Cloning of Kuruma Prawn Marsupenaeus Japonicus Crustin-Like Peptide Cdna and Analysis of Its Expression

Blackwell Science, LtdOxford, UK FISFisheries Science0919-92682004 Blackwell Science Asia Pty Ltd 705October 2004 869 Crustin-like peptide cDNA of kuruma prawn A Rattanachai et al. 10.1111/j.1444-2906.2004.00869.x Original Article765771BEES SGML

FISHERIES SCIENCE 2004; 70: 765–771

Cloning of kuruma prawn Marsupenaeus japonicus crustin-like peptide cDNA and analysis of its expression

Achara RATTANACHAI,1 Ikuo HIRONO,1 Tsuyoshi OHIRA,1 Yukinori TAKAHASHI2 AND Takashi AOKI1*

1Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477 and 2Department of Applied Aquaculture, National Fisheries University, Shimonoseki, Yamaguchi, Japan

ABSTRACT: Antimicrobial peptides serve as an important component of the innate immune system of all species by functioning to provide a rapid first line defense against infection. Arthropod antimicrobial peptides have been well described in insects, whereas only a few molecules have been identified in crustaceans. Five variants (types 1Ð5) of Marsupenaeus japonicus crustin-like peptide cDNA that were obtained from a hemocyte cDNA library and polymerase chain reaction (PCR) amplification are reported here. Marsupenaeus japonicus crustin-like peptide type 1, the predominant type, has a cDNA consisting of 679 nucleotides and an open reading frame consisting of 573 base pairs coding for 191 amino acid residues. Other types contain varying glycine-rich repeats at the N- terminal amino acid sequences. The deduced amino acid sequences of these variants are highly similar to those of Litopenaeus setiferus (80% identity), Litopenaeus vannamei (80% identity) and Carcinus maenas crustins (44% identity). Expression of Marsupenaeus japonicus crustin-like peptide mRNA was detected in hemocytes, but not in the heart, hepatopancreas, gill, fore-gut, mid-gut, muscle, subcuticular epithelium or ovary. The expression level of crustin-like peptide mRNA increased significantly 1, 3 and 7 days post-peptidoglycan feeding as determined by quantitative real-time PCR. These results suggest that crustin-like peptide could have an important role in shrimp defense mechanisms.

KEY WORDS: antimicrobial peptide, crustin-like peptide, hemocyte, Marsupenaeus japonicus, peptidoglycan, quantitative real-time polymerase chain reaction.

INTRODUCTION nisms by which immunostimulants induce the expression of biodefense genes are unclear. Basic The shrimp aquaculture industry is of significant knowledge of shrimp immunity at the molecular importance to the global economy. Since 1998, level is needed to develop strategies for the preven- world shrimp aquaculture production has been tion and control of diseases in shrimp aquaculture. estimated to be more than 1 million metric tons, Crustaceans do not have a specific humoral but the industry has suffered production losses defense system or a specific immune system mainly because of bacterial and viral diseases.1,2 against pathogens. However, they possess several Chemotherapeutics have been used to prevent different means of conferring innate immunity, these losses, yet the incidence of drug-resistant which include the production of antimicrobial pep- pathogens has become a serious problem in the tides and proteins, melanization, release of oxygen culture. In recent years, immunostimulants such radicals, production of lectins, and cell defense as peptidoglycan (PG), lipopolysaccharide (LPS) reactions. Antimicrobial peptides and proteins and b-1,3 glucan have been successfully used to serve as important components of the innate enhance the resistance of fish and crustaceans to immune system of all species by functioning to pro- bacterial or viral infection.3 However, the mecha- vide a rapid first line defense against a wide range of invading pathogens. Although a large number of *Corresponding author: Tel: 81-3-5463-0556. these molecules have been well studied in various Fax: 81-3-5463-0690. Email: [email protected] insect species, few have been isolated from crusta- Received 26 November 2003. Accepted 27 April 2004. ceans, these include lysozyme, penaeidins and

