Box 6, Bet Dagan 50250, Israel Received 12 January 2004; received in revised form 17 June 2005; accepted 20 June 2005 Abstract Growth and survival of postlarval abalone Haliotis discus h
Trang 1Effect of diatom diets on growth and survival of the abalone
Haliotis discus hannai postlarvae
Nurit Gordona, Amir Neoria,*, Muki Shpigela
a
Israel Oceanographic and Limnological Research, National Center for Mariculture, P.O Box 1212, Eilat 88112, Israel
b
Department of Biology, City College of City University of New York, New York, NY 10031, USA
c
Department of Aquaculture, Agricultural Research Organization, The Volcani Center, P.O Box 6, Bet Dagan 50250, Israel
Received 12 January 2004; received in revised form 17 June 2005; accepted 20 June 2005
Abstract
Growth and survival of postlarval abalone Haliotis discus hannai Ino fed different diatom diets were examined for one month from settlement Two diatoms, Amphora luciae Cholnoky and Navicula cf lenzii Hustedt, supported high postlarval growth and survival, especially when supplied in combination A third species, Nitzschia laevis Hustedt, did not support survival for more than two weeks as a unialgal diet and had limited value in mixed diets
Diatom mixtures were superior to single-species diets as of the first week after settlement The mixture of N cf lenzii and A lucia supported the highest survival, up to 50%, and growth rate up to 36Am of shell length per day, reaching a size of 1.4mm
30 days after settlement The three diatom species contained high levels of total lipids (6.4%–14.5% of dry weight) and fatty acids (16%–22% of lipids); from 39% to 48% of fatty acids were polyunsaturated (PUFA) The three diatoms were richer in n-3 PUFA than in n-6 PUFA The content of the essential fatty acid 20:5n-3 (EPA) was highest among the PUFAs and higher, though not significantly, in the two diatom species A luciae and N cf lenzii that produced the better results Among the free amino acids, arginine was dominant in N laevis, proline in N cf lenzii, and both free amino acids plus glutamic acid were equally dominant in A luciae The suitability of A luciae and N cf lenzii for enhancing growth and survival of postlarvae was attributed to their complementary balanced nutritional properties
D 2005 Elsevier B.V All rights reserved
Keywords: Abalone; Postlarvae; Biochemical composition; Growth; Survival; Diets; Diatoms; Fatty acids; Amino acids
1 Introduction Benthic diatoms are the principal food source for
Kawamura et al., 1998a) In spite of the increasing number of studies on the nutrition of newly settled
Kawa-0044-8486/$ - see front matter D 2005 Elsevier B.V All rights reserved.
doi:10.1016/j.aquaculture.2005.06.034
* Corresponding author Tel.: +972 8 6361445; mobile: +972 50
5993746; fax: +972 8 6375761.
E-mail address: aneori@shani.net (A Neori).
www.elsevier.com/locate/aqua-online
Trang 2mura, 1996; Kawamura et al., 1998a,b; Daume et al.,
1999, 2000; Roberts et al., 1999; Searcy-Bernal et al.,
postlarval stages as reported in the literature are
vari-able and generally low (Searcy-Bernal et al., 1992)
Poor and unpredictable performance is related to
variability in food (different diatoms and their
com-position), as well as to abalone species and the
grow-ing conditions in hatcheries (Kawamura et al., 1998a)
To improve growth and survival of abalone postlarval
stages in a specific growing system using specific
diatom species, a better understanding of their basic
diet requirements is necessary Cell density, digestion
efficiency, ingestibility, extra-cellular products, and
associated bacteria are known to affect food value of
Takami, 1995; Kawamura et al., 1995, 1998a,b;
Roberts et al., 1999; Searcy-Bernal et al., 2001)
The biochemical composition of algal cells is another
important factor (Dunstan et al., 1994), but its effect
Mer-cer et al., 1993; Mai et al., 1994, 1995a,b, 1996) rather
than in newly settled postlarvae
The biochemical composition of the diet is most
important once the postlarvae acquire the capability
to digest and benefit from diatom cell content (
Kawa-mura et al., 1998a) According to them, the diatom diet
has little impact on growth rates during the first two
weeks after settlement Diet-dependent postlarval
growth rates diverge at 800 Am SL, when the postlarvae
