aladeioleh et al 2008 effects of nor adrenaline on immunological activity in

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Effects of noradrenaline on immunological activity in

Sydney rock oysters

Saleem Aladaileh, Sham V. Nair, David A. Raftos

Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia

Received 28 June 2007; received in revised form 3 October 2007; accepted 4 October 2007Available online 22 October 2007

KEYWORDS

Sydney rock oyster;

Saccostrea glomerata;

Noradrenaline;

Environmental stress

Abstract

This study investigates the effects of noradrenaline injection on a range of immunologicalactivities in Sydney rock oysters (Saccostrea glomerata). Noradrenaline caused a decrease inmost of the immunological parameters tested. Phenoloxidase activities in both wholehemolymph and serum decreased significantly within the first 60 min of noradrenalineinjection, as did the total frequency of hemocytes in hemolymph, differential hemocytefrequencies, the frequency of phenoloxidase positive cells and phagocytic activity. All ofthese parameters started to return to normal levels within 120 min. In contrast, the total

protein content of hemolymph, which also decreased after noradrenaline injectioncontinued to decline throughout the experimental period. In vitro studies found thatsuperoxide and peroxide production by hemocytes increased in the presence of noradrena-line, but acid phosphatase activity decreased significantly. Additional experiments showedthat noradrenaline secretion was stimulated by altered salinity, altered temperature andphysical agitation. This suggests that stressors commonly associated with oyster farming mayresult in noradrenaline-based stress responses that suppress immunological activity.& 2007 Elsevier Ltd. All rights reserved.

1. Introduction

Hormone-based stress reactions are highly conserved be-tween taxa [1]. The endocrine system has an important rolein maintaining homeostasis by regulating behavioral andphysiological responses. Many environmental stressors inter-fere with hormone regulation, affecting biological functionsthat cause changes to the internal steady state.

Invertebrate endocrine systems seem to be more diversethan those of vertebrates [2]. However, many studies havealso shown similarities between invertebrate and vertebratehormonal responses [3,4]. The endocrine systems of

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0145-305X/$ - see front matter & 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.dci.2007.10.001

Abbreviations: ACTH, adrenocorticotropin hormone; CRH, corti-cotrophin-releasing hormone; DMSO, dimethyl sulfoxide; FSW,filtered sea water; L-DOPA, L-3, 4-dihydroxyphenylalanine; 4HA,hydroquinine monomethyl ether; MAC, marine anti-coagulant;MBTH, 3-methyl-2-benzothiazolinone hydrazone; NBT, nitrobluetetrazolium; NA, noradrenaline; OD, optical density.Corresponding author. Tel.: +61 2 98508402.

E-mail address: [email protected] (D.A. Raftos).

Developmental and Comparative Immunology (2008) 32, 627636
http://dx.doi.org/10.1016/j.dci.2007.10.001mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.dci.2007.10.001


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invertebrates are generally composed of neurosecretorycells with few true glands [2]. Invertebrates use a variety ofhormones to regulate a diverse array of both physiologicaland behavioral processes [5].

During stress responses in vertebrates, the hypothalamicpituitaryadrenal axis controls reactions to many externaland internal stressors. Corticotrophin-releasing hormone(CRH) and adrenocorticotropic hormone (ACTH) are themain mediators during these regulatory processes [4]. Inmolluscs, stress responses are also mediated by CRH andACTH [6,7]. CRH and ACTH control the release of catecho-lamines in both vertebrates and invertebrates [7]. In thefreshwater snail, Planorbarius corneus, ACTH and CRH causean increase in adrenaline, noradrenaline (NA) and dopaminelevels [8]. These catecholamines have been detected inmany invertebrates, such as gastropods (Helix aspersaand Crepidula fornicate), cnidarians (Renilla koellikeri),scallops (Placopecten magellanicus), snails (Biomphalaria

