Oral Immunization of Larvae and Juvenile of Lumpfish (Cyclopterus lumpus) against Vibrio anguillarum Does Not Influence Systemic Immunity

Vibrio anguillarum, a marine bacterial pathogen that causes vibriosis, is a recurrent pathogen of lumpfish (Cyclopterus lumpus). Lumpfish is utilized as a cleaner fish in the Atlantic salmon (Salmo salar) aquaculture in the North Atlantic region because of its ability to visualize and prey on the ectoparasite sea lice (Lepeophtheirus salmonis) on the skin of Atlantic salmon, and its performance in cold environments. Lumpfish immunity is critical for optimal performance and sea lice removal. Oral vaccine delivery at a young age is the desired method for fish immunization because is easy to use, reduces fish stress during immunization, and can be applied on a large scale while the fish are at a young age. However, the efficacy of orally delivered inactivated vaccines is controversial. In this study, we evaluated the effectiveness of a V. anguillarum bacterin orally delivered to cultured lumpfish and contrasted it to an intraperitoneal (i.p.) boost delivery. We bio-encapsulated V. anguillarum bacterin in Artemia salina live-feed and orally immunized lumpfish larvae. Vaccine intake and immune response were evaluated by microscopy and quantitative polymerase chain reaction (qPCR) analysis, respectively. qPCR analyses showed that the oral immunization of lumpfish larvae resulted in a subtle stimulation of canonical immune transcripts such as il8b, il10, igha, ighmc, ighb, ccl19, ccl20, cd8a, cd74, ifng, and lgp2. Nine months after oral immunization, one group was orally boosted, and a second group was both orally and i.p. boosted. Two months after boost immunization, lumpfish were challenged with V. anguillarum (7.8 × 105 CFU dose−1). Orally boosted fish showed a relative percentage of survival (RPS) of 2%. In contrast, the oral and i.p. boosted group showed a RPS of 75.5% (p < 0.0001). V. anguillarum bacterin that had been orally delivered was not effective in lumpfish, which is in contrast to the i.p. delivered bacterin that protected the lumpfish against vibriosis. This suggests that orally administered V. anguillarum bacterin did not reach the deep lymphoid tissues, either in the larvae or juvenile fish, therefore oral immunization was not effective. Oral vaccines that are capable of crossing the epithelium and reach deep lymphoid tissues are required to confer an effective protection to lumpfish against V. anguillarum


Introduction
Ectoparasitic sea lice infestations are a serious health issue for wild and cultured Atlantic salmon (Salmo salar) [1]. The sea louse (e.g., Lepeophtheirus salmonis) feeds on salmonid mucus and tissues leading to physiological and immune failures and may lead to death if left untreated [1,2]. Over the last few decades, the Atlantic salmon industry has used chemical and physical methods to control sea lice infestations [3,4]. The use of chemotherapeutants generates the emergence of resistance in sea lice [3] and enhances negative public opinion on the impacts of aquaculture activity on the environment. Routine mechanical treatments (e.g., abrasion and thermal treatment) used in the salmon industry to physically remove the lice have adverse effects on salmon health and welfare [4]. Currently, multiple non-medicinal methods are used in different combinations (e.g., curtains and skirts, anti-lice diets, laser, water jets, and ultrasound) as part of an integrated pest management (IPM) strategy [5]. Another current strategy to control sea lice in the North Atlantic farms is the use of cleaner fish species as a biological control [4,6]. The use of cleaner fish is considered an eco-friendly alternative, as it can reduce the use of chemotherapeutants, and it is less stressful to farmed fish [7]. In Atlantic Canada, both lumpfish (Cyclopterus lumpus) and cunner (Tautogolabrus adspersus) are used as cleaner fish [8,9]. However, lumpfish is the preferred species for the biocontrol of sea lice due to its performance in cold environments and biomass availability [10,11]. In contrast to other cleaner fish species, lumpfish actively remove sea lice from farmed salmon in cold environments, and they have been industrialized in the North Atlantic region [12][13][14][15]. Use and demand for lumpfish in salmon farms in Ireland, the United Kingdom (UK), Norway, the Faroe Islands, Iceland, and Canada have increased in recent years [13,16]. For example, 11.8 and 0.8 million juveniles were used in Norway and in the UK in 2015, respectively [17]. In 2018, approximately 30 million juvenile lumpfish were used in Norway [18]; in the UK, approximately 6 million lumpfish were used; in Iceland, approximately 3.5 million were used [19]; and approximately 300 thousand lumpfish were used in Ireland [20]. In Canada, the use of lumpfish is a very recent practice, and approximately 1 million lumpfish were used in Atlantic Canada in 2019 [19].
Similar to other finfish species, lumpfish is susceptible to different types of bacterial pathogens such as Aeromonas salmonicida, Pasteurella spp., Tenacibaculum spp., Pseudomonas anguilliseptica, and Moritella viscosa [21,22]. Vibriosis, typically caused by Vibrio anguillarum, is a prevalent pathogen in lumpfish in Newfoundland [23], but others such as V. ordalii, V. salmonicida, and V. splendidus also infect lumpfish elsewhere [12,24]. V. anguillarum is a Gram-negative, motile, rod-shaped bacterium and a ubiquitous marine pathogen of invertebrates and vertebrates [25,26]. Formalin-killed V. anguillarum (bacterins) serotypes O1 and O2 mixed with adjuvants are often used to formulate vaccines for different fish species, including Atlantic salmon, Atlantic cod (Gadus morhua) [27], gilthead seabream (Sparus aurata), and lumpfish [28]. Intraperitoneal (i.p.) injection is the most common method of vaccine delivery [29], but it cannot be applied to small fish. An oral-immersion vaccine is ideal for fish immunization, as it is needle-free and causes minimum stress during application [30]. However, oral vaccine delivery to small fish or larvae has been evaluated with controversial results [31]. Vaccine bio-encapsulation strategies, such as the used of Artemia salina fed with bacterin, can increase the delivery of the vaccine to small lumpfish larvae by increasing intake and can directly deliver the antigen to the fish gut [32][33][34].
Here, we evaluated the efficacy of a V. anguillarum bacterin bio-encapsulated in A. salina delivered as live-feed to lumpfish larvae. We determined that the oral V. anguillarum vaccine delivered in A. salina live-feed reached the lumpfish gut. Real-time quantitative polymerase chain reaction (qPCR) analyses of immune-relevant genes revealed that oral immunization modestly immune stimulated the lumpfish larvae. Nine months later, a lumpfish group was orally boosted, and an independent second group was orally and i.p. boosted. Two months later, the immunized fish groups were i.p. challenged with ten times the lethal dose 50 (LD 50 ) of V. anguillarum (7.8 × 10 5 colony forming units (CFU) dose −1 ). Oral immunization of lumpfish delayed mortality but did not confer protective immunity against the V. anguillarum challenge, which contrasts with the i.p. vaccination, which protected the fish against the lethal infection.

