Analysis of the white sturgeon ( Acipenser transmontanus ) immune response during immunostimulation and Veronaea botryosa infection

Systemic phaeohyphomycosis caused by Veronaea botryosa is regarded as an important emerging mycotic disease of sturgeon aquaculture. However, no vaccines or treatments are currently available. The effects of dietary β -glucan supplementation on resistance to V. botryosa infection was examined in controlled challenges by exposing immunostimulated and control fish to ~7.25 × 10 5 fungal spores/fish via intra-muscular injection. Six weeks post-challenge, cumulative mortality was determined and antibodies to acute phase-proteins (APP) were used to quantify the conserved APP peptides in the serum of challenged and control fish using Western blot. Transcript levels for all tested pro-inflammatory cytokines, APP, and regulatory cytokines in the spleen were similar amongst treatments at the end of the three-week feeding period. However, significantly higher survival occurred in fingerlings fed 0.3% β -glucans compared to non-immunostimulated fish groups ( p < 0.05) six weeks post-challenge. A strong proinflammatory response was detected in exposed treatment groups, and greater survival at 6 weeks was associated with higher transcript abundance of Il-17 in fish fed β -glucans. Findings support the important role of this cytokine in response to fungal infection.


Introduction
Infectious diseases pose a major threat to aquaculture production, causing millions of dollars in annual losses.In addition to direct mortalities, disease leads to reduced growth, increased labor costs, and additional treatment expenditures.The release of pathogens into the environment also poses a risk to wild fish populations.In California, sturgeon aquaculture is a multimillion-dollar industry that provides substantial revenues to producers and generates employment opportunities.The production of globally recognized, high quality caviar has made this product one of the few aquaculture-generated commodities exported by the US.Sturgeon culture is also ecologically important to the preservation of natural fish stocks, as wild sturgeon populations have been significantly affected by overfishing, habitat destruction, and pollution (Nelson et al., 2019;Monterey Bay Aquarium, 2020).
Phaeohyphomycosis caused by Veronaea botryosa results in systemic infection and high mortality in cultured adult white (Acipenser transmontanus) and Siberian sturgeon (A.baerii) (Steckler et al., 2014).In the Western United States, V. botryosa is responsible for a specific disease entity known locally to producers as "fluid belly".A cause of mortality in 5 to 8-year-old female white sturgeon, the phaeoid fungus infection results in significant economic losses for the caviar industry and is one of the most important emergent disease agents in sturgeon aquaculture (Steckler et al., 2014;Soto et al., 2017).Veronaea botryosa is also associated with disease in other aquatic animals and humans (Soto et al., 2017).In response to recent outbreaks in California and to gain a better understanding of disease pathogenesis, a laboratory-controlled V. botryosa challenge model was developed (Soto et al., 2017;Coleman et al., 2018).Intramuscular injection of V. botryosa spores in white sturgeon fingerlings maintained at 18 • C results in systemic phaeohyphomycosis where challenged fish exhibit mortality in association with widespread fungal dissemination, and clinical, gross, and histopathological changes emulating natural infections (Coleman et al., 2018).Serological and molecular tools developed to study the immune response of Atlantic sturgeon (Acipenser oxyrhynchus) have also been validated for use in white sturgeon (Fast et al., 2016;Soto et al., 2019).
β-glucans are high molecular-weight polysaccharides derived from D- glucose building blocks that are usually isolated from the cell walls of bacteria, algae, fungi, and plants (Meena et al., 2013;Caipang and Lazado, 2015;Rodrigues et al., 2020).β-glucans have been extensively used in animal production, including aquaculture, and several review papers have been published describing their mechanisms and potential uses in fish medicine (Meena et al., 2013;Petit and Wiegertjes, 2016;Rodrigues et al., 2020).Several beneficial outcomes have been reported after oral administration of β-glucans in fish, including: non-specific immunomodulatory effects enhancing both humoral and cellular immunity (Petit and Wiegertjes, 2016), protection against several infectious diseases, stress-reduction (Barros et al., 2014;Do Huu et al., 2016), anti-toxin effect (Dawood et al., 2020), and growth enhancement (Aramli et al., 2015;Do-Huu et al., 2018).
Few studies have investigated the immune responses of fish to fungal pathogens or the effects of immunostimulants on systemic disease caused by them.Investigations are limited to zebrafish models of mammalian mycoses and fungal-like pathogens (Rosowski et al., 2018).Although Guselle et al. (2006) demonstrated that intraperitoneal injection of β-glucans in rainbow trout (Oncorhynchus mykiss) reduced xenoma formation in the gills of fish experimentally challenged with the microsporidian Loma salmonae; there is a paucity of information on the effects of oral administration of β-glucans against fungal infections in fish.Therefore, the objectives of this study were to investigate aspects of the immune response of white sturgeon to β-glucan immunostimulation in feed and to investigate the protection conferred to white sturgeon fed β-glucans and challenged with V. botryosa.

