Molecular cloning of Salmo salar Toll-like receptors (TLR1, TLR22, TLR5M and TLR5S) and expression analysis in SHK-1 cells during Piscirickettsia salmonis infection.

In fish, the innate immune system is the primary response against infection. Toll-like receptors (TLRs) recognize pathogens through pathogen-associated molecular patterns (PAMPs), and some target molecules of TLRs are homologous between fish and mammals. Piscirickettsia salmonis is one of the main pathogens affecting the salmon industry in Chile. Better knowledge of mechanisms underlying its invasive capacity and recognition of target cells is crucial for vaccine development. Therefore, Salmo salar L. TLR1, TLR22, membrane TLR5M and soluble TLR5S sequences were cloned, and expression kinetics were analysed by RT-qPCR in salmon head kidney cells (SHK-1) infected with three different P. salmonis preparations: alive, formaldehyde treated, extract. Clearly, all analysed TLRs were expressed and transcription level changes were revealed at 2 hpi, 12 or 16 hpi and 24 hpi depending on P. salmonis infection scheme. Increased IL1-beta expression confirmed TLR pathway response. Furthermore, significant expression modulations of several members of the TLR pathway in this in vitro model suggest that P. salmonis extract rather than formaldehyde-inactivated bacteria might strengthen the salmon immune system.

Differential response of two somatolactin genes to zinc or estrogen in pituitary of Cyprinus carpio.

Environmental changes affect gene expression that we addressed in the pituitary, a central regulatory organ at the interface between the central nervous system and the endocrine system. With the aim to reveal effects of changes in the aquatic environment on the expression of hypothalamo-hypophyseal factors, we characterized somatolactin (SL) in Cyprinus carpio. SL, a fish specific pituitary hormone belonging to the prolactin (PRL) superfamily, is involved in background adaptation, osmoregulation, reproduction and fatty acid metabolism. Two sl genes, α and ß, were discovered in carp and transcripts of both were detected in pituitaries. Clearly, expression of slα and slß was modulated significantly in pituitary of male adult carp in response to treatment with ZnCl2 (Zn), but only slß responded to 17ß-estrogen (E2), relative to control carp as shown by RT-qPCR analyses. Furthermore, the amount of mRNA of related factors was assessed revealing variable effects on prl, growth hormone (gh), and factors involved in sl regulation: the pituitary transcription factor pit1 and hypothalamic pituitary adenylase cyclase activating peptide (pacap). In parallel, the physiological response of the experimental animals to Zn or E2 was confirmed by showing a significant increase of metallothionein (mt) or vitellogenin (vg) gene expression in liver, classical sentinels for exposure to heavy metal or estrogens. These data suggest that the sl genes seem to be involved in the response to Zn, as well as to estrogen, and could contribute to evaluate biological relevant changes in the aquatic environment.

Identification of the clpB and bipA genes and an evaluation of their expression as related to intracellular survival for the bacterial pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is the pathogen responsible for salmonid rickettsial septicemia (SRS), a disease that affects a wide variety of marine cultivated fish species and causes economic losses for the aquaculture industry worldwide. Many in vitro studies have reported on the capacity of this microorganism to replicate in the interior of cytoplasmic vesicles from varied fish cell lines. However, the mechanisms used by this bacteria to survive, replicate, and propagate in cell lines, especially in macrophages and monocytes, are unknown. A number of studies have described the diverse proteins in pathogens such as Legionella pneumophila, Coxiella burnetii, and Francisella tularensis which allow these to evade the cellular immune response and replicate in the interior of macrophages in different hosts. Some of these proteins are the virulence factor BipA/TypA and the heat shock protein ClpB, both of which have been widely characterized. The results of the current study present the complete coding sequence of the genes clpB and bipA from the P. salmonis genome. Moreover, the experimental results suggest that during the infectious process of the SHK-1 cellular line in P. salmonis, the pathogen significantly increases the expression of proteins ClpB and BipA. This would permit the pathogen to adapt to the hostile conditions produced by the macrophage and thus evade mechanisms of cellular degradation while facilitating replication in the interior of this salmon cell line.

At least two expressed genes for transcription factors Pitx2 and Rpx are present in common carp and are upregulated during winter acclimatization.

