Differential expression of immune-related biomarkers in primary cultures from Atlantic salmon (Salmo salar) exposed to processed Paecilomyces variotii with or without inactivated Moritella viscosa.

Paecilomyces variotii (a filamentous fungus), is a promising novel protein source in fish feeds due to its high nutritional value. Also, P. variotii has Microbial-Associated Molecular Patterns (MAMPs) such as glucans and nucleic acids that could modulate the host's immune response. To understand the potential bioactive properties of this fungus in Atlantic salmon (Salmo salar), our study was conducted to evaluate the gene expression of immune-related biomarkers (e.g., cytokines, effector molecules and receptors) on primary cultures from salmon head kidney (HKLs) and spleen leukocytes (SLs) exposed to either UV inactivated or fractions from P. variotii with or without inactivated Moritella viscosa (a skin pathogen in salmonids). Moreover, the effect of the fermentation conditions and down-stream processing on the physical ultrastructure and cell wall glucan content of P. variotii was characterized. The results showed that drying had a significant effect on the cell wall ultrastructure of the fungi and the choice of fermentation has a significant effect on the quantity of ß-glucans in P. variotii. Furthermore, stimulating Atlantic salmon HKLs and SLs with P. variotii and its fractions induced gene expression related to pro-inflammatory (tnfα, il1ß) and antimicrobial response (cath2) in HKLs, while response in SLs was related to both pro-inflammatory and regulatory response (tnfα, il6 and il10). Similarly, the stimulation with inactivated M. viscosa alone led to an up-regulation of genes related to pro-inflammatory (tnfα, il1ß, il6) antimicrobial response (cath2), intra-cellular signalling and recognition of M. viscosa (sclra, sclrb) and a suppression of regulatory response (il10) in both HKLs and SLs. Interestingly, the co-stimulation of cells with P. variotii and M. viscosa induced immune homeostasis (il6, tgfß) and antimicrobial response (cath2) in SLs at 48h. Thus, P. variotii induces immune activation and cellular communication in Atlantic salmon HKLs and SLs and modulates M. viscosa induced pro-inflammatory responses in SLs. Taken together, the results from physical and chemical characterization of the fungi, along with the differential gene expression of key immune biomarkers, provides a theoretical basis for designing feeding trials and optimize diets with P. variotii as a functional novel feed ingredient for Atlantic salmon.

Antigenic similarities and clinical cross-protection between variant and classic non-viscous strains of Moritella viscosa in Atlantic salmon in Norway.

Moritella viscosa (M. viscosa) is one of the major etiological agents of winter-ulcers in Atlantic salmon (Salmo salar) in Norway. Outbreaks of ulcerative disease in farmed fish occur across the North Atlantic region, causing reduced animal welfare and economical challenges, and are of hindrance for sustainable growth within the industry. Commercially available multivalent core vaccines containing inactivated bacterin of M. viscosa reduce mortality and clinical signs related to winter ulcer disease. It has previously been described two major genetic clades within M. viscosa, typical (hereafter referred to as classic) and variant, based on gyrB sequencing. In addition, there are phenotypical traits such as viscosity that may differ between different types of isolates. Western blot using salmon plasma showed that classic non-viscous strains are antigenically different from the classic viscous type included in core vaccines. Further, Western blot also showed that there are similarities in binding patterns between Norwegian variant and classic non-viscous isolates, indicating they may be antigenically related. Vaccination-challenge trials using Norwegian gyrB-classic non-viscous isolates of M. viscosa, demonstrate that the isolates from the classic clade that are included in current commercial multivalent core vaccines, provide limited cross protection against the emerging non-viscous strains. However, a vaccine recently approved for marketing authorization in Norway, containing inactivated antigen of a variant M. viscosa strain, demonstrates reduced mortality as well as clinical signs caused by infections with the classic non-viscous M. viscosa isolated from outbreaks in Norwegian salmon farms. The study shows that there are antigenic similarities between variant and classic non-viscous types of M. viscosa, and these similarities are mirrored in the observed cross-protection in vaccination-challenge trials.

New vaccination strategies are required for effective control of winter ulcer disease caused by emerging variant strains of Moritella viscosa in Atlantic salmon.

