Oral supplementation of low alginate dose in diet stimulates immune response of Litopenaeus vannamei at concrete circle pond mass culture

Litopenaeus vannamei is popular due to its ability to grow in different scales of rearing as a valuable export product. This research aimed to define the impact of alginate diet supplementation in a low dose as an immunostimulant strengthens the non-specific immunity of shrimps. The study was conducted in 20 tons concrete circle ponds with two treatments at the density of 400 ind.m-2. First, there were control A and B, and secondly, there was the addition of 1.0 g alginate in 1.0 kg of feed namely alginate 1.0 g kg-1 (A and B). Shrimps were reared for 30 days, and the non-specific immune parameters were assessed on 22 and 30 days of rearing. The parameters covered in this study were THC (total count of hemocyte), PA (activity of phagocytosis), PI (index of phagocytosis), PO (phenol-oxidase), SOD (superoxide dismutase), and LYZ (lysozyme) activity. Results showed that the THC/PA/PO, and LYZ enzyme activity of sodium alginate treatments were higher than the control (p<0.05), though the PI, SOD, and weight gain at the end of the experiment have not shown any differences. It is concluded that the supplementation of alginate at 1.0 g kg-1 enables to increase in the shrimps’ immune system and this application is projected to be useful in terms of blocking out the large-scale application of immunostimulants in ponds.


Introduction
The Whiteleg shrimp (Litopenaeus vannamei) is a valuable aquaculture cultivan global.Compare to animal protein, shrimps are the important aquatic species with the highest profits in the global market and currently, account for at least 55% of shrimp production [1].Global production increased totally up to 86% in the last decade, getting the price of approximately 40 million dollars U.S. [2].However, disease 2 outbreaks are a common occurrence in shrimp farming.For example, in 2013, the output of shrimp in disease impacted areas was cut down by more than 60%, resulting in annual worldwide collapses from over than 1billion dollars U.S. [3].Some of it's most severe diseases have been interconnected to bacterial pathogens such as Vibrio spp.[4; 5] , White Spot Virus [6], myonecrosis virus [7], as well as White Faecal Disease [8].Antibiotics can be used to control disease in shrimp culture; however, the negative impact of residues and resistance to cultured organisms and the environment are main points [5; 9].Vibrios were resistant to Amoxicillin, Ampicillin, and Co-Amoxiclav [9; 10], kanamycin (aphA-3) and chloramphenicol (catA2) [11].
L. vannamei is recognized as a shrimp species with a wide range of different scale of culture.Farmers have intensified the culture in effort to expand white shrimp production by developing small-scale areas at the highest density possible, as known as super-intensive farming [12].As a result, shrimp farming has observed issues related stress-stimulated disease caused by declining pond circumstances prevalence.Our previous study reported that innate immunity is highly related to disease resistance [5], [9].Furthermore, preventing pathogen aggregation and increasing shrimp resistance to pathogens are the crucial aspects.
Alginate is a non-toxic, inexpensive, and biodegradable biopolymer material [13], a compound of the cell wall structure of brown seaweeds.Our unique alginate is extracted from tropical Sargassum spp.which is abundant in our local coast and still under unexploited [14].Alginate c o n s i s t s o f two structural units namely Guluronic acid and Mannuronic acid and identified to be an immunomodulator [6; 15; 16].Supplementation alginate as oral administration in feed is dose dependent manner.Some experiments regarding with L. vannamei from Cheng et al [17] with commercial alginate, Montero-Rocha et al [18] with ergosan, and Yudiati et al [6]; Yudiati et al [15] with local Sargassum spp.has reported on their works that the lowest concentration (2.0 g kg -1 ) supplemented of alginate in the feed are able to improve its immune response.In this experiment, we administered the alginate supplementation even at lower dose ie.1.0 g kg -1 .This trial aimed to define the effect of alginate diet supplementation at 1.0 g kg -1 as immunostimulant to strengthen the non-specific immunity of shrimps.We examined the immune factors, namely THC (total count of hemocyte), PA (activity of phagocytosis), PI (index of phagocytosis), PO (phenol-oxidase), SOD (superoxide dismutase), and lysozyme activity.The weight gain, some water quality parameters as well as microalgae composition were also determined.

1 Research Design
The experiment was designed as completely randomized (CRD).There were 2 treatments of feed namely control (without supplementation) and 1.0 g alginate supplementation in 1kg feed (1.0g kg -1 ).Each treatment was replicated two times.The experiment was conducted over 30 days.Weights of shrimps were recorded at the beginning and at the end of shrimp experiment.

