Effects of exposure to Streptococcus iniae on microRNA expression in the head kidney of genetically improved farmed tilapia (Oreochromis niloticus).

BACKGROUND: Genetically improved farmed tilapia (GIFT, Oreochromis niloticus) are susceptible to infection by Streptococcus iniae when maintained in modern intensive culture systems. GIFT are commercially important fishes that are cultured widely in southern China. The role of microRNAs (miRNAs) in the regulatory response of GIFT to S. iniae infection has been underestimated and has not yet been well studied. Head kidney has an important immune function in teleost fishes. The main aim of this study was to determine the possible function of miRNAs in head kidney of S. iniae-infected GIFT. MiRNAs are small, non-coding RNAs that regulate gene expression by binding to the 3'-untranslated regions of their target mRNAs. MiRNAs are known to regulate immune-regulated signaling and inflammatory response pathways. RESULTS: High-throughput deep sequencing of two libraries (control group [CO] and infected group [IN]) of RNA extracted from GIFT head kidney tissues generated 12,089,630 (CO) and 12,624,975 (IN) clean reads. Bioinformatics analysis identified 1736 and 1729 conserved miRNAs and 164 and 165 novel miRNAs in the CO and IN libraries, respectively. Three miRNAs (miR-310-3p, miR-92, and miR-127) were found to be up-regulated and four miRNAs (miR-92d-3p, miR-375-5p, miR-146-3p, and miR-694) were found to be down-regulated in the S. iniae-infected GIFT. The expressions of these miRNAs were verified by quantitative real-time PCR. RNAhybrid and TargetScan were used to identify complementary miRNA and mRNA target sites, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used to annotate and predict potential downstream regulation of biological pathways. Seven target genes, which encode immune-related proteins (complement C3, cytidine deaminase, regulator of G-protein Rgs22, mitogen-activated protein kinase Mapk1, metabotropic glutamate receptorm GluR8, calcium-sensing receptor CaSR, and microtubule-associated protein Map1S) were predicted to play crucial roles in the GIFT response to S. iniae infection. CONCLUSIONS: S. iniae outbreaks have hindered the development of the tilapia industry in China. Understanding the miRNA transcriptome of S. iniae-infected GIFT is important for exploring the immune responses regulated by miRNAs as well as for studying novel regulated networks to prevent and treat S. iniae infections in the future.

Formation of the inner ear during embryonic and larval development of the cichlid fish (Oreochromis mossambicus).

BACKGROUND: The vertebrate inner ear comprises mineralized elements, namely the otoliths (fishes) or the otoconia (mammals). These elements serve vestibular and auditory functions. The formation of otoconia and otoliths is described as a stepwise process, and in fish, it is generally divided into an aggregation of the otolith primordia from precursor particles and then a growth process that continues throughout life. RESULTS: This study was undertaken to investigate the complex transition between these two steps. Therefore, we investigated the developmental profiles of several inner ear structural and calcium-binding proteins during the complete embryonic and larval development of the cichlid fish Oreochromis mossambicus in parallel with the morphology of inner ear and especially otoliths. We show that the formation of otoliths is a highly regulated temporal and spatial process which takes place throughout embryonic and larval development. CONCLUSIONS: Based on our data we defined eight phases of otolith differentiation from the primordia to the mature otolith.

gsdf is a downstream gene of dmrt1 that functions in the male sex determination pathway of the Nile tilapia.

Gonadal soma-derived factor (gsdf) is critical for testicular differentiation in teleosts, yet detailed analysis of Gsdf on testicular differentiation is lacking. In the present study, we knocked out tilapia gsdf using CRISPR/Cas9. F0 gsdf-deficient XY fish with high mutation rate (≥58%) developed as intersex, with ovotestes 90 days after hatching (dah), and become completely sex-reversed with ovaries at 180 and 240 dah. Those individuals with a low mutation rate (<58%) and XY gsdf(+/-) fish developed as males with normal testes. In F2 XY gsdf(-/-) fish, the gonads first expressed Dmrt1, which initiated the male pathway at 10 dah, then both male and female pathways were activated, as reflected by the simultaneous expression of Dmrt1 and Cyp19a1a in different cell populations at 18 dah, shifted to the female pathway expressing only Cyp19a1a at 36 dah, and finally developed into functional ovaries as adults. The male pathway and Dmrt1 expression was initiated, but failed to be maintained, in the absence of Gsdf. Aromatase-inhibitor treatment from 10 to 35 dah, however, rescued the phenotype, resulting in XY gsdf(-/-) with normal testes that expressed Dmrt1 and Cyp11b2. In vitro promoter analyses demonstrated that Dmrt1 activated gsdf expression in a dose-dependent manner in the presence of Sf1, even though Dmrt1 alone could not. Taken together, our results demonstrated that gsdf is a downstream gene of dmrt1. Gsdf probably inhibits estrogen production to trigger testicular differentiation. Mol. Reprod. Dev. 83: 497-508, 2016. © 2016 Wiley Periodicals, Inc.

