The temporary modulation of tyramine on immune responses, carbohydrate metabolism, and catecholamines in Macrobrachium rosenbergii

Fish and Shellfish Immunology 98 (2020) 1–9

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The temporary modulation of tyramine on immune responses, carbohydrate metabolism, and catecholamines in Macrobrachium rosenbergii

T

Chin-Chyuan Chang1, Hsin-Wei Kuo1, Chang-Chi Liu, Winton Cheng∗ Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan, ROC

A R T I C LE I N FO

A B S T R A C T

Keywords: Tyramine Macrobrachium rosenbergii Neuroimmunology Pharmacological modulation Neuroendocrine hormones Immune-endocrine

Tyramine (TA), a biogenic monoamine, plays various important physiological roles including immunological regulation in invertebrates. In this study, the effects of TA on the regulation of immune resistance, carbohydrate metabolism and biogenic monoamine, as well as its signaling pathway in Macrobrachium rosenbergii were determined. Results showed that total haemocyte count, hyaline cells, semigranular cells, and phenoloxidase activity per 50 μL of haemolymph and per granulocyte (the sum of semigranular and granular cells) at 0.5 h as well as phagocytic activity and clearance efficiency to Lactococcus garvieae at 1 h of prawn injected with TA at 1 nmol prawn−1 significantly increased, but the significantly decreased plasma lysozyme activity, phagocytic activity, clearance efficiency, and haemolymph glucose and dopamine were observed in prawn injected with TA at 10 nmol prawn−1 for 0.5 h. Respiratory bursts and haemolymph lactate in two TA-injection treatments at 0.5 h and 0.5–1 h, respectively, were significantly higher than those of the saline control, and in addition, TA depressed dopamine release in a dose-dependent manner after 0.5 h of TA injection. All the examined parameters returned to control levels after prawn injected with TA for 2 h. The inhibited effect of TA (at 10 nmol prawn−1 injection) on the phagocytic activity and clearance efficiency to pathogens was blocked by prazosin (an α1 adrenoceptors antagonist). For prawn received TA for 1 h then challenged with Lactococcus garvieae at 2 × 105 colony-forming units prawn−1, the survival ratio of TA 1 nmol prawn−1-injected prawn significantly increased by 20%, compared to the saline-challenged control or TA 10 nmol prawn−1-injected prawn after 144 h of challenge. These results suggested that the level of dopamine release suppression regulated by TA resulted in the immunoenhancing or immunosuppressive effects in prawn, and the signaling pathways of TA in mediating immune function were through octopamine (OA)/TA receptors.

1. Introduction In crustacean, catecholamines (CAs) were reported to be involved in several physiological and immunological functions regulation and behavior control [1–3]. The primary response of the stressed crustacean is the release of CAs, subsequently followed with the secondary responses of induction of hyperglycemia and suppression of immunity [1,3]. In insect, Monastirioti [4] reviewed two pathways of CA biosynthesis from tyrosine to dopamine and to octopamine, respectively. The conversion of tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA) was catalyzed by tyrosine hydroxylase then followed with the formation of dopamine (DA) and norepinephrine (NE) [5–7], and however, tyrosine catalyzed by tyrosine decarboxylase resulted in the formation of tyramine (TA), which was the precursor for octopamine (OA) [8]. In mollusk, the CA biosynthesis had been identified in haemocytes as a so-called “immune-

mobile-brain” [9,10], acting as revealing in macrophage in vertebrates [11], and the similar neuroendocrine-immune regulatory network was reported in haemocytes of Litopenaeus vannamei [12–14]. In Macrobrachium rosenbergii, the CA release had been assessed when they exposed to hypothermal stress [15,16], and the impact of CAs such as DA [16,17], NE [18,19], OA [20] on immunocompetence had been reported. Therefore, the two CA biosynthesis pathways might play crucial role in mediating immunocompetence in crustacean as shown in insect [4]. The applications of antagonists were conducted in M. rosenbergii to estimate the signaling transduction of NE [19,20] and DA [16] on immunological responses respectively through adrenoceptors and dopamine receptors, belonging to the superfamily of G protein-coupled receptors (GPCRs). OA and TA, sharing structural similarity, exert their activities by binding to G-protein-coupled receptors (GPCRs) [21,22]

∗

Corresponding author. E-mail address: [email protected] (W. Cheng). 1 These authors contributed equally as the first author to this work. https://doi.org/10.1016/j.fsi.2019.12.091 Received 10 September 2019; Received in revised form 25 December 2019; Accepted 28 December 2019 Available online 03 January 2020 1050-4648/ © 2020 Elsevier Ltd. All rights reserved.