The mechanisms of seasonal acclimatization in eurythermal fish such as common carp are not fully understood. Here, we concentrate on the regulation of pituitary factors, as this organ was shown to be highly affected by seasonal changes. We cloned and sequenced two different cDNAs for each of the transcription factors Pitx2 and Rpx, known to play a role in pituitary development. We show that these genes are conserved throughout evolution, to different degrees depending on the specific domain considered. Finally, we show that the cDNAs for both factors are clearly up-regulated during the winter season, in sharp contrast to other regulators such as Pit1 or pituitary hormone genes such as prolactin (prl) and growth hormone (gh). Our results suggest that increased expression of Pitx2 and Rpx contributes to seasonal adaptation of common carp to winter conditions.

Copeptin, derived from isotocin precursor, is a probable prolactin releasing factor in carp.

Control of prolactin (PRL) release is of crucial importance for the multiple functions exerted by PRL in vertebrates. Recently identified hypothalamic PRL-releasing peptides displayed additional neuromodulatory activities and in fish only few could be detected close to lactotrophs. Here we describe the C-terminal peptide processed from the carp isotocin precursor as probable physiologically relevant regulator of PRL release in carp. The amino acid sequence derived from the complete isotocin precursor gene of Cyprinus carpio, predicted a C-terminal peptide uncleaved between the neurophysin (Np) and copeptin (Cp) domain. Accordingly, antibodies against synthetic Np- and Cp-specific oligopeptides both immunodetected a 13kDa protein (cNpCp) in total pituitary proteins and showed abundant immunoreaction in hypothalamic axons in direct contact with lactotrophs in the rostral pars distalis of carp pituitary gland sections. Finally, incubation of cultured carp pituitary explants with purified carp cNpCp resulted in a potent stimulation of PRL release.

Modular changes of cis-regulatory elements from two functional Pit1 genes in the duplicated genome of Cyprinus carpio.

The pituitary-specific transcription factor Pit1 is involved in its own regulation and in a network of transcriptional regulation of hypothalamo-hypophyseal factors including prolactin (PRL) and growth hormone (GH). In the ectotherm teleost Cyprinus carpio, Pit1 plays an important role in regulation of the adaptive response to seasonal environmental changes. Two Pit1 genes exist in carp, a tetraploid vertebrate and transcripts of both genes were detected by RT-PCR analysis. Powerful comparative analyses of the 5'-flanking regions revealed copy specific changes comprising modular functional units in the naturally evolved promoters. These include the precise replacement of four nucleotides around the transcription start site embedded in completely conserved regions extending upstream of the TATA-box, an additional transcription factor binding site in the 5'-UTR of gene-I and, instead, duplication of a 9 bp element in gene-II. Binding of nuclear factors was assessed by electro mobility shift assays using extracts from rat pituitary cells and carp pituitary. Binding was confirmed at one conserved Pit1, one conserved CREB and one consensus MTF1. Interestingly, two functional Pit1 sites and one putative MTF1 binding site are unique to the Pit1 gene-I. In situ hybridization experiments revealed that the expression of gene-I in winter carp was significantly stronger than that of gene-II. Our data suggest that the specific control elements identified in the proximal regulatory region are physiologically relevant for the function of the duplicated Pit1 genes in carp and highlight modular changes in the architecture of two Pit1 genes that evolved for at least 12 MYA in the same organism.

Cloning of the prepro C-RFa gene and brain localization of the active peptide in Salmo salar.

In all vertebrates, the synthesis and release of prolactin (Prl) from pituitary lactotroph cells is tightly controlled by hypothalamic factors. We have cloned and characterized a hypothalamic cDNA from Atlantic salmon (Salmo salar) encoding C-RFa, a peptide structurally related to mammalian Prl-releasing peptide (PrRP). The deduced preprohormone precursor is composed of 155 amino acid residues presenting a 87.1% similarity to chum salmon C-RFa and a 100% similarity to all fish C-RFa in the bioactive precursor motifs. C-RFa-immunoreactive perikarya and fibres were located in the brain of S. salar, especially in the hypothalamus, olfactory tract, optic tectum and cerebellum. In contrast, immunolabelled fibres were not observed in the pituitary stalk or in the hypophysis. However, interestingly, we detected immunolabelled cells in the rostral pars distalis of the pituitary in the basolateral region in which Prl is synthesized. These results were confirmed by obtaining a strong signal by using reverse transcription/polymerase chain reaction (RT-PCR) on mRNA from both hypothalamus and pituitary. These data show, for the first time, by immunohistochemistry and RT-PCR, that C-RFa is produced in pituitary cells. Finally, based on these results, a possible function for C-RFa as a locally produced PrRP in this teleost is discussed.