glabrata) and oysters (Crassostrea gigas) [915]. Thephysiological and behavioral functions controlled by cate-cholamines in these species include feeding, ciliary activity,

metamorphosis, respiration, reproduction, locomotion,growth and development [1623].In addition to catecholamines, many other biogenic

amines have been reported among invertebrates. Forexample, tyramine and octopamine modulate activitiesincluding locomotion and egg laying [24,25]. They have alsobeen reported to regulate some immunological functions,such as nodule formation and phagocytosis, particularly ininsects [26,27]. This is part of a growing body of evidencethat invertebrate hormones affect immunological activity.Invertebrate immune systems involve innate cellular andhumoral responses similar to those that exist in vertebrates[28,29]. Cellular immunity involves phagocytosis, nodulationand encapsulation [30,31], whilst humoral effectors include

antimicrobial peptides, reactive oxygen species (ROS), andcoagulation and melanization factors [31,32]. Phenoloxidaseis the key enzyme in the melanization process. It issynthesized as inactive prophenoloxidase, which is con-verted to phenoloxidase by serine proteases [30,33,34].

The relationship between catecholamines and molluscanimmunity was first reported by Lacoste et al. [12,3538].They found that NA modulates the production of ROS andhemocyte phagocytosis in C. gigas [36,38]. Mechanicaldisturbance caused an increase in NA levels and resultedin decreased immunological activity [12,37]. Other environ-mental stressors, like salinity and temperature, also reduceimmunological activity among oysters, and increased susce-

ptibility to pathogens [35,39

42].The current study investigates the effects of NA onimmunological parameters (phagocytosis, phenoloxidaseactivity, hemocyte frequencies, total protein concentration,superoxide and peroxide production, and acid phosphataseactivity) in Sydney rock oysters. We also test the relation-ship between environmental stress and NA secretion.

2. Materials and methods

2.1. Oysters

Sydney rock oysters (Saccostrea glomerata) (4060 mm longfrom the hinge to the frontal edge of shell) were purchased

from the Sydney Fish Markets (Sydney, Australia). They weremaintained for 2 weeks in aerated aquaria supplied withcontinuously flowing seawater (50 l) at room temperature(25 1C). Oysters were feed regularly with a mix of fourmarine microalgae (Isochrysis, Pavlova, Thalassiosira weiss-

flogii and Tetraselmis, Shellfish Diet 1800, USA). Theseawater was collected from the Hawkesbury River, NSW,Australia.

2.2. Noradrenaline injection

NA (Sigma Aldrich, Castle Hill, NSW, Australia) wassuspended in sterile filtered seawater (FSW; 0.22mmfiltration; Millipore, North Ryde, NSW, Australia). Oysterswere injected with 70ng NA. Doses were based on theassumption that 3 ml of circulating hemolymph can becollected per oyster and that the total volume of hemo-lymph, including that in tissues, is approximately 7 ml peroyster (unpublished data). Therefore, 70 ng NA would yieldfinal concentrations of approximately 10 ng ml1 of hemo-lymph. Additional groups of 5 control oysters were injected

with sterile FSW without NA. The oysters were injectedthrough the shell hinge using 22-gauge needles fitted to 1 mlsyringes.

2.3. Hemolymph collection and preparation

At different times after injection, 5 oysters from eachtreatment were removed from aquaria and dried. Forcytology, phagocytosis assays and in vitro experiments,hemolymph was withdrawn from the foot sinus directlythrough a notch in the shell hinge. Oysters were thenshucked and the remaining hemolymph was collected fromthe shell cavity for phenoloxidase and protein assays.

Hemolymph samples were immediately placed in polypro-pylene tubes and held on ice. Serum was obtained bycentrifuging whole hemolymph at 700g for 3 min andcollecting the supernatant.

