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Article
Immunomodulatory and Antiviral Effects of Macroalgae
Sulphated Polysaccharides: Case Studies Extend Knowledge on
Their Importance in Enhancing Shellfish Health, and the Control
of a Global Viral Pathogen Ostreid Herpesvirus-1 microVar
Sharon A. Lynch 1,* , Rachel Breslin 1, Babette Bookelaar 1, Tawut Rudtanatip 2, Kanokpan Wongprasert 3
and Sarah C. Culloty 1,4
Citation: Lynch, S.A.; Breslin, R.;
Bookelaar, B.; Rudtanatip, T.;
Wongprasert, K.; Culloty, S.C.
Immunomodulatory and Antiviral
Effects of Macroalgae Sulphated
Polysaccharides: Case Studies Extend
Knowledge on Their Importance in
Enhancing Shellfish Health, and the
Control of a Global Viral Pathogen
Ostreid Herpesvirus-1 microVar.
Polysaccharides 2021,2, 202–217.
https://doi.org/10.3390/
polysaccharides2020014
Academic Editor: Cédric Delattre
Received: 19 February 2021
Accepted: 23 March 2021
Published: 1 April 2021
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Attribution (CC BY) license (https://
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1School of Biological, Earth and Environmental Sciences, Aquaculture Fisheries Development Centre and
Environmental Research Institute, University College Cork, Cork T23 XE10, Ireland;
114757835@umail.ucc.ie (R.B.); babette_bookelaar@hotmail.com (B.B.); s.culloty@ucc.ie (S.C.C.)
2Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand;
tawut@kku.ac.th
3Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand;
kanokpan.won@mahidol.edu
4Marine and Renewable Energy Research Centre, University College Cork, Cork P43 C573, Ireland
*Correspondence: s.lynch@ucc.ie; Tel.: +353-(21)-4904615
Abstract:
Macroalgae are the primary source of non-animal sulphated polysaccharides (SPs) in the
marine environment with fucoidans derived from brown algae (Phaeophyta) and carrageenans from
red algae (Rhodophyta). Much research has been carried out on SP effects on Asian shrimp species
(genera Penaeus and Metapenaeus) but their effect on commercially important bivalve mollusc
species is limited and in Pacific oyster Crassostrea gigas is unknown. Knowledge of their impact
on bivalve pathogens and Palaemon shrimp is unknown. The objectives of this study were to
assess the effects of Fucus vesiculosus (Phaeophyta), Mastocarpus stellatus (Rhodophyta) and algal
derivatives (fucoidan and
κ
-carrageenan) on C. gigas performance, and on ostreid herpesvirus-1
microvar (OsHV-1
µ
Var) and bacteria Vibrio spp. development. Both pathogens have been associated
with significant oyster mortalities and economic losses globally. The effects of sulphated galactan
from Gracilaria fisheri (Rhodophyta) on European common prawn Palaemon serratus, an important
fishery species, was also assessed. Findings indicate a rapid and prolonged increase in total blood
cell count, lysozyme (enzyme that destroys pathogens), and a difference in the ratio of blood cell
types in treated individuals compared to their control counterparts. A significantly lower OsHV-1
µ
Var prevalence was observed in treated oysters and
κ
-carrageenan was found to suppress viral
replication (loads), while OsHV-1
µ
Var was not detected in the fucoidan treated oysters from Day
8 of the 26-day trial. No antibacterial effect was observed however, the oysters did not succumb
to vibriosis. These findings contribute further knowledge to macroalgae sulphated polysaccharide
biotherapeutic properties, their twofold effect on animal health and viral suppression.
Keywords:
macroalgae sulphated polysaccharides; pacific oyster Crassostrea gigas; ostreid herpesvirus-1
microVar; European shrimp Palaemon serratus; antiviral
1. Introduction
Macroalgae (seaweed) are a prolific source of bioactive components in marine en-
vironments [
1
]. Such biocompounds boost vertebrate and invertebrate health by their
immunostimulating, anticancer, antiviral and antibacterial activities [
2
,
3
]. A sulphated
polysaccharide (SP) derived from brown algae (Phaeophyta) includes fucoidan [
4
,
5
] while
red seaweeds contain multiple SPs including sulphated galactans [
6
] and carrageenan [
7
]
both of which are successful in the treatment of herpesvirus in humans [
8
,
9
]. In more recent
Polysaccharides 2021,2, 202–217. https://doi.org/10.3390/polysaccharides2020014 https://www.mdpi.com/journal/polysaccharides
Polysaccharides 2021,2203
studies, antiviral effects of SPs against a variety of enveloped viruses, such as Herpes Sim-
plex Virus type 1 (HSV-1) and 2 (HSV-2), Human Immunodeficiency Virus (HIV), human
cytomegalovirus, dengue viruses, respiratory syncytial and influenza viruses have been
reported [10–12].
Alginic acid is generally the most abundant polysaccharide, with values reaching up
to 59% DW in F. vesiculosus. Alginates (salts of alginic acid) are polyuronic saccharides
consisting of
β
-D-mannuronic (M) and
α
-L-glucuronic (G) acid units linked together by
(1
→
4) bonds, arranged heteropolymeric (MG) and/or homopolymeric (M or G) [
13
]. Fu-
coidans are particularly abundant in F. vesiculosus, which can accumulate up to 26% DW.
