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Mariculture Research Laboratory, College of Fisheries, Ocean University of Qingdao, Qingdao 266003, Shandong, P. R. China
2To whom correspondence should be addressed. E-mail: kmai{at}mail.ouqd.edu.cn.
ABSTRACT
The experiments were conducted to investigate the effects of dietary myo-inositol on the survival, growth, proximate composition and de novo synthesis of myo-inositol in abalone, Haliotis discus hannai Ino. The possible inositol-synthesizing capacity of intestinal microflora was also examined. Seven semipurified diets were formulated to provide graded levels of myo-inositol (28.7–1020.1 mg/kg diet). A control diet, the basal diet supplemented with 4 g/kg tetracycline hydrochloride, was employed to suppress synthesis of myo-inositol by intestinal bacteria. Abalone juveniles of similar size (weight, 144.6 ± 0.8 mg; shell length, 10.92 ± 0.10 mm) were distributed in a flow-through system using a completely randomized design with eight treatments and three replicates per treatment. They were fed the appropriate diets once daily for 16 wk. Survival, growth, crude protein, lipid, moisture of whole soft body and visceral inositol content were independent of myo-inositol supplementation (P > 0.05). The addition of the antibiotic also did not affect the survival, growth and whole soft body composition. It indicated that intestinal microflora contributed little to the myo-inositol nutrition in abalone. The present study, for the first time, demonstrated de novo synthesis of myo-inositol in mollusks because the visceral tissue of abalone showed high levels of myo-inositol synthetase activities (combined activities of myo-inositol-1-phosphate synthetase and inositol-1-phosphatase), ranging from 74.0 to 98.2 µmol/(h·g protein). The enzyme activity significantly and negatively correlated with dietary myo-inositol level (r = -0.81). Hence, dietary myo-inositol is not essential for abalone because tissue synthesis of the vitamin appears to be sufficient to support normal growth and health of this mollusk.
KEY WORDS: • Haliotis discus hannai • myo-inositol • synthesis • mollusks
Myo-inositol is a structural constituent in living tissues and commonly
occurs as a component of phospholipids in animal cells. It plays an
important role in fat metabolism by promoting the export of fat from
the liver (1
). Recent advances in nutritional and
biochemical studies have demonstrated that phosphatidylinositol in cell
membranes is involved in regulation of cellular responses to external
stimuli and/or nerve transmission as well as mediation of enzyme
activity through interactions with various specific proteins
(2
). Furthermore, myo-inositol has psychoactive
effects by interacting with the second messenger system and ultimately
regulating the cytosolic concentration of calcium (3
, 4
).
The essentiality of myo-inositol for various species is generally
believed to be dependent upon the activity of myo-inositol
synthetase (combined L-myo-inositol-1-phosphate synthetase,
EC 5.5.1.4, and L-myo-inositol-1-phosphatase, EC
3.1.3.25) (5
). The intestinal microflora has also been
suggested to contribute to myo-inositol nutrition in some animals
(6
, 7
). In addition, dietary composition such as the levels
of carbohydrates and lipids appears to influence the severity of
myo-inositol deficiency (8
, 9
).
Dietary myo-inositol deficiency has been demonstrated in most
aquatic animals, including common carp, red sea bream, Japanese eel,
rainbow trout, chinook salmon and shrimp. In aquatic animals, the signs
of dietary inositol deficiency have involved mainly anorexia, poor
growth, edema, skin lesion, dark color, increased gastric emptying
time, distended stomachs, reduced cholinesterase and transaminase, and
the content of phosphotides (10
–15
). Interestingly,
channel catfish did not show obvious deficiency signs
(16
). It was further proved that de novo synthesis of
myo-inositol by fingerling channel catfish was sufficient to
support normal growth and a constant level of tissue myo-inositol,
and to prevent overt signs of myo-inositol deficiency
(17
).
