Discovery of an unrecognized nidovirus associated with granulomatous hepatitis in rainbow trout.

Rainbow trout (Oncorhynchus mykiss) is the principal species of inland-farmed fish in the Western hemisphere. Recently, we diagnosed in farmed rainbow trout a disease in which the hallmark is granulomatous-like hepatitis. No biotic agents could be isolated from lesions. Still, unbiased high-throughput sequencing and bioinformatics analyses revealed the presence of a novel piscine nidovirus that we named "Trout Granulomatous Virus" (TGV). TGV genome (28,767 nucleotides long) is predicted to encode non-structural (1a and 1 ab) and structural (S, M, and N) proteins that resemble proteins of other known piscine nidoviruses. High loads of TGV transcripts were detected by quantitative RT-PCR in diseased fish and visualized in hepatic granulomatous sites by fluorescence in situ hybridization. Transmission electron microscopy (TEM) revealed coronavirus-like particles in these lesions. Together, these analyses corroborated the association of TGV with the lesions. The identification and detection of TGV provide means to control TGV spread in trout populations.

Whole-Genome Inter-Sex Variation in Russian Sturgeon (Acipenser gueldenstaedtii).

The Russian sturgeon (Acipenser gueldenstaedtii, AG) is an endangered fish species increasingly raised on fish farms for black caviar. Understanding the process of sex determination in AG is, therefore, of scientific and commercial importance. AG lacks sexual dimorphism until sexual maturation and has a predominantly octoploid genome without a definite sex chromosome. A conserved short female-specific genomic sequence was recently described, leading to the development of a genetic sex marker. However, no biological function has been reported for this sequence. Thus, the mechanism of sex determination and the overall inter-sex genomic variation in AG are still unknown. To comprehensively analyze the inter-sex genomic variation and assess the overall inter-species variation between AG and A. ruthenus (AR, sterlet), a related tetraploid sturgeon species, we performed whole-genome sequencing on DNA from 10 fish-farm-raised adult AG (5 males and 5 females). We produced a partially assembled, ~2390 MBp draft genome for AG. We validated in AG the female-specific region previously described in AR. We identified ~2.8 million loci (SNP/indels) varying between the species, but only ~7400 sex-associated loci in AG. We mapped the sex-associated AG loci to the AR genome and identified 15 peaks of sex-associated variation (10 kb segments with 30 or more sex-associated variants), 1 of which matched the previously reported sex-variable region. Finally, we identified 14 known and predicted genes in proximity to these peaks. Our analysis suggests that one or more of these genes may have functional roles in sex determination and/or sexual differentiation in sturgeons. Further functional studies are required to elucidate these roles.

Cross-species conservation of a transposase-linked element enables genetic sexing of commercial populations of Russian sturgeon (Acipenser gueldenstaedtii).

All-female culture of sturgeon is essential for efficient caviar production. However, Russian sturgeon (Acipenser gueldenstaedtii) does not exhibit external sexual dimorphism, and therefore, commercial farms apply gonadal endoscopy or ultrasound at the earliest age of 4-5 years to separate the sexes, with ~90% accuracy. Recently, a dominant genomic marker (AllWSEX2) has been found with association to femaleness in sturgeons. We developed a duplex PCR (dAllWSEX2) with the adjacent bmp7 gene as an internal control, to validate an effective PCR. Robust amplification of control fragments was observed for all samples of our commercial A. gueldenstaedtii stock (n = 337). The dAllWSEX2 assay was significantly associated with sex (n = 43, p < 1.6 × 10-8 ), yet four (18%) of the endoscopy-determined females were genetic males. To examine whether some females display a male genetic profile, we tested 96 egg-producing females, which were all verified as genetic females, indicating that the observed mismatches may be attributed to wrong sexing by endoscopy. Application of dAllWSEX2 on 100 7-month-old fish showed no sex-dependent differences in body weight, indicating that weighing is not an applicable tool for sorting females at a young age. Sanger sequencing of the bmp7 fragment revealed octaploidy and sex-independent variation, suggesting that the critical sex-determining region harboring AllWSEX2 is small. In keeping with a model of a single-ploidy encoding female determination, AllWSEX2 showed no variation despite being a transposase-linked repetitive element. Cross-species conservation of AllWSEX2, and absence of annotated sex-determination genes in this region suggests that, in sturgeons, the sex-determining mechanism is different from mechanisms identified in other fish.

Molecular characterization of kisspeptin receptors and gene expression analysis during oogenesis in the Russian sturgeon (Acipenser gueldenstaedtii).