The involvement of Nile tilapia (Oreochromis niloticus) Neu4 sialidase in neural differentiation during early ontogenesis.

Neurogenesis is an important process for the formation of the central nervous system during ontogenesis. Mammalian sialidases are involved in neurogenesis through desialylation of sialo-glycoconjugates. However, the significance of fish sialidases, unlike that of mammals, in neurogenesis has not been investigated. The present study focuses on Nile tilapia (Oreochromis niloticus) because of its unique profiles of sialidases related to enzymatic properties, subcellular localization, and tissue-specific gene expression. First, the fish were cultured under aphotic condition, which is known to cause the delayed development of the retina and brain in various fish. Next, we investigate the effect of aphotic condition on the levels of tilapia sialidases. Our results revealed that the tilapia showed a decrease in the number of ganglion cell in the retina. The expression level of neu4 mRNA is up-regulated in the eyes from tilapia reared in Dark accompanied by the increase of retinal differentiation markers. These results indicated that tilapia Neu4 is involved in retinal development in Nile tilapia. Furthermore, we tried to clarify the function of tilapia Neu4 in the neuronal cells using two neuroblast cell lines (SH-SY5Y and Neuro2a cell lines). Tilapia Neu4 decreased sialic acid level of both nuclear glycoproteins as well as glycolipids. Moreover, tilapia Neu4 accelerated neurite formation in both two neural cell lines and, increased the acetylcholinesterase activity, but it did not affect cell proliferation. Collectively, these results suggest that Neu4 accelerates neurite differentiation during ontogenesis in tilapia.

Role of Hox PG2 genes in Nile tilapia pharyngeal arch specification: implications for gnathostome pharyngeal arch evolution.

Phylogenetic reconstructions suggest that the ancestral osteichthyan Hox paralog group 2 gene complement was composed of two genes, Hoxa2 and b2, both of which have been retained in tetrapods, but only one of which functions as a selector gene of second pharyngeal arch identity (PA2). Genome duplication at the inception of the teleosts likely generated four Hox PG2 genes, only two of which, hoxa2b and b2a, have been preserved in zebrafish, where they serve as functionally redundant PA2 selector genes. Evidence from our laboratory has shown that other telelosts, specifically striped bass and Nile tilapia, harbor three transcribed Hox PG2 genes, hoxa2a, a2b, and b2a, with unspecified function(s). We have focused on characterizing the function of the three Nile tilapia Hox PG2 genes as a model to examine the effects of postgenome duplication gene loss on the evolution of developmental gene function. We studied Hox PG2 gene function in tilapia by examining the effects of independent morpholino oligonucleotide (MO)-induced knockdowns on pharyngeal arch morphology and Hox gene expression patterns. Morphological defects resulting from independent MO-induced knockdowns of tilapia hoxa2a, a2b, and b2a included the expected PA2 to PA1 homeotic transformations previously observed in tetrapods and zebrafish, as well as concordant and unexpected morphological changes in posterior arch-derived cartilages. Of particular interest, was the observation of a MO-induced supernumerary arch between PA6 and PA7, which occurred concomitantly with other MO-induced pharyngeal arch defects. Beyond these previously unreported morphant-induced transformations, a comparison of Hox PG2 gene expression patterns in tilapia Hox PG2 morphants were indicative of arch-specific auto- and cross-regulatory activities as well as a Hox paralog group 2 interdependent regulatory network for control of pharyngeal arch specification.

DNA damage in liver cells of the tilapia fish Oreochromis mossambicus larva induced by the insecticide cyantraniliprole at sublethal doses during chronic exposure.

