Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV.

Cell-mediated cytotoxicity is a complex immune mechanism that involves the release of several killing molecules, being perforin (PRF) one of the most important effector players. Perforin is synthesized by T lymphocytes and natural killer cells in mammals and responsible for the formation of pores on the target cell membrane during the killing process. Although perforin has been extensively studied in higher vertebrates, this knowledge is very limited in fish. Therefore, in this study we have identified four prf genes in European sea bass (Dicentrarchus labrax) and evaluated their mRNA levels. All sea bass prf genes showed the typical and conserved domains of its human orthologue and were closely clustered by the phylogenetic analysis. In addition, all genes showed constitutive and ubiquitous tissular expression, being prf1.9 gene the most highly expressed in immune tissues. Subsequently, in vitro stimulation of head-kidney (HK) cells with phytohemagglutinin, a T-cell activator, showed an increase of all prf gene levels, except for prf1.3 gene. European sea bass HK cells increased the transcription of prf1.2 and prf1.9 during the innate cell-mediated cytotoxic activity against xenogeneic target cells. In addition, sea bass infected with nodavirus (NNV) showed a similar expression pattern of all prf in HK and brain at 15 days post-infection, except for prf1.3 gene and in the gonad. Finally, the use of a polyclonal antibody against PRF1.9 showed an increase of positive cells in HK, brain and gonad from NNV-infected fish. Taken together, the data seem to indicate that all prf genes, except prf1.3, appear to be involved in the European sea bass immunity, and probably in the cell-mediated cytotoxic response, with PRF1.9 playing the most important role against nodavirus. The involvement of the PRFs and the CMC activity in the vertical transmission success of the virus is also discussed.

Correlation of fetal heart rate dynamics to inflammatory markers and brain-derived neurotrophic factor during pregnancy.

OBJECTIVES: This study aims to show the relation between biomarkers in maternal and cord-blood samples and fetal heart rate variability (fHRV) metrics through a non-invasive fetal magnetocardiography (fMCG) technique. METHODS: Twenty-three women were enrolled for collection of maternal serum and fMCG tracings immediately prior to their scheduled cesarean delivery. The umbilical cord blood was collected for measurement of biomarker levels. The fMCG metrics were then correlated to the biomarker levels from the maternal serum and cord blood. RESULTS: Brain-derived neurotrophic factor (BDNF) had a moderate correlation with fetal parasympathetic activity (0.416) and fetal sympathovagal ratios (-0.309; -0.356). Interleukin (IL)-6 also had moderate-sized correlations but with an inverse relationship as compared to BDNF. These correlations were primarily in cord-blood samples and not in the maternal blood. CONCLUSIONS: In this small sample-sized exploratory study, we observed a moderate correlation between fHRV and cord-blood BDNF and IL-6 immediately preceding scheduled cesarean delivery at term. These findings need to be validated in a larger population.

Evidence of extraganglionic vagal mechanoreceptors in the mouse vagus nerve.

Vagal afferent neuronal somas are in the nodose and jugular ganglia. In this study, we identified extraganglionic neurons in whole-mount preparations of the vagus nerves from Phox2b-Cre-ZsGreen transgenic mice. These neurons are typically arranged in small clusters and monolayers along the cervical vagus nerve. Although infrequent, these neurons were sometimes observed along the thoracic and esophageal vagus. We performed RNAscope in situ hybridization and confirmed that the extraganglionic neurons detected in this transgenic mouse strain expressed vagal afferent markers (i.e., Phox2b and Slc17a6) as well as markers that identify them as potential gastrointestinal mechanoreceptors (i.e., Tmc3 and Glp1r). We also identified extraganglionic neurons in the vagus nerves of wild-type mice that were injected intraperitoneally with Fluoro-Gold, thereby ruling out possible anatomical discrepancies specific for transgenic mice. In wild-type mice, extraganglionic cells were positive for peripherin, confirming their neuronal nature. Taken together, our findings revealed a previously undiscovered population of extraganglionic neurons associated with the vagus nerve. Going forward, it is important to consider the possible existence of extraganglionic mechanoreceptors that transmit signals from the abdominal viscera in future studies related to vagal structure and function.