2.4. Phenoloxidase assays

Phenoloxidase activity in serum or whole hemolymph wasdetermined spectrophotometrically according to Peters andRaftos [43]. Assays were performed by recording eitherdiphenolase activity with the substrate, L-3,4-dihydroxy-phenylalanine (L-DOPA, ICN, Irvine, CA, USA), or monophe-nolase activity using hydroquinine monomethyl ether

(4HA, Fluka, Switzerland). The chromogen, 3-methyl-2-benzothiazolinone hydrazone (MBTH, Sigma Aldrich), wasadded to both substrates. MBTH increases spectrophoto-metric absorbance at 490 nm by trapping o-quinones. Assayswere carried out in 96-well flat-bottom microtiter plates(Sarstadt, Technology Park, SA). One hundred microliters ofserum or whole hemolymph was added per well followed bythe addition of 100 ml of L-DOPA (4 mg ml1 in FSW) or 100 ml4-HA (5mM in FSW), both containing 1 mM MBTH. Theabsorbance of the reaction mixture was measured at 490 nmimmediately after the addition of substrates using a Model550 microplate spectrophotometer (BioRad, Regents Park,NSW, Australia). A second reading was made after the plateshad been incubated for 1 h at room temperature. Enzyme

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activities are expressed as the change in optical density at490nm (OD492).

2.5. Phenoloxidase cytology

Whole hemolymph collected directly from the foot sinus wasimmediately mixed with an equal volume of marine anti-

coagulant (MAC; 0.1M glucose, 15 mM trisodium citrate,13 mM citric acid, 10 mM EDTA, 0.45 M NaCl, pH 7.0). Thirtymicroliter aliquots of the diluted hemolymph were placedon acid alcohol-washed microscope slides coated with poly-L-lysine and left to adhere for 10 min. The hemocytes werethen stained for phenoloxidase activity. The phenoloxidasestain was prepared in PBS and contained 5 mM 4-HA, 4 mML-DOPA and 5 mM MBTH. The adherent hemocytes wereoverlaid with 30 ml of the stain and allowed to stand for10min before a coverslip was added. Slides were thensealed, using nail polish, and incubated for a further 30 minat room temperature. An Olympus BH-2 microscope (Olym-pus, Tokyo, Japan) was used to examine randomly selectedfields of view on the slide. A total of 300 hemocytes were

examined so that the frequency of phenoloxidase-positivehemocytes (red stained) relative to unstained hemocytescould be determined.

2.6. In vitro phagocytosis assay

Phagocytic activity was assessed using bakers yeast,Saccharomyces cerevisiae (Sigma Aldrich), as target cells.Five milligrams of yeast was suspended in 5 ml FSW andmixed with an equal volume of filtered Congo red (SigmaAldrich; 0.8% in FSW). The suspension was autoclaved at120 1C for 15 min before being washed twice by centrifuga-tion at 1300g for 5 min and resuspended in 10 ml FSW.

To measure phagocytosis, 100ml of whole hemolymphfrom NA-injected or FSW-injected oysters (adjusted to1106 hemocytes ml1) was placed on glass coverslips(2222 mm) and incubated in a moist chamber. Hemocyteswere allowed to settle and adhere for 20min at roomtemperature (25 1C). The supernatants were then removedand the adherent hemocytes were rinsed 5 times with FSW.The coverslips were overlaid with 100ml of Congo red-stained yeast (0.7106 ml1) and incubated for 30 min atroom temperature. The coverslips were washed 10 timeswith FSW to remove non-phagocytosed yeast cells. Theywere then inverted over clean microscope slides and sealedwith nail polish. A minimum of 200 hemocytes on eachcoverslip were examined and the number of hemocytes that

had phagocytosed one or more yeast was recorded so thatthe percentage of phagocytic cells could be calculated.

2.7. Total and differential hemocyte counts

A Neubauer hemocytometer was used to determine the totalhemocyte frequencies in hemolymph after it had beendiluted 1:1 in MAC. Hemocyte monolayers were used tocalculate differential hemocyte frequencies for granulo-cytes and hyalinocytes. Monolayers were prepared byallowing hemocytes to attach to acid alcohol-cleaned slidesfor 25 min at room temperature. Differential interferencecontrast microscopy was then used to differentiate between

hemocyte types according to the presence or absence ofcytoplasmic granules.

