(vzw)
Fish & Shellfish
Immunology
ELSEVIER
F ish & S h ellfish Im m u n o lo g y 23 (20 0 7 ) 141 —153
w w w .e ls e v ie r.c o m /lo c a te /fsi
Influence of different yeast cell-wall mutants on performance
and protection against pathogenic bacteria (Vibrio campbellii)
in gnotobiotically-grown Artemia
Siyavash Soltanian a,b’*, Jean D h o n t3, Patrick Sorgeloos a, Peter B o ssier3
a L a b o ra to ry o f A q u a c u ltu re & A rte m ia R e fe ren c e C enter, F a c u lty o f B io sc ie n ce E n g in ee rin g , G h e n t U niversity,
R o z ie r 4 4, 9000 G ent, B elgium
b A q u a tic A n im a l H ea lth & D isea se s D ep a rtm en t, S c h o o l o f V ete rin a ry M edicine,
S h ira z U niversity, Isla m ic R e p u b lic o f Iran
R eceiv ed 2 2 M ay 2 0 0 6 ; re v ised 18 S e p te m b e r 2006; acc e p te d 2 9 S e p te m b e r 2006
A v ailab le o n lin e 11 O c to b e r 2006
A bstract
A s e le c tio n o f is o g e n i c y e a s t s t r a i n s ( w ith d e l e t i o n f o r g e n e s in v o lv e d in c e l l - w a l l s y n th e s is ) w a s u s e d t o e v a l u a te t h e i r n u tr i­
t i o n a l a n d i m m u n o s tim u la to r y c h a r a c t e r i s t i c s f o r g n o t o b io tic a lly - g r o w n A r te m ia . I n th e f ir s t s e t o f e x p e r i m e n t s th e n u tr i tio n a l v a lu e
o f i s o g e n i c y e a s t s tr a in s ( e f f e c te d in m a n n o p r o t e i n s , g l u c a n , c h itin a n d c e l l- w a ll b o u n d p r o t e i n s y n th e s is ) f o r g n o to b io tic a lly - g r o w n
A r t e m i a w a s s tu d ie d . Y e a s t c e l l- w a ll m u t a n t s w e r e a l w a y s b e t t e r f e e d f o r A r te m ia th a n t h e is o g e n i c w ild t y p e m a in ly b e c a u s e th e y
s u p p o r t e d a h i g h e r s u r v iv a l b u t n o t a s t r o n g e r in d iv id u a l g r o w th . T h e d if f e r e n c e in A r te m ia p e r f o r m a n c e b e tw e e n W T a n d m u ta n ts
f e e d i n g w a s r e d u c e d w h e n s ta t i o n a r y - p h a s e g r o w n c e l ls w e r e u s e d . T h e s e r e s u lts s u g g e s t t h a t a n y m u ta tio n a f f e c tin g th e y e a s t c e llw a ll m a k e - u p is s u f f ic ie n t to i m p r o v e th e d ig e s t i b i l i t y in A r te m ia . T h e s e c o n d s e t o f e x p e r i m e n t s , in v e s t ig a te s t h e u s e o f a s m a ll
a m o u n t o f y e a s t c e l l s in g n o to b io tic A r te m ia to o v e r c o m e p a th o g e n ic it y o f V ib r io c a m p b e ll ii (V C ) . A m o n g a ll y e a s t c e ll s tra in s
u s e d in th i s s tu d y , o n ly m n n 9 y e a s t ( le s s c e l l- w a ll b o u n d m a n n o p r o t e i n s a n d m o r e g lu c a n a n d c h itin ) s e e m s to c o m p le te l y p r o te c t
A r te m ia a g a i n s t t h e p a th o g e n . I n c o m p l e t e p r o t e c t i o n a g a i n s t th e p a th o g e n w a s o b t a i n e d b y th e g a s l a n d c h s 3 m u ta n ts , w h ic h a r e
l a c k i n g th e g e n e f o r a p a r tic u la r c e l l - w a l l p r o te in a n d c h i t i n s y n th e s is , r e s p e c tiv e ly , r e s u l t i n g in m o r e g lu c a n . T h e r e s u l t w ith th e
c h s 3 m u t a n t is o f p a r tic u la r i n te r e s t, a s its n u tr i tio n a l v a l u e f o r A r te m ia is c o m p a r a b l e to th e w ild ty p e . H e n c e , o n ly w'i.th th e c h s 3
s t r a i n , in c o n t r a s t to th e g a s l o r m n n 9 s tr a in s , t h e t e m p o r a r y p r o t e c t i o n to V C is n o t c o n c o m i t a n t w ith a b e t t e r g r o w th p e r f o r m a n c e
u n d e r n o n - c h a lle n g e d c o n d itio n s , s u g g e s t i n g n o n - i n t e r f e r e n c e o f g e n e r a l n u tr i tio n a l e f f e c ts .
© 2 0 0 6 E l s e v i e r L t d . A ll r ig h t s r e s e r v e d .
K ey w o rd s: A rte m ia ; G n o to b io tic c u ltu re ; S a c ch a ro m y ce s c e re visia e ; Iso g e n ic y e a st m u tants; Im m u n e ability ; V ib rio c a m pbellii
C orresp o n d in g author. L ab o rato ry o f A q u acu ltu re & A rte m ia R eferen c e C enter, Faculty o f B io scien ce E ngineering, G hent U niversity, R ozier 44,
9 0 0 0 G e n t, B elg iu m . T e l: 4-32 9 2 6 4 3 7 5 4 ; fax: + 3 2 9 2 6 4 4 1 9 3 .
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142
S. S o lta n ia n e t al. / Fish & S h e llfish Im m u n o lo g y 23 (2 0 0 7 ) 141—153
1. Introduction
Im m unom odulation o f larval fish has been proposed as a potential m eth o d for im proving larval survival by increas­
ing the innate responses o f the developing anim als until their adaptive im m une response is sufficiently developed to
increase an effective response to the pathogen [1].
Invertebrates are not equipped w ith cells that are analogous to antibody producing lym phocytes in vertebrates.
A ccording to R aa [2], invertebrates are apparently entirely dependent on non-specific im m une m echanism s to cope
w ith infections, as they lack the specific im m unological “ m em o ry ” that is found in fish and w arm -blooded anim als.
A s a result, it does not seem to m ake sense to vaccinate them against any specific diseases .Yet, a recent study in the
copepod M acrocyclops albidus show ed that the defence system o f this invertebrate species reacted m ore efficiently
after a previous encounter w ith an antigenically sim ilar parasite, im plying that a specific m em ory may exist [3].
F urtherm ore, exposure o f shrim p to inactivated Vibrio spp. has b een reported to p rovide som e protection [4—6].
T he use o f specific biological com pounds (im m unostim ulants) th at en hance im m une responses o f target organism s,
rendering anim als m ore resistant to diseases may be an excellent preventive tool against pathogens [7]. Such
substances m ay reduce the risk of disease outbreaks if adm inistered p rio r to a situation know n to result in stress
and im paired general perform ance (e.g. handling stress, change o f tem perature or o th er environm ental param eters,
w eaning from live to artificial feeds) or prior to an expected increase in exposure to pathogenic m icro-organism s
and parasites (e.g. spring and autum n bloom s in m arine environm ent, transfer to engrow ing system s).
Several im m unostim ulants have been used in vertebrate and invertebrate culture, to induce protection against
a w ide range o f diseases: i.e. ß-glucans [8—11], chitin [12—14], m annoproteins [15], lipopolysaccharides [16],
peptidoglycans [5,17] and dead bacteria [4,18,19].
M arques et al. [20,21] have recently developed and validated the usefulness o f an A rtem ia gnotobiotic test system
allow ing to study the effect o f food com position on survival and grow th in the presence or absence o f a pathogen.
B ak er’s yeast Saccharom yces cerevisiae, w hich has been found to be a good im m une enhancer in som e aquatic
organism , is an excellent source o f ß-glucans and chitin. T hese com pounds together w ith m annoproteins constitute
the m ajor com pounds o f the yeast cell w all [22]. T he present study aim s to identify the critical cell-w all com ponents
that induce pathogen-protection in Artem ia. T he effect o f isogenic yeast deletion m utants (eight strains), carrying
a null m utation in a gene involved in cell-w all synthesis, w as evaluated in a gnotobiotic A rtem ia test system . Firstly,
A rtem ia perform ance w as exam ined w ith the null-m utant y east cells, harvested in exponential and/or stationary
grow th phase. In a second stage, these feed sources w ere tested in com bination w ith a Vibrio cam pbellii challenge.