Evidence that fish death after Vibrio vulnificus infection is due to an acute inflammatory response triggered by a toxin of the MARTX family.

Vibrio vulnificus is an emerging zoonotic pathogen associated with fish farms that is capable of causing a hemorrhagic septicemia known as warm-water vibriosis. According to a recent transcriptomic and functional study, the death of fish due to vibriosis is more related to the inflammatory response of the host than to the tissue lesions caused by the pathogen. In this work, we hypothesize that the RtxA1 toxin (a V. vulnificus toxin of the MARTX (Multifunctional Autoprocessing Repeats in Toxin) family) is the key virulence factor that would directly or indirectly trigger this fatal inflammatory response. Our hypothesis was based on previous studies that showed that rtxA1-deficient mutants maintained their ability to colonize and invade, but were unable to kill fish. To demonstrate this hypothesis, we infected eels (model of fish vibriosis) by immersion with a mutant deficient in RtxA1 production and analyzed their transcriptome in blood, red blood cells and white blood cells during early vibriosis (0, 3 and 12 h post-infection). The transcriptomic results were compared with those obtained in the previous study in which eels were infected with the V. vulnificus parental strain, and were functionally validated. Overall, our results confirm that fish death after V. vulnificus infection is due to an acute, early and atypical inflammatory response triggered by RtxA1 in which red blood cells seem to play a central role. These results could be relevant to other vibriosis as the toxins of this family are widespread in the Vibrio genus.

Immunity elicited by AMP-encoding plasmids fails to increase the protection of European sea bass against nodavirus.

Antimicrobial peptides (AMPs) are a potent arm of the innate immune system that can directly kill pathogens and induce immunomodulation. In the marine aquaculture, European sea bass (Dicentrarchus labrax L.) is one of the most prosperous species but is highly susceptible to nodavirus (NNV), which produces high rates of mortality in larvae and juvenile stages. Thus, we aimed to evaluate whether AMPs exert immunomodulatory and/or NNV-preventive actions in sea bass. To do this, plasmids encoding the sea bass AMPs dicentracin (pDIC), beta-defensin (pDB1), hepcidin (pHAMP2) or NK-lysin (pNKL) were generated and intramuscularly injected into sea bass juveniles to evaluate their immunomodulatory and anti-NNV roles. Sea bass muscle transcribes the AMPs and produces an increase in their circulating levels, along with an increase of the antibacterial activity. Immune-related gene analysis revealed a great activation of the inflammatory response and the recruitment of neutrophilic granulocytes at the site of injection. However, AMP-encoding plasmids, namely pHAMP2, negatively affected to NNV disease by increasing fish mortality. In conclusion, plasmids encoding AMPs show immunostimulatory effects on European sea bass but do not improve the resistance to NNV.

A Sporothrix spp. enolase derived multi-epitope vaccine confers protective response in BALB/c mice challenged with Sporothrix brasiliensis.

Sporotrichosis is a cosmopolitan mycosis caused by pathogenic species of Sporothrix genus, that in Brazil is often acquired by zoonotic transmission involved infected cats with S. brasiliensis. Previous studies showed that the Sporothrix spp. recombinant enolase (rSsEno), a multifunctional protein with immunogenic properties, could be a promising target for vaccination against sporotrichosis in cats. Nevertheless, the considerable sequence identity (62%) of SsEno with its feline counterpart is a great concern. Here, we report the identification in silico, chemical synthesis and biological validation of six peptides of SsEno with low sequence identity to its cat orthologue. All synthesized peptides exhibit B-cell epitopes on the molecular surface of SsEno and proved to be highly reactive with the serum of infected mice with S. brasiliensis and sera of cats with sporotrichosis. Interestingly, our study revealed that anti-peptide sera did not react with the recombinant enolase from Felis catus (cats, rFcEno), thus, may not trigger autoimmune response in these felines if used as a vaccine antigen. The immunization with peptide mixture (PeptMix) formulated with Freund adjuvant (FA), induced high levels of antigen-specific IgG, IgG1 and IgG2b antibodies that conferred protection upon passive transference in infected BALB/c mice with S. brasiliensis. We also observed, that the FA+PeptMix formulation induced a Th1/Th2/Th17 cytokine profile ex vivo, associated with protecting effect against the experimental sporotrichosis. Our results suggest that the six SsEno-derived peptides here evaluated, could be used as safe antigens for the development of vaccine strategies against feline sporotrichosis, whether prophylactic or therapeutic.