Sturgeons belong to a subclass of fishes that derived from ray-finned fish ancestors preceding the emergence of teleosts. The Russian sturgeon (Acipenser gueldenstaedtii) is a late-maturing fish with the females reaching puberty under aquaculture conditions at 6-10 years of age. Since kisspeptin has been shown to be a key hormone involved in regulation of major reproductive processes of many vertebrate species, this study was conducted to better understand the kisspeptin receptor (KissR) in sturgeon. In this study we have cloned Russian sturgeon KissR1 from brain mRNA and observed the ontogeny of rsKissR1 mRNA expression in ovarian follicles. Multiple sequence alignment of KissR1, KissR4, and their orthologs revealed that the Russian sturgeon (rs) KissR1 sequence shares 64%-77% identity with elephant shark, coelacanth, and gar and 44-58% identity with tetrapod and teleost KissR1 sequences, while KissR4 seemed to share <65% identity to eel KissR2 and ~57% identity to Perciformes and Cypriniformes. Further rsKissR4 showed <97% identity to reed fish KissR4, <63% with Squamata (Reptiles) and gar KissR4. A phylogenetic analysis revealed that rsKissR1 is more closely related to coelacanth and gar KissR1 than teleost, while rsKissR4 was part of the KissR4 clade and shared higher similarity with Actinopterygiian sequences. We have further predicted homology models for both rsKiss receptors and performed in-silico analyses of their binding to a kiss-10 peptide. Both sturgeon and zebrafish Kiss1 and Kiss2 activated rsKissR1 via both PKC/Ca2+ and PKA/cAMP signal-transduction pathways, while rsKissR2 was found to be less effective and was not activated by stKiss peptides. Ovarian rsKissR transcript levels for 10 fishes were determined by real-time PCR and significantly increased concomitantly with oogenesis, where the highest level of expression was evident in black follicles. These data suggest that extra-neuronal expression of the kisspeptin receptor may be involved in sturgeon reproduction in a manner dependent on reproductive development.

Molecular characterization of two Russian sturgeon gonadotropin receptors: Cloning, expression analysis, and functional activity.

Sturgeons are being used in aquaculture because wild populations are now endangered due to overfishing for caviar. A challenge in working with sturgeon as an aquacultured species is its long and slow reproductive development. Reproduction is a hormonally regulated process that involves hierarchical signaling between the brain, pituitary gland, and gonads. In an effort to better understand the hormonal regulation of sturgeon reproduction, we have cloned the Russian sturgeon (st), Acipenser gueldenstaedtii, luteinizing hormone receptor (stLHR) and follicle stimulating hormone receptor (stFSHR) and measured their expression from previtellogenic to mature ovarian follicles. Sturgeon LHR and FSHR expression was elevated in early-vitellogenic and mature follicles compared with pre-vitellogenic and mid-vitellogenic follicles, and only LHR expression increased during late-vitellogenesis. Recombinant sturgeon FSH and LH both activated sturgeon LHR and FSHR in a cAMP reporter assay. Further molecular characterization of these receptors was accomplished by in silico modeling and cAMP reporter assays using heterologous recombinant gonadotropins from human and piscine species. There was no apparent trend in heterologous LH and/or FSH activation of the sturgeon LHR or FSHR. These data suggest that permissive activation of LHR and FSHR are a consequence of some yet undetermined biological characteristic(s) of different piscine species.

Sex-related gonadal gene expression differences in the Russian sturgeon (Acipenser gueldenstaedtii) grown in stable aquaculture conditions.

The Russian sturgeon (Acipenser gueldenstaedtii) is a primitive freshwater fish and a source of caviar. In the present study, the gonadal transcriptomes of male and female Russian sturgeons grown in stable aquaculture conditions were analyzed. RNA sequencing of whole-gonad transcriptomes from pools of 4-year old fish (five females, four males), resulted in the identification of 28,170 unique transcripts. Of these, 16,191 could be annotated by similarity to gene sequences from other species. There were 392 transcripts that showed differential abundance by a factor of 20-fold or more between the sexes; 272 of these were annotated; of these, 175 and 97 were in greater abundance in ovaries and testes, respectively. Functional annotation and clustering of the genes with differential abundances of mRNA allowed for identification of several clusters. Thus, a group of transcriptional regulators and factors involved in cell division, especially septins, were in greater abundance in the ovaries; while a different set of transcription factors (including sox6 and sox30) and a group of protein kinases were in greater abundance in the testes. The transcript abundances of nine highly abundant candidate transcripts, as well as of two additional genes previously known to be involved in reproduction, cyp19 (p450 aromatase) and foxl2, were assessed in the individual samples by qRT-PCR. Of these, five (including cyp19 and foxl2) were in greater abundance in ovaries, while the abundance of ighm1 mRNA was greater in testes. Phylogenetic analysis based on the k1c18 keratin gene placed the sturgeon sequence nearest those of other primitive fish species, supporting the ancient origin of the sturgeon. In conclusion, this study details transcriptome differences between male and female sturgeon and identifies key genes that may contribute to sexual determination and differentiation.