2. M ethodology
2.1. A xe ni c culture o f yeast
To verify the digestibility of live b aker’s yeast (S. cerevisiae) by A rtem ia, seven different null-m utants o f yeast (iso­
genic deletion strains derived from b aker’s yeast strain B Y 4741) an d the w ild type strain (W T) (genotype described in
Table 1) w ere fed to A rtem ia. All strains w ere provided by E U R O SC A R F (University o f F rankfurt, Germ any).
Yeast cultures w ere perform ed according to procedures previously described by M arques et al. [20], using m inim al
Y east N itrogen B ase culture m edium (YNB).
Yeasts w ere harvested by centrifugation (± 8 0 0 x g for 10 m in), either in the exponential growth p hase (after 20 h;
“ ex p .y east” ) o r in the stationary growth phase (after 3 days; “ stat.y east” ). Yeast cell concentrations w ere determ ined
w ith a B tirker haem ocytom eter. Yeast suspensions w ere sto red at 4 °C until the end o f each experim ent (m axim um
storage o f one w eek).
2.2. B acterial strains a n d grow th conditions
Two bacterial strains w ere selected, i.e. A erom onas hydrophila strain LVS3 [23—25] for its positive effect on
A rtem ia perform ance w hen fed sub-optim ally and Vibrio cam pbellii strain LM G 21363 (VC) for its pathogenic effect
tow ards A rtem ia and shrim p [25—27]. T he tw o bacterial strains w ere cultured and harvested according to procedures
previously described by M arques et al. [25], P ure cultures o f th e two bacterial strains w ere obtained from the
L aboratory o f M icrobial E cology and Technology, G ent U niversity, and from the L aboratory o f M icrobiology,
S. S o lta n ia n et al. / F ish & Shellfish Im m u n o lo g y 23 (2 0 0 7 ) 1 4 1 —153
143
T a b le 1
G e n o ty p e o f all y e a st strain s used as fe e d fo r A rte m ia an d d e sc rip tio n o f each gene m utation in th e dev elo p m en t o f c ell-w a ll co m p o n en ts
S train s
G en o ty p e
P h e n o ty p e
R eferen c e
(c e ll-w a ll ch anges)
WT
B Y 4 7 4 1 ; M a t a; h is 3A1; leu
C o n tro l y east
D a llie s e t al. [43]; K lis e t al. [35];
m nn9
2 A 0; m e t 15A 0; u ra 3 A0
B Y 4 7 4 1 ; M a t a; h is 3A1; leu
L e ss m a n n a n , h ig h er
M a g n e lli e t al. [22]; M arq u es e t al. [20,21]
K lis e t al. [35]; M ag n elli e t al. [22];
2A 0; m e t 15A 0; u ra 3 A0;
Y P L 0 5 0 c::k an M X 4
c h itin , h ig h e r ß-glucans
M a rq u es e t al. [20,21]
B Y 4 7 4 1 ; M a t a; h is 3 A l; leu
2A 0; m e t 15A 0; ura 3 A0;
L ess p h o sp h o m an n an
m nn6
K a rso n an d B allou [44]; W ang and N a k ay a m a [45];
J ig a ra i an d O dani [34]
Y P L 0 5 3 c::k an M X 4
fk s l
B Y 4 7 4 1 ; M a t a; his 3A1; leu
2A 0; m e t 15A 0; u ra 3A 0;
L e ss ß-1,3 g lucans,
h ig h e r ch itin
Y L R 3 4 2 w ::k an M X 4
k n r4
k re 6
chs3
A g u ila r-U sc an g a an d F ran c o is [29]
B Y 4 7 4 1 ; M a t a; his 3 A l; leu
L ess ß-1,3 g lucans,
2A 0; m e t 15 A0; u ra 3 A 0;
Y G R 2 2 9 c::k an M X 4
h ig h e r ch itin
B Y 4 7 4 1 ; M a t a; h is 3A Í; ¡eu
L ess ß -1 ,6 g lucans,
M ag n e lli e t al. [22]; A g u ila r-U sc an g a an d Francois
2A 0; m et 15A0; ura 3 A 0;
Y P R 1 5 9 w ::k an M X 4
h ig h e r ch itin
[29]; M artin -Y k e n e t al. [30]; P agé e t al. [46]
B Y 4 7 4 1 ; M a t a; h is 3A1; leu
L ess ch itin
V aldivieso e t al. [47]; C ab ib e t al. [31];
K lis e t al. [35]; M agnelli e t al. [22];
B Y 4 7 4 1 ; M a t a; h is 3A1; Ieu
L ess in teg ratio n o f y east
D e N obel e t al. [38]; P o p o lo e t al. [48];
2 A 0; m e t 15A0; ura 3 A0;
c ell a d h esio n p ro te in s
L ip k e a n d O v a lle [33]; M ag n e lli e t al. [22]
Y M R 3 0 7 w ::k an M X 4
in to th e c ell w all less
D a llie s e t al. [43]; M ag n elli e t al. [22];
M artin -Y k e n e t al. [30]; P a g é e t al. [46];
A g u ila r-U sc an g a and F ran c o is [29]
2 A 0; m e t 15A0; ura 3A 0;
Y B R 0 2 3 c::k an M X 4
gasl
D a llie s e t al. [43]; M agnelli e t al. [22];
M artin -Y k e n e t al. [30]; P a g é e t al. [46];
ß -1 ,3 g lu c a n s, h ig h e r ch itin
G ent U niversity. T he bacterial strains w ere stored at —80 °C and grown overnight at 28 °C on m arine agar, containing
D ifco™ m arine broth 2216 (37.4 g I- 1 , B D B iosciences) and agar bacteriological grade (20 g I- 1 , ICN ). For each
bacterial strain a single colony w as selected from the plate and incubated overnight at 28 °C in 50 ml Difco™ marine
broth 2216 on a shaker (150 rpm ). Stationary-grow n bacteria w ere harvested by centrifugation (15 m in; ± 2 2 0 0 x g);
the supernatant w ere discarded and the pellet resuspended in 20 ml filtered autoclaved sea w ater (FASW ). B acterial
densities w ere determ ined by spectrophotom etry (O D 550), assum ing that an optical density o f 1.000 corresponds to
1.2 x IO9 cells m l- 1 , according to M cFarland standard (B iom erieux, M arcy l ’E toile, France).
B acteria w ere resuspended in filtered autoclaved sea w ater (FASW ) and their densities w ere determ ined by spec­
trophotom etry (O D 550), assum ing that an optical density o f 1.000 corresponds to 1.2 x IO9 cells m l- 1 , according to
M cF arland standard (B iom erieux, M arcy 1’E toile, France).
A t d ay 3, challenge tests w ere p erform ed w ith live V C . F or that purpose, in a lam inar flow hood, the pathogen was
p ro v ided to each replicate at a density o f 5 x IO6 cells m l- 1 . D ead LVS3 was provided to A rtem ia using aliquots o f
au toclaved concentrated bacteria (autoclaving at 120 °C fo r 20 min). A fter autoclaving, bacteria w ere plated to check
if they w ere effectively killed by this m ethod. F or this purpose, 100 pi o f the culture m edium w ere transferred to m a­
rin e agar (M A; n — 3), containing Difco™ m arine broth 2216 (BD B iosciences, 3.74% w/v) and agar bacteriological
grade (ICN, 2% w /v). A bsence o f bacterial grow th w as m onitored after incubating plates for 5 days at 28 °C. A uto­
claving treatm ent was 100% effective, since no bacterial grow th w as observed on the M A after 5 days o f incubation.
D ead and live bacterial suspensions w ere stored at 4°C until the end o f each experim ent.
2.3. Y east a n d bacterial ash-fi-ee content
To determ ine the yeast and bacterial ash-free dry w eight (AFD W ), 50 ml o f each culture sam ple w ere filtered on
pre-d ried filters (pore size 0.45 pm , two replicate p er culture). Filters w ere subsequently dried at 60 °C for 24 h and
w eighed. A fterw ards they w ere com busted at 600 °C fo r 6 h to determ ine the ash content. T h e A FD W w as calculated
as the difference betw een dry w eight and ash w eight. T h e D W and A FD W o f control (filter only, n = 2) were sub­
tracted from all sam ples. T he A FD W o f the yeast strains and the bacteria is presented in Table 2.
