close

Вход

Забыли?

вход по аккаунту

?

j.fsi.2018.08.034

код для вставкиСкачать
Accepted Manuscript
Multi-strain probiotics enhance immune responsiveness and alters metabolic profiles
in the New Zealand black-footed abalone (Haliotis iris)
Roffi Grandiosa, Fabrice Mérien, Tim Young, Thao Van Nguyen, Noemi Gutierrez,
Eileen Kitundu, Andrea C. Alfaro
PII:
S1050-4648(18)30509-6
DOI:
10.1016/j.fsi.2018.08.034
Reference:
YFSIM 5492
To appear in:
Fish and Shellfish Immunology
Received Date: 29 April 2018
Revised Date:
13 August 2018
Accepted Date: 16 August 2018
Please cite this article as: Grandiosa R, Mérien F, Young T, Van Nguyen T, Gutierrez N, Kitundu E,
Alfaro AC, Multi-strain probiotics enhance immune responsiveness and alters metabolic profiles in the
New Zealand black-footed abalone (Haliotis iris), Fish and Shellfish Immunology (2018), doi: 10.1016/
j.fsi.2018.08.034.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Multi-strain probiotics enhance immune responsiveness and alters
2
metabolic profiles in the New Zealand black-footed abalone
3
(Haliotis iris)
4
5
Roffi Grandiosaa, Fabrice Mérienb, Tim Younga, Thao Van Nguyena, Noemi Gutierreza, Eileen
Kitundua, Andrea C. Alfaroa*
6
7
a
8
9
b
RI
PT
1
Aquaculture Biotechnology Research Group, School of Science, Auckland University of
Technology, Auckland, New Zealand
SC
AUT-Roche Diagnostics Laboratory, School of Science, Auckland University of Technology,
Auckland, New Zealand
10
EP
TE
D
Corresponding author
Andrea C. Alfaro
Phone: +64-9-921-9999 ext. 8197
Fax: +64-9-921-9743
andrea.alfaro@aut.ac.nz
M
AN
U
*
AC
C
11
12
13
14
15
ACCEPTED MANUSCRIPT
Abstract
17
We assessed whether dietary administration of a multi-strain probiotic (Exiguobacterium
18
JHEb1, Vibrio JH1 and Enterococcus JHLDc) led to enhanced immune responsiveness in
19
juvenile New Zealand black-footed abalone (Haliotis iris). Two groups of abalone were fed
20
(1% body weight per day) over a four-month period with different diets. The control diet
21
consisted of a standard commercial pellet feed (AbMax 16), whereas the treatment diet was
22
additionally enriched with the probiotic mix. At the end of the experiment, probiotic-fed
23
animals showed improved growth compared with control-fed abalone in (length (32.3% vs
24
22.3%), width (31.9% vs 20.7%) and wet weight (109.6% vs 72.8%), respectively.
25
Haemolymph sampling was conducted at the beginning of the experiment and after 2 and 4
26
months. Haemolymph samples were analysed for total haemocyte count (THC) and viability,
27
presence of apoptotic cells and production of Reactive Oxygen Species (ROS). Compared
28
with control abalone, probiotic-fed abalone had significantly higher THC (1.9x106 vs 5.6x105
29
cells), higher viability (90.8% vs 75.6%), higher percentage of ROS-positive cells (19.4% vs
30
0.5%) and higher numbers of non-apoptotic cells (88.0% vs 78.0%), respectively. These
31
results indicate that the probiotic-enriched diet enhanced the immunostimulatory
32
mechanisms, with a simultaneous low-level up-regulation of ROS production as a priming
33
mechanism of the antibacterial defence system. Metabolomics-based profiling of foot
34
muscle tissue additionally revealed that probiotic-fed abalone differentially expressed 16
35
unique metabolites, including amino acids, fatty acids and tricarboxylic acid related
36
compounds. These data suggest that the probiotic-supplemented diet can also alter central
37
carbon metabolic processes, which may improve the survival, as well as the growth of
38
abalone.
39
Keywords:
40
Probiotics; Abalone; Haliotis iris; Apoptosis; Oxidative stress; Metabolomics; Immunology;
41
Haemolymph
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
16
ACCEPTED MANUSCRIPT
1. Introduction
Introduction
43
A key focus to develop the New Zealand black-footed abalone (Haliotis iris) aquaculture
44
sector is to improve health and enhance growth. New Zealand is well known for its eco-
45
friendly production and strict quality assurance of shellfish aquaculture suitable for the
46
development of abalone aquaculture [1]. Previous studies have assessed optimal nutrition
47
and environmental parameters for the growth and health of cultured H. iris juveniles [2-9].
48
However, risks of disease outbreaks caused by pathogenic bacteria and viruses continuously
49
pose a potential threat to this growing industry [10]. Indeed, huge economic losses have
50
been experienced around the globe due to frequent occurrences of disease outbreaks in
51
farmed shellfish, such as abalone [11-13]. Antibiotics can be used as a valuable short-term
52
treatment to control bacterial infections. However, antibiotics come with food safety
53
concerns and negative health and environmental impacts, such as the emergence of
54
antibiotic resistant bacteria [14]. A scientifically demonstrable and ‘environmentally
55
friendly’ solution to overcome pathogenic infections lies in the application of probiotics [15-
56
17].
M
AN
U
SC
RI
PT
42
Probiotics are live microbial species, which when they are incorporated as feed
58
supplements, improve intestinal balance and enhance food digestibility, thus indirectly
59
benefiting the growth and the health of the host [18-19]. Probiotic bacteria may be isolated
60
from the aquatic environment (water column or substrate) or from the aquatic animal itself
61
by scraping the skin mucus or isolating the probiotic candidate from the digestive tract of a
62
healthy animal [20]. Bacterial species used as probiotics in aquaculture belong to a wide
63
range of Gram-positive and Gram-negative species [21] that have been suggested to possess
64
some kind of host specificity [19]. In most cases, the potential microbial organisms are
65
isolated from the host of the intended aquaculture species. Putative probiotic species used
66
in aquaculture are generally selected based on their ability to colonize the intestine, assist in
67
catabolism and uptake of nutrients (i.e. proteins, starch, alginate), produce acids, resist bile
68
salts, and/or improve survival through presumed immunostimulant activity [19-20].
AC
C
EP
TE
D
57
69
Although molluscs rely primarily on non-specific immunity, there is increasing
70
evidence to suggest that the abalone immune system can be modulated by probiotic
71
bacteria via an immuno-prophylactic control mechanism against pathogenic bacterial strains
72
[21-22]. Thus, the application of probiotics in abalone aquaculture has been successful for
ACCEPTED MANUSCRIPT
various species with significant benefits in improving health and disease resistance [22-25].
74
For example, in H. discus, specific immune enhancement in probiotic-fed abalone includes
75
an increase in haemocyte numbers, elevated haemocyte respiratory burst activity, and
76
enhanced lysozyme activity and higher levels of total protein concentration in centrifuged
77
haemolymph [22]. In H. rufescens, a probiotic-supplemented diet resulted in a reduction of
78
haemocyte apoptotic processes [25]. In addition, a transcriptomic study in H. rufescens [25]
79
was conducted to observe caspase-8, which is known to be an initiator of the caspase
80
cascade involved in apoptosis as a process for maintaining cellular and tissue homeostasis
81
[26]. It was observed that probiotic-fed abalone had a decreased caspase-8 expression
82
compared to basal-diet fed abalone [25]. Observations through proteomics analysis of
83
haemocytes also showed that probiotic-fed H. midae had stimulated haemocytes based on
84
the higher expression of Ras-related proteins and V+ ATPase proteins [27]. These
85
components are involved in the haemocyte maturation pathway [27].