Bacterin Preparation
The bacterin preparation was conducted according to established protocols and quantified using flow cytometry enumeration [37] with modifications. First, V. anguillarum J360 was grown in 500 mL of TSB supplemented with a final concentration of 150 µM 2,2-dipyridyl (Sigma-Aldrich, St. Louis, MO, USA) at 28 • C with aeration (180 rpm, in an orbital shaker) to induce synthesis of the iron-regulated outer membrane proteins (IROMPs) [38]. Bacterial growth was monitored spectrophotometrically until it reached 1 × 10 8 CFU −1 . V. anguillarum cells were harvested using centrifugation (4200× g at 4 • C for 10 min) and washed twice with PBS. V. anguillarum cells were fixed with buffered formalin 6% (Sigma) at room temperature for 3 d with gentle agitation in a rocker shaker. Cell viability was determined each day by plating onto TSA. Formalin was removed using centrifugation, and cells were then dialyzed (Spectrum™ Spectra/Por™ dialysis membrane 12-14,000 Dalton molecular weight cut-off, Thermo) in 2 L of PBS at 6 • C for 3 d with agitation. V. anguillarum bacterins were quantified using the BD FACS Aria II flow cytometer (BD Biosciences, San Jose, CA, USA) and BD FACS Diva v7.0 software as described previously [39]. Bacterin cells (1 × 10 10 CFU mL −1 ) were stored at 4 • C until use.
Next, we describe the optimization of V. anguillarum bacterin bio-encapsulation in A. salina. To optimize bacterin bio-encapsulation in A. salina, we used the method described by Campbell et al. [32] with modifications. Additionally, we developed a semi-quantitative method to estimate the levels of bacterin bio-encapsulation in A. salina. First, A. salina nauplii were hatched from cysts according to the supplier's instructions (INVE, Salt Lake City, UT, USA) ( Figure S1). After the A. salina nauplii hatched (~20 h at 20 • C), nauplii were washed with seawater for 30 min ( Figure S1). A. salina cultures were nutritionally supplemented with Ori-One (Skretting, Fontaine les Vervins, France) and Ori-Green (Skretting, Fontaine les Vervins, France) commercial dry microalgae extract at a ratio of Vaccines 2021, 9, 819 4 of 21 1:1 (Ori-One:Ori-green) for 3 h at 20 • C. To determine the optimal time for bacterin bioencapsulation in A. salnina, supplemented A. salina nauplii were inoculated into 6 well plates with a 3 mL total volume per well at a density of 1000 nauplii mL −1 . Additionally, a separate plate was inoculated with non-supplemented A. salina nauplii to determine the possible effect of nutrient supplementation on bio-encapsulation. Both groups were inoculated with DTAF-labeled V. anguillarum bacterin (5 × 10 7 cells mL −1 ). The control group was mock inoculated with seawater and used to evaluate autofluorescence. The nauplii were incubated at 20 • C for 48 h to determine the optimal time for bacterin bioencapsulation ( Figure S1A). A. salina samples (1 mL) were collected at 1, 3, 5, 24, 36, and 48 h, and fixed in 10% buffered formalin. The presence of V. anguillarum inactivated bacterin was examined and counted using confocal microscopy (Nikon Eclipse Ti, Melville, NY, USA) to determine the number of A. salina containing 0%, 25%, 50%, 75%, 100% bacterin ( Figure S1A). After determining the optimal time for bacterin bio-encapsulation in A. salina nauplii, we evaluated the bio-encapsulation stability at 6 • C for 6 d ( Figure S1B). A. salina nauplii were supplemented with Ori-One, Ori-Green, and DTAF-labeled V. anguillarum bacterin at 20 • C for 3 h and then placed at 6 • C for 6 d. The A. salina control group was mock inoculated with seawater. A. salina samples were collected each day and fixed with buffered 10% formalin. The levels of bio-encapsulation in A. salina were determined using confocal microscopy ( Figure S1B). DTAF-labeled V. anguillarum inactivated bacterin bio-encapsulated in A. salina nauplii were used to feed the lumpfish larvae ( Figure S2) and to determine the presence of V. anguillarum bacterin in the larvae gut compared to the non-orally immunized fish ( Figure S3). Fifty lumpfish larvae were orally immunized and maintained at 6 • C for 24 h. The larvae gut was observed at 0, 0.5, 1, 2, 4, 6, and 24 h post-oral immunization using epi-fluorescence microscopy (Optika, Italy) ( Figure S3).

V. anguillarum Bacterin Bio-Encapsulation in A. salina
Based on the vaccine bio-encapsulation optimization results, larger volumes of A. salina were prepared for lumpfish larvae immunization. A. salina nauplii were hatched and washed with seawater for 30 min and placed in 20 L buckets containing 15 L of seawater. A. salina nauplii were maintained at a density of~2.5 million A. salina per liter. A. salina cultures were enriched with nutritional supplements derived from microalgae, Ori-One (Skretting, France) and Ori-Green (Skretting, France) according to the manufacturer's instructions. A. salina cultures were inoculated with V. anguillarum bacterin (10 7 cells mL −1 ) and incubated at 20 • C for 3 h with aeration. Controls were mock inoculated with PBS. After 3 h of enrichment and bacterin bio-encapsulation, the A. salina cultures were maintained at 6 • C for 6 d under constant light. These cultures were used for the daily oral immunization of the lumpfish larvae ( Figure S4).

Fish Culture Conditions
All animal protocols required for this research were approved by the Institutional Animal Care Committee and the Biosafety Committee at Memorial University of Newfoundland (MUN). Experiments were conducted under protocols #18-01-JS, #18-03-JS, and biohazard license L-01. Lumpfish egg masses were in vitro fertilized and maintained in 5 L buckets containing UV treated (300 mW cm −2 ), filtered, flow-through seawater (33 ppt) at 10-10.5 • C, with 95-110% air saturation, and held under ambient photoperiod (springsummer) at the Joe Brown Aquatic Research Building (JBARB), Department of Ocean Sciences, Memorial University of Newfoundland, Canada. After embryo development was complete, the larvae were hatched and maintained in the same seawater conditions and air saturation at 10 • C until full egg yolk sac absorption and the establishment of independent feeding was achieved ( Figure S2).

