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Developmental and Comparative Immunology (2008) 32, 627636

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/devcompimm

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 2007 Available 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 immunological activities in Sydney rock oysters (Saccostrea glomerata). Noradrenaline caused a decrease in most of the immunological parameters tested. Phenoloxidase activities in both whole hemolymph and serum decreased signicantly within the rst 60 min of noradrenaline injection, as did the total frequency of hemocytes in hemolymph, differential hemocyte frequencies, the frequency of phenoloxidase positive cells and phagocytic activity. All of these parameters started to return to normal levels within 120 min. In contrast, the total protein content of hemolymph, which also decreased after noradrenaline injection continued to decline throughout the experimental period. In vitro studies found that superoxide and peroxide production by hemocytes increased in the presence of noradrenaline, but acid phosphatase activity decreased signicantly. Additional experiments showed that noradrenaline secretion was stimulated by altered salinity, altered temperature and physical agitation. This suggests that stressors commonly associated with oyster farming may result in noradrenaline-based stress responses that suppress immunological activity. & 2007 Elsevier Ltd. All rights reserved.

1. Introduction
Hormone-based stress reactions are highly conserved between taxa [1]. The endocrine system has an important role in maintaining homeostasis by regulating behavioral and physiological responses. Many environmental stressors interfere with hormone regulation, affecting biological functions that cause changes to the internal steady state. Invertebrate endocrine systems seem to be more diverse than those of vertebrates [2]. However, many studies have also shown similarities between invertebrate and vertebrate hormonal responses [3,4]. The endocrine systems of

Abbreviations: ACTH, adrenocorticotropin hormone; CRH, corticotrophin-releasing hormone; DMSO, dimethyl sulfoxide; FSW, ltered sea water; L-DOPA, L-3, 4-dihydroxyphenylalanine; 4HA, hydroquinine monomethyl ether; MAC, marine anti-coagulant; MBTH, 3-methyl-2-benzothiazolinone hydrazone; NBT, nitroblue tetrazolium; NA, noradrenaline; OD, optical density. Corresponding author. Tel.: +61 2 98508402. E-mail address: draftos@rna.bio.mq.edu.au (D.A. Raftos). 0145-305X/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2007.10.001

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628 invertebrates are generally composed of neurosecretory cells with few true glands [2]. Invertebrates use a variety of hormones to regulate a diverse array of both physiological and behavioral processes [5]. During stress responses in vertebrates, the hypothalamic pituitaryadrenal axis controls reactions to many external and internal stressors. Corticotrophin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) are the main mediators during these regulatory processes [4]. In molluscs, stress responses are also mediated by CRH and ACTH [6,7]. CRH and ACTH control the release of catecholamines in both vertebrates and invertebrates [7]. In the freshwater snail, Planorbarius corneus, ACTH and CRH cause an increase in adrenaline, noradrenaline (NA) and dopamine levels [8]. These catecholamines have been detected in many invertebrates, such as gastropods (Helix aspersa and Crepidula fornicate), cnidarians (Renilla koellikeri), scallops (Placopecten magellanicus), snails (Biomphalaria glabrata) and oysters (Crassostrea gigas) [915]. The physiological and behavioral functions controlled by catecholamines 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. For example, tyramine and octopamine modulate activities including locomotion and egg laying [24,25]. They have also been reported to regulate some immunological functions, such as nodule formation and phagocytosis, particularly in insects [26,27]. This is part of a growing body of evidence that invertebrate hormones affect immunological activity. Invertebrate immune systems involve innate cellular and humoral responses similar to those that exist in vertebrates [28,29]. Cellular immunity involves phagocytosis, nodulation and encapsulation [30,31], whilst humoral effectors include antimicrobial peptides, reactive oxygen species (ROS), and coagulation and melanization factors [31,32]. Phenoloxidase is the key enzyme in the melanization process. It is synthesized as inactive prophenoloxidase, which is converted to phenoloxidase by serine proteases [30,33,34]. The relationship between catecholamines and molluscan immunity was rst reported by Lacoste et al. [12,3538]. They found that NA modulates the production of ROS and hemocyte phagocytosis in C. gigas [36,38]. Mechanical disturbance caused an increase in NA levels and resulted in decreased immunological activity [12,37]. Other environmental stressors, like salinity and temperature, also reduce immunological activity among oysters, and increased susceptibility to pathogens [35,3942]. The current study investigates the effects of NA on immunological parameters (phagocytosis, phenoloxidase activity, hemocyte frequencies, total protein concentration, superoxide and peroxide production, and acid phosphatase activity) in Sydney rock oysters. We also test the relationship between environmental stress and NA secretion. S. Aladaileh et al. from the Sydney Fish Markets (Sydney, Australia). They were maintained for 2 weeks in aerated aquaria supplied with continuously owing seawater (50 l) at room temperature (25 1C). Oysters were feed regularly with a mix of four marine microalgae (Isochrysis, Pavlova, Thalassiosira weissogii and Tetraselmis, Shellsh Diet 1800, USA). The seawater was collected from the Hawkesbury River, NSW, Australia.

