EDITORIAL published: 17 March 2022 doi: 10.3389/fimmu.2022.850026 Editorial: Oral Immune-Enhancing Research in Fish Jorge Galindo-Villegas 1*, Peter Bossier 2* and Felipe E. Reyes-López 3,4,5* 1 Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway, 2 Laboratory of Aquaculture & Artemia Reference Center, Department of Animal Science and Aquatic Ecology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium, 3 Centro de Biotecnologı´a Acuı´cola, Facultad de Quı´mica y Biologı´a, Universidad de Santiago de Chile, Santiago, Chile, 4 Facultad de Medicina Veterinaria y Agronomía, Universidad de Las Américas, Santiago, Chile, 5 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Chile Keywords: teleost fish model, Omics, probiotics-immunobiotics, disease enhancement, endobolome, holistic analysis, selective manipulation, microbiome Editorial on the Research Topic Oral Immune-Enhancing Research in Fish INTRODUCTION Aquaculture is recognized globally as the fastest growing food-producing sector for human consumption (1). However, to satisfy increasing international demand, it is predicted that current production must be doubled by 2030 while improving the biosafety protocols (2). Such Edited and reviewed by: an expansion will necessarily involve implementing innovative actions in aquaculture production Gaetano Isola, while also targeting effective health control measures and considering animal welfare. University of Catania, Italy Diet, environment, and host genotype have been repeatedly demonstrated to play an essential *Correspondence: role in defining the fish gut composition (3). However, only a few studies have examined the Jorge Galindo-Villegas association between disease severity and degree of dysbiosis in fish. Likewise, the management of Jorge.galindo-villegas@nord.no Peter Bossier pathogens and parasites remains a sustainability challenge industry-wide due to poor understanding peter.bossier@ugent.be of the associated fish immune mechanism across species, and the effects of climate change on Felipe E. Reyes-López aquaculture remain uncertain and complex to validate (4). While these concepts are not necessarily felipe.reyes@uab.cat new, applying modern high-throughput sequencing approaches and state-of-the-art immune techniques will accelerate our understanding of the complex trophic (e.g., prokaryotic, Specialty section: eukaryotic) structures, the host recognition systems within aquaculture species and systems, and This article was submitted to the effect of an intervention on eventual health outcomes allowing aquaculture to benefit from Comparative Immunology, parallel advances in related disciplines (5). a section of the journal Among the latest therapies developed, orally administered health promoters have demonstrated Frontiers in Immunology a strong ability as critical mediators in enhancing the fish immune system. However, the interaction Received: 07 January 2022 between each element per fish species is highly complex and not always homogeneous along the Accepted: 25 February 2022 different developmental stages. Therefore, to assess the expected potential immunological Published: 17 March 2022 promotion and enhancement capacity, the use of dedicated, robust, and innovative molecular Citation: tools accompanied by keen strategies of analysis devoted to elucidating the mode of action for the Galindo-Villegas J, Bossier P and Reyes-López FE (2022) Editorial: Oral tested compounds are required. Immune-Enhancing Research in Fish. By collating evidence, eleven exciting research papers written by 79 authors in our Research Front. Immunol. 13:850026. Topic entitled “Oral Immune-Enhancing Research in Fish” are presented in this ebook. Together, doi: 10.3389/fimmu.2022.850026 they will provide a platform of discussion in the advancement of the actual knowledge and Frontiers in Immunology | www.frontiersin.org 1 March 2022 | Volume 13 | Article 850026 Galindo-Villegas et al. Editorial: Oral Immune-Enhancing Research in Fish understanding beyond the immediate immune effects and the was argued that the reduction in Pcna counts in the gills of the corresponding immune mechanisms in fish fed with functional fish-fed Phyto- and Gmos-supplemented diets might be feed additives, live supplemented feed, or even exposed to oral associated with reduced levels of oxidative species via pollutants that stimulates a differential immune response in the modulation of the endogenous antioxidant capacity. Moreover, animals under experimental or fish farm settings. Phyto supplementation reduced lamellar fusion, whereas Gmos supplementation reduced goblet