Immunomodulatory effects of a probiotic combination treatment to improve the survival of Pacific oyster (Crassostrea gigas) larvae against infection by Vibrio coralliilyticus

Abstract

Introduction: The culture of Pacific oysters (Crassostrea gigas) is of significant socio-economic importance in the U.S. Pacific Northwest and other temperate regions worldwide, with disease outbreaks acting as significant bottlenecks to the successful production of healthy seed larvae. Therefore, the current study aims to describe the mechanisms of a probiotic combination in improving the survival of C. gigas larvae. Specifically, we investigate changes in C. gigas larval gene expression in response to V. coralliilyticus infection with or without a pre-treatment of a novel probiotic combination.

Methods: Treatment groups consisted of replicates of Pacific oyster larvae exposed to a) a combination of four probiotic bacteria at a total concentration of 3.0 x 10⁵ CFU/mL at 18 hours post-fertilization (hpf), b) pathogenic V. coralliilyticus RE22 at a concentration of 6.0 x 10³ CFU/mL at 48 hpf, and c) the probiotic combination at 18 hpf and V. coralliilyticus RE22 at 48 hpf. RNA was extracted from washed larvae after 72 hpf, and transcriptome sequencing was used to identify significant differentially expressed genes (DEGs) within each treatment.

Results: Larvae challenged with V. coralliilyticus showed enhanced expression of genes responsible for inhibiting immune signaling (i.e., TNFAIP3, PSMD10) and inducing apoptosis (i.e., CDIP53). However, when pre-treated with the probiotic combination, these genes were no longer differentially expressed relative to untreated control larvae. Additionally, pre-treatment with the probiotic combination increased expression of immune signaling proteins and immune effectors (i.e., IL-17, MyD88). Apparent immunomodulation in response to probiotic treatment corresponds to an increase in the survival of C. gigas larvae infected with V. coralliilyticus by up to 82%.

Discussion: These results indicate that infection with V. coralliilyticus can suppress the larval immune response while also prompting cell death. Furthermore, the results suggest that the probiotic combination treatment negates the deleterious effects of V. coralliilyticus on larval gene expression while stimulating the expression of genes involved in infection defense mechanisms.

Keywords: Pacific oyster larvae; Vibrio coralliilyticus; aquaculture; immune response; probiotics.

Figure 1

Experimental timeline for larval assays. (A) In the survival assay, probiotic treatment groups were designated by the timing of probiotic addition. Following fertilization, probiotics were added at either 2 (2 hr PB Only), 6 (6 hr PB Only), 12 (12 hr PB Only), or 18 hpf (18 hr PB Only). At 48 hpf, Vibrio coralliilyticus was added to probiotic-treated larvae (“2 hr PB + Vcor”, “6 hr PB + Vcor”, “12 hr PB + Vcor”, “18 hr PB + Vcor”, and “24 hr PB + Vcor”). A positive control of larvae receiving only V. coralliilyticus was inoculated at 48 hpf. An additional treatment group (24 hr PB Only) received probiotics at 24 hpf, which remained within the larvae culture until 96 hpf. (see methods). (B) For RNA sequencing, larvae were sampled from a similar yet separate experimental setup from that shown in (A) – see details in section 2.4. Larvae used for RNA extractions were collected from the “Larvae Only” control, the “Vcor Only” control, the “2 hr PB Only”, “12 hr PB Only”, “18 hr PB Only”, and the “2 hr PB + Vcor”, “12 hr PB + Vcor”, and “18 hr PB + Vcor” treatment groups at 72 hpf.

Figure 2

Relative percent survival of 4-day-old D-larvae of C. gigas challenged with Vibrio coralliilyticus strain RE22 with or without probiotic supplementation. “Vcor Only” was the positive control for survival and did not receive any probiotics. A negative control (Larvae Only) did not receive any probiotics or the pathogen. Filled circles represent the average relative percent survival of replicate wells (n=18). The boxes indicate the upper and lower quartiles and the bar represents the median or middle quartile. The ends of the whiskers represent the most extreme values within the 1.5x interquartile range (IQR), and the empty circles indicate outliers. *** indicates statistical differences from “Vcor Only” at P ≤.001.

Figure 3

Numbers of differentially expressed genes in infection challenge and probiotic supplementation experiments. Venn diagrams represent the number of significant differentially expressed genes (DEGs) in each treatment group (“18 hr PB”, “18 hr PB + Vcor”, and “Vcor Only”) when compared to the “Larvae only” control. (A) The total numbers of all DEGs from each treatment, with the number of annotated genes listed in blue and the number of uncharacterized genes listed in red. (B) The total numbers of defence-related DEGs for each treatment compared to “Larvae only” controls.

Figure 4

Differential gene expression of defence-related DEGs between the treatment groups (“Vcor Only”, “18 hr PB + Vcor”, and “18 hr PB Only”) and the negative control (Larvae Only). The treatment type is indicated at the top of the heatmap, with black signifying the “Vcor Only” infection control, blue the “18 hr PB + Vcor” treatment, pink the “18 hr PB Only” treatment, and grey representing the “Larvae Only” negative control. All samples were taken at 72 hpf, 24 hours after the addition of V. coralliilyticus to the “Vcor Only” and “18 h PB + Vcor” treatment groups. Heatmap and hierarchical clustering of selected genes is based on normalized read counts. Each column represents a single sample, with each row indicating the expression level of each gene. The colours represent the individual read count data are normalized to its average expression across all samples, with blue indicating lower than the genes’ average (decreased gene expression) and red indicating higher than the average (increased gene expression) (Gene names with corresponding abbreviations are found in Supplementary Table 6 ).

Figure 5

Defence-related genes in various cellular locations are differentially expressed in response to infection by Vibrio coralliilyticus and pre-treatment of the probiotic combination at 18 hours post-fertilization in C. gigas larvae. The arrows represent either an increase or decrease in gene expression caused by the exposure to the probiotic combination alone, V. coralliilyticus alone, V. coralliilyticus in addition to the probiotic treatment, or all three bacterial treatment groups. Pattern recognition receptors bind to the bacteria, sending an inflammatory signal through the NF-kB and toll-like signalling cascades. The activated NF-kB transcription factor leads to the production of cytokines, antimicrobial peptides, and immune effectors. Meanwhile, the tumour necrosis factor alpha-induced protein 3-like prevents activation of NF-kB transcription by inhibiting upstream signalling. The 26S proteasome non-ATPase regulatory subunit 10 binds to the NF-kB transcription factor and retains it in the cytosol as a negative regulator of the NF-kB signalling pathway. Additionally, the serine/threonine-protein kinase RIO3 inhibits NF-kB transcription, regulating inflammatory signalling. The immune effector serine protease inhibitor Cvsi2 directly interacts with endocytosed bacteria. Cell-surface mucins are produced and used as an inflammatory barrier of the cell. Apoptosis is enabled by the cell death-inducing p53-target protein 1 but inhibited by the baculoviral IAP repeat-containing proteins 2, 3, and 7.