2.2. Noradrenaline injection


NA (Sigma Aldrich, Castle Hill, NSW, Australia) was suspended in sterile ltered seawater (FSW; 0.22 mm ltration; Millipore, North Ryde, NSW, Australia). Oysters were injected with 70 ng NA. Doses were based on the assumption that 3 ml of circulating hemolymph can be collected per oyster and that the total volume of hemolymph, including that in tissues, is approximately 7 ml per oyster (unpublished data). Therefore, 70 ng NA would yield nal concentrations of approximately 10 ng ml1 of hemolymph. Additional groups of 5 control oysters were injected with sterile FSW without NA. The oysters were injected through the shell hinge using 22-gauge needles tted to 1 ml syringes.

2.3. Hemolymph collection and preparation


At different times after injection, 5 oysters from each treatment were removed from aquaria and dried. For cytology, phagocytosis assays and in vitro experiments, hemolymph was withdrawn from the foot sinus directly through a notch in the shell hinge. Oysters were then shucked and the remaining hemolymph was collected from the shell cavity for phenoloxidase and protein assays. Hemolymph samples were immediately placed in polypropylene tubes and held on ice. Serum was obtained by centrifuging whole hemolymph at 700g for 3 min and collecting the supernatant.

2.4. Phenoloxidase assays


Phenoloxidase activity in serum or whole hemolymph was determined spectrophotometrically according to Peters and Raftos [43]. Assays were performed by recording either diphenolase activity with the substrate, L-3,4-dihydroxyphenylalanine (L-DOPA, ICN, Irvine, CA, USA), or monophenolase activity using hydroquinine monomethyl ether (4HA, Fluka, Switzerland). The chromogen, 3-methyl-2benzothiazolinone hydrazone (MBTH, Sigma Aldrich), was added to both substrates. MBTH increases spectrophotometric absorbance at 490 nm by trapping o-quinones. Assays were carried out in 96-well at-bottom microtiter plates (Sarstadt, Technology Park, SA). One hundred microliters of serum or whole hemolymph was added per well followed by the addition of 100 ml of L-DOPA (4 mg ml1 in FSW) or 100 ml 4-HA (5 mM in FSW), both containing 1 mM MBTH. The absorbance of the reaction mixture was measured at 490 nm immediately after the addition of substrates using a Model 550 microplate spectrophotometer (BioRad, Regents Park, NSW, Australia). A second reading was made after the plates had been incubated for 1 h at room temperature. Enzyme

2. Materials and methods


2.1. Oysters
Sydney rock oysters (Saccostrea glomerata) (4060 mm long from the hinge to the frontal edge of shell) were purchased

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Effects of noradrenaline on immunological activity activities are expressed as the change in optical density at 490 nm (OD492). 629 hemocyte types according to the presence or absence of cytoplasmic granules.

