Ana Teresa Gonçalves

Transcriptome immunomodulation of in-feed additives in Atlantic salmon Salmo salar infested with sea lice Caligus rogercresseyi.

One of the most significant threats to the Chilean salmon aquaculture industry is the ectoparasitic sea louse Caligus rogercresseyi. To cope with sea lice infestations, functional diets have become an important component in strengthening the host immune response. The aim of this study was to evaluate molecular mechanisms activated through immunostimulation by in-feed plant-derived additives in Atlantic salmon infected with sea lice. Herein, a transcriptome-wide sequencing analysis was performed from skin and head kidney tissues, evidencing that the immune response genes were the most variable after the challenge, especially in the head kidney, while other genes involved in metabolism were highly expressed individuals fed with the immunostimulants. Interestingly, defensive enzymes such as Cytochrome p450 and serpins were down-regulated in infested individuals, especially in skin tissue. Additionally, MHC-I and MHC-II genes were differentially expressed after the incorporation of the in-feed additives, giving some cues about the protection mechanisms of plant-derived compound as immunostimulants for infested salmons. This is the first published study that evaluates the transcriptomic response of sea lice-infested Atlantic salmon fed with in-feed additives.

Cathepsin Gene Family Reveals Transcriptome Patterns Related to the Infective Stages of the Salmon Louse Caligus rogercresseyi.

Cathepsins are proteases involved in the ability of parasites to overcome and/or modulate host defenses so as to complete their own lifecycle. However, the mechanisms underlying this ability of cathepsins are still poorly understood. One excellent model for identifying and exploring the molecular functions of cathepsins is the marine ectoparasitic copepod Caligus rogercresseyi that currently affects the Chilean salmon industry. Using high-throughput transcriptome sequencing, 56 cathepsin-like sequences were found distributed in five cysteine protease groups (B, F, L, Z, and S) as well as in an aspartic protease group (D). Ontogenic transcriptome analysis evidenced that L cathepsins were the most abundant during the lifecycle, while cathepsins B and K were mostly expressed in the larval stages and adult females, thus suggesting participation in the molting processes and embryonic development, respectively. Interestingly, a variety of cathepsins from groups Z, L, D, B, K, and S were upregulated in the infective stage of copepodid, corroborating the complexity of the processes involved in the parasitic success of this copepod. Putative functional roles of cathepsins were conjectured based on the differential expressions found and on roles previously described in other phylogenetically related species. Moreover, 140 single nucleotide polymorphisms (SNP) were identified in transcripts annotated for cysteine and aspartic proteases located into untranslated regions, or the coding region. This study reports for the first time the presence of cathepsin-like genes and differential expressions throughout a copepod lifecycle. The identification of cathepsins together with functional validations represents a valuable strategy for pinpointing target molecules that could be used in the development of new delousing drugs or vaccines against C. rogercresseyi.

Microbiome dynamic modulation through functional diets based on pre- and probiotics (mannan-oligosaccharides and Saccharomyces cerevisiae) in juvenile rainbow trout (Oncorhynchus mykiss)

This study used high‐throughput sequencing to evaluate the intestinal microbiome dynamics in rainbow trout (Oncorhynchus mykiss) fed commercial diets supplemented with either pre‐ or probiotics (0·6% mannan‐oligosaccharides and 0·5% Saccharomyces cerevisiae respectively) or the mixture of both.

Transcription expression of immune-related genes from Caligus rogercresseyi evidences host-dependent patterns on Atlantic and coho salmon.

The transcriptomic response of the sea louse Caligus rogercresseyi during the infestation on Atlantic salmon (Salmo salar) and coho salmon (Oncorhynchus kisutch) was evaluated using 27 genes related to immune response, antioxidant system and secretome. Results showed early responses of TLR/IMD signaling pathway in sea lice infesting Atlantic salmon. Overall, genes associated with oxidative stress responses were upregulated in both host species. This pattern suggests that reactive oxygen species emitted by the host as a response to the infestation, could modulate the sea louse antioxidant system. Secretome-related transcripts evidenced upregulation of trypsins and serpins, mainly associated to Atlantic salmon than coho salmon. Interestingly, cathepsins and trypsin2 were downregulated at 7 days post-infection (dpi) in coho salmon. The principal component analysis revealed an inverse time-dependent pattern based on the different responses of C. rogercresseyi infecting both salmon species. Here, Atlantic salmon strongly modulates the transcriptome responses at earlier infection stages; meanwhile coho salmon reveals a less marked modulation, increasing the transcription activity during the infection process. This study evidences transcriptome differences between two salmon host species and provides pivotal knowledge towards elaborating future control strategies.