Fish and husbandry conditions
All research was conducted under protocols approved by the University of California, Davis Institutional Animal Care and Use Committee.Seven hundred and twenty white sturgeon fingerlings (34 ± 11 g) were received from a local producer in Northern California and acclimatized in flow-through fresh water at 18 ± 1 • C for 2 weeks.Fish were maintained in aerated, 130 L tanks containing ~60 L of un-chlorinated freshwater.Oxygen levels (~9 mg/L) and water temperature were measured daily.

Fungal isolates
Archived stocks of Veronaea botryosa (F15-3-1) isolated from white sturgeon with fluid belly and frozen at − 80 • C in a sterile water solution were revived on potato flake agar (PFA) plates incubated at 25 • C under aerobic conditions for 15 days (Soto et al., 2017).On the day of challenge, plates were flooded with phosphate buffer saline (PBS) and spores purified by vacuum filtration through a Miracloth (EMD Millipore Corporation, Billerica, MA, USA) with varying pore sizes of 22-25 μm.Spore counts were confirmed using a hemocytometer and light microscope, then adjusted to ~10 7 spores/ml in PBS.Spore viability was confirmed by serial dilution, plating on PFA, and counting CFU/plate 7 days postinoculation.

Diets
Fish were fed a soft moist sturgeon feed with 45% protein (Rangen, Buhl, Idaho) at a rate of 2% body weight per day.The β-glucan diets consisted of the basal feed supplemented with 0.1% or 0.3% β-glucan derived from the yeast Saccharomyces cerevisiae (MacroGard®, Biorigin, Sao Paulo, Brazil).To prepare the diets, β-glucans and feed were thoroughly mixed, vegetable oil was slowly added, and the feed mixed for a further 15 min manually.The pellets were dried overnight at room temperature and stored at 4 • C until used.Glucan diets were prepared weekly.

Immunostimulation by β-glucans
Following the acclimatization phase, non-immunostimulated control groups were kept on the basal diet while the other groups were fed diets supplemented with β-glucans.Twenty fish/tank were randomly assigned to six replicate tanks for each of the three different diet groups and challenged with V. botryosa as detailed below.An additional six replicate tanks per diet group served as non-challenged negative controls.Fish were fed the different assigned diets for 21 days.After this period of time, all fish were fed the basal diet for the remainder of the experiment.

Fish challenge
After 21 days of immunostimulation with β-glucans, 12 fish from each group (two per tank) were anesthetized in 100 mg/L of tricaine methanesulfonate (MS-222) (Argent Chemical Laboratories, Redmond, Washington) buffered 1:1 with sodium bicarbonate.Blood was collected by caudal venipuncture and fish were then euthanized in 1000 mg/L of MS-222, weighed, and their total lengths recorded.Blood was allowed to clot at 4 • C for 1 h, centrifuged at 3000 for 15 min, and the separated serum removed for storage at − 80 • C until needed.Coelomic cavities were incised and approximately 20 mg of spleen removed and suspended in RNAlater (ThermoFisher Scientific) for molecular analysis as detailed below.Three fish from each treatment were fixed in 10% neutral buffered formalin, decalcified in Kristensen's solution, and trimmed serially into transverse slabs.Prepared tissues were processed routinely, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E) for light microscopic examination.
The remaining 18 fish per tank were used to investigate the protection conferred by dietary supplementation with β-glucans in V. botryosa challenged juvenile white sturgeon.Briefly, fish were anesthetized in 100 mg/L of buffered MS-222.Fish in half of the control diet and 0.1% or 0.3% β-glucan treatment tanks were injected in epaxial muscle with 7.2 × 10 5 V. botryosa spores per fish following published protocols (Coleman et al., 2018).Fish in the other half of the tanks served as negative controls and received intramuscular injections with physiologic buffered saline (PBS).Injected fish were returned to their respective tanks and observed twice daily during the six-week trial.Mortalities were recorded and the quantity of feed adjusted to avoid water quality deterioration.Moribund fish or any fish exhibiting abnormal swimming, lethargy, exophthalmia, skin lesions, or coelomic distention were euthanized with buffered MS-222 (1000 mg/L).Explants of spleen (~20 mg) were collected and plated on Sabouraud dextrose agar (University of California, Biological Media Services) to confirm infection.Six weeks post-challenge, cumulative mortality was calculated for each treatment.Ten randomly collected survivors per treatment were euthanized and splenic samples collected for V. botryosa isolation, quantification and gene expression analysis.Additionally, blood was collected and processed as above for serological analysis.All remaining fish were euthanized, weighed, and measured for growth comparison.