2.5. Phenoloxidase cytology


Whole hemolymph collected directly from the foot sinus was immediately mixed with an equal volume of marine anticoagulant (MAC; 0.1 M glucose, 15 mM trisodium citrate, 13 mM citric acid, 10 mM EDTA, 0.45 M NaCl, pH 7.0). Thirty microliter aliquots of the diluted hemolymph were placed on acid alcohol-washed microscope slides coated with polyL-lysine and left to adhere for 10 min. The hemocytes were then stained for phenoloxidase activity. The phenoloxidase stain was prepared in PBS and contained 5 mM 4-HA, 4 mM L-DOPA and 5 mM MBTH. The adherent hemocytes were overlaid with 30 ml of the stain and allowed to stand for 10 min before a coverslip was added. Slides were then sealed, using nail polish, and incubated for a further 30 min at room temperature. An Olympus BH-2 microscope (Olympus, Tokyo, Japan) was used to examine randomly selected elds of view on the slide. A total of 300 hemocytes were examined so that the frequency of phenoloxidase-positive hemocytes (red stained) relative to unstained hemocytes could be determined.

2.8. Total protein content of hemolymph


The total protein content of hemolymph was determined using 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 hemocyte pellets resuspended in 300 ml PBS before being freezethawed twice (80 1C/room temperature). The samples were centrifuged at 5000g for 30 min at 4 1C to remove cellular debris. The resulting hemocyte lysates were used to measure protein content interpolated from a standard curve generated with bovine serum albumen.

2.9. In vitro measurement of superoxide production, hydrogen peroxide and acid phosphatase
Superoxide anion, peroxide and acid phosphatase experiments were conducted in vitro because the timing required for sampling made in vivo experimentation difcult. A volume of 300 ml of whole hemolymph was mixed with equal volumes of NA (150 ng ml1 in FSW) and incubated for 30 min at room temperature. In controls, whole hemolymph was incubated with an equal volume of FSW or without any treatment. After incubation, the hemolymph samples were freeze-thawed twice (80 1C/room temperature). Superoxide production was determined by using nitroblue tetrazolium (NBT). One hundred microliter aliquots of hemolymph lysates were added to wells of 96-well microtiter plates. One hundred microliters of NBT (2 mg ml1 in FSW) was then added to each well and the plates were incubated for 1 h at room temperature. The plates were centrifuged at 300g for 10 min and the supernatants were removed. The formazan salts in each well were washed twice with 70% methanol before being air dried and dissolved in 200 ml dimethyl sulfoxide (DMSO). Absorbance was read at 620 nm. Hydrogen peroxide production was measured by the ferrous oxidationxylenol orange assay [44]. All reagents were from a Peroxidetect kit (Sigma Aldrich). Two hundred microliters of NA-treated or control hemolymph lysates was mixed with 1 ml color reagent. The color reagent was freshly prepared by mixing 100 volumes of 100 mM sorbitol and 125 mM xylenol orange with 1 volume of 25 mM ferrous ammonium sulfate in 2.5 M sulfuric acid. The plates were incubated for 1 h at room temperature. After incubation, absorbance was read at 595 nm and the hydrogen peroxide concentration was calculated using a standard curve obtained by incubating 116 nmol hydrogen peroxide with color reagent. Acid phosphatase activity in NA-treated and control hemolymph was measured using p-nitrophenylphosphate (pNPP) as a chromogenic substrate. Eighty microliters of hemolymph lysates was added to wells of 96-well microtiter plates followed by the addition of 120 ml of pNPP (2.5 mg ml1) to each well. The plates were incubated at 37 1C for 1 h before 50 ml of NaOH (0.05 M) was added to each