Lumpfish Immunization Assays
Lumpfish larvae were stocked in 4 tanks at a density of 2000 larvae per tank (20 L) with UV treated (300 mW cm −2 ), filtered, flow-through seawater at 8-10 • C, and with 95-110% air saturation ( Figure 1). Two tanks containing lumpfish larvae (1-week post-hatch (wph)) were orally immunized with the bio-encapsulated V. anguillarum vaccine daily for 4 weeks. Two tanks fed containing only A. salina served as controls ( Figure 1). Lumpfish larvae were fed with A. salina nauplii or bio-encapsulated vaccine 3 times per day (350 mL of A. salina culture per 2000 larvae) ( Figure S4). Thereafter, the fish were fed A. salina for additional 10 d and then fed with dry pelleted diet daily (0.75-2% body weight). Whole larvae pool samples were collected at 0, 2, and 4 weeks post-immunization (wpi) (Figure 1). At each time point, triplicate pools of 5-10 larvae were sampled from each tank and placed in a 1.5 mL RNase-free tube containing 300 µL of TRIzol Reagent (Invitrogen, Waltham, MA, USA), flash-frozen in liquid nitrogen, and stored at −80 • C until processing. After 8 weeks, the juvenile lumpfish were transferred into eight 500 L tanks ( Figure 1). The lumpfish were fed an assorted size pelleted diet for the remainder of the experiment (15 months). Nine months post-hatch, two tanks with 100 fish each (~72.1 ± 30 g) were orally boost immunized using commercial pellets coated with the V. anguillarum bacterin. Lumpfish were starved for 24 h pre-oral vaccination. Lumpfish were fed with V. anguillarum bacterin coated dry pellets three times (every 2 weeks for 3 days) at 0.75% body weight ( Figure 1). Two control tanks were mock-orally boosted with ficoll (vaccine vehicle) coated pellets ( Figure 1). Two independent groups orally boosted were additionally i.p. boosted at 40 wph (~145 ± 31.8 g) with V. anguillarum bacterin (6.3 × 10 8 cells dose −1 ). Control groups were mock-orally and i.p. boosted with the respective vaccine vehicle ( Figure 1). Two months later, the animals (~132-244 g) were transferred to the AQ3 aquatic biocontainment facility at the Cold-Ocean Deep-Sea Research Facility (CDRF) for challenge assays.

V. anguillarum J360 Challenge Assays in Lumpfish
The challenge assays were conducted at the CDRF AQ3 biocontainment facility under the Institutional Animal Care Committee approved protocol (18-02-JS) and established protocols [28]. First, after transfer to the AQ3 biocontainment facility, lumpfish were acclimated for 1 week under optimal conditions prior to the commencement of the challenge. Lumpfish were challenged by an i.p. injection with 7 times the lethal dose 50 (7.8 × 10 5 CFU dose −1 ) of V. anguillarum J360. Fish survival was monitored for 30 days post-challenge. The relative percent of survival (RPS) of the control and vaccinated fish was calculated using the formula: RPS = [1 − (% mortality in vaccinated fish/% mortality in control fish)] × 100 [47].

V. anguillarum J360 Challenge Assays in Lumpfish
The challenge assays were conducted at the CDRF AQ3 biocontainment facility under the Institutional Animal Care Committee approved protocol (18-02-JS) and established protocols [28]. First, after transfer to the AQ3 biocontainment facility, lumpfish were acclimated for 1 week under optimal conditions prior to the commencement of the challenge. Lumpfish were challenged by an i.p. injection with 7 times the lethal dose 50 (7.8 × 10 5 CFU dose −1 ) of V. anguillarum J360. Fish survival was monitored for 30 days postchallenge. The relative percent of survival (RPS) of the control and vaccinated fish was calculated using the formula: RPS = [1 − (% mortality in vaccinated fish/% mortality in control fish)] × 100 [47].

Total RNA Extraction.
RNA was extracted from the larvae pools pre-immunized (n = 3 individual pools of 10 larvae each), 2 wpi (n = 3 pools of 10 larvae each), and 4 wpi (n = 3 pools of 5 larvae each). RNA was also extracted from the larvae post mock immunization(control) at 2 and 4 wpi. Lumpfish larvae pools, previously flash frozen in a 1.5 mL RNase-free tube containing 300 μL of TRIzol reagent (Invitrogen), were homogenized using a micro-tube homogenizer (ThermoFisher Scientific, Waltham, MA, USA). An additional 700 μL of TRIzol was added to the tube, and the extractions were then completed following the manufacturer's instructions. The TRIzol extracted samples were then purified using the RNeasy ® Mini Kit (QIAGEN, Mississauga, ON, Canada) following the manufacturer's instructions. RNA samples were treated with 2 U mL −1 of TURBO DNase (TURBO DNA-free™ Kit, Invitrogen) following the manufacturer's instructions for the complete digestion of genomic DNA and the removal of the remaining DNase and divalent cations, such as magnesium and calcium. Purified RNA samples were quantified and evaluated

Immunization
Oral immunization with bioencapsulated V.anguillarum bacterin Figure 1. Immunization and challenge experimental design. Lumpfish larvae were immunized at 1 wph. At 8 wph, the fish were distributed in 500 L tanks. Lumpfish were boosted at 36 wph and challenged with V. anguillarum at 45 wph. * Larvae samples were collected at weeks 0, 2, 4 post-immunization for RNA extraction.

Total RNA Extraction
RNA was extracted from the larvae pools pre-immunized (n = 3 individual pools of 10 larvae each), 2 wpi (n = 3 pools of 10 larvae each), and 4 wpi (n = 3 pools of 5 larvae each). RNA was also extracted from the larvae post mock immunization(control) at 2 and 4 wpi. Lumpfish larvae pools, previously flash frozen in a 1.5 mL RNase-free tube containing 300 µL of TRIzol reagent (Invitrogen), were homogenized using a micro-tube homogenizer (ThermoFisher Scientific, Waltham, MA, USA). An additional 700 µL of TRIzol was added to the tube, and the extractions were then completed following the manufacturer's instructions. The TRIzol extracted samples were then purified using the RNeasy ® Mini Kit (QIAGEN, Mississauga, ON, Canada) following the manufacturer's instructions. RNA samples were treated with 2 U mL −1 of TURBO DNase (TURBO DNA-free™ Kit, Invitrogen) following the manufacturer's instructions for the complete digestion of genomic DNA and the removal of the remaining DNase and divalent cations, such as magnesium and calcium. Purified RNA samples were quantified and evaluated for purity using a Genova Nano spectrophotometer (Jenway, Staffordshire, UK) and evaluated for integrity using 1% agarose gel electrophoresis [35]. A PCR test was conducted using the 60S ribosomal protein L32 (rpl32) reference gene primers and the RNA as a template to rule out the presence of DNA. indicated cDNA quantity. Amplifications were performed using the QuantStudio 6 Flex Real-Time PCR system (384-well format) (Applied Biosystems, Waltham, MA, USA). The real-time analysis program consisted of 1 cycle of 50 • C for 2 min, 1 cycle of 95 • C for 10 min, 40 cycles of 95 • C for 15 s, and 60 • C for 1 min, with fluorescence detection at the end of each 60 • C step and followed by dissociation curve analysis.

