Expression Profiles of Branchial FXYD Proteins in the Brackish Medaka Oryzias dancena: A Potential Saltwater Fish Model for Studies of Osmoregulation

FXYD proteins are novel regulators of Na⁺-K⁺-ATPase (NKA). In fish subjected to salinity challenges, NKA activity in osmoregulatory organs (e.g., gills) is a primary driving force for the many ion transport systems that act in concert to maintain a stable internal environment. Although teleostean FXYD proteins have been identified and investigated, previous studies focused on only a limited group of species. The purposes of the present study were to establish the brackish medaka (Oryzias dancena) as a potential saltwater fish model for osmoregulatory studies and to investigate the diversity of teleostean FXYD expression profiles by comparing two closely related euryhaline model teleosts, brackish medaka and Japanese medaka (O. latipes), upon exposure to salinity changes. Seven members of the FXYD protein family were identified in each medaka species, and the expression of most branchial fxyd genes was salinity-dependent. Among the cloned genes, fxyd11 was expressed specifically in the gills and at a significantly higher level than the other fxyd genes. In the brackish medaka, branchial fxyd11 expression was localized to the NKA-immunoreactive cells in gill epithelia. Furthermore, the FXYD11 protein interacted with the NKA α-subunit and was expressed at a higher level in freshwater-acclimated individuals relative to fish in other salinity groups. The protein sequences and tissue distributions of the FXYD proteins were very similar between the two medaka species, but different expression profiles were observed upon salinity challenge for most branchial fxyd genes. Salinity changes produced different effects on the FXYD11 and NKA α-subunit expression patterns in the gills of the brackish medaka. To our knowledge, this report is the first to focus on FXYD expression in the gills of closely related euryhaline teleosts. Given the advantages conferred by the well-developed Japanese medaka system, we propose the brackish medaka as a saltwater fish model for osmoregulatory studies.     

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid–base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na⁺]_i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na⁺]_i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye–ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na⁺]_i, whereas no significant differences in branchial [Na⁺]_i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na⁺]_i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.     

Identification and pathway analysis of immediate hyperosmotic stress responsive molecular mechanisms in tilapia (Oreochromis mossambicus) gill

Salinity is a major environmental factor that strongly influences cellular and organismal function. We have used the euryhaline fish Oreochromis mossambicus to identify and annotate immediate hyperosmotic stress responsive molecular mechanisms and biological processes in gill epithelial cells. Using a suppression subtractive hybridization (SSH) approach, we have identified and cloned 20 novel immediate early genes whose mRNAs are induced in gill epithelial cells 4 h after transfer of fish from freshwater (FW) to seawater (SW). Full-length or partial sequences of open reading frames (ORFs) were obtained using the rapid amplification of cDNA ends (RACE) technique. Kinetics of induction was analyzed for all hyperosmotic stress-induced genes. Most genes show a robust transient increase in mRNA abundance characteristic of immediate early stress response genes with peak levels observed between 2 and 8 h after seawater transfer. The newly identified genes were classified according to their sequence similarity with other vertebrate homologs and based on their predicted functions. Pathway analysis revealed that more than half of the identified immediate hyperosmotic stress genes interact closely within a cellular stress response signaling network. Moreover, the 20 genes cluster together in six molecular processes that are rapidly activated in tilapia gills upon salinity transfer. These processes are (1) stress response signal transduction, (2) compatible organic osmolyte accumulation, (3) energy metabolism, (4) lipid transport and cell membrane protection, (5) actin-based cytoskeleton dynamics, and (6) protein and mRNA stability. Our identification and analysis of a set of novel osmo-responsive tilapia genes provides insight into critical physiological processes and pathways constituting the hyperosmotic stress adaptation program in gill epithelial cells of euryhaline fishes.     

Filter feeding,deviations from bilateral symmetry,developmental noise,and heterochrony of hemichordate and cephalochordate gills

We measured gill slit fluctuating asymmetry (FA), a measure of developmental noise, in adults of three invertebrate deuterostomes with different feeding modes: the cephalochordate Branchiostoma floridae (an obligate filter feeder), the enteropneusts Protoglossus graveolens (a facultative filter feeder/deposit feeder) and Saccoglossus bromophenolosus (a deposit feeder). FA was substantially and significantly low in B. floridae and P. graveolens and high in S. bromophenolosus. Our results suggest that the gills of species that have experienced a relaxation of the filter feeding trait exhibit elevated FA. We found that the timing of development of the secondary collagenous gill bars, compared to the primary gill bars, was highly variable in P. graveolens but not the other two species, demonstrating an independence of gill FA from gill bar heterochrony. We also discovered the occasional ectopic expression of a second set of paired gills posterior to the first set of gills in the enteropneusts and that these were more common in S. bromophenolosus. Moreover, our finding that gill slits in enteropneusts exhibit bilateral symmetry suggests that the left‐sidedness of larval cephalochordate gills, and the directional asymmetry of Cambrian stylophoran echinoderm fossil gills, evolved independently from a bilaterally symmetrical ancestor.     

