THE USE OF P-GLUCAN AS AN IMMUNOSTIMULANT TO
INCREASE THE NON-SPECIFIC IMMUNITY
AND THE PERFORMANCE OF
SHRIMP
(Perzaeus monodon,
Fab.)
UNDER ARTIFICIAL STRESS CONDITION
BY
EMILIANA DHIAN ANGGERAHENI
I'OSTGIIADUATE PROGRAM INSTITUT PERTANIAN BOGOR
ABSTRACT
EMlLlANA DHlAN ANGGERAHENI. The Use of (3-glucan as An Immunostimulant to Increase the Non-Specific Immunity and the Performance of Shrimp (Penaezts n~onodon, Fab.) Under Artificial Stress Condition. Under advisory cotlimittee AHMAD MAAD WIRAWlDJAJA and DARNAS DANA.
Environmental stress has been a colnmon risk in shrimp culture ponds. This stress could lead to the occurrence of diseases. The experiment was divided into three trials. The objective of the first trial was to investigate the effect of oxygen concentration. water salinity, and luniinous bacteria concentration on some parameters of stress condition i.e. hemocyte count, survival rate, and number of luminous bacteria (Kbrio splendidus biovar 1 ) in hemolymph. The result of this trial could be used as a fit stressor in an advanced trial. The second trial was to investigate the effect of P-glucan on some parameters of shrimp performance i.e. growth and feed conversion ratio. The third trial had an objective to study the affectivity of (3-glucan given in 1 and 2 months on some parameters of shrimp immune system i.e. hemocyte count. phagocytic activity, clearance bacteria, and survival rate at low oxygen concentration. The experiment was an advanced experiment using the worst condition resulted by trial 1.
The result suggests that feed containing e-glucan 0.1% affected the immune capacity of shrimp, such as clearance o f bacteria in hepatopancreas and hemolymph, total hemocyte and phagocytic activity of hemocyte. Its effect was better than the others. But the feed containing P-glucan had no significant effect on the performance of shri~np i.e. the growth and the feed conversion ratio. because the trial was conducted in short time to give evidence o f their effect on the performance of shrimp. Shrimp given feed containing P-glucan 0.1% could clear Vibrio splendidzrs biovar I in he~nolymph and hepatopancreas in less than 12 hours. Although the highest hemocyte count was found in shrimp fed by P-glucan 0.5%, their phagocytic activity had no significant difference with shrimp fed by P-glucan 0.1%. It indicated that the content of hyaline cells (phagocytic cells) in shrimp hemocyte fed by P-glucan 0.1% was higher than the other. Thus, the highest rate of phagocytic activity from the 7Ih up to the 14"' day after bacteria immersion was found in shrimp hemocyte fed by
IJ-
glucan 0.1 %.
DECLARATION
I declare that thesis using title:
THE USE OF P-GLUCAN AS AN IMMUNOSTIMULANT TO lNCREASE THE NON-SPECIFIC IMMUNITY AND THE PERFORMANCE OF
SHRIMF' (Perzaerrs ntortodort, Fab.) UNDER ARTIFICIAL STRESS CONDITION
is my writing, as the results of my experiment. The thesis has been not published as academic report at other universities or other institutions.
This thesis may not be reproduced in whole or part by photocopy or other means without permission o f t h e author.
THE USE OF P-GLUCAN AS AN IMMUNOSTIMULANT TO
INCREASE THE NON-SPECIFIC IMMUNITY
AND THE PERFORMANCE OF
SHRIMP
(Penae~cs monodon,Fab.)
UNDER ARTIFICIAL STRESS CONDITION
EMILIANA DHlAN ANGGERAHENI A thesis submitted in partial fulfillment o f the requirements for the degree o f
MASTER OF SCIENCE in the
Department of B i o l o ~ y
POSI'CRADUATE PROGRAM INSTITUT I'ERTANIAN BOGOR
APPROVAL
Title of Thesis : THE USE OF P-GIACAN AS AN IMMUNOSTIMULANT TO INCREASE THE NON-SPECIFIC IIVIMUNITY AND THE PERFORMANCE OF SHRIMP
(Penaeus monodon, Fab.)
UNDER ARTIFICIAL STRESS CONDITION
Name : EMILIANA DHIAN ANGGERAHENI
No. Reg. Student : 99440
Program : Biology
Approved by, I . Advisory Comtnittee
Dr. Ahmad Maad Wirawidiaia Dr. Darnas Dana. h4Sc.
Chairman Member
2. I-lead of Biology Department
Dr. Dedi Durvadi
CURRICULUM
VITAE
The writer, EMlLlANA DHlAN ANGGERAHENI, was born on January 5 ,
1971. in Semarang - Central Java. In September 1994, she graduated from the Faculty of Fisheries, Aquaculture Department, Institut Pertanian Bogor.
From November 1994 until May 1996, she worked at PT. Birulaut Khatulistiwa, the shrimp hatchery of PT. Dipasena Citra Darmaja in Kalianda- Lampung. Then, she worked at the shrimp hatchery of PT. Centralpertiwi Bahari in Suak Sidomulyo - Lampung from June 1996 until February 1999.
ACKNOWLEDGEMENTS
I gratefully acknowledge the financial support by the PT. Centralpertiwi Baliari. I would like to express illy deep appreciation to Mr. Djoko M. Basoeki. Dr. Sujint Tamniasart. and Mr. Johannes Kitono, the president director and directors of the PT. Centralpertiwi Bahari. Thanks are due also to Mr. Wayan Agus Edhy. Mr. Wahyudi, Mr. Cheawchan Ponpanitralsamee and Mr. Sarayuth for their expert advice; Mr. Januar Pribadi for his laboratory services; Mr. lsman Haryanto, Mr. Risdianro P.W, Ms. Maria Sisilia, and Ms. Eni for their attention; Dr. Dean Akiyama for his invaluable discussion and attention at the final stage of my thesis.
I wish to express thanks to ~ n y parents, iny sisters, and mas Joko Sulistyo for their loyal support throughout the study. Thanks also to the staff of PT Centralpertiwi Bahari (Pondsite) Larnpung, especially Yogi Sukirman, who helped me in the wet laboratory, Mr. Bastian Sitompul, Mr. Ilasan Solikin, Mr. Sutarto, all staff of Microbiology laboratory, Mr. Dani Yukasano, Mr. Tonif, Mr. Kristianto, Mr. Suherman, and all my friends at pond site.
TABLE O F CON'1'ENTS
PAGE
LIST OF TABLES
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viLIST OF FIGURES ...
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vii . . .LIST OF APPENDICES.. . .
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. V l l lINTRODUCTION
Justification and Problems.. .
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. 1Objective..
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. . . . 3Hypotheses.. . .
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3HISTORICAL REVIEW
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Luminous Vlbrio ... . ..
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Disease Trigger.. . .. . . .. . . .