cell count by 11% but did not affect the distribution of goblet cells along the primary lamellae. These protective effects could be considered in future prevention PHYTOGENICS AS SUSTAINABLE strategies against gill mucosal diseases in fish species, especially HEALTH ENHANCERS IN those sensitive to gill-associated pathogens. AQUACULTURE PRODUCTION In a different animal model, the gnotobiotic brine shrimp (Artemia franciscana), Zheng et al. showed that essential oils of The implementation of innovative and sustainable production M. alternifolia (containing mainly terpinen-4-ol and terpinene) strategies has proven that plants have the potential to enhance and L. citrata (containing mainly citral and limonene) could the general physiology of animals. Phytogenics, defined as significantly improve the survival of brine shrimp larvae when environmentally friendly plant-derived bioactive compounds, bath challenged with pathogenic V. campbellii. At the are used as functional feed additives that benefit animal growth concentrations tested, no direct effect of the essential oils on and health. These elements often comprise aromatic plants the pathogen could be detected as revealed by life/dead staining extracts and essential oils rich in biologically active and regrowth tests. The results rather indicated that compounds. In farmed fish, a broad spectrum of phytogenics supplementation of these two EOs enhanced immune gene has been studied mainly due to their vast repertoire of properties, expression, possibly contributing to protective immunity in including growth promotion, antimicrobial, immunostimulant, brine shrimp larvae against V. campbellii. Furthermore, the antioxidant, anti-inflammatory, and sedative activities. EOs-regulate the expression of bacterial virulence factors, Nevertheless, a pressing issue from using a phytogenic is the including decreased swimming motility and biofilm formation impact generated in the mucosal barrier and thus in the fish in vitro. This might contribute in part to protecting of the brine defense. Despite its enormous potential, establishing the shrimp larvae against pathogenic V. campbellii in vivo However, phytogenics mode of action in mucosal tissues is essential for further studies are needed to investigate the underlying their proper use and safe administration Firmino et al.. protective mechanism of EOs, such as analyzing the expression Firmino et al. used feed microcapsules containing a of a larger panel of immune-related genes. Taken together, this blend of garlic essential oil, carvacrol, and thymol. They study seems to indicate that essential oils can be part of disease analyzed the Gilthead seabream skin transcriptional profile intervention and/or prevention strategy in aquaculture. with some key skin mucus biomarkers and the resulting pathogenic bacterial growth capacity in the fish external live epithelium. Their results suggest that the phytogen- supplemented diet promoted the secretion of non-specific SELECTIVE DIETARY MANIPULATION immune molecules into the skin mucus, causing the decrease OF THE MICROBIOTA ANALYZED of bacterial growth capacity. Likewise, novel transcriptomic IN VITRO AND IN VIVO UNDER enrichment analysis is presented, showing some cellular VARIATED CONTEXTS elements like the phagocytosis linked to the phytogenic treatment. Unfortunately, the exact mechanisms behind the Complex microbial communities colonize virtually every surface action of the tested elements were not yet unveiled. Lastly, their exposed to the external environment. They include prokaryotes, results suggest that the tested additive could directly affect the eukaryotes, and viruses, which provide enormous enzymatic intestinal host-microbial community composition that may capacity and play a key role in controlling many aspects of influence the stress and immunological response in the skin. host physiology. Remarkably, the aquatic environment possesses This observation suggests that the effect of the experimental an intrinsic aggressiveness due to the impressive number and diet on skin phagocytes and phagocytosis potential are a hot diversity of microorganisms that coexist per square millimeter. research line that deserves further evaluation. Therefore, understanding the complex relationship between Torrecillas et al. investigated the effect of two commercial commensal microbes and fish is vital in maintaining healthy additives in gill health of European sea bass fed with a low fish physiological homeostasis. Any disruption of this symbiosis (i.e., meal (FM)/low oil (FO) diet. The diets contained galactomannan dysbiosis) can