Introduction
To keep up with an increasing demand for animal protein, the aquaculture sector has seen an increase in production over the past decades. The intensification of this sector has been accompanied by an increase in disease incidences. Besides prevention of diseases through vaccination and treatment of diseases by antibiotics, dietary supplementation with immunomodulators may provide an alternative route to maintain animal health in aquaculture. Among the better-known immunomodulatory feed additives are non-starch polysaccharides, with probably the best characterized compounds being β-glucans [as reviewed by:
(Dawood et al., 2018; Petit and Wiegertjes, 2016)
]. Another group of immunomodulatory substances are marine sulphated polysaccharides (MSPs) isolated from macroalgae. While MSP-rich extracts are gaining interest from the aquaculture sector as containing health-promoting compounds, their immunomodulatory effects are not always clearly defined.
Materials and methods
Green macroalgae (Ulva sp.) and red macroalgae (Solieria sp.) were processed to produce eight different crude extracts enriched for marine sulphated polysaccharides (MSP).
Antimicrobial activity of the MSP-rich extracts was investigated against 13 fish bacterial strains. Concentration of bacteria (CFU/mL) was determined and strains were diluted to 105 CFU/mL in Mueller Hinton Broth (MH broth). For all eight different MSP-rich extracts, extracts were first dissolved in ultra-pure water and sterilized by autoclaving, subsequently a two-times serial dilution was made from 12.5 mg/mL – 0.0061 mg mL−1. In a sterile culture plate, 100 µL bacterial suspension was combined with 100µL MSP solution dissolved in MH broth. Bacteria were incubated for 24h, or 72h for slow growing bacteria (i.e. Aeromonas salmonicida , Aliivibrio salmonicida , Streptococcus agalactiae, Streptococcus iniae ). Each plate included a positive control with bacterial strain alone in MH broth, and two negative controls: only MSP extract at the different dilutions, and MH broth alone. Following incubation, optical density was measured at O.D. 600 nm and plates were photographed. Antimicrobial effects were determined as the lowest MSP extract concentration required to completely inhibit visible growth (O.D. 600 nm, or visually determined from photographs) of tested microorganisms after 24h, or 72h of incubation. Three replicates were made for each microorganism.
Nile tilapia ( Oreochromis niloticus) were reared at 28 ± 2 °C temperature with a 12-12h light-dark cycle. The fish were fed a commercial diet twice per day. Rainbow trout (Oncorhynchus mykiss) were reared at 14 ± 0.5 °C temperature with a 12-12h light-dark cycle. The fish were fed a trout specific research diet twice per day.
Fish were killed with 0.3 g/L tricaine methanesulphonate in aquarium water buffered with 0.6 g L−1 sodium bicarbonate (Nile tilapia) or 2-phenoxyethanol (1 mL L-1) in aquarium water (Rainbow trout) and bled via the caudal vein. Head kidney was removed aseptically and total head kidney leukocytes (HKLs) were separated on a Percoll density gradient (51%), as previously described for Nile tilapia
, and for Rainbow trout
.
Production of ROS was determined by a real-time luminol-based luminescence assay, as previously described with minor modifications
(Petit et al., 2021)
. Cells were stimulated with one of the following: RPMI cell culture medium (control), zymosan (tlrl-zyd , 50 µg mL−1,) or one of the extracts at a concentration of 250, 500, 750, 1000 and 1500 µg mL−1. Chemiluminescence emission was measured in real time at 27˚C (Nile tilapia) or at 19°C (Rainbow trout) and expressed as area under the curve, as previously described
(Petit et al., 2019)
. Fold changes were calculated as the area under the curve of stimulated HKLs relative to unstimulated HKLs (treated with RPMI).
HKLs were seeded at a density of 4.5 x 106 per well in a 24-wells plates and stimulated with RPMI, zymosan (tlrl-zyd, 50 mg/ml,) or one of the extracts at a concentration of 500 µg mL-1 and incubated at 27°C (Nile tilapia) or at 19°C (Rainbow trout). At 3 and 6 hours post stimulation, cells were lysed in RLT lysis buffer and stored at -80˚C until RNA isolation.
Total RNA from cell lysate in RLT lysis buffer was isolated and stored at −80°C. Prior to cDNA synthesis, total RNA was treated with DNase I, Amplification Grade, and cDNA was synthesized using random primers (300 ng) and Superscript III First-Strand Synthesis for RT-PCR. cDNA samples were diluted in nuclease-free water prior to real-time quantitative PCR (RT-qPCR) analysis.
Gene expression was measured with RT-qPCR using ABsolute qPCR SYBR Green Mix in a Rotor-Gene Q, and fluorescence data were analysed using Rotor-Gene Analysis software version 2.3.5. The relative expression ratio (R) of each sample was calculated according to the Pfaffl method
based on the take-off deviation of sample versus each of the PBS controls and normalized relative to elongation factor 1α (elf1α) as reference gene.
Statistical analysis was performed in IBM SPSS statistical data editor version 26. Data presented as fold changes were transformed with natural logarithm, prior to statistical analyses. Subsequently, transformed data was tested for normality by using a Q-Q plot and performing a Shapiro-Wilk test. Data was then analysed using a repeated measures linear mixed model followed by a Bonferroni post hoc test . All values are means and expressed with their standard deviation (SD), and data were considered significant for p<0.05.
Results
While all extracts showed direct anti-bacterial effects to some degree, two red algal extracts in particular, had high activity against several pathogenic fish bacteria in vitro. Stimulation of head kidney leukocytes (HKLs) in vitro with MSP-rich extracts showed fish species specific differences. In Nile tilapia, HKLs showed a dose dependent reactive oxygen species (ROS) production following stimulation with Ulva-derived extracts, while stimulation with Solieria -derived extracts did not induce ROS production. In Rainbow trout, HKLs showed relatively high reactive ROS potential and Solieria -derived extracts could induce significant ROS production, albeit without clear dose dependent responses. Gene expression of in vitro stimulated HKLs showed a clear induction of most cytokines measured (il1b, il10, tnfa , ifng , il12p40). Although cytokine gene expressions were more prominent in Nile tilapia than in Rainbow trout, immunomodulatory effects of both, Ulva- and Soleria -derived extracts appeared evident. HKL might sense the presence of MSP via unknown pattern recognition receptors with known downstream signaling pathways. For genes associated with Toll-like Receptor (TLR) signaling , regulation of irak1 in Nile tilapia was evident. For genes associated with C-type Lectin Receptor (CLR) signaling , regulation of both card9 and bcl10 was found, again in Nile tilapia, confirming earlier observed fish species specific differences. Possibly, immunomodulatory effects of MSP could be regulated by CLR-mediated signaling, at least in Nile tilapia, for which most if not all red algae and green algae extracts induced changes in gene expression.
Conclusion
Overall, induction of ROS production and gene expression read-outs suggest immunomodulatory effects of MSP-rich extracts derived from green algae (Ulva sp.) and red algae (Solieria sp.), at least in vitro. The observed effects suggest clear fish species specific differences between the effects of MSP-rich extracts.