Moritella viscosa is one on the major etiological agents of winter-ulcers in Atlantic salmon (Salmo salar) in Norway. Outbreaks of ulcerative disease in farmed fish occurs across the North Atlantic region and is an impeding factor for sustainable growth within the industry. Commercially available multivalent core vaccines containing inactivated bacterin of M. viscosa reduce mortality and clinical signs related to winter ulcer disease. Two major genetic clades within M. viscosa have previously been described based on gyrB sequencing, namely typical (hereafter referred to as classic) and variant. Vaccination-challenge trials using vaccines including either variant and or classic isolates of M. viscosa show that classic clade isolates included in current commercial multivalent core vaccines provide poor cross-protection against emerging variant strains, while variant strains confer high level of protection against variant M. viscosa but to a lesser extent to classic clade isolates. This demonstrates that future vaccine regimens should include a combination of strains from both clades.

MLVA genotyping of Moritella viscosa reveals serial emergence of novel, host-specific clonal complexes in Norwegian salmon farming.

A Multi-Locus Variable number of tandem repeat Analysis (MLVA) genotyping scheme was developed for the epidemiological study of Moritella viscosa, which causes 'winter ulcer' predominantly in sea-reared Atlantic salmon (Salmo salar L.). The assay involves multiplex PCR amplification of six Variable Number of Tandem Repeat (VNTR) loci, followed by capillary electrophoresis and data interpretation. A collection of 747 spatiotemporally diverse M. viscosa isolates from nine fish species was analysed, the majority from farmed Norwegian salmon. MLVA distributed 76% of the isolates across three major clonal complexes (CC1, CC2 and CC3), with the remaining forming minor clusters and singletons. While 90% of the salmon isolates belong to either CC1, CC2 or CC3, only 20% of the isolates recovered from other fish species do so, indicating a considerable degree of host specificity. We further highlight a series of 'clonal shifts' amongst Norwegian salmon isolates over the 35-year sampling period, with CC1 showing exclusive predominance prior to the emergence of CC2, which was later supplanted by CC3, before the recent re-emergence of CC1. Apparently, these shifts have rapidly swept the entire Norwegian coastline and conceivably, as suggested by typing of a small number of non-Norwegian isolates, the Northeast Atlantic region as a whole.

Expression Analysis of Moritella viscosa-Challenged Atlantic Salmon Identifies Disease-Responding Genes, MicroRNAs and Their Predicted Target Genes and Pathways.

Moritella viscosa is a bacterial pathogen causing winter-ulcer disease in Atlantic salmon. The lesions on affected fish lead to increased mortality, decreased fish welfare, and inferior meat quality in farmed salmon. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional regulation by guiding the miRNA-induced silencing complex to specific mRNA transcripts (target genes). The goal of this study was to identify miRNAs responding to Moritella viscosa in salmon by investigating miRNA expression in the head-kidney and the muscle/skin from lesion sites caused by the pathogen. Protein coding gene expression was investigated by microarray analysis in the same materials. Seventeen differentially expressed guide-miRNAs (gDE-miRNAs) were identified in the head-kidney, and thirty-nine in lesion sites, while the microarray analysis reproduced the differential expression signature of several thousand genes known as infection-responsive. In silico target prediction and enrichment analysis suggested that the gDE-miRNAs were predicted to target genes involved in immune responses, hemostasis, angiogenesis, stress responses, metabolism, cell growth, and apoptosis. The majority of the conserved gDE-miRNAs (e.g., miR-125, miR-132, miR-146, miR-152, miR-155, miR-223 and miR-2188) are known as infection-responsive in other vertebrates. Collectively, the findings indicate that gDE-miRNAs are important post-transcriptional gene regulators of the host response to bacterial infection.

Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy.