766 FISHERIES SCIENCE A Rattanachai et al.

hemocyanin.4–7 Lysozyme, widely distributed Carlsban, CA, USA). Poly(A)+ RNA was then puri- among eukaryotes and prokaryotes, functions in fied from total RNA by using a QuickPrep micro hydrolyzing the b-1,4 linkages of bacterial cell wall mRNA purification kit (Amersham Bioscience, Pis- PG.8 Its lytic activity has been reported in crusta- catawy, NJ, USA). First strand cDNA was synthe- ceans including the freshwater crayfish Artemia sized with an oligo d(T) primer and ligated into the and cold water decapods.5,9,10 Recently, lysozyme l ZipLox phagemid vector using a SuperScript has been cloned in kuruma prawns and its bacterial Lamda system kit (Invitrogen). The ligation prod- lytic activity has been tested using recombinant uct was subjected to in vivo packaging as described lysozyme protein.11 Lysozyme in kuruma prawns in the manufacturer’s instructions. shows broad antibacterial lytic activity. The lysozyme cDNA of white shrimp Litopenaeus vannamei has also been cloned and characterized.12 Cloning of kuruma prawn crustin-like Penaeidins, originally isolated and characterized peptide cDNA from L. vannamei, have been cloned and well characterized in several shrimp species.13 These are Total RNA was prepared from prawn hemocytes as small peptides of a size range from approximately described above and subsequently treated with 5.5 to 6.6 kDa containing proline-rich domains and 1 unit of DNase I (Promega, Medison, WI, USA) for six cysteines, which form 3-disulfide bonds. They 30 min at 37∞C. First strand cDNA was synthesized display antimicrobial activity against fungi and with an oligi d(T) primer using an of AMV reverse Gram-positive bacteria. Crustin was first purified transcriptase first strand cDNA synthesis kit from the shore crab Carcinus maenas and is known (Life Science Technologies, St Petersburg, FL, USA) as an 11.5-kDa antibacterial peptide.14 This protein from 10 mg of total RNA. The final reaction volume is active only against marine Gram-positive bacte- of the cDNA synthesis reaction was 25 mL. Two oli- ria and appears to require high salinity to express gonucleotide primers, dAMP-F (5¢-ATGAAG this activity. Crustin is stable and retains activity GGCATCAGGCGGTGATTC-3¢) and dAMP-R even after boiling. Crustin is a cysteine-rich peptide (5¢-GCACACGTGTTCTCCCAAACACCTG-3¢) were and has 30% identity with some 4-disulfide core or designed based on the nucleotide sequence of whey acidic proteins. Proteins with this motif fre- L. vannamei crustin homologs identified by quently possess serine proteinase inhibitory prop- expressed sequence tag analysis.17 One microliter erties and can inhibit cell growth.15 Recently, crustin of reverse-transcribed reaction product was used homolog cDNA were also cloned from two species as the template in the polymerase chain reaction of penaeid shrimp L. vannamei and Litopenaeus (PCR) amplification. The PCR reaction was con- setiferus.16 These crustin cDNA encode a protein ducted with the initial denaturation step at 94∞C with a leader sequence, a hydrophobic glycine-rich for 2 min, followed by 30 cycles at 94∞C for 30 s, repeat amino-terminal region, and a unique car- 55∞C for 30 s and 72∞C for 1 min, and the final boxy-terminal region. The carboxy-terminal region extension step at 72∞C for 5 min. Polymerase chain was found to have sequence similarity to the shore reaction product was analyzed by a 1.5% agarose crab crustin.14 Thus far, however, information has gel electrophoresis stained with ethidium bromide. been obtained concerning only a few crustacean After gel purification using a DNA purification kit antimicrobial peptides and proteins. In addition, (Toyobo, Tokyo, Japan), the product was cloned knowledge at the molecular level concerning their into the pGEM-T Easy vector system (Promega). synthesis and mechanisms in penaeid prawns and Recombinant clones were identified and plasmid shrimp, especially kuruma prawns, remain limited. DNA was obtained by the alkaline lysis method.18 In the present study, five cDNA encoding different DNA sequencing of recombinant clones was per- crustin-like peptides were cloned from the kuruma formed on an automated DNA sequencer LC4200 prawn Marsupenaeus japonicus and their gene (Li-Cor, Lincoln, NE, USA) using a ThermoSeque- expressions were characterized. It was also shown nase fluorescent-labeled primer cycle sequencing for the first time that prawn crustin expression is kit (Amersham Bioscience). induced by the oral administration of PG. A partial cDNA fragment of M. japonicus crustin-like peptide amplified by reverse transcrip- tion (RT)-PCR was used as a probe for screening MATERIALS AND METHODS the cDNA library. The cDNA fragment was labeled with 32P-dCTP by using a random primer DNA Construction of hemocyte cDNA library labeling kit (Takara, Tokyo, Japan). Approximately 300 000 plaques were screened with the labeled Total RNA from 25 kuruma prawns was extracted probe. The recombinant plasmids were rescued from hemocytes using Trizol reagent (Invitrogen, from the bacteriophage clones by in vivo excision

Crustin-like peptide cDNA of kuruma prawn FISHERIES SCIENCE 767

using Escherichia coli strain DH10 B (ZIP) (Invitro- using b-actin-F and b-actin-R primers. The gen) according to the manufacturer’s protocols. hybridization was carried out as has been The isolated cDNA clones were subjected to reported previously.21 sequencing as described above.