2.8. Total protein content of hemolymph

The total protein content of hemolymph was determinedusing a BioRad protein assay kit. Hemocytes were collected

by centrifugation at 5000g for 5 min at room temperature.The supernatants were then removed and the hemocytepellets resuspended in 300 ml PBS before being freeze-thawed twice (80 1C/room temperature). The sampleswere centrifuged at 5000g for 30min at 4 1C to removecellular debris. The resulting hemocyte lysates were used tomeasure protein content interpolated from a standard curvegenerated with bovine serum albumen.

2.9. In vitro measurement of superoxide

production, hydrogen peroxide and acid

phosphatase

Superoxide anion, peroxide and acid phosphatase experi-ments were conducted in vitro because the timing requiredfor sampling made in vivo experimentation difficult.A volume of 300 ml of whole hemolymph was mixed withequal volumes of NA (150ng ml1 in FSW) and incubated for30 min at room temperature. In controls, whole hemolymphwas incubated with an equal volume of FSW or without anytreatment. After incubation, the hemolymph samples werefreeze-thawed twice (80 1C/room temperature).

Superoxide production was determined by using nitrobluetetrazolium (NBT). One hundred microliter aliquots ofhemolymph lysates were added to wells of 96-well micro-titer plates. One hundred microliters of NBT (2 mg ml1 in

FSW) was then added to each well and the plates wereincubated for 1 h at room temperature. The plates werecentrifuged at 300g for 10 min and the supernatants wereremoved. The formazan salts in each well were washedtwice with 70% methanol before being air dried anddissolved in 200 ml dimethyl sulfoxide (DMSO). Absorbancewas read at 620nm.

Hydrogen peroxide production was measured by theferrous oxidationxylenol orange assay [44]. All reagentswere from a Peroxidetect kit (Sigma Aldrich). Two hundredmicroliters of NA-treated or control hemolymph lysates wasmixed with 1 ml color reagent. The color reagent was freshlyprepared by mixing 100 volumes of 100mM sorbitol and

125mM xylenol orange with 1 volume of 25mM ferrousammonium sulfate in 2.5 M sulfuric acid. The plates wereincubated for 1 h at room temperature. After incubation,absorbance was read at 595 nm and the hydrogen peroxideconcentration was calculated using a standard curveobtained by incubating 116 nmol hydrogen peroxide withcolor reagent.

Acid phosphatase activity in NA-treated and controlhemolymph was measured using p-nitrophenylphosphate(pNPP) as a chromogenic substrate. Eighty microliters ofhemolymph lysates was added to wells of 96-well microtiterplates followed by the addition of 120 ml of pNPP(2.5mgml1) to each well. The plates were incubated at37 1C for 1 h before 50ml of NaOH (0.05 M) was added to each

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well to stop the reaction. The plates were then readat 405nm.

2.10. Stressors

Oysters were subjected to 3 types of stressextremetemperature, altered salinity and mechanical agitation.

For temperature stress, oysters were incubated at 12, 25, 35and 40 1C for 2 h. Another group of oysters was agitated for10min at 300 rpm/min using an MS1 Minishaker (CrownScientific, NSW, Australia). For salinity stress, oysters wereincubated in seawater adjusted to different salinities usingdeionized water and sea salt (10, 25, 32, 40 ppt) for 2 h atroom temperature (25 1C).