qPCR Primer Quality Assurance Testing
All primer pairs for the transcripts of interest (TOIs) and the endogenous control transcripts were designed and quality control (QC) tested using the larvae RNA samples generated herein. cDNAs were synthesized from the individual pooled larvae RNA samples, including pre-immunized control, mock-immunized control (2 and 4 wpi), and immunized larvae (2 and 4 wpi). The control and immunized cDNA samples were independently pooled and used for primer quality evaluation. To calculate amplification efficiencies for each primer pair [48], standard curves were generated for both cDNA pools (control and immunized) using a 5-point 1:3 dilution series starting with cDNA representing 10 ng of input total RNA. The reported efficiencies represent an average of the two values (Supplementary Table S1). Each primer pair was also tested to ensure that a single product was amplified and that there was no primer dimer present in the no-template control. Finally, amplicons were electrophoretically separated on 2% agarose gels and compared using a 1 kb plus ladder (Invitrogen) to verify that the correct size fragment was amplified.
Eighteen TOIs were well expressed in larvae, and as such, amplification efficiencies could be calculated using larvae cDNA template (see fluorescence threshold cycle (C T ) values for studies 1 to 3; Supplementary Table S2). However, fourteen of these transcripts were expressed at low levels in larvae (see C T values for studies 4 to 6; Supplementary  Table S2). For the latter TOIs, technical replicates and spacing were acceptable over the first three points of the cDNA dilution series. As the experimental input cDNA amount (8 ng) for these TOIs lies within the first 2 dilutions, these assays were deemed acceptable for analysis in larvae. However, for these fourteen transcripts, the amplification efficiencies reported in Supplementary Table S1 and inputted into the QuantStudio Real-Time PCR Software (version 1.3) (Applied Biosystems) were those that had been previously generated for head kidney samples.

Endogenous Control (Normalizer) Selection
Expression levels of the TOIs were normalized to the transcript levels of the two endogenous controls. To select these endogenous controls, 5 transcripts (rpl32, elongation factor 1-alpha (ef1a), eukaryotic translation initiation factor 3 subunit D (etif3d), polyadenylatebinding protein 1a (pabpc1a), and polyadenylate-binding protein 1b (pabpc1b)) were analyzed. First, the C T values of all 27 samples in the study were measured (in duplicate) for each of these transcripts using cDNA representing 3.25 ng of input total RNA and then analyzed using geNorm [49]. Based on this analysis, rpl32 (geNorm M = 0.169) and etif3d (geNorm M = 0.177) were selected as the two endogenous controls.

Experimental qPCR Analyses
To study the effects of oral immunization with the V. anguillarum bacterin bio-encapsulated in A. salina on the immunome of lumpfish larvae, expression levels of 32 TOIs with immunerelevant functional annotations were assessed (Supplementary Table S1). Individual larvae pools (n = 27 pools) were subjected to qPCR analyses ( Figure 1). In the qPCR analyses, cDNA representing 3.25 ng (study 1 to 3, see Supplementary Table S2) and 8 ng (study 3 to 6, see Supplementary Table S2) of input RNA was used as a template in the PCR reactions. The input RNA concentration was increased to 8 ng due to lower expression transcript levels in the larvae. In each qPCR study, expression levels of a given transcript were measured across two plates. On each plate, the TOIs and endogenous controls were tested in triplicate, and a notemplate control was included for every sample. A plate linker sample was also included to ensure that there was no plate-to-plate variability. The relative quantity (RQ) of each transcript was determined using the QuantStudio Real-Time PCR Software (version 1.3), with normalization to both the rpl32 and etif3d transcript levels and with the amplification and efficiencies incorporated. For each TOIs, the sample with the lowest normalized expression (mRNA) level was set as the calibrator sample (i.e., assigned an RQ value = 1) (Supplementary Table S2). Additionally, the transcript expression levels were determined using the comparative 2 −∆∆Ct method [50] with two reference genes [51] (Supplementary  Table S3).

Statistical Analysis
All data are expressed as the mean ± standard error (SE). Assumptions of normality and homogeneity were tested for variances. A one-way ANOVA followed by Tukey's multiple comparison post hoc test was used to determine significant differences between the survival of the control and infected groups. The Kaplan-Meier estimator was applied for the estimation of the survival fractions after the V. anguillarum challenges, and the log-rank test was used to identify differences between treatment groups (p < 0.0001). A twoway ANOVA was used to analyze the gene expression data followed by Sidak's multiple comparisons post hoc test to identify significant differences between each treatment in the control and immunized groups at each time point (2 weeks and 4 weeks). All statistical tests were performed using Graphpad Prism version 8.0 (Graphpad Software, USA, www.GraphPad.com (accessed on 15 June 2021)), and p-values < 0.05 were considered statistically significant.

Bio-Encapsulation of V. anguillarum Bacterin in A. salina Nauplii
We established a semi-quantitative method to estimate the levels of V. anguillarum bacterin bio-encapsulation in A. salina (Figure 2A). Autofluorescence was ruled out using A. salina nauplii inoculated with nutritional supplements (Ori-One and Ori-Green) ( Figure 2B). Using this method, we determined that approximately 100% of the A. salina nauplii reached the maximum capacity for V. anguillarum bacterin bio-encapsulation 3 h post-inoculation at 20 • C in both the absence ( Figure 2C) or presence ( Figure 2D) of nutritional supplements. In the absence of supplements, a significant decrease in the percentage of the A. salina nauplii with 100% bio-encapsulation levels was observed 24 h post-inoculation, which gradually declined thereafter ( Figure 2C). In the presence of nutritional supplements, a significant decrease occurred 36 h post-inoculation ( Figure 2D). We determined that the optimal V. anguillarum bacterin bio-encapsulation in A. salina method is the presence of nutritional supplementation with a bio-encapsulation time of 3 h at 20 • C. As we wanted to produce a bio-encapsulated vaccine batch that could be used for several days, we evaluated the stability of the V. anguillarum bacterin in the intestine of A. salina nauplii post bioencapsulation at 6 • C ( Figure S1B). This temperature was chosen as it is similar to the water temperature at which lumpfish are cultured. We determined that the bacterin concentration in A. salina nauplii remained stable for up to 6 d post-inoculation ( Figure 2E). DTAF-labeled V. anguillarum bacterin bio-encapsulated in A. salina nauplii were then fed to lumpfish larvae and fluorescence microscopy was used to determine if the V. anguillarum bacterin reached the larvae gut ( Figure S3). Fluorescence microscopy demonstrated the presence of the V. anguillarum bacterin in the gut of the lumpfish larvae after 6 and 24 h, whereas fluorescence was not detected in the gut of lumpfish larvae who had been fed A. salina nauplii only (Figure 3). were then fed to lumpfish larvae and fluorescence microscopy was used to determine the V. anguillarum bacterin reached the larvae gut ( Figure S3). Fluorescence microscop demonstrated the presence of the V. anguillarum bacterin in the gut of the lumpfish larva after 6 and 24 h, whereas fluorescence was not detected in the gut of lumpfish larvae wh had been fed A. salina nauplii only (Figure 3).