Morphological observations on the gills of dendrobranchiate shrimps

Gill morphology, traditionally, has played an important role in attempts to reconstruct the phylogenetic history of the Crustacea Decapoda. We examined the gills of dendrobranchiate shrimps (Crustacea, Decapoda, Dendrobranchiata) to test the assumption that all members of the clade have gills that are “dendrobranchiate” (highly branching) in form, from whence the taxon name Dendrobranchiata comes. Currently, the Dendrobranchiata consists of two superfamilies and seven families. Specimens from two genera in each of the known families were examined using light and scanning electron microscopy. Members of the family Luciferidae, all of which lack gills as adults, were not examined. Only one genus was examined for the Penaeidae (because they have been the subject of numerous previous studies) and Sicyoniidae (a monogeneric family). All gills examined have secondary branches that are further subdivided, conforming to existing and rather broad definitions of dendrobranchiate gills. Families with “typical” dendrobranchiate gills, which consist of curved secondary branches that in turn bear branched (dendritic) tubular tertiary elements on their distal surfaces, include the Penaeidae, Aristeidae, and Solenoceridae. In other families, secondary and tertiary gill elements are sometimes quite flattened, and the tertiary elements are not dendritic, giving the gill a distinctly non-dendrobranchiate appearance. Flattened biserial secondary branches and their flattened tertiary elements are particularly obvious in gills of the monogeneric family Sicyoniidae (Sicyonia). Within the family Sergestidae, gills of the genus Sergestes are unusual in having secondary branches that arise from the main gill axis in an alternating pattern; these gills also have distinctly oval tertiary elements that are not further subdivided and are directed basally rather than distally. Another sergestid genus, Petalidium, displays gills that differ from those of Sergestes; in Petalidium the secondary branches also come off the main gill axis in an alternating pattern, but these branches are more widely spaced and have relatively larger and broader tertiary elements when compared with gills of Sergestes. The family Benthesicyemidae also contains species with different gill types; Gennadas is shown to have flattened, plate-like tertiary elements, whereas Benthesicyemus has more typical dendrobranchiate gills. The significance of this variation in gill morphology within families and within the Dendrobranchiata as a whole is unclear at this point; rearrangements of the currently accepted phylogeny and resulting classification based solely on gill morphology are not recommended at this time.     

Response to environmental salinity of Na-KATPase activity in individual gills of the euryhaline crab Cyrtograpsus angulatus

The occurrence, localization and response to environmental salinity changes of Na⁺-K⁺ATPase activity were studied in each of the individual gills 4-8 of the euryhaline crab Cyrtograpsus angulatus from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). Na⁺-K⁺ATPase activity appeared to be differentially sensitive to environmental salinity among gills. Upon an abrupt change to low salinity, a differential response of Na⁺-K⁺ATPase activity occurred in each individual gill which could suggest a differential role of this enzyme in ion transport process in the different gills of C. angulatus. With the exception of gill 8, a short-term increase of Na⁺-K⁺ATPase specific activity was observed in posterior gills, which is similar to adaptative variations of this activity described in other euryhaline crabs. However, and conversely to that described in other hyperregulating crabs, the highest increase of activity occurred in anterior gills 4 by 1 day after the change to dilute media which could suggest also a role for these gills in ion transport processes in C. angulatus. The fact that variations of Na⁺-K⁺ATPase activity in anterior and posterior gills were concomitant with the transition to hyperregulation indicate that this enzyme could be a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab. The results suggest a differential participation of branchial Na⁺-K⁺ATPase activity in ionoregulatory mechanisms of C. angulatus. The possible existence of functional differences as well as distinct regulation mechanisms operating in individual gills is discussed.     

Exploration of polyacrylamide microplastics and evaluation of their toxicity on multiple parameters of Oreochromis niloticus

Due to the increased production of polyacrylamide microplastics in the environment impacting the adverse effects on aquatic organisms have become a global concern. The present study aimed to evaluate the toxicity of different concentrations (0.018, 0.03 0.09 g/1) of polyacrylamide microplastics on Oreochromis niloticus. Polyacrylamide microplastics were characterized by Fourier transform infrared and Raman spectroscopy. The FTIR technique revealed the spherical morphology and size range of polyacrylamide (0.1–0.4 mm) with 99 % purity. Raman Spectroscopy results showed peaks around (1100 cm^?1 and 1650 cm^?1) in gills, these peaks confirmed the presence of polyacrylamide microplastics in the gills of Oreochromis niloticus. Polyacrylamide microplastics significantly shortened the antioxidant enzymes (Catalase and Reduced glutathione) proceedings and the increase was observed in Malondialdehyde and Lipid peroxide levels in high-dose treated groups. Moreover, total protein contents were expressively increased, while other blood parameters (AST, ALP, ALT) were significantly decreased. Hemoglobin (g/dl), Erythrocyte (106/µl) and TLC (1x10⁹/l) levels were reduced significantly (p < 0.05) in high concentrations. The administration of polyacrylamide microplastics induced different histological changes in the gills, liver and intestine of O. niloticus. It was concluded that polyacrylamide microplastics are toxic agents having harmful effects on fish health.     