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, . . 5Shrin~p Defence Mechanism.. . .
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6P-glucan as An Immunostimulant ... ... .. . . .. . . .. ... 7
METHODOLOGY
Times Lines
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Experimental Design
Trial 1 : Artificial Stress Condition and Shrimp In~nii~nity . . .
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12 Trial 2: p-glucan and Shrimp I'erformance.. ..
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. . . .. 13Trial 3: Administration Period of b-glucan and
[image:86.595.80.468.15.794.2]RESULTS AND DISCUSSION
Results
Identification of Vibrio Bacteria in Hemolymph
and Hepatopancreas..
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. ....Trial 1: Artificial Stress Condition and Shrin~p Irmnuniry
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Trial 2: P-glucan and Shrimp Perfornlance... . . .
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Trial 3: Administration Period of P-glucan and
Shrimp Immunity..
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Discussion
Trial 1: Artificial Stress Condition and Shrimp Immunity.. Trial 2: p-elucan and Shrimp Perfornlance
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Trial 3: Administration Period of P-glucanand Slvimp Iminunity
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CONCLUSSION.. .
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BIBLIOGRAPHY..
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LIST OF TABLES
PAGE
1. Mean number of the hemocyte count, survival rate of shrimp and
number of Vibrio splendidus biovar 1 colony (cfu) in hemoplymph
at 18 hours after stressing condition..
...
182. Mean number of growth and feed conversion ratio caused
. .
by P-glucan adm~nlstration.. ... 21
3. Number of Vibrio splendidus biovar 1 colony in hepzropancreas
...
and hemolymph after challenge test.. 22
4. Mean number of hemocyte count at the 0,71h. 14"', and 21'' day
after challenge test.. ... 23
5. Mean number of phagocytic activity at the 0.
7"',
14u. and 21" day after challenge test. ... 246 . Survival rate of shrimp.. ... 25
LIST OF FIGURES
PAGE
1. The water quality in each treatment..
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26LIST OF APPENDICES
PAGE
1. The differential characteristics of the species and biovar of the
. .
Genus of fibrzo
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382. The analyses of variance for the hemocyte count in hemolyinph at
...
18 hours after stressing condition (Trial 1). 40
3. The analyses of variance for survival rate of shrimp at
18 hours after stressing condition (Trial 1). ... 4 1
4. The analyses of variance for number of luminous bacteria in
henlolymph at 18 hours after stressing (Trial 1).
...
425 . The analyses of covariance for the growth of sluimp (Trial 2) 43
6. The analyses of variance for the feed conversion ratio (Trial 2) 44
7. The analyses of variance for the hemocyte count in hemolymph on
the 0 day after challenge test..
...
458. The analyses of variance for the hemocyte count in hemolynlph on
the 7"' day after challenge test..
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469. The analyses of variance for the hemocyte count in hemolymph on
...
the 14"' day after challenge test 47
10. The analyses of variance for the heniocyte count in he~nolpmph on
...
the 21'' day after challenge test 45
11. The analyses of variance for the phagocytic activity on
the 0 day after challenge test ... 49
12. The analyses of variance for the pbagocytic activity on
the 7"' day after challenge test ... 50
13. The analyses of variance for the phagocytic activity on
11,
14
.
The analyses of variance for the phagocytic activity on the 21S1day after challenge test ...15 . Tile analyses of variance for the phagocytic activity on
...
the 50"' day after challenge test
16 . The analyses of variance for the s u ~ i v a l rate of shrimp
...
after challenge test
INTRODUCTION
,Justification and Problems
Shrimp has a less developed immune system than fish or other vertebrates. It is
probable that shrimp is relatively more dependent on the non-specific immune processes,
such as phagocytosis, than vertebrates (Raa et al., 1992). Thus, administration of
antibiotics and vaccines giving a specific immunity is an ineffective treatment (Anderson,
1992).
In~mu~lostimulant, a material that can elevate the non-specific defence
mechanism, i.e. P-glucan and lipopolysaccharide, may represent an alternative and
supplement to vaccines in the protection of farmed shrimp against diseases.
Lipopolysaccharide and P-glucan, present on the gram-negative bacterial and fungal
surface, can be recognized by shrimp henlolymph; thus, they.can increase the non-
specific in~nlune system of shrimp (Kondo et al., 1996; Vargas-Albores et al., 199s).
Although the im~nunostin~ulants have numerous positive effects on the imnlune
system in shrimp, they have some limitations and possible negati\.e effects, especially in
case of over dosage. Irnmunostiiuulants administration must notice the timing and
dosage of application (Anderson, 1992).
The timing of irnn~unostimulants application depends on the activating
characteristics of the ~naterial itself. Some result of trials indicate that P-glucan has a
short duration cffect on irnnlune system. Thus. P-glucan must be given exactly at the
The overdose or under dose of inlmunostimulants is an administration problem.
High doses of in~munostimulants can suppress the defence mechanism, and low doses
may not be effective (Anderson, 1992). High dietary levels of P-glucan (>I .5 %) in feed
may impair nutrient digestibility because they increase the viscosity of the ingesta
(Galeotti, 1998; Volpatti et al., 1998).
Generally, the experiment on im~~~unostimulant is conducted only to investigate
effectiveness of a material on animal immunity, and omit interaction of other factors in
environment that cannot be precisely controlled (Soderhall and Johansson, 1985, 1989;
Raa et al., 1992; Itami, 1994; Kondo et a/., 1996; Vargas-Albores el al., 1998; Clifford,
1999). The effect of imnlunostimulants on the interaction of disadvantageous conditions
of the environment causing stress has not been identified yet.
In fact, most diseases in aquaculture are caused by opportunistic pathogens i.e.
bacteria that cause disease when shrimp is weak or under stress. The phagocytic activity
is less affected by stress condition (Anderson, 1992). The stress is caused by temperature,
oxygen concentration, pH, salinity, etc, often exists under practical farming conditions
(Clifford, 1999).
Thus, this experinlent was done by combining some factors causing stress
condition i.e. oxygen concentration. salinity, and level of bacterial attack, to study the
Objcctivc
The experiment was divided into three trials with some objectives as follows:
1. To study the effect of oxygen concentration, water salinity, and luminous
bacteria concentration on sotne parameters of stress condition i.e. hetnocyte
count, survival rate, and number of luminous bacteria (Vibrio splendidus
biovar 1) in hemolympll.
2. To study the efkct of P-glucan on some parameters of shrimp performance
i.e. growth and feed conversion ratio.
3. To study the effectiveness of 0-glucan given in 1 and 2 months on some
parameters of shrimp immune system i.e. hemocyte count, phagocytic
activity, clearance bacteria, and survival rate at low oxygen concentration.