Gonadotropins in the Russian Sturgeon: Their Role in Steroid Secretion and the Effect of Hormonal Treatment on Their Secretion.

In the reproduction process of male and female fish, pituitary derived gonadotropins (GTHs) play a key role. To be able to specifically investigate certain functions of Luteinizing (LH) and Follicle stimulating hormone (FSH) in Russian sturgeon (Acipenser gueldenstaedtii; st), we produced recombinant variants of the hormones using the yeast Pichia pastoris as a protein production system. We accomplished to create in vitro biologically active heterodimeric glycoproteins consisting of two associated α- and ß-subunits in sufficient quantities. Three dimensional modelling of both GTHs was conducted in order to study the differences between the two GTHs. Antibodies were produced against the unique ß-subunit of each of the GTHs, in order to be used for immunohistochemical analysis and to develop an ELISA for blood and pituitary hormone quantification. This detection technique revealed the specific localization of the LH and FSH cells in the sturgeon pituitary and pointed out that both cell types are present in substantially higher numbers in mature males and females, compared to immature fish. With the newly attained option to prevent cross-contamination when investigating on the effects of GTH administration, we compared the steroidogeneic response (estradiol and 11-Keto testosterone (11-KT) in female and males, respectively) of recombinant stLH, stFSH, and carp pituitary extract in male and female sturgeon gonads at different developmental stages. Finally, we injected commercially available gonadotropin releasing hormones analog (GnRH) to mature females, and found a moderate effect on the development of ovarian follicles. Application of only testosterone (T) resulted in a significant increase in circulating levels of 11-KT whereas the combination of GnRH + T did not affect steroid levels at all. The response pattern for estradiol demonstrated a similar situation. FSH levels showed significant increases when GnRH + T was administered, while no changes were present in LH levels.

Identification of a novel RNA virus lethal to tilapia.

Tilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and it induced a cytopathic effect at 5 to 10 days postinfection. Electron microscopy revealed enveloped icosahedral particles of 55 to 75 nm. Low-passage TiLV, injected intraperitoneally in tilapia, induced a disease resembling the natural disease, which typically presents with lethargy, ocular alterations, and skin erosions, with >80% mortality. Histological changes included congestion of the internal organs (kidneys and brain) with foci of gliosis and perivascular cuffing of lymphocytes in the brain cortex; ocular inflammation included endophthalmitis and cataractous changes of the lens. The cohabitation of healthy and diseased fish demonstrated that the disease is contagious and that mortalities (80 to 100%) occur within a few days. Fish surviving the initial mortality were immune to further TiLV infections, suggesting the mounting of a protective immune response. Screening cDNA libraries identified a TiLV-specific sequence, allowing the design of a PCR-based diagnostic test. This test enables the specific identification of TiLV in tilapines and should help control the spread of this virus worldwide.

A pivotal role for the Streptococcus iniae extracellular polysaccharide in triggering proinflammatory cytokines transcription and inducing death in rainbow trout.

Streptococcus iniae is a major pathogen of fish, causing considerable economic losses in Israel, the United States and the Far East. Containment of mortalities through vaccination was recently compromised due to the emergence of novel vaccine-escape strains that are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide (EPS) that is released to the medium. In vitro and in vivo data now indicate that the EPS is a major virulence factor, capable of triggering the proinflammatory cytokine machinery and inducing mortality of fish. Streptococcus iniae EPS might therefore be considered to be responsible for sepsis and death just as lipopolysaccharide is for Gram-negative pathogens.

Emergence of novel Streptococcus iniae exopolysaccharide-producing strains following vaccination with nonproducing strains.

Streptococcus iniae is a major pathogen of fish, producing fatal disease among fish species living in very diverse environments. Recently, reoccurrences of disease outbreaks were recorded in rainbow trout (Oncorhynchus mykiss, Walbaum) farms where the entire fish population was routinely vaccinated. New strains are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide that is released into the medium. Present findings indicate that the extracellular polysaccharide is a major antigenic factor, suggesting an evolutionary selection of strains capable of extracellular polysaccharide production.

Transcytosis of Streptococcus iniae through skin epithelial barriers: an in vitro study.

