Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection

Fish & Shellfish Immunology 30 (2011) 877e885

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Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection Sunee Wanlem a, b, Kidchakan Supamattaya a, Chutima Tantikitti a, Poonsuk Prasertsan b, Potchanapond Graidist c, * a b c

Aquatic Animal Health Research Center, Department of Aquatic Science, Faculty of Natural Resources, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Tumor Biology Research Unit, Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 September 2010 Received in revised form 11 January 2011 Accepted 16 January 2011 Available online 25 January 2011

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1 h after the rLV-CHH injection and then returned to normal after 3 h. In addition to the effect of rLV-CHH administration (25 mg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBSinjected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

Keywords: Crustacean hyperglycemic hormone, CHH Glucose White shrimp Litopenaeus vannamei Vibrio harveyi

1. Introduction Physiological processes in crustaceans are mainly regulated by neuroendocrines of natural peptides, of which approximately fifty neuroendocrines have been described, although the functions of some hormones are still not well defined [1]. Crustacean hyperglycemic hormone (CHH), the most abundant neuroendocrine, is synthesized by X-organ (XO) and stored in the sinus gland (SG) prior to being released directly into the hemolymph [2]. The X-organ and sinus gland (XO-SG) are located in the optic ganglia in the eyestalk which synthesize and control neuropeptide hormones essential for the regulation of the physiology and metabolism of crustaceans. The major hormone family produced from XO-SG is composed of crustacean hyperglycemic hormone (CHH), moltinhibiting hormone (MIH), gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH) and mandibular organ-

* Corresponding author. Tel.: þ66 74281184; fax: þ66 74429584. E-mail address: [email protected] (P. Graidist).

inhibiting hormone (MOIH) [3e7]. These hormones are known as the CHH-family of peptides on account of the extensive similarities in their sequences between individuals in a species or among different species. The peptides of the CHH-family consist of 72e78 residues and contain six conserved Cys residues that form three disulfide bonds [8]. The sequence of CHH hormone from Carcinus maenus was first reported by Kegel and coworkers [9]. Subsequently, CHH peptides sequences have been described and characterized in lobster [10], crabs [11], crayfishes [12], prawns [13,14] and shrimp [8,15]. CHH functions mainly in the regulation of glucose levels, as well as in the metabolism of carbohydrates and lipids [16]. In addition, it has a significant role in the processes of molting [17], reproduction [18], and osmoregulation [19,20]. However, there is no report about its role on the activation of the immune response in crustacean. The objective of the present study is therefore to elucidate the characterization of the cDNA encoding CHH peptides of white shrimp (Litopenaeus vannamei) and to investigate the effect of the rLV-CHH protein on the induction of shrimp immunity against luminous bacteria, Vibrio harveyi by intramuscular injection.