Functional diets modulate the acute phase protein response in Oncorhynchus mykiss subjected to chronic stress and challenged with Vibrio anguillarum

Abstract The acute phase response to pathogens alters the production of proinflammatory cytokines that, in turn, activate the synthesis of acute phase proteins. These proteins neutralize, prevent, and indicate tissue damage, thereby influencing the specific immune response and allowing the organism to regain homeostasis. Functional diets based in pre‐ and probiotics are used in aquaculture to improve fish health and resistance to diseases, but there is an information gap on the mechanisms involved in these effects and if these diets are efficient when fish are raised under high stocking densities. This study aimed an evaluation of the acute phase response in Oncorhynchus mykiss fed functional diets supplemented with pre‐ and probiotics (i.e. mannan‐oligosaccharides and Saccharomyces cerevisiae, respectively) and challenged by either Vibrio anguillarum or chronic stress via maintenance under high stocking densities. For this, the relative expression of acute phase response related genes in liver, and of inflammatory response related genes in head kidney was evaluated by RT‐qPCR. The supplemented diets differentially modulated the acute phase protein response to the assessed challenge conditions, specifically evidencing an overexpression of the genes HAPT, SAA, LECT2, and IL‐1&bgr; under chronic stress and of HAPT, IL‐1&bgr;, IL8, and LECT2 at 24 h post‐challenge with V. anguillarum. The observed early‐stage regulation of acute phase proteins and of the immune response by the probiotic S. cerevisiae and by prebiotic mannan‐oligosaccharides suggests that both supplements have high immunostimulatory potentials for fish farmed under high stocking densities. HighlightsEffect of functional diets on acute phase response genes in fish was studied.Basal levels of acute phase protein genes increased with prebiotic.Pre‐ and probiotic diet increased expression of acute phase proteins genes.Acute response was faster in fish stocked at high density and fed functional diets.Response to V. anguillarum infection in fish at high density improved.

Transcriptome survey of the lipid metabolic pathways involved in energy production and ecdysteroid synthesis in the salmon louse Caligus rogercresseyi (Crustacea: Copepoda)

The goal of this study was to identify and analyze the lipid metabolic pathways involved in energy production and ecdysteroid synthesis in the ectoparasite copepod Caligus rogercresseyi. Massive transcriptome sequencing analysis was performed during the infectious copepodid larval stage, during the attached chalimus larval stage, and also in female and male adults. Thirty genes were selected for describing the pathways, and these were annotated for proteins or enzymes involved in lipid digestion, absorption, and transport; fatty acid degradation; the synthesis and degradation of ketone bodies; and steroid and ecdysteroid syntheses. Differential expression of these genes was analyzed by ontogenic stage and discussed considering each stages feeding habits and energetic needs. Copepodids showed a low expression of fatty acid digestion genes, reflected by a non-feeding behavior, and the upregulation of genes involved in steroid biosynthesis, which was consistent with a pathway for cholesterol synthesis during ecdysis. The chalimus stage showed an upregulation of genes related to fatty acid digestion, absorption, and transport, as well as to fatty acid degradation and the synthesis of ketone bodies, therefore suggesting that lipids ingested from the mucus and skin of the host fish are metabolized as important sources of energy. Adult females also showed a pattern of high lipid metabolism for energy supply and mobilization in relation to reproduction and vitellogenesis. Adult females and males revealed different lipid metabolism patterns that reflected different energetic needs. This study reports for the first time the probable lipid metabolic pathways involved in the energy production and ecdysteroid synthesis of C. rogercresseyi.

Intestinal transcriptome modulation by functional diets in rainbow trout: A high-throughput sequencing appraisal to highlight GALT immunomodulation

ABSTRACT Functional ingredients such as pre‐ and probiotics are used in aquaculture to improve fish condition, modulating microbiota and promoting a healthy intestinal functioning. They also exert an active effect on the gut associated lymphoid tissue (GALT), stimulating the immune system. However, the molecular underpinnings of pre‐ and probiotics effect on intestinal mucosa are still unknown. This study investigated the intestinal mucosa transcriptome modulation when fish were fed functional diets and kept at different stocking densities. Juvenile rainbow trout were kept at low (LD‐3Kgm‐3) and high density (HD‐40 kgm‐3) and fed for 30 days functional diets with the prebiotic mannanoligosaccharide (PRE‐0.6%), the probiotic Saccharomyces cerevisiae (PRO‐0.5%), the mixture of both (MIX) and a control diet (CTRL). Intestinal transcriptome was evaluated by high‐throughput sequencing and blood plasma for biochemical parameters. Fish fed functional diets presented better condition regardless density, and that functional diets modulate intestinal transcriptome in different manner depending on the stocking density. At LD, fish from PRO presented stronger modulation with the majority of transcripts being down‐regulated, including the immune related ones, whereas at HD both PRO and MIX groups were more modulated, when comparing to the respective CTRL groups. Density had an overwhelming suppressive effect on the immune‐related genes, but this effect was counteracted by feeding functional diets, especially in fish fed with probiotics. This study shows for the first time the intestinal transcriptomic modulation when fish are fed functional diets at different stocking densities, and it shows the mitigating effect of these diets against deleterious conditions such as high density. HIGHLIGHTSFish Intestinal transcriptome modulation with probiotic, prebiotic and mix was accessed.Transcripome is modulated by functional feeds but depending on stocking density.Intestinal immunity related genes are downregulated after 3o days with functional diets at low density.High density suppresses immune related genes.Functional diets increase intestinal mucosa integrity and defense at transcriptomic level at high density.