Detection of kuruma prawn crustin-like peptide Sequence data analysis mRNA in hemocytes stimulated with peptidoglycan by real-time The nucleotide, the deduced amino acid polymerase chain reaction sequences and multiple alignment were analyzed by using GENTYX WIN ver. 3.1 (SDC Software Peptidoglycan was prepared from Bifidobacte- Development, Tokyo, Japan). Each obtained cDNA rium thermophilum as has been reported sequence was compared with all sequences depos- previously.22 Cultivated kuruma prawns with a ited in DDBJ/EMBL/GenBank using BLASTN and sex ratio of 1:1 and an average weight of 21.8 g BLASTX programs.19 The cleavage site of the signal had been acclimated for 1 week in a tank under peptide was predicted by the SignalP program.20 laboratory conditions at 20 ± 1∞C and fed a com- mercial pelleted diet at 5% of body weight. The prawns were then fed daily with a diet containing Tissue distribution of kuruma prawn crustin-like 0.2 mg PG/kg shrimp body weight for 7 days. peptide mRNA Three shrimps were randomly sampled on day 1, 3 and 7 after PG feeding. Samples taken on day 0 Total RNA was extracted from healthy kuruma were used as non-stimulated controls. Their prawn hemocytes, heart, hepatopancreas, gill, whole hemolymphs were individually collected fore-gut, mid-gut, muscle and subcuticular epithe- by heart puncture using anticoagulant buffer lium and treated with DNase I as mentioned above. (0.1 M HEPES, 0.1 M EDTA with 3.2% NaCl, First strand cDNA synthesis was then performed by pH 7.0) at a ratio of 1:1 v/v. Hemocytes from each using oligo d(T) primer as described in the cloning sample (averaging 8 ¥ 106 cells/mL) were sepa- of kuruma prawn crustin-like peptide cDNA. rated from plasma by centrifugation at 1000 ¥g Two universal primers for the amplification of for 15 min at 4∞C. Total RNA were prepared and M. japonicus crustin-like peptides, rtAMP-F (5¢- treated with DNase I as mention above. First GACTGCAGGTACTGGTGCAAGA-3¢) and rtAMP-R strand cDNA were synthesized as described (5¢-CCTGCTCCAACGACTACAAGTG-3¢) were above and were used as templates in the follow- designed. A set of b-actin primers, b-actin-F ing real-time PCR. (5¢-TTCCCCTCCATCGTGGGCCG-3¢) and b-actin-R To make a standard curve, serial standard (5¢-TGTAAGGTCACGTCCAGCCA-3¢), served as a DNA of M. japonicus crustin-like peptide and control for the amount and quality of each cDNA. b-actin were prepared as has been described The PCR reaction was conducted with an initial previously.23 Primers for M. japonicus crustin-like denaturation step at 95∞C for 5 min, 20 cycles at peptides (rlAMP-F [5¢- CTGCTGCGAAAGGTCT- 95∞C for 30 s, 56∞C for 30 s and 72∞C for 30 s, fol- CACGAG-3¢] and rtAMP-R) and for b-actin (rtb- lowed by a final extension step at 72∞C for 5 min. actin-F [5¢-TGCTGGACTCTGGCGATGGCGT-3¢] The reaction products were electrophoresed on a and rtb-actin-R [5¢-TGTAAGGTCACGTCCAGCCA- 1.5% gel containing ethidium bromide. 3¢]) were used. Real-time PCR was carried out using SYBR Green PCR core reagent (Perkin- Elmer Biosystems, Forter City, CA, USA) accord- Northern blot analysis ing to the manufacturer’s protocols. Polymerase chain reactions were conducted with the initial Total RNA was prepared from M. japonicus denaturation step at 50∞C for 2 min and 95∞C for hemocytes as described above. Ten micrograms 10 min, followed by 40 cycles at 95∞C for 15 s of the sample was loaded for RNA electrophore- and 60∞C for 60 s using a GeneAmp5700 sis on a 1% agarose gel containing 17% formal- sequence detector (Perkin-Elmer Biosystems). All dehyde and transferred to Hybond-N+ samples were run in triplicate. Kuruma prawn nitrocellulose membrane (Amersham Bioscience) b-actin content in a standard sample (105) was according to the manufacturer’s protocols. An used as an internal positive control and the M. japonicus crustin-like peptide cDNA fragment normalizing reference for individual variation. obtained by RT-PCR with specific primers Dunnett’s multiple comparison test was used for (rtAMP-F and rtAMP-R) was used as a probe. A statistical analysis. Values were considered to be b-actin probe was also amplified by RT-PCR significant at P < 0.05. 768 FISHERIES SCIENCE A Rattanachai et al.