Phylogenetic characterization of endosymbionts of the hydrothermal vent mussel Bathymodiolus azoricus by analysis of the 16S rRNA, cbbL, and pmoA genes

In order to assess the phylogenetic diversity of the endosymbiotic microbial community of the gills of marine bivalve Bathymodiolus azoricus, total DNA was extracted from the gills. The PCR fragments corresponding to the genes encoding 16S rRNA, ribulose-bisphosphate carboxylase (cbbL), and particulate methane monooxygenase (pmoA) were amplified, cloned, and sequenced. For the 16S rDNA genes, only one phylotype was revealed; it belonged to the cluster of thiotrophic mytilid’s symbionts within the Gammaproteobacteria. For the RuBisCO genes, two phylotypes were found, both belonging to Gammaproteobacteria. One of them was closely related to the previously known mytilid symbiont, the other, to a pogonophore symbiont, presumably a methanotrophic bacterium. One phylotype of particulate methane oxygenase genes was also revealed; this finding indicated the presence of a methanotrophic symbiont. Phylogenetic analysis of the pmoA placed this endosymbiont within the Gammaproteobacteria, in a cluster including the methanotrophic bacterial genus Methylobacter and other methanotrophic Bathymodiolus gill symbionts. These results provide evidence for the existence of two types of endosymbionts (thioautotrophic and methanotrophic) in the gills of B. azoricus and demonstrate that, apart from the phylogenetic analysis of 16S rRNA genes, parallel analysis of functional genes is essential.     

Hydrodynamics of water flow in front of and through the gills of skipjack tuna

• 1.1. The structure of the gills of skipjack tuna (Katsuwonus pelamis) is reviewed and the pattern of water flow in front of, and through, the gills is described. The tuna system is attractive for analysis of this type because water flow is continuous rather than pulsatile and the gill system is more rigid than that of other fish. In addition, oxygen uptake rates are the extreme case (i.e. highest values) for water-breathers.
• 2.2. Flow into the slits between the gill bars is rectilinear and nearly uniform, and therefore irrigation at the gill sieve should be nearly uniform.
• 3.3. Reynolds numbers are so low that turbulent flow is unlikely and entrance effects are negligible.

     

Identifications of Captive and Wild Tilapia Species Existing in Hawaii by Mitochondrial DNA Control Region Sequence

Background

The tilapia family of the Cichlidae includes many fish species, which live in freshwater and saltwater environments. Several species, such as O. niloticus, O. aureus, and O. mossambicus, are excellent for aquaculture because these fish are easily reproduced and readily adapt to diverse environments. Historically, tilapia species, including O. mossambicus, S. melanotheron, and O. aureus, were introduced to Hawaii many decades ago, and the state of Hawaii uses the import permit policy to prevent O. niloticus from coming into the islands. However, hybrids produced from O. niloticus may already be present in the freshwater and marine environments of the islands. The purpose of this study was to identify tilapia species that exist in Hawaii using mitochondrial DNA analysis.

Methodology/Principal Findings

In this study, we analyzed 382 samples collected from 13 farm (captive) and wild tilapia populations in Oahu and the Hawaii Islands. Comparison of intraspecies variation between the mitochondrial DNA control region (mtDNA CR) and cytochrome c oxidase I (COI) gene from five populations indicated that mtDNA CR had higher nucleotide diversity than COI. A phylogenetic tree of all sampled tilapia was generated using mtDNA CR sequences. The neighbor-joining tree analysis identified seven distinctive tilapia species: O. aureus, O. mossambicus, O. niloticus, S. melanotheron, O. urolepies, T. redalli, and a hybrid of O. massambicus and O. niloticus. Of all the populations examined, 10 populations consisting of O. aureus, O. mossambicus, O. urolepis, and O. niloticus from the farmed sites were relatively pure, whereas three wild populations showed some degree of introgression and hybridization.

Conclusions/Significance

This DNA-based tilapia species identification is the first report that confirmed tilapia species identities in the wild and captive populations in Hawaii. The DNA sequence comparisons of mtDNA CR appear to be a valid method for tilapia species identification. The suspected tilapia hybrids that consist of O. niloticus are present in captive and wild populations in Hawaii.