The hypotheses of this study were as follows:
1. The lolv oxygen concentration, high water salinity, and high luminous bacteria
concentration could decrease hemocyte count, survival rate, and number of
luminous bacteria (Vibrio sl7lendidus biovar 1) in hemolympli of shrimp.
2. The administration of 0-glucan could increase the immune systeni and
performance of shrimp (Penaeus ~izorzodon Fab.) on disadvantageous
HISTORICAL REVIEW
Luminous Vibrio
The description information about genus Vibrio was reported by Baumann et al.
(1984). Vibrios were straight or curve rods, which did not form endospores or
mycrocysts. They were gram negative, facultative anaerobes capable of both
fermentative and respiratory metabolism. All were chemoorganothrophs;most grew in a
mineral medium containing D-glucose. Most species were oxidase positive. ~ a +
stilllulated growth of all species of Vibrio. Species-of Vibrio vmy with respect to the
temperature at which growth occurred. All grew at 20°C and most did at 30°C; some
grew at 4°C and 45°C; none grew at 50°C. Many strains of marine Vibrio were able to
swarm on solid media. Until recently we. only know four species of Vibrio contained
lumi~lous strain; Vibrio harveyi, Vibrio splendidus biovar I, Vibriojschery, and Vibrio
logei (Lavilla-Pitogo et al., 1990). She further explained some luminous bacteria have
the capacity of entering a symbiotic association with marine animals.
Lightner (1988) reported that vibriosis has become the most common bacterial
disease in sluimp culture, and in some tropical countries, the diseases occur throughout
the year. Rukyani and Sunarto (1998) observed that luminous bacteria caused disease on
shrimp. Vibrio spp were contagious disease agents as they were able to spread and infcct
Disease Trigger
The interaction of host, pathogen, and environment has become a common
relationship and could not be avoided in shrimp culture, a classic three-cycle system. But
disease could only occur when the system was an unequal interaction of host, pathogen,
and environment (Anderson, 1992).
Disease outbreak usually occurred on stressed shrimp during water exchange,
temperature and climate changes, larvae transportation, etc. Direkbusarakom and
Danayadol (1998) reported that one of important states of the environment was the
presence of sufficient oxygen to maintain system vital to the health of an aquatic
ecosystem. Generally, the grade of oxygen concentration in a pond fluctuated; therefore
it has become a good indicator of water quality that affecting health of shrimp.
Hernolymph
Hemolymph or shrimp blood has become one of sonle shrimp health indicators
affected by stress condition i.e. oxygen concentration. Maynard (1960) reported that the
color of heniolymph was affected by hemocyanine (blood pigment). The hemocyanine
contained Cu and their tasks were to bind and to distribute the oxygen, and they play
important roles in blood osmotic.
The type of henlocytes (blood cells) in Crustacean was observed by Soderhall and
Johansson (1989). Based on the type of granules in helnocyte cytoplasn~, they reportcd
that crustacean generally contained three types of circulating hemocytes: hyaline cells (no
granules), semi granular cells (small granules), and granular cells (large granules).
and Pet~aeus by Graves and Gary (1985). But they were called agranular cells, small-
granule cells, and large-granule cells.
Further they reported that the mean number of hemocytes per ml in Sicyo17ia and
Penaeus was similar. In both species, the proportion of agranular cells and the large
granule cells were very low, and the small granule cells composed approximately 75% of
all hemocytes.
Based on the phagocytic capability Soder!lall and Johansson (1989) noted that the
hyaline cells were typical phagocytic cells; the semi granular cells also had some
phagocytic capacity, but the granular cells had no phagocytic activity.
Shrimp Defence Mechanism
Soderhall and Johansson (1989) reported that arthropods and invertebrates in general
did not possess imn~unoglobulins. But with an open circulatory system, they had defence
and coagulation mechanism to entrap parasites and prevent blood loss upon wounding.
The cellular defence reaction in invertebrates was most often accompanied by
melanization involving prophenoloxidase (proPO) as an inactive pro-enzyme in
hemocyte. Some rllicrobial products could affect activation of prop0 to phenoloxidase.
The prophenolosidase-activating (ppA) system stimulated several cellular defence
reactions including phagocytosis. Both prophenoloxidase and inactive propllenoloxidase-
activating enzynle were stored in hemocyte, from where the): were released by
degranulation. Further they reported that the active enzyme, phenoloxidase, oxidized
An additional systcm to activate prophenoloxidase was reported by Vargas-
Albores ef al. (1998). They observed that the activation of prophenoloxidase involved
two steps. The first was the degranulation that occurred when bacteria stimulated
hen~ocytes, and the second required the participation of Calciun~ for the conversion of
inactive prophenoloxidase-activating enzyme to an active proteinase that, in turn,
transform prophenoloxidase to active-phenoloxidase.
Microbial products affected coagulation in hemolymph. The mechanism has
become an essential defence response for crustacean to prevent loss of hemolymph
through a break in the exoskeleton and dissenliriation of bacteria throughout the body.
Vargas-Albores et al. (1998) observed that shrimp plasma had type C coagulation. It has
been named clotting protein. The clotting process occurred when prophenoloxidase-
activating enzyme was activated by the presence of calcium. Calcium triggering cellular
Transglutarninase (Tgase) enzyme was supplied by hyaline cells, on clotting protein to
build clot formation (like fibrin formation in vertebrates).
P-glucan as, An Imrnunostirnulant
Anderson (1992) defined that an imrnunostimulant is a chemical, drug, stressor, or
action that elevated the non-specific defence mechanism. Long-chain polysaccharide
extracted from yeast, P-glucan, has been known as one of some imniunos~imulant agcnts
stimulating non-specific defence mechanism in animals including shrimps. Vargas-
Albores e/ al. (1998) reported that P-glucan stimulated the propllenolosidase-activatillg
words, P-glucan is capable of stimulating shrimp hemocytes to release cellular
components.
The recognition of nlicrobial product in shrimp hemolymph has been reported by
Vargas-Albores ef cl. (1998). Two kinds of protein were involved in recognition of
microbial products in shrimp. The first group, lectins or hemaglutinins, was multivalent
sugar-binding agglutinins, which were able to react with bacterial lipopolysaccharide and
form some lipopolysaccharide-binding agglutinins (LPSBA). The second group,
monovalent: was able to react with P-glucan, and therefore, it was called P-glucan-
binding protein (BGBP). The existence of BGBP and LPSBA in shrimp plasma indicated
capability of shrimp immune system to detect, and then to bind P-glucan and
lipopolysaccharide given in order to trigger shrimp defence mechanism, especially in
2
METHODOLOGY
Time Lines
The experiment was conducted on January 22 up to June 30, 2001 at PT.
Centralpertiwi Bahari (Pondsite), in Lampung-Sumatera.