M
AN
U
SC
RI
PT
73
Previous research by our group resulted in the development of multi-strain bacterial
87
supplemented feeds, which display probiotic activity in the New Zealand black-footed
88
abalone [20]. Three probionts (Exiguobacterium JHEb, Vibrio JH1, and Enterococcus JHLDc)
89
isolated from the digestive organs of adult H. iris increased growth and survival of juvenile
90
animals and enhanced the digestibility of proteinaceous components. The observed
91
improvement in health and reduced mortality suggested that the beneficial probiotic
92
activity might be related, in part, to immunostimulatory mechanisms [20].
EP
TE
D
86
In abalone, haemocytes are the main cell types involved in immune responses, such as
94
the production of stress mediators [12] and phagocytosis activity [28]. According to their
95
morphology and functions, H. iris haemocytes are classified into lymphocyte-like and
96
monocyte-like haemocytes [28]. The development of an innovative approach to characterize
97
abalone haemocytes using a miniaturized fluorescent detection and micro-capillary
98
technology flow cytometer platform (Muse® Cell Analyser [Merck Millipore]) [28] made an
99
important contribution to the study, since we could measure non-specific immune
100
AC
C
93
parameters of haemocytes in an efficient and quantitative manner.
101
The methodological approach taken in this study to investigate the effect of probiotic-
102
supplemented diets on the growth and health of abalone is a mixed methodology based on
103
measurements of a variety of non-specific cellular immune parameters of haemocytes,
ACCEPTED MANUSCRIPT
including total haemocyte cell counts, haemocyte viability, generation of reactive oxygen
105
species (ROS), and different stages of apoptosis. We also applied untargeted metabolite
106
profiling, or metabolomics-based analysis [29-31] of foot muscle tissues in an attempt to
107
gain additional insights into the health status and nutritional condition of the animals. This
108
study aims to advance the understanding of the role of probiotics to support the
109
development of H. iris aquaculture in New Zealand.
110
2. Methods
111
2.1 Experimental animals
112
about a total of 160 juvenile abalone (H. iris) (shell length = 27.5 ± 5 mm; weight = 2.14 ±
113
1.19 g) were obtained from a commercial hatchery (Moana Ltd: Ruakaka, Northern New
114
Zealand). Animals were acclimatized at the Auckland University of Technology aquaculture
115
facility for one week prior to commencement of the feeding trial. Animals were individually
116
labelled with waterproof tags and were divided into four 40 L tanks (40 animals per tank) on
117
a recirculating seawater system (5 µm filtered and UV-treated seawater [35 ppt]),
118
maintained in dark conditions at 16°C, and fed daily to satiation thrice per day with feeding
119
at 1% of body weight.
120
2.2 Probiotic bacteria preparation
121
The probionts used in this study were previously isolated from the gastrointestinal tract of
122
healthy abalone (selected for their proteolytic and amylolytic capabilities, and ability to alter
123
the acid environment [12]) and comprised the strains Exiguobacterium JHEb1, Vibrio JH1,
124
and Enterococcus JHLDc. Bacteria isolates were previously stored at – 80°C in sterile marine
125
broth (Difco) containing 20% [v/v] glycerol. The isolates were re-cultured in suitable
126
enriched media for 48 hr at 22°C. Exiguobacterium JHEb1 was propagated in 200 mL marine
127
broth (Difco) supplemented with 1% yeast extract; Vibrio JH1 was grown in 200 mL marine
128
broth (Difco) supplemented with 0.5% glucose; Enterococcus JHLDc was prepared
129
anaerobically in 100 mL MRS broth (Difco) containing 2% NaCl. The three strains of bacteria
130
were prepared and cultured weekly.
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
104
ACCEPTED MANUSCRIPT
131
2.3 Experimental diets
132
The control diet consisted of a commercial pelletized abalone feed (AbMax 16; E.N.
133
Hutchinson Ltd, Auckland, New Zealand). The feed was shown to be free of culturable
134
bacteria via microbiological evaluation every two weeks using general nutrient agar (Difco).
The probiotic-enhanced diet was prepared weekly by supplementation of commercial
136
feed (AbMax 16) with the probiotic mix. The bacterial strains cultured in broth media were
137
harvested via centrifugation (Sorvall RC- 5C; Sorvall Instruments) at 5000 rpm at 10°C for 10
138
minutes. Bacterial pellets (probiotic biomass) were collected, transferred to a single bottle
139
containing 25 mL of sterile nutrient broth (Difco), and then vortexed to get a homogeneous
140
suspension. The probiotic mixture was inoculated on 20 g of commercial feed by spraying
141
the feed pellets spread on plastic trays. The pellets were air-dried for 5 hours in a sterile
142
microbiological cabinet at room temperature (19°C), then placed in sterile Petri dishes and
143
stored at 10°C. Using the marine agar plating method [12], the average total viable bacterial
144
count in the pellets was found to be around 2×109 CFU.g-1. New batches of feed were
145
prepared weekly and stored at 10°C until use. The stability of the probiotic-supplemented
146
diet during semi-long term storage was tested by assessing bacterial viability after three
147
months of storage at 10°C to assure that the probionts were still active in the feed.
148
2.4 Experimental design
149
After acclimation, shell length, shell width and total body wet weight of all animals were
150
measured. Allometric data were then collected monthly for the four-month feeding trial,
151
and cumulative daily mortalities recorded. Six static 20 L tanks (5 µm filtered seawater;
152
salinity 35 ppt; temperature 16°C) were allocated, with triplicate tank systems for each of
153
the two dietary treatments. Twenty healthy abalone were randomly distributed to each
154
tank. Remaining animals were divided into two additional tanks and maintained under
155
similar experimental conditions to be used as replacements for dead individuals in the
156
feeding trial to maintain the same densities, but these animals were not used in the
157
analyses.
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
135
158
Water exchanges (50% by volume) were performed daily, while 100% water
159
exchanges and thorough tank cleaning were performed every three days. Uneaten food was
160
siphoned prior to each feeding event. Each tank contained a black coloured plastic tray that
161
provide the abalone with shelter for favourable living conditions.
ACCEPTED MANUSCRIPT
2.5 Microbial analysis
163
Microbial quantification of probiotic strains within digestive tracts were determined after
164
one and three months by sacrificing three animals per treatment (one animal per tank) for
165
each microbial sampling. Digestive tracts were aseptically excised with sterile forceps and
166
blades, homogenized and 10-fold serially diluted with peptone water. From each dilution,
167
100 µL aliquots were spread plated on selective agar. ChromAgarTM Vibrio (CHROMAgar,
168
France), Oxoid™ Kanamycin Aesculin Azide Agar Base (Thermo ScientificTM) and Marine Agar
169
pH 8 (Fort Richard Laboratories Ltd) were used for the selective cultivation and enumeration
170
of Vibrio sp., Enterococcus sp., and Exiguobacterium sp., respectively. Difco Marine Agar
171
(Thermo ScientificTM) was used for the enumeration of the mixed probiotic species.
172
2.6 Haemocyte analysis
173
Haemocyte parameters, including counts and viability, apoptosis and percentage of cells
174
exhibiting ROS were assessed in haemolymph samples with the Muse® Cell Analyzer at the
175
beginning of the trial and after two and four months. At each sampling event, three abalone
176
per tank (nine abalone per treatment) were used to obtain haemolymph. Haemolymph was
177
collected from the anterior arterial sinus of the abalone for each sampling event using a 1
178
mL sterile syringe (25 G x 5/8 in), and immediately mixed with Alsever’s solution in a 1:1
179
ratio.
180
2.6.1. Haemocyte cell count and viability
181
Haemocyte counts and viability were measured using the Muse® Cell Cycle Assay Kit (Merck
182
Millipore) according to the manufacturer’s instructions. Twenty microliters of diluted
183
sample in Alsever’s solution (SIGMA) were mixed with 380 μL assay reagent in 1.5 mL micro-
184
centrifuge tubes, incubated for 5 min in the dark at room temperature, briefly vortexed and
185
analysed.