Molecular analysis
Assembled transcriptomes for Atlantic sturgeon were used as a database source for generation of white sturgeon quantitative reversetranscription PCR assays (Fast et al., 2016).The sequences of haptoglobin, serotransferrin, serum amyloid A (SAA), cathelicidin, tumor necrosis alpha (TNF-α), interleukin 17 predicted protein (pp) (IL-17), interferon regulatory factor 8 (IRF8), transforming growth factor beta (TGF-β) and major histocompatibility class II (MHCII) were used to investigate pro-inflammatory, regulatory, adaptive and innate immune responses (Soto et al., 2019).The sequences of all primers used in this study are listed in Table 1.Primer efficiencies were determined using 10fold dilution series of cDNA and genomic DNA as template for qPCR reactions.Total RNA was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.A 15 min on-column DNase digestion with RNase-Free DNase (Qiagen) was performed to ensure removal of contaminating genomic DNA as suggested by the manufacturer.RNA concentration and purity, determined by 260/280, were measured with a spectrophotometer (Cytation 5, Biotek).Reverse transcription of 1 μg extracted RNA in 20 μl reactions was performed using the Superscript III First Strand Synthesis System (Thermofisher) according to the manufacturer's instructions.A control with omitted reverse transcriptase was performed for each extraction to check for the presence of contaminating genomic DNA.After reverse transcription, the samples were used as templates for quantitative PCR analysis.
In order to quantify and compare the expression levels of the white sturgeon cytokines, relative quantification by qRT-PCR was made using the β-actin and elongation factor reference genes for normalization in a QuantStudio 5 Real-Time PCR System (Thermofisher).The PCR mixture contained 1 μl of template cDNA, 1× SYBR Green PCR Master Mix (Applied Biosystems) and 20 μM of the appropriate forward and reverse primers (Invitrogen).The thermal cycling conditions for the PCR were as follows: 1 cycle at 95 • C 10 min, 45 cycles of amplification at 95 • C for 15 s and annealing at 60 • C for 1 min.The data were collected during each elongation step.Negative (DEPC-treated H 2 O) and no-reverse transcriptase controls were included in each run.All qPCR were assayed on every biological replicate (n = 12) and each sample was run in triplicate.
Relative gene expression of haptoglobin, serotransferrin, SAA, cathelicidin, TNF-α, IL 17, IRF8, TGF-β and MHCII was calculated using the 2 -∆∆Ct method and normalized against the expression of the average of β-actin and elongation factor reference genes (Livak and Schmittgen, 2001).Tissues from control fish were used as calibrators for all genes.
Total genomic DNA was extracted from recovered spleens from challenged and control fish using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, California) according to the manufacturer's instructions, followed by elution in 100 μl of elution buffer.Veronaea botryosa beta tubulin (tub2) gene was quantified following protocols by Yazdi et al. (2021).qPCR assays were run and analyzed using the QuantStudio3 qPCR System (Thermo Fisher Scientific, MA, USA).The 12 μl reaction mixture consisted of 7 μl TaqMan Environmental Master Mix (EMM; Applied Biosystems, Foster city, CA, USA), 400 nM of each primer, 80 nM of probe and 5 μl of DNA extracted sample (1 μg).Environmental Master Mix was used in all qPCR reactions to minimize potential PCR inhibitors.All samples, including diluted standards and negative controls were run in triplicate.Template controls containing PCR grade water and seven serial dilutions of PCR products generated with the Vbot1F and Vbot1R primers purified using QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) following manufacturer's instructions and quantified spectrophotometrically.The online tool from the University of Rhode Island, Kingston, Rhode Island, USA (http://cels.uri.edu/gsc/cndna.html) was used to approximate the number of copies of the purified 125 bp template.Purified PCR products diluted in PCR grade water were included in each PCR run.The lower limit of detection of this assay is 10 conidia (~500 gene copies) per reaction.Tissue samples, as well as the diluted standards and negative controls were run in triplicate.