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tons of aerated fresh water at 28 ± 1 °C for 2 weeks before experimentation. Only healthy prawn in the intermolt stage (stage C) were used for this study. The molt stage was determined according to retraction of the epithelium within the setal base interface of the antennal scale under a stereomicroscope [34]. Six studies were conducted. For the susceptibility experiment (1), test or control group comprised 10 prawn each in triplicate. To determine immunological parameters (2), phagocytic activity and the clearance efficiency (3), glucose and lactate (4), DA, NE and OA (5), and pharmacological modulation of the clearance efficiency and phagocytic activity (6), test or control group consisted of six prawn rearing each in 20-L PVC tank containing 10 L of aerated fresh water as six replicates. No significant difference in weight was observed among the treatments. Prawns were fed twice daily with a formulated prawn diet (Grobest Feeds, Pingtung, Taiwan) during acclimation and the experiments, and the water temperature was maintained at 28 ± 1 °C, and pH at 7.1–7.5.

then activate a second-messenger cascade, and furthermore, they differed in a variety of physiological and behavioral processes [23]. Most members of the conserved family of seven transmembrane GPCRs are activated by both OA and TA, and those differ in affinity for TA and/or OA, location and action, and pharmacology [24]. Evans and Maqueira [25] indicated three classes of octopaminergic receptors including αadrenergic-like (OAα-R), β-adrenergic-like (OAβ-R), and octopaminergic/tyraminergic (OA/TA-R) or tyraminergic (TA-R) receptors, and Farooqui [24] proposed that OA/TA-R might be further classified into TA receptor type 1 and 2, and those were therefore concluded into four families of OA receptors and TA receptors. In M. rosenbergii, ReyesColón et al. [26] identified a putative OA/TA receptor in ganglia, and its sequence closely clustered with the OA/TA receptor family in insects. These implied that the OA and TA signaling transduction pathways might also present in crustacean. Evans and Maqueira [25] indicated that OA and TA receptors were pharmacologically similar to vertebrate adrenoceptors, and following the concept, the immunocompetence modulated by OA in M. rosenbergii [20] and TA in Litopenaeus vannamei [27] through signaling transduction were estimated with the traditional adrenergic antagonists. To combat the potential invading microbe, shrimps rely on the effective innate immune system, and the major immune reactions occur in haemolymph, where three different types of haemocytes can be observed and defined as the hyaline, granular and semigranular haemocytes [28]. The prophenoloxidase (proPO) system, storing in granulocytes as an inactive form, serves an important role as a non-selfrecognition system in innate immune responses, and accompanies cellular responses via hemocyte attraction and inducing phagocytosis, melanization, cytotoxic reactant production, particle encapsulation, and the formation of nodules and capsules [29,30]. In addition, reactive oxygen species (ROS) including superoxide anions (O2−), hydrogen peroxide (H2O2), singlet oxygen, and the hydroxyl radical (OH−) are produced known as respiratory bursts (RBs) to eliminate invading microorganisms during phagocytosis [31], and the homeostasis was maintained by antioxidant system such as superoxide dismutase and glutathione peroxidase [32]. For estimating immunocompetence, PO activity, RBs, phagocytic activity, and the clearance efficiency were suggested as health indicators [33], and these indicators were successfully applied to evaluate the potential influence of CAs on immunocompetence of M. rosenbergii [15–20]. The giant freshwater prawn, M. rosenbergii, is a commercially important cultured freshwater prawn, and consistently encounters various environmental stressors in intensive cultivation resulting in severe impact on economy. Therefore, to clarify the linkage between the stressinduced neuroendocrine and immunocompetence is of primary concern in disease prevention of prawn cultivation. The aims of present study were to examine the effect of TA on the susceptibility of M. rosenbergii against Lactococcus garvieae infection, and the immune responses of prawn injected with TA. Immune parameters of the total haemocyte count (THC), differential haemocyte count (DHC), PO activity, RBs, lysozyme activity, and the clearance efficiency and phagocytic activity of prawn against L. garvieae were used as indicators. Furthermore, plasma glucose and lactate levels, and haemolymph DA, NE and OA levels were used to evaluate the effects of TA on physiologic and neuroendocrinologic functions, and the various adrenoceptor antagonists were used to evaluate the potential modulation of TA on the clearance efficiency and phagocytic activity of prawn against L. garvieae by their pharmacological inhibition.