A g-type lysozyme from the scallop Argopecten purpuratus participates in the immune response and in the stability of the hemolymph microbiota.

Lysozymes are antimicrobial acid hydrolases widely distributed in nature. They are located inside the cells in lysosomes, or they are secreted to the extracellular space, where they can lyse the cell wall of certain species of bacteria via hydrolysis of the peptidoglycan. Thus, lysozymes are bacteriolytic enzymes and play a major biological role in biodefense, as these enzymes can act as antibacterial and immune-modulating agents. In this study, we characterized a g-type lysozyme from the scallop Argopecten purpuratus named ApGlys. The cDNA sequence comprises an open reading frame (ORF) of 600 nucleotides, codifying for a putative protein of 200 amino acids with a signal peptide of 18 amino acids. The deduced mature protein sequence displays a molecular weight of 20.07 kDa and an isoelectric point (pI) of 6.49. ApGlys deduced protein sequence exhibits conserved residues associated with catalytic activity and substrate fixation in other g-type lysozymes. The phylogenetic analysis revealed a high degree of identity of ApGlys with other mollusk g-type lysozymes, which form a restricted and separated clade from the vertebrate lysozymes. ApGlys transcripts were constitutively and highly expressed in the digestive gland, and it was induced in hemocytes and gills of scallops after an immune challenge. Furthermore, the ApGlys protein was located inside hemocytes of immunostimulated scallops, determined by immunofluorescence analysis. Finally, the transcript silencing of ApGlys by RNA interference led to an increase of total culturable bacteria from the scallop hemolymph. Furthermore, we detected a higher diversity of the bacterial community in ApGlys-silenced scallops and an imbalance of certain bacterial groups present in the hemolymph by 16S rDNA deep amplicon sequencing. Overall, our results showed that ApGlys is a new member of scallop lysozymes that is implicated in the immune response and in the microbial homeostasis of A. purpuratus hemolymph.

Big defensin from the scallop Argopecten purpuratus ApBD1 is an antimicrobial peptide which entraps bacteria through nanonets formation.

Big defensins is a large family of antimicrobial peptides found in restricted groups of invertebrates, in particular mollusks where they have highly diversified. Big defensins are composed of a highly hydrophobic N-terminal region and a C-terminal region containing six cysteine residues whose arrangement is identical to that of vertebrate ß-defensins. They have been shown to be active against both Gram-positive and Gram-negative bacteria and fungi. Antimicrobial aggregates called nanonets entrapping and killing bacteria have been recently described for the hydrophobic N-terminal region of the Cg-BigDef1 from the oyster Crassostrea gigas. To determine whether nanonets formation is a conserved trait of mollusk big defensins, we assessed the potential entrapping of bacteria through nanonets of the big defensin from the scallop Argopecten purpuratus, ApBD1. Recombinant ApBD1 was produced with a thrombin-cleavable N-terminal His6 tag, followed by the mature peptide carrying a mutation of the last cysteine residue of the C-terminal region by and arginine, named rApBD1(C87R). This mutation did not apparently affect the three-dimensional structure and the biological properties of rApBD1(C87R), as evidenced by in silico modeling and in vitro antimicrobial assays. Strong immune staining of rApBD1(C87R) in numerous areas surrounding bacteria was observed by confocal microscopy, suggesting that rApBD1(C87R) entraps bacteria in peptide aggregates similar to those reported to the oyster big defensin. This study suggests the conservation of bactericidal activity and nanonet formation across big defensins from bivalve mollusks.

Comparison of Circulating Markers and Mucosal Immune Parameters from Skin and Distal Intestine of Atlantic Salmon in Two Models of Acute Stress.