Acute phase protein quantification
Following determination of protein concentration using the Pierce BCA Protein Assay Kit (Thermo Scientific, IL, USA) 20 μg of total protein from white sturgeon serum was denatured in Laemmli buffer (Bio-Rad) for 5 min at 95 • C. The sample was then loaded into Mini-PROTEAN® TGX Stain-Free™ 4-15% gels (Bio-Rad Laboratories Inc., CA, USA) and separated using the Mini-PROTEAN® Tetra Cell gel apparatus (Bio-Rad) for approximately 50 min at 150 Volts, and total protein was imaged using the stain-free application on the ChemiDoc MP (Bio-Rad) imager.
Following protein separation gels were immediately activated and then transferred onto 0.22 μM nitrocellulose serotransferrin membranes (Azure Biosystems, Dublin, CA, USA), while the haptoglobin protein gels were transferred onto TransBlot® Turbo™ Mini-size PVDF (Bio-Rad) membranes as in Soto et al. (2019).Both transfers used the Trans-Blot® Turbo™ Mini-size Transfer Stacks and the Trans-Blot® Turbo™ Transfer System (Bio-Rad) for 3 min and total protein was imaged using the stainfree application on the ChemiDoc MP imager.Membranes for serotransferrin protein were immediately transferred to the blocking buffer of 5% Non-Fat Dry Milk in PBS with Tween (PBSTw; 1.37 M NaCl, 27 mM KCl, 100 mM Na 2 HPO 4 , 18 mM KH 2 PO 4 , pH 7.4, 0.1% (w/v) Tween 20) and incubated with gentle agitation for 2 h at room temperature.Membranes for haptoglobin protein were immediately transferred to the blocking buffer of 3% bovine serum albumin in tris buffered saline (TBS) with Tween (TBSTw; 20 mM Tris, 500 mM NaCl, pH 7.5, 0.1% (w/v) Tween 20) and incubated with gentle agitation for 2 h at room temperature.Each blot was then incubated with the following primary/ secondary antibody combination: 1. Chicken IgY ++ (IgG) (H + L) (1:50,000 Jackson ImmunoResearch Laboratories, Inc.).All primary antibodies were diluted in their respective blocking buffer and incubated overnight at 4 • C. All secondary antibodies were diluted in their respective blocking buffers and incubated for 1 h at room temperature with gentle agitation.Protein bands were visualized using Clarity™ Western ECL Substrate chemiluminescent detection reagent (Bio-Rad) prior to image acquisition and visualized using the ChemiDoc MP imager.Image analysis was performed using the Image Lab™ Software (BioRad).Data is expressed as ratio of protein of interest expressed as a "fold difference" to the loading control, normalized to total protein.Representative fish from each treatment group at each time are shown in results.

Statistical analysis
Cumulative mortality, fish weight and total length after challenge, and fungal abundance (Log10 number of V. botryosa gene copies detected per microgram of white sturgeon spleen) comparisons were performed using GraphPad Prism (version 8.3.0,GraphPad Software, La Jolla, CA, USA).Statistical significance was determined by one-way ANOVA and Tukey's test.
For gene expression studies, the statistical significance between mean values were determined by one-way ANOVA and Tukey's test for pairwise comparison of means using the JMP pro 14 (Version 14.2.0,SAS Institute, Cary, NC, USA).Fold change of target genes in different time points, and treatments were conducted according to Ct values  based on 2 − ΔΔCt method when the PBS-treatment used as a control.The log transformation was used to transform skewed data to approximately normal distribution.Therefore, the response variable is Log2 fold change with a wide theoretical range of negative to positive real numbers.For the analysis to posses a power of 80%, assuming a Type I error of 0.05, and a significant difference to be detected = 1 standard deviation, at least 10 replicate samples were planned to be included at each time point per treatment.Due to poor RNA quality and/or quantity a few samples (<5%) were not carried through to qRT-PCR.