Mining of Phospholipase D (PLD) with high activity and stability has attracted strong interest for investigation. A novel PLD from marine Moritella sp. JT01 (MsPLD) was biochemically and structurally characterized in our previous study; however, the short half-life time (t1/2) under its optimum reaction temperature seriously hampered its further applications. Herein, the disulfide bond engineering strategy was applied to improve its thermostability. Compared with wild-type MsPLD, mutant S148C-T206C/D225C-A328C with the addition of two disulfide bonds exhibited a 3.1-fold t1/2 at 35 °C and a 5.7 °C increase in melting temperature (Tm). Unexpectedly, its specific activity and catalytic efficiency (kcat/Km) also increased by 22.7% and 36.5%, respectively. The enhanced activity might be attributed to an increase in the activation entropy by displacing more water molecules by the transition state. The results of molecular dynamics simulations (MD) revealed that the introduction of double disulfide bonds rigidified the global structure of the mutant, which might cause the enhanced thermostability. Finally, the synthesis capacity of the mutant to synthesize phosphatidic acid (PA) was evaluated. The conversion rate of PA reached about 80% after 6 h reaction with wild-type MsPLD but reached 78% after 2 h with mutant S148C-T206C/D225C-A328C, which significantly reduced the time needed for the reaction to reach equilibrium. The present results pave the way for further application of MsPLD in the food and pharmaceutical industries.

A New Phospholipase D from Moritella sp. JT01: Biochemical Characterization, Crystallization and Application in the Synthesis of Phosphatidic Acid.

A new phospholipase D from marine Moritella sp. JT01 (MsPLD) was recombinantly expressed and biochemically characterized. The optimal reaction temperature and pH of MsPLD were determined to be 35 °C and 8.0. MsPLD was stable at a temperature lower than 35 °C, and the t1/2 at 4 °C was 41 days. The crystal structure of apo-MsPLD was resolved and the functions of a unique extra loop segment on the enzyme activity were characterized. The results indicated that a direct deletion or fastening of the extra loop segment by introducing disulfide bonds both resulted in a complete loss of its activity. The results of the maximum insertion pressure indicated that the deletion of the extra loop segment significantly decreased MsPLD's interfacial binding properties to phospholipid monolayers. Finally, MsPLD was applied to the synthesis of phosphatidic acid by using a biphasic reaction system. Under optimal reaction conditions, the conversion rate of phosphatidic acid reached 86%. The present research provides a foundation for revealing the structural-functional relationship of this enzyme.

Global gene expression responses of Atlantic salmon skin to Moritella viscosa.

Moritella viscosa is a Gram-negative pathogen that causes large, chronic ulcers, known as winter-ulcer disease, in the skin of several fish species including Atlantic salmon. We used a bath challenge approach to profile the transcriptome responses of M. viscosa-infected Atlantic salmon skin at the lesion (Mv-At) and away from the lesion (Mv-Aw) sites. M. viscosa infection was confirmed through RNA-based qPCR assays. RNA-Seq identified 5212 and 2911 transcripts differentially expressed in the Mv-At compared to no-infection control and Mv-Aw groups, respectively. Also, there were 563 differentially expressed transcripts when comparing the Mv-Aw to control samples. Our results suggest that M. viscosa caused massive and strong, but largely infection site-focused, transcriptome dysregulations in Atlantic salmon skin, and its effects beyond the skin lesion site were comparably subtle. The M. viscosa-induced transcripts of Atlantic salmon were mainly involved in innate and adaptive immune response-related pathways, whereas the suppressed transcripts by this pathogen were largely connected to developmental and cellular processes. As validated by qPCR, M. viscosa dysregulated transcripts encoding receptors, signal transducers, transcription factors and immune effectors playing roles in TLR- and IFN-dependent pathways as well as immunoregulation, antigen presentation and T-cell development. This study broadened the current understanding of molecular pathways underlying M. viscosa-triggered responses of Atlantic salmon, and identified biomarkers that may assist to diagnose and combat this pathogen.

Comparative genomic analysis of obligately piezophilic Moritella yayanosii DB21MT-5 reveals bacterial adaptation to the Challenger Deep, Mariana Trench.