drive inflammation, and even worse, the oligosaccharides (Gmos) and a blend of garlic and labiatae-plant development of inflammatory diseases (6). Indeed, studies oils (Phyto), while an unsupplemented group served as a control. using the 16s rRNA amplicon sequencing strategy have The morphological, histopathological, immunohistochemical, provided evidence that some pathologies are complex and biochemical parameters of the fish gills of sea bass given multifactorial diseases characterized by a profound dysbiosis of supplemented diets were protected from oxidative stress by the microbial community. As a good example, in Atlantic modulating the expression patterns of oxidative enzyme- salmon, it has been demonstrated that yellow mouth disease is related genes. The diets also lowered the Pcna+ cell counts. It dominated by distinct amplicon sequenced variants of Frontiers in Immunology | www.frontiersin.org 2 March 2022 | Volume 13 | Article 850026 Galindo-Villegas et al. Editorial: Oral Immune-Enhancing Research in Fish Tenacibaculum maritimum, together with opportunistic taxa, and immunity, Castejó n et al. investigated the integral role of including Vibrio spp., play a role in the clinical disease the microbiota upon immunity and antibody processing in progression (7). Several strategies to generate a positive loop in response to an extended endocrine disruptor in the aquatic maintaining host health through microbiota management have environment in the Gilthead seabream. Specifically, the 17a- been proposed. Among the diverse strategies to selectively ethinylestradiol (EE 2) and the agonist G protein-coupled manipulate host-microbe interactions, diet is a significant factor estrogen receptor agonist G-1 (G1) in the gut of gilthead that shapes the proportional representation of the microbiota seabream. Humoral lytic and oxidative stress response, present, particularly in the gut and their relative gene content (8). essential mucosal inflammatory marker genes, and goblet cells Reciprocally, the microbiota configuration influences the contribution to immunity at the gut mucosal surface were nutritional value of food Montalban-Arques et al. Although these explored. Additionally, the microbiome landscape on the approaches are particularly promissory, a critical issue determining immune repertoire following the dietary treatment and a a successful output from the potential promoters expected on priming immunization protocol using a model antigen (KLH) providing solid immune indicators and showing a reliable disease was revealed. Minor changes were observed on the stress markers enhancement at the time of dissecting responses is the robustness of or the gene expressions. However, the dietary treatment with EE2 the employed assays. In this special issue, we put forward solid combined with the immunization promoted remarkable changes pieces of research providing reliable evidence on the host-microbe in the bacterial class Betaproteobacteria and, unexpectedly, on relationships and the dietary manipulatory capacity. the estrogen-degrading genus Novosphingobium a major The study performed by Firmino et al. presents an integrative member of the endobolome. Overall, this study highlighted the analysis linking the gene transcriptional activity with the intrinsic relationship between xenoestrogens and their associated microbiota responses in the gut of gilthead seabream. The fish receptors in the ubiquitous fish immune regulation. Moreover, were fed with a regular diet supplemented with a functional feed the subtle but significant crosstalk with the fish gut endobolome containing microencapsulated essential oils from garlic, was described in fish for the first time. carvacrol, and thymol. The formula displayed an extensive There is an increasing consensus of the relevance for evaluating holistic capacity of modulating the immune response and the in vitro the biological mode of action of isolated compounds before microbial composition towards a beneficial profile characterized moving forward to in vivo trials Firmino et al.. From the by the decrease in the classes Bacteroidia and Clostridia. immunomodulatory point of view, amino acids (AA) are crucial Likewise, the feed additives increased the bacterial sequences components in the biosynthesis of immune support-associated associated with glutathione and lipid metabolism. Therefore, the molecules. In fish, several articles reported the effect of the dietary study provides new insights into host-microbial metabolism’s administration of AA on a spectrum of immune-related functions shared role in building