Cyantraniliprole can effectively control lepidopteran pests and has been used all over the world. In general, the risk of cyantraniliprole seems low for fish, but the toxicity selectivity among different fish species was not clear. Here the acute toxicity and chronic effects of cyantraniliprole to juvenile tilapia (Oreochromis mossambicus) were assessed. The results showed that 96 h LC50 of cyantraniliprole to tilapia was 38.0 mg/L. After exposed for 28 days, specific growth rates of the blank control, solution control, and the treatments of 0.037, 0.37 and 3.7 mg/L of cyantraniliprole were 1.14, 0.95, 0.93, 0.82 and 0.70% per day, respectively. The results of micronucleus experiment and single cell gel electrophoresis showed that cyantraniliprole damaged DNA in liver cells of tilapia larvae. Quantitative PCR results showed that cyantraniliprole could induce the up-regulation of Rpa 3 that is responsible for the DNA repair. The significantly down-regulation of Chk 2 gene was related to p53 pathway. It is therefore proposed that cyantraniliprole causes DNA damage in liver cells of tilapia and activates DNA damage and repair pathways.

Ontogenic change in tissue osmolality and developmental sequence of mitochondria-rich cells in Mozambique tilapia developing in freshwater.

We investigated a change in tissue fluid osmolality and developmental sequences of mitochondria-rich (MR) cells during embryonic and larval stages of Mozambique tilapia, Oreochromis mossambicus, developing in freshwater. Tissue osmolality, representing body fluid osmolality, ranged from 300 to 370 mOsm/kg during embryonic and larval stages. This suggests that tilapia embryos and larvae are also able to regulate body fluid osmolality to some extent, although the levels are somewhat higher and fluctuate more greatly in embryos and larvae than in adults. Na(+)/K(+)-ATPase-immunoreactive MR cells were first detected in the yolk-sac membrane 3 days before hatching (day -3), followed by their appearance in the body skin on day -2. Subsequently, MR cells in both the yolk-sac membrane and body skin increased in number, and most densely observed on days -1 and 0. Whereas yolk-sac and skin MR cells decreased after hatching, MR cells in turn started developing in the gills after hatching. Thus, the principal site for MR cell distribution shifted from the yolk-sac membrane and body skin during embryonic stages to the gills during larval stages, and tilapia could maintain continuously their ion balance through those MR cells during early life stages.

The effects of maternal Cd on the metallothionein expression in tilapia (Oreochromis mossambicus) embryos and larvae.

The purpose of this study was to identify the factor(s) which would enhance the Cd resistance as assessed by the metallothionein (MT) expression in tilapia larvae. Larvae were collected from parents that were pretreated respectively with Cd or saline. At the end of the 12-week experiment, the hepatic MT and Cd contents in the breeding female fish were recorded. Our results indicated that a significant relationship between Cd and MT contents can be found in the offspring from the parent fish treated with Cd. However, the higher Cd resistance, Cd contents, and MT expression were limited to those larvae from parent fish bred within 4 weeks of the injection. By week 12, the Cd-treated fish still contained high levels of MT in their hepatic tissues. However, the MT and Cd contents in the larvae from these adult fish were not significantly different from those from the controls. In summary, we suggest that the higher Cd resistance of larvae from the egg stage was a result of the Cd contamination of the parent female, as evidenced by an increase in MT expression induced in tilapia embryos and larvae.

Gene structure and promoter function of a teleost ribosomal protein: a tilapia (Oreochromis mossambicus) L18 gene.

We have cloned and characterized a tilapia (Oreochromis mossambicus) L18 ribosomal protein gene, including the complete transcribed region and 488 bp of upstream regulatory sequences. We have also isolated two L18 cDNAs from another tilapia (Oreochromis niloticus) with a few conservative nucleotide differences. Our results suggest the presence of two genes in both species. Reporter constructs were tested for transient expression in CV1 cells and in microinjected zebrafish and tilapia embryos. The tilapia L18 promoter was able to drive expression of the reporter gene in all three experiments, with no apparent preference for a particular tissue. The tilapia L18 promoter is therefore likely to be a powerful tool to drive tissue-independent gene expression in fish.

Isolation and characterisation of tilapia beta-actin promoter and comparison of its activity with carp beta-actin promoter.