Fucoidans are complex polysaccharides, ranging from 100 to 1600 kDa, being composed
mainly of fucose and sulfate, although other monosaccharides (mannose, galactose, glu-
cose, xylose, etc.), uronic acids or even acetyl groups and proteins may be present [
13
].
Laminarin is another polysaccharide of brown seaweeds and is a
β
-1,3-glucan, consisting of
β
-1,3-d-glucopyranose units interspersed with
β
-1,6-linked D-glucopyranose units forming
branch-points or interchain residues [
14
]. Mastocarpus stellatus along with Irish moss Chon-
drus crispus were the first seaweed used for carrageenan extraction [
15
]. Carrageenans are
a family of water soluble, linear, and sulfated galactans. They are composed of alternating
3-linked
β
-D-galactopyranose (G-units) and 4-linked
α
-D-galactopyranose (D-units) or
4-linked 3,6-anhydro-
α
-D-galactopyranose (DA-units), forming the disaccharide repeating
unit of carrageenans [
15
]. The structure of fucoidans typically consist of a backbone of
(1
→
3)- and (1
→
4)-linked
α
-L-fucopyranose residues, which can be separated in two types.
Fucoidans from Fucus spp. have a type II arrangement and are essentially composed of
fucose and sulfate, although small amounts of other monosaccharides may occur. Type II
fucoidans consist of alternating (1→3)- and (1→4)-linked α-L-fucopyranose residues [16].
The Pacific oyster Crassostrea gigas is one of the most commercially important aqua-
culture species worldwide (http://www.fao.org/fishery/culturedspecies/Crassostrea_
gigas/en, accessed 26 March 2021). C. gigas are filter feeding bivalves that ingest plankton
and organic particles from the water column [
17
]. C. gigas stocks have suffered significant
summer mortalities globally due to a complex aetiology, primarily associated with disease-
causing pathogens such as ostreid herpesvirus-1 microVar (OsHV-1
µ
Var) and variants, as
well as Vibrio spp. bacteria, [
18
–
21
]. The common prawn Palaemon serratus is found in the
Atlantic Ocean from Denmark to Mauritiana, and the Mediterranean and Black Seas, and
is a commercially valuable trap fishery primarily in the UK and Ireland [
22
]. P. serratus are
omnivorous and will feed on macroalgae [22].
Shellfish species rely on an innate immune system, which can be directly and/or
indirectly affected by their environment [
23
,
24
]. Their effective innate immune responses
and mechanisms involve immune cells, genes and proteins [
25
] and is responsible for
providing resistance to microbial pathogens and preventing disease outbreaks [
26
–
28
].
Vaccination against infectious pathogens and diseases in these species is not possible [29].
Haemocytes (blood cells) are the primary defence mechanism in shellfish [
30
] and the cel-
lular protein lysozyme is an enzyme linked with the destruction of pathogens or “nonself”
and is also an indicator of physiological condition and vitality [
31
–
33
]. Two types of haemo-
cytes, hyalinocytes/hyaline and granulocytes/granular, are involved in immunological
defence [
30
,
34
]. Lysozymes support the immune system by lysing cell walls of invading
particles, which lead to osmotic and mechanical stress in the cell and often death [35].
In recent years, it has been highlighted that seaweeds can be a beneficial diet source for
bivalve molluscs [
36
]. Porphyra haitanensis (Rhodophyta) was found to be a successful substitute
diet in the nursery production of Belcher’s cupped oyster Crassostrea belcheri [
37
,
38
]. Extracted
SPs from Chondrus crispus (Rhodophyta) induced health enhancing activities in the blue mussel
Mytilus spp. at a cellular, humoral and molecular level [
39
]. Extracted SPs from red seaweed
spp. have been successfully used in Asian aquaculture of giant tiger prawns Penaeus monodon
and Whiteleg shrimp Litopenaeus vannamei in the fight against infection with white spot syn-
drome virus (WSSV) [
40
,
41
]. Significantly greater hyaline cells (HC), granular cells (GC), and
total haemocyte count (THC) were reported in L. vannamei after they were fed a diet incorpo-
Polysaccharides 2021,2204
rated with
λ
-carrageenan [
42
]. Carrageenan has been found to increase the immune-related
expression in shrimp L. vannamei through oral
administration [43,44]
. Dietary administration
of SPs from Asparagopsis taxiformis,Gracilaria tenuistipitata, and
Gracilaria verrucosa
resulted in
significantly higher HC, GC, and THC in P. monodon and L. vannamei [
43
,
45
,
46
]. P. monodon
treated with a diet of Asparagopsis spp. (Rhodophyta) were able to suppress and control
infection with Vibrio spp. [47].