up a local immune response and crosstalk (10), with no particular attention on the cell-specific modulatory between gut and microbiota. mechanisms. Thus, this necessitates in vitro studies that provide fast So far, the major challenge has been to understand the poorly lights about the response dynamic for specific immune cell subsets studied signals and mechanisms of leukocytes after the addition of prior to the in vivo stage. Machado et al. showed that methionine novel feed ingredients and their interaction with the microbiome at and tryptophan perform distinct roles in the immune response of mucosal interphases in fish. Due to the unique characteristics head kidney leukocytes (HKL). While L-methionine increased the displayed by the zebrafish model, exciting and solid progress in modulation of the expression of pro-inflammatory gene markers, the recognition of new dietary elements and the associated tryptophan augmented the expression of the anti-inflammatory mechanisms in the complex development and function of counterparts. However, the increase was only perceived when the leukocytes and other types of cells with immune capacity has HKL were exposed to lipopolysaccharides-extracted or UV- been documented (9). Here, Xie et al. using the powerful inactivated Photobacterium damselae subsp. pisicida (Phdp). zebrafish as a model of intestinal inflammation show that Exposing HKL to Phdp produces and releases an exotoxin that excessive recruitment of leukocytes triggered by soybean meal can induces apoptosis. Therefore, the exposure of HKL to the exotoxin be reverted by applying a dietary sinomenine therapy. Sinomenine resulted in a diminished signal of apoptosis in the L-methionine- is an agonist of alpha7 nicotinic acetylcholine receptor (a7nAchR), incubated cells. Meanwhile, the L-tryptophan group showed a pro- a critical mediator controlling neuro-immune functions with anti- apoptotic activity by the elevated expression of the initiator caspase inflammatory properties through nicotine. Treated animals 8 and an increased activity of caspase 3, suggesting a lowered resulted with a higher proportion of CD4+ or Foxp3+ resilience in the L-tryptophan-treated HKL in response to the lymphocytes in the base of the intestinal folds, suggesting a infection by Phdp. synergy between a7nAchR and regulatory T-cells (Treg) in the response. Moreover, reduced enteritis and increased glucose metabolism were associated with the shift of Sphingomonas and CLASSIC PROBIOTIC BACTERIA, Shigella composition and the increase in Cetobacterium biomass, IMMUNOSTIMULANTS, AND NOVEL suggesting that sinomenine can prevent fish foodborne enteritis at RECOMBINANT PROTEINS the immune or microbiota levels. To gain further insights on the impact that dietary Probiotics and immunostimulants have been used as preventive modulators have over the host commensal microorganisms strategies for limiting and controlling fish diseases. Probiotics Frontiers in Immunology | www.frontiersin.org 3 March 2022 | Volume 13 | Article 850026 Galindo-Villegas et al. Editorial: Oral Immune-Enhancing Research in Fish supplementation is aimed to maximize the profit from the crosstalk role in immunomodulation, showing differences between short- between the host and the commensal microbiota for improving the and long-treatments, including the modulation of stress-associated fish robustness and resilience. The study of Muñoz et al. used the mechanisms and immunosuppression. Cornet et al. proposed the Lactococcus lactis – a commensal bacteria found in mammals and use of Gas1a b-glucan produced by the null-mutant yeasts Gas1 of salmonids, for producing the recombinant type Ia interferon (rIFN- Saccharomyces cerevisiae, as new immunostimulant to protect Ia). Likewise, its role in protecting against the infectious pancreatic rainbow trout (Oncorhynchus mykiss) juveniles against necrosis virus (IPNV) following in vitro and in vivo strategies was Aeromonas salmonicida achromogenes infection. For comparison assessed. The study showed in vitro upregulation of antiviral genes purposes, the study included three b-glucan preparations: wild-type (mx; pkr) after the administration of different doses of rIFN-IA and (WT), MacroGard, and the novel Gas1, evaluated at two time points a reduction of the IPNV viral load determined through qPCR. The after feeding the animals. Unexpectedly, after 36 days, the dietary oral administration of Atlantic salmon (Salmo salar) with the L. supplementation with MacroGard resulted in the downregulation lactis rIFN-Ia, revealed a higher expression of antiviral-related genes of immune genes