Transcript Expression Profile of the Immunome of Orally Immunized Lump
Expression levels of the transcripts related to the innate and adap sponse were measured in pre-immunized larvae and at 2 and 4 wpi with rum bacterin bio-encapsulated in A. salina nauplii. The transcript expres analyzed using both the 2 −ΔΔCt (Figures 4 and 5) and the RQ methods Figures S5 and S6). Both methods showed similar results, with the except tical significance demonstrated for toll-like receptor 7 (tlr7) and immun chain b (ighb) (Figures 4-6; Supplementary Figures S5-S7). Transcript e were compared statistically in orally immunized compared to control l wpi only. Comparisons over time could not be assessed due to develop siderable size differences of the larvae over time. Significant up-regulatio 8b (il8b) ( Figure 4A and Figure S5A), immunoglobulin heavy chain a (igh munoglobulin mu heavy chain c (ighmc) ( Figure 5A and Figure S6A), ch and ccl20) ( Figure 5B and Figure S6B), cluster of differentiation 8 alpha class II histocompatibility antigen gamma chain (cd74) ( Figure 5C and Fi feron-gamma (ifng) (Figure 6 and Figure S7), and ATP-dependent RN (lgp2) (Figure 6 and Figure S7) occurred at 2 wpi. Significant up-regulatio 10 (il10) occurred at 4 wpi ( Figure 4A and Figure S5A). Significant do lymphocyte antigen 6 family member G6F (ly6g6f) (Figure 6 and Figu ( Figure 6B and Figure S6B) occurred at 2 and 4 wpi, respectively. There we differences in the expression levels of the remaining transcripts at either

Transcript Expression Profile of the Immunome of Orally Immunized Lumpfish Larvae
Expression levels of the transcripts related to the innate and adaptive immune response were measured in pre-immunized larvae and at 2 and 4 wpi with the V. anguillarum bacterin bio-encapsulated in A. salina nauplii. The transcript expression levels were analyzed using both the 2 −∆∆Ct (Figures 4 and 5) and the RQ methods ( Supplementary Figures S5 and S6). Both methods showed similar results, with the exception of the statistical significance demonstrated for toll-like receptor 7 (tlr7) and immunoglobulin heavy chain b (ighb) (Figures 4-6; Supplementary Figures S5-S7). Transcript expression levels were compared statistically in orally immunized compared to control larvae at 2 and 4 wpi only. Comparisons over time could not be assessed due to developmental and considerable size differences of the larvae over time. Significant up-regulation of interleukin 8b (il8b) ( Figure 4A and Figure S5A), immunoglobulin heavy chain a (igha), ighb and immunoglobulin mu heavy chain c (ighmc) ( Figure 5A and Figure S6A), chemokines (ccl19 and ccl20) ( Figure 5B and Figure S6B), cluster of differentiation 8 alpha (cd8a) and HLA class II histocompatibility antigen gamma chain (cd74) ( Figure 5C and Figure S6C), interferon-gamma (ifng) (Figure 6 and Figure S7), and ATPdependent RNA helicase lgp2 (lgp2) (Figure 6 and Figure S7) occurred at 2 wpi. Significant upregulation of interleukin 10 (il10) occurred at 4 wpi ( Figure 4A and Figure S5A). Significant downregulation of lymphocyte antigen 6 family member G6F (ly6g6f ) (Figure 6 and Figure S7) and ccl20 ( Figure 6B and Figure S6B) occurred at 2 and 4 wpi, respectively. There were no significant differences in the expression levels of the remaining transcripts at either time point. Toll-like receptors. Transcript expression levels were assessed pre-immunization (T0 control, n = 3 individual pools of 10 larvae each), 2 wpi (n = 3 pools of 10 larvae each), and 4 wpi (n = 3 pools of 5 larvae each). Time point controls post-mock immunization were collected in a similar fashion at 2 and 4 wpi. Relative expression was calculated using the 2 (−∆∆Ct) method and normalized using log2; etif3d and rpl32 were used as endogenous controls. A two-way ANOVA test, followed by the Sidak multiple comparisons post hoc test was used to assess significant differences between the treatments (control and vaccinated) at each individual time point. Asterisks (*) represent significant differences (* p < 0.05, ** p < 0.01). Toll-like receptors. Transcript expression levels were assessed pre-immunization (T0 control, n = 3 individual pools of 10 larvae each), 2 wpi (n = 3 pools of 10 larvae each), and 4 wpi (n = 3 pools of 5 larvae each). Time point controls post-mock immunization were collected in a similar fashion at 2 and 4 wpi. Relative expression was calculated using the 2 (−∆∆Ct) method and normalized using log 2 ; etif3d and rpl32 were used as endogenous controls. A two-way ANOVA test, followed by the Sidak multiple comparisons post hoc test was used to assess significant differences between the treatments (control and vaccinated) at each individual time point. Asterisks (*) represent significant differences (* p < 0.05, ** p < 0.01).
Vaccines 2021, 9, x FOR PEER REVIEW 12 of 21 Figure 5. Transcript expression levels of immunoglobulin heavy locus genes, cytokine CC genes and interferon-induced GTP-binding proteins genes, and cluster of differentiation genes in lumpfish larvae orally immunized with the V. anguillarum bacterin bio-encapsulated in A. salina nauplii (A-E). Immunoglobulin heavy locus genes (F-J). Cytokine CC genes and interferon-induced GTP-binding proteins genes (K-N). Cluster of differentiation genes. Transcript expression levels were assessed pre-immunization (T0 control, n = 3 individual pools of 10 larvae each), 2 weeks post-immunization (n = 3 pools of 10 larvae each), and 4 weeks post-immunization (n = 3 pools of 5 larvae each). Time point controls post-mock immunization were collected in a similar fashion to those collected at weeks 2 and 4. Relative expression was calculated using the 2 (−∆∆Ct) method and normalized using log2; etif3d and rpl32 were used as endogenous controls. A two-way ANOVA test followed by the Sidak multiple comparisons post hoc test were used to assess significant differences between the treatments (control and vaccinated) at each individual time point. Asterisks (*) represent significant differences (** p < 0.01, *** p < 0.001). . Transcript expression levels of immunoglobulin heavy locus genes, cytokine CC genes and interferon-induced GTP-binding proteins genes, and cluster of differentiation genes in lumpfish larvae orally immunized with the V. anguillarum bacterin bio-encapsulated in A. salina nauplii (A-E). Immunoglobulin heavy locus genes (F-J). Cytokine CC genes and interferon-induced GTP-binding proteins genes (K-N). Cluster of differentiation genes. Transcript expression levels were assessed pre-immunization (T0 control, n = 3 individual pools of 10 larvae each), 2 weeks post-immunization (n = 3 pools of 10 larvae each), and 4 weeks post-immunization (n = 3 pools of 5 larvae each). Time point controls post-mock immunization were collected in a similar fashion to those collected at weeks 2 and 4. Relative expression was calculated using the 2 (−∆∆Ct) method and normalized using log 2 ; etif3d and rpl32 were used as endogenous controls. A two-way ANOVA test followed by the Sidak multiple comparisons post hoc test were used to assess significant differences between the treatments (control and vaccinated) at each individual time point. Asterisks (*) represent significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001). Transcript expression levels were assessed pre-immunization (T0 control, n = 3 individual pools of 10 larvae each), 2 weeks post-immunization (n = 3 pools of 10 larvae each), and 4 weeks post-immunization (n = 3 pools of 5 larvae each). Time point controls post-mock immunization were collected in a similar fashion to those collected at weeks 2 and 4. Relative expression was calculated using the 2 (−∆∆Ct) method and normalized using log2; etif3d and rpl32 were used as endogenous controls. A two-way ANOVA test followed by the Sidak multiple comparisons post hoc test were used to assess significant differences between the treatments (control and vaccinated) at each individual time point. Asterisks (*) represent significant differences (** p< 0.01, *** p< 0.001).