2.11. Noradrenaline assay

Hemolymph from stressed oysters was extracted from thefoot sinus and freeze-thawed twice (80 1C/room tempera-ture). The samples were then centrifuged at 5000g for

30min at 4 1C to remove cellular debris. A NA ELISA kit(IBL, Hamburg, Germany) was then used to determine NAconcentrations in hemolymph lysates. According to themanufacturers instructions, 500ml of hemolymph lysateswas added to extraction plates. After extraction, bound NAwas eluted using release buffer and transferred to a 96-wellELISA plate. Fifty microliters of NA antiserum was thenadded into each well and incubated for 2 h. After incuba-tion, the plates were washed and 100 ml of enzymeconjugate was added per well and incubated for 1 h. Theywere then washed, followed by the addition of 200 ml ofsubstrate solution into each well. The plates were incubatedfor 40 min at room temperature before the addition of 50 mlof pNPP stop solution per well. The optical densities of eachwell were measured at 405 nm. A standard curve wasgenerated using a range of NA standards (0, 5, 15, 50,150, 500 ng ml1).

2.12. Statistical analysis

All experiments were conducted three times. Data areshown for one representative experiment from each trial. Ineach experiment, different groups of 5 oysters were testedat each time point. Data were analyzed using the MicrosoftExcel package with pop tools (Version 2.7.1). One-wayanalysis of variance was used to determine the significance

of differences between mean values. Differences wereconsidered to be significant if Po0.05.

3. Results

3.1. Phenoloxidase activity decreases in response

to noradrenaline injection

Hemolymph phenoloxidase activities decreased after theinjection of NA. Fig. 1A shows that whole hemolymphphenoloxidase activities (monophenolase and diphenolase)were reduced significantly (Po0.05) 30min after NAinjection.

Diphenolase activity in whole hemolymph started todecrease within 30 min of NA injection, reaching a minimumof 0.3670.04 OD492 after 60 min compared with 0.6770.03OD492 for FSW-injected oysters. After 60 min, diphenolase

activity started to recover.Whole hemolymph monophenolase activity followeda similar pattern after NA injection. It decreased signi-ficantly (Po0.05) within 30min of injection. After60min, monophenolase activity was 0.2370.04 OD492compared with 0.4670.04 OD492 for FSW-injectedoysters. Monophenolase activity recovered faster thandiphenolase and had almost returned to control levelsafter 120 min.

Serum phenoloxidase activities followed a similar trend tothose of whole hemolymph (Fig. 1B). Diphenolase activity inserum reached a minimum of 0.2770.04 OD492 60 min afterNA injection. Serum monophenolase activity started todecrease immediately after NA injection and reached a

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0.1

0.3

0.5

0.7

0.8

0 30 60 90 120

time after injection (min.)

OD49

2nm

NA + 4HA

NA + L-DOPA

FSW + 4HA

FSW + L-DOPA


OD492nm

0.1

0.2

0.3

0.4

0.5

0.6

0 30 60 90 120

Fig. 1 Effect on phenoloxidase activities of injecting Sydney

rock oysters with 70 ng noradrenaline (NA) or FSW (control). (A)

Whole hemolymph. (B) Serum. Phenoloxidase activities were

measured using 4HA as a monophenolase substrate and L-DOPA

as a diphenolase substrate. n 5, bars SEM.



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minimum of 0.1870.01 OD492 60 min post-injection. As inwhole hemolymph, serum monophenolase recovered fasterthan diphenolase.

3.2. Frequency of phenoloxidase-positive

hemocytes decreases after noradrenaline injection

Fig. 2A demonstrates that the frequency of phenoloxidase-positive hemocytes decreased significantly (Po0.05) within30 min of NA injection (39%74%) relative to FSW-injectedoysters (47%72%). After 60 min, the frequency of phenolox-idase-positive hemocytes had dropped to a minimum of28%76% in NA-injected oysters compared with FSW-injectedoysters (41%75%). The frequency of phenoloxidase-positivehemocytes started to increase after 60 min but was stillsignificantly lower than that of FSW-injected controls after120min (Po0.05).