Materials
Shrimp Penaeus n2onodon Fab. (DOC: 50.and 30), Saccharonzyces cerevisiae Type
11 from SIGMA, Staphylococcus aureus (lo6 cfdml), isolate of Vibrio spp., Trypan Blue
solution, Saline solution, Triptych Soy brothlagar, Thiosulfate Citrate Bile Salt Sucrose
agar, Gelatin agar, Arginine, Ornithine, Lysine, Citrate agar, L-Arabinose, Sucrose,
Salicine, D-mannitol, D-sorbitol, Amylum, Kovacs solution, Sodium Chloride, D-
Glucose, Peptone agar, Trisodium Citrate, Citric Acid, EDTA, Chloride Acid, Ethanol,
Aquadest, Paraplast, Methanol, Giemsa stain, Gram stain. Xylol, H E stain, Entelan, Silk
screen 200 S, tank and aeration system, syringe Tuberculin 1 ml and needle, object glass,
cover glass, hemocytometer, petridisc, ose, Bunsen lamp. hotplate, stirrer, Becker glass,
Erlenilleyer glass, autoclave,n~icrotray. glass tubes, DO and pH meter, disposable
pipette, microtome. and microscope.
Mcthods
Before running the experiment, extraction of P-glucan from cell \valls of
Srrcchcrronzyces cerevisiue was done by alkaline - acid nisthod (William el al., 1991).
Saccharomyces cerevisiae in 3.5 L of 0.75 M (3%) Sodium hydroxide. The process of
separating residue from supernatant was conducted by keeping the mixture overnight and
discarding the dark brown supernatant. The NaON digestion was repeated twice. Then
the process of extraction was continued by acid digestion using Chloride acid 3.5 L of
2.45 M, 1.75 M, and 0.94 M. Separating process conducted at each step of acid digestion
was the same as alkaline digestion. The purification of P-glucan was continued by
bleaching process using 2 L of distilled water to gain white and flocculent residue. The
process was conducted ten times by boiling and separating process. The colorless
process was continued by adding 1.5. L of ethanol to the residue and the process was
conducted tluee times by boiling and separating process. Before filtering the particle of
P-glucan by using fine silkscreen, the washing process using 2 L of distilled water was
conducted three times by boiling and separating process. Then the particle of P-glucan
was dried by freeze-lyophilize process.
Mixing of P-glucan in feed was condxted by using albumin as a binder. The
com~nercial feed was mixed into the mixture of P-glucan and albumin, and then the blend
of this feed was baked in an oven in the temperature of 60°C for 2 hours to get the
nloisture 5 - 8.
Isolate of lulllinous bacteria used in this experiment came from hatchery of PT.
Ce~itralpertiwi Bahari. The bacteria have beconle a prob!cm in mortality of shrimp in
hatchery. Type of luminous bacteria used for challenge test and type of bacteria in
shrimp hernolymph and hepatopancreas were identified by Baumann el CII., (1984)
The number of bacteria in shrimp hemolymph and hepatopancreas, to examine the
clearance process, was counted by using disc-counting method (Thompson et al., 1997).
One gram of shrimp hepatopancreas was mixed into 9 ml of saline solution. The blend
was spread into petridisc containing Thiosulfate Citrate Bile Salt Sucrose agar and held
12 hours before counting the bacteria. Counting bacteria in hemolymph was conducted
by the same method. The shrimp hemolymph 0.02 ml was drawn from thc ventral
abdominal artery of shrimp and then was spread into petridisc containing Thiosulfate
Citrate Bile Salt Sucrose agar.
The number of hemocyte was counted by Blaxhall and Daisley (1973) abbreviation.
Hemolymph 0.1 ml was drawn from the ventral abdominal artery of shrimp into syringe
containing 0.1 ml Trypan Blue solution. To make a homogenized fluid, the mixture was
shaken carefully. One drop of the solution was added to a hemocytometer and the
number of cell per n1m3 was counted.
Total of h e n ~ o c ~ t e / m n ~ ~ =Number of counted-cell x diluting factor x 100 x 10
Number of counted-block 1 mn1 x 4
The ability of phagocytosis by hemocyte was evaluated by Anderson and Siwicki
(1995) method. Hen~olympll 0.1 ml was drawn from the ventral abdominal artery of
shrimp into syringe containing 0.1 ml anticoagulant (0.14 M Sodium chloride, 0.1 M
Glucose, 30 mM Trisodium citrate, 26 n1M Citric acid, and 10 inM EDTA pH 4.6).
Then, the hcmolymph was transferred into microtray and 0.1 ml S/c~~~lzylococc~i.s uureu.5
luinutes the suspension was spread on glass slide, stained with Giemsa, and then
observed under the light microscope. Phagocytic index was counted based on percentage
of phagocyte cells showing phagocytosis activity.
Phagocytic index (%) = Number of cell showing phagocytosis activity x 100
I
Number of cell hemocyte in glass slideI
The survival rate, growth, and rate of feed conversion ratio were counted by using
formula Zonneveld el al., 1991:
I
Survival Rate (%) = Number of final populationloo
I
Number of initial population
Growth = Average of final growth - Average of initial growth
Feed Conversion Ratio = Total given feed - total uneaten feed
Expcrinlental Design
Trial 1: Artificial Stress Condition and SIlrimp Immunity
The objcctive of this trial was to study the effect of oxygen concentration, water
survival rate, and nunlber of luminous bacteria in hemolymph. The result of this trial
could be used as a fit stressor in advanced trial. .
The trial was conducted with 3 replications and used factorial design. The
statistical model was factorial 2
x
2 x 2. The first factor I stressor was oxygen concentration which had levels of 3 and 6 ppm, the second factor I stressor was watersalinity which had levels of 20 and 70 ppt, and the third factor / stressor was luminous
bacteria concentration with levels of 0 and 10' cfu/ml. Statistical analysis for survival
rate data used the arcsine transformation method (Steel and Torrie, 1991).
One hundred and forty-four healthy shrimps (DOC: 50) were randomly divided
and put into 24 tanks: 6 shrimpsltank. The fattors were given in three steps; the first
stressor was given until the shrinip attained weak condition, then the secolid stressor was
given in 10 minutes (Baticados ct al., 1986), finally the third stressor was given until the
shrimp attained weak condition.
The temperature during the experimental period was maintained at 25 - 27 'C.
Oxygen concentration was made and maintained by regulating aeration. To avoid the
negative effect of feed in the environment. shrimp was not fed.
After shrimp attained weak condition, three shrimps from each tank were selected
to cxanline the number of lun~inous bacteria in shrimp hemolymph, total hemocyte, and
survival rate of shrimp.
Trial 2: P-glucan and Shrimp Performance
. .
Ihc objective of this trial was to study the effect of P-glucan on some parameterswith 3 replications and used randomized coluplete design. The treatments in this trial
were the dozes of P-glucan mixed in feed: 0%, 0.1%, and 0.5%. Statistical analyze for
growth data used the analyzes of covariance method (Steel and Torrie, 1991).