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 and mixed with an equal volume of ltered Congo red (Sigma Aldrich; 0.8% in FSW). The suspension was autoclaved at 120 1C for 15 min before being washed twice by centrifugation at 1300g for 5 min and resuspended in 10 ml FSW. To measure phagocytosis, 100 ml of whole hemolymph from NA-injected or FSW-injected oysters (adjusted to 1 106 hemocytes ml1) was placed on glass coverslips (22 22 mm) and incubated in a moist chamber. Hemocytes were allowed to settle and adhere for 20 min at room temperature (25 1C). The supernatants were then removed and the adherent hemocytes were rinsed 5 times with FSW. The coverslips were overlaid with 100 ml of Congo redstained yeast (0.7 106 ml1) and incubated for 30 min at room temperature. The coverslips were washed 10 times with FSW to remove non-phagocytosed yeast cells. They were then inverted over clean microscope slides and sealed with nail polish. A minimum of 200 hemocytes on each coverslip were examined and the number of hemocytes that had phagocytosed one or more yeast was recorded so that the percentage of phagocytic cells could be calculated.

2.7. Total and differential hemocyte counts


A Neubauer hemocytometer was used to determine the total hemocyte frequencies in hemolymph after it had been diluted 1:1 in MAC. Hemocyte monolayers were used to calculate differential hemocyte frequencies for granulocytes and hyalinocytes. Monolayers were prepared by allowing hemocytes to attach to acid alcohol-cleaned slides for 25 min at room temperature. Differential interference contrast microscopy was then used to differentiate between

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630 well to stop the reaction. The plates were then read at 405 nm.
NA + 4HA

S. Aladaileh et al.

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2.10. Stressors
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Oysters were subjected to 3 types of stressextreme temperature, altered salinity and mechanical agitation. For temperature stress, oysters were incubated at 12, 25, 35 and 40 1C for 2 h. Another group of oysters was agitated for 10 min at 300 rpm/min using an MS1 Minishaker (Crown Scientic, NSW, Australia). For salinity stress, oysters were incubated in seawater adjusted to different salinities using deionized water and sea salt (10, 25, 32, 40 ppt) for 2 h at room temperature (25 1C).

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2.11. Noradrenaline assay


Hemolymph from stressed oysters was extracted from the foot sinus and freeze-thawed twice (80 1C/room temperature). The samples were then centrifuged at 5000g for 30 min at 4 1C to remove cellular debris. A NA ELISA kit (IBL, Hamburg, Germany) was then used to determine NA concentrations in hemolymph lysates. According to the manufacturers instructions, 500 ml of hemolymph lysates was added to extraction plates. After extraction, bound NA was eluted using release buffer and transferred to a 96-well ELISA plate. Fifty microliters of NA antiserum was then added into each well and incubated for 2 h. After incubation, the plates were washed and 100 ml of enzyme conjugate was added per well and incubated for 1 h. They were then washed, followed by the addition of 200 ml of substrate solution into each well. The plates were incubated for 40 min at room temperature before the addition of 50 ml of pNPP stop solution per well. The optical densities of each well were measured at 405 nm. A standard curve was generated using a range of NA standards (0, 5, 15, 50, 150, 500 ng ml1).
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2.12. Statistical analysis


All experiments were conducted three times. Data are shown for one representative experiment from each trial. In each experiment, different groups of 5 oysters were tested at each time point. Data were analyzed using the Microsoft Excel package with pop tools (Version 2.7.1). One-way analysis of variance was used to determine the signicance of differences between mean values. Differences were considered to be signicant if Po0.05.

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.

3. Results
3.1. Phenoloxidase activity decreases in response to noradrenaline injection
Hemolymph phenoloxidase activities decreased after the injection of NA. Fig. 1A shows that whole hemolymph phenoloxidase activities (monophenolase and diphenolase) were reduced signicantly (Po0.05) 30 min after NA injection.