Hadal trenches are the deepest but underexplored ecosystems on the Earth. Inhabiting the trench bottom is a group of micro-organisms termed obligate piezophiles that grow exclusively under high hydrostatic pressures (HHP). To reveal the genetic and physiological characteristics of their peculiar lifestyles and microbial adaptation to extreme high pressures, we sequenced the complete genome of the obligately piezophilic bacterium Moritella yayanosii DB21MT-5 isolated from the deepest oceanic sediment at the Challenger Deep, Mariana Trench. Through comparative analysis against pressure sensitive and deep-sea piezophilic Moritella strains, we identified over a hundred genes that present exclusively in hadal strain DB21MT-5. The hadal strain encodes fewer signal transduction proteins and secreted polysaccharases, but has more abundant metal ion transporters and the potential to utilize plant-derived saccharides. Instead of producing osmolyte betaine from choline as other Moritella strains, strain DB21MT-5 ferments on choline within a dedicated bacterial microcompartment organelle. Furthermore, the defence systems possessed by DB21MT-5 are distinct from other Moritella strains but resemble those in obligate piezophiles obtained from the same geographical setting. Collectively, the intensive comparative genomic analysis of an obligately piezophilic strain Moritella yayanosii DB21MT-5 demonstrates a depth-dependent distribution of energy metabolic pathways, compartmentalization of important metabolism and use of distinct defence systems, which likely contribute to microbial adaptation to the bottom of hadal trench.

Absolute quantification of priority bacteria in aquaculture using digital PCR.

Modern aquaculture systems are designed for intensive rearing of fish or other species. Both land-based and offshore systems typically contain high loads of biomass and the water quality in these systems is of paramount importance for fish health and production. Microorganisms play a crucial role in removal of organic matter and nitrogen-recycling, production of toxic hydrogen sulfide (H2S), and can affect fish health directly if pathogenic for fish or exerting probiotic properties. Methods currently used in aquaculture for monitoring certain bacteria species numbers still have typically low precision, specificity, sensitivity and are time-consuming. Here, we demonstrate the use of Digital PCR as a powerful tool for absolute quantification of sulfate-reducing bacteria (SRB) and major pathogens in salmon aquaculture, Moritella viscosa, Yersinia ruckeri and Flavobacterium psychrophilum. In addition, an assay for quantification of Listeria monocytogenes, which is a human pathogen bacterium and relevant target associated with salmonid cultivation in recirculating systems and salmon processing, has been assessed. Sudden mass mortality incidents caused by H2S produced by SRB have become of major concern in closed aquaculture systems. An ultra-sensitive assay for quantification of SRB has been established using Desulfovibrio desulfuricans as reference strain. The use of TaqMan® probe technology allowed for the development of multi-plex assays capable of simultaneous quantification of these aquaculture priority bacteria. In single-plex assays, limit of detection was found to be at around 20 fg DNA for M. viscosa, Y. ruckeri and F. psychrophilum, and as low as 2 fg DNA for L. monocytogenes and D. desulfuricans.

Structure and Mechanism of the Ketosynthase-Chain Length Factor Didomain from a Prototypical Polyunsaturated Fatty Acid Synthase.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential ingredients of the human diet. They are synthesized by LC-PUFA synthases (PFASs) expressed in marine bacteria and other organisms. PFASs are large enzyme complexes that are homologous to mammalian fatty acid synthases and microbial polyketide synthases. One subunit of each PFAS harbors consecutive ketosynthase (KSc) and chain length factor (CLF) domains that collectively catalyze the elongation of a nascent fatty acyl chain via iterative carbon-carbon bond formation. We report the X-ray crystal structure of the KS-CLF didomain from a well-studied PFAS in Moritella marina. Our structure, in combination with biochemical analysis, provides a foundation for understanding the mechanism of substrate recognition and chain length control by the KS-CLF didomain as well as its interaction with a cognate acyl carrier protein partner.

Sequence analysis of nonulosonic acid biosynthetic gene clusters in Vibrionaceae and Moritella viscosa.

Nonulosonic acid (NulO) biosynthesis in bacteria is directed by nab gene clusters that can lead to neuraminic, legionaminic or pseudaminic acids. Analysis of the gene content from a set mainly composed of Aliivibrio salmonicida and Moritella viscosa strains reveals the existence of several unique nab clusters, for which the NulO products were predicted. This prediction method can be used to guide tandem mass spectrometry studies in order to verify the products of previously undescribed nab clusters and identify new members of the NulOs family.

Measuring protein stability in the GroEL chaperonin cage reveals massive destabilization.