186
2.6.2. Haemocyte oxidative stress
187
Production of Reactive Oxygen Species (ROS) in haemocytes was evaluated using the MUSE®
188
Oxidative Stress Kit (Merck Millipore), with minor modifications to the manufacturer’s
189
instructions. Briefly, diluted haemocyte samples were prepared by mixing 100 μL of 1:1
190
haemolymph to Alsever’s solution with 100 μL of 1X Assay Buffer (Merck Millipore). The
191
Muse® Oxidative Stress working solution was first diluted 100X with the Assay Buffer to
192
make an intermediate solution. This intermediate solution was further diluted 80X with
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
162
ACCEPTED MANUSCRIPT
Assay Buffer to make Muse® Oxidative Stress working solution. The final sample solution to
194
be processed via flow cytometry was prepared by mixing 190 μL of Muse® Oxidative Stress
195
working solution with 10 μL of the diluted haemocyte sample in 1.5 ml micro-centrifuge
196
tubes, followed by incubation at 37°C for 30 minutes.
197
2.6.3. Haemocyte apoptosis
198
Haemocyte apoptosis profiling was performed using the Muse® Annexin V and Dead Cell
199
Assay Kit (Merck Millipore), with minor modification to the manufacturer’s instructions. A
200
total of 100 μL of diluted sample in Alsever’s solution was mixed with 100 μL of assay
201
reagent in 1.5 mL micro-centrifuge tubes, incubated for 20 min in the dark at room
202
temperature, briefly vortexed and analysed.
203
2.7
204
Nine randomly selected animals from each dietary treatment group (probiotics and control)
205
were sacrificed at the end of the feeding experiments to obtain tissue samples for
206
metabolomics analysis. Approximately 150 mg wet weight of foot muscle were dissected
207
from each individual, placed in 2 mL cryogenic vials, snap frozen in liquid nitrogen, and
208
stored at –80°C until metabolite extraction.
209
Tissue samples were lyophilised overnight then ground in liquid nitrogen using a mortar and
210
pestle. Approximately 10 mg of powdered tissue were placed in 1.5 mL Eppendorf tubes and
211
co-extracted with an internal standard (L-Alanine-2,3,3,3-d4) using a cold methanol-water
212
method according to Villas-Bôas et al. [32], with modifications. L-Alanine-2,3,3,3-d4, sodium
213
hydroxide, pyridine and methyl chloroformate (MCF) were purchased from Sigma Aldrich
214
(St. Louis, MO, USA). Methanol and chloroform were purchased from Merck (Darmstadt,
215
Germany). Sodium bicarbonate and anhydrous sodium sulphate were purchased from Ajax
216
Finechem (NSW, Australia). One control sample was accidently contaminated during the
217
extraction process, and thus excluded from further processing. Polar extracts were
218
lyophilised, re-suspended in NaOH followed by derivatization via MCF alkylation
219
(Supplementary Methods). A solvent blank was similarly processed. Derivatised extracts
220
were analysed using a gas chromatograph GC7890 (Agilent Technologies, USA) coupled to a
221
quadrupole mass spectrometer MSD 5975 (Agilent Technologies, USA) according to the
222
protocol described by Smart et al. [33]. Deconvolution of chromatographic data was
223
performed using the Automated Mass Spectral Deconvolution and Identification System
AC
C
EP
TE
D
M
AN
U
Metabolomics analysis
SC
RI
PT
193
ACCEPTED MANUSCRIPT
(AMDIS v2.66) software. According to the electron impact fragmentation mass spectrum
225
and retention time, metabolites were identified using Chemstation software (Agilent
226
Technologies) and customised R xcms-based scripts [34] to interrogate an in-house library of
227
MCF derivatised compounds. Data were manually checked for the presence of contaminants
228
(i.e., derivatization artefacts and non-biologically derived compounds) with and aberrant
229
records being removed, and remaining data were blank corrected to control for background
230
contamination [33]. The resulting QC-filtered peak intensity values were normalised by the
231
internal standard to compensate for potential technical variations (e.g. variable metabolite
232
recoveries), and then by sample-specific biomass prior to statistical analyses.
233
2.8
234
Differences between bacterial cell counts, treatments of total haemocyte cell counts, cell
235
viability, oxidative stress and apoptosis were identified with the Student’s t-test (p < 0.05)
236
and ANOVA with Duncan post-hoc analysis using the SPSS Statistics for Windows (Version
237
19.0. Armonk, NY: IBM Corp.). Bacterial cell count data were log-transformed prior to
238
analysis to meet parametric assumptions.
239
The normalised metabolite data matrix was auto-scaled and analysed using Metaboanalyst
240
3.0 [35]. A Student’s t-test with an alpha level of 0.05 was performed to identify differences
241
in foot muscle metabolite profiles between abalone fed with the two experimental diets.
242
Combined heat map and hierarchical cluster analysis (Euclidean distance; Wards criterion)
243
of the top 20 metabolites ranked by their t-test statistics was performed to assist
244
visualisation of major group differences and between-sample variability.
245
3.
246
3.1 Microbial analysis of probiotic bacteria in the feed post-storage
247
The viability of three selected bacterial species was observed by plate culture and remained
248
viable at a concentration optimal for probiotic function after three months of storage at 4°C
249
(Figure 1). Enterococcus spp., Vibrio spp. and Exiguobacterium spp. were detected at an
250
average bacterial count of 4.15×108 CFU.g-1, 7.00×107 CFU.g-1 and 2.25×107 CFU.g-1,
251
respectively, while the count for mixed species in marine agar was 1.30×109 CFU.g-1.
252
Observation of non-probiotic supplemented control feed showed no presence of microbial
253
contamination.
SC
RI
PT
224
AC
C
Results
EP
TE
D
M
AN
U
Statistics
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
254
257
Figure 1. Viability of probiotic bacteria in the probiotic supplemented feed after three months of storage at
4°C.
TE
D
255
256
3.2 Microbial analysis of digestive tracts
259
After one and three months of feeding, probiotic bacteria had already been established
260
within the digestive tracts of abalone fed with probiotic-supplemented feed in substantially
261
higher concentrations than those found in abalone fed with the control diet (except for
262
Vibrio spp. after one month) (Figure 2). Enterococcus spp. nor Exiguobacterium spp. were
263
detected in the non-probiotic fed control animals at the time points assessed. Since our
264
culture-based techniques could not distinguish the specific species of Vibrio, it is unknown if
265
control animals had our probiotic Vibrio JH1 or another commonly found Vibrio species in
266
the digestive tracts of the animals.
AC
C
EP
258
Figure 2. Quantity of probiotic bacteria present in the digestive tracts of abalone (H. iris) after 1 month of
feeding (A), and 3 months of feeding (B). Asterisks indicate statistical significance (p < 0.05) between the
different dietary groups (n.p. = bacteria not present).
271
M
AN
U
267
268
269
270
SC
RI
PT
ACCEPTED MANUSCRIPT
3.3 Effect of probiotics on mortality
273
Survival of abalone was monitored throughout the experiment. All animals fed with the
274
probiotic-supplemented diet survived after four months of feeding, whereas five animals
275
from the control treatment died after the first three months (ca. 10% mortality). All of the
276
abalone that died showed signs of poor growth, appearance of pigment loss in the foot
277
tissue and epithelial erosion of the foot organ.
278
3.4 Effect of probiotics on growth
279
A general increase in length, width and weight of the probiotic-fed H. iris was observed
280
throughout the four-month feeding period (Figure 3). In general, the probiotic-fed animals
281
had higher values of length, width and weight compared to the control animals. However,
282
the only statistically significant growth difference between the probiotic-fed and the control
283
animals was recorded in length and width after 3 months of feeding. At the end of the four-
284
months feeding experiment, the mean growth increase length, width and wet weight
285
percentage (± SD) of the probiotics fed animals were 32.3 ± 1.1%, 31.9 ± 0.3%, 109.6 ±
286
6.5%, respectively, while the values for the control fed abalone were 22.3 ± 9.6%, 20.7 ±
287
11.5% and 72.8 ± 41.3%, respectively.