Results
No mortalities or other adverse effects were observed during the three-week immunostimulatory period during which time feeding behavior was similar for all control and treatment groups.No significant differences in weight, length, splenic transcript levels of haptoglobin, serotransferrin, SAA, cathelicidin, TNF-α, IL-17, IRF8, TGF-β or MHCII, or serum haptoglobin and serotransferrin protein were detected amongst the different treatments at the conclusion of the immunostimulatory period (Figs. 1 and 5).
Microscopic examination revealed no observable differences or pathological changes in any tissues between control and glucan treated fish at either dose level.The livers of all fish possessed large polygonal hepatocytes with central nuclei and abundant, pale staining, wispy cytoplasm indicating plentiful energy stores in the form of glycogen.A single layer of tall, slender enterocytes lined closely apposed, elongate mucosal folds with occasional short branches in the anterior intestine.Enterocytes possessed an oval basal nucleus and apical microvilli formed a prominent brush border.Goblet cells, occurring individually or in small groups, were scattered throughout the mucosa.Numbers varied dramatically between individuals and even within mucosal folds of the same individual, regardless of treatment group.Apoptotic cells and lymphocytes were rare within the epithelium.Mucosal folds of the spiral intestine were shorter, blunter, more widely spaced, and exhibited no branching compared to those in the proximal segment.Enterocytes were broader with abundant, finely vacuolated, pale eosinophilic cytoplasm.Goblet cells were subjectively larger and more numerous.
Significantly greater mortality was observed in all V. botryosa exposed treatment groups in comparison to non-exposed controls (Fig. 2).Although both β-glucan diets resulted in lower mortality than in fish fed control diets, only fish fed the 0.3% β-glucan diet exhibited significantly lower mortalities than controls at the end of the study (p = 0.0057) (Fig. 2).No significant differences were detected between the 0.1% and 0.3% β-glucan fed treatments (p > 0.05).Lower amounts of V. botryosa DNA was quantified in the spleens of survivors fed glucans when compared to the inoculated control fish (Fig. 3A).However, only those treated with 0.1% β-glucan were significantly different (p = 0.0035).Additionally, fish fed 0.3% β-glucan were significantly larger at the end of the study compared to control and groups 0.1% β-glucan (Fig. 3B).
Similar transcript levels of MHCII, TGF-β and TNF-α were quantified in control and treatment groups six weeks post-challenge (Fig. 4).Haptoglobin, serotransferrin, SAA, and cathelicidin transcript levels were significantly greater in challenged fish compared to non-exposed controls 6 weeks post-challenge (p ≤0.05).Significantly greater transcript levels of Il-17 were detected in V. botryosa exposed fish fed 0.1% and 0.3% β-glucan compared to non-stimulated exposed controls 6weeks post-challenge, as well as in non-exposed controls (p < 0.05).All other comparisons were similar (p > 0.05).Haptoglobin protein values were similar between control and exposed treatments regardless of immunostimulation (Fig. 5C).However, haptoglobin serum levels in the non-exposed non-immunostimulated treatment were significantly higher than in other treatments (Fig. 5C).On the other hand, serotransferrin levels in exposed treatments fed basal diet or 0.1% β-glucan were significantly greater (p ≤0.05) than those in non-exposed treatments (Fig. 5D).