RESULTS region (Fig. 1). The deduced amino acid sequence of M. japonicus crustin-like peptide type 2 was Cloning of kuruma prawn crustin-like identical to that of type 1 except that it had an addi- peptide cDNA tional 28 amino acids in the repeat region (Fig. 2). Three other variants of M. japonicus crustin-like A PCR product of approximately 250 base pairs peptide were also found (types 3, 4 and 5: GenBank (bp) was amplified using the oligonucleotide AB121742, AB121743 and AB121744, respectively) primers and found to have high homology to the by using specific PCR primers designed on the crustin homolog of L. setiferus and L. vannamei.16 type 1 and 2 nucleotide sequences. The deduced This PCR fragment was then used as a probe for amino acid sequences of these three variants were cDNA library screening. Several positive clones identical to that of type 1 except for the number of were sequenced and two variants (type 1 [GenBank glycine-rich repeats (Fig. 2). The repeat regions in AB121740] and type 2 [GenBank AB121741]) were the type 3, 4 and 5 peptides were 20 residues obtained. Marsupenaeus japonicus crustin-like longer, 16 residues longer and 8 residues shorter, peptide type 1 has a cDNA of 694 nucleotides and respectively, than the repeat region in the type 1 an open reading frame of 573 bp coding for 191 peptide. Type I peptide was the predominant amino acid residues (Fig. 1). The N-terminal seg- molecule among the five crustin-like peptides, as ment included a high proportion of hydrophobic nine of 11 positive clones (81.8%) encoded type I amino acid residues and therefore the first 18 peptide, by screening of the cDNA library. amino acids were predicted to be a signal peptide by the SignalP program. The deduced amino acid sequence of M. japonicus crustin-like peptide Expression analysis of kuruma prawn type 1 contained a number of glycine-rich repeats crustin-like mRNA between positions 28 and 103 in the amino acid sequences. Following the repeat region is a Pro/ The tissue distribution of M. japonicus crustin-like Cys-rich region, which possesses 12 Cys residues peptide mRNA was examined by RT-PCR using that might participate in the formation of 6-disul- universal primers for all of five types. Polymerase fide bonds. Two potential proline-directed arginyl chain reaction products were detected only in cleavage sites were found at the carboxy-terminal hemocytes (Fig. 3). The PCR product was directly

ACTGCAGGCAACCATGAAGGGCTTCAAAGCGGTGGTTCTGTGCAGCCTTCTTGCCTCAGCTTTAGCTGGCAAGCTCCGTTTTGTTCGGAG 90 M K G F K A V V L C S L L A S A L A G K L R F V R S 26

TTTCGGAGGCGGTTACGGAGGCGGGGTAGGGGGTGTTCATGGTGGTGGCTTAGGAAACGGTTTCGGAGGCGTCCAAGGCGGCGGGGTAGG 180 F G G G Y G G G V G G V H G G G L G N G F G G V Q G G G V G 56

AGGTGTTCATGGTGGTGGCTTAGGAAACGGTTTCGGAGGCGGCTTTGGAGGTGGCTTTGGAGGCCCTCAAGGAGGTGGCTTTGGAGGCCT 270 G V H G G G L G N G F G G G F G G G F G G P Q G G G F G G L 86 rtAMP-F TCAAGGAGGTGGCTTTGGAGGCCTTCAAGGAGGTGGCTTAGGTGGTAGTGGGTCGAGCGACTGCAGGTACTGGTGCAAGACCCCAGGGGG 360 Q G G G F G G L Q G G G L G G S G S S D C R Y W C K T P G G 116 rlAMP-F GCAAAACTACTGCTGCGAAAGGTCTCACGAGCCTGAGGGGCCTGTTGGTACCAAGCCACTGGACTGCCCTCAAGTCCGTCCCACTTGCCC 450 Q N Y C C E R S H E P E G P V G T K P L D C P Q V R PT C P146 rtAMP-R ACGCTTCCAGGGCGGGGGCCCCGTAACCTGCTCCAACGACTACAAGTGTGCTGGCATCGATAAATGTTGCTTCGACACGTGTTTGCAAGA 540 RF Q G G G P V T C S N D Y K C A G I D K C C F D T C L Q E 176