3.3. Total and differential hemocyte counts

The injection of NA caused a significant decrease in the totalnumber of hemocytes in whole hemolymph compared withFSW-injected oysters. Fig. 2B shows that the number ofhemocytes in NA-injected oysters decreased significantlywithin the first 15min. At this time, the total number ofhemocytes per milliliter was 6.3 105 in NA-injectedanimals compared with 8.4 105 for FSW-injected oysters(Po0.05). The frequency of hemocytes continued todecline, reaching a minimum after 60 min of 4.0105 ml1

compared with 8.0 105ml1 in FSW-injected oysters.There were also significant decreases in the frequencies

of both granulocytes and hyalinocytes (Fig. 2C) in hemo-lymph after the injection of NA, relative to FSW-injectedoysters. Hyalinocyte frequency decreased significantly inthe first 30 min after injection. Granulocytes respondedslower, so that decreases in the frequency of these cells

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%h

emoc

ytes

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35

45

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g + NAg + FSW

70


0 30 60 90 120


%

PO-positivehemocytes

15

25

35

45

55

0

hem

ocytefrequency(x104/ml)

0

40

80

120NA

FSW

NA

FSW

30 60 90 120


0 30 90 12060

Fig. 2 Effect of injecting Sydney rock oysters with 70 ng of noradrenaline (NA) or FSW (control) on (A) percentage of phenoloxidase

(PO)-positive hemocytes, (B) total hemocyte frequency and (C) differential hemocyte frequencies of hyalinocyte (h) and granulocyte

(g) at various times after the injection. n 5, bars SEM.



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became significant (Po0.05) only after 60min. The fre-quencies of both cell types then started to increase in theperiod 60120 min after NA injection.

3.4. Phagocytic activity is inhibited by

noradrenaline

Fig. 3A reveals that NA injection decreased the phago-cytic activity of hemocytes. After 30min, 32711% ofhemocytes from NA-injected oysters had ingested yeast,compared with 4976% of hemocytes from FSW-injectedanimals (Po0.05). The frequency of phagocytic hemo-cytes continued to decrease so that after 60min, 2079%of hemocytes from NA-injected oysters had ingestedyeast, compared with 4574% for FSW-injected oysters(Po0.05). Phagocytic activities started to recover within120min.

3.5. Total protein concentration decreases after

noradrenaline injection

Fig. 3B shows that the total protein content of hemolymphdecreased significantly compared with FSW-injected oysterswithin 60 min of injecting NA (Po0.05). Protein concentra-tion continued to decline throughout the experimentalperiod, so that it was only 70% of its starting level after120min.

3.6. Peroxide and superoxide anion production,

and acid phosphatase activity

Fig. 4A shows that NA increased H2O2 production byhemocytes significantly (Po0.05) compared with untreatedhemocytes. FSW-treated hemocytes did not show a signifi-cant increase in H2O2 production compared with untreatedhemocytes. H2O2 concentration was 6.870.09 nmol ml

1

among NA-stimulated hemocytes, compared with5.670.19 and 5.270.3 nmol ml1 for FSW-treated anduntreated hemocytes, respectively.

A similar response was evident for superoxide anionproduction (Fig. 4B), even though the superoxide responseto NA stimulation was stronger than the response ofperoxide production. NA-stimulated hemocytes showed analmost two-fold increase in superoxide anion activity(0.7970.08, OD620), when compared with untreatedhemocytes (0.4870.1, OD620) and FSW-treated controls(0.4670.05, OD620) (Po0.05).

Fig. 4C demonstrates that the acid phosphatase activityof whole hemolymph was also reduced by in vitro NAtreatment. Acid phosphatase activity decreased significantlyrelative to both untreated hemocytes and FSW-treatedcontrols (Po0.05). It was 0.0670.01mg ml1 in NA-treated

hemocytes compared with 0.3570.05 and 0.3770.05mg ml1 for untreated hemocytes and FSW-treatedcontrols, respectively.