S. S o lta n ia n et al. / F ish & S h e llfish Im m u n o lo g y 2 3 (2 0 0 7 ) 1 4 1 —153
144
T able 2
A verage ash -free d ry w e ig h t (A F D W ) o f sev en d ifferen t n u ll-m u tan ts o f y e a st (iso g e n ic stra in s d e riv e d from B Y 47 4 1 ) a n d th e w ild ty p e strain (W T )
h a rv e ste d in the e x p o n en tial a n d statio n ary g ro w th p h a se , to g e th e r w ith A F D W o f d e ad L V S 3 and live V C b a c te ria e x p re sse d in m g /1 0 9 cells
Strains
A F D W (m g /IO9 cells)
A F D W (m g /F a lc o n tu b e)
E x p o n en tial
S tatio n ary
p h a se
phase
E x p o n e n tia l
ph a se
p - v alue
S ta tio n a ry
ph a se
E x ponential
vs stationary
ph a se A FD W
(m g /F alco n tube)
WT
15.24 ± 0.18 f
13.69 ± 0 . 0 7 d
1.60 ± 0 . 0 2 '
1.44 ± 0 . 0 1de
0.014
m nn9
3 6 .4 0 ± 7.2 3 a
5 .7 4 ± 0 . 17a
3 .8 2 ± 0 . 7 5 “
0.161
m nnó
5 4 .6 7 ± 1.66a
17.09 ± 0 . 3 7 '
11.83 ± 0 . 1 0 de
1.79 ± 0.04d
1.24 ± 0 . 0 1 '
fk s l
knr4
1 8 .9 0 ± 1.41d
14.77 ± 0 . 2 6 f
17.73 ± 0 .2 8 °
13.17 ± 0.13d
1.98 ± 0 .1 3 d
1.86 ± 0.0cd
0.013
0.644
1.55 ± 0 . 0 3 '
3 4 .5 4 ± 1.41b
2 4 .5 2 ± 1 . 2 5 b
3.63 ± 0 .2 8 b
1.38 ± 0 . 1 0 “®
2 .5 7 ± 0.13b
0.037
kre6
chs3
16.4 ± 0 . 1 2 '
1.72 ± 0.01d
1.15 ± 0 . 0 4 '
g asl
2 9 .0 9 ± 0.86c
11.0 ± 0 . 4 0 '
2 0 .3 0 ± 2 . 6 0 bc
L ive LV S3
D ead LV S3
-
0 .2 1 8 6 ± 0 . 0 2 f
-
-
0.2725 ± 0.0 2 f
-
0.023 ± 0 . 0 1 f
0 .0 2 9 ± 0.011
0.020
0.1
-
L ive V C
-
0 .1 1 3 4 ± 0 .0 1 *
-
0 .0 3 4 ± 0 . 0 1 r
13.05 ± 0 . 0 9 '
2 .1 3 ± 0.28bc
0.076
-
V alues o f A F D W a re p re se n te d w ith th e re sp ec tiv e stan d ard d e v iatio n (m e a n ± S D ). V alues in th e sam e c o lu m n sh o w in g th e sa m e su p erscrip t le tte r
a re n o t sig n ific a n tly d iffe re n t (p Xllk<,y > 0.05). p -v a lu e s o b ta in e d fo r d ire c t c o m p a riso n o f A F D W (m g /F alco n tube) o f d ifferen t y e a st cell strains,
h a rv e ste d in e x p o n en tial a n d statio n ary gro w th p h a se w e re in clu d ed . S ig n ifican t d ifferences w e re o b ta in e d w hen p Tukey < 0 .0 5 .
2.4. A rtem ia gnotobiotic culture
E xperim ents w ere perform ed w ith A rtem ia fra nciscan a cysts, originating from G reat S alt L ake, U tah, U SA (EG ®
type, IN V E A quaculture, B elgium ). B acteria-free cysts and nauplii w ere obtained using the procedure described by
M arques el al. [20]. A fter hatching, 20 nauplii (Instar II) w ere p icked and transferred to F alcon lubes containing 30 ml
o f FASW together w ith the am ount o f feed scheduled fo r day 1. F eeding rates w ere intended to provide a d libitum
ratios but avoiding excessive feeding in order not to affect the w ater quality in the test tubes, except in Experim ents
4 and 5 (treatm ents 19 and 20) w here nauplii were overfed (5.74 m g A FD W /FT ) (Table 5, feeding regim e: (d) in order
to verify the effect o f overfeeding. E ach treatm ent consisted o f four Falcon tubes (replicates). Falcon tubes were
placed on a rotating rod at 4 cycles p er m in, exposed to constant incandescent light ( ± 41 p E m -2 ) at 28 °C. Tubes
w ere being transferred to the lam inar flow ju s t once p er day for feeding.
2.5. M ethod used to verify axenity
A xenity o f feed, decapsulated cysts and Artemia cultures w ere checked at the end o f each experim ent using a com bi­
nation o f plating (MA) and live counting (using tétrazolium salt M T T staining following the procedure described by
M arques e t al. [20,21]. In challenge treatm ents, the axenity o f A rtem ia culture w as always checked before challenge using
the sam e methods. Contam inated culture tubes w ere not considered fo r further analysis and the treatm ent w as repeated.
2.6. E xperim ental design
In E xperim ent 1, all live and axenic yeast strains (W T and seven null m utants) w ere harvested in the exponential
grow th phase and used as feed for the Artem ia.
In E xperim ent 2, stationary-grow n live and axenic yeast strains (the sam e strains as used in Exp. 1) w ere used as
feed fo r nauplii. In both experim ents, a m odified feeding schedule w as adopted from Coutteau et al. [28] and M arques
et al. [20]. T he feeding schedule resulted in an equal am ount o f yeast-cell particles p er treatm ent being offered to
A rtem ia. B oth experim ents w ere perform ed tw ice (A and B), to verify the reproducibility o f the results.
In E xperim ent 3, an equal am ount o f feed w as provided to A rtem ia (Table 5). A s the A FD W per cell o f the yeast
m utants is different (see Table 2), this resulted in different am ount o f yeast cells being offered. E ach feed w as tested in
four replicates.
S. S o lta n ia n e t a i / F ish <£ Shellfish Im m u n o lo g y 23 (2 0 0 7 ) 1 4 1 —153
145
In E xperim ents 4 and 5, all treatm ents w ere fed w ith an equal am ount o f y east (in term s o f A FD W ). Yeast strains (in
exponential and/or stationary grow th phase) w ere provided daily in sm all but equal am ounts, in com bination with dead
LVS3 (as a m ajor part o f the feed) to A rtem ia (Table 5 — feeding regim e for E xps. 4 and 5). A s a control, A rtem ia was
fed only dead LVS3 (Table 5 — feeding regim e: (c). C hallenge tests were perform ed w ith live V C at a density of
5 x IO6 cells m l-1 added at day 3.
2.7. Survival a n d grow th o f A rtem ia
Survival and grow th of A rtem ia nauplii w ere determ ined according to procedures described by M arques et al.
[20,21]. A t the end o f E xperim ents 1, 2 and 3 (day 6 after hatching) the n um ber o f sw im m ing larvae w as determ ined
and survival percentage was calculated. L iving larvae w ere fixed w ith L u g o l’s solution to m easure their individual
length (grow th calculation), using a dissecting m icroscope equipped with a draw ing mirror, a digital plan measure
and the softw are A rtem ia l.O0 (M am ix Van D om m e). In order to integrate th e results o f survival and grow th, the cri­
terion “ total le n g th ” was introduced, i.e. total m illim eters o f A rtem ia per Falcon tube or m m /FT = num ber o f survi­
vors x m ean individual length.
In E xperim ents 4 and 5 the survival percentage for each treatm ent was determ ined daily. F or this purpose, the num ­
ber o f live A rtem ia w as registered before feeding (or adding any bacteria) by exposing each transparent F alcon tube to
an incandescent light w ithout opening the tube to preserve the axenity.
Values o f larval survival (percentage) w ere arcsin transform ed, w hile values o f individual length and total length
w ere logarithm ic or square root transform ed to satisfy norm al distribution and hom ocedasticity requirem ents. D iffer­
ences o n survival, individual length and total length o f A rtem ia fed with different feeds, w ere studied w ith analysis of
v ariances (ANOVA) and m ultiple com parisons o f T ukey’s range, tested at 0.05 level o f probability, using the softw are
Spss 11.5 for W indows.