ACACGTGTGCAAGCCTCCATCAGTCTTTGGGAAACCTCTTTTCGGATAAAGGGAAAAAAACAAAAGGCTTGACTGACATCCAAGAATTTC 630 H V C K P P S V F G K P L F G * 191

AAATCAGAGTGATCACAACGTCAACTACACAAGAGTAAAACAATCTGTTAAAAAAAAAAAAAAA 694

Fig. 1 Nucleotide and deduced amino acid sequences of Marsupenaeus japonicus crustin-like peptide type 1 (Gen- Bank AB121740), the predominant crustin-like peptide clone isolated from the hemocyte cDNA library. The first methionine (M) is numbered as the first deduced amino acid. The arrowhead (5) indicates the putative signal cleavage site. Cysteine residues that may participate in the formation of disulfide bonds are in bold. Potential proline-directed arginine cleavage motives are underlined. The stop codon is indicated by an asterisk (*). Arrows indicate primers used in the study. Crustin-like peptide cDNA of kuruma prawn FISHERIES SCIENCE 769

crustin-like peptide type 1 1:MKGFKAVVLCSLLASALAGKLRFVRSFGGGY------GGGVGGVHG 40 crustin-like peptide type 2 1:MKGFKAVVLCSLLASALAGKLRFVRSFGGGYGGGVGGVHGGGLGNGFGGVQGGGVGGVHG 60 crustin-like peptide type 3 1:MKGFKAVVLCSLLASALAGKLRFVRSFGGGYGGGVGGVHGGGLGNGFGGVQGGGVGGVHG 60 crustin-like peptide type 4 1:MKGFKAVVLCSLLASALAGKLRFVRSFGGGYGGGVGGVH------GGGVGGVHG 48 crustin-like peptide type 5 1:MKGFKAVVLCSLLASALAGKLRFVRSFGGGY------GGGVGGVHG 40 ******************************* ********* crustin-like peptide type 1 41:GGLGNGFGGVQGGGVGGVHGGGLGNGFGGGFGGGFGGP------QGGGFGGLQGGGFG 92 crustin-like peptide type 2 61:GGLGNGFGGVQGGGVGGVHGGGLGNGFGGGFGGGFGGPQGGGFGGLQGGGFGGLQGGGFG 120 crustin-like peptide type 3 61:GGLGNGFGGVQGGGVGGVHGGGLGNGFGGGFGGGFGGP------QGGGFGGLQGGGFG 112 crustin-like peptide type 4 49:GGLGNGFGGVQGGGVGGVHGGGLGNGFGGGFGGGFGGPQGGGFGGLQGGGFGGLQGGGFG 108 crustin-like peptide type 5 41:GGLGNGFGGVQGGGVGGVHGGGLGNGFGGGFGGGFGGP------QGGGFG 84 ************************************** ****** crustin-like peptide type 1 93:GLQGGGLGGSGSSDCRYWCKTPGGQNYCCERSHEPEGPVGTKPLDCPQVRPTCPRFQGGG 152 crustin-like peptide type 2 121:GLQGGGLGGSGSSDCRYWCKTPGGQNYCCERSHEPEGPVGTKPLDCPQVRPTCPRFQGGG 180 crustin-like peptide type 3 113:GLQGGGLGGSGSSDCRYWCKTPGGQNYCCERSHEPEGPVGTKPLDCPQVRPTCPRFQGGG 172 crustin-like peptide type 4 109:GLQGGGLGGSGSSDCRYWCKTPGGQNYCCERSHEPEGPVGTKPLDCPQVRPTCPRFQGGG 168 crustin-like peptide type 5 85:GLQGGGLGGSGSSDCRYWCKTPGGQNYCCERSHEPEGPVGTKPLDCPQVRPTCPRFQGGG 144 ************************************************************ crustin-like peptide type 1 153:PVTCSNDYKCAGIDKCCFDTCLQEHVCKPPSVFGKPLFG 191 crustin-like peptide type 2 181:PVTCSNDYKCAGIDKCCFDTCLQEHVCKPPSVFGKPLFG 219 crustin-like peptide type 3 173:PVTCSNDYKCAGIDKCCFDTCLQEHVCKPPSVFGKPLFG 211 crustin-like peptide type 4 169:PVTCSNDYKCAGIDKCCFDTCLQEHVCKPPSVFGKPLFG 207 crustin-like peptide type 5 145:PVTCSNDYKCAGIDKCCFDTCLQEHVCKPPSVFGKPLFG 183 ***************************************