begin digesting and utilizing the cell content
Accord-ing toDaume et al (1999), differences in growth rates
by postlarvae fed different diatoms can already be
observed earlier, a week following settlement
The nutritional value of microalgae as a feed is
influenced to a great extent by the fatty acid
composi-tion of their lipids (Brown et al., 1997; Renaud et al.,
(Chu et al., 1982) The protein amino acid
composi-tion of microalgae is generally conserved (Brown et
al., 1996) and is unlikely to account for major
differ-ences in the nutritional value of a particular species
(Brown, 1991; Brown and Jeffrey, 1995; Brown et al.,
1997) Free amino acids (FAA) may constitute a
sig-nificant proportion of the total amino acids in the algal
et al., 1998) FAA are easily absorbed by postlarvae
(Manahan and Jaeckle, 1992), a fact that is especially important in very early life stages, before the complete development of the gut enzymes involved in protein digestion (Takami et al., 1998) For this reason the diatom composition phase of the present study has focused on FAA and fatty acids
Diatoms, as a class, offer high levels of lipids and PUFAs, especially the essential PUFA 20:5(n-3) (Dunstan et al., 1994; Brown et al., 1997), and there-fore may fulfill the nutritional requirements of abalone postlarvae better than other algae Polyunsaturated fatty acids (PUFA) of both n-3 and n-6 families are essential for growth of juvenile Haliotis discus hannai (Mai et al., 1996) Their primary function is consid-ered to be structural (Mai et al., 1995a; Floreto et al.,
the most to faster growth of juvenile H discus hannai (Mai et al., 1996)
The aim of this research was to investigate growth and survival of H discus hannai postlarvae fed differ-ent diets of diatoms (including local species), which had previously been shown to induce larval settlement (Gordon et al., 2004), and verify whether these could
be correlated to the diatoms’ nutritional quality
2 Materials and methods 2.1 Preparation of abalone postlarvae Larvae of H discus hannai were obtained from an indoor abalone hatchery in Eilat (Red Sea, Israel) Adults were induced to spawn using ultraviolet light (Kikuchi and Uki, 1974) Fertilized eggs were collected and transferred into 20-L aquaria at a concentration of
12 eggs/ml To control bacterial growth, an antibiotic (Rafamycin) was added at a concentration of 1.5 mg/L Larvae were kept at 22 8C with a 12 L:12 D photo cycle (60–70 Amol photons m 2 s 1), for 4–5 days, until reaching competence Larval competency on day 5 was assessed by observing the swimming behavior, as
larvae were used for the growth experiments
2.2 Diatom cultures Benthic diatoms were isolated from the Red Sea (Eilat, Israel) and from the Atlantic Ocean
Trang 3(Massachu-setts, USA) (Table 1) Axenic cultures were prepared
were cultured in 1-L Erlenmeyer flasks filled with
with silica (Na2SiO3) and aerated with CO2
Tempera-ture was maintained at 22 8C and light intensity was
60–70 Amol photons m 2s 1throughout the growth
experiment
2.3 Growth experiments with abalone postlarvae
Competent larvae of 280 F 12 Am in size were
transferred to 90-mm petri dishes, filled with 32 ppt
Red Sea water (diluted with DDW from 40 ppt) To
reduce handling damage to the larvae, the number of
larvae transferred was calculated according to samples
taken from culture bottles Each petri dish was
stocked with 82 F 17 larvae Settlement was induced
by adding 1 AM gamma-amino butyric acid (GABA)
(Morse, 1992), to petri dishes, whose media included
50 mg/l (each) penicillin and streptomycin (Sigma)
(Morse and Morse, 1984) This approach was
pre-ferred over natural settlement induction by the
dia-toms, to obtain a better reproducibility of larval
settlement across diatom treatments After 24 h,
GABA was rinsed out of the dishes and algae, as
Five replicate dishes were made for each diet Algal
cell concentration was adjusted to obtain a similar cell
volume rather than cell number throughout all the
experiments Water in the dishes was exchanged
daily, and algae were replaced with fresh cultures
once a week During the week algae were added to
each dish once clear patches (consumed diatoms)
developed around the postlarvae, to keep them
sup-plied Larval survival was measured as a percentage of