AC
C
EP
TE
D
272
RI
PT
ACCEPTED MANUSCRIPT
288
Figure 3. Abalone (H. iris) growth in shell length (A), shell width (B) and wet weight (C) for control (grey bars)
and probiotics (black bars) fed animals over four months. Data represents the mean± SD (n=60 per treatment)
from nine abalone per treatment. Asterisks indicate statistical significance (p < 0.05) between the different
dietary treatment of control and probiotic.
295
Total haemocyte counts (x106 cell.ml-1) and cell viability (%) showed no statistical
296
differences (p > 0.05) between control-fed and probiotic-fed animals at week 1 and week 8
297
(Figure 4). However, after 16 weeks (four months), total haemocyte counts and cell viability
298
were significantly higher (p < 0.05) than those in the control group, with relative mean fold
299
changes of 3.4 and 1.2, respectively.
M
AN
U
SC
289
290
291
292
293
294
300
301
302
303
304
305
AC
C
EP
TE
D
3.5 Effect of probiotics on haemocyte immune parameters
Figure 4. Immune responses of \ abalone (H. iris) fed with probiotic supplemented and non-supplemented
control diets, including A) total cell counts and B) haemocyte viability. Data represent the mean ± SD from nine
abalone per treatment. Asterisks indicate statistical significance (p < 0.05) between the different dietary
groups of control and probiotic within the same sampling week.
ACCEPTED MANUSCRIPT
Direct quantitative measurements of haemocyte cells exhibiting ROS (namely superoxide
307
radicals) showed a low percentage (ca. 1%) of ROS-positive cells at the beginning of the trial
308
(Figure 5). After 4 months of feeding, the probiotics-fed abalone exhibited a significantly
309
higher percentage (19.4 ± 23.3%) of ROS-positive cells compared with those of the control
310
group (0.5 ± 0.7%) (Student’s t-test, p < 0.05).
M
AN
U
SC
RI
PT
306
311
312
313
314
315
316
Figure 5. Percentage of abalone (H. iris) haemocytes exhibiting Reactive Oxygen Species (ROS) before and after
feeding with probiotic supplemented and non-supplemented control diets. Data represent the mean ± S.D
from nine abalone per treatment.
317
example of an apoptosis profile (Figure 6), presents the population of cells based on their
318
apoptotic status; non-apoptotic live cells (lower left quadrant: Annexin V–/7-AAD–), cells
319
exhibiting early stages of apoptosis (lower right quadrant: Annexin V+/7-AAD−), cells during
320
late stages of apoptosis (upper right quadrant: Annexin V+/7-AAD+), and dead cells (upper
321
left quadrant: V–/7-AAD+). Apoptosis were quantitatively analysed and the results are
322
presented in a bar graph format (Figure 7).
324
325
326
TE
D
EP
AC
C
323
Haemocytes at different stages of apoptosis were measured for each treatment. An
SC
RI
PT
ACCEPTED MANUSCRIPT
327
M
AN
U
Figure 6. Apoptotic analysis of abalone (H. iris) haemocytes using the MUSE® Cell Analyzer produced cellular
profiles, which displayed various stages of apoptosis. Red lined quadrants indicate boundaries of the four cell
populations (live, early apoptotic, late apoptotic, and dead cells).
331
AC
C
EP
TE
D
328
329
330
332
Figure 7. Percentage of non-apoptotic, early apoptotic, late stage apoptosis and dead cells (mean ± S.D from 9
333
abalone). Difference in notation above bar graphs indicate statistical significance (p < 0.05).
334
335
Apoptosis analyses were conducted at the beginning and at the end of the experiment. Early
336
apoptotic haemocyte percentage were present at a significantly greater value (ANOVA, p <
ACCEPTED MANUSCRIPT
0.05) during the initial stage of the experiment (32.4%). The early apoptotic cell values
338
obtained after four months of feeding from both probiotic supplemented (8.4%) and non-
339
supplemented control diets were 8.4% and 2.0%, respectively. The percentage of non-
340
apoptotic haemocyte cells in control-fed animals (78.0%) and in probiotic fed animals
341
(88.0%) did not differ significantly from each other. However, it was observed that the
342
percentage of non-apoptotic haemocyte cells in the initial stage of the experiment was
343
significantly lower (58.9%).
344
3.6 Metabolomics
345
GC-MS analysis of foot tissue polar extracts detected 102 unique metabolites after QC
346
filtering of the data. Of these, 82 were attributed to specific chemical identities by matching
347
chromatographic and mass-spectral information against our in-house metabolite library.
348
The remaining 20 features are currently listed as ‘unknowns’ since no matches were found.
349
Univariate statistical analysis showed a number of differences in the metabolite profiles
350
between animals receiving the two experimental diets (Figure 8 and Table 1). Five amino
351
acids (serine, glutamine, asparagine, lysine, proline), two fatty acids (palmitoleic acid,
352
adrenic acid), four organic acid derivatives (alpha-ketoglutarate, succinic acid, lactic acid,
353
oxalic acid), and six unknowns, but unique, metabolites were over-expressed (t-test; p <
354
0.05) in the probiotic-fed abalone, compared to those which received the non-probiotic
355
supplemented control feed.
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
337
356
357
358
359
360
Figure 8. Volcano plot of metabolites comparing probiotic supplemented diet vs control diet; where mean fold
change (FC) values = probiotic / control. Each point represents a metabolite. Points in black are significantly
different between treatments (t-test; p < 0.05).
ACCEPTED MANUSCRIPT
Table 1. List of significantly (t-test) altered metabolites (Fold change = Probiotic/Control).
a
Metabolite
1.45
0.009
Proline
1.47
0.016
Asparagine
1.51
0.023
Glutamine
1.52
0.038
Serine
1.31
0.046
Adrenic acid
1.45
0.022
Palmitoleic acid
1.29
0.049
Lactic acid
1.78
0.025
Succinic acid
1.52
0.038
Oxalic acid
1.31
0.009
α-ketoglutarate
1.33
0.013
Unknown 1
2.30
0.009
Unknown 2
1.26
0.011
Unknown 3
1.28
0.021
Unknown 4
1.53
0.025
Unknown 5
1.39
0.027
1.37
0.039
362
TE
D
Unknown 6
M
AN
U
Lysine
RI
PT
Fold change p-value
SC
361
A heatmap with combined hierarchical cluster analysis of the top 20 metabolites ranked by
364
their t-test statistics was used to assist visualisation of the major group differences (Figure
365
9). Although variations in levels of some metabolites were observed among individuals, a
366
general pattern of enhanced metabolite expressions in probiotics-fed abalone can easily be
367
identified.
AC
C
EP
363
SC
RI
PT
ACCEPTED MANUSCRIPT
369
370
371
372
M
AN
U
368
Figure 9. Heatmap with combined hierarchical cluster analysis (Euclidean distance; Ward’s criterion) of
metabolites. Columns represent samples (orange = Control diet, blue = Probiotic diet) and rows represent
metabolites. The green/black/red colour scale represents standardised (autoscaled) abundance data,
where red = higher values, and green = lower values.
373
4 Discussion
375
Our study is the first to report the effect of probiotics in modulation of various properties of
376
the immune system of H. iris abalone fed with probiotics compared to control abalone.
377
Following the dietary inclusion of multi-strain probiotics (Exiguobacterium JHEb, Vibrio JH1,
378
and Enterococcus JHLDc) in the feed, there was evidence of the enhanced innate-immunity
379
in the probiotic-fed animals observed from the total haemocyte count, cell viability,
380
apoptosis and production of reactive oxygen species (ROS) parameters. In addition,
381
metabolic profiles from foot tissues of abalone showed differences between treatments.