The objectives of this study were to assess the biotherapeutic and antimicrobial effects
of red and brown seaweed spp. and their SP derivatives on C. gigas and two associated
pathogen groups of significance, ostreid herpesvirus-1 microVar and Vibrio spp., and
P. serratus for the first time in laboratory-based trials. The seaweed species chosen are
commonly found at oyster culture and near shore coastal locations where both shellfish
species occur [
48
,
49
]. Findings from these studies will not only fill the knowledge gaps that
exist with regards the benefits of macroalgae spp. and their biocompounds on commercially
important marine invertebrate species, but also on a significant global marine virus.
2. Materials and Methods
2.1. The Effects of (a) Intact Seaweed and (b) Extracted Derivatives on Pacific Oyster Crassostrea
Gigas Performance and Pathogen Development
(a) This study investigated the effects of crude seaweed (Figure 1) and extracted sea-
weed derivatives on the innate immunity of C. gigas and associated pathogen development.
Polysaccharides2021,2,4
Figure1.Seaweedspecies(A)BladderwrackFucusvesiculosus,(B)falseIrishmossMastocarpus
stellatus, and (C) PacificoysterCrassostreagigas.
Threereplicateswereused pertreatment,i.e., (a) oystersheldwithF. vesiculosus,(b)
oystersheldwith M. stellatusand(c)controloystersheldwithnoseaweed.Thirtyoysters
consistingofseedandadultswereusedpertank,resultingin90controloystersand90
oysterspertreatmentintotal.Theoysters andseaweedspecieswereheldinstand-alone
aquaria(10L)treatedwith4mLofabiofiltertreatmentSeachemStability
®
thatrapidly
andsafelyestablishesabio‐filterandprevents“NewTankSyndrome”atthestartofeach
trialtoensureoptimalwaterqualityinallofthetanksfor26days atasalinityof34.During
thefirst19daystheoysterswereexposedto14°C,afterDay19thetemperaturewentup
to19°C for7days(upuntilDay26) toapplyathermalshocktotheoystersandencourage
viralreplicationinthesystem.Thetankswereexposedto12hofdarknessand12hof
light.Nowaterchangeswereconductedforthedurationofthetrialandoysterswerefed
2mLofShellfishDiet1800(ReedMariculture,AmixeddietofIsochrysis,Pavlova
Thalassiosira,Tetraselmis,size: ~5–12microns,dryweight8%,moreinformation:
(https://reedmariculture.com/product_shellfish_diet_1800.phplastaccessed26thMarch
2021) atDay7,14and21.
Aninitial/baselinesampleofoysters(n=30) wasprocessedpriortothetrial
commencingforpathogenscreening.Theoysterswerecheckedformortalityseveraltimes
dailyandanymoribundordeadindividualswereremovedimmediatelyandprocessed.
OnDays2,5,7,12,19and22threeoysterspertankwererandomlyselectedandprocessed,
resultinginatotalof9oysterspertreatmentandcontrolatfixedtimepointsduringthetrial.
Allremainingoystersdeadoralivewereprocessedonthefinaldayofthetrial(Day26).
ForthedetectionofOsHV-1μVar, DNAwasextracted fromoystergilltissue
(approximately5mm
2
) usingtheChelex-100method(10gofChelexin100mLofddH
2
O)
[52].The oyster gill tissue was added to 100μLofChelexsolutionina0.5mLEppendorf
safelocktube.SampleswerethenplacedinaHybaidthermocyclerandheatedata
temperatureof99°Cfor1h15mintofacilitatecelllysis.Apolymerasechainreaction(PCR)
[18] andquantitativePCR(qPCR)[53] wascarriedout todeterminepresence/absenceof
OsHV-1μVaranddeterminetheviralloadinoysters,respectively.In the PCR [18],2μL
genomicDNAtemplateperindividualwasscreened.ExpectedsizeofamplifiedPCR
productsforOsHV‐1μVarwas385bpandPCRwascarriedoutin25μLcontaining12.9μL
ddH
2
0,5μL,5×buffer,5μLdNTPs(0.2mM),0.5μLMgCl
2
(25mMstock),0.25μLofeach
primerOHVA/OHVB(100pmolmL−1stock),and0.1μLTaqDNApolymerase.Positive
controls(triplicate)consistingofOsHV‐1μVarinfectedoystertissueandnegativecontrols
(triplicate)ofdoubledistilledwater(ddH
2
O)wereusedforeachPCR.Thermocycling
conditionswereperformedbyinitialdenaturationof1minof95°C,followingby35cycles
includingadenaturationstepof20sat94°C,anannealingstepof30sat56°Candan
elongationstepat72°Candfinishingwithafinalelongationstepof7minat72°Cbyusing
athermoHybaidPCRexpressthermalcycler. PresenceofamplifiedPCRproductswas
Figure 1.
Seaweed species (
A
) Bladder wrack Fucus vesiculosus, (
B
) false Irish moss Mastocarpus stellatus,
and (C) Pacific oyster Crassostrea gigas.