2.2. Lactococcus garvieae The bacterium, L. garvieae, isolated from diseased M. rosenbergii (Pingtung, Taiwan), which displayed symptoms of an opaque and whitish musculature, was used in the present study [35,36]. Stocks were plated on tryptic soy agar (TSA; 211825, Difco, USA) for 24 h at 28 °C before being transferred to 10 mL tryptic soy broth (TSB; 21152, Difco, USA). After 24 h of cultivation at 28 °C, the bacterium was harvested by centrifugation at 7155 ×g for 15 min at 4 °C. The supernatant was removed, and the bacterial pellet was suspended in a saline solution at concentrations of 107 and 109 colony-forming units (cfu) mL−1 as respective stock bacterial suspensions for the susceptibility study and for the phagocytic activity and clearance efficiency studies. 2.3. Effect of tyramine on the susceptibility of prawn to L. garvieae Tyramine (1099914, Sigma-Aldrich, USA) was dissolved in sterile saline to concentrations of 5 × 10−4 and 5 × 10−5 mol L−1 as TA stock solution. Initially, 20 μL of a TA solution was injected into the ventral sinus of the cephalothorax of individual M. rosenbergii (9.2 ± 0.5 g) to reach respective doses of 1 and 10 nmol prawn−1. After 1 h of TA injection, 20 μL of a bacterial suspension (107 cfu mL−1) was injected into the ventral sinus of the cephalothorax resulting in 2 × 105 cfu prawn−1 for the challenge test. Prawn that received 20 μL of sterile saline and then L. garvieae at 2 × 105 cfu prawn−1 served as the challenged controls, and those received TA at 10 nmol prawn−1 and then were injected with 20 μL of sterile saline served as the unchallenged control (Table 1). Test and control prawn (10 prawn aquarium−1) were kept in 60-L glass aquaria containing 40 L of fresh water at 28 °C. Therefore, there were four treatments in total. Each treatment was conducted with 30 prawn. The experiment lasted 168 h. 2.4. Effects of tyramine on immune parameters of M. rosenbergii Prawn (12.3 ± 0.7 g) was individually injected with 5 × 10−4 or 5 × 10−5 mol L−1 of a TA solution into the ventral sinus of the cephalothorax to reach respective doses of 1 and 10 nmol prawn−1. Prawn received 20 μL of saline served as the control. There were three treatments (saline (0), 1, and 10 nmol prawn−1) with five sampling times (0.5, 1, 2, 4, and 8 h) for determination of immune parameters in which the lysozyme activity was only determined at 0.5 and 1 h. Six prawn from each treatment and time were used for the studies. In addition, another six prawn with no treatment were used as the initial group. At the beginning (0 h) and at 0.5, 1, 2, 4 and 8 h after the injection, haemolymph was withdrawn from the ventral sinus of each prawn into a 1-mL sterile syringe (25 gauge), and was diluted 10-fold with

2. Materials and methods 2.1. Macrobrachium rosenbergii Prawn, M. rosenbergii, obtained from an aquafarm of National Pingtung University of Science and Technology (Pingtung, Taiwan), were acclimated in an indoor concrete pond (5 × 5 × 1 m) with 12 2

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Table 1 Effect of tyramine on the survival ratio of Macrobrachium rosenbergii challenged with Lactococcus garvieae. Bacterial dose (cfu prawn−1)

saline 2 × 105 2 × 105 2 × 105

Tyramine (nmol prawn−1)

10 saline 1 10

No. of prawn

30 30 30 30

Survival ratio after challenge (hrs) 24

48

72

96

120

144

168

100 93.3 ± 5.7a 96.7 ± 5.7a 96.7 ± 5.7a

100 86.7 ± 5.7a 90.0 ± 0.0a 83.3 ± 5.7a

100 70.0 ± 0.0b 86.7 ± 5.7a 73.3 ± 11.5ab

100 53.3 ± 5.7b 76.7 ± 5.7a 56.7 ± 5.7b

100 46.7 ± 5.7b 70.0 ± 0.0a 46.7 ± 5.7b

100 46.7 ± 5.7b 66.7 ± 5.7a 46.7 ± 5.7b

100 46.7 ± 5.7b 66.7 ± 5.7a 46.7 ± 5.7b

Data in the same column with different letters are significantly different (p < 0.05) among treatments. Values are mean ± S.E.

of a sterile anticoagulant solution. This mixture was divided into two equal subsamples: one to measure phagocytic activity and the other to measure the clearance efficiency. Phagocytic activity was evaluated as previously described by Weeks-Perkins et al. [43]. Briefly, 200 μL of the haemolymph mixture was mixed with 200 μL of 0.1% paraformaldehyde for 30 min at 4 °C to fix the haemocytes. It was then centrifuged at 400 ×g and 4 °C, washed, and resuspended in 0.4 mL of a saline solution. A 50-μL sample of the suspension was spread on a glass slide. The slide was placed in a cytospin centrifuge (Model Cytospin 3, Shandon, UK) and centrifuged at 113 ×g for 3 min. The slide was then air-dried, stained with Liu's staining method, and observed under a light microscope. In total, 200 haemocytes were counted, and haemocytes engulfing stained bacteria were considered phagocytic haemocytes. Phagocytic activity, defined as the percentage phagocytosis (PR), was expressed as:

anticoagulant solution (0.114 M trisodium citrate and 0.1 M sodium chloride, at pH 7.45, osmolality adjusted with glucose to 490 mOsm kg−1). A drop of the anticoagulant-haemolymph mixture was loaded in a haemocytometer to enumerate the THC and DHC (Leica DMIL, Leica Microsystems, Wetzlar, Germany). One milliliter of the haemolymph mixture was centrifuged at 400 ×g and 4 °C for 20 min, and the supernatant as plasma was used for the lysozyme activity assay. The haemocyte pellet was washed twice with cacodylate buffer (0.01 M sodium cacodylate, 0.45 M sodium chloride, 0.01 M calcium chloride, and 0.26 M magnesium chloride; pH 7.0). The resultant haemocyte pellet was then used for the PO activity assay. Another 100 μL of the haemolymph mixture was used for the RB analysis. According to the method of Mason [37] and Hernández-López et al. [38], PO activity of haemocytes was spectrophotometrically measured at 490 nm by recording the formation of dopachrome produced from L3,4-dihydroxyphenylalanine (L-DOPA; D-9628, Sigma-Aldrich, USA). LDOPA and trypsin respectively served as the substrate and elicitor. The procedure was formerly described in detail by Li et al. [17]. The optical density (OD) was expressed as dopachrome formation in haemocytes per 50 μL of haemolymph. PO activity was further estimated with the amount of 107granulocytes (the sum of GCs and SGCs) because the proPO system was synthesised and localized in granulocytes. As described previously [39,40], RBs of haemocytes were quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion (O2−) formation. Details of the measurements were previously described [17]. The OD at 630 nm was measured in triplicate reactions using a microplate reader (Model VERSAmax, Molecular Devices, Sunnyvale, CA, USA). RBs are expressed as NBT reduction in haemocytes per 10 μL of haemolymph or per 107 haemocytes. Plasma lysozyme activity was modified as described by Ellis [41] and Obach et al. [42]. Briefly, 10 μL of individual plasma was mixed with 200 μL of a Micrococcus luteus (M3770, Sigma-Aldrich, USA) suspension at 0.2 mg mL−1 in 0.05 M sodium phosphate buffer (pH 6.2). The mixture was incubated at 27 °C, and its OD was detected after 1 and 6 min at 530 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader. One unit of lysozyme activity was defined as the amount of enzyme producing a decrease in absorbance of 0.001 min−1 mL−1 serum. Lysozyme concentrations were calculated from a standard curve of known lysozymes from chicken egg white (L4631, Sigma-Aldrich, USA) concentrations.