382
After 3 months of feeding, growth and survival rates of probiotic-fed abalone were also
383
found to be higher than that in control-fed animals.
384
The high numbers of Exiguobacterium JHEb, Vibrio JH1 and Enterococcus JHLDc found in the
385
gastrointestinal tract of H. iris was suggested to have contributed to the growth and health
386
of juvenile abalone by the bacterial-mediated improvement on feed digestibility and feed
387
utilization [20]. The active role of the multi-strain probiotic may have reduced their energy
388
consumption to digest feed, hence providing improved growth and immunologic condition.
AC
C
EP
TE
D
374
ACCEPTED MANUSCRIPT
A previous study in H. rufescens [25] showed that probiotic-fed abalone were associated
390
with a decrease in expression of genes related to energy metabolism. Probiotics may
391
provide additional digestive enzymes, which may increase the growth and immune state of
392
abalone by reducing the energy needed for digestion.
393
Haemocytes are the main defence cell of molluscs and are capable of antigen recognition,
394
attachment followed by agglutination, phagocytosis, and elimination of antigens by
395
respiratory burst or exocytosis of antimicrobial factors [36]. Our study showed an increase
396
of haemocyte numbers circulating inside the body possibly due to the effect of the
397
probiotics. Previous studies confirm our finding where probiotics feeding also increased the
398
total haemocyte count (THC) in H. discus hannai [22], H. midae [23] and H. rufescens [25].
399
These increases also occurred in other invertebrate phyla, such as Litopenaeus stylirostris
400
where animals were fed probiotics daily [37]. In some cases, probiotics feeding did not
401
necessarily increase the THC in abalone (H. midae), but provided an enhanced ability for
402
haemocyte proliferation to occur post-challenge to pathogenic Vibrio anguillarum [23].
403
Increased THC is associated with haematopoiesis processes, that involve components such
404
as haemocyte precursor cells and haematopoietic tissue. However, the site of
405
haematopoiesis in abalone is still unclear. Previous studies on molluscs have indicated that
406
the haematopoiesis process involves a complicated mechanism of signalling pathways,
407
receptors, cytokines, growth factors, and transcription factors [38]. Given that haemocyte
408
proliferation remains high after probiotics feeding, and considering that haemocytes remain
409
in the circulation for a certain period, this may indicate a process of immunological priming
410
to protect the animal against future pathogenic stress [39]. However, the mechanisms and
411
consequences of immune priming in invertebrates still need to be properly demonstrated as
412
to whether these immune responses are stable following immune system stimulation.
413
The lower cell viability observed in the control-fed animals at the end of the experiment
414
may reflect stress status, and suggests that the dietary probiotics provide a protective
415
effect. Haemocyte viability in invertebrates can be influenced by environmental factors,
416
such as sudden temperature fluctuations [40], pH stress [41], handling [42], air exposure
417
[42], and presence of heavy metals [43]. However, it is thought that the low viability
418
observed in the control animals was caused by stress from an opportunistic pathogen, since
419
animals that died in the control tanks showed symptoms of a disease caused by a bacterial
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
389
ACCEPTED MANUSCRIPT
pathogen. The effect of antigens on invertebrate haemocyte viability has been studied in
421
vitro where bacterial pathogens, bacterial extracellular product, and LPS have reduced cell
422
viability [43-44]. Furthermore, in vivo pathogenic bacterial challenges can also reduce
423
haemocyte viability [45]. The reduction of invertebrate haemocyte viability might be
424
induced by oxidative damage or by other pathways leading to apoptosis [46]. It is possible
425
that the multi-strain probiotics actively inhibit the colonization of potential pathogens in the
426
digestive tract through antibiosis, competition for nutrients and/or space, and alteration of
427
microbial metabolism [19].
428
Most of the previously mentioned studies regarding haemocyte cell viability did not report
429
the specific necrotic status, since cell death could manifest non-apoptotic features [47].
430
Although a difference in the apoptotic status of haemocytes was not found between
431
control-fed and probiotics-fed animals, we detected apoptotic symptoms from animals
432
during the initial sampling. Activation of apoptosis in mollusc haemocytes can be stimulated
433
by various inducing factors, for different purposes. For example, apoptosis provides a
434
protective host defence mechanism during viral infections by reducing cell longevity to stem
435
viral proliferation, it can be induced by phagocytised bacterial pathogens to impair the host
436
defence system, and it can be activated as a cell death mechanism during exposure to
437
environmental stressors to reduce pro-inflammatory necrosis and maintain good health [48-
438
49]. A higher percentage of early apoptotic haemocyte cells at the start of the experiment
439
may indicate that the abalone were mildly stressed.
440
Probiotics-fed abalone showed higher production of ROS in haemocytes compared with
441
control-fed animals. The presence of ROS, such as superoxide anion (O2-) and hydrogen
442
peroxide (H2O2), may indicate an immune response mechanism specifically related to the
443
phagocytosis activity of the haemocyte [50]. The specific signal transduction pathways that
444
mediate this production are not been thoroughly understood. However, a recent proteomic
445
study on H. midae observed that the haemocytes in probiotics-fed abalone showed
446
phagosomal maturation and also exhibited significant expression of fundamental proteins,
447
such as V-type proton ATPase and Ras-related protein Rab in haemocytes [27]. Although
448
production of cytotoxic ROS molecules is employed by abalone, it does not necessarily
449
indicate oxidative stress and may only be a sign that a stimulus factor is threatening the
450
homeostasis of the abalone [50]. Environmental stressors may also have a role in the
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
420
ACCEPTED MANUSCRIPT
production of these molecules [51]. However, it is unlikely to be the cause of ROS
452
production in haemocytes of probiotics-fed animals since environmental parameters were
453
similar in tanks with both control- and probiotics-fed abalone. The multi-probiotics
454
supplemented diet may enhance the immune capacity potentially by influencing or priming
455
the ROS regulatory system. However, further work is needed to test this hypothesis,
456
including an analysis of ROS-regulatory enzyme expression and/or activities.
457
Metabolite profiling of foot tissue revealed variations in the relative abundance of 17
458
unique metabolites between control-fed and probiotics-fed abalone. Enriched levels of
459
FFA’s (e.g., lysine, proline, asparagine, glutamine, serine) in probiotics-fed abalone reflect
460
differential capacities for protein turnover. These observations could be a result of
461
enhanced digestibility of dietary protein attributable to the high proteolytic abilities of the
462
selected probionts, as reported by Hadi et al. [20]. Irrespective of their origin, higher levels
463
of FAA’s in abalone foot muscle tissue reflect good animal health status [52-53] and some
464
specific amino acids are associated with non-specific immunity. For example, proline
465
appears to play a role in modulating immune responses of fish towards bacterial infections
466
and can be used as a therapeutic agent to improve survivability [54]. Free proline also serves
467
an important function in protecting organisms through its ability to act as a free radical
468
scavenger [55], and by stabilizing proteins from in vivo aggregation [56]. Up-regulation of
469
glutamine and asparagine in probiotics-fed abalone may stimulate growth and improve
470
immunocapacity [57]. Glutamine is a key nutrient for dividing cells since it functions as non-
471
toxic nitrogen vehicle and respiratory fuel [58–60] and serves as a precursor for purine and
472
pyrimidine synthesis, which is essential for immune cells to replicate [61]. Glutamine
473
synthase in abalone muscle is down-regulated following environmental stress [62] and
474
bacterial infections [63]. Thus, glutamine content in foot muscle may indicate the health
475
status of abalone. Elevated levels of free asparagine also appear to be beneficial for
476
mounting a successful immune response in higher taxa [64]. However, the mechanisms
477
involved are poorly understood, and a functional role in molluscan immunity is yet to be
478
investigated.
