Exopolysaccharide from Porphyridium cruentum (purpureum) is Not Toxic and Stimulates Immune Response against Vibriosis: The Assessment Using Zebrafish and White Shrimp Litopenaeus vannamei

Abstract

Exopolysaccharides, or extracellular polysaccharides (EPS, sPS), represent a valuable metabolite compound synthesized from red microalgae. It is a non-toxic natural agent and can be applied as an immunostimulant. The toxicity test of exopolysaccharides from Porphyridium has been done in vivo using zebrafish (Danio rerio) embryonic model, or the ZET (zebrafish embryotoxicity test). The administration of extracellular polysaccharides or exopolysaccharides (EPS) from microalgae Porphyridium cruentum (synonym: P. purpureum) to shrimps Litopenaeus vannamei was investigated to determine the effect of this immunostimulant on their non-specific immune response and to test if this compound can be used as a protective agent for shrimps in relation to Vibrio infection. For immune response, exopolysaccharides were given to shrimps via the immersion method on day 1 and booster on day 8. Shrimp hemocytes were taken on day 1 (EPS administration), day 7 (no treatment), day 8 (EPS booster) and day 9 (Vibrio infection) and tested for their immune response on each treatment. The result shows that the EPS is not toxic, as represented by the normal embryonic development and the mortality data. In the Pacific white shrimps, an increase in the values of all immune parameters was shown, in line with the increasing EPS concentration, except for the differential hemocyte count (DHC). In detail, an increase was noted in total hemocytes (THC) value, phagocytotic activity (PA) and respiratory burst (RB) in line with the EPS concentration increase. These results and other previous studies indicate that EPS from Porphyridium is safe, enhances immune parameters in shrimp rapidly, and has the ability to act as an immunostimulant or an immunomodulator. It is a good modulator for the non-specific immune cells of Pacific white shrimps, and it can be used as a preventive agent against vibriosis.

Keywords: Danio rerio; Vibrio harveyi; extracellular polysaccharide; hemocytes; immunomodulator; innate immune cells; microalgae; phagocytic activity; respiratory burst; toxicity; white shrimp.

Figure 1

Median number of individual deaths of zebrafish embryos observed following exposure to different exopolysaccharide concentrations according to the zebrafish embryotoxicity test (ZET) method. Box represents 25th–75th percentiles; bars represent minimum and maximum values; Twenty embryos were treated in each concentration treatment (N = 20) in triplicate.

Figure 2

Zebrafish (Danio rerio) embryonic development exposed to exopolysaccharide from Porphyridium cruentum (purpureum) at different EPS concentrations (v/v). Photos taken under a microscope Olympus CE-21 with 100 times magnification.

Figure 3

Morphology of Vaname (Litopenaeus vannamei). Healthy (A) and shrimp infected by Vibrio harveyi (B) showing vibriosis as indicated by smoky body and organ coloration, and reddish color change at the cephalotorax and caudal fin parts (arrows). Left: whole body; center: uropod. (C) Hemocyte preplacement with a needle. Scale bars: 1 cm. (Photos: Intan Hasanah).

Figure 4

Total hemocyte count (THC) of Litopenaeus vannamei after EPS administration on day 1 (EPS administration), day 8 (EPS booster), day 9 (post-infection with Vibrio harveyi, 10⁷ cells/mL). Bars represent mean with SD, and different letters (a, b, c, d) between the bars indicate highly significant differences (p < 0.05).

Figure 5

Percentage of Hyalin cells of Litopenaeus vannamei after EPS administration with different immersion concentrations. Day 1: EPS administration; Day 7: no treatment; Day 8: EPS booster; Day 9: post-infection with Vibrio harveyi, 10⁷ cells/mL. Bars represent mean with SD, different letters (a, b, c, d) between the bars indicate highly significant differences (p < 0.05).

Figure 6

Semi-granular cells of Litopenaeus vannamei after EPS administration with different immersion concentrations. Day 1: EPS administration; Day 7: no treatment; Day 8: EPS booster; Day 9: post-infection with Vibrio harveyi, 10⁷ cells/mL. Bars represent mean with SD; different letters (a, b, c, d) between the bars indicate highly significant differences (p < 0.05).

Figure 7

Percentage of granular cells of Litopenaeus vannamei after EPS administration with different immersion concentration. Day 1: EPS administration; Day 7: no treatment; Day 8: EPS booster; Day 9: post-infection with Vibrio harveyi, 10⁷ cells/mL. Bars represent mean with SD, different letters (a, b, c, d) between the bars indicate highly significant differences (p < 0.05).

Figure 8

Phagocytic activity (PA) of hemocyte cells of vaname post 24 h. Vibrio harveyi bacterial infection. Arrows show the cells phagocyte yeast activity. Photo was taken under a light microscope (750 magnifications).

Figure 9

Phagocytosis activity on Litopenaeus vannamei after EPS administration with different immersion concentrations. Day 1: EPS administration; Day 7: no treatment; Day 8: EPS booster. Day 9: post-infection with Vibrio harveyi, 10⁷ cells/mL. Bars represent mean with SD, different letters (a, b, c, d) between the bars indicate highly significant differences (p < 0.05).

Figure 10

Respiratory burst activity on Litopenaeus vannamei after EPS administration with different immersion concentrations. Day 1: EPS administration; Day 7: no treatment; Day 8: EPS booster; Day 9: post-infection with Vibrio harveyi, 10⁷ cells/mL. Different letters show statistically significant difference at 0.05.