Diphenolase activity in whole hemolymph started to decrease within 30 min of NA injection, reaching a minimum of 0.3670.04 OD492 after 60 min compared with 0.6770.03 OD492 for FSW-injected oysters. After 60 min, diphenolase activity started to recover. Whole hemolymph monophenolase activity followed a similar pattern after NA injection. It decreased signicantly (Po0.05) within 30 min of injection. After 60 min, monophenolase activity was 0.2370.04 OD492 compared with 0.4670.04 OD492 for FSW-injected oysters. Monophenolase activity recovered faster than diphenolase and had almost returned to control levels after 120 min. Serum phenoloxidase activities followed a similar trend to those of whole hemolymph (Fig. 1B). Diphenolase activity in serum reached a minimum of 0.2770.04 OD492 60 min after NA injection. Serum monophenolase activity started to decrease immediately after NA injection and reached a

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Effects of noradrenaline on immunological activity minimum of 0.1870.01 OD492 60 min post-injection. As in whole hemolymph, serum monophenolase recovered faster than diphenolase. 631

3.3. Total and differential hemocyte counts


The injection of NA caused a signicant decrease in the total number of hemocytes in whole hemolymph compared with FSW-injected oysters. Fig. 2B shows that the number of hemocytes in NA-injected oysters decreased signicantly within the rst 15 min. At this time, the total number of hemocytes per milliliter was 6.3 105 in NA-injected animals compared with 8.4 105 for FSW-injected oysters (Po0.05). The frequency of hemocytes continued to decline, reaching a minimum after 60 min of 4.0 105 ml1 compared with 8.0 105 ml1 in FSW-injected oysters. There were also signicant decreases in the frequencies of both granulocytes and hyalinocytes (Fig. 2C) in hemolymph after the injection of NA, relative to FSW-injected oysters. Hyalinocyte frequency decreased signicantly in the rst 30 min after injection. Granulocytes responded slower, so that decreases in the frequency of these cells

3.2. Frequency of phenoloxidase-positive hemocytes decreases after noradrenaline injection


Fig. 2A demonstrates that the frequency of phenoloxidasepositive hemocytes decreased signicantly (Po0.05) within 30 min of NA injection (39%74%) relative to FSW-injected oysters (47%72%). After 60 min, the frequency of phenoloxidase-positive hemocytes had dropped to a minimum of 28%76% in NA-injected oysters compared with FSW-injected oysters (41%75%). The frequency of phenoloxidase-positive hemocytes started to increase after 60 min but was still signicantly lower than that of FSW-injected controls after 120 min (Po0.05).

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25 0 30 60 90 time after injection (min.) 120

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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632 became signicant (Po0.05) only after 60 min. The frequencies of both cell types then started to increase in the period 60120 min after NA injection. S. Aladaileh et al.

3.5. Total protein concentration decreases after noradrenaline injection


Fig. 3B shows that the total protein content of hemolymph decreased signicantly compared with FSW-injected oysters within 60 min of injecting NA (Po0.05). Protein concentration continued to decline throughout the experimental period, so that it was only 70% of its starting level after 120 min.

3.4. Phagocytic activity is inhibited by noradrenaline


Fig. 3A reveals that NA injection decreased the phagocytic activity of hemocytes. After 30 min, 32711% of hemocytes from NA-injected oysters had ingested yeast, compared with 4976% of hemocytes from FSW-injected animals (Po0.05). The frequency of phagocytic hemocytes continued to decrease so that after 60 min, 2079% of hemocytes from NA-injected oysters had ingested yeast, compared with 4574% for FSW-injected oysters (Po0.05). Phagocytic activities started to recover within 120 min.

