Extracellular Polysaccharide from Porphyridium cruentum (purpureum) stimulates the non-specific immune activities on white shrimp (Litopenaeus vannamei)

White shrimps are susceptible to outbreaks of vibriosis because they do not have any adaptive immune system, they only have a non-specific innate immune system. The administration of EPS from microalgae Porphyridium cruentum (synonym: P. purpureum) on shrimps Litopenaeus vannamei was investigated to determine the effect of this immunostimulant on their non specific immune response and to test if EPS can be used as a protective agent for shrimp related to Vibrio infection. EPS was given to shrimps by 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. Result shows an increase in values of all immune parameters in line with the increasing EPS concentration, except the Differential Haemocyte Count (DHC). In detail, an increase was noted in total hemocytes (THC) value, Phagocytotic Activity (PA), Respiratory Burst (RB) in line as the EPS concentration increase. Although there is a decrease after the infection, the value obtained is not lower than the control value. These results indicate that EPS from Porphyrydium enhances immune parameters in shrimp rapidly and has the ability 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 Vibrio.

immunoglobulins, but they are able to recognize and destroy invading or parasitic microorganisms [21]. Cellular response as biomarker for immune systems of aquatic animals has been studied as an effect not only associated to bacterial infection or disease, but also related to environmental changes [18; 39; 40].
Invertebrates cell wall have proteins such as lipopolysaccharide (LPS), β-1,3-glucans (BG) and a specific protein called beta glucan binding protein (BGBP). BGBP in shrimp appears to be the main plasma protein right after binding to β-glucans, where β-glucan is a compound composed of polysaccharides, which react with the surface of the hemocytes and stimulate the formation of granule cells. Granule cells are one of the hemocyte cells that are responsible for immune system in shrimps [21].
For immune system in shrimp, this study used EPS from Porphyridium cruentum. Porphyridium cruentum is a single-celled red microalgae belonging to the class of Rhodophyceae, currently has other synonym: Porphyridium purpureum (Bory) . It is capable of living freely or colonizing in marine waters, and can be cultivated in a fast life cycle. The round shaped Porphyridium cruentum cells is 4 to 9 µm, and almost 60% is composed of carbohydrates. The cells are bound in mucilage, a compound constantly excreted by the cell forming a capsule, surrounding the cell, containing polysaccharide sulfate, known as EPS. EPS is one of the important components in the function of Porphyridium cruentum as an antioxidant, antibacterial, antiviral, and anti-hyperglycemic substance [14; 15].
Various studies on the benefits of Porphyridium cruentum as an anti-bacterium have been widely carried out, but the use of EPS from this species as immunostimulant in Vibrio harveyi-infected shrimp has not been done. Therefore, in addition to becoming the initial foundation of research related to future problems, this study is needed to determine the effectiveness of the Extracellular Polysaccharide Sulfate (EPS) of Porphyridium cruentum as a source of immunostimulant.

Vaname, the Pacific white shrimp (Litopenaeus vannamei) when it is infected by Vibrio harveyi
showing vibriosis as shown in Figure 1. All treatments almost showed the same symptoms, where the shrimps experienced a change in color from the carapace -cephalotorax to the caudal parts. The whole body and the hepatopancreas organ show smoky-pale coloration and the lateral cephalotorax and caudal fin parts indicate reddish orange.
The result shows that the EPS modulates all immune cells,in this paper they were expressed in Total Hemocyte Count (THC), Diferential Hemocyte Count (DHC) including hyaline cells number, granular and semi granular cells number, Phagocytic activity (PA) and Respiratory Burst. Hemocyte prelevement with a noodle.  (Figure 3).

Semi-granular and granular cells
The lowest number of semi-granular cells for all days was obtained from the treatment of 14 ppt EPS; 11.50% on day 1, 16.80%on day 7, 14.30% on day 8,and 8.10%on day 9. (Figure 4).
The highest granular cell value was obtained from all 14 ppt EPS treatment: 64.20% on day 1 immunostimulant administration, 46.60% on day 7 without immunostimulant administration, 54.60% on day 8 after the booster, and 47.20 % on day 9 after infection. On the 7th day without EPS addition, the value of granular cells appeared to decrease; this indicates a decrease in shrimp's immunity ( Figure 5). In all treatments, the phagocytosis level in day 7 is lower than that in day 1, except the control, probably due to no immunostimulant treatment. On day 8 after booster administration, the phagocytosis level is higher than the level in day 7, except for control (EPS= 0 ppt).    Semi Granular cells (%) 0 ppt 10 ppt 12 ppt 14 ppt It was found that respiratory burst activity increases along EPS concentration. An interesting phenomenon was on the 9th day post-infectious treatment with Vibrio harveyi, where the respiratory burst (RB) value is lower than of the 1st, 7 th , and 8th (0.511, 0.395, 0.537, and 0.383 respectively) ( Figure 8).

