Immunostimulatory effect of a marine yeast Candida sake S165 in Fenneropenaeus indicus

TL;DR: The study has demonstrated that marine yeast C. sake at 10% in diet (w/w) may be used as an effective source of immunostimulants in F. indicus and their enhancement could be observed on the second and third day following challenge with the virus.

About: This article is published in Aquaculture.The article was published on 2006-06-30 and is currently open access. It has received 79 citations till now.

Summary

1. Introduction

• The immune system of crustaceans ismainly non-specific and relies on phagocytosis, encapsulation and agglutination alongside the phenoloxidase-mediated production ofmelanin through thepro-phenoloxidase cascade (Smith andSoderhall, 1983).
• Only a few studies have been reported on the use of yeast as a source of immunostimulants in penaeids and investigations have not been conducted with marine yeasts.
• Scholz et al. (1999) compared the efficacy of five different yeast-supplemented diets in shrimp and reported that Phaffia rhodozyma incorporated into the feed gave better performance in terms of bacterial clearance and increased phenoloxidase activity in haemolymph.

2.1. Yeast strain

• C. sake S165 was isolated from coastal waters off Cochin and maintained in the Microbiology laboratory of the Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences.
• Selection of this isolate was based on the preliminary tests with F. indicus that showed growth increments and improved survival upon WSSV challenge for postlarvae fed on a diet supplementedwith sevenmarine yeasts (Sajeevan et al., 2003).
• A lawn of C. sake was prepared using malt extract agar (malt extract, 20 g; mycological peptone, 5 g; agar, 20 g, 20 ppt seawater, 1 L; pH 6) and cell biomass was harvested at the exponential phasewith sterile seawater (20 ppt).

2.2. Experimental diet

• Three experimental dietswerepreparedby incorporating yeast biomass (wet weight) at concentrations of 1%, 10%, and 20% (w/w) to a standard shrimp diet.
• Diet without supplementation of yeast was used as control.
• In the experimental diets wheat flour was substituted by yeast biomass at different quantities.
• Ingredients except the yeast biomass were mixedwell into adoughwith100mlwater andwas steamed for 10min in an autoclave.
• The feeds were air dried for 2 h and stored at−20 °C until used.

2.3. Animals used

• A batch of apparently healthy adult F. indicus (mean body weight 15.6±1.5 g) was brought to the laboratory from a shrimp farm located at Kannamali, Cochin.
• The shrimps (60 Nos), after seven days quarantine, were transferred to four aquarium tanks of 500 L capacity and acclimatized for a week.

2.4. Feeding experiment

• Group 1 received the control diet, Group 2, the feed containing 1% yeast, Groups 3 and 4 the diets containing 10% and 20% yeast, respectively.
• Physico-chemical parameters of the rearing water such as salinity, NH3–N, NO2–N, NO3–N and dissolved oxygen were monitored regularly (APHA, 1995) and maintained at optimal levels by water exchange (Table 1).

2.5. Challenge test

• After 28 days of feeding the animals were challenged with white spot syndrome virus by feeding white spot virus infected frozen tissue at the rate of 1 g/animal.
• Thereafter they were maintained on their respective diets and the immune parameters assayed at defined intervals.
• The percentage survival in each group was also recorded for a period of seven days.

2.6.1. Collection of haemolymph

• Haemolymph was withdrawn aseptically from the rostral sinus using specially designed sterile capillary tubes having a diameter of 0.5 mm and pre-rinsed with anticoagulant (Song and Hsieh, 1994).
• It was transferred to sterile microcentrifuge tubes containing cooled shrimp anticoagulant.
• The haemolymph collected from five shrimps (n=5) of each treatment group was assayed separately.
• Sampling was carried out at the beginning of the feedingexperiment (0day/base line), atday15, day28, and post-challengeatdays1(PCD1),2(PCD2),3(PCD3)and5 (PCD5).

2.6.3. Phenoloxidase activity assay

• Phenoloxidase activity was measured spectrophotometrically using L-3,4-dihydroxyphenylalanine (L-DOPA) as the substrate (Soderhall, 1981).
• The dopachrome formed was measured at 495 nm and phenoloxidase activity was then expressed as the increase in absorbance per minute per 100 μl haemolymph.

2.6.4. Superoxide anion (NBT reduction) assay

• Respiratory burst activity of haemocytes was measured spectrophotmetrically as described by Song and Hsieh (1994) using nitro blue tetrazolium (NBT) as the substrate.
• Ontaining graded levels of yeast for 28 days and then challenged with y different (pb0.05).
• Blue formazan compound is formed due to O2 − reduction during phagocytosis by haemocytes.
• The absorbance at 620 nm was recorded and expressed as NBT activity per 100 μl haemolymph.