Three hundred healthy shrimps (DOC: 30) were randomly divided and put into 30
tanks: 10 shrimpsltank. Before dividing, shrimp weight was counted. To avoid
cannibalism, approximately, the shrimp weight in each tank was similar. The trial needed
30 days and feed for treatment was given adlibitum everyday.
After 30 days, shrimp weight and uneaten feed were counted to investigate shrimp
growth and feed conversion ratio. Uneaten feed was collected every 6 hours and was
baked in an oven with temperature 60°C.
Trial 3: Administration Period of P-glucan and Shrimp Immunity
The objective of this trial was to study the effectivity of 0-glucan given in 1 and 2
months on some parameters of shrimp immune system i.e. hemocyte count, phagocytic
activity, clearance of bacteria, and survival rate at a low oxygen concentration. The
experiment was an advanced experinieilt using the worst condition resulted by trial 1.
Tlie experiment was conducted with 3 replicatioils and used factorial design. The
statistical lnodel was factorial 3 x 2 x 2. The first factor was doze of P-glucan mixed in
feed which had levels 0%, 0.1% and O.j%, the second hctor was administration period of
0-giucan which had levels I aiid 2 months, and the third factor was luminous bactcria
concentration which had levels 0 and 10' c f d ~ i i l . Tlie number of luminous bacteria in
for phagocytic activity and survival rate data used the arcsine transformation method
(Steel and Torrie, 1991).
Tluee hundred healthy shrimps (DOC: 30) were randomly divided and put into 30
tanks: 10 shrimpltank. Before dividing, shrimp weight was counted. To avoid
cannibalism, approximately, the shrimp weight in each tank was similar. Based on the
result of the stressor test, shrimp was kept in low water concentration (3 ppm). The water
in each tank was changed every 3 days and the temperature during the experimental
period was maintained at 25 -27 OC. Besides, the stopping of aeration for about 6 hours
everyday, the stocking of low oxygen water, to water exchange process, was conducted to
maintain the low oxygen concentration in tank.
The feed for treatment given everyday was adlibitum. The commercial feed was
used as a continued feed given at the treatment using 1-month administration period of
P-
glucan.
Immersion of luminous bacteria (challenge test) was conducted when shrimp had
been kept and had been given P-glucan dozes for 30 days. The process of challenge test
in this experiment was the same as process conducted in the stressor test.
After shrimp attained weak condition caused by challenge test, three shrimps from
each tank were selected to examine the number of lulninous bacteria in hemolymph and
hepatopancreas, hemocyte count, and ~hagocytic activity of shrimp. Then, to investigate
bacteria clearance, the nu~nber of luminous bacteria in hemolymph and hepatopancreas
Continued examination of hemocyte count was conducted in the 711', 14'11, and 21''
day after challenge test. Continued examination of phagocytic activity was conducted in
the 711', 14", 21L', and 30''' day after challenge test. The survival rate of shrimp was
examined in the 30"' day after challenge test. ,
As additional examination, the water quality in each treatment, including total
ammonia nitrogen, nitrite, nitrate, and alkalinity concentration, was examined weekly. In
order to complete the data, the bacteria found in shrimp hemolymph and hepatopancreas
RESULTS AND DISCUSSION
Results
Identification of Vibrio Bacteria in Hemolymph and Hepatopancreas
The characteristic and biovar of Vibrio from henlolymph and hepatopancreas
were reported (Appendix I). Based on the Baumann et al. (1984) method these yellow,
green, and luminous bacteria indicated the characteristic of Vibrio alginolyticus, Vibrio
parahaemolyticus, and Vibrio splendidus biovar 1.
Trial 1: Artificial Stress Condition and Shrimp Immunity
Hemocyte count of shrimp was affected by individual and interaction of the
factors in treatment (Table 1). The significant effects (Pi0.05) on hemocyte count
occurred as the result of individual factors i.e. water salinity and Vibrio splendidus biovar
1 concentration and as the result of interaction factor involving oxygen, water salinity and
Vibrio splendidus biovar 1 concentration.
Although interaction among factors gave significant effect on hemocyte count, the
interaction between water salinity factor and ~ i b r i o splendidus biovar 1 concentration
factor gave the biggest effect. Qxygen factor only gave significant effect on hemocyte
count when it was interacted with water salinity factor and Vibrio splendidus biovar 1
conce~~tration factor.
The highest llemocyte count occurred at oxygen concentration
G
~ P I I I , waterTable 1. Mean number of the hemocyte count, su:vival rate of shrimp and number of
Vibrio splendidus biovar 1 colony (cfu) in hemolymph at 18 hours after
stressing condition.
Oxygen @pm)
3
Note *: Significant at P < 0.05
Salinity
@PO
20 70 Vibrio splendidusbiovar I (~fU/llll)
0
1 0'
0
Mean of parameters
Heinocyte count (cellln~mj)
448.00
526.67*
221 .OO*
Survival Rate (%)
82.98
78.68
82.98
Numb. Of lum. Bac. In
hemolymph (cfidml)
-
3.89
[image:109.595.56.507.132.795.2]As an individual factor, Vibrio splendidus biovar 1 and oxygen concentration
affected the survival rate of shrimp significantly (P < 0.05). The lowest survival rate
occurred at low oxygen concentration (3 p p n ~ ) and Vibrio splendidus biovar 1
concentration lo7 cfdml.
Significantly, the number of Vibrio splendidus biovar 1 in hemolymph was
affected by water salinity. The highest concentration of Vibrio splendidus biovar 1 in
hemolymph was found at water salinity 70 ppt.
Trial 2: P-glucan and Shrimp Performance
The result of trial 2 indicated that the growth and feed conversion ratio of shrimp
were not affected by the treatment.. All treatment had no significant effect on the
performance of shrimp (P < 0.05) (Table 2).
Trial 3: Administration Period of p-glucan and Shrimp Immunity
One of some results of trial 3 indicated that the most rapid bacteria clearance in
hemolymph occused in shrimp fed by feed containing P-glucan 0.1%, in less than 12
hours after challenge test. The clearance of bacteria in hepatopancreas for all treatment
occurred in less than 12 hours after the challenge test (Table 3).
Star~stical analyze for hemocyte count indicated as individual factor, P-glucan and
Vibrio sl)lei7cf1dzrs biovar 1 concentration gave significant effect on heliiocyte count in the
7"' day afier challenge test. The highest hemecyte count occurred in levcl of P-glucan
In 14"' day, as individual factor, P-glucan and administration period gave
significant effect on hemocyte count. The level of P-glucan 0%, 0.1%, and 0.5% could
affect significantly, but the biggest effect was given by 0-glucan 0.5%. The same result
was given by the level of 1-month administration period.