PR = [(phagocytic haemocytes) (total haemocytes)

−1

] × 100

The clearance efficiency was measured following the method of Adams [44]. The haemolymph mixture was further diluted with a sterile saline solution. Three 50-μL portions of each diluted haemolymph sample were spread on separate TSA plates and incubated at 28 °C for 18 h before the colonies were counted using a colony counter. The number of colonies of prawn receiving saline was expressed as the control group, and the numbers of colonies of prawn receiving TA after 0.5, 1, 2, 4 and 8 h were expressed as the test groups. The clearance efficiency toward L. garvieae, defined as the percentage inhibition (PI), was calculated as: PI = 100 − [(cfu in test group) (cfu in control group)−1] × 100 2.6. Estimation of glucose and lactate of M. rosenbergii After prawn received saline or TA, haemolymph was collected and treated as described above except for the osmolality of anticoagulant solution was adjusted with NaCl (0.114 M trisodium citrate and 0.1 M sodium chloride, at pH 7.45, osmolality adjusted with NaCl to 490 mOsm kg−1). Glucose concentrations of plasma were measured using a coupled glucose oxidase and peroxidase reaction with the Glucose kit (GL2623, Randox, UK). The OD at 505 nm was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader, and glucose concentrations were calculated from the standard curve of known glucose concentrations. Lactate concentrations of plasma were measured using the colorimetric lactate oxidase and peroxidase method with a Lactate kit (BXC0621A, Fortress diagnostic, UK). The OD at 550 nm was measured using an ELISA plate reader, and lactate concentrations were calculated from the standard curve of known lactate concentrations.

2.5. Phagocytic activity and clearance efficiency against L. garvieae in M. rosenbergii Prawn received saline or TA as described above. Tests were carried out on six prawn (replicates) at each treatment and sampling time. For phagocytic activity and the clearance efficiency assay, 20 μL of a bacterial suspension (109 cfu mL−1) resulting in 2 × 107 cfu prawn−1 was injected into the ventral sinus of each prawn after receiving saline or two dosages of TA at 0.5, 1, 2, 4 and 8 h. After injection of the bacterial suspension, the prawn were kept for 90 min in a separate tank containing 40 L of fresh water at 28.0 ± 0.5 °C. Then, 200 μL of haemolymph was collected from the ventral sinus and mixed with 200 μL

2.7. Estimation of DA, NE and OA of M. rosenbergii Prawn received saline or TA for 0, 0.5 and 1 h, and then haemolymph was collected as described above for DA, NE and OA determination. The haemolymph was diluted 5-fold with anticoagulant solution (0.114 M trisodium citrate and 0.1 M sodium chloride, at pH 7.45, 3