Research Procedures
Alginate was provided by Tropical Mar.Biotech (TMB) Laboratory, Diponegoro University.Alginate was supplemented at the dose of 1.0g kg -1 in the commercial diet (Evergreen ® ).The feeding frequency was four times daily, at satiation.Shrimps were reared in concrete circle tank in 20 tons volume (5.0m diameter and 1.2m depth).All shrimps in the treatment tanks were never been touched to avoid stress, and surely adequate feed for their metabolism and growth as well as water quality requirements.
Water quality was managed by siphoning out and filling in the sterilized seawater.Water quality parameters namely pH, salinity, Dissolved Oxygen, temperature, and alkalinity observed daily, while ammonia, nitrite, and nitrate, were observed weekly.Microalgae composition (total plankton, green algae, blue green algae, protozoa, and Dinoflagellates) were also determined weekly.

Total Haemocyte
Count.Six L. vannamei from every treatment were selected for determining the immune factors.Using a sterile syringe, hemolymph was extracted.Two microliters of hemolymph were diluted with eight milliliters of PBS before being assessed with a hemocytometer to count THC.This observation was conducted under a microscope.Shrimps were treated carefully and gently to reach at a minimun stress.When collecting haemolymph was performed, it was administered as fast as possible, using the new sharp sterile syringe, and as much as necessary.All the haemolymph treated shrimps were then quarantine in a single certain 10 L tank, aerated and feeded well before moved to the normal tanks, in the following day.

Phagocytic activity (PA) and Phagocytic Index (PI).
Activity of phagocytosis and index were determined in a well plate by mixing 10 μL hemolymph and 10 μL PBS.The mixture was then added up with 10 μl killed Bacillus sp. 5 μL of these mixtures were then smeared in an object glass, fixed with 95 percent ethanol, and stained with 10 percent Giemsa for approximately 15 minutes.The slides were thoroughly rinsed and dried.A light microscope was used to observe the slides and count the hemocyte.

Phenol-Oxidase
Activity.The activity of PO was done by measuring the configuration of dopachrome from L-DOPA. 100 µl of hemolymph was combined with same volume of saline PBS and separated by centrifugation at 700 g for 20 minutes at low temperature.The supernatant was separated once again in 100 μl cacodylate buffer.After that, 100 μl of trypsin was inserted, and followed 10 mins incubations in room temperature.Finally, 50 μl volume L-DOPA was then added.A spectrophotometer was used to measure at 490 nm optical density [19].

Superoxide-Dismutase
Activity.The activity of SOD was analyzed by riboflavin production with nitroblue tetrazolium [20].The 20 μl of hemolymph was mixed with 180 μl of PB prior to centrifugation at 6000g for 7 minutes (4 °C), then followed by supernatant heating for 5 mins.At last, 50 µl NBT was put in.A spectrometer was used to determine SOD activity by recording the absorbance at the 630 nm optical density.

Lysozyme
Activity.LYZ activity was assessed using a turbidimetric principal [21] with lyophilized, and using M. lysodeikticus and lysozyme of chicken egg white (Sigma, USA) as standard.So, 150 µL of M. lysodeikticus was pipetted into 96-well plates at a dose of 0.2 mg mL -1 (w/v) in 0.02 M acetate buffer, and the optical density was observed using a microplate reader (450 nm).After one hour of incubation (25°C), the final optical density was then recorded.The LYZ activity was represented as g mL -1 , which is equivalent to the activity of a standard chicken white egg.

2.3.6
The parameters of water quality.pH, dissolved oxygen, water temperature, alkalinity, and salinity was recorded daily (Hanna Instruments), while ammonia, nitrate, and nitrite, was checked by commercial kit weekly.Microalgae composition was monitored every week.Blue green algae, Dinoflagellates, Diatomae, and Green algae was also checked routinely.

Data Analysis
The entire data were statistically analyzed using ANOVA (R Studio) using a level significance of (0.05).

THC, PA, and PI
THC and PA of shrimps supplemented with Alg 1.0 kg -1 (Figure 1a, b) was higher than control, but nonsignificant in PI (Figure 1c).Immune reactions occur primarily in hemolymph and hemocytes, though some take place in immune tissues as well as organs for instance gills, intestines, and lymphoid organs [22].THC increases the immune system response in Whiteleg shrimp and is associated with an improvement in the body's defense mechanism [6; 15].Phagocytic activity is a criterion of foreign particle control and destruction.The phagocytosis defense route is divided into three different phases: chemotaxis, followed by recognition, and internalization [23].Hemocytes are a major element of the non-specific cellular defense system.The capacity of phagocytic activity, which arises during infection, demonstrates that hemocytes are a cellular defense of shrimp's body [24].