The regulatory sequence including proximal promoter, untranslated exon 1 and intron 1 of the beta-actin gene from tilapia (Oreochromis niloticus) has been isolated and spliced to a beta-galactosidase reporter gene to test its activity. Comparisons of promoter activity have been carried out with three different constructs: (1) 1.6 kb tilapia beta-actin regulatory sequence, (2) 1.5 kb carp beta-actin regulatory sequence, and (3) 4.7 kb carp beta-actin regulatory sequence. Although the 1.6 kb tilapia beta-actin regulatory sequence gave slightly different expression patterns in tilapia embryos assayed by in situ X-gal staining, no difference was observed in expression level when the tilapia sequence was compared with the 4.7 kb carp beta-actin regulatory sequence by quantitative assay. In comparison with the 1.5 kb carp beta-actin regulatory sequence, the 1.6 kb tilapia beta-actin regulatory sequence gave higher expression levels in tilapia embryos, while a reverse result was observed in zebrafish embryos. In cell transfection experiments, the 1.6 kb tilapia beta-actin regulatory sequence showed three to four times better activity in blue gill cells than either the 4.7 kb carp beta-actin or the 1.5 kb carp beta-actin regulatory sequences. The 1.6 kb tilapia beta-actin regulatory sequence also drove higher reporter gene activity in somatic cells of tilapia than did the 4.7 kb carp beta-actin regulatory sequence following direct injection of constructs into muscle. Therefore, taken together, the data demonstrate that the tilapia beta-actin promoter can be used as an efficient regulatory sequence to produce autotransgenic tilapia.

A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus).

We have constructed a genetic map for a tilapia, Oreochromis niloticus, using DNA markers. The segregation of 62 microsatellite and 112 anonymous fragment length polymorphisms (AFLPs) was studied in 41 haploid embryos derived from a single female. We have identified linkages among 162 (93.1%) of these markers. 95% of the microsatellites and 92% of the AFLPs were linked in the final map. The map spans 704 Kosambi cM in 30 linkage groups covering the 22 chromosomes of this species. Twenty-four of these linkage groups contain at least one microsatellite polymorphism. From the number of markers 15 or fewer cM apart, we estimate a total map length of approximately 1000-1200 cM. High levels of interference are observed, consistent with measurements in other fish species. This map is a starting point for the mapping of single loci and quantitative traits in cichlid fishes.

Two isoforms of vasa homologs in a teleost fish: their differential expression during germ cell differentiation.

Two isoforms of vasa mRNA and protein are present in a teleost fish, tilapia. One (vas-s) lacks a part of the N-terminal region found in the other isoform (vas). Both isoforms are expressed in oocytes through the embryonic stage when primordial germ cells (PGCs) localize in the lateral plate mesoderm. After PGC localization in the gonadal anlagen, vas-s expression increased and vas expression became undetectable. Expression of both isoforms was observed again after morphological gonadal sex differentiation, irrespective of genotypic sex. In ovary, compared with vas expression vas-s expression predominated throughout oogenesis. In testis, vas expression was predominant compared with vas-s during spermatogenesis. These results indicate that relative expression of two vasa isoforms is dependent upon germ cell differentiation and sex.

Development of the dorsal root ganglion in a teleost, Oreochromis mossambicus (Peters).

The precursor crest cells of the spinal dorsal root ganglia (DRG) in the tilapia, Oreochromis mossambicus, were analysed by HNK-1 antibody staining, scanning electron microscopy, and DiI labeling techniques. The ontogeny of the DRG was followed in the embryos and young fry of the fish. Neural crest cells which contribute to the formation of the DRG were observed to commence their migration in the trunk region after 40 hours postfertilization. They do not penetrate the somites but travel through the space between the neural tube and the somite. Crest cells destined to become the DRG accumulate at the midsomitic region where the ventral root exits. At 50 to 80 hours postfertilization, they differentiate and become bipolar sensory cells. The DRG continues to grow and develop right through hatching at 115 hours. During the early larval stages, crest cells accumulate around the ventral root and the DRG eventually fuses with the motor root, giving rise to a situation in which the DRG contains not only the sensory cells but also motor fibres. The mixed nature of the DRG was confirmed by HRP retrograde labeling. We believe that this is the first report in describing the formation of the DRG in a teleost.

The development of NADPH-diaphorase and nitric oxide synthase in the visual system of the cichlid fish, Tilapia mariae.