3. Results
3.1. A rtem ia perform ance fe d live ye a st cells
A rtem ia nauplii w ere fed w ith seven different isogenic m utant strains o f b ak er’s yeast (Saccharom yces cerevisiae)
(Table 1) and com pared w ith nauplii fed w ild type yeast u nder gnotobiotic condition. In all cases equal am ounts o f
y east cells w ere offered. T he results presented in Tables 3 and 4 (results obtained in Exps. 1 and 2) show that inde­
p endently o f the grow th stage, the yeast genetic background has a big influence on A rtem ia perform ance. Com pared
w ith W T yeast, total biom ass production o f nauplii w as significantly im proved w hen the exp-grow n isogenic yeast
m utant strains w ere used as feed, due to both significant h igher survival and/or individual length (Table 3). A m ong
them , th e m nn9 yeast strain supported the best nauplii perform ance.
T a b le 3
E x p e rim e n t 1: av erag e survival (% ), in d iv id u al le n g th (m m ) and to ta l le n g th (m m p e r F alcon tube-F T ) o f A rte m ia na u p lii fe d w ith liv e y e a st cells
(h a rv e ste d in ex p o n en tial gro w th p h ase) after 5 days: e ffe c t o f gro w th sta g e a n d g e n etic b a ckground
S train s
Su rv iv al (Vo)
WT
3 2 ± 6C
m nn9
87 ± 9 “
6 4 ± 7 hc
m nn6
fk s l
B
A
In d iv id u al
T otal le n g th
le n g th (m m )
(m m /F T )
1.3 ± 0 . 1 f
4 .0 ± 0 .4a
8 .6 ± 1.7d
7 0 .7 ± 7 .7 a
1.8 ± 0 . 1 de
gasl
61 ± 5 b
2.1 ± 0 . 1 c
3.2 ± 0 . 2 ”
kn r4
62 ± 6b
2.0 ± 0 .2cd
k re 6
ch s3
4 5 ± 4 bc
2.0 ± 0 .1cd
55 ± 5b
1.7 ± 0 . 2 °
55 ± 10b
Survival (% )
In d iv id u al
T otal length
le n g th (m m )
(m m /F T )
29 ± T
2 .2 ± 0. l cd
3.9 ± 0 .2 “
12.5 ± 3.3d
68.7 ± 5 . 0 “
2 3 .5 ± 2 . 6 °
87 ± 6 “
52 ± 6 hc
2 3 .6 ± 4 .6 C
50 ± 12 bc
1.8 ± 0 .2 d
2.2 ± 0.3cd
2 0 .0 ± 9.0cd
39 .0 ± 3.0b
25 .0 ± 3.4C
64 ± 5b
3.3 ± 0 .1 b
5 0 ± 12bc
2.3 rib 0.1c
4 2 .0 ± 3.0b
2 3 .0 ± 5.6cd
58 ± 13b
4 2 ± 5 bc
2.5 ± 0 . Ie
29.0 ± 6.0°
2 .0 ± 0.2cd
17.0 ± 2.0cd
18 ± 1.6°
18.5 ± 2.0°
19.0 ± 2.3cd
M ea n s w e re p u t to g e th e r w ith th e stan d a rd d e v ia tio n (m e a n ± S D ) . E a c h e x p erim e n t w as repeated tw ic e A and B. E a c h fe e d w as tested in four
re p lic a te s . V alues in th e sa m e co lu m n sh o w in g the sam e su p ersc rip t le tte rs a re n o t significantly d ifferen t (Arukey > 0.05).
S. S o lta n ia n e t a i / F ish & Shellfish Im m u n o lo g y 23 (2007) 1 4 1 —153
146
T able 4
E x p e rim en t 2: av erag e su rv iv a l (% ), in d iv id u a l le n g th (m m ) and to ta l le n g th (m m p e r F a lc o n tu b e -F T ) o f A rte m ia n a u p lii fe d w ith live y east cells
(h arv ested in th e s ta tio n ary gro w th p h a se ) a fte r 5 days: effe c t o f gro w th stag e and g e n etic b a ck g ro u n d
Strains
B
A
S u rv iv al (% )
Individual
Total length
le n g th (m m )
(m m /F T )
Individual
length (m m )
T otal length
(m m /FT )
5 .2 ± 2.5°
25 ± 4 °
1.7 ± 0 . 2 ^
8.5 ± 1.4°
4 5 .6 ± 3 .2 a
14.4 ± 1.5b
71 ± 4 a
2.8 ± 0.1a
4 0 .0 ± 2.1a
3 8 ± % hc
2 8 ± 1 3 bc
1.3 ± 0.1d
14.3 ± 1.7b
4 0 ± 7b
1.8 ± 0.1b
9.4 ± 1 .9 * *
14.9 ± 2.6b
1.3 ± 0 .2d
13.2 rib 1.3b
3 5 ± 4 bc
1.6 ± 0 .1cd
1.7 ± 0 .2 bcd
1 2 . 6 ± 4 .0 h
9 .7 ± 2 .9 bc
32 ± 15b
1.5 ± 0.1cd
1.6 ± 0.2bcd
10.4 ± 1 .2 * *
12.2 ± 2bc
2 5 ± 6 bc
1.9 ± 0.1b
WT
15 ± 7 d
m nn9
68 ± 5 a
m nn6
4 0 ± 4 bc
fk sl
18 ± 3 d
1.8 ± 0 .1bc
1.7 ± 0 .2bcd
g a sl
35 ± 4 bc
2 .0 ± 0 .3b
knr4
48 ± 5b
kre6
4 0 ± 13hc
2 9 ± 8cd
chs3
S urv iv al (% )
1 . 7 5 ± 0 .2 bcd
3 .3 ± 0 .2a
6 .6 ± 0.9C
9.8 ± 1.9**
1.6 ± 0.2bc
9.3 ± 2.2bc
M eans w e re p u t to g e th e r w ith the stan d a rd d e v ia tio n (m ean ± SD ). E ach e x p erim e n t w a s re p e a te d tw ic e A and B. E a c h fe e d w as tested in fo u r
replicates. V alues in th e sa m e c o lu m n sh o w in g th e sam e su p erscrip t le tte rs a re no t sig n ific a n tly d ifferen t (pjukcy > 0.05).
A lso the use o f the m m n6 m utant resulted in a significantly im proved total biom ass production o f Artem ia. In this
treatm ent a higher biom ass production w as obtained due to a considerable increase in survival. U sing knr4 and fks 1 (less
ß -1,3 glucans) and kre6 (less ß - 1,6 glucans) as feed resulted in less A rtem ia biom ass production com pared to the mnn9
yeast strain but significantly m ore A rtem ia biom ass production com pared to the W T yeast. T he chitin-defective yeast
strain (chs3) supported a sm all increase in A rtem ia biom ass production com pared to the W T -treatm ent, m ainly due to
better nauplii survival. Finally, using the g a sl m utant as food (this strain is lacking an im portant cell-w all protein in­
volved in cross-linking the m ajor cell-w all com ponents) resulted in b etter nauplii perform ance com pared to W T yeast.
H igher total biom ass (except for fk s l fed A rtem ia) production (com pared to W T) in A rtem ia fed m utant stat-grow n
yeast cells w ere m ainly due to higher nauplii survival rather than stronger individual growth. Only w ith stat-grow n
m nn9 cells higher survival and stronger individual growth contributed to m ore biom ass production. W hen exp-yeast
cells w ere fed to the nauplii, significant higher total A rtem ia biom ass production (m ostly due to higher nauplii sur­
vival) values w ere observed in all cases com pared w ith stat-yeast cells possibly because the A FD W o f yeast cells
in the exponential grow th phase w as higher than in the stationary grow th phase (Table 2) (although n ot significantly
different in the m nn9, g a s l and fk sl yeast strains).
In the experim ents described above equal am ounts o f yeast cell particles (Table 5 — feeding regim e for Exp. 3)
were supplied as food, resulting, how ever, in different am ounts o f A FD W o f food offered to A rtem ia (see Table 2).