479
Alterations in metabolites involved in glycolysis and the tricarboxylic acid (TCA) cycle likely
480
reflect differential energetic statuses. Alpha-ketoglutarate is a rate-determining
481
intermediate in the TCA cycle and has a crucial role in cellular energy metabolism. Succinate
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
451
ACCEPTED MANUSCRIPT
is also a crucial intermediate and is positioned at the crossroads of several metabolic
483
pathways [66], including the formation and elimination of ROS [67]. Although higher levels
484
of lactic acid in tissues of probiotics-fed abalone could signal differences in glycolytic
485
metabolism, the relative increase in this metabolite may reflect input from the selected
486
lactic acid producing probiont (Enterococcus JHLDc) which was incorporated into the
487
treatment diet. Other differential metabolite signatures in probiotics-fed abalone tissue
488
included increased levels of the FFAs palmitoleic acid and adrenic acid, and higher levels of
489
oxalic acid. Palmitoleic acid is a common mono-unsaturated fatty acid produced from
490
desaturation of palmitic acid, whereas adrenic acid is an eicosanoid derived from elongation
491
of arachidonic acid and is involved in prostaglandin synthesis [68]. Although defined
492
functional roles for oxalic acid in animals are not yet firmly established, this metabolite has
493
been marked as a key regulator or precursor of various branches of amino acid synthesis, an
494
antioxidant, and a mediator of apoptotic processes [69-71].
495
5 Conclusions
Conclusions
496
The present study was designed to determine the effect the enrichment of feed with multi–
497
strain probiotics on survival, growth, selected non-specific immune parameters and
498
metabolite profiles of the New Zealand black-footed abalone. The results of this
499
investigation show that probiotics increased the survival rate and growth of abalone, and
500
stimulate haemocyte immune parameters. In addition, metabolites up-regulated in the
501
probiotics-fed abalone are associated with enhancement of the non-specific immune
502
response. These findings have significant implications for the understanding of how
503
probiotic enrichment affects the abalone. Therefore, the findings of this study strengthen
504
future development and application of probiotics in aquaculture of New Zealand black-
505
footed abalone (H. iris).
SC
M
AN
U
TE
D
EP
AC
C
506
RI
PT
482
507
Acknowledgement
508
We are thankful to Ryan Lanauze, Lynette Suvalko and Moana Ltd. for supplying abalone
509
samples and for their ongoing support of our research activities. We are also grateful to
510
Evan Brown, Sonya Popoff and Rachel Boyle (technical staff at the School of Science,
511
Auckland University of Technology) for their assistance with technical logistics, maintenance
ACCEPTED MANUSCRIPT
of the aquaculture lab and animal transport and holding permits. We also are grateful to the
513
members of the Aquaculture Biotechnology Research Group for the multiple fruitful
514
discussions that improved this work along the way, especially Ming Li who assisted in the
515
graphical display of the metabolomics data. This work was supported by an Indonesian
516
DIKTI – AUT doctoral scholarship to R. Grandiosa under the supervision of A. C. Alfaro and F.
517
Mérien.
RI
PT
512
518
519
References
[1]
A. ALFARO, A. JEFFS & N. KING. Enabling and driving aquaculture growth in New
Zealand through innovation. New Zealand Journal of Marine and Freshwater
Research. 48 (2014) 311-313.
523
524
525
[2]
C. TUNG & A. ALFARO. Effect of dietary protein and temperature on the growth and
health of juvenile New Zealand black-footed abalone (Haliotis iris). Aquaculture
Research. 42 (2011) 366-385.
526
527
528
[3]
C. TUNG & A. ALFARO. Alternative Protein Sources in Artificial Diets for New
Zealand's Black-footed Abalone, Haliotis iris, Martyn 1784, Juveniles. Journal of the
World Aquaculture Society. 43 (2011) 1-29.
529
530
531
[4]
C. TUNG & A. ALFARO. Effects of dietary protein source and amount on shell
morphology of juvenile abalone Haliotis iris. Journal of Fisheries and Aquatic
Science. 6 (2011) 107-118.
532
533
[5]
M. STUART & M. BROWN. Growth and diet of cultivated black-footed abalone,
Haliotis iris (Martyn). Aquaculture. 127 (1994) 329-337.
534
535
536
[6]
V. ALLEN, I. MARSDEN, N. RAGG & S. GIESEG. The effects of tactile stimulants on
feeding, growth, behaviour, and meat quality of cultured Blackfoot abalone,
Haliotis iris. Aquaculture. 257 (2006) 294-308.
537
538
[7]
T. SEARLE, R. ROBERTS & P. LOKMAN. Effects of temperature on growth of juvenile
blackfoot abalone, Haliotis iris Gmelin. Aquaculture Research. 37 (2006) 1441-1449.
539
540
541
[8]
542
543
544
[9]
M. BEWICK, R. WELLS & R. WONG. Free amino acid and nucleotide concentrations
in New Zealand abalone (paua), Haliotis iris, fed casein-based, macroalgal, or wild
diets. Journal of Aquatic Food Product Technology. 6 (1997) 57-69.
545
546
547
[10]
S. WEBB. Assessment of Pathology Threats to the New Zealand Shellfish Industry,
2013, p. 69. Prepared for Ministry of Science and Innovation, Programme
CAWX0802. Cawthron Report No. 1334.
AC
C
EP
TE
D
M
AN
U
SC
520
521
522
M. PREECE & P. MLADENOV. Growth and mortality of the New Zealand abalone
Haliotis iris Martyn 1784 cultured in offshore structures and fed artificial
diets. Aquaculture Research. 30 (1999) 865-877.
ACCEPTED MANUSCRIPT
[11]
S. WANG, Y. WANG, Z. ZHANG, R. JACK, Z. WENG, Z. ZOU & Z. ZHANG. Response of
innate immune factors in abalone Haliotis diversicolor supertexta to pathogenic or
nonpathogenic infection. Journal of Shellfish Research. 23 (2004) 1173-1177.
551
552
553
554
[12]
C. HOOPER, R. DAY, R. SLOCOMBE, J. HANDLINGER & K. BENKENDORFF. Stress and
immune responses in abalone: limitations in current knowledge and investigative
methods based on other models. Fish and Shellfish Immunology. 22 (2007) 363379.
555
556
557
[13]
T. SAWABE, S. INOUE, Y. FUKUI, K. YOSHIE, Y. NISHIHARA & H. MIURA. Mass
mortality of Japanese abalone Haliotis discus hannai caused by Vibrio harveyi
infection. Microbes and Environments. 22 (2007) 300-308.
558
559
560
561
[14]
J. HANDLINGER, J. CARSON, L. DONACHIE, L. GABOR & D. TAYLOR. Bacterial
infection in Tasmanian farmed abalone: causes, pathology, farm factors and control
options, in: Diseases in Asian Aquaculture V, Australia, 2002. pp. 289-300.
Proceedings of the 5th Symposium on Diseases in Asian Aquaculture. 2005.
562
563
564
[15]
A. KESARCODI-WATSON, H. KASPAR, M. LATEGAN & L. GIBSON. Probiotics in
aquaculture: the need, principles and mechanisms of action and screening
processes. Aquaculture. 274 (2008) 1-14.
565
566
[16]
A. NEWAJ-FYZUL, A. AL-HARBI & B. AUSTIN. Review: developments in the use of
probiotics for disease control in aquaculture. Aquaculture. 431 (2014) 1-11.
567
568
569
[17]
L. VERSCHUERE, G. ROMBAUT, P. SORGELOOS & W. VERSTRAETE. Probiotic bacteria
as biological control agents in aquaculture. Microbiology and Molecular Biology
Reviews. 64 (2000) 655-671.
570
571
[18]
R. FULLER. Probiotics in man and animals. Journal of Applied Bacteriology. 66 (1989)
365-378.