The thermodynamics of protein folding in bulk solution have been thoroughly investigated for decades. By contrast, measurements of protein substrate stability inside the GroEL/ES chaperonin cage have not been reported. Such measurements require stable encapsulation, that is no escape of the substrate into bulk solution during experiments, and a way to perturb protein stability without affecting the chaperonin system itself. Here, by establishing such conditions, we show that protein stability in the chaperonin cage is reduced dramatically by more than 5 kcal mol-1 compared to that in bulk solution. Given that steric confinement alone is stabilizing, our results indicate that hydrophobic and/or electrostatic effects in the cavity are strongly destabilizing. Our findings are consistent with the iterative annealing mechanism of action proposed for the chaperonin GroEL.

All cells contain molecules known as proteins that perform many essential roles. Proteins are made of chains of building blocks called amino acids that fold to form the proteins' three-dimensional structures. Many proteins fold spontaneously into their well-defined and correct structures. However, some proteins fold incorrectly, which prevents them from working properly, and can lead to formation of aggregates that may harm the cell. To prevent such damage, cells have evolved proteins known as molecular chaperones that assist in the folding of other proteins. For example, a molecular chaperone called GroEL is found in a bacterium known as Escherichia coli. This molecular chaperone contains a cavity which prevents target proteins from forming clumps by keeping them away from other proteins. However, it remained unclear precisely how GroEL works and whether enclosing target proteins in its cavity has other effects. Moritella profunda is a bacterium that thrives in cold environments and, as a result, many of its proteins are unstable at room temperature and tend to unfold or fold incorrectly. To study how GroEL works, Korobko et al. used a protein from M. profunda called dihydrofolate reductase as a target protein for the chaperone. A clever trick was then used to determine the folding state of dihydrofolate reductase when inside the chaperone cavity. The experiments revealed that the environment within the cavity of GroEL strongly favors dihydrofolate reductase adopting its unfolded state instead of its folded state. This suggests that GroEL helps dihydrofolate reductase and other incorrectly folded target proteins to unfold, thus providing the proteins another opportunity to fold again correctly. Parkinson's disease, Alzheimer's disease and many other diseases are caused by proteins folding incorrectly and forming aggregates. A better understanding of how proteins fold may, therefore, assist in developing new therapies for such diseases. These findings may also help biotechnology researchers develop methods for producing difficult-to-fold proteins on a large scale.

Impact of co-infection with Lepeophtheirus salmonis and Moritella viscosa on inflammatory and immune responses of Atlantic salmon (Salmo salar).

This study was conducted to determine the effects of a co-infection with Moritella viscosa at different exposure levels of sea lice Lepeophtheirus salmonis in Atlantic salmon (Salmo salar). M. viscosa (1.14 × 106  cfu/ml) was introduced to all experimental tanks at 10 days post-lice infection (dpLs). Mean lice counts decreased over time in both the medium lice co-infection (31.5 ± 19.0 at 7 dpLs; 16.9 ± 9.3 at 46 dpLs) and high lice co-infection (62.0 ± 10.8 at 7 dpLs; 37.6 ± 11.3 at 46 dpLs). There were significantly higher mortalities and more severe skin lesions in the high lice co-infected group compared to medium lice co-infected group or M. viscosa-only infection. Quantitative gene expression analysis detected a significant upregulation of genes in skin from the high lice co-infection group consistent with severe inflammation (il-8, mmp-9, hep, saa). Skin lesions retrieved throughout the study were positive for M. viscosa growth, but these were rarely located in regions associated with lice. These results suggest that while M. viscosa infection itself may induce skin lesion development in salmon, co-infection with high numbers of lice can enhance this impact and significantly reduce the ability of these lesions to resolve, resulting in increased mortality.

A Truncated Galectin-3 Isolated from Skin Mucus of Atlantic Salmon Salmo salar Binds to and Modulates the Proteome of the Gram-Negative Bacteria Moritella viscosa.