1050-4648/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.01.014

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2. Materials and methods 2.1. Animals Subadult shrimp, L. vannamei (size 10e15 g) were obtained from a local farm in Satun province, Thailand. The shrimp were transported to the laboratory in aerated plastic tanks and maintained for two weeks in seawater (15 ppt) at an ambient temperature (28  2  C). During the acclimation period, the shrimp were fed on a commercial diet 4 times daily at 7:00 am, 12:00 am, 4:00 pm and 11:00 pm. After acclimatization, intermolt shrimp were collected for use in each treatment and feeding was stopped for 24 h before starting the experiment. 2.2. Bacterial strains V. harveyi, a luminous bacterium, was isolated from hepatopancreas of infected-juvenile L. vannamei cultured in an earthen pond in Satun Province, Southern Thailand in 2005. Prior to the study, the bacterium was re-challenged for increasing virulence in L. vannamei by intramuscular injection and re-isolated on thiosulfate citrate bile salt sucrose agar (TCBS) as a Vibrio e selective media. Purified bacteria were cultured on tryptic soy agar (TSA) with 1.5% NaCl and incubated at 37  C for 18 h. One loopful of bacteria was transferred into a sterile saline solution (1.5% NaCl). Turbidity was adjusted to an optical density of 640 nm to 0.09e0.10 and counted on TSA agar with 1.5% NaCl. A bacterial suspension of 1.5  106 CFU/ml was used as the stock solution for the challenge and clearance ability test. A mean lethal dose (LD50) of bacterial suspension, 1.5  106 CFU/ml, was checked by preliminary testing. This suspension was used as the stock solution for the challenge and clearance ability test. 2.3. Isolation of total RNA and RT-PCR Total RNA was extracted from the eyestalk using TRIzol reagent (Invitrogen Corporation, USA) according to the manufacturer’s protocol. A partial sequence of the CHH gene of L. vannamei (GenBank accession no. AY434016) was used to amplify a 783 bp segment of CHH cDNA sequence. The gene-specific oligonucleotide primers were as follows; forward 50 -TCACCAAACGCTCGCTCTTC-30 and reverse 50 -TGCATGCCAGTGCTTTATTCG-30 . Primers were synthesized by Invitrogen Corporation (Carlsbad, CA, USA). RT-PCR product was carried out with total RNA (500 ng) by the QIAGENÒ OneStep RT-PCR kit (QIAGEN, Germany). The initial reaction started at 50  C for 30 min and 95  C for 15 min. The PCR reactions were performed as follows: 30 cycles of 94  C for 1 min, 50  C for 1 min and 72  C for 1 min, followed by a final extension at 72  C for 10 min. The RT-PCR product was analyzed on 1.8% agarose gel electrophoresis and visualized by ethidium bromide staining under ultraviolet light. The identity of PCR products was confirmed by cloning into pGEMÒ-T Easy vector (Promega Corporation, USA) and the sequence was analyzed using the ABI prism 377 apparatus. The data on the nucleotide sequence of CHH was used to design specific primers for cloning and expression. 2.4. Construction of the recombinant LV-CHH plasmid CHH gene was amplified by specific oligonucleotide primers using RT-PCR technique. For forward primer, nucleotide sequences CACC were added for coupling with the site of T-overhang, located in pET-TOPO vector (Invitrogen Corporation, USA) as an expression vector. Oligonucleotide primers were as follows: forward primer 50 -CACCATGACCAAACGCTCGCTCTTC-30 and reverse primer 50 -TTT CCCGACCATCTGGACAGCTAG-30 . The amplified PCR product (size