Discussion
This paper showed the first study of EPS application as a "stimulator" or "modulator" of immune system in Litopenaeus vannamei from Vibrio harveyi infection. We revealed that the EPS from The increase of hyaline cells is associated with phagocytic activity; where in hyaline cells are infected, it will get a significant increase, as well as when immunostimulant, which can stimulate the body's defense activities, is given so that hyaline cells increase as the first defense response [22].
Hyaline cells decrease after being given immunostimulant. This can be caused by granular cell formation through the process of hyaline cell maturation. Semi granular cells are the cellular type between hyaline cells and granular cells that play an active role in the encapsulation of larger-size foreign bodies that cannot be phagocyted by hyaline [24]. The effect of EPS after infection can be assessed as unfavorable or, in the other words, the infection attacks and reduces the number of semi-granular cells and granular cells increased at day 9, although in all treatment, the higher concentration of EPS influenced the modulation of immune cells.
Granular cells are the largest hemocyte cell type whose the nucleus is active in the process of storing and releasing prophenoloxydase and cytotoxicity systems [24]. These cells are characterized by the presence of granules in their cytoplasm. They are able to respond to polysaccharides from bacterial cell walls or β-glucan derived from fungi [25]. They play a role in phenoloxidase enzymes production for non-specific body defense activities, which are driven by the influence of immunostimulatory components such as β-glucan, composed of polysaccharides [26].
They are involved in a fast defense response to the virus attack two hours post infection [31].
A significant increase of phagocytic activity was noted in shrimps post infected with Vibrio harveyi as seen in this study. The results above reveal that shrimp has phagocytic activity against Vibrio harveyi infection, where the phagocytic activity itself is a reaction of cellular defense and is an important process to maintain and eliminate microorganisms or other foreign particles that enter the body [23; 18]. In general, phagocytic activity and respiratory burst increase in line with the increasing EPS concentration.
The measurement of phagocytic activity aims to determine the level of phagocytosis that occurs through several treatments. The entry of microbial components in the body can activate the body's defense response cellularly [18]; this can be observed through phagocytic activity, which is the main activity in the process of defending against foreign infections. Respiratory bursts were related, and they are monitored to identify the body's defense level associated with the activity of superoxide anion (O2-) which was characterized by the ability of blood cells to reduce NBT (nitrobluetetrazolium). In addition, the value of RB is related to the level of phagocytosis. The higher the respiratory burst value, the better the shrimp's defense system [24].
Respiratory burst is an advanced activity of the phagocytosis process, where particles planted in phagolysosomes will be destroyed by digestive respiratory burst enzymes until free radical release occurs in phagolysosomes. In line with this study, RB activity of turbot phagocytes increases in high concentration water soluble seaweed extracts [28]. While, RB activity decreased when the treatment is added with serum, it might cause suppressive effects on cellular parameter. In contrast, RB increased when aquatic animal is in under osmotic stress [29].
A question can be arising regarding the toxicity of the compound for the testing animals. It exist various methods, approaches and animal models to analyze toxicity, from biochemical, cellular or mortality assays [39]. Indeed, the toxicity test using rat model shows that Phorphyridium biomass was not toxic [41]. Moreover, EPS has been evaluated to be not toxic, our study using EPS didn't show any toxicity as has been revealed in our work (unpublished reports).
Various preventive efforts have been done; among others using macroalgae extracts, to overcome vibriosis in shrimps particularly in black tiger shrimps [32] and in white shrimps [42].
Algae has polysaccharides contents which is potential for various purposes [43], among other, EPS.
EPS has been tested to have the antimicrobial activities, particularly for HSV virus, types 1 and 2, Vaccinia virus and Vesicular stomatitis virus and two Gram-negative (Escherichia coli and Salmonella enteritidis) and one Gram-positive (Staphylococcus aureus) bacteria. All EPS extracts revealed a strong activity against V. stomatitis virus, higher than the activity of all chemical compounds tested [27]. In this study, we revealed that the EPS stimulates or modulates all immune biomarkers rapidly, and this indicates that EPS from microalgae is a good modulator for the non specific immunity of Pacific white shrimps. In spite of the fact that the EPS can modulate immune response of white shrimps rapidly, there is still a need for further research on the function of EPS not only as an immunostimulant for preventive objective but also as curative material with the addition of higher doses of EPS or with a longer time treatment post Vibrio infection.