PO, SOD, and Shrimp Weight Gain
PO activity shrimps supplemented with Alg 1.0 kg -1 (Figure 2) at 22 and 30 days of experiment were higher than control.Alginate contains high amounts of polysaccharides built in from mannuronic and guluronic acid monosaccharides [14].This increase in PO activity is primarily caused by lipopolysaccharide stimulation, which activates proPO.PO is a critical component of L. vannamei immune system [25].In contrast, the control treatment, without any supplementation of alginate, the PO activity was significantly lower.Study by Yudiati et al [6]; Yudiati et al [15]; Setyawan et al [24] on L. vannamei supporting these facts.Different from PO activity, the SOD activity was similar to both treatments (Figure3).SOD activity is related to the antioxidant activity.Our previous study noted that low viscosity alginate [26], produced by thermal heating [27], microwave irradiation [6], and the addition of some chemical reagents [28] were able to produce stronger antioxidant activity than raw alginate.Additionally, supplementing L. vannamei with alginate at a dose up to 2.0g kg -1 accelerated SOD activity.This is demonstrated by the production of non-excessive superoxide anions, which can lead to an increase in SOD activity [6].The supplementation of raw and low doses of alginate was postulated to be insufficient to improve the SOD activity.This may result the similar weight gain of L. vannamei in control and alginate 1.0g kg -1 (Figure 4).

LYZ activity
LYZ activity in alginate 1.0g.kg - was higher than control (Figure 5).In the non-specific defense mechanism, Lysozyme is a protein-like hydrolytic enzyme that is critical.A raise in serum lysozyme activity is agreed to be a natural protective mechanism [21].This study discovered an increase in lysozyme activity in L. vannamei fed low alginate concentration.In aggreement, Novriadi et al [29] reported considerably increased lysozyme activity in L. vannamei after consuming a feed containing nucleotides In terms of increasing THC and lysozyme activity.We suggest that alginate can be used as a functional ingredient in feed to optimize immune defense systems.

Parameters of Water Quality
Temperature, pH, dissolved oxygen, alkalinity, salinity, and other water quality parameters measured on a daily basis were in tolerable range (Figure 6 a, b, c, d) [30].Alkalinity, ammonia, nitrite, and nitrate (Figure 7 a, b, c) was acceptable, nitrate was undetected due to the usage of healthy microplankton.de Lourdes Cobo et al [31] suggested that the level of TAN does not surpass 0.42 mg L -1 TAN, equal to 0.06 mg L -1 NH3-N.At initial rearing, generally, the plankton was in good condition (Figure 7a).As the rearing time progressed, in accordance with the amount of feed, Dinoflagellates (Figure 7b), blue green algae (Figure 7c) as some species categorized responsible for the red tide phenomenon and harmful algae [30].As the rearing time continued, Protozoa (Figure 7d) is detected to grow higher.Adversely, Diatomae (Figure 7e), as well as green algae (Figure 7f) were decreased according to the rearing time.

Figure 1 .
Figure 1.(a) Total of Hemocyte Counts, (b) Activity of Phagocytosis, and (c) Index of Phagocytosis of Whiteleg shrimp fed diets supplemented with control and alginate 1.0 g kg -1 at 30 days.Significant differences (p < 0.05) are noted with different alphabets.

Figure 2 .
Figure 2. Whiteleg shrimp PO activity in feed supplemented with control and alginate 1.0 g kg -1 on 21 st and 30 th day.Significant differences (p < 0.05) are noted by with different alphabets.

Figure 3 .
Figure 3. Whiteleg shrimp SOD activity in feed supplemented with control and alginate 1.0 g kg -1 on 21 st and 30 th day.Significant differences (p < 0.05) are noted with different alphabets.

1 )Figure 4 .
Figure 4. Weight gain of whiteleg shrimp fed diets supplemented with control and alginate 1.0 g kg -1 on 30 th day of rearing.Significant differences (p < 0.05) are noted with different alphabets.

Figure 5 .
Figure 5. LYZ activity of Whiteleg shrimp fed diets supplemented with control and alginate 1.0 g kg -1 at 30 th day of rearing.Significant differences (p < 0.05) are noted with different alphabets.