Seasonal modulation of growth hormone mRNA and protein levels in carp pituitary: evidence for two expressed genes.

Adaptation of eurythermal fish to naturally varying environmental conditions involves modulation of expressions of various factors in the hypothalamo-hypophyseal axis. Here we used three complementary approaches to assess the seasonal variation of growth hormone (GH) protein and mRNA levels in pituitary glands of acclimatized carp fish. First, a polyclonal antibody raised against an oligopeptide derived from the carp GH sequence was used for immunohistochemistry; second, oligonucleotides specific for GH transcripts were used for in situ hybridization. Specific immunodetection of GH coincides with visualization of GH mRNA in the proximal pars distalis, the specific location of somatotroph cells in carp pituitary gland. Finally, competitive RT-PCR analyses confirmed that GH expression exhibits seasonal cyclical reprogramming with higher levels in summer- than in winter-adapted fish. The expression pattern suggests an important role for GH in the molecular mechanisms underlying the acclimatization process. In parallel, amplification of sequences from the fourth intron and adjacent sites from exons IV and V demonstrates the existence of a new GH gene previously undescribed. The detection of transcripts corresponding to each gene suggests that both GH gene copies are active in the duplicated carp genome and that they are similarly affected by seasonal adaptation.

Gradual and quantum genome size shifts in the hystricognath rodents.

We assessed genome size variation by flow cytometry within and among 31 species of nine families of African and South American hystricognath rodents. Interspecific variation was extensive and genome size was relatively high among the South American radiation whereas only moderate variation and smaller estimates of genome size were observed in the African counterparts. The largest genome size, indicating tetraploidy was recorded in the South American octodontid, Tympanoctomys barrerae (16.8 pg DNA). This quantum shift in DNA content represents a novel mechanism of genome evolution in mammals. As expected in polyploid organisms, varying nucleotypic effects were observed in the dimensions of the sperm cells and lymphocytes of T. barrerae. The role of control mechanisms that influence cell dimensions in polyploid organisms is discussed.

Glandular kallikreins in the teleost Cyprinus carpio: tissue distribution, possible involvement in prolactin processing, and effect of 17 beta-estradiol in vivo.

We examined glandular kallikrein (GK), a putative prolactin processing protease, in the teleost Cyprinus carpio. When employing an anti-Centropristis striata GK antibody proteins of 39 kDa in muscle, 52 kDa in gill, 52 kDa in kidney, and two proteins of 46 and 72 kDa in pituitary gland were detected. Immunoreactive kallikreins were recognized in intermuscle cell tissue, epithelial gill cells, apical region of tubular cells, and prolactin producing lactotrophs in pituitary gland, suggesting a osmoregulatory role for this enzyme. We found three prolactin (PRL) variants using anti-tilapia PRL antibodies, in pituitary gland 23 and 16 kDa, and in plasma 23 and 22 kDa forms. Clearly co-localization of GK and PRL in lactotrophs could be demonstrated. In winter-acclimatized male carp, where the pituitary PRL level is low, 17beta-estradiol treatment increased PRL but not GK immunoreactivity. In contrast to GK and PRL co-regulation by estrogen in mammalian pituitary gland, no similar effect on immunoreactive PRL and GK was observed in the ichtyc pituitary. No changes in GK immunostaining occurred in gill or muscle tissue in response to estrogen treatment. These results, taken with the observation of significantly increased GK immunoreactivity in the apical region of kidney tubular cells in estrogen treated male carp, indicate that the regulation of GK expression in pituitary and kidney could be different in fish with respect to mammals.

In situ hybridization of somatolactin transcripts in the pituitary glands from acclimatized carp (Cyprinus carpio).

We isolated and cloned a carp somatolactin SL DNA fragment, of which 78% of the nucleotides were identical to the corresponding salmon SL sequence. The results obtained upon Northern blot hybridization of carp pituitary RNA allowed the identification of two transcripts as described for other fish. When the content of SL transcripts in pituitary sections from summer- and winter-acclimatized carp was quantified by in situ hybridization assays, we found no significant differences between the two seasons. In salmonids, plasma SL reaches higher levels in summer than in winter in synchrony with the water temperature cycle; in the eurythermal carp, however, the complex adaptive responses imposed by seasonal environmental changes do not seem to include the regulation of the somatolactin detected with the probe used at the transcriptional level in pituitary glands.

Environmental reprogramming of the expression of protein kinase CK2beta subunit in fish.