3.7. Noradrenaline concentrations increase in

response to altered temperature, salinity and

agitation

Fig. 5A shows that salinity affected the production of NA byoyster hemocytes. NA concentrations in hemolymph lysatesincreased in response to both high (40 ppt) and low (10 ppt)salinities. NA concentrations were 2 times greater inhemocytes incubated at high salinity (Po0.05) compared

with those incubated at 32 and 25 ppt. The response to lowsalinity was stronger. NA concentrations were 3 timesgreater in oysters held at 10 ppt than in those incubatedat 25 and 32 ppt (Po0.05).

Responses to temperature extremes followed a similarpattern to those for salinity. Fig. 5B demonstrates that NAproduction increased in response to high (401C) and low(12 1C) temperature. At 401C, NA concentrations were2-fold greater than in oysters incubated at 251C, and1.7-fold greater than in oysters incubated at 35 1C (Po0.05).Low temperature (121C) caused similar increases in NAconcentrations relative to 25 and 35 1C (Po0.05).

NA production was also stimulated by agitation (Fig. 5C).NA production increased significantly (Po0.05) in oysters

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120


protein(OD590nm)

00.3

0.4

0.5

0.6

0.7 FSW

NA

30 60 90


%p

hagocyticcells

0

20

40

60

80

0

FSW

NA

30 60 90 120

Fig. 3 (A) Percentage of hemocytes that had ingested one or

more yeast (% phagocytic cells) and (B) total protein concen-

tration of whole hemolymph at various times after oysters had

been injected with either 70 ng noradrenaline (NA) or FSW.

n 5, bars SEM.



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subjected to agitation for 10 min. The concentration of NA inthese oysters was 3 times greater than in undisturbedoysters.

4. Discussion

There is an urgent need to study the relationship betweenenvironmental stress and the immune systems of marineinvertebrates, particularly those used in aquaculture. Thefrequency of infectious diseases in invertebrate aquaculturehas increased in the last few decades [45]. Many studiessuggest that environmental stressors, such as pollutants,extreme temperature, altered salinity, hypoxia and mechan-ical disturbance, have negative impacts on invertebrateimmune systems leading to increased disease susceptibility[4650]. Recent evidence suggests that molluscs respond tothis stress by releasing corticosteroid and catecholamine(noradrenaline (NA) and adrenaline) hormones [12,51]and that these hormones are responsible for suppressingimmunological activity.

In the current study, in vivo experiments showed that NAsecretion is elevated by environmental stress. Agitation,altered temperature and salinity extremes all caused anincrease in the NA concentration of hemolymph. Thesefindings agree with those of Lacoste et al. [12], who foundthat in C. gigas the same stressors caused increases in bothNA and dopamine concentrations. Mechanical disturbance

has also been shown to cause NA and dopamine concentra-tions to increase in abalone (Haliotis tuberculata) [46].Our study also indicates that NA can inhibit immune

function. We found that injecting NA into oysters atconcentrations equivalent to those induced in vivo by stressdecreased most of the immunological parameters tested,including phenoloxidase activity. Phenoloxidase activitydecreased to a minimum level within 60 min of NA injection.However, activity started to return to normal levels within120 min, suggesting that NA-mediated effects are transient.Similar decreases in phenoloxidase activity have beenreported in the shrimp, Litopenaeus vannamei, after NAinjection [52], indicating that NA may affect phenoloxidaseactivity in a broad range of invertebrate species.

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0

1

2

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8

control

peroxide(nmole/ml)

0

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control

superoxide(OD620nm)

FSWNA FSWNA

0

0.1

0.2

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0.4

0.5

control

acidphosphatase(g/ml)

FSWNA

Fig. 4 (A) Peroxide concentration (nmol ml1), (B) NBT reduction (OD 620 nm) and (C) acid phosphatase activity (mgpNPPml1) in

hemocytes incubated in vitro with 150ng ml1 noradrenaline, FSW or without any treatment (control). Responses were measured

30 min after exposure. n 5, bars SEM.