Ensuring salmon health and welfare is crucial to maximize production in recirculation aquaculture systems. Healthy and robust mucosal surfaces of the skin and intestine are essential to achieve this goal because they are the first immunological defenses and are constantly exposed to multistressor conditions, such as infectious diseases, suboptimal nutrition, and environmental and handling stress. In this work, Atlantic salmon, split from a single cohort, were subjected to acute hypoxia stress or 15-min crowding stress and observed over a 24-h recovery period. Samples were collected from fish at 0, 1, 3, 6, 12 and 24 h post-stress to analyze plasma-circulating markers of endocrine function (cortisol), oxidative stress (glutathione peroxidase) and immune function (interleukin 10 (IL-10), annexin A1). In addition, mucosal barrier function parameters were measured in the skin mucus (Muc-like protein and lysozyme) and distal intestine (simple folds, goblet cell size and goblet cell area). The results showed that both acute stress models induced increases of circulating cortisol in plasma (1 h post-stress), which then returned to baseline values (initial control) at 24 h post-stress. Moreover, the hypoxia stress was mostly related to increased oxidative stress and IL-10 production, whereas the crowding stress was associated with a higher production of Muc-like protein and lysozyme in the skin mucus. Interestingly, in the distal intestine, smaller goblet cells were detected immediately and one hour after post-hypoxia stress, which could be related to rapid release of the cellular content to protect this organ. Finally, the correlation of different markers in the hypoxic stress model showed that the circulating levels of cortisol and IL-10 were directly proportional, while the availability of Muc-like proteins was inversely proportional to the size of the goblet cells. On the other hand, in the crowding stress model, a proportional relationship was established between plasma cortisol levels and skin mucus lysozyme. Our results suggest key differences in energy partitioning between the two acute stress models and support the need for further investigation into the interplay of multistressor conditions and strategies to modulate immunological aspects of mucosal surfaces.

Behavioural Fever Promotes an Inflammatory Reflex Circuit in Ectotherms.

BACKGROUND: The communication between the brain and the immune system is a cornerstone in animal physiology. This interaction is mediated by immune factors acting in both health and pathogenesis, but it is unclear how these systems molecularly and mechanistically communicate under changing environmental conditions. Behavioural fever is a well-conserved immune response that promotes dramatic changes in gene expression patterns during ectotherms' thermoregulatory adaptation, including those orchestrating inflammation. However, the molecular regulators activating the inflammatory reflex in ectotherms remain unidentified. METHODS: We revisited behavioural fever by providing groups of fish a thermal gradient environment during infection. Our novel experimental setup created temperature ranges in which fish freely moved between different thermal gradients: (1) wide thermoregulatory range; T° = 6.4 °C; and (2) restricted thermoregulatory range; T° = 1.4 °C. The fish behaviour was investigated during 5-days post-viral infection. Blood, spleen, and brain samples were collected to determine plasmatic pro- and anti-inflammatory cytokine levels. To characterize genes' functioning during behavioural fever, we performed a transcriptomic profiling of the fish spleen. We also measured the activity of neurotransmitters such as norepinephrine and acetylcholine in brain and peripheral tissues. RESULTS: We describe the first set of the neural components that control inflammatory modulation during behavioural fever. We identified a neuro-immune crosstalk as a potential mechanism promoting the fine regulation of inflammation. The development of behavioural fever upon viral infection triggers a robust inflammatory response in vivo, establishing an activation threshold after infection in several organs, including the brain. Thus, temperature shifts strongly impact on neural tissue, specifically on the inflammatory reflex network activation. At the molecular level, behavioural fever causes a significant increase in cholinergic neurotransmitters and their receptors' activity and key anti-inflammatory factors such as cytokine Il10 and Tgfß in target tissues. CONCLUSION: These results reveal a cholinergic neuronal-based mechanism underlying anti-inflammatory responses under induced fever. We performed the first molecular characterization of the behavioural fever response and inflammatory reflex activation in mobile ectotherms, identifying the role of key regulators of these processes. These findings provide genetic entry points for functional studies of the neural-immune adaptation to infection and its protective relevance in ectotherm organisms.

Tea Bags for Fmoc Solid-Phase Peptide Synthesis: An Example of Circular Economy.