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osmolality adjusted with glucose to 490 mOsm kg−1), and then was centrifuged at 10000 ×g for 15 min at 4 °C. The supernatant was used to measure DA, NE and OA levels. Manual and automated enzyme immunoassays were performed for the in vitro diagnostic quantitative determination of DA and NE in haemolymph using a 2-CAT (N-D) Research ELISA™ (E−5500, LDN, Germany). This solid-phase ELISA is based on the sandwich principle according to the manufacturer's instructions, and the determinations were modified following Mapanao et al. [48]. Briefly, for both DA and NE determinations, 100 μL each of haemolymph supernatant mixture was added into the Extraction Plates. After extraction, bound DA or NE was eluted using release buffer and then transferred to Dopamine Norepinephrine Microtiter Strips. Fifty microliters of Dopamine or Norepinephrine Antiserum was then added to each well and incubated at 4 °C overnight. After incubation, the plate was washed, and 100 μL of Enzyme Conjugate was added into each well and incubated at room temperature for 30 min. The reaction wells were then washed, followed by the addition of 100 μL of substrate solution into each well. The plate was placed in dark for 30 min at room temperature before the addition of 100 μL Stop Solution. The optical density was measured at 450 nm using ELISA reader (SPECTRAmax 190, Molecular Devices). The sample concentrations were directly determined from the standard curve. The concentration is expressed in nmole L−1. OA level in haemolymph was measured following the method of Châtel et al. [45] with slight modification. For OA determination, flatbottom 96-well ELISA plates (SpectraPlate™-96HB, PerkinElmer, Massachusetts, USA) were coated with 100 μL well−1 haemolymph supernatant (5 μg protein) and incubated for 3 h under gentle shaking at room temperature, then was washed three times with 150 μL washing buffer (0.1% Tween 20 in PBS). One hundred microliter of blocking buffer (3% BSA in PBS) was then added to each well and incubated at 4 °C overnight. After incubation, the plate was washed with 150 μL washing buffer three times, and 100 μL of the anti-OA polyclonal antibody (1:1000 in dilution buffer contained 0.3% BSA in PBS; ab37092, Abcam, France) was added into each well and incubated at room temperature for 1 h under gentle shaking. The reaction wells were then washed three times with 150 μL washing buffer, followed by the addition of 100 μL of the peroxidase-conjugated secondary antibody (1:10000 dilution; A0545, Sigma-Aldrich, USA) into each well, and incubated at room temperature for 3h under gentle shaking. The plate was then washed three times with 150 μL washing buffer, and 100 μL 3,3,5,5-tetramethylbenzidine (T8665, Sigma-Aldrich, USA) were added to each well. After 20 min of incubation under shaking at room temperature, the reaction was stopped by addition of 10 μL stopping buffer (1 M H2SO4 in distilled water). The optical density was measured at 450 nm using ELISA reader (SPECTRAmax 190, Molecular Devices). Each sample was executed in triplicate, and all steps were performed under dim light. The concentration is expressed in μmole L−1.

antagonists at 50 nmol prawn−1. Prawn that received 20 μL of saline served as the negative controls, and those received a single injection of TA at 10 nmol prawn−1 served as the positive controls. Six treatments were conducted including saline, TA + saline, TA + Phe, TA + Pra, TA + Prop, and TA + Meto. The clearance efficiency and phagocytic activity were determined at 0.5 h after the injection as described above. Each treatment comprised of six prawns. Therefore, 36 prawn were used for the phagocytic activity and clearance efficiency assays. 2.9. Statistical analysis A one-way analysis of variance (ANOVA) was used to analyze the data. When the ANOVA identified differences among groups, a multiple-comparisons (Duncan) test was conducted to compare significant differences among treatments using the SAS computer software (SAS Institute, Cary, NC, USA). Percent data were normalized using an arcsine-transformation before analysis. A statistically significant difference required that p < 0.05. 3. Results 3.1. Effects of TA on the susceptibility of M. rosenbergii to L. garvieae All of the unchallenged control prawn received TA at 10 nmol prawn−1 then injected with saline survived. The survival ratio of prawn received TA at 1 nmol prawn−1 were significantly higher than that of prawn received TA at 10 nmol prawn−1 and saline control after 96–168 h of infection. After 144 h of challenge, survival ratio of prawn received TA at 1 nmol prawn−1 were obvious higher by 20% than that of prawn received TA at 10 nmol prawn−1 or in the saline-challenged control (Table 1). 3.2. Effect of TA on the immunological parameters of M. rosenbergii The THC, HCs, and SGCs of prawn received TA at 1 nmol prawn−1 were significantly higher at 0.5 h than those of prawn received saline (Fig. 1A–C). The same tendency was observed in PO activities in haemocytes per 50 μL of haemolymph and in per granulocyte (Fig. 2A and B). However, no significant differences were observed in THCs, HCs, SGCs and PO activity at 1–8 h and in GCs at 0.5–8 h among the three treatments (Fig. 1A–D). Between TA treatment of 10 nmol prawn−1 and saline control, THCs, HCs SGCs and PO activity showed no significant difference at 0.5 h (Fig. 1A–C and 2A,B). For prawn received TA at 1 and 10 nmol prawn−1, RBs in haemocytes per 10 μL of haemolymph and per haemocyte were significantly higher than those of prawn received the saline at 0.5 h. No significant difference in RBs of haemocytes per 10 μL of haemolymph or in per haemocyte was observed among the three treatments at 1–8 h or between the two TA treatments at 0.5 h (Fig. 3A and B). Phagocytic activity and the clearance efficiency of prawn received TA at 10 nmol prawn−1 were significantly lower than those of prawn received TA at 1 nmol prawn−1 or saline at 0.5 h. The significantly increased phagocytic activity and clearance efficiency were detected in prawn received TA at 1 nmol prawn−1 at 1 h, compared to those of prawn received TA at 10 nmol prawn−1 and saline. However, no significant difference in phagocytic activity or the clearance efficiency was observed among the three treatments at 2–8, or between the treatments of TA at 1 nmol prawn−1 and saline at 0.5 h, and the treatments of TA at 10 nmol prawn−1 and saline at 1 h (Fig. 4A and B). Plasma lysozyme activity of prawn received TA at 10 nmol prawn−1 at 0.5 h were significantly lower than that of prawn received TA at 1 nmol prawn−1 and saline. However, no significant difference in lysozyme activity was observed among the three treatments at 1 h, or between the treatments of TA at 1 nmol prawn−1 and saline at 0.5 h (Fig. 4C).