Fig. 2 Multiple alignment of the deduced amino acid sequences of all identified Marsupenaeus japonicus crustin-like peptides. The signal peptide sequences are shown in italic letters at the N-terminal part. Cysteine residues are indicated in bold. Residues that are identical in all sequences are indicated by asterisks (*). sequenced and found to have the same sequence copy number of crustin-like peptide after 7 days of as the cloned cDNA. Polymerase chain reaction PG feeding was significantly different from that of products using the b-actin primer set were day 0 (P < 0.05). observed in all tested tissues. In northern blot hybridization using total RNA from hemocytes, a cDNA probe containing a common sequence of all DISCUSSION five types hybridized to a single band (Fig. 4). The size of this band was approximately 700 bp (Fig. 4), This is the first report of cDNA cloning of a crustin- which is consistent with the lengths of the five like peptide in kuruma prawn M. japonicus and crustin-like peptide cDNA. characterization of its gene expression. Five cDNA variants of the crustin-like peptides (types 1–5) were found that differed in the number of glycine- Kinetics of kuruma prawn crustin-like peptide rich repeats at the amino-terminal region. The mRNA expression deduced amino acid sequences of these five variants show high similarity to those of L. setiferus The effect of PG on the expression of crustin-like (80% identity), L. vannamei (80% identity) and peptide mRNA was examined by real-time PCR C. maenas (44% identity) crustins. All the variants using universal primers. The average copy num- contain a putative signal/leader sequence, a gly- bers of M. japonicus crustin-like peptide mRNA cine-rich repeat region and a carboxy-terminal before (day 0) and after PG feeding for 1, 3 and region, which are also present in the white prawn 7 days were 2.00 ¥ 104, 4.24 ¥ 105, 1.96 ¥ 106 and crustins.16 In addition, the carboxy-terminal region 9.57 ¥ 106, respectively. Marsupenaeus japonicus of the kuruma prawn contains conserved crustin-like peptide expression was gradually sequences that have putative proteinase inhibitory induced after PG feeding (Fig. 5). The copy number activity, which is also found in shore crab and white of crustin-like peptide after 1, 3 and 7 days of PG prawn crustins.14,16 Kuruma prawn crustin-like feeding was found to be 28.86-, 137.02- and 672.90- peptide possess 12 cysteine residues at the car- fold higher, respectively, than that of the control boxy-terminal region and their positions are the (day 0, unstimulated group). However, only the same as those of shore crab and white prawn 770 FISHERIES SCIENCE A Rattanachai et al.

(Kb) 1 2 3 4 5 6 7 8 9

2 Fig. 3 Tissue distribution of 2.3 Marsupenaeus japonicus crustin- 0.5 Crustin-like peptide like peptide mRNA. Lane 1, hemocytes; lane 2, heart; lane 3,

2 hepatopancreas; lane 4, gill; 2.3 lane 5, fore-gut; lane 6, mid-gut; 0.5 b-actin lane 7, muscle; lane 8, subcuticular epithelium; lane 9, ovary.