Discussion
Results of this study suggest that β-glucan supplementation in the diets of white sturgeon produces a positive immunostimulatory effect that can be exploited as a prophylactic tool against systemic phaeohyphomycosis caused by V. botryosa.Overall, β-glucan supplementation resulted in lower cumulative mortality, lower fungal abundance in survivors, and better growth compared to fungus challenged controls fed an unsupplemented commercial diet.The positive effects of dietary β-glucan supplementation in cultured Persian sturgeon (Acipenser persicus) have also been investigated recently.Persian sturgeon fed diets containing 0.1%, 0.2% and 0.3% β-glucans exhibited significantly higher lysozyme and alternative complement activity as well as better growth compared to controls (P < 0.05) (Aramli et al., 2015), providing further justification for investigation into the potential use of immunostimulants to prevent losses in the caviar industry.
A host of soluble immune and inflammatory mediators are associated with fungal infections in mammals and previous work has shown increases in several of these in response to V. botryosa challenge in white sturgeon (Rosowski et al., 2018;Soto et al., 2019).As a result, haptoglobin, serotransferrin, SAA, cathelicidin, TNF-α, IL-17, IRF8, TGF-β and MHCII were chosen for evaluation in the study.Immunostimulated and control fish produced similar transcript levels for all evaluated immune mediators after the three-week period prior to challenge.Similar findings have been reported in striped catfish (Pangasianodon hypophthalmus) (Sirimanapong et al., 2015) and Atlantic cod (Gadus morhua L.) (Lokesh et al., 2012) fed β-glucan supplemented diets.In contrast,    2014) reported some changes in the expression of Creactive protein (crp1 and crp2) and complement (c1rs, bf/c2, c3 and masp2) related genes in the head kidney, mid-gut, and liver tissues of common carp (Cyprinus carpio) after 7 or 25 days of β-glucan feeding (0.1% MacroGard).However, direct comparisons between the studies is difficult as different fish species, different tissues, time points, and genes were compared.
Veronaea botryosa challenge induced robust expression of APP and the antimicrobial peptide cathelicidin in the spleens of sturgeon regardless of their diet (Figs. 4 and 5).Increases in the pro-inflammatory cytokine TNF-α, although insignificant, were detected in glucan fedfungal challenged treatment groups compared to exposed controls and non-exposed treatments, possibly the result of only a single time point being used in the gene expression analysis 6 weeks post-challenge.Less likely, fungal interference with immune function or the presence of TNFα orthologues in white sturgeon not detected by the primers used may have obscured the data.Different TNF-α orthologues have been reported in other fish species (Rodríguez et al., 2009) and are a consideration in sturgeon as a consequence of polyploidy (Rajkov et al., 2014).
The observed pro-inflammatory response detected in sturgeon upon V. botryosa challenge, was similar to those reported in snakehead murrel (Channa striatus) (Kumaresan et al., 2018) and zebrafish (Chao et al., 2010;Chen et al., 2013;Gratacap et al., 2013;Voelz et al., 2015) challenged with oomycetes, yeast or mucormycetes.Possible explanations for the discrepancies between different studies include the use of whole fish and different challenge methods.In transcriptomic analyses, patterns of gene expression in an entire organism may differ from and mask those of individual tissues (Chen et al., 2013;Gratacap et al., 2013).Additionally, different challenge methods, even when using the same host and pathogen, likely result in different gene expression patterns (Voelz et al., 2015).
Higher expression of IRF 8 occurred in all V. botryosa exposed treatment groups and was correlated with greater survival, although only glucan supplementation and fungal challenge stimulated significant upregulation (Fig. 4).Interferon-regulatory factors regulate transcription of IFN genes and are used in signaling pathways involved with innate immunity.IRF8 is also a major determinant of monocyte/ macrophage fate during hematopoiesis in fish and mammals (Shiau et al., 2015).On the other hand, MHC-II transcripts in challenged sturgeon were lower than those in non-challenged controls, although the difference was not significant.Fungal induced downregulation of MHC-II and interference with the adaptive immune response have been proposed in both in-vivo (Kumaresan et al., 2018) and in-vitro studies (Kales et al., 2007).
TGF-β transcripts were similar in all exposed and non-exposed treatments.TGF-β is a multifunctional cytokine involved in the regulation of immune responses (Zou and Secombes, 2016).While most studies refer to TGF-β as an immunosuppressive cytokine, TGF-β in combination with IL6 and IL21 are known to drive Th17 differentiation (Zou and Secombes, 2016;Zhang, 2018).Thus, cautious interpretation of the current results is necessary as the expression of many of these cytokines is known to differ between organs, and only spleen was evaluated in the current study.
Serum haptoglobin and serotransferrin have previously been shown to be elevated in V. botryosa infected (32 dpi) white sturgeon, held at 18 • C.This matched spleen transcript levels for these acute phase proteins in the same study (Soto et al., 2019).In the current work, serum Fig. 5. Expression relative to total protein (ERTP) of serum haptoglobin (A and C) and serotransferrin (B and D) of white sturgeon (Acipenser transmontanus) after 21 days of control or β-glucan (0.1% or 0.3% yeast, Saccharomyces cerevisiae) feeding (A and B), or 42 days (6 weeks) post-challenge with Veronaea botryosa or PBS (unexposed) of white sturgeon fed diets containing β-glucan (0.1% or 0.3% yeast) or control diet for 21d (C and D).Data represents mean ± SE (n = 8).Treatments with different letters are significantly different amongst each other, one-way ANOVA and Tukey's test for pairwise comparison p ≤ 0.05.serotransferrin again, was elevated in infected animals, regardless of diet, and much higher than reported previously (Soto et al., 2019).While, serotransferrin protein in the serum matched transcript expression of serotransferrin and other APP in the spleen (SAA and haptoglobin), this was not the case for haptoglobin.Haptoglobin was depressed in all exposed animals at 42 dpi.Haptoglobin levels in preand unchallenged controls were again similar to those previously reported, but significantly dropped following exposure.Although blood samples collected throughout the study were processed identically, for unclear reasons all serum from exposed fish exhibited a high degree of hemolysis.This observation in conjunction with low haptoglobin levels is compatible with intravascular hemolysis (Phillips and Henderson, 2018).Elevated transferrin levels are suggestive of concurrent iron deficiency, but in the absence of additional confirmatory testing remains speculative (Peng and Uprichard, 2017).