2.8. Modulation of the phagocytic activity and clearance efficiency of M. rosenbergii in vivo via ARs OA and TA receptors were pharmacologically similar to vertebrate adrenoceptors, and the signaling transduction were potential to be estimated with the traditional adrenergic antagonists [25]. Therefore, the traditional adrenergic antagonists including phentolamine (Phe, an α1+2 AR antagonist, P-7547), prazosin (Pra, an α1 AR antagonist, P7791), propranolol, (Prop, a β1+2 AR antagonist, P-0884), and metoprolol (Meto, a β1 AR antagonist, M − 5391), purchased from Sigma–Aldrich, were used to evaluate the potential mediating pathway of TA on phagocytic activity and clearance efficiency against L. garvieae through OA/TA receptors. The TA and adrenergic antagonists were individually dissolved in sterile saline for this experiment. Tyramine (in a 10 μL solution) was mixed with each AR antagonist (in a 10 μL solution), then co-injected into the ventral sinus of the cephalothorax of individual prawn to reach doses of TA at 10 nmol prawn−1 and AR 4

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Fig. 1. Total haemocyte count (A), hyaline cells (B), semigranular cells (C) and granular cells (D) of Macrobrachium rosenbergii received saline or tyramine at 1 or 10 nmol prawn−1. Each bar represents the mean value from 6 samples with the standard error. Bars with different letters significantly differ (p < 0.05) among treatments at the same sampling time.

no significant difference among three treatments in DA at 1 h, in NE at 0.5–1 h and in OA at 0.5–1 h; and between the treatments of saline and TA at 1 nmol prawn−1 or of TA at 1 and 10 nmol prawn−1 (Fig. 7A–C).

3.3. Pharmacological modulation of immunological parameters Phagocytic activity and the clearance efficiency in prawn were assessed after they received saline, TA (at 10 nmol prawn−1), and TA (at 10 nmol prawn−1) co-injected with various AR antagonists for 0.5 h. Phagocytic activity and the clearance efficiency of prawn received saline and TA + Pra were significantly higher than those of prawn received TA, TA + Phe, TA + Prop or TA + Meto. However, no significant differences in phagocytic activity and the clearance efficiency were observed among the TA, TA + Phe, TA + Prop and TA + Meto, or between the saline and TA + Pra groups (Fig. 5A and B). These results indicated that TA reduced the phagocytic activity and the clearance efficiency, which was blocked by Pra.

4. Discussion Tyrosine, a precursor for CA biosynthesis, converts to DA or OA through two different pathways, in which tyrosine hydroxylase and tyrosine decarboxylase play crucial role as a rate-limiting enzyme, respectively, in invertebrates [4,24]. In recent, these amines involving in the two biosynthesis pathways had been identified to influence the immunocompetence in decapods. In shrimp, the L-DOPA, DA, and NE produced from tyrosine in one of the two CA biosynthesis pathways might depress the immune resistance abilities [16–19,46,47], and in the other way, TA and OA were capable to promote the immune resistance abilities [20,27,48,49]. The immunosuppressive or immunoenhancing effect of OA derive from TA was considered to be dose-dependent in insect [43]. The obviously enhanced immune responses and resistance to V. alginolyticus were observed in L. vannamei receiving TA at 100 and 1000 pmol shrimp−1 [27]. In the present study, the preliminary assessment on the phagocytic activity and clearance efficiency of prawn received TA at 25–250 pmol prawn−1 showed the consistent level as those in the control group (unpublished data); however, those of prawn receiving TA at 1 and 10 nmol prawn−1 for 1 h revealed the significant enhancement and insignificant difference, respectively, compared to the control. Those correlated well with the finding in the susceptibility of prawn after receiving TA for 1 h then challenging with L. garvieae. These phenomena implied that when a threshold of TA was crossed, the enhanced immune function was reversed, which indicated that TA might possess biphasic effects on immunity. To clarify the potential finding, further challenge tests for estimating the immunocompetence of prawn received TA higher than 10 nmol prawn−1 and their

3.4. Effect of TA on physiological parameters Glucose level in plasma was significantly lower in prawn received TA at 10 nmol prawn−1 than those of prawn received TA at 1 nmol prawn−1 or saline at 0.5 h. Lactate level of plasma was significantly higher in TA-injected prawn than that of prawn received saline at 0.5–1 h. However, no significant difference was observed among three treatments in glucose at 1–8 h and in lactate at 2–8 h; and between the treatments of TA at 1 nmol prawn−1 and saline in glucose at 0.5 h; and between two TA-injected prawns in lactate at 0.5–1 h (Fig. 6A and B). 3.5. Effect of TA on DA, NE and OA levels of haemolymph Haemolymph DA, NE and OA levels were determined in prawn received saline, and TA at 1 and 10 nmol prawn−1 after 0.5 and 1 h. DA levels in haemolymph gradually decreased with the dosages of TA administration, and was significantly lower in prawn received TA at 10 nmol prawn−1 than that of prawn received saline at 0.5 h. However, 5

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Fig. 2. Phenoloxidase (PO) activity in haemocytes per 50 μL of haemolymph (A) and in per 107 granulocytes (the sum of granular and semigranular cells) (B) of Macrobrachium rosenbergii received saline or tyramine at 1 or 10 nmol prawn −1 . Statistical descriptions are the same as those in Fig. 1.