Vaccine Challenge
Immunized lumpfish were challenged at 45 wpi with 7 times the LD50 dose for V. anguillarum J360 (7.8 × 10 5 CFU dose −1 ) (Figure 1). Mortality in lumpfish who had been orally mock immunized as larvae and then mock-orally boosted as juveniles started at 3 days post-challenge, with 100% mortality by day 10 post-challenge. Mortality in lumpfish that had been orally immunized as larvae and then orally boosted as juveniles was delayed and started at 3 days post-challenge, with a final RPS of 2% ( Figure 7A). Mortality in lumpfish that had been mock orally immunized as larvae and then both mock orally and i.p. boosted as juveniles started at 7 days post-challenge, with 100% mortality by 20 days post-challenge ( Figure 7B). Lumpfish that had been orally immunized as larvae and then both orally and i.p. boosted as juveniles survived the i.p. challenge with V. anguillarum, with a RPS of 76.5% (p < 0.0001) ( Figure 7B). Transcript expression levels were assessed pre-immunization (T0 control, n = 3 individual pools of 10 larvae each), 2 weeks post-immunization (n = 3 pools of 10 larvae each), and 4 weeks post-immunization (n = 3 pools of 5 larvae each). Time point controls post-mock immunization were collected in a similar fashion to those collected at weeks 2 and 4. Relative expression was calculated using the 2 (−∆∆Ct) method and normalized using log 2 ; etif3d and rpl32 were used as endogenous controls. A two-way ANOVA test followed by the Sidak multiple comparisons post hoc test were used to assess significant differences between the treatments (control and vaccinated) at each individual time point. Asterisks (*) represent significant differences (** p< 0.01, *** p< 0.001).

Vaccine Challenge
Immunized lumpfish were challenged at 45 wpi with 7 times the LD 50 dose for V. anguillarum J360 (7.8 × 10 5 CFU dose −1 ) (Figure 1). Mortality in lumpfish who had been orally mock immunized as larvae and then mock-orally boosted as juveniles started at 3 days post-challenge, with 100% mortality by day 10 post-challenge. Mortality in lumpfish that had been orally immunized as larvae and then orally boosted as juveniles was delayed and started at 3 days post-challenge, with a final RPS of 2% ( Figure 7A). Mortality in lumpfish that had been mock orally immunized as larvae and then both mock orally and i.p. boosted as juveniles started at 7 days post-challenge, with 100% mortality by 20 days post-challenge ( Figure 7B). Lumpfish that had been orally immunized as larvae and then both orally and i.p. boosted as juveniles survived the i.p. challenge with V. anguillarum, with a RPS of 76.5% (p < 0.0001) ( Figure 7B). 9, x FOR PEER REVIEW ure 7. Cumulative survival rate of orally and i.p. immunized lumpfish after i.p. challenge with V. anguillarum (7.8 × 1 dose −1 ). (A) Survival (%) of orally immunized and orally boosted lumpfish after V. anguillarum challenge. Lumpfi e orally immunized as larvae and then orally boosted as juveniles. Control groups were mock-vaccinated using t e inoculation route. After 45 weeks post-initial immunization, the animals were then i.p. challenged; each treatme sisted of two tanks (see Figure 1). (B) Survival of orally immunized and i.p. boosted lumpfish. Lumpfish were ora unized as larvae and then orally boosted as juveniles (see Figure 1). Control groups were mock-vaccinated using t e inoculation route. After 45 weeks post-initial immunization, the animals were then i.p. challenged; Each treatme sisted of two tanks (see Figure 1). Survival was assessed for 30 days. RPS: relative percentage survival; p < 0.0001.

Discussion
Vibriosis is one of the most common bacterial diseases affecting lumpfish aq ture [39]. As mentioned previously, immunization of fish at an early age with a n free vaccine and with minimal stress during immunization is the ideal vaccine d method for finfish aquaculture [30]. However, the effectiveness of bath and oral ina vaccine delivery to small fish or larvae has been evaluated with varied results [52] mercial bath vaccines have been used in lumpfish with low effectiveness against l anguillarum isolates [28]. However, the efficacy of an orally administered vaccine in fish during the early-life stages remains unknown. Here, we evaluated the efficac orally delivery V. anguillarum bacterin bio-encapsulated in A. salina nauplii in lu larvae. The molecular immune response to oral immunization was evaluated in lar qPCR. Approximately 9 months after the initial oral immunization, lumpfish we either orally or both orally and i.p. boosted, and the effectiveness of the vaccines wa uated by assessing the RPS after a lethal i.p. V. anguillarum challenge.
First, we evaluated the V. anguillarum bacterin uptake in A. salina nauplii and after this bio-encapsulated bacterin, in the gut of lumpfish larvae. Our observation cated that the V. anguillarum bacterin was fully bio-encapsulated by the A. salina after 3 h and was maintained for at least 6 d at 6 °C ( Figure 2). Similar results w served by Campbell et al. [32], where the V. anguillarum bacterin showed maximu encapsulation after 1 h or 2 h using 1.5 × 10 7 CFU mL −1 or 1.5 × 10 6 cells mL −1 , respec Vaccine bio-encapsulation in A. salina nauplii protects the antigens from the int tract of the fish and facilitates the recognition of the antigens using the macroph the mucosal layer of the hindgut [53]. The effectiveness of protecting the antige gastrointestinal digestion and its delivery to the hindgut of fish larvae has been d Lumpfish were orally immunized as larvae and then orally boosted as juveniles. Control groups were mock-vaccinated using the same inoculation route. After 45 weeks post-initial immunization, the animals were then i.p. challenged; each treatment consisted of two tanks (see Figure 1). (B) Survival of orally immunized and i.p. boosted lumpfish. Lumpfish were orally immunized as larvae and then orally boosted as juveniles (see Figure 1). Control groups were mock-vaccinated using the same inoculation route. After 45 weeks post-initial immunization, the animals were then i.p. challenged; Each treatment consisted of two tanks (see Figure 1). Survival was assessed for 30 days. RPS: relative percentage survival; p < 0.0001.