369 bp) was cloned into the pET-TOPO vector, forming a sequence encoding a fusion protein with an NH2-terminal hexa-histidine (6xHis) tag. The recombinant plasmid was termed LV-CHH-pET. The cloned fragment was sequenced using the ABI prism 377 apparatus to ensure authenticity of the cloned nucleotide sequence. Then, the LV-CHH-pET was transformed into competent Escherichia coli BL21 (DE3) using ChampionÔ pET-101 Directional TOPO expression kit (Invitrogen Corporation, USA). 2.5. Expression of recombinant LV-CHH in E. coli The E. coli BL21 (DE3) harboring LV-CHH-pET was grown in LB medium containing 100 mg/ml ampicillin with vigorous shaking at 37  C until the OD600 reached 0.6e0.8. The recombinant protein was induced by the addition of 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) with gentle shaking at room temperature for 5 h. After induction, the bacterial cells were harvested by centrifugation at 10,000g (4  C) for 15 min and washed with phosphate buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). The pellet was collected by centrifugation at 8000g (4  C) for 10 min and resuspended in lysis buffer (100 mM NaH2PO4, 10 mM Tris-Cl, pH 8.0, 8 M Urea). The cells were lyzed by sonication on ice, freezing and heating treatment according to Bangrak et al. [21]. The lysate was centrifuged at 18,000g for 30 min at 4  C. The pellet and supernatant were stored at 80  C until analysis. 2.6. Purification of recombinant LV-CHH (rLV-CHH) protein using Ni2þ-column The rLV-CHH was purified using Ni-NTA column. Briefly, the supernatant was mixed gently with a slurry of Ni2þ-resin on a rocking shaker for 1 h at room temperature. The mixture was packed into the column and washed with binding buffer (20 mM NaH2PO4, 500 mM NaCl, 5 mM imidazole, pH 7.4) before elution. The 6xHis tagged-fusion protein was eluted by elution buffer (20 mM NaH2PO4, 500 mM NaCl, 500 mM imidazole, pH 7.4). The fractions of eluted protein were analyzed by 12% SDS-PAGE gel. Authenticity of the rLV-CHH was confirmed by Western blot analysis using an anti-V5 antibody as primary antibody (Invitrogen Corporation, USA) and anti-mouse IgG conjugated to horse peroxidase serum as secondary antibody (GE Healthcare, UK). The total protein yield after purification was measured by the modified Lowry’s method [22]. The rLV-CHH protein was stored at 70  C until used. The bacterial protein from E. coli was used as a negative control, namely Neg-protein, according to the same method as mentioned above. 2.7. Effect of rLV-CHH on physiological parameters in L. vannamei One hundred and sixty eight of the white shrimp (10e15 g) were divided into three groups including rLV-CHH protein, Neg-protein and control group. Individual shrimp in each treatment group were injected with 25 mg of rLV-CHH or Neg-protein through the abdominal muscle. PBS buffer was used to inject the control group. After the injection at 0, 0.5, 1, 3, 6, 12 and 24 h, hemolymph samples of eight individual shrimp were withdrawn from the ventral sinus at the 3rd segment of walking leg using a 1-ml syringe with a 25 Gneedle and then transferred into a 1.5-ml microtube for physiological parameters analysis as follows. 2.7.1. Hyperglycemic activity One hundred microliters of hemolymph from eight individual shrimp in each time point of each treatment were separately well mixed with 900 ml of 3% trichloroacetic acid (TCA) as in

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deproteinization. The hyperglycemic activity assay was performed by the modified method of Dubowski [23]. Briefly, 300 ml of supernatant after deproteinization with TCA was mixed with 2.7 ml of color reagent (0.6% o-Toluidine with 1.5% Thiourea in glacial acetic acid) and then tubes were immersed in boiling water for 8 min. Distilled water and glucose solution were used as a blank and a standard, respectively. All tubes were removed and rapidly cooled on ice. The optical density at 630 nm was measured by a spectrophotometer (Shimadzu UV-1201, Japan) and the glucose concentration was calculated from the calibration curve of the standard. Glucose was reported in milligrams per 100 ml of blood sample (mg/dL). 2.7.2. Total hemocytes count After withdrawal, 50 ml of hemolymph from each shrimp was immediately diluted with 450 ml of 0.5% trypan blue in 2.6% NaCl. Hemocytes were counted using a haemocytometer under a microscope [24] and calculated as the number of total hemocytes (cell/ ml) following the equation below: Total hemocyte count (cell/ml) ¼ ((A þ B)/2)  dilution  103 mm3)/ 0.1 mm3  1 ml whereas A, B ¼ number of hemocyte cells in each side of haemocytometer. 2.8. Administration of rLV-CHH in shrimp against V. harveyi Forty five shrimp (10e15 g) were divided into three groups, and injected with either rLV-CHH protein or Neg-protein or PBS (control group). V. harveyi were used as a pathogenic organism. Three hours after the injection, shrimp were challenged with 0.1 ml of bacterial suspension (1.5  106 CFU/ml) resulting in 1.5  105 CFU/shrimp. Hemolymph from each shrimp was collected by using a 1-ml syringe at the ventral sinus at 3 h after bacterial infection. Each sample was measured for immune parameters analysis by the following methods.