Oysters with a whole wet weight range of 0.35–7.36 g were sourced from an Irish
oyster culture site where ostreid herpesvirus-1 microVar (OsHV-1
µ
Var) is endemic. Prior
to the trial commencing, the seaweed was washed with tap water to remove all attached
organisms including pathogens. A total of 100 g of each seaweed species (water squeezed
out and blotted dry) that were attached to oyster culture bags was used per experimental
tank (Figure 1). Although sulphate concentrate was not analysed, the amount of seaweed
that the oysters were exposed to was based on seaweed density attached to the oyster trestle
bags in the field. The mineral content, including sulphate concentrations varied between
2.4–11.5 g/100 g dry weight and 3.75 g
±
0.04/100 g dry weight for F. vesiculosus [
50
] and
0.6–3.9 g/100 g for M. stellatus [51].
Three replicates were used per treatment, i.e., (a) oysters held with F. vesiculosus, (b) oys-
ters held with M. stellatus and (c) control oysters held with no seaweed. Thirty oysters
consisting of seed and adults were used per tank, resulting in 90 control oysters and 90 oysters
per treatment in total. The oysters and seaweed species were held in stand-alone aquaria (10 L)
treated with 4 mL of a biofilter treatment Seachem Stability
®
that rapidly and safely establishes
a bio-filter and prevents “New Tank Syndrome” at the start of each trial to ensure optimal wa-
ter quality in all of the tanks for 26 days at a salinity of 34. During the first 19 days the oysters
were exposed to 14
◦
C, after Day 19 the temperature went up to 19
◦
C for 7 days (up until Day
Polysaccharides 2021,2205
26) to apply a thermal shock to the oysters and encourage viral replication in the system. The
tanks were exposed to 12 h of darkness and 12 h of light. No water changes were conducted
for the duration of the trial and oysters were fed 2 mL of Shellfish Diet 1800 (Reed Mariculture,
A mixed diet of Isochrysis,Pavlova Thalassiosira,Tetraselmis, size: ~5–12 microns, dry weight
8%, more information: (https://reedmariculture.com/product_shellfish_diet_1800.php, last
accessed 26 March 2021) at Day 7, 14 and 21.
An initial/baseline sample of oysters (n= 30) was processed prior to the trial com-
mencing for pathogen screening. The oysters were checked for mortality several times
daily and any moribund or dead individuals were removed immediately and processed.
On Days 2, 5, 7, 12, 19 and 22 three oysters per tank were randomly selected and processed,
resulting in a total of 9 oysters per treatment and control at fixed time points during the trial.
All remaining oysters dead or alive were processed on the final day of the trial (Day 26).
For the detection of OsHV-1
µ
Var, DNA was extracted from oyster gill tissue (approxi-
mately 5 mm
2
) using the Chelex-100 method (10 g of Chelex in 100 mL of ddH
2
O) [
52
]. The
oyster gill tissue was added to 100
µ
L of Chelex solution in a 0.5 mL Eppendorf safe lock
tube. Samples were then placed in a Hybaid thermocycler and heated at a temperature
of 99
◦
C for 1 h 15 min to facilitate cell lysis. A polymerase chain reaction (PCR) [
18
] and
quantitative PCR (qPCR) [
53
] was carried out to determine presence/absence of OsHV-1
µ
Var and determine the viral load in oysters, respectively. In the PCR [
18
], 2
µ
L genomic
DNA template per individual was screened. Expected size of amplified PCR products for
OsHV-1
µ
Var was 385 bp and PCR was carried out in 25
µ
L containing 12.9
µ
L ddH
2
0,
5
µ
L, 5
×
buffer, 5
µ
L dNTPs (0.2 mM), 0.5
µ
L MgCl
2
(25 mM stock), 0.25
µ
L of each
primer OHVA/OHVB (100 pmol mL
−
1 stock), and 0.1
µ
L Taq DNA polymerase. Positive
controls (triplicate) consisting of OsHV-1
µ
Var infected oyster tissue and negative controls
(triplicate) of double distilled water (ddH
2
O) were used for each PCR. Thermo cycling
conditions were performed by initial denaturation of 1 min of 95
◦
C, following by 35 cycles
including a denaturation step of 20 s at 94
◦
C, an annealing step of 30 s at 56
◦
C and an
elongation step at 72
◦
C and finishing with a final elongation step of 7 min at 72
◦
C by
using a thermo Hybaid PCR express thermal cycler. Presence of amplified PCR products
was confirmed by electrophoresis using a 2% agarose gel stained with ethidium bromide
(10 mg/L stock) and was run with an electrical charge of 110 V for 60 min.
In the qPCR [
53
], all qPCRs used a total of 5
µ
L genomic DNA template per individual
(duplicate). The qPCR mix was carried out in 25
µ
L containing 12.5
µ
L 2
×
Brilliant Sybr
Green
®
Q PCR Master Mix, 2.5
µ
L HVDP-F (5
µ
M) and 2.5
µ
L HVDP-R (
µ
M) primers and
2.5
µ
L ddH
2
O. Standards were used to detect the exact number of viral copies in tested
samples. Standard curves were prepared by diluting a viral DNA suspension of 10
8
viral
copies of OsHV-1 (supplied by IFREMER). qPCR plates included 5 dilutions of 10
5
, 10
4
, 10
3
,
10
2
and 10
1
viral copies. Thermo cycling conditions were performed by initial denaturation
of 2 min of 50
◦
C and 10 min at 95
◦
C, following by 40 cycles of 15 s at 95
◦
C and 1 min at
60
◦
C and a melt curve of 95
◦
C for 15 s, 60
◦
C for 1 min, 95
◦
C for 30 s and 60
◦
C for 15 s in
a 7500-thermal cycler (Life Technologies).