Peptide synthesis is an area with a wide field of application, from biomedicine to nanotechnology, that offers the option of simultaneously synthesizing a large number of sequences for the purpose of preliminary screening, which is a powerful tool. Nevertheless, standard protocols generate large volumes of solvent waste. Here, we present a protocol for the multiple Fmoc solid-phase peptide synthesis in tea bags, where reagent recycling steps are included. Fifty-two peptides with wide amino acid composition and seven to twenty amino acid residues in length were synthesized in less than three weeks. A clustering analysis was performed, grouping the peptides by physicochemical features. Although a relationship between the overall yield and the physicochemical features of the sequences was not established, the process showed good performance despite sequence diversity. The recycling system allowed to reduce N, N-dimethylformamide usage by 25-30% and reduce the deprotection reagent usage by 50%. This protocol has been optimized for the simultaneous synthesis of a large number of peptide sequences. Additionally, a reagent recycling system was included in the procedure, which turns the process into a framework of circular economy, without affecting the quality of the products obtained.

Impact of treatment plant management on human health and ecological risks from wastewater irrigation in developing countries - case studies from Cochabamba, Bolivia.

Wastewater irrigation is a common practice in developing countries due to water scarcity and increasing demand for food production. However, there are health risks and ecological risks associated with this practice. Small-scale wastewater treatment plants (WWTPs) intend to decrease these risks but still face management challenges. This study assessed how the management status of five small-scale WWTPs in Cochabamba, Bolivia affects health risks associated with consumption of lettuce and ecological risks due to the accumulation of nutrients in the soil for lettuce and maize crops. Risk simulations for three wastewater irrigation scenarios were: raw wastewater, actual effluent and expected effluent. Results showed that weak O&M practices can increase risk outcomes to higher levels than irrigating with raw wastewater. Improving O&M to achieve optimal functioning of small-scale WWTPs can reduce human health risks and ecological risks up to 2 log10 DALY person-1 year-1 and to 2 log10 kg nitrogen ha-1 accumulated in soil, respectively.

NK-lysin from skin-secreted mucus of Atlantic salmon and its potential role in bacteriostatic activity.

NK-lysin, despite being a direct effector of cytotoxic T and natural killer cells, is an antimicrobial peptide (AMP) with known antibacterial function in vertebrates and so in fish. Its presence has been described in different tissues of teleost fish. One of the strongest antimicrobial barriers in fish is skin-secreted mucus; however, this mucus has been found to contain only a small number of AMPs. The present study describes for the first time the constitutive expression of NK-lysin in Atlantic salmon (Salmo salar) mucus produced by the skin, recording the AMP at a higher concentration than in serum with greater bacteriostatic activity. Hepcidin may be involved to a greater extent in systemic responses since it was expressed to a higher degree in serum which was more potent for alternative complement and peroxidase activities.

Biochar filters as an on-farm treatment to reduce pathogens when irrigating with wastewater-polluted sources.

Microbial contamination of vegetables due to irrigation with wastewater-polluted streams is a common problem around most cities in developing countries because wastewater is an available source of water and nutrients but wastewater treatment is often inadequate. On-farm treatment of polluted water is a feasible option to manage microbial risks in a multi-barrier approach. Current evidence indicates good suitability of biochar filters for microbe removal from wastewater using the hydraulic loading rate (HLR) designed for sand filters, but their suitability has not been tested under on-farm conditions. This study evaluated the combined effect of several variables on removal of microbial indicators from diluted wastewater by biochar filtration on-farm and the correlations between removal efficiency and HLR. Columns of biochar with three different effective particle diameters (d10) were fed with diluted wastewater at 1x, 6x, and 12x the design HLR and two levels of water salinity (electrical conductivity, EC). Influent and effluent samples were collected from the columns and analyzed for bacteriophages (ɸX174 and MS2), Escherichia coli, Enterococcus spp., and Saccharomyces cerevisiae. Microbe removal decreased with increasing HLR, from 2 to 4 to 1 log10 for bacteria and from 2 to 0.8 log10 for viruses, while S. cerevisiae removal was unaffected. Effective particle diameter (d10) was the main variable explaining microbe removal at 6x and 12x, while EC had no effect. Correlation analysis showed removal of 2 log10 bacteria and 1 log10 virus at 3x HLR. Thus biochar filters on-farm would not remove significant amounts of bacteria and viruses. However, the design HLR was found to be conservative. These results, and some technical and management considerations identified, can assist in the development of a scientific method for designing biochar filters for on-farm and conventional wastewater treatment.