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2.8.1. Total hemocyte count (THC) After withdrawal, 50 ml of hemolymph from each shrimp was immediately diluted with 450 ml of 0.5% trypan blue in 2.6% NaCl. The hemocytes were counted using a haemocytometer [24] and calculated as the number of total hemocytes (cell/ml) following the same equation as above. 2.8.2. Protein concentration in serum The total protein concentration of the shrimp serum was determined using the modified Lowry’s method [22]. Bovine serum albumin served as a protein standard. Briefly, hemolymph in each sample was clotted on ice and then homogenized with a hand homogenizer. Then, samples were centrifuged at 10,000g for 15 min at 4  C. Forty microliters of the supernatant was used to react with 120 ml of Folin reagent (1:15 in distilled water) to produce an unstable product that reduced molybdenum/tungsten blue under alkaline conditions and the optical density of 640 nm was read using a microplate reader (BioTek PowerWaveX, US). 2.8.3. Phenoloxidase activity in serum Fifteen shrimp from each experimental group were used for PO activities determination. Phenoloxidase activity was measured by recording the formation of the red pigment dopachrome from L-dihydrophenylalanine (L-DOPA) using a microplate reader [25]. Briefly, 100 ml of hemolymph from individual shrimp was mixed with cacodylate-citrate (CAC) buffer (0.01 M sodium cacodylate, 0.45 M NaCl, 0.1 M sodium citrate; pH 7.0) and kept immediately in liquid nitrogen before assays. Hemolymph samples were homogenized and centrifuged at 6000g at 4  C for 10 min. The supernatant was diluted (1:6) with chilled CAC buffer and 25 ml of this solution was pre-incubated with an equal volume of 1 mg/ml of trypsin solution for 5 min at 25  C in 96-microwell plate. For blank assays, hemolymph was replaced by CAC buffer. After incubation, 50 ml of L-DOPA (4 mg/ml in CAC buffer) was added to the wells as a substrate and the enzyme activity was measured the optical density at the wavelength of 490 nm every 2 min for 10 min. The protein content in hemolymph was determined by Lowry’s method

Fig. 1. Nucleotide and amino acid sequence of CHH gene from L. vannamei. The stop codon is indicated by asterisk. The position corresponding to the sequences of the primers (forward, reverse) used in the RT-PCR are underlined. The nucleotide sequence has been submitted in GenBank (accession no. FJ598023).

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Fig. 2. Multiple alignments of nucleotide (A) and amino acid sequence (B) of eyestalk CHH gene from L. vannamei (CHH4: accession no. FJ59823) with that of various CHHs; L. vannamei (CHH1: accession no. AY434016), P. monodon (CHH2: accession no. GQ221085), P. vannamei (CHH3: accession no. X99731). Numbers on the right margin represent nucleotide and amino acid positions.

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using bovine serum albumin as a standard. One unit of PO activity was defined as an increase in absorbance of 0.001 min1 mg protein1 [26]. 2.8.4. Bacterial clearance ability The ability of shrimp to remove foreign particles from the blood circulation system was measured by the modified method of Martin and coworkers [27]. Fifteen shrimp from each group were tested. Hemolymph of challenged-shrimp with 0.1 ml of V. harveyi for 3 h was collected from each shrimp without using an anticoagulant. Twenty microliters of whole blood (without dilution) was dropped on TCBS agar. A two-fold dilution of whole blood was made using a sterile 1.5% NaCl solution. The number of bacteria was counted on the TCBS as above. The bacterial cells in the hemolymph of each shrimp were reported as CFU/ml. 2.9. Challenge test The challenge test was conducted in triplicate with 15 shrimp per replicate in each treatment. Individual shrimp was injected with rLV-CHH protein or Neg-protein or PBS (control) on the sixth segment of the abdominal muscle for 3 h and then re-injected with 0.1 ml of bacterial suspension (1.5  106 CFU/ml) resulting in 1.5  105 CFU/shrimp. After the injection, shrimp were kept in a separate 80-L glass aquaria. Shrimp mortality was recorded every day for a period of 14 days. 2.10. Statistical analysis Data was analyzed by one-way ANOVA. When significant differences existed among groups, multiple comparisons among means were tested by the Tukey’s test. The P  0.05 was considered statistically significant. 3. Results 3.1. CHH gene of L. vannamei The CHH segment was amplified from total RNA derived from eyestalk of L. vannamei, and cloned into a pET-TOPO vector. The nucleotide sequences of CHH (Fig. 1) were submitted to GenBank (accession no. FJ598023). The partial cDNA of CHH contains an open reading frame (ORF) of 369 bp encoding a putative protein of 122 amino acid residue. A putative peptide was identified using MLC