Besides as individual factor, factor of P-glucan and administration period,
together with Vibrio splendidus biovar 1 factor affected the hemocyte count of shrimp on
21'' day. The highest hemocyte count occurred in shrimp fed by feed containing
P-
glucan 0.5% given in 1 month and it was given under no bacteria condition (Table 4).
Statistical analyze for phagocytic activity indicated that as an individual factor
P-
glucan gave significant effect on phagocytic activity in the 7"' up to 3oth day after
challenge test. Although feed containing P-glucan gave better result in phagocytic
activity than other, both levels of P-glucan in feed (0.1% and 0.5%) had no significant
difference (Table 5).
Statistical analyze for the survival rate of sluimp indicated that all treatment save
the same effect. But, s!witnp fed by feed containing P-glucan gave survival rate higher
than shrimp fed by no P-glucan feed (Table 6).
Table 2. Mean number of growth and feed conversion ratio caused P-glucan administration
Treatment
Glucan 0 %
Glucan 0.1 %
Glucan 0.5%
Mean of parameters
Growth
5.00
4.98
5.31
Feed Conversion Ratio
1.19
1.12
[image:112.842.39.705.118.551.2]Table 3. Number of l/iDrio splendidus biovar 1 colony in hepatopancreas and hemolymph after challenge test
Treatment Number of Vibrio splendidzls biovar I ( c f u h l )
Hepatopancreas Hemolyniph
The 1
hour
1500
1500
975
'The 1'' hour
300
150
225 l'he
G"'
hour
--
-420
505
750
The 6"' hour
100
100
150 The 12"'
[image:113.842.158.681.121.338.2]Table 4. Mean number of hemocyte count at tllc 0. 7"', 14th. and 2ISL day after challenge test
Note *: Significant at P < 0.05
A: % p-glucan in feed
B: Administration period (month)
C: Vibrio splendidrr.: biovar I (cfulml)
Treatment
7
B2
1
2
C 0 1
o7
0 1
o7
0 10:
Meall of hemocvte count (cell/mni.') The 0 day
7166 7833 7900 8100 7566 7000 0.5
The 71h day 6966 8600 7550 9616 7900 9333 1 2
0
1
8!00 1091614516 10900 11233
The 14"' day 8000 8216 13850 11666 11400 9166
1
o7
0 1o7
[image:114.595.77.481.97.805.2]Table 5 . Mean number of phagocytic activity at the 0, 71h, 141h, 21s', and 30"' day after challenge test
Note
*:
Significant at - P < 0.05 A: % P-glucan in feedB: Administration period (month)
[image:115.595.71.527.104.800.2]Table 6 . Survival rate of shrimp
B: ~dlninistration period (month)
[image:116.595.81.479.77.798.2]TAN i parameter .. ~~~~ ~-~~~ I i
!
N l T R l T N I T R A T A L K P H I
parameters
. -. -. --
@ C O N T R O L - B A C T E R I A = C O N T R O L - N O B A C T E R I A
U G L U C . 0.1% - 1 M O N T H - B A C T E R I A U G L U C . 0.1% - 1 M O N T H - N O B A C T E R I A P G L U C . 0.1% - 2 M O N T H S - B A C T E R I A
I I CIGLUC. 0.1% - 2 M O N T H S - N O BACTERIA
I B G L U C . 0.5% - 1 M O N T H - B A C T E R I A
I !
O G L U C . 0.5% - 1 M O N i H - N O B A C T E R I A1
( I G L U C . 0.5% -2 M O N T H S - B A C T E R I A H G L U C . 0.5% - 2 M O N T H S - N O B A C T E R I A
I I -...-..-.._---d- ~
[image:117.602.82.479.57.794.2].. .... ~~ -. .. -
Trial 1: Artificial Stress Condition and Shrimp Immunity
The number of hemocyte in he~nolympli indicated their ability to respond some unadvantages condition e.g. stress condition. Anderson and Siwicki (1995) reported that the highest heinocyte count wandering in hernolymph, .the biggest respond could be given to hold the stress condition (Anderson and Siwicki, 1995).
111 this trial, the stress condition caused by concentration 10' cfu/ml of Vibrio splendidus biovar I gave the biggest effect on helnocyte count.
Besides affecting the hemocyte count, this concentration gave the worst effect on survival rate of shrimp.
Besides the highest concentration of Vibrio splendidus biovar I , the worst effect on survival rate of shrimp was caused by low oxygen concentration (3 ppm). Direkbusarakom and Danayadol (1998) reported that one of ilnportant states of the environlnent was the presence of sufficient oxygen to maintain a system vital to the health of an aquatic ecosystein. and the critical concentration for shrinip ponds was 3.7 pp111.
Salinity 70 ppt gave effect on the introduction of bacteria in shrimp body. It occurred easily when the oxygen concentration was low enough and especially wlieti the carapace of shrimp was soft. The low oxygen concentration increased the shrimp metabolism, thus to fulfill the osygen consumption, the shrimp lnlust more active to take oxygen in water. At tlie same time, high concentration of bacteria in water
would enter tlie shrimp body. Balicados el crl. (1986) observed that a hypet osmotic
All results in trial 1 indicated that shrimp im~nunity could be triggered by a stress condition involving stressor factors such as low oxygen concentration (3 ppm),
IGrio .sp/endiidr(s biovar I with concentralion 20' cfu/inl, and high salinity (70 ppi). Therefore, the interaction of these factors could bc a challenge test for the other trial. The challenge test was a test to give a challenge to investigate the animal capability after given a treatment.
Trial 3: P-glucan and Sl~rirnp Performance
Feed containing P-glucan had no effect on the performance of shrimp i.e. the shrimp growth and the feed conversion ratio. It indicated that no additional nutritious materials affecting the growth cells of shrimp body. It also indicated that adding
P-
gluca~i did not change the shrimp respond to the feed.
A proximate analysis conducted to analyze the contain of protein, lipid, ash, fibber? and nitrogen free materials indicated that all feed had no different level at their nutrient content (Table 7).
Trial 3: Administration Period of P-glucan and Shrimp Imniunity
Martin and arenda (1985) reported that gram positive and ~iegative bacteria in hemolymph could be cleared from hemalymph rapidly by gills in circulation process. In the first time, bacteria entered the shrimp body and before it was distributed to hemolymph the bacteria would stay in hepatopancreas a li~tle while. In this trial,
shrimp fed by P-glucan O,1% cleared Vibrio sp1ri~dicili.s biovav 1 i n their hepatopancreas and Iiemolymph more quickly than the otiisrs (Table 3). It indicated
that p-glucan O. I % \\,as a good bacleria-clearable substance.
day after challenge test \\,as the effect of P-glucan and bacteria factor individually
(Table 4 and 5 ) . Anderson and Siwicki (1995) reported that the increasing of heniocyie count in hemoly~npli was caused by infection, stress. and blood disease. The infection caused an inflammation; a non-specitic characteristic triggered by some factors e.g. parasite and bacteria (Person et al., 1987; Itami et a/., 1996).