For any species of mollusk, the essentiality of dietary
myo-inositol and the inositol-synthesizing capacity remain
unknown. Abalone are large algivorous marine mollusks of the genus,
Haliotis (Gastropoda, Prosobranchia,
Archaeogastropoda, Haliotidae). They are the most commercially
important gastropods in aquaculture. Due to the widening gap between
supply and demand in the abalone market, interest has been growing in
the development of nutritionally balanced and effective artificial
feeds for abalone farming. With decades of study, the requirements of
abalone for dietary protein, lipid, carbohydrate and essential fatty
acids have been estimated (18
–24
). More recently, some
reports have begun to focus on the requirements, interaction and
availability of minerals in abalone (25
–28
). However,
very few studies have reported on the vitamin nutrition for any mollusk
species. Only in our laboratory have the effects of dietary vitamins A,
K and C on the growth, survival and tissue concentration of the
vitamins of the abalone H. tuberculata and/or H.
discus hannai been investigated (29
–31
). For the
sake of safety, vitamins are usually added to abalone feeds based on
the requirements of fishes, and generally in excess
(19
–32
). Undoubtedly, this status could not obtain
maximum efficiency of nutrient utilization and profit with minimum
ecological effect.
The objective of the present study was to investigate the effects of dietary myo-inositol on the survival, growth and proximate compositions of abalone, H. discus hannai Ino. The possible de novo biosynthesis of myo-inositol by abalone and/or by intestinal microflora in abalone was also investigated.
MATERIALS AND METHODS
Diet preparation.
The basal diet formulation (Table 1
) is similar to that of Mai et al. (23
), which has been
shown to be adequate to support normal growth and health of H.
tuberculata and H. discus hannai. Dietary treatments
were prepared by replacing the dextrin with graded levels (0, 100, 200,
400, 600, 800 and 1000 mg/kg diet) of myo-inositol (Sigma, St.
Louis, MO). Before addition to the basal diet, the crystalline inositol
and other water-soluble vitamins were microencapsulated with sodium
alginate, according to a modification of the method of Bodmeier and
Wang (33
). Briefly, a portion of sodium alginate solution
(20 g/L) containing 20 g/L vitamins was mixed with another portion of
oil containing Span80 (6.74 g/L) and stirred at 400 rpm for 15 min. The
emulsified solution was slowly poured into 1 g/L
CaCl2 solution, with continuously stirring for 1
min, then filtered below normal pressure. The harvested microcapsules
were washed with cyclohexane and absolute alcohol, in turn, to remove
oil and water, dried below normal pressure and kept at -20°C until
use.
|
). Our preliminary study showed that the intestinal
bacteria of abalone could be effectively suppressed by 4 g/kg diet of
tetracycline hydrochloride, whereas no negative effects were observed.
Procedures for diet preparation and storage were the same as those
described by Mai et al. (23
, 26
). The myo-inositol in
the test diets was extracted with HCl and
Na2SO4 to determine the
real levels of dietary inositol by colormetric method
(35
, 36
). Briefly, 10 µL of feed extract was
added in 250 µL of distilled water. Then, 50
µL sulfuric acid (8 mol/L) was added, followed by 20
µL hydroquinone solution (0.3 mmol/L) and 100
µL
Na2Cr2O7-CrCl3
mixed reagent (containing 20 mmol/L
Na2Cr2O7
and 10 mmol/L CrCl3). The mixture was shaken and
immersed in a boiling water bath for 8 min. The preparation was cooled.
Then, 20 µL arabinose (0.13 mol/L) and 0.6 mL sulfuric
acid (9 mol/L) were added. The reaction solution was immersed in
boiling water for 10 min. At the end of the reaction, the absorption
was determined at 450 nm. The inositol contents of the experimental
diets were estimated to be 28.7, 141.2, 233.6, 423.7, 627.5, 816.9,
1020.1 and 28.3 mg/kg diet for
D0,3
D100, D200, D400, D600, D800, D1000 and the control diet,
respectively.
The leaching test of dietary myo-inositol was modified from the
method used by Marchetti et al. (37
). Each feed (10 g),
packaged in a nylon mesh bag (100 mesh), was immersed in a beaker
containing 2 L of seawater maintained at 20.0 ± 1°C and
mechanically stirred. Thimerosal was added at 100 mg/L to reduce
bacterial activity. At the end of the allotted time (3, 6 and 12 h, respectively), the remaining feed was removed from the bags and
dried overnight at 60°C in an oven. Dried feed was submitted for
analysis of total dietary myo-inositol by the colormetric method
(36
). The leaching of dietary myo-inositol was
reflected as retention efficiency (RE), which is defined as follows:
 |
 |
Experiment procedures.