By constructing a biological model based on in vitro culture of polarized rainbow trout primary skin epithelial cell monolayers, the series of early events that precede Streptococcus iniae infection, particularly colonization and translocation through external barriers, were analyzed. Streptococcus iniae promptly invades skin epithelial cells, but the rapid decline of viable intracellular bacteria points out the limited capability of intracellular survival for this bacterium. Translocation assays, supported by electron microscopy microphotographs, demonstrate that following successful in vitro invasion of skin epithelial cell, the bacterium exists free in the cytoplasm after release from the endosome, and translocates through the skin barrier. Bacterial invasion and transcytosis is not accompanied by apparent cell-line damages or disruption of host cells' tight junctions. It is hypothesized that the phenomenon of epithelial invasion coupled to the rapid translocation through the barrier plays a crucial role in Streptococcus iniae infection.

Anatomical, hormonal and histological descriptions of captive Russian sturgeon (Acipenser gueldenstaedtii) with intersex gonads.

Sturgeons are known throughout the world as the source of black caviar. Their declining populations in their native habitats, mainly the Caspian Sea, due to over-fishing for meat and caviar production, destruction of their spawning grounds and water pollution, have led to their introduction into aquaculture in areas with suitable conditions, including Israel. Recently, we noticed an unusual phenomenon in these normally gonochoristic species. Several 5-year-old female sturgeons were found to have one or more testicular sections in each of their two gonads, forming an intersexual gender. Further examination of other fish from the same age group revealed 14% fish with intersex gonads among a population of 5000 fish that had been pre-selected as females. This phenomenon has not been found however in other age groups of Russian sturgeons, cultured at the same facility. Sturgeons are a generally gonochoristic species, and hermaphroditism is only very infrequently observed under natural or normal breeding conditions. Moreover, these rare cases have all been from polluted habitats. The present work is the first description of fish containing intersex gonads in Russian sturgeon (Acipenser gueldenstaedtii). We describe the phenomenon anatomically and histologically, and examine plasma steroid levels and pituitary gonadotropin gene expression by comparing fish with intersex gonads with normal females and males of the same age group. Intersex gonads were typical female ovaries with one or more white testicular components embedded in each. The testis components were not uniform in size or location among the two gonads of each fish or among different fish, and they showed marked differences in distribution. The ovarian component of the intersex gonad was at the pre-vitellogenic stage as in normal females, and the testis component contained spermatids and mature spermatozoa as in normal males of the same age. However, in terms of estradiol and 11-ketotestosterone plasma levels, as well as of pituitary gonadotropin (betaLH and betaFSH) gene-expression levels, the fish with intersex gonads were more similar to the normal males than to the normal females, even though the testis part of the intersex gonad was smaller than the ovarian part. To examine the possibility that the fish containing intersex gonads were hybrids, phylogenetic trees were constructed from the consensus sequences of Cytochrome b and control region (D-loop) genes. Results indicated no differences between the fish with intersex gonads and normal males or females of the same age group. However, statistically significant differences were found between different age groups of Russian sturgeon, as well as of white sturgeons (A. transmontanus), grown under the same culture conditions.

Studies on retinol-binding protein during vitellogenesis in the Rainbow Trout (Oncorhynchus mykiss).

Retinoids are important regulatory signaling molecules during embryonic development and therefore, should be present in the eggs of oviparous animals that develop independently of the maternal organism. Studies were initiated in Rainbow Trout to elucidate the role of retinol-binding protein (RBP), the specific retinol carrier protein in vertebrate plasma, during vitellogenesis. Plasma levels of RBP in pre-vitellogenic, vitellogenic, and post-vitellogenic females were compared to plasma of male trout, using a rabbit polyclonal antiserum that was generated to His-tagged RBP recombinant protein. Western-blot analyses showed that there were no differences in the relative plasma levels of RBP between pre-vitellogenic, vitellogenic or post-vitellogenic females and similar levels were also found in males. In contrast, strong elevation of vitellogenin (VTG) was observed in the plasma from vitellogenic females. Northern-blot analysis of hepatic mRNA revealed that there were no dramatic changes in the abundance of RBP transcripts in the liver of females during vitellogenesis, but showed a significant increase in the expression of VTG in the livers of vitellogenic females. These results indicate differences in the regulation of RBP and VTG during vitellogenesis, suggesting that RBP may not be the main transporter protein for retinoids to fish egg. Recent publications on the association of retinal with VTG in fish and the occurrence of RBP transcripts in ovarian tissues raise the need for reevaluation of the role of RBP during vitellogenesis in oviparous non-mammalian vertebrate species.