Discussion
Vibriosis is one of the most common bacterial diseases affecting lumpfish aquaculture [39]. As mentioned previously, immunization of fish at an early age with a needle-free vaccine and with minimal stress during immunization is the ideal vaccine delivery method for finfish aquaculture [30]. However, the effectiveness of bath and oral inactivate vaccine delivery to small fish or larvae has been evaluated with varied results [52]. Commercial bath vaccines have been used in lumpfish with low effectiveness against local V. anguillarum isolates [28]. However, the efficacy of an orally administered vaccine in lumpfish during the early-life stages remains unknown. Here, we evaluated the efficacy of an orally delivery V. anguillarum bacterin bio-encapsulated in A. salina nauplii in lumpfish larvae. The molecular immune response to oral immunization was evaluated in larvae by qPCR. Approximately 9 months after the initial oral immunization, lumpfish were then either orally or both orally and i.p. boosted, and the effectiveness of the vaccines was evaluated by assessing the RPS after a lethal i.p. V. anguillarum challenge.
First, we evaluated the V. anguillarum bacterin uptake in A. salina nauplii and, thereafter this bio-encapsulated bacterin, in the gut of lumpfish larvae. Our observations indicated that the V. anguillarum bacterin was fully bio-encapsulated by the A. salina nauplii after 3 h and was maintained for at least 6 d at 6 • C (Figure 2). Similar results were observed by Campbell et al. [32], where the V. anguillarum bacterin showed maximum bio-encapsulation after 1 h or 2 h using 1.5 × 10 7 CFU mL −1 or 1.5 × 10 6 cells mL −1 , respectively. Vaccine bio-encapsulation in A. salina nauplii protects the antigens from the intestinal tract of the fish and facilitates the recognition of the antigens using the macrophages in the mucosal layer of the hindgut [53]. The effectiveness of protecting the antigen from gastrointestinal digestion and its delivery to the hindgut of fish larvae has been demonstrated in previous studies [33,53]. Here, we confirmed the presence of the V. anguillarum bacterin in the A. salina nauplii and in the gut of fish larvae 6 h post-oral immunization ( Figure 3). These results validated the internalization of the V. anguillarum bacterin in the lumpfish gut.
The expression profiles of 32 TOIs related to innate and adaptive immunity were evaluated in larvae pre-immunization and at 2 and 4 wpi. In pre-immunized larvae, we did not see any expression of the TOIs, which was expected. When considering orally immunized compared to larvae who had been orally mock immunized at 2 and 4 wpi, there were no significant differences in the levels of pro-inflammatory cytokines (tnfa, il1b, il8a; Figure 4A and Figure S6A), toll-like receptors (tlr3, tlr5a, tlr5b; Figure 4B and Figure S6B), immunoglobulin heavy chain transcripts (ighma, ighd; Figure 5A and Figure S7A), interferon-induced effectors (mxa, mxb, mxc; Figures 5B and 7B), cluster of differentiation transcripts (cd4a, cd4b; Figure 5C and Figure S7C), or other immune-related transcripts (cox2, irf7, lgp2, stat1, rsad2, hamp, saa5; Figure 6 and Figure S8). In contrast, levels of il8b, igha, ighmc, ighb, ccl19, ccl20, cd8a, cd74, if ng, and lgp2 were significantly upregulated, and levels of ly6g6f and tlr7 were significantly downregulated at 2 wpi ( Figures 4A, 5 and 6, Figures S6A, S7 and S8). Levels of il10 were significantly upregulated, and levels of ccl20 were significantly downregulated at 4 wpi ( Figures 4A and 5B, Figures S6A and S7B). These results indicate that 35 d old lumpfish larvae are not highly immune stimulated by oral immunization, suggesting that the interaction between the lymphoid tissues and the vaccine was not enough to trigger adaptive immune protection.
il8 and ifng play important roles in the recruitment of monocytes and neutrophils to sites of inflammation. Whereas il10 acts as anti-inflammatory cytokine and as such, plays a crucial role in the regulation of the inflammatory response [54]. Although there are studies on the expression of il8 and il10 in fish, the role of these interleukins in the early developmental stages of lumpfish is still unknown. In the current study, in orally immunized larvae compared to larvae who had been orally mock immunized, il8 and ifng were significantly upregulated at 2 wpi, il10 significantly upregulated at 4 wpi (Figures 4A, 5 and 6; Figures S6 and S8A). Similar expression profiles for ifng and il10 have been observed in Atlantic salmon that had been infected with the salmonid alphavirus subtype-3 (SAV-3) [55] or immunized with the A. salmonicida vaccine [56]. lgp2 is a member of the RLR family that participates in the recognition of viral RNA pathogen-associated molecular patterns (PAMPs) in the cytoplasm and induces the synthesis of if ng [57][58][59][60]. Similarly, our results showed that lgp2 and if ng were significantly upregulated at 2 wpi ( Figure 6 and Figure S8). The results suggest that the oral immunization of lumpfish larvae triggers an innate immune response that is later regulated via the canonical anti-inflammatory cytokine il10.
TLRs play an important role in early innate and adaptive immunity by detecting PAMPs in bacteria and viruses [61][62][63]. TLRs activate the transcription factor NF-κB, resulting in the production of several pro-inflammatory cytokines such as il1b, tnfa, il8, il10, il6, il12, il17, if ng, and the tumour necrosis factor (tnf ) [64,65]. Expression levels of tlr3, tlr5, and tlr7 were not significantly different in orally immunized compared to larvae who had been orally mock immunized at either time point ( Figure 4B), however tlr7 was significantly downregulated at 2 wpi in the RQ statistical analysis ( Figure S6B). These results suggest that the vaccine does not induce a full immune response in larvae.
ly6g6f is a member of the superfamily lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) [66]. Ly6/uPAR proteins have functions in cell proliferation, migration, cell-cell interaction, immune cell maturation, macrophage activation, T lymphocyte development, differentiation, and cytokine production [67][68][69]. The function of ly6g6f in fish is not yet defined, and in this study, the expression of ly6g6f was significantly downregulated at 2 wpi in lumpfish larvae ( Figure 6 and Figure S8). These results agree with the low level of immune protection.
The igh (immunoglobulin heavy locus) encodes the IgM heavy chains, and these loci have been characterized in several fish species, including fugu, rainbow trout, zebrafish, and Atlantic salmon [70][71][72]. It has been established that igh plays a role during the adaptive immune response by recognizing foreign antigens for phagocytosis, and the complement system [73]. Here, we found that igha, ighd, and ighmc expression were significantly upregulated in orally immunized compared to larvae who had been orally mock immunized at 2 wpi ( Figure 5A and Figure S7A). These results suggest the oral immunization of larvae triggers some level of an adaptive immune response, but it seems insufficient to trigger memory immune protection.
ccl19 is a chemokine known to orchestrate the migration of dendritic cells (DCs) and T cells into lymphoid tissue or vaccination sites and is also involved in immune tolerance and inflammatory responses [74,75]. ccl20 attracts lymphocytes and DCs towards epithelial cells to mucosal immune sites under inflammatory conditions [76]. In orally immunized compared to larvae who had been orally mock immunized, there was a significant increase in levels of ccl19 and ccl20 at 2 wpi, while there was a significant decrease in ccl20 levels at 4 wpi ( Figure 5B and Figure S7B). These results aligned with the expression patterns of other transcripts evaluated here, supporting the idea that the lumpfish larvae did respond to the oral immunization.
CD4 (a classical marker of T helper cells) and CD8 (a marker of cytotoxic lymphocytes) are polypeptides that play an important role in signal transduction and the activation of T-helper cells and cytotoxic T cells, respectively [77]. We found that cd8 was significantly upregulated in orally immunized compared to larvae who had been orally mock immunized at 2 wpi ( Figure 5C and Figure S7C). However, cd4 was not significantly dysregulated. These results suggest that CD8 cellular-mediated adaptive immunity, but not the CD4 response, was activated in lumpfish larvae aligning with the lack of immune protection triggered by the oral immunization ( Figure 7A).
CD74 is the MHC class II-associated invariant chain, which plays a role in antigen presentation [78,79]. CD4 and CD74 lost their original functions in anglerfish (Lophius piscatorius) and Atlantic cod (Gadus morhua) [80][81][82][83]. Here, we found that these transcripts are present in lumpfish, and although cd74 was upregulated in orally immunized compared to larvae who had been orally mock immunized at 2 wpi, cd4a and cd4b were not ( Figure 5C and Figure S7C). These results revealed that oral immunization in lumpfish larvae triggers a partial adaptive immune response.
The lumpfish larvae were vaccinated after the yolk sac was absorbed, after which the larvae exhibited an active feeding behavior. Although there is no literature about the immunity of lumpfish larvae, it is well known that lumpfish larvae are more mature and active than other marine fish [84]. It has also been shown that the main immune organs of lumpfish develop after hatching [85]. These reports, in addition to our current results, suggest that lumpfish larvae are immune competent, and antigens need to be delivered across the epithelia to trigger full immunity. The transcript expression levels (Figures 4-6) also indicated that lumpfish larvae are immune stimulated by oral immunization, but not enough to trigger immune protection. For instance, the expression of il8b, il0, igha, ighmc, ighb, cd8, and c74 was upregulated in orally immunized larvae (Figures 4-6). It seems that oral immunization with V. anguillarum bacterin in lumpfish larvae triggered Th1-like immune response and cellular immunity, which is related to il10 and cd8 upregulation. This is the first study on lumpfish larvae molecular immunity and provides novel knowledge and a baseline to study the ontogeny of the immune system in lumpfish.
The effectiveness of vaccination in fish depends on the delivery, vaccine design, and the fish species. For instance, mortality in lumpfish bath immunized and i.p. boosted with a commercial polyvalent formalin-inactivated V. anguillarum O1 and O2 vaccine was only delayed in an i.p. challenge using V. anguillarum [28]. Similar to our current results, a commercial bivalent whole-cell V. anguillarum O1 and O2 vaccine delivered by immersion and followed by an i.p. boost immunization in European sea bass (Dicentrarchus labrax) conferred approximately 99% survival against a V. anguillarum i.p. challenge [86]. In this study, we observed that lumpfish orally immunized as larvae and then orally boosted as juveniles did not survive the V. anguillarum i.p. challenge ( Figure 7A). Nevertheless, we determined that oral vaccination delayed mortality in lumpfish challenged with V. anguillarum, suggesting that the oral vaccination did stimulate fish immunity, but not enough to confer protection. Similar results were found in salmonids orally immunized against Yersinia and V. anguillarum, where oral immunization conferred no or low immunity to juvenile immunized fish [87][88][89][90]. In contrast, lumpfish orally immunized as larvae and then both orally and i.p. boosted as juveniles showed a significant RPS (76.5%) to the V. anguillarum i.p. challenge ( Figure 7B). This suggests that the orally administered vaccines were not reaching the deep lymphoid tissues, either in the larvae or juvenile fish, and as such, oral immunization was not effective in contrast to the i.p. delivered vaccine. Therefore, it is suggested that inactivated V. anguillarum vaccines for lumpfish should be administered using the i.p. route to confer acceptable levels of immune protection.