Juvenile abalone used in this experiment were derived from a spawning
in June 1999, at Mashan Fisheries, Rongcheng, P. R. China. The
present study on abalone complied with the NIH (38
).
Before initiation of the experiment, the abalone underwent a 1-wk
conditioning period during which they readily acclimated to the
environmental conditions. Before trial, shell length was measured with
calipers to the nearest 0.02 mm and the abalone were weighed to the
nearest 0.1 mg using an electronic balance. They were kept in acrylic
square cages (35 x 28 x 20 cm). Each rearing unit was
stocked with 40 abalone juveniles. Similar size juveniles of H.
discus hannai Ino (weight, 144.6 ± 0.8 mg; shell length,
10.92 ± 0.10 mm) were assigned to the rearing system using a
completely randomized design with eight treatments and three replicates
per treatment. The system was flow-through, with water filtered to
30 µm by primary sand filters, then to 10
µm by secondary composite sand filters. The flow rate
was 
0.5 L/(min · cage). Cages were kept in dim light by screening
with black plastic drapes. During the experimental period, water
temperature was 18.2–22.0°C, salinity 30–34 parts per thousand, pH
7.6–7.9. Dissolved oxygen was not <7.0 mg/L, and there were
negligible levels of free ammonia and nitrite determined by the
conventional procedures (39
). Abalone were hand-fed
the test diets at a rate equaling 5–10% of abalone wet body weight
once daily at 1700 h. Every morning, uneaten feed and feces were
cleaned to maintain the water quality. The feeding trial was conducted
for 16 wk.
At the termination of the feeding trials, abalone were not fed for
3 d to deplete digestive canal contents. The procedure was done
first to obtain an accurate body weight and second, to prepare visceral
samples for the determinations of tissue inositol and inositol
synthetase activity. All abalone were removed from the rearing system,
weighed, measured and counted. Then, 30 abalone from each replicate
were quickly frozen (-70°C) for subsequent analysis. Growth was
reported as specific growth rate (SGR, %/d) and daily increment in
shell length (DISL, µm/d). The calculation formulae
are as follows:
 |
 |
where Wt, Wi are final and initial mean weight (mg), respectively, SLt, SLi are final and initial mean shell length (mm), respectively, and t is the feeding trial period (d).
The frozen samples were slightly thawed, and shell and soft body were
separated. An aliquot of whole soft bodies (including mantle, foot
muscle and viscera) was finely cut and homogenized for proximate
analyses to determine the crude protein, lipid and moisture contents
using conventional procedures (39
). The visceral tissues
(including all inner organs, mainly digestive canal and hepatopancreas
in weight) were separated from another aliquot of soft bodies and
homogenized. The homogenate was freeze-dried in vacuo;
myo-inositol was extracted from the visceral tissue with boiling
water (40
) and assayed by the colormetric procedure
described above (36
).
Another aliquot of visceral tissue was used for the measurement of
myo-inositol synthetase activity. Samples were homogenized for 5
min in 5 volumes of 0.02 mol/L phosphate buffer (pH 7.2) containing 0.5
mmol/L glutathione in an ice bath. The homogenate was heated (60°C)
for 2 min to destroy glucose-6-phosphatase and then diluted with 10 mL
of phosphate buffer and centrifuged for 10 min at 900 x g. The
supernatant was further centrifuged at 100,000 x g at 4°C for
3 h. The resulting supernatant was dialyzed overnight against
0.005 mol/L Tris-HCl (pH 7.2) at 4°C. The protein concentration
of supernatant was assayed using bovine serum albumin as the standard
(41
). Then, the conversion of glucose-6-phosphate to
inositol by enzymes (inositol-1-phosphate synthetase and
inositol-1-phosphatase) in the viscera extracts was measured
(42
). Briefly, 50 µL of 0.02 mol/L
D-[1-14C]-glucose-6-phosphate
(specific radioactivity, 1.95 GBq/mol, NEN, Boston, MA), together with
25 µL each of nicotinamide adenine dinucleotide (8.0
mmol/L) (Sigma), MgCl2 (1.2 mmol/L) and
NH4Cl (0.14 mmol/L), 50 µL of enzyme
solution and water to a final volume of 250 µL, was
incubated for 1 h at 29°C. Immersing in boiling water for 1 min
stopped the reaction. The preparation was cooled and centrifuged (900
x g, 10 min). Supernatant (10 µL) was
chromatographed on Whatman No.1 paper using acetone/water (85:15, v/v)
as solvent. The inositol area was determined by Isotope-imaging
Analyzer (BAS-1800II, Fujifilm, Tokyo, Japan) and cut out, then
counted in 5 mL of scintillation fluid (Packard Instrument, Meriden,
CT) in a liquid scintillation analyzer (Winspectral-1414, Wallac,
Germany). Preliminary work with each assay ensured that
enzyme-saturating conditions were achieved. The enzyme activity was
expressed as units per gram of protein, where 1 U equals 1
µmol myo-inositol synthesized/h at 29°C. All samples
were determined in triplicate.