Figure 2. The relationship between phagocytic activity and hemocyte count
FA: Pliagocytic activity, HC: He~nocyte count, -0, -7, -14, -21, -30: the 0: 7'h, 1411', 21s', and 30"' day after challenge test, A: p-glucan 0%
+
7 months+
0 cfulml K splendidzrs biovar 1, B : p-glucan 0%+
2 months+
lo7 cfulml V. splendidzrs biovar 1, C: P-glucan 0.1% + 1 months+
0 cfulinl V. splendidzrs biovar 1, D: P-glucan 0.1% + [image:121.599.84.474.80.360.2]31
Table 7. The nutrient content (% wet basis) of the trial diets.
Crude Protein
1
Crude LipidCrude Fibber
Moisture
Nitrogen-Free Materials
Feed containing
P-
glucan 0%Feed containing
P-
ylucan 0.1% [image:122.602.83.506.65.799.2]The phagocytic cells could phagocyte a peculiar substance or bacteria that entered the host. The process of phagocytosis involved some mechanism of attachment. engulfing, degranulation, and lyscsbacleria (Gudkovs, 1988). T h e cells which running phagocytic activity could be observed by light niicroscope. The process could be differed with the accuinulation of bacteria on the cells by observing the characteristic of color absorption.
Although the highest hemocyte count was found in shrimp fed by 6-glucan
O.j%, the phagocytic activity in shrimp fed by P-glucan 0.5% had no a significant
difference with the phagocytic activity in shrimp fed by P-glucan 0.1% (Table 4 and
5 ) . It indicated that the composition of hyaline cells in hemocyte of shrimp fed by
P-
glucan 0.1% was higher than in shrimp fed by P-glucan 0.5%. Thus, shrimp fed by P-glucan 0.1% was more resistant than the others because of their ability in phagocytic activity.
The p-glucan in feed could be digested by P-I, 3-glucanase enzymes in crustacean digestion system (Dall and Moriarty, 1983) before it \\,as distributed to he~nolymph system. Recognition protein in hemocyte plasnia could recognize the characteristic of P-glucan and lipopolysaccharide (Vargas-Albores e1 al., 1998).
Figure 2 showed that the increasing of phagocytic activity was caused by adding P-glucan in feed. Fecd containing P-glucan could increase the phagocytic
activity higher than commercial feed that had no P-glucan inside.
All treatnlenrs did not give significant difference on the survival rate o f shrimp. However, the survival rate of shrimp fed by P-glucan was higher than the other (Table 6).
Galeotti (1998) reported that prolonged im~nunostiniulant administration, in doze and time, did not give a better effect. In this trial, (3-glucan adtiiinistration in 2
CONCLUSSJON
The general conclusion of all trial was the low oxygen and high bacteria concentration gave a bad effect on shrimp defence mechanism. The high water salinity affected the entering of bacteria in shrimp body.
P-glucan extracted from Saccharon~yces cerevisiae by alkali-acid method
(William el al., 1991) could increase some parameters o f the non-specific immune syste~ii of shrimp that was kept in low oxygen concentration (3 ppm), but they had no effect on the performance of shrimp, because the trial was conducted in short time to give evidence of their effect on the performance of shrimp. Feed containing P-glucan
0.1% gave a better effect than P-glucan 0.5% in their role to increase the non-specific
immune system of shrimp by increasing phagocytic activity.
The administration of P-glucan in 2 months did not give a better result than the
ad~ninistration o f P-glucan in I month. Thus. the best administration period o f P-
Andcrson, D.P. 1992. Iinmunostimulants. Adjuvant, and Vaccine Carriers in Fish: Applications to Aquaculture. .Ann. Rev. Fish Dis.. 2: 281- 307.
and Siwicki. 1995. Basic Hematology and Serology for Fish Health Programs, p. 185-202. Inshariff, M.. R.P. Subasinghe and J. Arthur (ed.). Diseases in Aquaculture 11. Fish Healrh Secr. Asian Fish Soc., Manila.
Baticados, M.A.C.L., R. M. Coloso, and R.C. Duremdez. 1986. Studies on the
Chronic Soft-Shell Syndrome in the Tiger Pra\tn, Periaelcs
nzonodon Fabricius, from Brackish water Ponds. .Squaculture. 56:271-285
Bauman, P., A.L. Furniss and J.V. Lei. 1984. Facultatively Anaerobic Gram- Negative Rods, p. 518 - 536.
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Tansill, B. (ed.). Bergey's Manual of Systematic Bacteriolog. William and Wilkis. Baltimore.Blaxhall, P. and K. Daisley. 1973. Some Blood Parameters of the ~ n b o f i Trout I. The Kamloops Variety. J. Fish Biol.. 5:l-8.
Clifford, H. 1999. Immunostimulailts, p. 243. InWorld Shrimp Farmi~g 1999
Number 12 Published Allnually. Shrimp N e x j Intemationa!.
Dall, W. and D.J.W. Moriarty. 1983. Functional Aspects of Sutritian and
Digestion, p: 215 - 261. Mantel
L.H.
(ed). Internal A?atom].- and Physiological Regulation. The Biology of Crustacean. Vol 5. Academic Press. Toronto.i o n on Direltbusarakoni, S. and Y. Danayadol. 19.98. Effect of Oxygen Deplsz-
Soine Parameters of the In1mune System in Black Tiger Shrimp
L \ ailcss (Pe!me~rs nzoriodon), p. 147-149. Flegel. T.W. (ed.). A' .
in Shrimp Biotechnology. National Center for Genetic Englxeerin~ and Biotechnology, Bangkok.
Galcolti, M. 1998. Some Aspects of the Application of Immunostirnu1~.-7ts and
a Critical Review of Methods for Their Evaluation. J. Appl.
Ichthyol., 14: 189-199.
Graves, U.L. and G.M. Gary. 1985. Fine Srructure and Classificzrion of
Gudkovs.
N.
:988. Fish Imniunology Fish Diseases Refresher Course For Vcrerinarians. Proc.Itami, T. 1994. Body Defencc System of Pe~iaeid Shrimp. Department of Aquaculture and Biology. Bulletin of Shimonoseki-slii University of Fisheries Japan, 759-65.
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M. Kondo. and Y. Takahashi. 1996. Enhancement of DiseaseResistatice of Kuruma Prawn, P L ~ ~ ~ O E I I S jal~onicus After Oral Administration of Peptidoglycan. National Fisheries University, Japan. 70 p.
Kondo, M., Itanii, Y. Takahasyi, R. Fujii, and S. Tomonaga. 1996.
Ultrastructure and Cytocheniical Characteristics of Phagocytes in
Kuruma Prawn Penaeus japonicus. Shinio-ono National Fisheries
University, Japan. 13:754-759.