To evaluate oyster immune function, haemolymph was collected and lysozyme levels
and activity were measured [
32
] according to [
54
]. The haemolymph serum was defrosted
and 30
µ
L of serum was placed in triplicate into a 96 well round flat-bottomed plate. 170
µ
L
of the Micrococcus lysodeikticus suspension (0.2 mg
·
mL
−1
sodium phosphate buffer pH 6.4,
store at
−
20
◦
C) was added to the serum and the decrease in absorbance was recorded at
450 nm every minute for the following 4 min using a Spectra III SLT plate reader.
(b) In the laboratory trial to assess the effects of commercial seaweed biocompounds
fucoidan and kappa (
κ
)-carrageenan, oysters from the same age cohort (approximately
1 year old, 3.835 g
±
1.19 (SD) with a weight range of 2.01–8.69 g) were used from an oyster
culture site where both pathogens are endemic.
κ
-carrageenan and fucoidan (MERCK-
SigmaAldrich Ireland Ltd., Arklow, Co., Wicklow, Ireland) were used in the trial.
Prior to the trial starting, 30 oysters from the initial batch of 300, were processed and
screened for the presence of OsHV-1
µ
Var and Vibrio spp. The experiment consisted of
Polysaccharides 2021,2206
nine stand-alone plastic aquaria (10 L volume). Three of the tanks acted as controls (no
algal dose), whilst the two algal treatments had 3 tanks each, with each tank containing
20 C. gigas. The dose amount added was 300
µ
g of algal extract/g of oyster per tank, i.e.,
for every 1 g (wet weight) of oyster 300
µ
g of
κ
-carrageenan or fucoidan was added to
each tank for example if the wet weight of oysters in a tank was 30 g than 9000
µ
g of the
algal extract was added to that tank, as this dose amount was observed to have an effect
when native oysters O. edulis were administered a dose of zymosan [
33
]. The trial ran for
a duration of 14 days, excluding the first 5 days prior to the trial to allow the animals to
acclimate. The oysters were kept at ambient room temperature, with salinity being kept
at 33 ppt and tank water temperature ranging from 14.9 to 16.2
◦
C. A biofilter treatment
consisting of 4 mL of Seachem Stability
®
was added to each of the tanks to ensure optimal
water quality. The tanks were checked three times daily, and any dead individuals were
removed and processed. Living specimens were sampled at 12 h, 24 h, 48 h, and on days 5,
8, and 10, with the remaining oysters being collected and screened on Day 14. Throughout
the study, the tank water was not changed, and the oysters were not fed.
During the processing of the oysters, gill tissue (approximately 5 mm
2
) was taken for
DNA extraction (chelex) to screen for OsHV-1
µ
Var and V. aestuarianus. PCR and qPCR were
carried out to screen for OsHV-1
µ
Var [
18
,
53
], V. aestuarianus [
55
] and Vibrio spp. [
56
,
57
]
validated by Sanger sequencing and qPCR). In the V. aestuarinaus qPCR [
58
], a total of
5
µ
L of DNA per invertebrate was used to detect Vibrio aestuarianus prevalence. The
amplification was conducted in 20
µ
L of the reaction mixture containing: 5.62
µ
L ddH
2
O,
0.75
µ
L of 5
µ
M forward primer (dnaJ f420), 0.75
µ
L of 5
µ
M reverse primer (dnaJ r456),
0.38
µ
L of 5
µ
M of probe (dnaJ p441 3) and 12.5
µ
L of 2x Taqman mastermix, using a
7500-thermal cycler (Life Technologies). qPCR was completed using a thermal profile of
50
◦
C for 2 min and 95
◦
C for 10 min, followed by 40 cycles of 95
◦
C for 15 s and 60
◦
C for
30 s. All samples were performed in triplicate. Negative controls (n= 3) of 5
µ
L double
distilled water (ddH
2
O) were used; likewise, positives controls (n= 3) contained 5
µ
LVibrio
infected DNA. C
T
values were used to determine real-time TaqMan PCR quantitation and
detection limits, a tested sample is considered positive if its mean Ct value was below 37.
In the Vibrio spp. [
56
,
57
] PCR. The PCR reaction mix contained 2.5
µ
L of DNA, 0.25
µ
L
each of forward and reverse primers (Vibf/Vibr, 100 pmol/mL), 0.1
µ
L of GoTaq, 0.5
µ
L
MgCl
2
(25 mM), 1.5
µ
L of DMSO, 5
µ
L of 5
×
Green buffer, 5
µ
L of DNTPs (0.2 mM),
and 12.9
µ
L of ddH
2
O per each individual oyster being screened. Negative controls used
were ddH
2
O while positive controls of purified Vibrio aestuarianus DNA (2.5
µ
L) (Marine
Institute, Ireland) was used. The thermocycling conditions for the PCR consisted of 1 min
at 95
◦
C denaturation phase, 35 cycles of 94
◦
C for 20 s, 56
◦
C for 30 s, 72
◦
C for 30 s,
for denaturation, annealing and elongation, respectively. This was followed by a final
elongation phase of 7 min at 72 ◦C. Visualisation of PCR products (286 bp) by agarose gel
electrophoresis using SYBR safe dye was recorded. For the latter diagnostic tool, the PCR
primers were specific to Vibrio spp., as confirmed by Sanger sequencing.