572
573
[19]
A. IRIANTO & B. AUSTIN. Probiotics in aquaculture. Journal of Fish Diseases. 25
(2002) 633-642.
574
575
576
[20]
J. HADI, N. GUTIERREZ, A. ALFARO & R. ROBERTS. Use of probiotic bacteria to
improve growth and survivability of farmed New Zealand abalone (Haliotis iris).
New Zealand Journal of Marine and Freshwater Research. 48 (2014) 1–11.
577
578
579
[21]
580
581
582
583
[22]
584
585
[23]
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
548
549
550
M. CROSS, A. GANNER, D. TEILAB & L. FRAY. Patterns of cytokine induction by grampositive and gram-negative probiotic bacteria. FEMS Immunology & Medical
Microbiology. 42 (2004) 173-180.
H. JIANG, X. LIU, Y. CHANG, M. LIU & G. WANG. Effects of dietary supplementation
of probiotic Shewanella colwelliana WA64, Shewanella olleyana WA65 on the
innate immunity and disease resistance of abalone, Haliotis discus hannai Ino. Fish
& Shellfish Immunology. 35 (2013) 86-91.
B. MACEY & V. COYNE. Improved growth rate and disease resistance in farmed
Haliotis midae through probiotic treatment. Aquaculture. 245 (2005) 249-261.
ACCEPTED MANUSCRIPT
[24]
S. IEHATA, T. INAGAKI, S. OKUNISHI, M. NAKANO, R. TANAKA & H. MAEDA.
Improved gut environment of abalone Haliotis gigantea through Pediococcus sp.
Ab1 treatment. Aquaculture. 305 (2010) 59-65.
589
590
591
592
[25]
F. SILVA-ACIARES, D. MORAGA, M. AUFFRET, A. TANGUY & C. RIQUELME.
Transcriptomic and cellular response to bacterial challenge (pathogenic Vibrio
parahaemolyticus) in farmed juvenile Haliotis rufescens fed with or without
probiotic diet. Journal of Invertebrate Pathology. 113 (2013) 163-176.
593
594
595
[26]
D. HILDEMAN, T. JORGENSEN, J. KAPPLER & P. MARRACK. Apoptosis and the
homeostatic control of immune responses. Current Opinion in Immunology. 19
(2007) 516–521.
596
597
598
[27]
V. DIAS. Investigation of the effect of a probiotic-supplemented diet on the
haemocyte proteome of the abalone Haliotis midae. Doctoral dissertation.
University of Cape Town. 2016.
599
600
601
[28]
R. GRANDIOSA, F. MÉRIEN, K. PILLAY & A. ALFARO. Innovative application of classic
and newer techniques for the characterization of haemocytes in the New Zealand
black-footed abalone (Haliotis iris). Fish & Shellfish Immunology. 48 (2016) 175-184.
602
603
[29]
T. YOUNG & A. ALFARO. Metabolomic strategies for aquaculture research: a
primer. Reviews in Aquaculture. 10 (2016) 26-56.
604
605
[30]
A. ALFARO & T. YOUNG. METABOLOMICS: An Innovative and Powerful Tool That
Will Revolutionize Aquaculture. World Aquaculture. (2015).
606
607
[31]
A. ALFARO & T. YOUNG. Showcasing metabolomic applications in aquaculture: a
review. Reviews in Aquaculture. 10 (2016) 135-152.
608
609
610
[32]
S. VILLAS-BÔAS, K. SMART, S. SIVAKUMARAN & G. LANE. Alkylation or silylation for
analysis of amino and non-amino organic acids by GC-MS? Metabolites. 1 (2011) 320.
611
612
613
[33]
K. SMART, R. AGGIO, J. VAN HOUTTE & S. VILLAS-BÔAS. Analytical platform for
metabolome analysis of microbial cells using MCF derivatization followed by gas
chromatography-mass spectrometry. Nature Protocols. 5 (2010) 1709–1729.
614
615
616
[34]
R. AGGIO, S. VILLAS− BÔAS & K. RUGGIERO. Metab: an R package for highthroughput analysis of metabolomics data generated by GC-MS. Bioinformatics. 27
(2011) 2316-2318.
617
618
[35]
619
620
621
[36]
C. ADEMA, W. VAN DER KNAAP & T. SMINIA. Molluscan hemocyte-mediated
cytotoxicity: the role of reactive oxygen intermediates. Reviews in Aquatic Science.
4 (1991) 201- 223.
622
623
624
625
[37]
M. CASTEX, P. LEMAIRE, N. WABETE, & L. CHIM. Effect of probiotic Pediococcus
acidilactici on antioxidant defences and oxidative stress of Litopenaeus stylirostris
under Vibrio nigripulchritudo challenge. Fish & Shellfish Immunology. 28 (2010)
622-631.
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
586
587
588
J. XIA, I. SINELNIKOV, B. HAN & D. WISHART. MetaboAnalyst 3.0—making
metabolomics more meaningful. Nucleic Acids Research. 43 (2015) 251-257.
ACCEPTED MANUSCRIPT
[38]
E. PILA, J. SULLIVAN, X. WU, J. FANG, S. RUDKO, M. GORDY & P. HANINGTON.
Haematopoiesis in molluscs: a review of haemocyte development and function in
gastropods, cephalopods and bivalves. Developmental & Comparative Immunology.
58 (2016) 119-128.
630
631
632
[39]
T. LITTLE & A. KRAAIJEVELD. Ecological and evolutionary implications of
immunological priming in invertebrates. Trends in Ecology & Evolution. 19 (2004)
58-60.
633
634
635
636
637
[40]
H. HÉGARET, G. WIKFORS & P. SOUDANT. Flow cytometric analysis of haemocytes
from eastern oysters, Crassostrea virginica, subjected to a sudden temperature
elevation: II. Haemocyte functions: aggregation, viability, phagocytosis, and
respiratory burst. Journal of Experimental Marine Biology and Ecology. 293 (2003)
249-265.
638
639
640
641
642
[41]
W. WANG, J. ZHOU, P. WANG, T. TIAN, Y. ZHENG, Y. LIU, W. MAI & A. WANG.
Oxidative stress, DNA damage and antioxidant enzyme gene expression in the
Pacific white shrimp, Litopenaeus vannamei when exposed to acute pH stress.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 150
(2009) 428-435.
643
644
645
646
[42]
M. CARDINAUD, C. OFFRET, S. HUCHETTE, D. MORAGA & C. PAILLARD. The impacts
of handling and air exposure on immune parameters, gene expression, and
susceptibility to vibriosis of European abalone Haliotis tuberculata. Fish & Shellfish
Immunology. 36 (2014) 1-8.
647
648
649
650
[43]
E. MOTTIN, C. CAPLAT, M. MAHAUT, K. COSTIL, D. BARILLIER, J. LEBEL & A.
SERPENTINI. Effect of in vitro exposure to zinc on immunological parameters of
haemocytes from the marine gastropod Haliotis tuberculata. Fish & Shellfish
Immunology. 29 (2010) 846-853.
651
652
653
654
[44]
M. PRADO-ALVAREZ, A. ROMERO, P. BALSEIRO, S. DIOS, B. NOVOA & A. FIGUERAS.
Morphological characterization and functional immune response of the carpet shell
clam (Ruditapes decussatus) haemocytes after bacterial stimulation. Fish & Shellfish
Immunology. 32 (2012) 69-78.
655
656
657
658
[45]
B. ALLAM, C. PAILLARD, M. AUFFRET, S. FORD. Effects of the pathogenic Vibrio
tapetis on defence factors of susceptible and non-susceptible bivalve species: II.
Cellular and biochemical changes following in vivo challenge. Fish & shellfish
immunology. 20 (2006) 384-397.