Statistical analysis.
All percentage data were square-root arcsine transformed before analysis. Data from each treatment were subject to one-way ANOVA. When overall differences were significant at less than the 5% level, Tukey’s test was used to compare the means between individual treatments. Statistical analysis was performed using STATISTICA package (Stat Soft, Tulsa, OK).
RESULTS
The results of the 12-h leaching test of the supplemented
myo-inositol are presented in Table 2
. The supplemented myo-inositol content in all diets decreased with
the increase in immersion time. After 3- and 6-h immersions in sea
water, the RE was 85.4–92.6 and 70.4–77.9%, respectively; this
value decreased to 38.4–56.1% after a 12-h immersion in all diets. In
the first 6 h, there were no significant differences in RE among
all dietary treatments. After a 12-h immersion, however, the RE of the
all diets were significantly different (P < 0.05), and
were negatively correlated with the supplemented level of
myo-inositol (r = -0.96; P < 0.05).
|
). Similarly, the soft-body moisture (736.3–761.7 g/kg), crude
protein (540.7–560.3 g/kg), crude lipid (61.3–80.7 g/kg) and visceral
myo-inositol concentration (2.17–2.22 µmol/g) were
independent of myo-inositol supplementation (Table 4
). Compared with other groups, the control diet with tetracycline
(4g/kg) did not affect survival, growth, proximate composition or the
visceral inositol concentration in abalone.
|
|
. Myo-inositol synthetase
activity was the highest in abalone fed the basal diet (D0),
intermediate in those fed the diet containing 141.2, 233.6, 423.7 and
627.5 mg inositol/kg diet and lowest in abalone fed the diets
supplemented with 816.9 and 1020.1 mg/kg diet. The differences between
the highest group and the lowest groups were significant (P
< 0.05). The myo-inositol synthetase activity of abalone fed
the antibiotic diet (control) was not different from that of those fed
the basal diet.
DISCUSSION
A previous study in our laboratory demonstrated that leaching of
water-soluble vitamins from the experimental diets of abalone was
significantly reduced by microencapsulation (43
). In the
current study, the RE of dietary myo-inositol ranged from 85 to
92% after a 3-h immersion in seawater, and there was no significant
difference observed in the RE after the first 6-h immersion (Table 2)
.
Abalone usually reach satiation in 2 h (44
). Thus,
the leaching here should not significantly influence the results and
conclusions. In practice, most farmers expect an average growth rate of
2–3 mm/mo (67–100 µm/d) to ensure that abalone reach
50 mm in 2 y and 70–80 mm in 3 y (45
). In
comparison to the figures reported by other authors (0.66–1.36%/d in
SGR, 53–95 µm/d in DISL) (20
–24
), the
satisfactory growth of abalone (1.29–1.34%/d in SGR, 66.4–76.0
µm/d in DISL) in this study indicated that the diets and
water-soluble vitamins encapsulated by calcium alginate could be
utilized efficiently by the abalone.