General
This study uses experimental methodwith a simple Complete Randomized Design (CRD) and

Culture condition
Vaname (Litopenaeus vannamei) of the young stage, aged 45 days or more, with the length of 7 cm and weight of 5 grams are maintained in aquariums. Acclimatization was done one day before treatment, using pellet feeding and cultured in laboratory condition. Temperature, pH, dissolved oxygen (DO) and salinity were controlled during the study. Daily temperature was between 25 and 26oC, pH was 7.5-7.8, DO value was between 5.64 and 6.5 mg/L, and salinity was 35 ppt.

Extracellular Polysaccharides Extraction
Extracellular Polysaccharides (EPS) can be obtained from microalgae as a supernatant. The EPS supernatant was obtained from the centrifugation process of Porphyridium cruentum together with the medium of its life. Centrifugation was carried out at a speed of 10,000 rpm for 15-20 minutes. The acquired supernatant was separated using microalgae pellets; only the supernatant was used. The combination of all Porphyridium cruentum supernatants was carried out by maceration using Ethanol 96% solvent with a solvent and media ratio of 1: 0.75 v/v. The samples were stored and allowed to stand at room temperature for 72 hours until white EPS deposits were formed. After 72 hours, the sample was put in a water bath for 1 hour at 80 o C. The samples were filtered using simple filter paper, and precipitation was carried out using cold ethanol. EPS suspensions obtained in the freeze dryer and dialysis were carried out by resuspending the dry EPS into distilled water, and this process was carried out several times.
We have reported the Chemical functional group of EPS in our previous study which used the Fourier Transformed Infrared (FTIR) method to determine the chemical groups of the compounds contained in EPS as initial information about the chemical composition of EPS [16].

Shrimp Treatment
The test animals were white shrimps (Litopenaeus vannamei), 7 cm in length and 5 gram in weight. The shrimps were acclimatized before treatment at laboratory in a controlled condition. The test treatment was carried out by challenging the vaname shrimp (L. vannamei) using Vibrio harveyi with the density of 10 7 cells/ml using immersion method. The test shrimp samples were maintained by administering EPS as immunostimulant with variations in dosages of 10 ppt, 12 ppt, and 14 ppt.
Immunostimulant addition was carried out on the 1st day. The shrimps were then left alive without Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 December 2020 doi:10.20944/preprints202012.0193.v1 immunostimulant until the 7th day and were given a booster immunostimulant on the 8th day. On the 8th day (3-4 hours after immunostimulant was given), the shrimps were infected with 10 7 cells/ml of Vibrio harveyifor 24 hours. Hemolymph was taken from the shrimps at each treatment as the parameter test material for the study of immune cells.

Immune parameters
The parameters which included Total Hemocyte Count (THC), Differential Hemocyte Count (DHC), Phagocytotic Activity (PA) and Respiratory Burst (RB) were calculated using a haemocytometer with the help of a light microscope with 400x magnification. Total Hemocyte Count (THC), Differential Hemocyte Count (DHC), and Phagocytotic Activity (PA) were based on the procedure as described on the previous studies [17,18]. The respiratory burst activity was measured using the reduction of nitro-blue tetrazolium (NBT) assay [19].

Toxicity Test
To test the toxicity of EPS, other experimaental study has been performed [44]. Mortality test shows that the the EPS is safe up to concentration of 15% (v/v), thus, the concentration used in this study is appropriate.

Data Analyses
The data was analyzed using one-way Analysis of Variance (ANOVA) with a confidence interval of 95%. This analysis was used to analyze differences in the average value between groups of treatments or variations obtained between test groups. Thus, if F count > F table 5% and F table  1%, it can be concluded that the results of this research are significantly different. Then, the test was continued with the Smallest Significant Difference test (LSD) and Tukey test.

Ethical Consideration
The approval according to the regulation on the use of animals in our study is not necessary because our research used a limited number of common shrimps frequently consumed by the wider community.

Conclusions
The provision of immunostimulant EPS from Porphyridium cruentum can stimulate the non-specific immune activity of Litopenaeus vannamei, with the best dose being 14 ppt. Immune activity in this paper is characterized by an increase in THC value, DHC (hyaline and granular cells), Phagocytotic Activity (PA), and Respiratory Burst (RB) before being infected with Vibrio harveyi.
Post-infection, decreases were identified in all parameters, except PA, but the treatment of EPS was higher than the control. All these results indicate that the EPS from Porphyrydium is a good modulator for the non specific immune cells of Pacific white shrimps, and it can be used as a preventive agent against Vibrio.