659
660
661
[46]
662
663
[47]
I. SOKOLOVA. Apoptosis in molluscan immune defense. Invertebrate Survival
Journal. 6 (2009) 49-58.
664
665
[48]
T. KISS. Apoptosis and its functional significance in molluscs. Apoptosis. 15 (2010)
313-321.
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
626
627
628
629
R. FRANCO, R. SÁNCHEZ-OLEA, E. REYES-REYES, & M. PANAYIOTIDIS. Environmental
toxicity, oxidative stress and apoptosis: menage a trois. Mutation Research/Genetic
Toxicology and Environmental Mutagenesis. 674 (2009) 3-22.
ACCEPTED MANUSCRIPT
[49]
K. TERAHARA & K. TAKAHASHI. Mechanisms and immunological roles of apoptosis
in molluscs. Current Pharmaceutical Design. 14 (2008) 131-137.
668
669
[50]
A. NAPPI. & E. OTTAVIANI. Cytotoxicity and cytotoxic molecules in invertebrates.
Bioessays. 22 (2000) 469-480.
670
671
672
[51]
L. MARTELLO, C. FRIEDMAN & R. TJEERDEMA. Combined effects of
pentachlorophenol and salinity stress on phagocytic and chemotactic function in
two species of abalone. Aquatic Toxicology. 49 (2000) 213-225.
673
674
675
[52]
M. VIANT, E. ROSENBLUM & R. TJEERDEMA. NMR-based metabolomics: a powerful
approach for characterizing the effects of environmental stressors on organism
health. Environmental Science & Technology. 37 (21) 4982-4989.
676
677
678
679
[53]
E. ROSENBLUM, M. VIANT, B. BRAID, J. MOORE, C. FRIEDMAN & R. TJEERDEMA.
Characterizing the metabolic actions of natural stresses in the California red
abalone, Haliotis rufescens using 1 H NMR metabolomics. Metabolomics. 1 (2005)
199-209.
680
681
682
[54]
X. ZHAO, Y. HAN, S. REN, Y. MA, H. LI & X. PENG. L-proline increases survival of
tilapias infected by Streptococcus agalactiae in higher water temperature. Fish and
Shellfish Immunology. 44 (2015) 33–42.
683
684
685
[55]
R. PANIELLO, R. HAYDEN & S. BELLO. Improved survival of acute skin flaps with
amino acids as free radical scavengers. Archives of Otolaryngology–Head & Neck
Surgery. 114 (1988) 1400-1403.
686
687
[56]
M. FISHER. Proline to the rescue. Proceedings of the National Academy of
Sciences. 103 (2006) 13265-13266.
688
689
[57]
P. HOCHACHKA (Ed.). Mollusca: Metabolic
Biomechanics. Academic Press. (2003).
690
691
[58]
J. ALEDO. Glutamine breakdown in
investment? Bioessays. 26 (2004) 778-785.
692
693
694
[59]
J. CARRASCOSA, P. MARTÍNEZ & I. DE CASTRO. Nitrogen movement between host
and tumor in mice inoculated with Ehrlich ascetic tumor cells. Cancer Research. 44
(1984) 3831–3835.
695
696
697
[60]
698
699
[61]
700
701
702
703
[62]
Biochemistry
rapidly
dividing
and
cells:
Molecular
waste
or
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
666
667
R. MOREADITH & A. LEHNINGER. The pathways of glutamate and glutamine
oxidation by tumor cell mitochondria. Role of mitochondrial NAD (P)+-dependent
malic enzyme. Journal of Biological Chemistry. 259 (1984) 6215-6221.
N. WALSH, A. BLANNIN, P. ROBSON & M. GLEESON. Glutamine, exercise and
immune function. Sports Medicine. 26 (1998) 177-191.
M. CARDINAUD, C. OFFRET, S. HUCHETTE, D. MORAGA & C. PAILLARD. The impacts
of handling and air exposure on immune parameters, gene expression, and
susceptibility to vibriosis of European abalone Haliotis tuberculata. Fish & Shellfish
Immunology. 36 (2014) 1-8.
ACCEPTED MANUSCRIPT
[63]
M. TRAVERS, A. MEISTERTZHEIM, M. CARDINAUD, C. FRIEDMAN, S. HUCHETTE, D.
MORAGA & C. PAILLARD. Gene expression patterns of abalone, Haliotis
tuberculata, during successive infections by the pathogen Vibrio harveyi. Journal of
Invertebrate Pathology. 105 (2010) 289-297.
708
709
[64]
P. LI, Y. YIN, D. LI, S. KIM & G. WU. Amino acids and immune function. British
Journal of Nutrition. 98 (2007) 237-252.
710
711
[65]
J. LU, Y. SHI, S. CAI & J. FENG. Metabolic responses of Haliotis diversicolor to Vibrio
parahaemolyticus infection. Fish & Shellfish Immunology. 60 (2017) 265-274.
712
713
714
715
716
717
[66]
G. TANNAHILL, A. CURTIS, J. ADAMIK, E. PALSSON-MCDERMOTT, A. MCGETTRICK,
G. GOEL, C. FREZZA, N. BERNARD, B. KELLY, N. FOLEY, L. ZHENG, A. GARDET, Z.
TONG, S. JANY, S. CORR, M. HANEKLAUS, B. CAFFERY, K. PIERCE, S. WALMSLEY, F.
BEASLEY, E. CUMMINS, V. NIZET, M. WHYTE, C. TAYLOR, H. LIN, S. MASTERS, E.
GOTTLIEB, V. KELLY, C. CLISH, P. AURON, R. XAVIER, L. O’NEILL. Succinate is a
danger signal that induces IL-1β via HIF-1α. Nature. 496 (2013) 238.
718
719
720
[67]
L. TRETTER, A. PATOCS & C. CHINOPOULOS, C. Succinate, an intermediate in
metabolism, signal transduction, ROS, hypoxia, and tumorigenesis. Biochimica et
Biophysica Acta (BBA)-Bioenergetics. 1857 (2016) 1086-1101.
721
722
723
724
[68]
W. CAMPBELL, J. FALCK, J. OKITA, A. JOHNSON & K. CALLAHAN. Synthesis of
dihomoprostaglandins from adrenic acid (7, 10, 13, 16-docosatetraenoic acid) by
human endothelial cells. Biochimica et Biophysica Acta (BBA)-Lipids and Lipid
Metabolism. 837 (1985) 67-76.
725
726
727
728
729
730
731
732
733
734
[69]
T. KAYASHIMA & T. KATAYAMA. Oxalic acid is available as a natural antioxidant in
some systems. Biochimica et Biophysica Acta (BBA)-General Subjects. 1573 (2002)
1-3.
[70]
GREGORC, A. POGACNIK & I. BOWEN. Cell death in honeybee (Apis mellifera) larvae
treated with oxalic or formic acid. Apidologie. 35 (2004) 453-460.
[71]
LEHNER, A., MEIMOUN, P., ERRAKHI, R., MADIONA, K., BARAKATE, M., & BOUTEAU,
F. Toxic and signalling effects of oxalic acid: Oxalic acid—Natural born killer or
natural born protector? Plant Signaling & Behaviour. 3 (2008) 746-748.
SC
M
AN
U
TE
D
EP
AC
C
735
RI
PT
704
705
706
707
ACCEPTED MANUSCRIPT
Highlights
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
1. A multi-strain probiotic enhanced immune responsiveness in juvenile abalone
2. Probiotic-fed abalone had higher haemocyte cell counts and viability
3. Probiotic-fed abalone had higher numbers of ROS-positive and non-apoptotic cells
4. Metabolomics profiling resulted in differential expression of metabolites in foot
5. Probiotics diet can alter central carbon metabolism to improve growth and survival
1
Документ
Категория
Без категории
Просмотров
1
Размер файла
2 697 Кб
Теги
fsi, 034, 2018
1/--страниц
Пожаловаться на содержимое документа