Cloning of FSHbeta, LHbeta, and glycoprotein alpha subunits from the Russian sturgeon (Acipenser gueldenstaedtii), beta-subunit mRNA expression, gonad development, and steroid levels in immature fish.

The Russian sturgeon, Acipenser gueldenstaedtii, is a late-maturing Acipenseriformes. To elucidate the role of FSH and LH in its reproduction, we cloned its glycoprotein alpha-subunit (GPalpha) and gonadotropin beta-subunits (FSHbeta and LHbeta) using 5' and 3' RACE-PCR. The nucleotide sequences of the Russian sturgeon (st) GPalpha, FSHbeta, and LHbeta are 345, 384, and 411 bp long, encoding peptides of 91, 115, and 114 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with those of other teleosts. Sequence analysis showed that stFSHbeta is more similar to higher vertebrate FSHbetas (35-37%) than to higher vertebrate LHbetas (26-30%). The next objective of this work was to compare the development of sturgeon gonads at the very first stages of their growth with the expression of their gonadotropins. Sturgeons at ages 1, 2, 3 or 4 years were sacrificed. The expression of their gonadotropin beta-subunits was determined using quantitative real-time PCR, and their gonads were examined histologically, followed by a determination of the plasma levels of estradiol in females and 11-ketotestosterone (11-KT) in males. The expression levels of stFSHbeta subunit was found to be higher in fish at 3 and 4 years of age than in 1-year olds. mRNA levels of stLHbeta were higher than those of stFSHbeta in both genders. Moreover mRNA levels of stFSHbeta detected in females were significantly higher than those found in males. Even at age 4 years, all female Russian sturgeons tested contained gonads at the pre-vitellogenic stage, with small oocytes and very low levels of estradiol in the plasma. However, among the males, at ages 3 and 4 years, we found testes that contained spermatids and spermatozoa. Those males were found to have significantly high GSI (gonadosomatic index; gonadal weight as a percentage of BW) levels, stLHbeta expression and 11-KT levels.

Cloning and developmental expression of the cytochrome P450 aromatase gene (CYP19) in the European eel (Anguilla anguilla).

To characterize the involvement of the aromatase gene during the process of sex determination in the European eel (Anguilla anguilla), the expression of its gonadal form was determined during various developmental stages. The cloned cDNA from the European eel gonad (EeCYP19) contains an open reading frame of 1539 bp, encoding a deduced protein of 513 residues. The predicted amino acid sequence shows 97% identity with that of the Japanese eel, and 59-69% of identity with those of the CYP19 gonadal and brain forms of other teleost fish. Two potential initiation sites (ATG) were found downstream of the first ATG codon. A fluorescent-based method of real-time PCR was developed to quantify EeCYP19 expression. The expression levels of EeCYP19 in the gonads of adult males were approximately 12- and 30-fold lower than the levels in adult females and juvenile eels previously treated with E2, respectively. Expression of aromatase was found only in a single specimen in the control group. In contrast, no difference was found among sexes in the aromatase expression in the brain. Treatment with aromatase inhibitor (AI) of juvenile eel resulted in the total loss of aromatase expression in the gonads and brains. The results of this work revealed that AI treatment not only reduces the synthesis of estradiol, but reduces the expression levels of EeCYP19 as well. No evidence for the presence of a distinct extra-gonadal (brain) form of aromatase in the European eel could be provided.

Trojan horse effect: phagocyte-mediated Streptococcus iniae infection of fish.

The salmonid macrophage-like cell line RTS-11 and purified trout pronephros phagocytes were used to analyze in vitro entry and survival of two Streptococcus iniae serotypes. Efficient invasion by S. iniae occurred in both cells, but only the type II strain persisted in pronephros phagocytes for at least 48 h. Ex vivo models of opsonin-dependent phagocytosis by pronephros phagocytes demonstrated increased phagocytosis efficacy. Analysis of phagocytes collected from diseased fish demonstrated that approximately 70% of the bacteria contained in the blood during the septic phase of the disease were located within phagocytes, suggesting an in vivo intracellular lifestyle. In addition to the augmented levels of bacteremia and enhanced survival within phagocytes, S. iniae type II induces considerable apoptosis of phagocytes. These variabilities in intramacrophage lifestyle might explain differences in the outcomes of infections caused by different serotypes. The generalized septic disease associated with serotype II strains is linked not only to the ability to enter and multiply within macrophages but also to the ability to cause considerable death of macrophages via apoptotic processes, leading to a highly virulent infection. We assume that the phenomenon of survival within phagocytes coupled to their apoptosis plays a crucial role in S. iniae infection. In addition, it may provide the pathogen an efficient mechanism of translocation into the central nervous system.