postlarvae surviving from all the larvae introduced into the petri dishes at the beginning of the experi-ment Larval shell length (SL) was measured once a week for all postlarvae that were on the bottom of the dish, with the aid of a calibrated ocular micrometer, and then averaged Daily growth rate (DGR) was calculated according to the formula: Lf Li/ t, where
Lf= final shell length (Am), Li= initial shell length (Am) and t = time in days The duration of each experi-ment was 31 days
2.4 Biochemical analysis of algal cells Fatty acid analysis was carried out for the three algal species (Nitzschia laevis, Amphora luciae and Navicula cf lenzii) on batches harvested during the late logarithmic growth phase Centrifugally concen-trated algal cells were lyophilized and the lipids were extracted (Folch et al., 1957) The lipid extracts were then transmethylated to fatty acids methyl esters (FAME) by acidified methylation overnight at 50 8C
concentrated in hexane and injected into an
et al., 2001)
For free amino acid analysis, late logarithmic phase cells from the three diatom species were centrifuged and homogenized with an ultrasonic cell disruptor (Microson) for 5 min Cell disruption was confirmed microscopically Free amino acid analysis was carried out with an HPLC (Biotronik LC-5000 Amino Acid
Dry matter was calculated from weight loss after drying for 24 h at 105 8C Crude protein was calculated from Kjeldahl nitrogen multiplied by 6.25 Crude lipid was measured gravimetrically after 5 min
homogeniza-Table 1
Details of the diatoms in the seven diets used in this study
(total cells/cm2) Length (Am) Width (Am)
3 Amphora luciae Lake Tashmoo, Martha’s Vineyard Island, MA, USA 10 5 1.9 104
Trang 4tion of the sample in chloroform-methanol (2:1),
separation and vacuum drying (Folch et al., 1957)
Ash content was calculated from weight loss after
incineration of samples in a muffle furnace for 24 h
at 550 8C Carbohydrates were calculated as:
crude protein þ lipid þ ashð Þ:
2.5 Statistical analyses
The data were compared using ANOVA (one way)
with Duncan’s multiple range tests The results, in
percentages, were arc-sine transformed prior to
and Rohlf, 1969)
3 Results
3.1 Survival of abalone postlarvae
Survival of larvae/postlarvae during the first month
(Table 2) varied from as low as 4% when fed on a diet
of N laevis (diet 2) to a high of 49% when fed on a
diet of N cf lenzii and A luciae (diet 6) Survival of
postlarvae with a diet of N laevis was significantly
lower than with all the other diets ( P b 0.05); these
postlarvae were excluded from later data analysis
3.2 Growth of abalone postlarvae
With the exception of the diet based on N laevis,
the postlarvae grew steadily on all the diets offered
The highest growth rates of postlarvae were obtained
on a mixture diet of N cf lenzii and A luciae (diet 6) and a mixture of the three diatoms (diet 4) After one month in culture, the postlarvae fed these diets reached mean SL of 1.4 mm and 1.3 mm, respectively (Fig 1) Growth rates in these treatments were significantly higher ( P b 0.05) than in all the other diets The SL
of the postlarvae increased by an average of 35.5 F 1.1
Am day 1on the diet of A luciae and N cf lenzii and
combined In the first 12 days, postlarvae grew faster when fed the two-diatom mixture of A luciae and N laevis (diet 7) than those fed most other diets (Table 2) Diet 7 also supported the largest ( P b 0.05) postlarval
Table 2
Survival rates and daily growth rates (DGR based on shell length) of the postlarvae fed the seven different diets of diatom listed in Table 1 Diet # Initial individuals
per dish (# F SD)
Survival rate at
31 days (% F SE* )
DGR, % day 1F SE (*)
* Data with the same letter indicate treatments that are not significantly different from each other within columns (ANOVA, Duncan’s multiple range test, critical p = 0.05).
500 1000
1500
1 N cf lenzii
2 N laevis
3 A luciae
4 Mix 1+2+3
5 Mix 1+2
6 Mix 1+3
7 Mix 2 + 3
Diets
a b c d
d
a c b
e
Time from larval introduction (days)
Fig 1 Growth of postlarval shell length (SL F SE) during 31 days post settlement More information on the diets is provided in Table
1 Significant differences (ANOVA, Duncan’s multiple range test,
p b 0.05) between data points are letter-labeled only on days 24 and
31, to reduce clutter.