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In addition to phenoloxidase activity, total hemocytefrequencies also decreased significantly after NA injection.Again this effect was rapid, reducing hemocyte frequenciesby one half within 60 min of NA injection before starting torecover within 120 min. Differential hemocyte counts alsovaried significantly after NA injection. Both granulocytesand hyalinocytes decreased in relative frequency, withhyalinocytes being more susceptible to NA injection than

granulocytes. These two cell types are major immunologicaleffectors, so their rapid decline is likely to have an overallinhibitory effect on immune function. Similar declines inhemocyte frequency have been reported in many inverte-brates as a result of pathogen injection. In the shrimp,Penaeus monodon, hemocytes rapidly settle in the tubulewalls of the lymphoid organ before they phagocytosepathogens [53], whereas in the crab, Carcinus maenas,hemocytes become adherent and form numerous smallcell clumps in the gills, heart and hepatopancreas as aresponse to bacterial injection [54]. Evidence to bepresented in a subsequent publication indicates that oneeffect of NA in Sydney rock oysters is to induce the apoptosisof hemocytes.

As well as altering hemocyte frequencies, NA has beenshown to affect the phagocytic activities of hemocytes. NAmodulates hemocyte functions, such as phagocytosis andassociated reactive oxygen species (ROS) production inC. gigas and L. vannamei [36,38,52]. Phagocytosis inC. gigas was inhibited in a dose-dependent manner as aresponse to NA. Lacoste et al. [36,38] found that this effecton phagocytosis was regulated by b-adrenergic receptors,

which are present on hemocyte surfaces. Activation of thesereceptors may decrease phagocytic ability by modifyingcytoskeletal activity and the formation of pseudopodia. Theresults of the current study suggest that Sydney rock oysterhemocytes possess similar b-adrenergic receptors. InjectingNA into Sydney rock oyster led to decreases in phagocyticactivity comparable to those observed in C. gigas. NAinjection also inhibited acid phosphatase activity in Sydneyrock oyster hemocytes. Similar decreases in the acidphosphatase activity have been demonstrated in manyinvertebrates under stress conditions [55,56].

Even though the phagocytic and acid phosphataseactivities of hemocytes were inhibited by NA, in vitroexperiments showed that superoxide anion and peroxide

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40 32 25 10

salinity (ppt)

noradrenaline(ng/ml)

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35 25 12

temperature (C)

noradrenali

ne(ng/ml)

agitation

0

2

4

6

8

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before after

noradrenaline(ng/ml)

Fig. 5 Noradrenaline concentrations (ng ml1) in oysters subjected to (A) altered salinity, (B) altered temperature or (C) 10 min of

physical agitation. n 5, bars SEM.



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production by Sydney rock oyster hemocytes increased afterincubation with NA. The superoxide anion and peroxideexperiments were conducted in vitro because it was morelogistically expedient. However, the results for superoxideanion and peroxide production contrast studies of C. gigasand L. vannamei. In C. gigas, NA inhibited the production ofROS [38] and in L. vannamei the release of superoxide wasdecreased after NA treatment. The decrease in superoxideproduction in L. vannamei was due to the decreases in totalhemocyte counts and lower superoxide dismutase activity[52]. These differences suggest that there may be somespecies-specificity regarding action with NA.

Our ability to show that environmental stress stimulatesNA release in Sydney rock oysters, and that NA inhibits manyimmune functions in this species, indicates that there mightbe a causal relationship between stress and diseasesusceptibility. The relationship between environmentalstress and decreased immune competency has been impliedin many invertebrates, such as Mytilus edulis and Crassos-trea virginica [57,58]. Our own work has also shown thatenvironmental stress decreases phenoloxidase activity in

Sydney rock oysters, and that infection by the protozoanparasite, Marteilia sydneyi, occurs as a result of thisimmunosuppression [43]. Our next step will be to investigatethe relationship between NA and disease susceptibility inSydney rock oysters.

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