3.6. Peroxide and superoxide anion production, and acid phosphatase activity
Fig. 4A shows that NA increased H2O2 production by hemocytes signicantly (Po0.05) compared with untreated hemocytes. FSW-treated hemocytes did not show a signicant increase in H2O2 production compared with untreated hemocytes. H2O2 concentration was 6.870.09 nmol ml1 among NA-stimulated hemocytes, compared with 5.670.19 and 5.270.3 nmol ml1 for FSW-treated and untreated hemocytes, respectively. A similar response was evident for superoxide anion production (Fig. 4B), even though the superoxide response to NA stimulation was stronger than the response of peroxide production. NA-stimulated hemocytes showed an almost two-fold increase in superoxide anion activity (0.7970.08, OD620), when compared with untreated hemocytes (0.4870.1, OD620) and FSW-treated controls (0.4670.05, OD620) (Po0.05). Fig. 4C demonstrates that the acid phosphatase activity of whole hemolymph was also reduced by in vitro NA treatment. Acid phosphatase activity decreased signicantly relative to both untreated hemocytes and FSW-treated controls (Po0.05). It was 0.0670.01 mg ml1 in NA-treated hemocytes compared with 0.3570.05 and 0.377 0.05 mg ml1 for untreated hemocytes and FSW-treated controls, respectively.

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3.7. Noradrenaline concentrations increase in response to altered temperature, salinity and agitation
Fig. 5A shows that salinity affected the production of NA by oyster hemocytes. NA concentrations in hemolymph lysates increased in response to both high (40 ppt) and low (10 ppt) salinities. NA concentrations were 2 times greater in hemocytes incubated at high salinity (Po0.05) compared with those incubated at 32 and 25 ppt. The response to low salinity was stronger. NA concentrations were 3 times greater in oysters held at 10 ppt than in those incubated at 25 and 32 ppt (Po0.05). Responses to temperature extremes followed a similar pattern to those for salinity. Fig. 5B demonstrates that NA production increased in response to high (40 1C) and low (12 1C) temperature. At 40 1C, NA concentrations were 2-fold greater than in oysters incubated at 25 1C, and 1.7-fold greater than in oysters incubated at 35 1C (Po0.05). Low temperature (12 1C) caused similar increases in NA concentrations relative to 25 and 35 1C (Po0.05). NA production was also stimulated by agitation (Fig. 5C). NA production increased signicantly (Po0.05) in oysters

protein (OD 590 nm)

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Fig. 3 (A) Percentage of hemocytes that had ingested one or more yeast (% phagocytic cells) and (B) total protein concentration 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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Effects of noradrenaline on immunological activity 633

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Fig. 4 (A) Peroxide concentration (nmol ml ), (B) NBT reduction (OD 620 nm) and (C) acid phosphatase activity (mg pNPP ml1) in hemocytes incubated in vitro with 150 ng ml1 noradrenaline, FSW or without any treatment (control). Responses were measured 30 min after exposure. n 5, bars SEM.

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

4. Discussion
There is an urgent need to study the relationship between environmental stress and the immune systems of marine invertebrates, particularly those used in aquaculture. The frequency of infectious diseases in invertebrate aquaculture has increased in the last few decades [45]. Many studies suggest that environmental stressors, such as pollutants, extreme temperature, altered salinity, hypoxia and mechanical disturbance, have negative impacts on invertebrate immune systems leading to increased disease susceptibility [4650]. Recent evidence suggests that molluscs respond to this stress by releasing corticosteroid and catecholamine (noradrenaline (NA) and adrenaline) hormones [12,51] and that these hormones are responsible for suppressing immunological activity.

In the current study, in vivo experiments showed that NA secretion is elevated by environmental stress. Agitation, altered temperature and salinity extremes all caused an increase in the NA concentration of hemolymph. These ndings agree with those of Lacoste et al. [12], who found that in C. gigas the same stressors caused increases in both NA and dopamine concentrations. Mechanical disturbance has also been shown to cause NA and dopamine concentrations to increase in abalone (Haliotis tuberculata) [46]. Our study also indicates that NA can inhibit immune function. We found that injecting NA into oysters at concentrations equivalent to those induced in vivo by stress decreased most of the immunological parameters tested, including phenoloxidase activity. Phenoloxidase activity decreased to a minimum level within 60 min of NA injection. However, activity started to return to normal levels within 120 min, suggesting that NA-mediated effects are transient. Similar decreases in phenoloxidase activity have been reported in the shrimp, Litopenaeus vannamei, after NA injection [52], indicating that NA may affect phenoloxidase activity in a broad range of invertebrate species.