This could have been the reason for the considerable higher A rtem ia biom ass production e.g. w ith m nn9 yeast. Hence,
the feeding experim ent was repeated, this tim e offering equal am ounts o f food expressed as AFDW . In all cases, feed­
ing m utant-yeast cells resulted in better nauplii survival than feeding W T yeast. A lso in this experim ent, the m nn9-fed
A rtem ia presented the h ighest biom ass production (Table 6). A rtem ia b iom ass production w as equal after feeding W T,
T able 5
F eeding re g im e s in th e 3 e x p erim e n ts (E x p .) p erfo rm ed . D aily and a v erag e total a sh -fre e dry w e ig h t (A F D W ), e x p re sse d in p g /F T o f y east cells and
dead b a cteria (L V S3) su p p lie d to A rte m ia in E x p e rim en ts 3, 4 and 5
F e e d in g re g im e
D ayi
Y
E x p .3
T otal A FD W
D ay 2
DB
124
0
(a)
25
(b)
50
(c)
(d)
0
Y
D ay4
Day3
DB
248
0
221
50
197
100
246
492
0
0
Y
DB
Y
DB
Y
324
0
442
62
592
394
124
525
492
0
0
656
1312
248
0
442
50
394
100
492
0
0
(p g /F T ) o ffered
D ay5
DB
496
0
1440
100
886
2870
200
786
2870
0
984
0
1968
2870
5740
E xps. 4 a n d 5
0
984
984
C h allen g e tests w e re p e rfo rm ed w ith liv e V ib rio c a m p b ellii (V C ) a t a d e n sity o f 5 x IO6 cells m l-1 a dded at d a y 3 in E x p e rim e n ts 4 and 5.
(a) D ead LV S3 + y e a s t 5 % ; (b) D ead LV S3 + y e a st 10% ; (c) T h e tre a tm e n t d e ad LV S3 X ; an d (d) T h e treatm ent: d e a d LV S3 2X.
X = th e to ta l a m o u n t o f fe e d o ffered (2 8 7 0 p g A F D W /F T ); Y = y e a st (w ild ty p e an d iso g en ic y e a st m utants a d d ed a t 5% o r 10% ); DB = dead
b a cteriu m LVS3.
.S. So lta n ia n e t al. i F ish & Shellfish Im m u n o lo g y 2 3 (2 0 0 7 ) 141—153
147
T able 6
E x p e rim e n t 3: a v erag e survival (To), in d iv id u al le n g th (m m ) a n d total le n g th (m m /F T ) o í A r te m ia nauplii fe d w ith liv e y east cells (h arv ested in the
s ta tio n a ry gro w th p h ase) fo r 5 days: effe c t o f g e n etic b a ck g ro u n d
S train s
Survival (So)
In d iv id u al
T otal length
le n g th (m m )
(m m /FT )
WT
18 ± 6 °
1 .7 4 ± 0 .1 °
m nn9
63 ± 6 a
2.44 ± 0 . I a
30.6 ± 3.2a
m nn6
35 ± 9 b
28 ± 7 bc
1.8 ± 0 . 1 c
1.67 ± 0. I e
38 ± 6b
43 ± 6b
2.11 ± 0 . 1 b
1.53 ± 0 . 1 d
12.6 ± 3.3bc
9.2 ± 2.2cd
15.8 ± 2 . 7 b
kre6
33 ± 7 b
1.33 ± 0. I e
8.6 ± 1 . 7 “ *
chs3
33 ± 6b
1.81 ± 0 . 1 c
11.8 ± 2 . 3 bcd
fk s l
gasl
knr4
6.1 ± 2 . 2 d
13.0 ± 2.0bc
A ll tre a tm e n ts w e re fe d w ith a n equal a m o u n t o f fe e d c o rre sp o n d in g to a n A F D W (1 .4 4 ± 0.01 m g /F T ) (see T a b le 5 — E xp. 3) (for in sta n ce fed w ith
W T -Y N B y e a s t c ells). M ea n s w e re p u t to g e th e r w ith th e stan d a rd d e v ia tio n (m e a n ± S D ). E ach fe e d w as tested in fo u r replicates. V alues in the sam e
c o lu m n sh o w in g th e sa m e su p erscrip t le tte rs a re n o t sig n ifican tly d ifferen t (p-rukey > 0.05).
f k s l, kre6 and chs3 cells although the m utants (kre6 and knr4) display, respectively, a higher or equal A FD W as com ­
pared w ith W T yeast.
3.2. Protection effect by bacteria (LVS3) a n d yeast
T h e effect o f feeding two different am ounts o f dead bacteria (autoclaved LV S3) to the nauplii (either challenged or
not w ith a live pathogen) is presented in Table 7 (Exp. 4). U nchallenged, there is no effect on survival in the two feed­
ing regim e. Yet, nauplii fed with dead LVS3 (5.74 mg A FD W /FT ) (Table 5, feeding regim e: (d) yielded a significantly
higher survival in the challenge test as com pared to the nauplii fed w ith only h alf o f this am ount (Table 7, lines 20 and
22). T he results o f supplying a low am ount o f W T or isogenic yeast m utants to th e nauplii fed dead LVS3 are presented
in Tables 7 and 8. M ost o f the non-challenged nauplii fed solely w ith dead LV S3 or both dead LVS3 and yeast cells
survived until day 6 (68 % o r higher). In m ost treatm ents, challenged nauplii fed only w ith d ead LVS3 or both dead
LVS3 and yeast cells (WT, mnn6, fk s l, knr4, kre6) died before day 5. Yet, exceptions occurred w hen the nauplii were
fed both dead LVS3 and m nn9, gas 1 and chs3 (only exp-yeast) yeast strains. O nly m m n9 yeast cells can protect nauplii
against the live pathogen until day 6 (Tables 7 and 8, line 4). H ow ever, the addition o f 5% o f the total A FD W in the
form o f m nn9 yeast cells was not sufficient to keep the nauplii alive until day 6 (Tables 7 and 8, line 6). In all yeast
strains, the exp-yeasts provided a better protection against the pathogen than stat-yeasts, but in m ost cases, this pro­
tection lasted only for a short period (one day after challenge). T h e mnn9 cells supported a high protection until the
end o f the experim ents against the VC pathogen w hen offered as exp-grow n o r stat-grow n cells (Tables 7 and 8, line 4).
4. Discussion
M arques et al. [20,21] have show n that yeast digestion by A rtem ia can be significantly im proved by m anipulating
the genetic characteristics o f the yeast, the grow th stage and the m edium used. T his study confirm s that the genetic
background o f the yeast strain used, has a strong influence on the A rtem ia perform ance (Exps. 1, 2 and 3).
In this study, a yeast strain containing low concentrations o f m annoproteins in the cell w all, such as the m nn9
m utant, alw ays supported a high A rtem ia biom ass production (i.e. best nauplii grow th as w ell as highest survival
rate). A ccording to Coutteau et al. [28], ß-glucanase activity is detected in the digestive tract o f A rtem ia b ut no
m annase activity, m aking the external m annoprotein layer o f the yeast cell w all probably the m ajor barrier for the d i­
gestion o f the yeast cell by the A rtem ia nauplii. T herefore, it is likely that the digestive enzym es o f A rtem ia (such as
ß-glucanase) can easily enter and provide suitable digestion o f yeast cells w ith reduced m annoprotein content.