Conclusions
Oral immunization of lumpfish larvae using bio-encapsulated bacterin demonstrated that it reached the gut and immune, stimulating the fish larvae. However, oral immunization did not trigger an evident adaptive immune response, even after oral boost immunization. V. anguillarum bacterin that had been orally administered delayed mortality and did not confer protection against the i.p. V. anguillarum. In contrast, i.p. immunization conferred significant immune protection. These results suggest the need for oral vaccines that have the capability of crossing the epithelium and reaching the deep lymphoid tissues to trigger immune protection.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/vaccines9080819/s1, Figure S1: Experimental design for optimization of V. anguillarum bacterin bio-encapsulation in A. salina nauplii, Figure S2: Lumpfish culture conditions and embryonic development, Figure S3: Oral immunization of lumpfish larvae with the DTAF-labeled V. anguillarum bacterin bio-encapsulated in Artemia nauplii, Figure S4: V. anguillarum bacterin bio-encapsulation in A. salina nauplii for industrial application, Figure S5: Transcript expression levels of cytokines (A) and toll-like receptors (B) in lumpfish larvae orally immunized with the V. anguillarum bacterin bioencapsulated in A. salina nauplii, Figure S6: Transcript expression levels of immunoglobulin heavy locus genes (A), cytokine CC genes and interferon-induced GTP-binding proteins (B), and cluster of differentiation genes (C) in lumpfish larvae orally immunized with the V. anguillarum bacterin bio-encapsulated in A. salina nauplii, Figure S7: Transcript expression levels of other immune-related genes in lumpfish larvae orally immunized with the V. anguillarum bacterin bio-encapsulated in A. salina nauplii, Table S1: Sequences of primer pairs used in qPCR analyses of transcript expression levels in lumpfish (Cyclopterus lumpus) larvae, Table S2: Ct, ∆Ct, and RQ values of transcript expression levels of immune-related transcripts in lumpfish (Cyclopterus lumpus) larvae orally immunized with the V. anguillarum bacterin bio-encapsulated in A. salina nauplii, Table S3: Conversion and normalization of Ct values using the 2 (−∆∆Ct) method.