Alpha-helical domain from IL-8 of salmonids: Mechanism of action and identification of a novel antimicrobial function.

In this work, the potential antimicrobial role and mechanism of action of α-helix domain of trout and salmon IL-8 against Eschericia coli, Pseudomonas aeruginosa and Staphylococcus aureus was investigated. By an in silico analysis of the primary structure of IL-8 from Oncorhynchus mykiss and salmo salar, it was evidenced that γ-core motif was present, as in the vast majority of kinocidins. The α-helix domain of IL-8 (αIL-8) was synthesized by solid phase peptide synthesis and showed a tendency to form an α-helix conformation, as revealed by circular dichroism. Additionally, it was demonstrated that αIL-8 from both species showed antimicrobial activity against E. coli, P. aeruginosa and S. aureus. Membrane permeabilization and co-localization assay, as well as scanning electron microscopy, showed that these peptides were accumulated on the cell surface and in the cytoplasm, suggesting that they were capable of permeabilizing and disrupt the bacterial membranes and interact with cytoplasmic components. Our results represent the first analysis on the antimicrobial function of IL-8-derived peptide from salmonids.

Chronic stress inhibits growth and induces proteolytic mechanisms through two different nonoverlapping pathways in the skeletal muscle of a teleost fish.

Chronic stress detrimentally affects animal health and homeostasis, with somatic growth, and thus skeletal muscle, being particularly affected. A detailed understanding of the underlying endocrine and molecular mechanisms of how chronic stress affects skeletal muscle growth remains lacking. To address this issue, the present study assessed primary (plasma cortisol), secondary (key components of the GH/IGF system, muscular proteolytic pathways, and apoptosis), and tertiary (growth performance) stress responses in fine flounder ( Paralichthys adspersus) exposed to crowding chronic stress. Levels of plasma cortisol, glucocorticoid receptor 2 ( gr2), and its target genes ( klf15 and redd1) mRNA increased significantly only at 4 wk of crowding ( P < 0.05). The components of the GH/IGF system, including ligands, receptors, and their signaling pathways, were significantly downregulated at 7 wk of crowding ( P < 0.05). Interestingly, chronic stress upregulated the ubiquitin-proteasome pathway and the intrinsic apoptosis pathways at 4wk ( P < 0.01), whereas autophagy was only significantly activated at 7 wk ( P < 0.05), and meanwhile the ubiquitin-proteasome and the apoptosis pathways returned to control levels. Overall growth was inhibited in fish in the 7-wk chronic stress trial ( P < 0.05). In conclusion, chronic stress directly affects muscle growth and downregulates the GH/IGF system, an action through which muscular catabolic mechanisms are promoted by two different and nonoverlapping proteolytic pathways. These findings provide new information on molecular mechanisms involved in the negative effects that chronic stress has on muscle anabolic/catabolic signaling balance.

The expression of TRPV channels, prostaglandin E2 and pro-inflammatory cytokines during behavioural fever in fish.