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workbench 5.7 with a calculated molecular mass of 14.72 kDa and a predicted isoelectric point (pI) of 8.64. Multiple alignments of LV-CHH with other CHH homolog using ClustalW program are shown in Fig. 2. The percentages of nucleotide sequence identities of CHH had a similarity with Penaeus monodon (GQ221085), L. vannamei (AY434016) and P. vannamei (X99731) at 94%, 99% and 100%, respectively. 3.2. Expression and purification of rLV-CHH The rLV-CHH was expressed in E. coli strain BL21 (DE3). The protein was purified in a Ni-NTA affinity column and separated on 12% SDS-PAGE (Fig. 3A). The purified protein was further analyzed by Western blot analysis using V5-epitope monoclonal antibody. A specific blot band was detected at the corresponding position as shown in Fig. 3B. The predicted molecular mass of the rLV-CHH protein was 14.72 kDa with additional 6xHis-tag and V5-epitope in the N-terminal of the expressed fusion protein, the extra amino acids increased the molecular mass of the expressed target protein by w3.81 kDa. The apparent molecular mass of the purified protein was approximately 18.53 kDa. The final yield of the total protein was 1.3 mg/ml after a single Ni2þ affinity chromatographic step. 3.3. Biological activity in white shrimp (L. vannamei) on rLV-CHH administration After an injection of rLV-CHH or Neg-protein or PBS, the hemolymph glucose level showed an increase at 0.5 h post injection (Fig. 4A). In the group that injected with rLV-CHH, the glucose level increased to a maximum level at 1 h after the injection. However, the glucose level in both the PBS and Neg-protein group was changed rapidly at 0.5 h and then decreased toward the resting state level within 6 h after the injection (Fig. 4A). These results show that rLV-CHH can enhance glucose levels in shrimp hemolymph. The total hemocyte count in shrimp after injection with rLVCHH or Neg-protein or PBS shows the same trend as the glucose level in hemolymph. The THC of L. vannamei that injected with rLVCHH was significantly higher than those of shrimp that injected with Neg-protein and PBS (control group) at 1 h after injection (Fig. 4B). The THC of shrimp injected with rLV-CHH has the highest value of 22.08  1.24 cell/ml at 3 h post injection. On the other hand, shrimp injected with Neg-protein and PBS has a value of 16.38  1.46 cell/ml and 13.70  0.92 cell/ml, respectively.

Fig. 3. The rLV-CHH profile was performed by SDS-PAGE and Western immunoblot technique. (A) Coomassie blue stained SDS-PAGE of the rLV-CHH protein after purification by Ni2þ-column; Lane M: molecular weight marker, lane 1: Neg-protein, lane 2: rLV-CHH at first elution, lane 3: rLV-CHH at second elution and lane 4: rLV-CHH at fourth elution. (B) The recombinant LV-CHH protein was analyzed by Western immunoblot technique using anti-V5 epitope (Lane M: molecular, lane 1: Neg-protein, lane 2: rLV-CHH protein).