The pattern of NADPH-diaphorase expression was studied in the retina and optic tectum of the cichlid fish Tilapia mariae during the first developmental stages. NADPH-diaphorase activity was seen early, at hatching. In the retina a few cell bodies of the retinal inner nuclear layer showed a faint labeling. Scattered labeled cells were found in the stratum periventriculare of the optic tectum, while the optic nerve was unlabeled. Two days after hatching, the number of labeled neurons increased in the inner nuclear layer and a few stained cell bodies were also scattered in the ganglion cell layer. Both the inner and outer plexiform layers showed a diffuse staining and the optic nerve was devoid of labeling. In the optic tectum several positive cells in the periventricular layer, with their dendritic trees extending in the superficial fibrous layer, were found. In 1-month-old Tilapia, NADPH-diaphorase staining and nitric oxide synthase immunoreactivity were found to overlap in both the retina and optic tectum. The density of NADPH-diaphorase labeled neurons in the inner nuclear layer of the retina and in the stratum periventriculare of the optic tectum was largely reduced in comparison with 2 days posthatching embryos. These findings indicated an early and transient production of nitric oxide in the retina and optic tectum of Tilapia, suggesting a functional role for nitric oxide in the development of visual structures in aquatic vertebrates.

Histological study of the development of the embryo and early larva of Oreochromis niloticus (Pisces: Cichlidae).

The developmental stages of Oreochromis niloticus are similar to those described in other mouth-breeding tilapias except that, as in zebrafish, no cavity was found in the blastula. Variation in the rate of development of the embryo and larva of O. niloticus was found within a clutch of eggs as well as between clutches. Hatching glands are described for the first time in tilapias. They are widely distributed within the ectoderm covering the head, body, tail, and surface of the yolk sac near its attachment to the embryo. Timing of larval development is similar to that in other mouthbrooding tilapias, but is slower than that found in substrate-spawning tilapias. A pneumatic duct connects the swimbladder to the digestive tract and swimbladder inflation and initiation of feeding occurs at about the same time. The digestive tract of the larva 8 and 9 days after fertilization is similar to that found in the adult, except that there are no digestive glands. An endocrine pancreatic islet was first seen 76 h after fertilization. A prominent thymus gland is present at 100 h. Hematopoietic tissue develops in the vicinity of the pronephros during early larval development. A spleen develops later, 7 days after fertilization.

Comparative studies of the development and differentiation of chloride cells in tilapine fish with different reproductive styles.

Using light and electron microscopy and fluorescent probes, we followed the ontogenesis of selected organs in embryos of several species of tilapia (Cichlidae, Pisces) with emphasis on chloride cell differentiation in species with two different reproductive styles: we compared the substrate-brooder Tilapia zillii and the mouth-brooders Oreochromis niloticus, O. aureus, Sarotherodon galilaeus, and Tristramella sacra. In all species a transitory blood network system nurtured by the vena caudalis inferiores supplied the yolk sac and preanal finfold during the advanced stages of embryonic and initial stages of larval development. During these stages chloride cells occurred on the yolk sac, as a part of the abdominal epithelium. The cells and their associated blood plexus remained active here until the gill-lamellae, operculum, and mouth became functional. The chloride cells of their epithelium and blood system then took over, concomitant with a gradual degradation of the transitory blood system on the yolk sac. Ontogenesis of these systems (transitory and permanent) progressed at a higher rate in substrate-brooders than in mouth-brooders and was correlated with the earlier functioning of the gill-operculum system. Thus, at a constant temperature of 26 degrees C, the more exposed T. zillii progeny completed metamorphosis at 7-8 days after fertilization, calculated around 5,000 +/- 80 h/temp, whereas juveniles of more protected mouth-brooders attained a similar stage only 15 +/- 1 days after fertilization and around 9,000 +/- 200 h/temp. This earlier development of chloride cells and other pivotal organs in environmentally exposed progeny of substrate-brooders, as compared to the protected progeny of mouth-brooders, shows that their ontogeny was selected for the optimal survival style under specific etho-ecological conditions.

Fine structure of tooth germs during the formation of enameloid matrix in Tilapia nilotica, a teleost fish.