(Kb) Copy number

1.00E+08

* 1.00E+07

1.00E+06 Crustin-like peptide 1 1.00E+05 0.5 1.00E+04 0 day 1 day 3 days 7 days

Fig. 5 Expression pattern of Marsupenaeus japonicus crustin-like peptide mRNA in hemocytes from M. japonicus administered with peptidoglycan (PG). b- actin Shrimps were collected at 0, 1, 3 and 7 days post-PG feeding. Marsupenaeus japonicus crustin-like peptide mRNA Fig. 4 Northern blot hybridization of Marsupenaeus levels were determined by real-time polymerase chain japonicus crustin-like peptide and b-actin using reaction and standardized according to the respective hemocyte total RNA. b-actin mRNA levels. Data are presented as mean ± standard error (n = 3). The y-axis uses a logarith- mic scale. Points marked with an asterisk (*) indicate crustins. This indicates that these cysteine residues values significantly different from that of day 0 (P < 0.05, might be important for antimicrobial activity. Dunnett’s multiple comparison test). Six variants of crustin-like peptide were found in L. vannamei and three were found in L. setiferus.16 such as mussels, shrimps and horseshoe crabs.25–27 In another group of prawn antimicrobial peptides, The presence of crustins in hemocytes might con- the penaeidins, four variants were found from tribute to the fact that hemocytes are the main site these two shrimp species.13 This suggests that anti- for precursor production of antimicrobial peptides microbial peptides of shrimp exist in multiple cop- in these invertebrate species. Moreover, in crusta- ies in contrast to human antimicrobial peptides cean species, hemocytes are systemically circu- such as defensin and cathelicidin, which are lated and are able to target pathogenic agents; thus encoded by single copy genes.24 The presence of they efficiently provide antimicrobial peptides in multiple copies of antimicrobial peptides in response to invading pathogens or they activate shrimp could mean that they exist as pseudogenes the host’s biodefense mechanisms. or that the antimicrobial peptides in shrimps are Previously, there have been several reports on more important than in humans. Shrimps do not the effect of immunostimulants, including PG, for have a sophisticated immune system and must rely prevention of fish and shellfish infectious disease.3 only on innate immunity, and whether the differ- Orally administered PG derived from Bifidobacte- ent types of antimicrobial peptides like penaeidins rium thermophilum has been shown to enhance and crustins are expressed simultaneously or in resistance to Vibrio penaeicida, a highly virulent subsets is unknown. bacterium in shrimps.22 In that study, PG stimula- The expression of M. japonicus crustin-like tion increased the phagocytic index, but the peptide mRNA was observed in hemocytes in the molecular factors responsible for the increased present study. This expression pattern is also found resistance were not elucidated. In the present in the antimicrobial peptides of other invertebrates study, it had also been shown through real-time Crustin-like peptide cDNA of kuruma prawn FISHERIES SCIENCE 771