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3.4. The effect of rLV-CHH on immune response of L. vannamei against V. harveyi infection The immune response in L. vannamei injected with rLV-CHH or Neg-protein or PBS was checked at 3 h after challenging with V. harveyi. The total hemocyte count of shrimp that injected with rLVCHH was significantly higher (P < 0.05) than those of Neg-protein and PBS groups (Fig. 5A). In the rLV-CHH group, the THC increased significantly by 12.33  1.27 (106 cell/ml) after 3 h post bacterial challenging. No significant difference in THC was observed among the shrimp that received Neg-protein (8.45  2.19 (106) cell/ml) and PBS (9.01 1.53 (106) cell/ml). In addition, a similar trend in serum protein levels was recorded where it was significantly higher (P < 0.05) in rLV-CHH group (169.30  12.08 mg/ml) compared to Neg-protein (137  10.91 mg/ml) and PBS (121.80  15.36 mg/ml) as the control group (Fig. 5B). For the PO activity test, rLV-CHH injected shrimp showed a significant (P < 0.05) increase at 4.20  0.50 unit/min/mg protein in comparison with the Negprotein group (2.10  0.74 unit/min/mg protein) and PBS group (1.93  0.50 unit/min/mg protein), respectively (Fig. 5C). To study the clearance ability to remove foreign particles in shrimp hemolymph after injection with V. harveyi (1.5  105 CFU/shrimp) for 3 h. Bacterial cells in shrimp injected with rLV-CHH decreased significantly by 45.50  4.26 (102) cell/ml when compared to Negprotein (130.69  21.66 (102) cell/ml) and PBS group (142.17  28.97 (102) cell/ml). No significant difference in clearance ability was observed between the Neg-protein and PBS group (Fig. 6). Fig. 4. The effect of rLV-CHH on physiological changes in L. vannamei. (A) Hyperglycemic activity and (B) the total hemocyte count (THC) of shrimp hemolymph after rLVCHH administration (25 mg/shrimp) in comparison with Neg-protein and PBS group at different time. Each value represents Mean  SEM of 8 individual assays. Significant differences between time-points for PBS, Neg-protein and rLV-CHH injected shrimp were assessed using one-way ANOVA. The different characters indicate P  0.05.

3.5. The effect of rLV-CHH on the resistance of L. vannamei to V. harveyi The death of shrimp occurred after 18 h post bacterial infection and no shrimp mortality occurred after 7e14 days in the treatments of rLV-CHH or Neg-protein or PBS. At 14 days post bacterial infection, survival rate of the rLV-CHH group was significantly highest

Fig. 5. The effect of rLV-CHH on immunological changes in L. vannamei. (A) Phenoloxidase specific activity, (B) serum protein level and (C) total hemocytes count in hemolymph of white shrimp post infection with V. harveyi for 3 h. Values are shown as Mean  SE of 15 individual assays. Significant differences are indicated with different character at P  0.05.

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Fig. 6. Clearance ability of V. harveyi in hemolymph of white shrimp (L. vannamei) after injection with rLV-CHH protein (25 mg/shrimp), Neg-protein (25 mg/shrimp) and PBS as a control group by intramuscular injection for 3 h. Each shrimp was challenged with 100 ml of V. harveyi suspension (1.5  105 CFU/shrimp). Clearance ability was analyzed at 3 h after bacterial infection. Each value represents (Mean  SE) of 15 individual assays. Significant differences are indicated with different character at P  0.05.

(P < 0.05) at 66.0%, whereas survival rate of shrimp injected with Neg-protein and PBS group was 33.3% and 28.9%, respectively (Fig. 7). The period of time for mortality checking was 14 days which is enough to ensure that the shrimp survived from bacterial infection. 4. Discussion Crustacean hyperglycemic hormone was identified in the eyestalk of L. vannamei. Its mRNA was present at a high level in crustacean eyestalks. In previous work, the expression of the CHH gene in P. monodon was not only restricted to the eyestalk but also detectable in the heart and in the gill [14]. In crustacean, neuroendocrine centers are located in different structures of the nervous system and one of these structures, the eyestalk of X-organ-sinus gland complex (XO-SG), is traditionally considered as the major endocrine system of decapods [6,28]. The XO-SG complex produced several neuropeptides including the CHH/MIH/GIH family [6,29e31]. The CHH/MIH/GIH family consists of two kinds of neuropeptides, the CHH hormone, and the molt-inhibiting hormone (MIH), the gonad-inhibiting hormone (GIH) or the vitellogenin-inhibiting hormone (VIH) and the mandibular organinhibiting hormone (MIOH) [6,9,32,33]. Although the biochemical and molecular study of the CHH-family of neuropeptides has been performed on a large number of crustacean species, including

Fig. 7. The effects of bacterial infection (V. harveyi) on survival rate of injected shrimp (L. vannamei) with rLV-CHH compared to Neg-protein and PBS group. Survival shrimp was observed for 14 days after bacterial infection.

