebrafish but absent in mammals, seems to be specific to fishes. Six mammalian Sox genes are represented by two copies each in Fugu, indicating a large-scale gene duplication in the fish lineage. These findings point to recent Sox gene loss, duplication and divergence occurring during the evolution of tetrapod and teleost lineages, and provide further evidence for large-scale segmental or a whole-genome duplication occurring early in the radiation of teleosts. (C) 2004 Elsevier B.V. All rights reserved.

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.

Partitioning of respiration between the gills and air-breathing organ in response to aquatic hypoxia and exercise in the Pacific tarpon, Megalops cyprinoides

The evolution of air-breathing organs (ABOs) is associated not only with hypoxic environments but also with activity. This investigation examines the effects of hypoxia and exercise on the partitioning of aquatic and aerial oxygen uptake in the Pacific tarpon. The two-species cosmopolitan genus Megalops is unique among teleosts in using swim bladder ABOs in the pelagic marine environment. Small fish ( 58 - 620 g) were swum at two sustainable speeds in a circulating flume respirometer in which dissolved oxygen was controlled. For fish swimming at 0.11 m s(-1) in normoxia (Po-2 = 21 kPa), there was practically no air breathing, and gill oxygen uptake was 1.53 mL kg(-0.67) min(-1). Air breathing occurred at 0.5 breaths min(-1) in hypoxia ( 8 kPa) at this speed, when the gills and ABOs accounted for 0.71 and 0.57 mL kg(-0.67) min(-1), respectively. At 0.22 m s(-1) in normoxia, breathing occurred at 0.1 breaths min(-1), and gill and ABO oxygen uptake were 2.08 and 0.08 mL kg(-0.67) min(-1), respectively. In hypoxia and 0.22 m s(-1), breathing increased to 0.6 breaths min(-1), and gill and ABO oxygen uptake were 1.39 and 1.28 mL kg(-0.67) min(-1), respectively. Aquatic hypoxia was therefore the primary stimulus for air breathing under the limited conditions of this study, but exercise augmented oxygen uptake by the ABOs, particularly in hypoxic water.

Prosorhynchoides lamprelli n. sp (Digenea : Bucephalidae) from the brassy trevally, Caranx papuensis (Teleostei : Carangidae), from off Lizard Island on the Great Barrier Reef, Australia

Prosorhynchoides lamprelli n. sp. ( Digenea: Bucephalidae) is described from the intestine of the brassy trevally, Caranx papuensis (Carangidae) from off Lizard Island, Great Barrier Reef, Australia. The new species is differentiated from other species of Prosorhynchoides Dollfus, 1929 by the shape and distribution of its vitelline follicles, the shape and extent of its uterus and the configuration of its digestive system. This is the first bucephalid to be described from Caranx papuensis; we have not encountered this species from other carangids or from over 1,500 individuals of other teleosts species we have found to be infected with bucephalids from the Great Barrier Reef.

Sanguinicola maritimus n. sp (Digenea : Sanguinicolidae) from Labridae (Teleostei : Perciformes) of southern Australian waters

A new species of Sanguinicola Plehn, 1905 is described from the marine teleosts Notolabrus parilus (Richardson) and N. tetricus (Richardson) (Perciformes: Labridae) from Western Australian and Tasmanian waters. This host distribution is strikingly anomalous; however, the present material fulfils the morphological criteria of Sanguinicola. S. maritimus n. sp. differs from previously described species in having the combination of a body 1,432-1,701 mu m long, the oesophagus 18.3-21.7% of the body length, the testis occupying 42.8-52.3% of the body length, an oviducal seminal receptacle and Mehlis' gland present, ovoid eggs, and vitelline follicles that extend anteriorly past the nerve commissure, laterally past the lateral nerve chords and posteriorly to the anterior margin of the cirrus-sac. S. maritimus also lacks a protrusible anterior proboscis. It also differs in the combination of host and geographical location, being the first Sanguinicola species from a marine teleost and the first from Australian waters.