Heart smears were carried out to conduct the total blood cell count (TBC) and differen-
tial blood cell count (DBC). Estimated Total blood cell count was found by examining heart
smear slides under 40
×
magnification and obtaining the average number of blood cells
for 10 high power fields (40
×
objective), and multiplying the average number found by
2000 to obtain an estimated total white blood cell count per microliter [
58
,
59
]. Differential
blood cell (DBC) counts were carried out by using two hand-held clickers, and counting
the number of hyalinocytes and granulocytes present, until 100 blood cells were counted in
total [60,61].
2.2. Immunological Effects of Naturally Extracted Sulphated Polysaccharides from a Red Seaweed
Gracilaria Fisheri on Shrimp (European Common Prawn) Palaemon Serratus
Wild P. serratus with a weight range of 3.0–4.1 g and a length range of 7.0–7.8 cm
were collected from pots at Lough Hyne Marine Nature Reserve, Co., Cork, Ireland
(51◦3007.4300 N–9◦18019.8200 W) (Figure 2).
Polysaccharides 2021,2207
Polysaccharides2021,2,6
IntheVibriospp.[56,57]PCR.ThePCRreactionmixcontained2.5μLofDNA,0.25
μLeachofforwardandreverseprimers(Vibf/Vibr,100pmol/mL),0.1μLofGoTaq,0.5
μLMgCl
2
(25mM),1.5μLofDMSO,5μLof5×Greenbuffer,5μLofDNTPs(0.2mM),
and12.9μLofddH
2
Opereachindividualoysterbeingscreened.Negativecontrolsused
wereddH
2
OwhilepositivecontrolsofpurifiedVibrioaestuarianusDNA(2.5μL)(Marine
Institute,Ireland)wasused.ThethermocyclingconditionsforthePCRconsistedof1min
at95°Cdenaturationphase,35cyclesof94°Cfor20s,56°Cfor30s,72°Cfor30s,for
denaturation,annealingandelongation,respectively.Thiswasfollowedbyafinal
elongationphaseof7minat72°C.VisualisationofPCRproducts(286bp)byagarosegel
electrophoresisusingSYBRsafedyewasrecorded.For the latter diagnostic tool, the PCR
primerswerespecifictoVibriospp.,asconfirmedbySangersequencing.
Heartsmearswerecarriedouttoconductthetotalbloodcellcount(TBC) and
differentialbloodcellcount(DBC).EstimatedTotalbloodcellcountwasfoundby
examiningheartsmearslidesunder40×magnificationandobtainingtheaveragenumber
ofbloodcellsfor10highpowerfields(40×objective),andmultiplyingtheaveragenumber
foundby2000toobtainanestimatedtotalwhitebloodcellcountpermicroliter[58,59].
Differential blood cell (DBC)countswerecarriedoutbyusingtwohand‐heldclickers,
andcountingthenumberofhyalinocytesandgranulocytespresent,until100bloodcells
werecountedintotal[60,61].
2.2.ImmunologicalEffectsofNaturallyExtractedSulphatedPolysaccharidesfromaRed
SeaweedGracilariaFisheri onShrimp(EuropeanCommonPrawn) PalaemonSerratus
WildP. serratuswithaweightrangeof3.0–4.1gandalengthrangeof7.0–7.8cm
werecollectedfrompotsatLoughHyneMarineNatureReserve,Co. Cork,Ireland(51°
30′7.43″N–9°18′19.82″W)(Figure2).
Figure2.TheEuropeancommonprawnPalaemonserratus.
Sulphatedgalactans (Sg) wereextractedfromtheredseaweedG.fishericollected
fromtheShrimpGeneticImprovementCenter(SCIG),Chaiyadistrict,SuratThani
Province,Thailand.ThestructureofSgsconsistedofthelinearbackboneofalternating3‐
linkedβ‐
D
‐galactopyranoseand4‐linked3,6‐anhydro‐α‐
L
‐galactopyranoseorα‐
L
‐
galactose6sulfateunits[62].
Theshrimpwereheldinstand-aloneplasticaquaria(10L),fedwithpelletdiets and
acclimatedfor7dayspriortothetrialcommencing atasalinityof34inaconstant
temperatureroom(10°C). Thetankswereexposedto12hofdarknessand12hoflight.
A biofilter treatment consisting of 4mLofSeachemStability
®
wasaddedtoeachofthetanks
toensureoptimalwaterquality.
Thetreatmentsincluded(a) shrimpnotexposedtosulphatedgalactans,(b) shrimp
exposedtoalowdoseofSg(finalconcentrationof5μgofSg/mLoftankseawater,i.e.,50
μgofSgpertank) and(c) shrimpexposedtoahighdoseofsulphatedgalactans(final
concentrationof50μgofSg/mLoftankseawater,i.e.,500μgofSgpertank). Each
Figure 2. The European common prawn Palaemon serratus.