3. Results

• The groups of shrimp fed a diet with 10% (w/w) yeast exhibited a significantly higher immune response compared to thecontrolandother treatmentgroups(pb0.05) (Figs.1,2 and 3).
• The highest total haemocyte count, phenoloxidase activity and NBT reduction were observed on the 15th and 28th day of feeding prior to challenge with WSSV.
• Subsequent challengewithWSSVresulted in temporal increase in the above responses in groups of shrimp fed on different percentages of yeast.
• These responses were significantly higher (pb0.05) on the 3rd day post-challenge ing graded levels of yeast for 28 days against experimental infection of y different (pb0.05).
• Meanwhile a uniform decrease in all immunological parameters was seen on the 5th day post-challenge in all challengegroups.

4. Discussion

• Yeast is generally considered a good source of proteins, nucleic acids, vitamins and polysaccharides.
• Information regarding the synthesis and metabolism of nucleotides in fish and crustaceans is extremely limited (Li and Gatlin, 2003).
• A probable explanation could be the infiltration of haemocytes, especially semigranular cells into connective tissues, stomach and gills upon WSSV infection (Munoz et al., 2002).
• A very prominent elevation in NBT level on day 3 after challenge could be attributed to an increase in phagocytosis and the resulting production of more superoxide anions.
• It could be proposed that yeast might act both as a source of an immunostimulant and a nutritional supplement in penaeid shrimp.

Figures

Fig. 1. Mean (±S.D.) THC of F. indicus fed on diets containing graded levels of yeast for 28 days and then challenged with WSSV. Data at the same exposure time with different letters are significantly different (pb0.05). 1Y=1% yeast, 10Y=10% yeasts, 20Y=20% yeasts. PCD = post-challenge day.

Fig. 2. Mean (±S.D.) phenoloxidase (PO) values of F. indicus fed on diets c WSSV. Data at the same exposure time with different letters are significantl

Table 1 Rearing conditions and water quality

Fig. 4. Mean (±S.D.) post-challenge survival of F. indicus fed on diets contain WSSV. Data at the same exposure time with different letters are significantl

Fig. 3. Mean (±S.D.) Superoxide anion values of F. indicus fed on diets containing graded levels of yeast for 28 days and challenged with WSSV. Data at the same exposure time with different letters are significantly different (pb0.05).

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Immunostimulatory effect of a marine yeast candida sake s165 in fenneropenaeus indicus" ?

Ten per cent C. sake in the diet was found to support an optimum immune response in the animals in general and their enhancement could be observed on the second and third day following challenge with the virus. The study has demonstrated that marine yeast C. sake at 10 % in diet ( w/w ) may be used as an effective source of immunostimulants in F. indicus. 

Q2. What is the immune system of crustaceans?

The immune system of crustaceans ismainly non-specific and relies on phagocytosis, encapsulation and agglutination alongside the phenoloxidase-mediated production ofmelanin through thepro-phenoloxidase cascade (Smith andSoderhall, 1983). 

Q3. What is the way to test the immune system?

An exogenous source of nucleotides may optimize the functions of rapidly dividing cells, such as those of the immune system, that lack the capacity to synthesize nucleotides and therefore must depend on a pre-formed source (Carver andWalker, 1995). 

Q4. How many days did the animals receive the white spot virus?

After 28 days of feeding the animals were challenged with white spot syndrome virus by feeding white spot virus infected frozen tissue at the rate of 1 g/animal. 

Q5. What is the reason for the elevation of NBT?

A very prominent elevation in NBT level on day 3 after challenge could beattributed to an increase in phagocytosis and the resulting production of more superoxide anions. 

Q6. What is the reason for the increase in adipose activity in the ly?

Signal transduction in the prophenoloxidase-activating system of Macrobarchium rosenbergii and intracellular phenoloxidase activity in haemocyte lysate supernatant (HLS) were found to be increased after treating with CpG oligonucleotides (Chuo et al., 2005). 

Q7. How long did the shrimps stay in the aquarium?

The shrimps (60 Nos), after seven days quarantine, were transferred to four aquarium tanks of 500 L capacity and acclimatized for a week. 

Q8. what is the halotolerent property of the yeast C. sake?

The halotolerent property of the yeast C. sake would be an advantage in this context and it can be used in brackish water and seawater aquaculture where it would not result in cell lysis and associated water quality deterioration. 

Q9. Who is the author of this article?

The authors are grateful to the Department of Ocean Development, Govt. of India for a research grant with which the work was carried out. 

Q10. What is the diet for shrimp?

Shrimpsfed thediet containing10%yeast showed 44±2% survival while groups fed on diets containing 1% and 20% yeast showed only 11±2% and 23±3% survival respectively (Fig. 4). 

Q11. What was the diet of C. sake?

Control diet: fish meal, 28 g; prawn shell powder, 20 g; rice bran, 10 g; soyabean meal, 10g;groundnutoil cake,8g;vitaminmix,2g; refinedwheat flour, 20 g. 

Q12. How was the yeast biomass added to the test feeds?

Into the test feeds yeast biomass was added at a graded levels 1, 10 and 20 g and pelletised using a laboratory model pelletiser having 1 mm die. 

Q13. Which diet was used for the treatment of white spot syndrome?

Group 1 received the control diet, Group 2, the feed containing 1% yeast, Groups 3 and 4 the diets containing 10% and 20% yeast, respectively.