Trang 5SL for the first 2 weeks (Fig 1) However, later, diet 6
and diet 4 became the significantly better diets with
respect to both DGR and SL of the postlarvae, while
the performance of diet 7 deteriorated by day 24 and
became even worse by day 31 (Fig 1) Growth of the
postlarvae on the single alga N laevis (diet 2) was
worst ( P b 0.05) of all diets after the first week (Fig 1)
The diet of A luciae (diet 3) was best of the
single-diatom diets, yet significantly ( P b 0.05) inferior to all
but one of the mixed-diatom diets
3.3 Lipid content and composition of diatoms
Lipids comprised between 6.4% and 14.5% of the
dry weight of the diatoms analyzed (Table 3)
Poly-unsaturated fatty acids (PUFA) constituted the largest
fraction (between 41% and 47%) of the total fatty
acids (TFA) The proportion of the various PUFAs
varied among the diatom species Although all of the
analyzed diatoms had significant quantities (between
14% and 21% of TFA) of 20:5n-3 (EPA), N laevis
had slightly lower percentages of this fatty acid and
higher quantities (9.7% of TFA) of 20:4n-6
arachido-nic acid (AA) Shorter chain PUFAs, 16:2n-4 and
16:3n-4, were also present in significant quantities
3.4 Amino acid content and composition of diatoms
The three diatoms varied in their total free amino
acid (TFAA) composition (Table 5) Proline was the
main free amino acid (2.8 fmol cell 1, 49% of TFAA)
in N cf lenzii but only a minor constituent (0.13 fmol
cell 1, 8% of TFAA) in N laevis The proline content
of A luciae was intermediate (0.8 fmol cell 1, 30% of
TFAA) The share of arginine in the TFAA fluctuated
even more than proline among the analyzed species
In N laevis arginine was the main free amino acid
(0.7 fmol cell 1, 43% of TFAA), while in N cf lenzii
luciae arginine appeared in between these extremes
glutamic acid in N laevis was lesser in the other two species but it contained glutamine, which was
Table 3
Biochemical composition of the three diatoms used in this study
Diatom Protein (% in DW) Lipids (% in DW) Fatty acids (% in Lipids) Carbohydrates (% in DW) Ash (% in DW)
a A missing value (technical reason).
Table 4 The content of specific fatty acids as fractions of total fatty acids (TFA) in the three diatoms used in this study (n = 4 in a, n = 2 in b) Type of fatty acid Diatom species
N laevisa (% F SD)
A luciaea (% F SD)
N cf lenziib (% F SD) Saturated
16:0 14.7 F 0.5 13.7 F 0.9 13.9 F 0.1
Monounsaturates 15:1n-8 0.3 F 0.3 0.1 F 0.2 0.0 F 0.0 16:1n-7 22.4 F 1.6 18.4 F 2.4 18.8 F 0.4 16:1n-9 2.7 F 0.9 2.8 F 0.3 3.5 F 0.1 18:1n-7 1.0 F 0.7 0.8 F 0.8 1.5 F 0.2 18:1n-9 2.7 F 2.6 3.6 F 2.6 4.5 F 0.0
Polyunsaturates 16:2n-4 4.2 F 0.2 4.2 F 0.6 5.6 F 0.4 18:2n-6 1.7 F 0.7 3.4 F 0.1 1.5 F 0.1
18: 3n-3 0.6 F 0.4 0.5 F 0.2 0.9 F 0.6 18:3n-4 0.3 F 0.4 0.1 F 0.3 0.0 F 0.0 18:3n-6 0.8 F 1.1 0.8 F 1.2 1.8 F 0.0 18:4n-3 0.0 F 0.0 1.1 F 0.9 0.0 F 0.0 20:2n-6 0.4 F 0.5 0.5 F 0.7 0.7 F 0.1 20:3n-3 0.3 F 0.6 0.0 F 0.0 0.5 F 0.1 20:4n-6 (AA) 9.7 F 1.3 5.0 F 1.4 2.8 F 0.1 20:4n-3 0.0 F 0.0 0.3 F 0.4 0.0 F 0.0 20:5n-3 (EPA) 14.3 F 3.5 18.5 F 2.9 21.2 F 0.2
22:5n-3 0.4 F 0.5 0.8 F 0.2 0.85 F 0 22:6n-3 (DHA) 1.6 F 0.2 0.5 F 0.6 1.8 F 0.9 Sum PUFA 42.4 F 3.4 41.2 F 2.6 46.6 F 1.1 Sum n-3 PUFA 17.2 F 2.9 21.7 F 3.2 25.1 F1.9 Sum n-6 PUFA 12.6 F 0.9 9.7 F 3.1 6.8 F 0.1
Trang 6not found in the other species The protein content
was the same in N cf lenzii and A luciae (32% of
DW) and higher in N laevis (38% of DW) (Table 3)
The carbohydrate content was lowest in N laevis
(18%) and highest in N cf lenzii (25%) The ash
content was highest in A luciae (41%)
4 Discussion
4.1 Growth and survival of abalone postlarvae
The growth experiments with H discus hannai
postlarvae fed the different diatom diets, together
with biochemical analysis of these diatoms,
estab-lished that those diatoms that are attractive for the
larval settlement usually also support postlarval
et al., 2004) we found that the three diatom species
used in this research, N laevis, N cf lenzii and A
luciae, induced settlement of H discus hannai larvae
We examined the suitability of these diatoms to
sup-port early postlarval growth, concurring with other
studies, where conditions that induced a good larval