Sulphated galactans (Sg) were extracted from the red seaweed G. fisheri collected from
the Shrimp Genetic Improvement Center (SCIG), Chaiya district, Surat Thani Province,
Thailand. The structure of Sgs consisted of the linear backbone of alternating 3-linked
β
-D-
galactopyranose and 4-linked 3,6-anhydro-
α
-L-galactopyranose or
α
-L-galactose 6 sulfate
units [62].
The shrimp were held in stand-alone plastic aquaria (10 L), fed with pellet diets
and acclimated for 7 days prior to the trial commencing at a salinity of 34 in a constant
temperature room (10
◦
C). The tanks were exposed to 12 h of darkness and 12 h of light. A
biofilter treatment consisting of 4 mL of Seachem Stability
®
was added to each of the tanks
to ensure optimal water quality.
The treatments included (a) shrimp not exposed to sulphated galactans, (b) shrimp
exposed to a low dose of Sg (final concentration of 5
µ
g of Sg/mL of tank seawater,
i.e., 50
µ
g of Sg per tank) and (c) shrimp exposed to a high dose of sulphated galactans
(final concentration of 50
µ
g of Sg/mL of tank seawater, i.e., 500
µ
g of Sg per tank). Each
treatment was performed in duplicate. Sixteen shrimp were used per tank, giving 32 control
shrimp and 32 shrimp per treatment in total. Sulphated galactans was added into the tank
and no water changes were conducted for the duration of the trial (10 days) to ensure that
the amount of sulphated galactans added to the water was not altered in the system. On
Days 3, 4, 5, 7 and 10 of the trial, three shrimp were arbitrarily selected from each tank to
determining the total haemocyte count (THC). An initial/baseline sample of shrimp (n= 5)
was processed prior to the trial commencing.
To determine the THC, 100
µ
L of haemolymph was withdrawn from the ventral sinus
and mixed with 100
µ
L of 10% formalin in 0.45 M NaCl. After incubation for 10 min, 20
µ
L
of haemocyte mixture was stained with 20
µ
L of Rose Bengal solution (1.2% Rose Bengal
in 50% ethanol) and further incubated at room temperature for 20 min before being used
to determine THC by a hemocytometer (improved Newbauer bright line) under a light
microscope at 20×magnification [63].
2.3. Statistics
2.3.1. Crassostrea gigas Exposed to Intact Seaweed
Oyster mortality and prevalence of infection were analysed by Chi-square test. Dif-
ferences in lysozyme amount and activity were tested by Student’s t-test. For both tests,
significant differences at p≤0.05 were considered.
2.3.2. Crassostrea gigas Exposed to Extracted Derivatives
The data were presented as mean
±
SD and analysed by Chi-square test, and statis-
tically significant difference was required at p-value
≤
0.05. Statistical significance was
calculated via one-way ANOVA analysis followed by a Tukey test were performed on data
in Rstudio. Values of p< 0.05 were considered to be statistically significant.
Polysaccharides 2021,2208
2.3.3. Palaemon serratus Exposed to Extracted Sulphated Galactans
All assays were performed in triplicate. The data were presented as mean
±
SD and
analysed by one-way ANOVA followed by Tukey’s multiple comparison. Statistically
significant difference was required at p≤0.05.
3. Results and Discussion
3.1. Exposure of Crassostrea Gigas to Intact Seaweed Species Fucus Vesiculosus and
Mastocarpus Stellatus
Results indicated that oysters exposed to either of the seaweed species showed sig-
nificantly lower OsHV-1
µ
Var prevalence and viral loads compared to control oysters
throughout the 26-day trial (Figure 3). The initial oyster sample from the C. gigas culture
site had a prevalence of 53.33% for OsHV-1
µ
Var. An overall OsHV-1
µ
Var prevalence
of 22.0% was detected in oysters exposed to F. vesiculosus while it was 23.1% for oysters
exposed to M. stellatus. Control oysters had a significantly higher overall prevalence of
39.8% compared with the oysters exposed to the seaweed species (X
2
= 9.1, df = 2, p= 0.01).
Minimal mortalities were observed in all oyster groups.
Polysaccharides2021,2,8
Figure3.Prevalenceofostreidherpesvirus-1microVar(OsHV-1μVar)andviralload(white
roundmarker: livingoysters,blackroundmarker: deadoysters) inoystersexposedtoseaweed
speciesandcontroloysters.
Inthebloodlysozymeamountanalyses,theinitialoystersampleshowedanaverage
ODabsorbanceof0.221foruninfectedindividualsand0.199forinfectedindividualsin
thebaselinesample,indicatingalowerbloodlysozymeamountinuninfectedoysters
(Figure4). Duringthefirstweekofthetrialthe bloodlysozymeamountwashigherbut
notsignificantlysoin uninfectedoystersheldwithbothseaweedspeciescomparedwith
thecontroloysters (Figure4).AsimilartrendwasobservedinOsHV-1μVarinfected
oystersupuntilDay5ofthetrialforoystersheldwithM.stellatusandbetweenDays12
and19whiletheeffectsweremorelongterminoystersheldwithF. vesiculosus(<Day22).