The mucus of fish skin plays a vital role in innate immune defense. Some mucus proteins have the potential to incapacitate pathogens and/or inhibit their passage through the skin. In this study the aim was to isolate and characterize galectin(s), ß-galactosides binding proteins, present in skin mucus. A novel short form of galectin-3 was isolated from Atlantic salmon skin mucus by α-lactose agarose based affinity chromatography followed by Sephadex G-15 gel filtration. Mass spectrometric analysis showed that the isolated protein was the C-terminal half of galectin-3 (galectin-3C). Galectin-3C showed calcium independent and lactose inhabitable hemagglutination, and agglutinated the Gram-negative pathogenic bacteria Moritella viscosa. Galectin-3 mRNA was highly expressed in skin and gill, followed by muscle, hindgut, spleen, stomach, foregut, head kidney, and liver. Moritella viscosa incubated with galectin-3C had a modified proteome. Proteins with changed abundance included multidrug transporter and three ribosomal proteins L7/12, S2, and S13. Overall, this study shows the isolation and characterization of a novel galectin-3 short form involved in pathogen recognition and modulation, and hence in immune defense of Atlantic salmon.

Fish pathogen binding to mucins from Atlantic salmon and Arctic char differs in avidity and specificity and is modulated by fluid velocity.

Disease outbreaks are limiting factors for an ethical and economically sustainable aquaculture industry. The first point of contact between a pathogen and a host occurs in the mucus, which covers the epithelial surfaces of the skin, gills and gastrointestinal tract. Increased knowledge on host-pathogen interactions at these primary barriers may contribute to development of disease prevention strategies. The mucus layer is built of highly glycosylated mucins, and mucin glycosylation differs between these epithelial sites. We have previously shown that A. salmonicida binds to Atlantic salmon mucins. Here we demonstrate binding of four additional bacteria, A. hydrophila, V. harveyi, M. viscosa and Y. ruckeri, to mucins from Atlantic salmon and Arctic char. No specific binding could be observed for V. salmonicida to any of the mucin groups. Mucin binding avidity was highest for A. hydrophila and A. salmonicida, followed by V. harveyi, M. viscosa and Y. ruckeri in decreasing order. Four of the pathogens showed highest binding to either gills or intestinal mucins, whereas none of the pathogens had preference for binding to skin mucins. Fluid velocity enhanced binding of intestinal mucins to A. hydrophila and A. salmonicida at 1.5 and 2 cm/s, whereas a velocity of 2 cm/s for skin mucins increased binding of A. salmonicida and decreased binding of A. hydrophila. Binding avidity, specificity and the effect of fluid velocity on binding thus differ between salmonid pathogens and with mucin origin. The results are in line with a model where the short skin mucin glycans contribute to contact with pathogens whereas pathogen binding to mucins with complex glycans aid the removal of pathogens from internal epithelial surfaces.

Atlantic salmon challenged with extracellular products from Moritella viscosa.

Skin ulcers in Atlantic salmon Salmo salar in the Canadian east coast salmon aquaculture industry lead to high mortality rates. This condition is clinically similar to winter ulcer disease in Norway with the exception that it occurs at temperatures above 10°C. Moritella viscosa is thought to be the causative agent for winter ulcer disease in Norway, and it is occasionally also isolated from skin ulcer cases in Atlantic Canada. This bacterium is known to produce cytotoxins. The objective of this study was to determine if extracellular products (ECP) from an Atlantic Canadian strain of M. viscosa could induce a tissue response similar to what is observed with M. viscosa infections in Atlantic salmon in eastern Canada. We injected fish subcutaneously with ECP and monitored the development of skin lesions. We sampled fish with early skin lesions and ulcers to describe the pathology associated with the condition. Samples were taken for histopathology, bacterial culture, and quantitative PCR (qPCR). All experimental fish expressed early skin lesions, with 5 fish (8.3%) developing deep skin ulcers after 12 d post-exposure. Our results suggest the ECP of M. viscosa from the east coast of Canada induces a similar tissue response to what is described in ulcer disease in Atlantic salmon. These extracelluar products may partially explain the pathology associated with M. viscosa.

Effects of Pressure and Temperature on the Atomic Fluctuations of Dihydrofolate Reductase from a Psychropiezophile and a Mesophile.