The dramatic segregation of the nucleolar components in winter-acclimatized carp is the most striking cellular-phenotypical feature observed during the seasonal adaptation of this fish toward the circannual changes in its habitat. Our studies also show that the carp habitat temperature and photoperiod winter conditions provoke a remarkable reduction of both rRNA transcription and the processing of their precursors. To gain knowledge on the mechanisms involved in the regulation of nucleolar activity during the seasonal adaptation process, we studied the behavior of some genes, specifically snoRNA U3 and protein kinase CK2. Consistent with the reduction in the synthesis and processing of pre-rRNA observed during the cold season, the level of CK2beta expression decreases in winter when compared to that attained in summer. Similarly, in winter, liver and kidney cells contain lower levels of CK2beta subunit protein compared to summer. CK2 is associated with or modifies different factors and enzymes involved in the nucleolar activity; therefore, its higher or lower content could be part of the molecular mechanisms underlying the nucleolar seasonal changes that occur during the compensatory acclimatization process.

Environmental acclimatization of the carp modulates the transcription of beta-actin.

A cascade of mechanisms involving changes in gene expression are substantial to shape the adaptive responses that a eurythermal fish requires upon environmental changes in its habitat. We have previously shown that the cyclical reprogramming of rRNA transcription is a remarkable feature in carp under seasonal acclimatization. Using in situ hybridization and competitive RT-PCR we found significant differences in beta-actin transcripts, generally accepted to be coded by a typical housekeeping gene, in tissues from summer- and winter-acclimatized carp. The physiological differential beta-actin transcription herein reported places us on the alert for the reference genes estimated to be constitutive to quantitatively assess gene transcripts.

Seasonal adaptation modulates the expression of the protein kinase CK2 beta subunit gene in the carp.

Carp fish seasonal acclimatization induces a cyclical transcriptional modulation of several genes. Its most dramatic expression results in a concomitant structural rearrangement of the nucleolar components that phenotypically represents profound shifts in the level of ribosomal RNA synthesis. In connection with the recent studies that implicate CK2 in the control of rRNA synthesis in vertebrates, we characterized the cDNA of carp protein kinase CK2beta subunit and assessed its transcriptional behavior in winter- and summer-acclimatized fish. We found a remarkable differential gene expression of CK2beta subunit between summer- and winter-acclimatized carp which correlates with the modulatory pattern observed in rRNA transcription.

Transcription factor pit-1 expression is modulated upon seasonal acclimatization of eurythermal ectotherms: identification of two pit-1 genes in the carp.

A second Pit-1 gene in carp (Cyprinus carpio), including the complete structural gene and 1.1 kb of promoter region, was identified and completely sequenced. The exon-intron structure was determined, and reverse transcription-polymerase chain reaction (RT-PCR) experiments suggest that only one Pit-1 splice variant is present in carp pituitary. The effect of seasonal acclimatization on the extent of Pit-1 gene expression was studied in summer- and winter-acclimatized carp. Quantitative RT-PCR analysis revealed a clear increase of Pit-1 mRNA in the pituitaries from summer-acclimatized carp compared with the winter-adapted fish. In situ hybridization of pituitary gland sections with riboprobes representing the complete 5'-transactivating region of carp Pit-1 depicted a significantly higher Pit-1 mRNA level in the rostral pars distalis of the summer-acclimatized fish where prolactin is expressed in a manner that resembles the seasonal increase observed in the proximal pars distalis and the pars intermedia. The cell- and temporal-specific transcription of Pit-1 supports its role in the molecular mechanisms that underly the acclimatization process undergone by eurythermal fish as a result of the physical effects of seasonal changes on their habitat.

Effect of seasonal acclimatization on the expression of the carp transcription factor Pit-1.

We isolated a clone comprising four exons of the carp Pit-1 gene. Using synthetic oligonucleotide probes derived from the carp Pit-1 sequence Pit-1 expression was assessed by in situ hybridization in pituitary sections from summer- and winter-acclimatized carp. Semiquantitative analyses of the hybridization signals revealed a significant higher Pit-1 expression in the proximal pars distalis (PPD) and pars intermedia (PI) of the pituitary glands from summer-acclimatized carp, compared to the winter-acclimatized fish. In both adaptive states, relative to the PPD and PI, only a basal Pit-1 expression was detected in the rostral pars distalis. Thus, during seasonal acclimatization of an eurythermal fish, Pit-1 seems to be involved in the mechanisms that underlie the compensatory response.