A n im proved A rtem ia perform ance w as also obtained w ith yeast m utants w ith reduced ß-glucans (fk sl, knr4, kre6
and g a s l) and chitin (chs3) levels as com pared to the nauplii fed W T-yeast cells (especially w ith exp-grow n yeast cells
as food). A ccording to A guilar-U scanga and F rancois [29], ß-1,3 glucans and especially ß-1,6 glucans provide an ­
chorage to m ost cell-w all m annoproteins and are also covalently linked w ith chitin, contributing to the m odular struc­
ture o f th e cell w all, ß-1,3 glucans also contribute to the rigidity and integrity o f the cell w all, and determ ine the cell
S. S o lta n ia n e t al. / F ish & Shellfish Im m u n o lo g y 2 3 (2 0 0 7 ) 1 4 1 —153
148
T able 7
E x p e rim en t 4: m ean d a ily su rv iv al (% ) o f A rte m ia fed d aily w ith d e ad LV S3 an d y e a s t cell stra in s h a rv e ste d in th e ex p o n en tial grow th phase
A — survival (% )
TN
D ay 2
1
2
D ay 3
B — survival (% )
D ay 4
D ay 5
D ay 6
D ay 2
D ay 3
D ay 4
D ay 5
D ay 6
66 ± 3a
0b
D ead LV S3 + W T 10%
93 ± 3 a
86 ± 3 a
81 ± 5 a
74 ± 4a
88 ± 3 a
83 ± 3a
7 3 ± 3a
91 ± 3 a
8 8 ± 3a
5 6 ± 5b
0b
68 ± 3a
0b
91 ± 3 a
D ead LV S3 + W T 10%
88 ± 3a
86 ± 3a
55 ± 4b
0b
98 ± 3a
96 ± 3“
94 ± 3a
90 ± 4 a
86 ± 3a
83 ± 3 a
96 ± 3a
93 ± 3 a
80 ± 4
86 ± 3 “
80 ± 4a
76 ± 3 a
98 ± 3a
93 ± 3 a
91 ± 3 a
88 ± 3a
84 ± 3a
9 3 ± 3a
81 ± 5 a
76 ± 3a
76 ± 3a
0b
+ V C D3
3
4
D ead LVS3 + m n n 9 10%
5
D ead LVS3 + m n n 9 5%
98 ± 3a
93 ± 3*
86 ± 3 a
80 ± 4 a
75 ± 4 a
98 ± 3a
93 ± 3 a
88 ± 3 “
84 ± 3a
6
D ead LV S3 + m n n 9 5 % +
V C D3
99 ± 3a
89 ± 3a
74 ± 3 a
31 ± 5 b
0b
98 ± 3a
91 ± 3 a
81 ± 3 a
35 ± 4b
7
D e ad LVS3 4- m n n 6 10%
93 ± 3a
89 ± 3a
83 ± 5 a
79 ± 3a
73 ± 3 a
91 ± 3 a
89 ± 3 a
81 ± 3 a
78 ± 3
8
D ead LV S3 + m n n 6 10%
91 ± 3 a
88 ± 3a
66 ± 5b
0b
0b
96 ± 3a
86 ± 3 a
70 ± 4 b
0b
9
+ V C D3
D e ad LV S3 + f k s l 10%
93 ± 3a
86 ± 3a
70 ± 4a
93 ± 3a
86 ± 3 a
D e ad LVS3 + f k s l 10%
93 ± 3 a
88 ± 3a
83 ± 3 a
64 ± 5 b
79 ± 3a
10
0b
0b
91 ± 3 a
88 ± 3 a
81 ± 3 a
6 6 ± 5b
78 ± 3a
0b
11
+ V C D3
D ead LV S3 + k n r4 10%
9 4 ± 3a
88 ± 3a
79 ± 3a
78 ± 4 a
73 ± 3a
9 3 ± 3a
86 ± 3 a
81 ± 3 a
75 ± 4 a
69 ± 3a
12
D e ad LV S3 + k n r4 10%
93 ± 3a
8 6 ± 3a
63 ± 3 b
0b
0b
9 3 ± 3a
88 ± 3a
6 6 ± 3b
0b
0b
13
-1-V C D3
D ead LV S3 + k re 6 10%
80 ± 4 a
75 ± 4 a
71 ± 3 a
83 ± 3a
7 8 ± 3a
88 ± 3a
60 ± 4b
0b
0b
9 3 ± 3a
9 3 ± 3a
88 ± 3a
D ead LV S3 + k re 6 10%
9 4 ± 3a
94 ± 3a
88 ± 3a
14
86 ± 3a
65 ± 4 b
0b
70 ± 4 a
0b
93 ± 3 a
88 ± 3a
84±3a
81 ± 3 a
73 ± 3a
94 ± 3a
89 ± 3a
71 ± 5 a
8 6 ± 3a
50 ± 4b
36 ± 4 b
0b
9 3 ± 3a
88 ± 3a
86 ± 3a
53 ± 6 b
7 8 ± 3a
94 ± 3a
33 ± 6h
0b
D ead LVS3 + m n n 9 10%
+ V C D3
70 ± 4 a
0b
73 ± 3a
0b
+ V C D3
15
16
D ead LV S3 + g a s l 10%
D ead LVS3 + g a s l 10%
+ V C D3
17
D ead LVS3 + chs3 10%
94 ± 3 “
86 ± 3a
79 ± 5 a
79 ± 5 a
75 ± 3a
96 ± 3a
88 ± 3 a
81 ± 5 a
75 ± 4a
74 ± 3a
18
D ead LVS3 + ch s3 10%
94 ± 3a
88 ± 3 a
75 ± 6a
34 ± 5 b
0b
98 ± 3a
86 ± 3a
61 ± 5 a
28 ± 3b
0b
19
+ V C D3
D ead LV S3(2X )
98 ± 3a
93 ± 3 a
84 ± 3 a
98 ± 3
89 ± 3a
85 ± 4 a
78 ± 3a
74 ± 3a
D e ad LV S3(2X )
98 ± 3 “
86 ± 3a
77 ± 3a
78 ± 3a
53 ± 3b
73 ± 3a
20
44 ± 3b
99 ± 3a
8 6 ± 3a
78 ± 3a
65 ± 4 b
51 ± 3 b
+ V C D3
D ead LVS3 (X )
98 ± 3 a
7 6 ± 3a
69 ± 5a
9 6 ± 3a
75 ± 4 6
56 ± 5b
0b
0b
98 ± 3a
91 ± 3 a
93 ± 3a
83 ± 3a
96 ± 3a
9 0 ± 3a
89 ± 3 “
83 ± 3 a
D ead LV S3 (X )
60 ± 4 b
0b
21
22
8± 3
0b
+ V C D3
T h e y e a st c ells co n stitu ted e ith e r 5 7 4 ± 0 .2 p g /F T o r 287 ± 0 .2 p g /F T o f th e to ta l A F D W supplied. T h e c h allen g ed te st w as p erfo rm ed w ith V ibrio
c a m pbellii (V C ) a d d ed a t d a y 3. E ach e x p erim e n t w as re p e ated tw ice: A an d B. E a c h fe e d w as te sted in fo u r re p lic a tes. T h e to ta l a m o u n t o f feed
o ffered is e q u al to 2 8 7 0 p g A FD W /FT . M ea n s w'ere p u t to g e th e r w ith th e stan d ard d e v ia tio n (m e a n ± SD ). Survival in th e c h allen g e test w as co m ­
p a re d d ire c tly to th e su rv iv a l o f n o n -c h a lle n g ed A rtem ia . V alues show ing th e sa m e su p e rsc rip t le tte r are n o t s ig n ific a n tly d ifferen t (p > 0.05).
shape [30]. As a consequence, a lack o f ß-glucans in the yeast cell w all m ight resu lt in less covalent linkage betw een
the three cell-w all com pounds, resulting in a m ore perm eable and digestible cell w all in com parison to the W T strain.
A lthough in a W T -yeast strain chitin concentration m akes up only 1—2% o f the cell-w all dry m ass [22], it plays a key
role in yeast cell grow th and division and is attached covalently to ß-1,3 glucans, ß-1,6 glucans and m annoproteins
[31], T herefore, the better results obtained w ith nauplii fed chitin-defective yeast could also b e due to an enhanced
digestibility o f chs3 cells by A rtem ia, caused by reduced linkage betw een the three cell-w all com ponents.