Fig. 3. Respiratory bursts in haemocytes per 10 μL of haemolymph (A) and in per 107 haemocytes (B) of Macrobrachium rosenbergii received saline or tyramine at 1 or 10 nmol prawn −1. Statistical descriptions are the same as those in Fig. 1.

susceptibility against L. garvieae infection were necessary to be conducted. A transient increase in THC, DHC and PO activity in haemocytes per unit of haemolymph, and decrease in PO activity in per granulocyte of L. vannamei that received TA at 100 and 1000 pmol shrimp−1 was observed [27], and the similar pattern was reported in those of OA treatment [49]. Moreover, THC in L. vannamei received OA was returned to the initial level later than those of TA treatments [27,49]. In M. rosenbergii, after receiving OA at 25 and 250 pmol prawn−1 for 2 h, the elevated THC, DHC and PO activity in haemocytes per unit of haemolymph, and decrease in PO activity in per granulocyte returned to the initial level [20]. In the present study, the preliminarily assessment of the immunocompetence in M. rosenbergii receiving TA at 25 and 250 pmol prawn−1 remained consistent as those in the control (unpublished data), and however, the increase of THC, HCs, SGCs, and PO activity in haemocytes per 50 μL of haemolymph and per granulocyte were observed in prawn received TA at 1 nmol prawn−1 at 0.5 h, and meanwhile, no significant difference in THC, DHC and PO activity were observed between the treatments of TA at 10 nmol prawn−1 and saline. Those indicated that OA and TA at same levels induced the immunoenhancement in L. vannamei; however, the similar finding was assessed when M. rosenbergii receiving OA or TA at fourfold OA level, which implied that the influence of TA and OA, deriving from tyrosine through tyrosine decarboxylase, might differ according to the species. In decapod, haemocytes played an important roles in immunity, and furthermore, serotonin, a monoamine neurotransmitter, was found to affect the release of differential haemocytes from haematopoietic tissue into haemolymph with astakin 1 stimulation [50,51]. The findings in the present study suggested that an appropriate concentration of TA induced the release of SGCs and HCs regenerated from haematopoietic

tissue into the circulation, which caused the increase in THC, and furthermore, the increase in PO activity resulted from an increase in SGCs in 1 nmol prawn−1 TA-injected prawn. In crustaceans, the production of species of reactive oxygen intermediates during phagocytosis is a key cellular defense mechanism, which originates from the membrane-bound NADPH oxidase [52], and the clearance efficiency is an important humoral defence mechanism [53]. Clearance from the circulation is induced by humoral factors such as agglutinins, lectins, cytotoxic factors [54], and antimicrobial factors [55]. Significant elevation in RBs, and phagocytic activity and clearance efficiency toward pathogens were observed in L. vannamei or M. rosenbergii that injected with OA at 100 and 1000 pmol shrimp−1 [20,49], and in L. vannamei that injected with TA at 100 and 1000 pmol shrimp−1 [27]. In the present study, prawn received TA at 10 nmol prawn−1 for 0.5 h significantly depressed the clearance efficiency and phagocytic activity against pathogens, and lysozyme activity accompanied with the promoted RBs, and those subsequently recovered to the level of saline control, and in addition, the significantly elevated clearance efficiency and phagocytic activity against pathogens were assessed in prawn received TA at 1 nmol prawn−1 for 1 h, which agreed with the test of susceptibility to pathogens. The facts indicated that TA possessed a potential biphasic effects on regulating immune function, and induced NADPH oxidase activity to elevate O2− level in M. rosenbergii. Actions of CAs were shown to be mediated via the activation of Gprotein-coupled receptors (GPCRs). Pharmacologic antagonist drugs compete the binding site of an intermediate macromolecule, which do not trigger the usual receptor-mediated intracellular effect [56]. Kuo and Cheng [20] reported that the promoted effect of OA in immunity was mediated via OAαRs and OAβRs using pharmacological depletions 6

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Fig. 5. Clearance efficiency (A) and phagocytic activity (B) of Macrobrachium rosenbergii received saline or tyramine at 10 nmol prawn −1 and co-injected with various adrenergic receptor antagonists, Phe, phentolamine (an α1+2 adrenergic receptor antagonist); Pra, prazosin (an α1 adrenergic receptor antagonist); Prop, propranolol (a β1+2 adrenergic receptor antagonist); and Meto, metoprolol (a β1 adrenergic receptor antagonist). An adrenoceptor antagonist interferes with the physiological actions of TA by combining with and blocking its receptor. Statistical descriptions are the same as those in Fig. 1.