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shrimp, crab, crayfish and lobster, the expression pattern and the evolutionary relationship of the peptides have not been completely resolved. CHH peptides belonging to CHH/MIH/GHH family are polymorphic due to changes in the amino acid sequence, and to the isomerization of one single amino acid residue from L- to D-configuration [6]. The different peptides of the CHH/MIH/GHH family are expressed in single cells [4,34]. Some studies have revealed that these peptides express in different neurons [35,36]. However, in this study we expressed rLV-CHH from eyestalk in E. coli and characterized the hyperglycemic activity of the protein in L. vannamei. After purification, we found a band of non-target protein contaminate in the protein solution, even though it had been passed through the Ni2þ column. Some of the bacterial protein existed in the target protein solution, indicating that the result of rLV-CHH purification in this study has a little contamination of residual bacterial protein. Therefore, Neg-protein was used as a control in the experiments. In this study, the rLV-CHH protein demonstrated a function in stimulating the hyperglycemic activity in shrimp by intramuscular injection. The THC was also increased to maximum level at 3 h after rLV-CHH administration. It may be possible that the hemolymph in this period is composed of glucose or other proteins at a high concentration which is stimulated by rLV-CHH. However, THC increased to a maximum level at 3 h and then decreased to a normal level within 12 h when compared to the control group. For the short term response from rLV-CHH administration, excess glucose will not cause organ damage. It is possible that rLV-CHH can cause excessive urination to remove the excess blood sugar in the body system. However, hyperglycemia in crustacean is still lacking in information about the effects of a high blood sugar on the physiological response and regulating excess blood sugar in the circulating system. The ability to elevate the glucose level in hemolymph of rLV-CHH protein indicated that rLV-CHH function as crustacean hyperglycemic hormone (CHH) in L. vannamei. Even though the purified target protein was contaminated by the residual protein from E. coli, it had no effect on the hyperglycemia property of the rLV-CHH. Neg-protein is expressed proteins from E. coli which is transformed with an expression vector without CHH gene. Neg-protein was used in this study to confirm that contaminated protein in E. coli does not affect shrimp immunity. In fact, crustacean need to temporarily regulate their blood glucose concentration in hemolymph to ensure that adequate glucose levels are available for all cells and that excess glucose can be stored as glycogen in the hepatopancreas [37]. D-glucose is the major component of hemolymph carbohydrate [38,39] and is controlled within a strict range. The balanced secretion of several hormones is an essential part of the controlled glucose homeostasis. In particular, CHH is the important signaling molecule that played a role in the regulation of glycemia by increasing the glucose level in hemolymph via mobilization from the hepatopancreas and muscle glycogen stores [6,37]. The effects of hormones other than CHH on glucose levels in crustaceans are not well understood. Although, the major function of CHH is to regulate glucose metabolism in crustaceans, several studies have indicated that it may also regulate the reproductive and the molting cycles [15,40]. For example, the hemolymph CHH varies during molting cycles in Astacus leptodactylus [41] and Carcinus maenas [42]. The functional specificities of CHH hormone and other related hormones are not clearly defined because of the overlapping of their biological activities [2]. In addition, the possibility of using rLV-CHH to induce shrimp immune responses was tested. Immunostimulants have been administered to marine fish and shellfish through oral, immersion and injection methods [43]. In this study, the rLV-CHH was performed by intramuscular injection to ensure the amount of injected