and the lysozyme activity, immunoglobulin in spleen rather than head kidney. In addition, it showed a lower production and neutrophils proliferation in comparison to Gas1. IPNV load in live Atlantic salmon even up to 60 days post-infection, The resulting immune status produced by Gas1 correlated with a suggesting that this oral strategy improved the antiviral response. higher survival rate against A. salmonicida. Moreover, the survival Adopting a similar strategy, Santibañez et al. evaluated the effect of rate difference between Gas1 and WT suggests lower degree of the L. lactis recombinant rIFN-g-producer oral administration on branching and shorter side chain length may be the responsible in rainbow trout (Oncorhynchus mykiss) through a challenge against the differences observed in the efficiency of the immuno- 108 CFU of F. psychrophilum. The higher survival rate on rainbow stimulation between both b-glucan preparations. trout fed L. lactis rIFN-g suggests that this application would be a Overall, the contributions gathered in this Research Topic useful novel strategy for conferring oral protection to fish against provide holistic evidence on various mechanisms associated with the infection of some extracellular bacterial pathogens. However, oral immune enhancement of fish at the cellular and immune further evaluations of the system in vivo and using variated level, propose methods on the selective manipulation of the gut developmental stages are required. microbiota, and more importantly, deal on how to provide an On the other hand, yeasts are considered immune modulators increased health status and disease resistance in cultured teleost since they contain b-glucan, mannans, chitin, and nucleic acids, that fish. By putting this information together, we hope to contribute can be recognized as microbial-associated molecular patterns to improving fish aquaculture by providing new concepts and (MAMPs) by pattern recognition receptors (PRRs) with high research trends. Notably, we intend to provide evidence that by capacity of training the innate memory in fish myelocytes Zhang utilizing robust methodologies accompanied with robust et al. The study of Morales-Lange et al. used the Cyberlindnera analytical strategies, functional, exciting novel findings and jadinii yeast in a 40% soybean meal (SBM) diet. The perspective of future research perspectives could undoubtedly be achieved. the study is interesting because they propose the use of the spleen to evaluate the effect of the novel diet fed in Atlantic salmon (Salmo salar). The study includes the evaluation of the immune response by RNAseq and indirect ELISA. Furthermore, the C. jadinii was AUTHOR CONTRIBUTIONS exposed to two different down-stream processing conditions: All authors listed have made a substantial, direct, and intellectual heat-inactivated (ICJ), or autolysation (ACJ). In line with contribution to the work, and approved it for publication. previous effects reported in the intestine after the dietary administration of SBM, these authors confirmed the pro- inflammatory status in spleen, being characterized by the increased TNFa and IFNg levels recorded. Notably, ACJ response ACKNOWLEDGMENTS was characterized by the increasing level of IL-10 and the diminished content of TNFa. hus, the authors suggest using ACJ To the Frontiers in Immunology Journal for allowing us to serve as a component in the formulation of diets for sustainable Atlantic as guest editors on the present special issue targeting the oral salmon farming. immune-enhancing research in fish. To all the different b-glucans have been extensively used in the aquaculture international authors who independently of the output showed industry. However, the species-specificity, length of their side interest and participated with the external reviewers and chains, and frequency of administration have depicted a variable appointed editors in this exciting journey. 3. Bozzi D, Rasmussen JA, Carøe C, Sveier H, Nordøy K, Gilbert MTP, et al. REFERENCES Salmon Gut Microbiota Correlates With Disease Infection Status: Potential 1. FAO. The State of World Fisheries and Aquaculture 2020. In:. Sustainability for Monitoring Health in Farmed Animals. Anim Microbiome (2021) 3:30. in Action Rome. (2020). doi: 10.4060/ca9229en doi: 10.1186/s42523-021-00096-2 2. Acosta F, Montero D, Izquierdo M, Galindo-Villegas J. High-Level Biocidal 4. Naylor RL, Hardy RW, Buschmann AH, Bush SR, Cao L, Klinger Products Effectively Eradicate Pathogenic g-Proteobacteria Biofilms From DH, et al. Troell M. 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