In the g asl yeast m utant, the production o f the glycosylphosphatidylinositol (GPI) anchored protein is inhibited
resulting in a non-proper fiber assem bly o f the cell w all (defective architecture) as w ell as in reduced ß-glucans
[32,33]. T his apparently results in an eventual digestibility o f g a s l cells by Artemia. C om pared to W T-fed nauplii,
yeast strains w ith reduced phosphom annan levels in the cell w all (m nn6) alw ays gave higher A rtem ia perform ance
m ainly due to a higher nauplii survival. This fact could be due to interference o f phosphom annans in phosphodiester
cross-linking o f m annoproteins to ß-glucans [34], In the experim ents show n in Tables 3 and 4, equal am ounts o f yeast
cell particles (feeding schedule o f Exps. 1 and 2) are offered. T his results in different am ounts o f A FD W being sup­
plied (Table 2). W e therefore, in a further experim ent, supplied exactly equal am ount o f A FD W (see Table 5 — feeding
regim e for Exp. 3) o f the different yeast cells to A rtem ia (Table 6). C onsequently, in th ese experim ents, the feed
S. S o lta n ia n e t al. / F ish & S h e llfish Im m u n o lo g y 23 (2 0 0 7 ) 1 4 1 —153
149
T able 8
E x p e rim e n t 5 : m e a n d a ily survival (% ) o f A rte m ia fe d d a ily w ith d e ad LV S3 an d y e a st c ell stra in s h a rv e ste d in th e statio n ary grow th phase
A — survival (% )
TN
1
2
D e a d LV S3 + W T 10%
D e a d LV S3 + W T 10%
B — survival (%)
D ay 2
D ay 3
D ay 4
D ay 5
D ay 6
D ay 2
D ay 3
D ay 4
D ay 5
93 ± 3 a
93 ± 3a
88 ± 3 a
79 ± 5a
75 ± 3a
65 ± 4 a
0b
0b
88 ± 3a
89 ± 3a
73 ± 3 a
46 ± 5b
91 ± 3 a
9 4 ± 3a
76 ± 3 a
89 ± 3 a
45 ± 6b
0b
0b
9 9 ± 3a
96 ± 3 a
9 5 ± 3a
91 ± 3 a
86 ± 3 a
83 ± 3 a
98 ± 3a
94 ± 3 a
89 ± 5 a
74 ± 3a
69 ± 3 “
98 ± 3a
93 ± 3 a
86 ± 3 a
86 ± 3 a
73 ± 3 a
80±4a
8 6 ± 3a
81 ± 5 a
61 ± 5 a
75 ± 4 a
73 ± 3 a
98 ± 3a
91 ± 3 a
80 ± 6a
0b
9 6 ± 3a
90 ± 4 a
64 ± 3b
75 ± 4 a
25 ± 4 b
71 ± 3 a
2 3 ± 3b
D ay 6
6±5a
+ V C D3
3
4
D e a d LV S3 + m n n 9 10%
D e a d LV S3 + m n n 9 10%
98 ± 3 a
69 ± 3 a
+ V C D3
5
D e a d LV S3 + m n n 9 5 %
94 ± 3a
6
D e a d LV S3 + m n n 9 5%
91 ± 3 a
8 9 ± 3a
88 ± 3a
7
+ V C D3
D e a d LV S3 + m n n 6 10%
99 ± 3a
91 ± 3 a
84 ± 3 a
7 8 ± 3a
70 ± 4 a
99 ± 3 a
93 ± 3 a
83 ± 3a
7 6 ± 5a
69 ± 5 a
8
D e a d LV S3 + m n n 6 10%
98 ± 3a
90 ± 4 a
4 3 ± 3b
0b
0b
9 9 ± 3a
91 ± 3 a
45 ± 4 b
0b
0b
0b
+ V C D3
9
D e a d LV S3 + f k s l 10%
98 ± 3a
91 ± 3 a
81 ± 3 a
84 ± 3a
75 ± 4 a
69 ± 3 “
96 ± 3a
90±4a
41 ± 5 a
0b
98 ± 3 a
96 ± 3a
91 ± 3 a
D e ad LVS3 + f k s l 10%
76 ± 3a
0b
68 ± 3 a
10
90 ± 4 a
39 ± 5 b
0b
0b
D e a d LV S3 + k n r4 10%
95 ± 3a
89 ± 3a
85 ± 4 a
78 ± 3a
71 ± 5 a
96 ± 3a
91 ± 3 a
84 ± 3a
76 ± 5a
74 ± 3 a
D e a d LV S3 + k n r4 10%
99 ± 3a
88 ± 3a
46 ± 5b
0b
0b
98 ± 3 a
93 ± 3a
50 ± 4b
0b
0b
+ V C D3
11
12
+ VC D3
13
D e ad LVS3 + k re 6 10%
98 ± 3a
84 ± 3a
7 9 ± 3a
78 ± 3 a
69 ± 5a
9 6 ± 3a
88 ± 3a
78 ± 3a
79 ± 3a
71 ± 5 a
14
D e ad LVS3 + k re 6 10%
96 ± 3 “
86 ± 3a
41 ± 5 b
0b
0b
9 8 ± 3a
8 6 ± 3a
38 ± 3b
0b
0b
84 ± 3
43 ± 3b
73 ± 3 a
69 ± 3 a
93 ± 3a
89 ± 3 a
83 ± 3 a
74 ± 5a
71 ± 3 a
25 ± 4 b
0b
93 ± 3a
8 6 ± 3a
46 ± 3b
28 ± 3b
0b
89 ± 5 a
88 ± 3 a
84 ± 3a
78 ± 3a
66 ± 5b
0b
70 ± 4 a
0b
+ V C D3
15
D e a d LVS3 + g a s l 10%
91 ± 3 a
16
D e ad LV S3 + g a s l 10%
90 ± 3a
88 ± 3a
89 ± 3a
17
+ V C D3
D e ad LV S3 + ch s3 10%
98 ± 3 a
88 ± 3a
83 ± 3 a
75 ± 4 a
68 ± 3 a
9 6 ± 3a
18
D e ad LVS3 + chs3 10%
98 ± 3 a
89 ± 3 a
68 ± 3b
0b
0b
9 8 ± 3a
+ V C D3
T h e y e a st cells c o n stitu te d e ith e r 5 c7o o r 10% o f the total A F D W su p p lied . T h e c h allen g e d te st w a s p erfo rm ed w ith V ib rio c a m p b ellii (V C ) added at
day 3. E a c h e x p e rim e n t w as re p e ated tw ice: A and B. E a c h feed w as te ste d in fo u r replicates. M eans w ere p u t to g e th e r w ith the stan d a rd deviation
(m e a n ± S D ). S u rv iv al in th e c h allen g e te s t w as c o m p a re d d ire c tly to th e su rv iv a l o f n o n -c h a lle n g ed A rtem ia. V alues sh o w in g th e sa m e su p erscrip t
le tte r a re n o t sig n ific a n tly d ifferen t (p > 0.05).
p article concentration, ju s t after the feeding, w as different w ith the various m utants. A lso in this case m nn9-fed A rte­
m ia outperform ed W T-fed A rtem ia (both in survival an d individual growth). W ith the other m utants, A rtem ia biom ass
p roduction im proved m ainly through higher survival. W ith two m utants, nam ely fk sl and chs3, A rtem ia biom ass p ro ­
duction was equal as in the experim ent w here W T -cells w ere offered.
In the p resent study the Artem ia nauplii displayed a h ig h er perform ance w hen fed w ith an exp-grow n cells com ­
pared w ith A rtem ia nauplii fed w ith stat-grow n yeast strains. A ccording to K lis et al. [35], yeast cells entering the
stationary ph ase o f grow th w ill form different cell w alls, i.e. thicker, m ore resistant to enzym atic breakdow n and
less perm eable to m acrom olecules. The level o f m annosyl phosphorylation o f cell-w all proteins increases in the lateexponential and stationary phase o f grow th [36]. In addition m ore extensive cross-linking (through disulfide bridges)
betw een the polysaccharide com ponents o f the cell w all (m annoproteins, glucans and chitin) is taking place in the
stationary phase [31,37,38]. In conclusion, it seem s that the density o f covalent linkage betw een the three cell-w all
com pounds o f th e yeast cell plays an im portant role in their digestibility by Artemia. In addition to that, high am ounts
o f cell-w all chitin and glucans in com bination w ith low am ounts o f m annoproteins favour A rtem ia biom ass production
u nder gnotobiotic condition [20,21].