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protein per shrimp. THC has been reported to be a potential indicator of immune responses in crustacean especially in shrimp [44] and a higher-than-normal number of circulating hemocytes in crustaceans correlate with an increased resistance to pathogens [45]. The role in melanization related to the PO activity system, is to stimulate several cellular defense reactions, including phagocytosis, nodule formation, encapsulation and hemocyte migration [46]. The activation of the PO system has been used as an indicator to measure immunostimulation in shrimp [47]. The experiment showed that the injecting of shrimp with rLV-CHH resulted in an increase in their ability to survive after being infected with V. harveyi, in comparison with Neg-protein and PBS as the control group. Besides, injecting rLV-CHH in shrimp significantly increased the blood parameters such as THC, PO activity and serum protein, including clearance ability than those of Neg-protein and PBSinjected shrimp. The clearance ability and PO activity are important humoral defense mechanisms against pathogens in crustaceans [48,49]. The PO activity results from the activation of the pro-PO enzyme. The pro-PO activating system has been well studied in crustaceans such as crayfish, shrimp, crab and lobster [26,44,50,51]. The cumulative effect of elevated THC, serum protein and PO activity coupled with the rapid clearance ability of bacteria in hemolymph reflected a higher survival rate in the rLV-CHH injected group compared to that in the Neg-protein and PBS-injected group, which suggests the possibility that rLV-CHH could enhance the immunity in L. vannamei against luminous bacteria, V. harveyi. In order to get more information of the mechanism on the immune system of shrimp by rLV-CHH administration, more experiments and specific parameters (such as lysozyme activity, complement, antioxidant enzymes activity) are required to understand how recombinant CHH can stimulate shrimp immune against pathogens. Acknowledgments This work was financially supported by The Royal Golden Jubilee Program and The Thailand Research Fund (PHD/0259/2545), and the Graduate School, Prince of Songkla University, Hat Yai, Songkhla, Thailand. References [1] Lago-Lèston A, Ponce E, Munoz ME. Cloning and expression of hyperglycemic (CHH) and molt-inhibiting (MIH) hormones mRNAs from the eyestalk of shrimps of Litopenaeus vannamei grown in different temperature and salinity conditions. Aquaculture 2007;7:343e57. [2] Böcking D, Dirksen H, Keller R. The crustacean neuropeptides of the CHH/MIH/ GIH family: structures and biological activities. In: Wiese K, editor. The crustacean nervous system. Berlin: Springer; 2002. p. 84e97. [3] Lacombe C, Grève P, Martin G. Overview on the sub-grouping of the crustacean hyperglycemic hormone family. Neuropeptides 1999;33:71e80. [4] Chan SM, Gu PL, Chu KH, Tobe SS. Crustacean neuropeptide genes of the CHH/ MIH/GIH family: implications from molecular studies. Gen Comp Endocrinol 2003;134:214e9. [5] Chen SH, Lin CY, Kuo CM. In silico analysis of crustacean hyperglycemic hormone family. Mar Biotechnol 2005;7:193e206. [6] Fanjul-Moles ML. Biochemical and functional aspects of crustacean hyperglycemic hormone in decapods crustaceans: review and update. Comp Biochem Physiol C 2006;142:390e400. [7] Treerattrakool S, Panyim S, Chan SM, Withyachumnarnkul B, Udomkit A. Molecular characterization of gonad-inhibiting hormone of Penaeus monodon and elucidation of its inhibitory role in vitellogenin expression by RNA interference. FEBS J 2008;275:970e80. [8] Huberman A, Aguilar MB, Navarro-Quiroga I, Ramos L, Fernandez I, White FM, et al. A hyperglycemic peptide hormone from the Caribbean shrimp Penaeus (litopenaeus) schmitti. Peptides 2000;21:331e8. [9] Kegel G, Reichwein B, Weese S, Gaus G, Peter-Katalinic J, Keller R. Amino acid sequence of the crustacean hyperglycemic hormone (CHH) from the shore crab, Carcinus maenas. FEBS Lett 1989;255(1):10e4. [10] Tensen CP, De Kleijn DPV, Van Herp F. Cloning and sequence analysis of cDNA encoding two crustacean hyperglycemic hormone from the lobster Homarus americanus. Eur J Biochem 1991;200:103e6.

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