settlement were usually followed by high growth rates
and survival of the settled postlarvae (Daume et al.,
dsettlement-inductiveT diatoms differed greatly in
their nutritive value A luciae and N lenzii were indeed highly nutritious A luciae was the best among unialgal diets and, combined with N cf lenzii
in a two-diatom diet, supported the best postlarval growth and survival in this study On the other hand, N laevis, the preferred species for settlement (Gordon et al., 2004), was unsuitable as a sole diet for early postlarval growth or survival This finding is reminiscent of the results with the diatom Cocconeis scutellum var parva, when fed to postlarvae of H discus hannai byTakami et al (1997) Both diatoms induced good larval settlement, but were poor food for newly settled postlarvae The latter authors attributed their observations to the scarce mucus secretion and the highly adhesive strength of C scutellum var parva These properties made it an unsuitable diet for the postlarvae, which eventually starved In con-trast, N laevis, being small, poorly silicified and attached only weakly, is probably more easily edible Yet it did not support growth, and 96% of the post-larvae began to avoid the algae after several days and eventually died within 2 weeks This observation suggests that the abalone postlarvae do not like N laevis as a sole food for extended periods, for reasons yet to be determined It could be that N laevis secretes unfavorable or toxic substances that gradually accu-mulate in the dishes (Wen and Chen, 2002)
A good growth of the postlarvae was apparently related to a wholesome diet, as indicated by the synergism between the two bbestQ diatoms, A luciae and N cf lenzii, in the sustenance of the fastest growth rates and largest SL when administered together in this study, as also suggested by Epifanio (1979) However, Kawamura et al (1998a) showed the benefit of a good diet is not necessarily steady and sustained, as we have shown with the mixture of A luciae and N laevis An inconstant nutritional value
of this mixture may reflect a dual function, wherein N laevis apparently provides the postlarvae feeding sti-mulation right after settlement, while A luciae pro-vides the required balanced nutrition for more sustained growth This diet combination could there-fore support the best growth only for the first 2 weeks after settlement Afterwards, however, the same rea-sons that caused the postlarvae to avoid feeding on a uni-algal N laevis diet after several days apparently came into play, making this mixture unsuitable for further growth
Table 5
The content of free amino acids (FAA) in cells of the three diatoms
used in this study
Amino acid Diatom species
N laevis A luciae N cf lenzii
fmol
cell 1
cell 1
cell 1
%
Aspartic
acid
0.12 7.11 0.21 9.56 0.59 10.43
Glutamic
acid
0.21 12.69 0.45 20.14 1.08 19.24
Glutamine 0.24 14.21 0.00 0.00 0.00 0.00
Proline 0.13 7.87 0.68 30.38 2.79 49.46
Alanine 0.16 9.90 0.21 9.56 0.66 11.69
Ornithine 0.01 0.76 0.06 2.73 0.07 1.26
Arginine 0.71 42.89 0.50 22.18 0.18 3.24
Trang 7Nutritional developments with postlarval age seem
to include increases in the rate and efficiency of
feeding and digestion, leading to the acceleration in
the postlarval growth rates with most diets during
days 13–24 post-settlement; this phenomenon was
already noted by other investigators (Martinez-Ponce
and Searcy-Bernal, 1998; Kawamura et al., 1998a;
Roberts et al., 1999) The reduced postlarval growth
rates in the fourth week of the experiment, when they
were already over 1 mm long, probably resulted from
excessive biomass densities (Kawamura et al., 1998a)
reached in the petri dishes
4.2 Nutritional value of diatoms to abalone
postlarvae
The three diatoms contained protein levels that
al., 1995b) They were probably also similar to each
other in their high protein quality (Brown and Jeffrey,
conditions (nutrients, light and temperature) and
har-vested in the same phase of growth, it can be
assumed they had similar nutritional value with
respect to their amino acid profiles (Brown and
Jef-frey, 1995; Brown et al., 1997) Conversely, as in