Lavilla-Pitogo, C.R.L., M.C.L. Baticados. E.R. CIUZ Laci-erda, and L.D. Dela
Pena. 1990. Occurrence of Lu~iiinous Bacterial Disease of
Penaeus n~onodon Larvae in the Philippines. Aquaculture, 91: 1- 13.
Lightner, D.V. 1988. Disease of Cultured Penaeid Shrimp and Prawns, p. S- 127. ~ S i n d e r m a n n , C.J. and D.V. Lightner (eds.). Diseases Diagnosis and Co~itrol in Sorth American Marine Aquaculture. Elsevier. Amsterdam.
Marlin, G.G. and G.L. Brenda. 1985. Clearance of Bacteria Injected into the
I-Icmoly~iipli of the Ridgeback Prawn, Sicyonia ingentis
(Crustacea:Decapoda): Role of Haematopoietic Tissue. J. Morph.,
227: 227-233
Maynard. D.M. 1960. Circulations and Heai-t Function, p. 81-87.
Watermanu,
T.N.
(ed.). The Pfiysiology of Crustacean I.Academic Press. Nen York.
Person, M., L. Cerenius, and K. Soderhall. 1987. The Influence of Haemocyte
Number oil the Resistance of the Freshwater Crayfish Pqcifasltts lei7iuscztl~cs llaua, to the Parasitic Fungus A/1hnnoil7yce.s nsfaci. J. Fish Diseases. 10: 471-477.
and J. Arthur (eds.). Diseases in Asiail Aquaculture I. Fish I-Iealth Sect. Asian Fish Soc., Manila.
Itukyani. A. and A. Sunarto. 1998. Manayeinen Kesehatan Udang dcngan
Meuggunakan In~munostimulall LPS. PT Apperindo - JICA ATA-
379. Bali. Prosiding
Soderliall. K. and M.W. Johansson. 1985. Exocytosis of the Prophenoloxidase Activating Spstenl from Crayfish Haemocytes. J. Comp. Pllysiol B.. l56:175-181.
and V.J. Smith. 1989. Internal Defence Mechanism, p. 213-
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Holdich, D.M. and R.S. Lowwery (ed.j. FreshwaterCrayfish. Biology, Management, and Exploitatiou. Croom Helm Timber Press. London.
Steel. R.G.D. and J.H. Torrie. 1991. Principles and Procedures of Statistics. Teriemahan. Sumantri, B. Prinsip dan Prosedur Statistika, Suatu ~ e i d e k a t a n Biometrik. Gra~nedia. Jakarta. 748 p.
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Vargas-Albores, F., Fernandes-I;opez, J., Gollas-Galvan, T. Montano-Perez,
K.
Jimenez-Vega, F., and Yepiz-Plascencia, G. 1998. Activation of
Shrimp Cellular Defence Functions by Microbial Product. Flegel, T.W (ed.). Advances in Shrinlp Bio:cclinology. Nationa! Center for Genetic Engineering and Biotechnology. Bangkok.
Volpatti. D., L.D. Angelo. G. Jeney, Z. h e y , D.P. Anderson, and M. Galeotii. 1998. Nonspecific Immune Response in Fish Fed Glucan Diets Prior to Induced 'Transporration. .I. Appl. lchthyoi., 14:201-206.
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Soiubilizalion of a (I,.?)-P-d-Glucan Isolated from S~cchnrorl~j.ces
cerevisine. Carbohydv Res., 219: 203-21 3.
Appendix 1. The differential characteristics of the species and biovar of the
Discrepancies
+
-t -+
+
- i+
21 - 22 23 24 25 26 273 0
3 1
32 28 29 Sucrose Salicine D-rnannitol D-glucose D-Sorbitol Production of Gelatinase Anlylase
I
MediaNitrate to Nitrite
MR VP -
+
+
-+
+
IndoleSwamling on Solid
+
+
-+
-+
+
i
+
Appendix 2. The analyses of variance for the hemocpte count in hemoiymph at
18 hours after stressi~lg condition (Trial 1).
Source
SAL
OX
BAC
SAL
*
OXSAL
*
BACOX
*
BACSAL
*
OX*
BAC15rror
Total
Corrected Total
Appendix 3. The analyses of variance for survival rate of shrimp at 18 hours
after stressing condition (Trial 1).
Source
S AL
OX
BAC
SAL
*
OXSAL
*
BACOX
*
BACSAL
*
OX*
BACE11.o~ Total Corrected Total df 1 1 1 1 1 1 1 16 24 23 Type I11 Sum
43
Appendix 5. The analyses of covaria~lce for the growth of shrimp (Trial 2)
Source X GLUC Error Total Corrected Total
44
Appendix 7.' The analyses of variance for the hemocyte count in hemolympl~ on the 0 day after challenye test.
Source GLUCAN MONTH BAC GLU* MONTH Corrected Total
Appendix 8. The analyses of variance for the hemocyte count in liemolynip11
cn the 7"' day after clial!enge test.
Source
GLUCAN
MONTH
BAC
GLU
*
MONTHGLU
*
BACMONTH
*
BACGLU
*
MONTH*
BAC Error
Total
Corrected Total
Appendix 9. The analyses of variance for thc hcmocytc count in hernolymph
oe test. on the 14"' day after cliallen,
Source
GLUCAN
MONTH
BAC
GLU
*
MONTHGLU
*
BACMONTH I' BAC
GLU
*
MONTH*
B AC Error Total Corrected Total Mean Square 44896458.333 22041 666.667 772278 1.532 1870416.667 3181250.000 93750.000 60000.000 2106416.667 Type 111 Sum of
Appendix 10. The analyses of variance for the hei~~ocyte coullt in l~ernolymph
me test. on the 21" day after challen,
Source
GLUCAN
MONTI-l
B AC
GLU
*
MONTHGLU
*
BACMONTH
*
BACGLU
*
MONTH"
BAC ErrorTotal
Corrected Total
Type I11 Sum of Squares 53580069.444
805041 6.667
526357.786
1550416.667
694 104 1.667
Appendix 11. The analyses of variance for the phagocytic activity on the 0 day
after challenge test.
Source
GLUCAN
MONTH
BAC
GLU
"
MONTHGLU
*
BACMONTH
*
BACGLU
*
MONTH*
BAC Error Total Corrected Total df 2 1 1 1 2 1 1 20 30 29 Type I11 Sun1
of Squares 9.924E-02 1.707E-02 1.035E-02 2.940E-02 3.851E-02 2.407E-02 1.067E-03 ,370 3039.796 .669 Mean Square 4.962502 1.707E-02 1.03jE-02 2.940E-02 1.926E-02 2.407E-02 1.067E-03 1.SjOE-02 F 2.683 ,923 559 1.589 1.041 1.301 ,058 Sig. .093 248 .463 222 ,371
2 6 7