Tooth germs were examined by light and transmission electron microscopy. Collagen fibrils were relatively dispersed and thin at the early and middle stages of formation of the enameloid matrix, when the enameloid layer was thin. At the late stage, the fibrils became thicker, reaching nearly 30 nm dia, and formed the interwoven thick bundles that are characteristic of teleost cap enameloid. Abundant flocculent and/or fine, network-like material, probably representing glycosaminoglycans or proteoglycans, was located between the collagen fibrils. Tall, columnar, inner dental epithelial cells contained abundant rough endoplasmic reticulum and many mitochondria, and a well-developed Golgi apparatus was seen around the nuclei at the late stage. Elongated vesicles enclosing fine, filamentous material that resembled procollagen granules, and large granules containing fibril-like structures that were 150 nm in thickness and had periodic cross-banding at 32-nm intervals, were usually observed near the Golgi apparatus. The contents of the large granules were well stained with phosphotungstic acid, which suggests that inner dental epithelial cells synthesize collagen fibrils. At this time, odontoblasts also contained abundant rough endoplasmic reticulum and mitochondria, a well-developed Golgi, several kinds of granule including those that probably contained procollagen, and many microtubules. It is proposed that odontoblasts are involved in the formation of a considerable portion of the enameloid matrix, including collagen fibrils.

Developmental pattern of NADPH-diaphorase activity in the peripheral nervous system of the cichlid fish Tilapia mariae.

The distribution of NADPH-diaphorase activity was studied in the cichlid fish Tilapia mariae, during the first developmental stages by means of the tetrazolium salt technique. The reaction product was first found, 48 hours after fertilization (stage 10), in the cells of the olfactory placodes and in the superficial neuromasts. A faint positivity was seen in some hair cells of the otic vesicles. The epithelial cells of the most caudal part of the intestinal tract showed a strong labeling. At stage 12 (hatch), the reaction product was in addition detected in scattered enteric neurons surrounding the digestive tract. At stage 13 (4.5 days after spawning), the reaction product was also found in the putative sympathetic trunk, which supplies the gill arches and digestive tract. The epithelial cells of the gastrointestinal canal showed a more strong positive labeling and two large clusters of cells near the pronephritic tubules (the putative adrenomedullar tissue) were also labeled. The present results indicate an early activity of NADPH-diaphorase during the development of the peripheral nervous system of Tilapia and reveal a gradual maturation of NADPH-diaphorase positive structures.

Effects of cortisol on ion regulation in developing tilapia (Oreochromis mossambicus) larvae on seawater adaptation.

The yolk diameter of cortisol-treated tilapia (Oreochromis mossambicus) larvae, immersed in freshwater (FW) containing 10 mg L-1 cortisol from 48 h postfertilization to 12 d posthatching, was significantly larger than that of control larvae after 8 d of treatment, suggesting that inhibition on larval growth occurred only after a long-term treatment with cortisol. Tilapia embryos or larvae treated with 1-10 mg L-1 cortisol for 1-2 d and then transferred to 20-30 g L-1 seawater (SW) showed reduced cumulative larval mortality in SW compared with controls. Moreover, 4-5 d of cortisol treatments significantly diminished the degree of increase in larval body Na content after the transfer to SW. Significant effect of cortisol on body Na content of larvae occurred as early as 4-8 h after the transfer to SW, while no significant difference was found in the ouabain binding of yolk-sac epithelia between control and cortisol-treated larvae even 12 h after the transfer. Cortisol may be involved in the early phase of SW adaptation in developing larvae, and this mechanism may be achieved by other means than increasing the Na-K-ATPase of yolk-sac epithelia.

Presence of Na-K-ATPase in mitochondria-rich cells in the yolk-sac epithelium of larvae of the teleost Oreochromis mossambicus.

The purpose of this study is to provide biochemical evidence for the functions of the mitochondria-rich cell (MR cell) in the yolk-sac epithelium of the developing larvae of tilapia Oreochromis mossambicus. Western blotting with the antibody (6F) raised against avian Na-K-ATPase alpha1 subunit demonstrated the presence of Na-K-ATPase in yolk-sac epithelium of tilapia larvae and about 1. 46-fold more of the enzyme in seawater larvae than in freshwater ones. The yolk-sac MR cells were immunoreacted to the antibody (alpha5) against the alpha subunit of avian Na-K-ATPase and were double-labeled with anthroylouabain and dimethylaminostyrylethyl-pyridiniumiodine, suggesting the existence and activity of Na-K-ATPase in these cells. Binding of 3H-ouabain in the yolk sac of seawater larvae was much higher than in that of freshwater larvae (4.183+/-0.143 pmol/mg protein versus 1.610+/-0. 060 pmol/mg protein or 0.0508+/-0.0053 pmol/yolk sac versus 0. 0188+/-0.0073 pmol/yolk sac). These biochemical results are further evidence that yolk-sac MR cells are responsible for a major role in the osmoregulatory mechanism of early developmental stages before the function of gills is fully developed.