An exceptionally rich complex of Sanguinicolidae von Graff, 1907 (Platyhelminthes : Trematoda) from Siganidae, Labridae and Mullidae (Teleostei : Perciformes) from the Indo-West Pacific Region

We describe an unprecedented radiation of sanguinicolid blood flukes ( Digenea: Sanguinicolidae) from two species of Labridae (Choerodon venustus and C. cauteroma), seven species of Mullidae (Mulloidichthys vanicolensis, Parupeneus barberinoides, P. barberinus, P. bifasciatus, P. cyclostomus, P. indicus and P. multifasciatus) and ten species of Siganidae (Siganus argenteus, S. corallinus, S. doliatus, S. fuscescens, S. lineatus, S. margaritiferus, S. puellus, S. punctatus, S. virgatus and S. vulpinus) from sites off Australia and Palau. The flukes were morphologically similar in having the combination of a long thread-like body, tegumental spines in lateral transverse rows, a vestigial oral sucker bearing concentric rows of fine spines, an H-shaped intestine, a cirrussac, a notch level with the male genital pore, a lateral or post-ovarian uterus, a uterine chamber and separate genital pores. These species are divided into two genera on the basis of testis number. Sanguinicolids from Siganus fuscescens have a single large testis between the intestinal bifurcation and the ovary and are placed in Ankistromeces Nolan & Cribb, 2004. Species from the remaining nine species of Siganidae, Labridae and Mullidae are placed in Phthinomita n. g.; these species have two testes, the anterior testis being large and between the intestinal bifurcation and the ovary whereas the small posterior testis is at the posterior end of the body and appears rudimentary or degenerate and probably non-functional. The second internal transcribed spacer (ITS2) of ribosomal DNA ( rDNA) from 29 host/parasite/location combinations (h/p/l) was sequenced together with that of Ankistromeces mariae Nolan & Cribb, 2004 for comparison. From 135 samples we found 19 distinct genotypes which were interpreted as representing at least that many species. Replicate sequences were obtained for 25 of 30 h/p/l combinations ( including A. mariae); there was no intraspecific variation between replicates sequences for any of these. Interspecific variation ranged from 1 - 41 base differences (0.3 - 12.7% sequence divergence). The 19 putative species were difficult to recognise by morphological examination. We describe 13 new species; we do not describe (= name) six species characterised solely by molecular sequences and three putative species for which morphological data is available but for which molecular data is not. We have neither morphological nor molecular data for sanguinicolids harboured in five hosts species ( Siganus margaritiferus, S. puellus, Choerodon cauteroma, Parupeneus indicus and P. multifasciatus) in which we have seen infections. Where host species were infected in different localities they almost always harboured distinct species. Some host species ( for example, S. argenteus and S. lineatus from Lizard Island) harboured two or three species in a single geographical location. This suggests that, for parts of this system, parasite speciation has outstripped host speciation. Distance analysis of ITS2 showed species from each host family ( Siganidae, Mullidae and Labridae) did not form monophyletic clades to the exclusion of species from other host families. However, a host defined clade was formed by the sequences from sanguinicolids from S. fuscescens.

The number, morphology, and distribution of retinal ganglion cells and optic axons in the Australian lungfish Neoceratodus forsteri (Krefft 1870)

Australian lungfish Neoceratodus forsteri may be the closest living relative to the first tetrapods and yet little is known about their retinal ganglion cells. This study reveals that lungfish possess a heterogeneous population of ganglion cells distributed in a horizontal streak across the retinal meridian, which is formed early in development and maintained through to adult stages. The number and complement of both ganglion cells and a population of putative amacrine cells within the ganglion cell layer are examined using retrograde labelling from the optic nerve and transmission electron-microscopic analysis of axons within the optic nerve. At least four types of retinal ganglion cells are present and lie predominantly within a thin ganglion cell layer, although two subpopulations are identified, one within the inner plexiform and the other within the inner nuclear layer. A subpopulation of retinal ganglion cells comprising up to 7% or the total population are significantly larger (> 400 mu m(2)) and are characterized as giant or alpha-like cells. Up to 44% of cells within the retinal ganglion cell layer represent a population of presumed amacrine cells. The optic nerve is heavily fasciculated and the proportion of myelinated axons increases with body length from 17% in subadults to 74% in adults. Spatial resolving power, based on ganglion cell spacing, is low (1.6-1.9 cycles deg(-1), n = 2) and does not significantly increase with growth. This represents the first detailed study of retinal ganglion cells in sarcopterygian fish, and reveals that, despite variation amongst animal groups, trends in ganglion cell density distribution and characteristics of cell types were defined early in vertebrate evolution.