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634 S. Aladaileh et al.

7 6 noradrenaline (ng / ml) 5 4 3 2 1 0 40 32 25 salinity (ppt) 10 10 noradrenaline (ng / ml)

6 5 4 3 2 1 0 40 35 25 temperature (C) 12

8 noradrenaline (ng / ml)

0 before agitation after

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.

In addition to phenoloxidase activity, total hemocyte frequencies also decreased signicantly after NA injection. Again this effect was rapid, reducing hemocyte frequencies by one half within 60 min of NA injection before starting to recover within 120 min. Differential hemocyte counts also varied signicantly after NA injection. Both granulocytes and hyalinocytes decreased in relative frequency, with hyalinocytes being more susceptible to NA injection than granulocytes. These two cell types are major immunological effectors, so their rapid decline is likely to have an overall inhibitory effect on immune function. Similar declines in hemocyte frequency have been reported in many invertebrates as a result of pathogen injection. In the shrimp, Penaeus monodon, hemocytes rapidly settle in the tubule walls of the lymphoid organ before they phagocytose pathogens [53], whereas in the crab, Carcinus maenas, hemocytes become adherent and form numerous small cell clumps in the gills, heart and hepatopancreas as a response to bacterial injection [54]. Evidence to be presented in a subsequent publication indicates that one effect of NA in Sydney rock oysters is to induce the apoptosis of hemocytes.

As well as altering hemocyte frequencies, NA has been shown to affect the phagocytic activities of hemocytes. NA modulates hemocyte functions, such as phagocytosis and associated reactive oxygen species (ROS) production in C. gigas and L. vannamei [36,38,52]. Phagocytosis in C. gigas was inhibited in a dose-dependent manner as a response to NA. Lacoste et al. [36,38] found that this effect on phagocytosis was regulated by b-adrenergic receptors, which are present on hemocyte surfaces. Activation of these receptors may decrease phagocytic ability by modifying cytoskeletal activity and the formation of pseudopodia. The results of the current study suggest that Sydney rock oyster hemocytes possess similar b-adrenergic receptors. Injecting NA into Sydney rock oyster led to decreases in phagocytic activity comparable to those observed in C. gigas. NA injection also inhibited acid phosphatase activity in Sydney rock oyster hemocytes. Similar decreases in the acid phosphatase activity have been demonstrated in many invertebrates under stress conditions [55,56]. Even though the phagocytic and acid phosphatase activities of hemocytes were inhibited by NA, in vitro experiments showed that superoxide anion and peroxide

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Effects of noradrenaline on immunological activity production by Sydney rock oyster hemocytes increased after incubation with NA. The superoxide anion and peroxide experiments were conducted in vitro because it was more logistically expedient. However, the results for superoxide anion and peroxide production contrast studies of C. gigas and L. vannamei. In C. gigas, NA inhibited the production of ROS [38] and in L. vannamei the release of superoxide was decreased after NA treatment. The decrease in superoxide production in L. vannamei was due to the decreases in total hemocyte counts and lower superoxide dismutase activity [52]. These differences suggest that there may be some species-specicity regarding action with NA. Our ability to show that environmental stress stimulates NA release in Sydney rock oysters, and that NA inhibits many immune functions in this species, indicates that there might be a causal relationship between stress and disease susceptibility. The relationship between environmental stress and decreased immune competency has been implied in many invertebrates, such as Mytilus edulis and Crassostrea virginica [57,58]. Our own work has also shown that environmental stress decreases phenoloxidase activity in Sydney rock oysters, and that infection by the protozoan parasite, Marteilia sydneyi, occurs as a result of this immunosuppression [43]. Our next step will be to investigate the relationship between NA and disease susceptibility in Sydney rock oysters. 635
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