Although myo-inositol is generally categorized as a
water-soluble vitamin, most warm-blooded animals including
humans do not require a dietary source of myo-inositol. It has been
found that de novo synthesis of myo-inositol occurs in liver,
kidney, testis, brain and other tissues of these animals
(5
, 8
, 46
–50
). In contrast to warm-blooded
animals, most aquatic animals depend upon dietary myo-inositol, and
the dietary requirements are usually very high, e.g., salmon, 400 mg/kg
diet; common carp, 440 mg/kg diet; red sea bream, 300 mg/kg diet; sea
bass, 800 mg/kg diet; rainbow trout, 250–500 mg/kg diet; and
yellowtail, 423 mg/kg diet (1
, 10
, 12
, 51
, 52
). Nevertheless,
some aquatic animals have demonstrated the ability to synthesize
myo-inositol. The possible biosynthesis of myo-inositol was
suggested in carp, although it is likely insufficient at a younger
stage (12
). Additionally, the lack of response to dietary
myo-inositol was attributed to the de novo synthesis of
myo-inositol in channel catfish, Ictalurus punctatus
(17
). To our knowledge, the biosynthesis of
myo-inositol was first reported in rat and chick embryos
(53
). Since then, a series of studies have been designed
to elucidate the pathway for the biosynthesis of myo-inositol
(9
, 42
, 54
). Two enzymes believed to be responsible for
myo-inositol synthesis are
L-myo-inositol-1-phosphate synthetase (EC
5.5.1.4) and L-myo-inositol-1-phosphatase (EC
3.1.3.25). Through the effects of these two enzymes, glucose can be
converted into myo-inositol in vivo or in vitro. However, the
ability differs from species to species. For example, under similar in
vitro conditions, the myo-inositol synthetase system in yeast had
29,000 U of activity (42
), and rat testis
homogenate had 13 U (54
). The myo-inositol
synthetase activity of either liver or brain in fingerling channel
catfish had 39.8 and 67.3 U, respectively
(17
). In the current study, a high activity of
myo-inositol synthetase (74.0–98.2 U) was detected in
abalone viscera. Furthermore, at the termination of the present
experiment, abalone in all treatment groups grew normally, did not show
overt signs of deficiency, and maintained high and similar levels of
tissue myo-inositol (Table 4)
. Hence, the present study clearly
showed that abalone, one of the mollusk species, can synthesize
myo-inositol de novo to meet physiologic needs. Compared with other
organisms, the ability of abalone to synthesize myo-inositol
(74.0–98.2 U) is lower than that of yeast (29,000
U), but higher than those of chick embryos (5.6
U), rats (13 U), gerbils (27 U) and
channel catfish (39.8–67.3 U) (17
, 46
–48
, 53
).
The intestinal synthesis of myo-inositol by microflora of the
gastrointestinal tract also provides considerable inositol for some
species. It was reported that the suppression of intestinal bacteria
growth produced hepatic lipid accumulation in rats, and this was
prevented by adding inositol to the diet (6
). In aquatic
animals, the biosynthesis of myo-inositol by intestinal microflora
is still uncertain. However, intestinal microflora have been shown to
contribute folic acid, B-12 and biotin to some fishes
(55
–59
). In the present study, the survival, growth and
carcass proximate compositions of abalone fed the antibiotic diet were
not significantly different from those of abalone fed the basal diet.
This indicates that the intestinal microflora probably contribute
little to myo-inositol nutrition for juvenile abalone. This is
similar to the case in channel catfish (17
).
Dietary myo-inositol supplementation significantly affected
myo-inositol synthetase activity (Table 4)
, and a negative
correlation between the enzyme activity and dietary myo-inositol
levels was observed (r = -0.81). This implies that
there probably is a feedback regulation system between myo-inositol
synthetase activity and exogenous myo-inositol, which warrants
further investigation.
In conclusion, the present study clearly demonstrates that dietary myo-inositol is not essential for abalone because tissue synthesis of this vitamin is sufficient to support normal growth and health. It is the first time that the capacity of myo-inositol synthesis in mollusks has been demonstrated.
ACKNOWLEDGMENTS
We would like to thank Q. F. Ye (Institute of Nuclear Agriculture, University of Zhejiang, China), W. Xu and Z. G. Liufu (Ocean University of Qingdao, China) for their excellent technical assistance.
FOOTNOTES
1 Supported by grants from the National Science
Fund for Distinguished Young Scholars (NSFC, no. 39925029) and from the
Excellent Young Teachers Program of MOE, P. R. China. 
3 Abbreviations used: D0, D100, D200, D400, D600,
D800, D1000, diets containing 28.7, 141.2, 233.6, 423.7, 627.5, 816.9
and 1020.1 mg/kg inositol, respectively; DISL, daily increment in shell
length; RE, retention efficiency; SGR, specific growth rate.
Manuscript received 21 February 2001. Initial review completed 6 April 2001. Revision accepted 20 August 2001.
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