Influence of teleost abundance on the distribution and abundance of sharks in Florida Bay, USA

Understanding the factors that influence the distribution and abundance of predators, including sharks, is important for predicting the impacts of human changes to the environment. Such studies are particularly important in Florida Bay, USA where there are planned large-scale changes to patterns of freshwater input from the Everglades ecosystem. Studies of many marine predators suggest that links between predator and prey habitat use may vary with spatial scale, but there have been few studies of the role of prey distribution in shaping habitat use and abundance of sharks. We used longline catches of sharks and trawls for potential teleost prey to determine the influence of teleost abundance on shark abundance at the scale of regions and habitats in Florida Bay. We found that shark catch per unit effort (CPUE) was not linked to CPUE ofteleosts at the scale of sampling sites, but shark CPUE was positively correlated with the mean CPUE for teleosts within a region. Although there does not appear to be a strong match between the abundance of teleosts and sharks at small spatial scales, regional shark abundance is likely driven, at least partially, by the availability of prey. Management strategies that influence teleost abundance will have cascading effects to higher trophic levels in Florida Bay.

The role of teleost grazers in a relatively pristine seagrass ecosystem

Trophic downgrading of ecosystems necessitates a functional understanding of trophic cascades. Identifying the presence of cascades, and the mechanisms through which they occur, is particularly important for seagrass meadows, which are among the most threatened ecosystems on Earth. Shark Bay, Western Australia provides a model system to investigate the potential importance of top-down effects in a relatively pristine seagrass ecosystem. The role of megagrazers in the Shark Bay system has been previously investigated, but the role of macrograzers (i.e., teleosts), and their importance relative to megagrazers, remains unknown. The objective of my dissertation was to elucidate the importance of teleost macrograzers in transmitting top-down effects in seagrass ecosystems. Seagrasses and macroalgae were the main food of the abundant teleost Pelates octolineatus, but stable isotopic values suggested that algae may contribute a larger portion of assimilated food than suggested by gut contents. Pelates octolineatus is at risk from numerous predators, with pied cormorants (Phalacrocorax varius) taking the majority of tethered P. octolineatus. Using a combination of fish trapping and unbaited underwater video surveillance, I found that the relative abundance of P. octolineatus was greater in interior areas of seagrass banks during the cold season, and that the mean length of P. octolineatus was greater in these areas compared to along edges of banks. Finally, I used seagrass transplants and exclosure experiments to determine the relative effect of megagrazers and macrograzers on the establishment and persistence of three species of seagrasses in interior microhabitats. Teleost grazing had the largest impact on seagrass species with the highest nutrient content, and these impacts were primarily observed during the warm season. My findings are consistent with predictions of a behaviorally-mediated trophic cascade initiated by tiger sharks (Galeocerdo cuvier) and transmitted through herbivorous fishes and their predators.

Expression of Bf/C2 Gene(S) in the Japanese Medaka Fish

Complement factor B and C2 are two central serine proteases of the alternative and classical complement pathways, respectively, that serve as the catalytic subunits of the C3 convertase. Research has been completed using a female Japanese medaka fish, (Oryzias latipes), and other teleost and elasmobrach species in order to isolate eDNA clones and perform linkage analysis of the Bf/C2 gene(s). To further analyze the evolution of the complement system in teleosts, different tissues than the ones from previous studies of medaka fish were analyzed for the constitutive gene expression of factor B and C2. Bf/C2 sequences were amplified by reverse transcription-polymerase chain reaction with primers corresponding to the common amino acid sequences shared by mammalian Bf and C2. Agarose gel electrophoresis was used to visualize sample bands and to calculate the concentration of gene expression of the Bf/C2 gene(s) in each tissue. All five tissue types, kidney, liver, muscle, testis, and spleen from a male medaka fish demonstrated Bf/C2 gene(s) expression, confirming that the messages of Bf/C2 gene(s) are distributed throughout the medaka fish. Tissues of the spleen, liver, and kidney contained the highest concentrations of expression of Bf/C2 gene( s ), while tissues of the muscle and testis contained the lowest concentrations. This research also determined that RT-PCR allowed for more sensitive analysis of gene expression than other molecular biology techniques such as Northern blotting analysis.