PCR that crustin-like peptide expression in 13. Cuthbertson BJ, Shepard EF, Chapman RW, Gross PS. hemocytes of kuruma prawns is induced by Diversity of the penaeidin antimicrobial peptides in two administration of PG. This explains the possible shrimp species. Immunogenetics 2002; 54: 442–445. role of PG in the enhancement of some of the genes 14. Relf JM, Chisholm JRS, Kemp GD, Smith VJ. Purification and responsible for the innate immune response in characterization of a cysteine-rich 11.5-kDa antibacterial protein from the granular hemocytes of the shore crab, shrimps. Crustin-like peptides, together with the Carcinus maenas. Eur. J. Biochem. 1999; 264: 350–357. other inducible genes, likely contribute to the 15. Larsen M, Ressler SJ, Lu B, Gerdes MJ, McBride L, Dang TD, enhancement of resistance to infectious diseases Rowley DR. Molecular cloning and expression of ps20 by immunostimulants. growth inhibitor-A novel WAP-type for-disulphide cor domain protein expressed in smooth muscle. J. Biol. Chem. 1998; 273: 4574–4584. ACKNOWLEDGMENT 16. Bartlet TC, Cuthbertson BJ, Shepard EF, Chapman RW, Gross PS, Warr GW. Crustins, homologues of an 11.5-kDa This research was supported in part by a Grant-in- antibacterial peptide, from two species of penaeid shrimp, Aid for Scientific Research (S) from the Ministry of Litopenaeus vannamei and Litopenaeus setiferus. Mar. Education, Culture, Sports, Science, and Technol- Biotechnol. 2002; 4: 278–293. ogy of Japan. 17. Gross PS, Bartlet TC, Browdy CL, Chapman RW, Warr GW. Analysis of tissues specific expressed sequence tag libraries from the hemocytes and hepatopancreas from two related REFERENCES litopenaeid species, the Pacific white shrimp, Litopenaeus vannamei, and the Atlantic white shrimp, L. setiferus. Dev. 1. Lightner DV, Redman RM. Shrimp disease and current diag- Comp. Immunol. 2001; 25: 565–577. nostic methods. Aquaculture 1998; 164: 201–220. 18. Sambrook I, Russel D. Molecular Cloning: A Laboratory 2. Takahashi Y, Itami T, Maeda M, Kondo M. Bacterial and Manual, 3rd edn. Cold Spring Harbor Laboratory Press, viral diseases of kuruma shrimp (Penaeus japonicus) in New York. 2001. Japan. Fish Pathol. 1998; 33: 357–364. 19. Altschul SF, Madden TL, Schofer AA, Zhang Z, Miller W, 3. Sakai M. Current research status of fish immunostimulants. Lipman DJ. Gapped BLAST and PSI-BLAST: a new genera- Aquaculture 1999; 172: 63–92. tion of protein database search programs. Nucleic Acids Res. 4. Lehrer RI, Ganz T. Antimicrobial peptides in mammalian 1997; 25: 3389–3402. and insect host defence. Curr. Opin. Immunol. 1999; 11: 23– 20. Nielsen H, Engelbrecht J, Brunak S, Heijne G. Identification 27. of prokaryotic and eukaryotic signal peptides and prediction 5. Fenouil E, Roch P. Evidence and characterization of of their cleavage sites. Protein Engineering 1997; 10: 1–6. lysozyme in six species of freshwater crayfishes from Ast- 21. Hirono I, Nam B, Kurobe T, Aoki T. Molecular cloning, char- acidae and Cambaridae families. Comp. Biochem. Physiol. acterization, and expression of TNF cDNA and gene from 1991; 99B: 43–49. Japanese flounder Paralichthys olivaceus. J. Immunol. 2000; 6. Destoumieux D, Bulet P, Loew D, Dorsselaer AV, Rodriguez 165: 4423–4427. J, Bachere E. Penaeidins, a new family of antimicrobial 22. Itami T, Asano M, Tokushige K, Kubono K, Nakagawa A, peptides isolated from the shrimp Penaeus vannamei Takeno N, Nishimura H, Maeda M, Kondo M, Takahashi Y. (Decapod). J. Biol. Chem. 1997; 272: 28 398–28 406. Enhancement of disease resistance of kuruma shrimp, 7. Destoumieux D, Saulnier D, Garnier J, Jouffrey C, Bulet P, Penaeus japonicus, after oral administration of peptidogly- Bachere E. Crustacean immunity: antifungal peptides are can derived from Bifidobacterium thermophilum. Aquacul- generated from C-terminus of shrimp hemocyanin in ture 1998; 164: 277–288. response to microbial challenge. J. Biol. Chem. 2001; 276: 47 23. Park C, Kurobe T, Hirono I, Aoki T. Cloning and character- 070–47 077. ization of cDNAs for two distinct tumor necrosis factor 8. Smith VJ, Chisholm JRS. Antimicrobial proteins in crusta- receptor superfamily genes from Japanese flounder Parali- ceans. Adv. Exp. Med. Biol. 2001; 484: 95–112. chthys olivaceus. Dev. Comp. Immunol. 2003; 27: 365–375. 9. Stabili L, Miglietta AM, Belmonte G. Lysozyme-like and 24. Hancock REW, Diamond G. The role of cationic antimicro- trypsin-like activities in the cysts of Artemia franciscana bial peptides in innate host defences. Trends Microbiol. Kellog, 1906: Is there a passive immunity in a resting stage? 2000; 8: 402–410. J. Exp. Mar. Biol. Ecol. 1999; 237: 291–303. 25. Mitta G, Vandenbulcke F, Roch P. Original involvement of 10. Haug T, Kjuul AK, Stensva K, Sandsdalen E, Strvold OB. antimicrobial peptides in mussel innate immunity. FEBS Antibacterial activity in four marine crustacean decapods. Lett. 2000; 486: 185–190. Fish Shellfish Immunol. 2002; 12: 371–385. 26. Destoumieux D, Munoz M, Cosseau C, Rodriguez J, Bulet P, 11. Hikima S, Hikima J, Rojtinnakorn J, Hirono I, Aoki T. Char- Comps M, Bachere E. Penaeidins, antimicrobial peptides acterization and function of kuruma shrimp lysozyme with chitin-binding activity, are produced and stored in possessing lytic activity against Vibrio species. Gene 2003; shrimp granulocytes and released after microbial challenge. 316: 187–195. J. Cell Sci. 2000; 113: 461–469. 12. Sotelo-Mundo RR, Islas-Osuna MA, Re-Vega E, Hernandes- 27. Shigenaga T, Muta T, Toh Y, Tokunaga F, Iwanaga S. Anti- Lopez J, Vargas-Albores F, Yepiz-Plascencia G. cDNA clon- microbial peptide tachyplesin peptide precursor: cDNA ing of the lysozyme of the white shrimp Penaeus vannamei. cloning and cellular localization in the horseshoe crab (Tac- Fish Shellfish Immunol. 2003; 15: 325–331. hypleus tridentatus). J. Biol. Chem. 1990; 265: 21 350–21 354.