A ccording to R aa [2] im provem ents in the h ealth status o f aquatic organism s can be achieved by balancing the diet
w ith regards to nutritional factors. This phenom enon is identified as nutritional im m unology, since som e nutritional
factors are so closely linked w ith biochem ical processes o f the im m une system that significant health benefits can be
o btained by adjusting the concentration o f such factors. Inadequate food or im balances in the nutrient com position
o f the d iet w ill affect grow th and general perform ance o f an anim al, m ost likely, also the biochem ical process o f
the im m une system [2]. In this study nauplii fed w ith dead LVS3 (5.74 m g A FD W /FT) (Table 5 — feeding
5. S o lta n ia n e t al. / F ish & Shellfish Im m u n o lo g y 2 3 (2 0 0 7 ) 1 4 1 —153
150
regim e: (d), presented significantly higher survival after challenge in com parison to nauplii fed solely w ith h alf o f this
am ount (Table 7). T his experim ent clearly illustrated that the outcom e o f the challenge w ith V. cam pbellii under gno­
tobiotic condition is very m uch dependent on the overall condition o f the nauplii. T hese results are also consistent with
the perception that Vibrio spp. are opportunistic pathogens. T herefore in all challenge experim ents, in w hich the effect
o f yeast mutants w ere tested in sm all quantities, the total A FD W supplied was kept constant. T h e m nn9-fed Artemia
could resist detrim ental effect o f pathogenic V C until the end o f the experim ents as previously reported by M arques
et al. [25] (Tables 7 and 8). N evertheless, the addition o f 5% o f m nn9 y east w as n ot ab le to protect nauplii against VC
until day 6. The m nn9 yeast has a null m utation resulting in phenotypically increased am ounts o f cell-w all bound chi­
tin and glucans in com bination w ith reduced am ount o f m annose linked to m annoproteins, an d probably a reduced
density o f covalent linkages betw een these three yeast cell-w all constituents and/or nature o f the covalent bonds,
in com parison to the W T strain [22,29]. T he protection provided by the m nn9 yeast could be the effect o f general
im provem ents in A rtem ia health condition due to extra (or better quality) nutrients available in this yeast or due to
a stim ulation o f a non-specific im m une response by som e com pounds, such as ß-glucans or chitin that are present
in the yeast cell w all. V ism ara et al. [39] considerably increased A rtem ia resistance to stress conditions, such as
poor growth m edium quality and daily handling, by adm inistering daily nauplii w ith a m utant o f E uglena gracilis
presenting high am ount o f ß-glucans (and thus enabling its response against disease). In contrast to m nn9 yeast, which
has both strong nutritional and/or im m unologic characteristics, g a sl cells have good nutritional effects, and protect
nauplii tem porarily against the pathogen in the challenge test. F urtherm ore, w eak o r no nutritional and protection
effects w ere observed w ith fk s l, knr4, m nn6 and kreó yeast cells (Tables 9 and 10). Yet, interestingly, tem porary
protection against VC w as obtained by adding chs3 in the diet, w hile this m utant has hardly any effect on individual
grow th (Table 7). U sing the described set o f yeast m utants, a full or partial protection against V C can be associated
w ith increased glucan and chitin in the cell w all (e.g. m nn9 but also g a s l) and reduced chitin an d increased glucan in
the cell w all (chs3). T his seems to suggest that chitin as such is not involved in the protection against VC. R ather the
results indicate that glucan as such is the potential active com pound.
ß-glucans have b een identified as specific im m unostim ulants activating the aquatic organism s im m une system and
protecting them from adverse conditions [7]. For exam ple, yeast Saccharom yces cerevisiae has been found to be
T a b le 9
S u m m ary ta b le o f re su lts o b ta in e d in th e c h a lle n g e d an d n o n -c h a lle n g ed ex p erim en ts
Y east
P h en o ty p e
WT
C o n tro l y e a st
m nn9
L ess m an n an ,
N o n -c h a lle n g ed ex p erim en ts (E xps. 1—3)
C h allen g e d ex p erim e n ts (E xps. 4 a n d 5)
S u rv iv al
(D 6 ) (rio)
Survival
(D 4) (rio)
IL
(m m )
TBP
(m m /F T )
S u rv iv al
(D 5) (rio)
Survival
(D 6) (rio)
C
A
C
A
C
-
-
A
-F
+
+
h ig h er chitin,
h ig h er ß -g lu can s
m nn6
L ess p h o sp h o m an n an
B
C
C
—
-
fk s l
L ess ß-1,3 g lu can s,
B
C
C
+
—
—
h ig h er chitin
k n r4
L ess ß-1,3 g lu can s,
h ig h er chitin
B
C
c
+
—
—
k re 6
L ess ß -1 ,6 g lu can s,
h ig h er chitin
B
c
c
+
—
—
chs3
L ess ch itin
B
c
c
-
Less in teg ratio n o f y east
B
B
B
+
T
®
g asl
ffi
cell a d h esio n , p ro tein s
in to th e c ell w all, less
ß-1,3 g lu can s, h ig h e r chitin
A an d B m ean resp ectiv ely sta tistic a lly d ifferen t (p < 0 .0 5 ) stro n g and m o d e ra te p o sitiv e effe c t o f y e a s t fe e d on A rte m ia p e rfo rm a n c e in com parison
to th e w ild ty p e y east strain . C m eans n o s tatistic a l d iffe re n t e ffe c t o f fe e d on A rte m ia p e rfo rm an c e in com p ariso n to th e w ild ty p e y east strain.
“ + ” m ean s p ro te c tio n (n o sig n ific a n t d ifferen c e in survival rate b etw een c h allen g e d an d n o n -c h a lle n g ed treatm en ts) p ro v id e d b y sm all am ounts
o f y e a st fe e d (5 o r 10% ) o n A rte m ia p e rfo rm a n c e w h e n fe d w ith d e ad LV S3 an d c h allen g e d w ith V ibrio cam pbellii .
” m e a n s no protection
(sig n ifica n t d ifferen ce in su rv iv al ra te b e tw ee n c h allen g e d and n o n -ch allen g ed treatm en ts). ® m ean s th at the fe e d w a s only p rotecting p artially
a g a in st th e pathogen. D 4 , D 5 and D 6 co rre sp o n d , resp ectiv ely , to day 4 , 5 and 6.
S. S o lta n ia n e t al. / F ish & Shellfish Im m u n o lo g y 2 3 (2 0 0 7 ) 141—153
151
T a b le 10
E x p e rim en ta l d e sig n o f th e 5 ex p erim en ts (E xp.) p erfo rm ed
D ay 1
D ay 2
D ay 3
D ay 4
D ay 5
(start)
E x p s. 1—3
E x p s. 4 an d 5
D ay 6
(harvest)
(a)
Y
—>
(b)
DB + Y
-
Y
DB + Y
DB + Y
->
(c)
DB+Y
->
(d)
-*•
->
D B (X )
->
(e)
DB (X)
D B (X)
DB (X )
-»
(f)
D B (2X )
-»
D B (2X )
(g)
D B (2X )
-*
D B (2X )
-*
Y
-
Y
DB + Y
-
DB + Y
DB + Y + P
->
DB + Y
D B (X )
-*■
D B (X )
D B (X ) + P
->
—►
D B (X )
D B (2X )
D B (2 X ) + P
—
>
D B (2X )
D B (2X )
Y
-
DB + Y
—►
DB + Y
DB (X )
—►
—»
->
DB (X )
—►
DB (2X )
->
—►
-
D B (2X )
(a—g) c o rre sp o n d to th e tre a tm e n ts p erfo rm ed ; Y = y e a st stra in s (w ild ty p e o r iso g en ic y e a st m u ta n ts). Y east stra in s w e re a d d ed e ith e r a t an e qual
a m o u n t o f y e a st c ell p a rtic le s (E x p s. 1 an d 2) o r an e q u al a m o u n t o f fe e d (E x p . 3 ), o r 5 o r 10c,o (E x p s. 4 and 5); DB = d e ad b a cteriu m LV S3; X = the
to tal a m o u n t o f d e a d LV S3 o ffered o v e r the fu ll e x p erim e n tal p e rio d (2 8 7 0 p g A F D W /F T ); P = p ath o g e n ( V ibrio cam pbellii). S e e T a b le 1.
a good enhancer o f the trout im m une system [40]. P atra and M oham ed [41] show ed that A rtem ia supplem ented with
Saccharom yces boulardi w ere protected against Vibrioi harveyi. Im m unostim ulant properties o f w ild type yeast (WT)
and fk sl m utant strain (resulting in fivefold higher cell-w all bound chitin) w ere adm inistrated to the diet to gilthead
sea bream (Sparus aurata L .) for six w eeks under non-axenic conditions [42]. T h e results show ed that chitin-enrichm ent in the fk sl strain m ay be responsible for increasing the innate im m une responses resulting in beneficial effects on
fish perform ance. T he latter findings are not supported by our results.
In conclusion, the m nn9 yeast strain, even in sm all quantities, can protect A rtem ia nauplii against pathogenic bac­
teria, suggesting that this yeast strain is stim ulating the innate im m une response. It seem s probable that m nn9 cells
protect nauplii eith er through their higher concentration o f ß-glucans in the cell w all and/or the higher availability
o f ß-glucans to nauplii. H ow ever, an overall nutritional stim ulation by m nn9 w ith positive effect on the im m unological
status cannot b e excluded. U sing chs3 strain (in com parison to W T) as feed has very little extra effect on the growth
and survival. Yet, this feed can tem porarily protect A rtem ia against V C.
Acknowledgments
T he M inistry o f Science, R esearch and T echnology o f Iran fo r supporting this study through a doctoral grant to the
first author. T he B elgian Foundation for Scientific R esearch (FW O ) for financing the project “ F unctional role and
characteristics o f m icro-organism s in the larviculture o f aquatic organism s: A rtem ia as preferred test organism ”
(no. 350230.02) and the project “ N utritional and im m unostim ulatory characteristics o f isogenic yeast m utants in
A rtem ia ” (no. 1.5.125.04).
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