Nosignificantdifferenceswereobservedbetweenseaweedexposedandcontrol
uninfectedoysters(Figure4A).Significantdifferenceforlysozymeamountbetween
controlandseaweedexposedoysterswasobservedinoystersexposedtotheF.vesiculosus
onDay22(p<0.01)(Figure4B).
Overthe26Daystudyperiodnosignificantdifferencesinoysterperformance,such
asmortalityandlysozymeactivity,wereobservedbetweenthetreatments.
(A)
0
0.1
0.2
0.3
0.4
0.5
Init ial D ay2Day5Day7Day12 Day19 Day22 Day26
ODabsorptionat450nm
F.vesiculosus M.stellatus Control
Figure 3.
Prevalence of ostreid herpes virus-1 microVar (OsHV-1
µ
Var) and viral load (white round
marker: living oysters, black round marker: dead oysters) in oysters exposed to seaweed species and
control oysters.
In the blood lysozyme amount analyses, the initial oyster sample showed an average
OD absorbance of 0.221 for uninfected individuals and 0.199 for infected individuals in the
baseline sample, indicating a lower blood lysozyme amount in uninfected oysters (Figure 4).
During the first week of the trial the blood lysozyme amount was higher but not significantly
so in uninfected oysters held with both seaweed species compared with the control oysters
(Figure 4). A similar trend was observed in OsHV-1
µ
Var infected oysters up until Day 5 of
the trial for oysters held with M. stellatus and between Days 12 and 19 while the effects were
more long term in oysters held with F. vesiculosus (<Day 22). No significant differences were
observed between seaweed exposed and control uninfected oysters (Figure 4A). Significant
difference for lysozyme amount between control and seaweed exposed oysters was observed
in oysters exposed to the F. vesiculosus on Day 22 (p< 0.01) (Figure 4B).
Polysaccharides 2021,2209
Polysaccharides2021,2,8
Figure3.Prevalenceofostreidherpesvirus-1microVar(OsHV-1μVar)andviralload(white
roundmarker: livingoysters,blackroundmarker: deadoysters) inoystersexposedtoseaweed
speciesandcontroloysters.
Inthebloodlysozymeamountanalyses,theinitialoystersampleshowedanaverage
ODabsorbanceof0.221foruninfectedindividualsand0.199forinfectedindividualsin
thebaselinesample,indicatingalowerbloodlysozymeamountinuninfectedoysters
(Figure4). Duringthefirstweekofthetrialthe bloodlysozymeamountwashigherbut
notsignificantlysoin uninfectedoystersheldwithbothseaweedspeciescomparedwith
thecontroloysters (Figure4).AsimilartrendwasobservedinOsHV-1μVarinfected
oystersupuntilDay5ofthetrialforoystersheldwithM.stellatusandbetweenDays12
and19whiletheeffectsweremorelongterminoystersheldwithF. vesiculosus(<Day22).
Nosignificantdifferenceswereobservedbetweenseaweedexposedandcontrol
uninfectedoysters(Figure4A).Significantdifferenceforlysozymeamountbetween
controlandseaweedexposedoysterswasobservedinoystersexposedtotheF.vesiculosus
onDay22(p<0.01)(Figure4B).
Overthe26Daystudyperiodnosignificantdifferencesinoysterperformance,such
asmortalityandlysozymeactivity,wereobservedbetweenthetreatments.
(A)
0
0.1
0.2
0.3
0.4
0.5
Init ial D ay2Day5Day7Day12 Day19 Day22 Day26
ODabsorptionat450nm
F.vesiculosus M.stellatus Control
Polysaccharides2021,2,9
(B)
Figure4.Lysozymeamountfor(A) uninfectedand(B) OsHV-1μVar infectedoystersbasedon
average(+SE) ODabsorbance.Alowvalueindicatesahighbloodlysozymeamountandahigh
valueindicatesalowbloodlysozymeamount.
3.2.EffectsofFucoidanandKappa(κ)CarrageenanonCrassostreaGigasandPathogens
(OsHV-1μVarandVibriospp(V.splendidus).
Forthedurationoftrialperiod,noC. gigasmortalitieswereobservedinthecontrol
andalgaltreatedtanks,includingindividualswhowereselectedforprocessingatthe
allocatedsampletimeperiods.Nomortalitiesoccurredinthetanksbyweek9(posttrial
period)inallthreeoystergroups.
Theinitialoystersample(n=30)testednegativeforOsHV-1μVarbyPCR,andwas
negativeforV. aestuarianusbyqPCR,howeverinthegenericVibriospp. PCR,26.7%ofthe
oystersamplifiedaproduct(subsequentlyconfirmedtobeVibriosplendidusbySanger
sequencing).Overall,forthedurationofthetwo‐weektrialthecontroloystershadthe
highestprevalenceofherpesvirus(43.05%) whileoysterstreatedwitheachofthealgal
extracts (carrageenanandfucoidan) hadasignificantlylowerherpesvirusprevalence