pharmacologically similar characteristics to vertebrate adrenoceptors. Stress-induced neuroendocrine changes are thought to divert an organism's energy resources away from physiological functions including reproduction, growth, and certain immune processes to allow metabolic and behavioral adaptations that may help the animal overcome a threat and survive [9,57]. Glucose is oxidized by glycolysis, an energy-generating pathway that converts it to pyruvate. In the absence of oxygen, pyruvate is converted to lactate, and when oxygen is present, pyruvate is further to form CO2 and H2O. Elevated blood glucose can result from a reduced utilization of glucose or stimulation of gluconeogenesis and/or glycogenolysis. M. rosenbergii injected with OA at 25.0 or 250.0 pmol prawn−1 showed a significantly increased glucose accompanied with a significantly decreased lactate in haemolymph, implying that OA resulted in gluconeogenesis and/or glycogenolysis to provide a metabolic energy source under aerobic glycolysis [20]. In the present study, glucose levels in haemolymph significantly decreased in prawn received TA at 10 nmol prawn−1, but lactate significantly increased in both TA-injected treatments, suggesting that TA induced anaerobic glycolysis to maintain homeostasis. In the experiment of neural circuit mediating food response in Caenorhabditis elegans, Suo et al. [58] indicated that DA depressed OA signaling via two D2-like DA receptors and the G protein Gi/o; moreover, the D2-like receptors worked in both the octopaminergic neurons and the octopamine-responding SIA neurons. Therefore, they suggested that DA suppressed OA release as well as OA-mediated downstream signaling. Friggi-Grelin et al. [59] reported that DA was absolutely required for cuticle biosynthesis and thus survival of insects. Mutant Drosophila melanogaster without tyrosine hydroxylase in the central

Fig. 4. Phagocytic activity (A), clearance efficiency (B) and lysozyme activity of Macrobrachium rosenbergii received saline or tyramine at 1 or 10 nmol prawn −1 . Statistical descriptions are the same as those in Fig. 1.

by biogenic amines co-injected with traditional adrenergic antagonists of Phe, Pra, Prop and Meto; moreover, the improved THC, HCs, SGCs, PO activity in haemocytes per unit of haemolymph, and phagocytic activity and clearance efficiency to pathogens in L. vannamei received TA at 1000 pmol prawn−1 were notably down-modulated via OAαRs [27]. Chang et al. [19] reported that in M. rosenbergii, norepinephrine modulated the phagocytic activity through β1 adrenergic receptor, and in the present study, the obviously reduced phagocytic activity and clearance efficiency in prawn receiving TA at 10 nmol prawn−1 were able to be blocked by Pra (an α1 adrenergic receptor antagonist), which indicated TA and NE modulating phagocytic activity through different pathways and the potential influence of NE can be ignored in the present study. Those suggested that adrenergic receptor antagonists possessed the potential to compete with TA and/or OA receptors against TA to block intracellular transduction because of their 7

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Fig. 6. Haemolymph glucose (A) and lactate (B) of Macrobrachium rosenbergii received saline or tyramine at 1 or 10 nmol prawn −1. Statistical descriptions are the same as those in Fig. 1.

nervous system confirmed that dopamine deficiency caused hypophagy [60]. The immune ability of M. rosenbergii injected with DA at 0.5–50 pmol prawn−1 showed a transient immunosuppression following the decreased immunity including phagocytic activity and clearance efficiency, and these led to a temporary increase in susceptibility to L. garvieae infection [17]. In the present study, prawn received TA at 1 nmol prawn−1 in short term significantly increased immune responses and resistance ability, but the significantly decreased lysozyme activity, and phagocytic activity and clearance efficiency to L. garvieae were detected in prawn received TA at 10 nmol prawn−1, and meanwhile, TA-injected prawn showed a dose-dependent decline in DA levels in haemolymph. The facts suggested that the regulation of immunity induced by TA related with the depressed DA release in M. rosenbergii, and both exogenous DA and TA-induced DA deficiency might cause immunosuppression. Tyrosine was conversed into L-DOPA by tyrosine hydroxylase then followed with the formation of DA and NE, and however, tyrosine was also the precursor for TA and OA, catalyzed by tyrosine decarboxylase. The two pathways of CA biosynthesis from tyrosine to DA and to OA, respectively, were reported in insect [4–8] and in L. vannamei [12–14]. In M. rosenbergii, DA [16] and NE [15,19] were estimated in haemolymph, and the effects of CAs such as DA [16,17], NE [18,19], OA [20] on immunocompetence were reported. Although the studies on key enzymes involving in CA biosynthesis were absent, yet the DA, NE and OA were detectable in haemolymph of M. rosenbergii, which indicated that the two pathways of CA biosynthesis were presented in prawn and played a crucial role in mediating stress-induced immunity. In conclusion, the present study documented that M. rosenbergii received TA by injection at a suitable dose (1 nmol prawn−1) resulted in immunoenhancing effect, but at an excessive dose (10 nmol prawn−1)

Fig. 7. Dopamine (A), norepinephrine (B) and octopamine (C) concentration in haemolymph of Macrobrachium rosenbergii received saline or tyramine at 1 or 10 nmol prawn −1. Statistical descriptions are the same as those in Fig. 1.

injection caused immunosuppressive effect. The present of immunoenhancing and immunosuppressive effects correlated with the degree of DA release downregulation induced by TA, and TA mediated immune function through OA/TA receptors signaling pathways.

Acknowledgements This research was supported by a grant from the Ministry of Science and Technology, Taiwan, ROC (MOST 107-2313-B-020-010-MY3).

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