Determining the effects of extreme conditions on proteins from "extremophilic" and mesophilic microbes is important for understanding how life adapts to living at extremes as well as how extreme conditions can be used for sterilization and food preservation. Previous molecular dynamics simulations of dihydrofolate reductase (DHFR) from a psychropiezophile (cold- and pressure-loving), Moritella profunda (Mp), and a mesophile, Escherichia coli (Ec), at various pressures and temperatures indicate that atomic fluctuations, which are important for enzyme function, increase with both temperature and pressure. Here, the factors that cause increases in atomic fluctuations in the simulations are examined. The fluctuations increase with temperature not only because of greater thermal energy and thermal expansion of the protein but also because hydrogen bonds between protein atoms are weakened. However, the increase in fluctuations with pressure cannot be due to thermal energy, which remains constant, nor the compressive effects of pressure, but instead, the hydrogen bonds are also weakened. In addition, increased temperature causes larger increases in fluctuations of the loop regions of MpDHFR than EcDHFR, and increased pressure causes both increases and decreases in fluctuations of the loops, which differ between the two.

The transcriptional response of the Pacific oyster Crassostrea gigas under simultaneous bacterial and heat stresses.

Bacterial infection and heat stress are considered as two major environmental threats for the aquaculture industry of oyster Crassostrea gigas. In the present study, the expression profiles of mRNA transcripts in the hemocytes of oysters under bacterial challenge and heat stress were examined by next-generation sequencing. There were 21,095, 21,957 and 21,141 transcripts identified in the hemocytes of oysters from three groups, respectively, including control group (designated as Con group), Vibrio splendidus challenge group (Bac group), and bacterial and heat stress combined treatment group (BacHeat group). There were 4610, 5093 and 5149 differentially expressed transcripts (DTs) in the three pairwise comparisons Con/Bac, Con/BacHeat and Bac/BacHeat, respectively. The main enriched GO terms in biological process category of the DTs included the metabolic processes, cellular process, response to stimulus and immune system process. The expression patterns of DTs involved in pattern recognition, immune signal transduction and energy metabolic indicated that the immune response to bacterial challenge was disturbed under acute heat stress, which was also confirmed by quantitative real-time PCR. The neuroendocrine immunomodulation, especially the catecholaminergic regulation, played indispensable roles in stress response. The total energy reserves as well as cellular energy allocation (CEA) in hepatopancreas of oysters decreased remarkably especially in BacHeat group, while the energy consumption generally increased, suggesting that the immune defense against the simultaneous stimulation of pathogen and heat stress imposed greater costs on oyster's energy expenditure than a single stressor. These results above indicated that, the heat stress disturbed the normal expression of genes involved in immune response and energy metabolism, accelerated energy consumption and broke the metabolic balance, leading to a decline in resilience to infection and mass mortality of oyster in summer.

A time-course study of gene expression and antibody repertoire at early time post vaccination of Atlantic salmon.

The majority of studies of vaccine responses in Atlantic salmon have focused on several weeks after vaccination, and employed a limited number of marker genes. In this study, novel techniques were used to examine a broad panel of expressed genes and antibody repertoire of Atlantic salmon following vaccination. Salmon parr were vaccinated with a multivalent oil-based vaccine, and blood plasma and head kidney were sampled at several time-points between 0-35 days post vaccination. Saline-injected fish were used as control at all time-points. Microarray analyses showed increased expression of immune genes from the first day to the end of study in the head kidney of vaccinated fish. Genes up-regulated in the late phase included several leukocyte markers and components of the oxidative burst complex. A suite of genes that can take part in B cells differentiation were up-regulated from day 14, at which time secretory IgM transcripts also peaked. This coincided with marked increased plasma titres of non-vaccine specific antibodies binding to a hapten-carrier antigen DNP-KLH, while antibodies to bacterial components of the vaccine, Moritella viscosa and Aeromonas salmonicida, first showed significantly elevated antibody levels at day 21, and at a markedly lower magnitude than the non-vaccine specific titres. Sequencing of the variable region of IgM heavy chain (CDR3) revealed higher cumulative frequencies of unique clonotypes in vaccinated salmon starting from day 14 when specific antibodies were first detected. Reduced sequence variance of CDR3 suggested expansion of recently emerged clonotypes. Overall, the results presented here follow a broad panel of gene expression, immunoglobulin sequencing and plasma antibody titres in the first few weeks after vaccination of Atlantic salmon, pointing to a potentially important contribution of non-vaccine specific antibody responses early in the vaccine response.