Martin-Jezequel et al (1988), De Roeck-Holtzhauer
et al (1993)and Derrien et al (1998), the
composi-tion of free amino acid (FAA) varied between the
diatoms and was dominated in each diatom by
dif-ferent FAAs
In A luciae, our bbestQ unialgal diet, three
predo-minant FAAs (proline, arginine and glutamic acid)
were in equal amounts In N cf lenzii, which
sup-ported moderate yet steady growth of the postlarvae,
proline was predominant Proline is considered an
essential amino acid for molluscs (Harrison, 1975)
and a major component in the FAA pool in the tissues
of early developmental stages of abalone (Litaay et
al., 2001) However, arginine, an often limiting
essen-tial amino acid for abalone postlarvae (Mai et al.,
which was not as good a diet as the other two species
Good lipid complements also contribute to the
nutritional value of a diatom In the three diatoms
studied, total lipid content was about double the
value reported to be required for maximal growth of
juvenile abalone (Uki et al., 1986; Mai et al., 1995a)
The relative PUFA content in our three diatoms was high in comparison to diatoms of similar size ( Volk-man et al., 1989; Renaud et al., 1999) and so was the ratio of n-3 to n-6 PUFA Both of these fatty acid groups are considered essential for the growth of H discus hannai juveniles (Uki et al., 1986; Mai et al.,
1996) The essential fatty acid 20:5n-3 (EPA) was the predominant n-3 PUFA in the diatoms studied here, as
in other diatoms (Dunstan et al., 1994) This PUFA is reported to promote a fast growth in H discus hannai juveniles (Mai et al., 1995a, 1996; Dunstan et al.,
1996) Indeed, our two dbetterT diatoms, N cf lenzii and A luciae, contained larger fractions of n-3 PUFA, especially EPA, than N laevis (though the latter still had a higher EPA content than other Nitzschia species reported for instance by Renaud et al., 1999) On the other hand, in our worst diatom, N laevis, PUFAs were dominated by n-6 and particularly by arachido-nic acid 20:4n-6; these PUFAs are important for larval stages of fish but have no special reported importance
in abalone The fatty acid 22:6n-3 (DHA), which is low in abalone tissue and therefore presumed of a lesser quantitative importance (Dunstan et al., 1996; Fleming et al., 1996; Mai et al., 1996), was also low in the three diatoms studied here
Carbohydrate content in our three diatoms was
Jeffrey, 1995; Renaud et al., 1999; Simental-Trinidad
et al., 2001) and within the range needed for juvenile abalone diet (Mercer et al., 1993)
The fact that no gross composition nor single chemical component (fatty acid or free amino acid) could be decisively correlated with postlarval growth
or survival was, as suggested by others (Chu et al., 1982; Mai et al., 1996; Brown et al., 1997), probably due to the multitude of components that determine the nutritional value of a diatom
5 Conclusions Carefully controlled, mixed and administered diets
of selected diatoms have provided consistently good growth and survival of abalone postlarvae during their most critical stage of life, when mortalities are high-est The results presented here substantiate the nutri-tional basis proposed for low performance of abalone postlarvae in their natural habitat and in certain
Trang 8arti-ficial settings; the biochemical composition of the
diatoms has been shown to affect their suitability as
feed for abalone postlarvae Differences in n-3 PUFA
and in FAA composition of diets used in this study
can partly explain differences in diatom nutritional
value, as reflected in postlarval growth and survival
The results can be of practical help in the reproduction
of abalone in culture
Acknowledgements
This work was supported by the Israeli Ministry for
National Infrastructures (N.G., M.S and A.N.), by
several grants from the European Commission (M.S
and A.N.) and NIH /NIGMS 08168-22 (J.J.L.) We
are grateful to E Chernova, D Malka, B Koven, H
Krogliak, I Lupatsch, R Weiss, and V Zlatnikov for
their help during the experiments; to M Ben-Shaprut,
A Colorni and several anonymous reviewers for help
in preparation of the manuscript and bringing it to its
final form
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