Conclusion
Our results suggest feed supplementation with β-glucans could be used to help prevent losses due to V. botryosa in cultured white sturgeon.This protective response is at least partially associated with a higher expression of IL17 and IRF8 in β-glucan immunestimulated fish.In addition to conferring protection against fungal pathogens through dietary supplementation, other studies provide evidence of a potential role for β-glucan particles as an adjuvant and oral antigen delivery system by inducing a Th17-biased response with strong mucosal antibody production (De Smet et al. 2013).Future research investigating appropriate β-glucan feeding protocols through the production cycle of the fish and the efficacy of β-glucans as an adjuvant in sturgeon vaccinology is warranted for use in the management of this important pathogen and to mitigate its impacts on the caviar industry.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Genes expression profiles in the spleen of white sturgeon (Acipenser transmontanum) after 21 days of control or β-glucan (0.1% or 0.3% yeast, Saccharomyces cerevisiae) feeding.Expression levels are shown as LOG-fold change for β-glucan fed fish compared to the control fed fish for each gene.Data represents mean ± SD (n = 10).*, one-way ANOVA and Tukey's test for pairwise comparison p ≤ 0.05, when comparing the data to the control group.

Fig. 3 .
Fig. 3. A. Infectivity in white sturgeon (Acipenser transmontanus) presented in LOG Veronaea botryosa gene equivalents per 1 μg of spleen of fish fed diets containing β-glucan (0.1% or 0.3% yeast, Saccharomyces cerevisiae) or control diet for 21d and challenged with V. botryosa.The error bars represent standard error of ten fish per treatment.Treatments with different letters are significantly different from one another at p < 0.05.B. Mean total length 42 days (6 weeks) post-challenge with Veronaea botryosa or PBS (unexposed) of white sturgeon fed diets containing β-glucan (0.1% or 0.3% yeast, Saccharomyces cerevisiae) or control diet for 21d.*, oneway ANOVA and Tukey's test for pairwise comparison p ≤ 0.05, when comparing group.

Fig. 4 .
Fig. 4. Genes expression profiles in the spleen of white sturgeon (Acipenser transmontanus) 42 days (6 weeks) post-challenge with Veronaea botryosa or PBS (unexposed) of white sturgeon fed diets containing β-glucan (0.1% or 0.3% yeast, Saccharomyces cerevisiae) or control diet for 21d.Expression levels are shown as LOGfold change for β-glucan fed fish compared to the control fed fish for each gene.Data represents mean ± SD (n = 10).Treatments with different letters are significantly different amongst each other, two-way ANOVA and Tukey's test for pairwise comparison p ≤ 0.05.
Primers used in this study for gene expression analysis.