A fever, or increased body temperature, is a symptom of inflammation, which is a complex defence reaction of the organism to pathogenic infections. After pathogens enter the body, immune cells secrete a number of agents, the functions of which stimulate the body to develop a functional immune and fever response. In mammals it is known that PGE2 is the principal mediator of fever. The extent to which PGE2 and other pro-inflammatory cytokines such as TNF-α, IL-6, or IL-1ß could be involved in the induction of behavioural fever in fish remains to be clarified. Several members of the transient receptor potential (TRP) family of ion channels have been implicated as transducers of thermal stimuli, including TRPV1 and TRPV2, which are activated by heat. Here we show that members of the TRP family, TRPV1 and TRPV4, may participate in the coordination of temperature sensing during the behavioural fever. To examine the behavioral fever mechanism in Salmo salar an infection with IPNV, infectious pancreatic necrosis virus, was carried out by an immersion challenge with 10 × 105 PFU/mL-1 of IPNV. Behavioural fever impacted upon the expression levels of both TRPV1 and TRPV4 mRNAs after the viral challenge and revealed a juxtaposed regulation of TRPV channels. Our results suggest that an increase in the mRNA abundance of TRPV1 is tightly correlated with a significant elevation in the expression of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and PGE2) in the Pre-Optic Area (POA) and cytokine release in plasma. Together, these data indicate that the reduction of TRPV4 expression during behavioural fever may contribute to the onset of behavioural fever influencing movement toward higher water temperatures. Our data also suggest an effect of TRPV channels in the regulation of behavioural fever through activation of EP3 receptors in the central nervous system by PGE2 induced by plasma-borne cytokines. These results highlight for first time in mobile ectotherms the key role of pro-inflammatory cytokines and TRPV channels in behavioural fever that likely involves a complex integration of prostaglandin induction, cytokine recognition and temperature sensing.

Cloning and molecular characterization of two ferritins from red abalone Haliotis rufescens and their expressions in response to bacterial challenge at juvenile and adult life stages.

Ferritins are ubiquitous proteins with a pivotal role in iron storage and homeostasis, and in host defense responses during infection by pathogens in several organisms, including mollusks. In this study, we characterized two ferritin homologues in the red abalone Haliotis rufescens, a species of economic importance for Chile, USA and Mexico. Two ferritin subunits (Hrfer1 and Hrfer2) were cloned. Hrfer1 cDNA is an 807 bp clone containing a 516 bp open reading frame (ORF) that corresponds to a novel ferritin subunit in H. rufescens. Hrfer2 cDNA is an 868 bp clone containing a 516 bp ORF that corresponds to a previously reported ferritin subunit, but in this study 5'- and 3'-UTR sequences were additionally found. We detected a putative Iron Responsive Element (IRE) in the 5'-UTR sequence, suggesting a posttranscriptional regulation of Hrfer2 translation by iron. The deduced protein sequences of both cDNAs possessed the motifs and domains required in functional ferritin subunits. Expression patterns of both ferritins in different tissues, during different developmental stages, and in response to bacterial (Vibrio splendidus) exposure were examined. Both Hrfer1 and Hrfer2 are most expressed in digestive gland and gonad. Hrfer1 mRNA levels increased about 34-fold along with larval developmental process, attaining the highest level in the creeping post-larvae. Exogenous feeding is initiated at the creeping larva stage; thus, the increase of Hrfer1 may suggest and immunity-related role upon exposure to bacteria. Highest Hrfer2 expression levels were detected at trochophore stage; which may be related with early shell formation. Upon challenge with, the bacteria an early mild induction of Hrfer2 (2 h post-challenge), followed by a stronger induction of Hrfer1 at 15 h post-challenge, was observed in haemocytes from adult abalones. While maximal upregulation of both genes in the whole individual occurred at 24 h post-challenge, in juveniles. A significant increase in ferritin protein levels from 6 h to 24 h post-challenge was also detected. Our results suggest an involvement of Hrfer1 and Hrfer2, and of ferritin proteins in the immune response of H. rufescens to bacterial infection.

HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress.

BACKGROUND: Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown. METHODS: We amplified and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neumayeri, experimentally exposed to heat stress (5  and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression. RESULTS: The sequence obtained from S. neumayeri showed high identity with Hsp70 members. Several Hsp70 family features were identified in the deduced amino acid sequence and they indicate that the isolated Hsp70 is related to the cognate heat shock protein type. The corresponding 70 kDa protein, called Sn-Hsp70, was immune detected in the coelomocytes and the digestive tract of S. neumayeri using a monospecific polyclonal antibody. We showed that S. neumayeri do not respond to acute heat stress by up-regulation of Sn-Hsp70 at transcript and protein level. Furthermore, the Sn-Hsp70 protein expression was not induced in the digestive tract. CONCLUSIONS: Our results provide the first molecular evidence that Sn-Hsp70 is expressed constitutively and is non-induced by heat stress in S. neumayeri.