K. T sukam oto , T. K aw anuira, T. Takeuchii, T. D. B eard, Jr. a n d M. J. Kaiser, eds.Fisheries fo r C lo b a l W elfare a n d E nvironm ent, 5 th W o rld Fisheries Congress 200 8 , pp . 221 -2 3 9 .© b y TERRAPUB 2 0 0 8 .

Biotechnology of Marine Invertebrates —Recent Advances in Shrimp

and Shellfish

Anchalee Tassanakajon,1 Tipachai Vatanavicharn,1 Premruethai Supungul,1'2 Sureerat Tang,2 Piti A m paryup,1'2

Kunlaya S om boonw iw at,1 Sirinit Tharn tada,1 Jun Takahashi3 and Haruhiko Toyohara3*

'S h rim p M o le c u la r B io lo g y a n d G enom ics L ab o ra to ry D e p a rtm e n t o f B io ch e m is try , Facu lty o f Science

C hu la lo n g ko rn U n ive rs ity , B angkok 10330, T ha iland

2N a tio n a l C en te r fo r G e n e tic E ng ineering a n d B io te c h n o lo g y (BIOTEC)N a tio n a l S cience a n d T e ch n o lo gy D e v e lo p m e n t A g e n c y (N S TD A )

P a th u m th an i 12120, T ha iland

3D iv is io n o f A p p lie d B iosc iences, G radua te S c h o o l o f A g r ic u ltu re K yo to U nive rs ity , K yo to 6 08 -8502 , ja p a n

* E-mai I : toyohara@ kai s .kyo to -u .ac.j p

M arine inver tebra tes possess a var ie ty of interesting func tions a n d s o m e of th em have b e e n well s tud ied from the s ta n d p o in t of b io techno logy . Shrimp and shellfish are useful animals a m o n g m arine invertebrates . In this sec tion , w e d e sc r ib e the rece n t ad v an c es in biological d e fen se system of shrim p and the b iom ineral iza tion system of shellfish as exam ples of w h ich m olecu la r m ec h an ism s have b e e n well e luc idated .

First, w e fo cu s on shrimp antimicrobial p e p t id e (AMPs) w hich are c o n s id e red to play a m ajor role in the innate im m unity against invading m icrobes . A total of 767 ESTs e n c o d in g pu tat ive AM Ps w e re identified in th e Penaeus m onodon EST d a ta b a s e (h t tp : / /p m o n o d o n .b io t e c .o r . t h ), w h ich w e re co m p r ise d of h o m o lo g u e s of the th ree AMP families; penaeid ins, crustins and antil ipopolysaccharide factors. S e q u e n ce analysis revealed that e ach AMP family had s e q u e n c e diversity and const itu ted multiple isoforms or subgroups . The re levance of this s e q u e n c e variabil ity to the potentia l antimicrobial func tion w a s investigated. T hese shrimp AM Ps with high se lectivity to severe p a th o ­gens are po ten tia l c a n d id a te s as an alternative to antibiotics in aquacu ltu re .

As a se c o n d exam ple , w e p re sen t the rece n t a d v an c es in the m olecu la r m e c h a ­nism of shell form ation . A varie ty of animals and p lants have th e ability to m ake hard biological s truc ture called biominerals m a d e of calcium, silicon and o th e r minerals. M olluscan shell is a typical biomineral c o m p o s e d of C a C 0 3, b u t a small a m o u n t of organ ic matrix p ro te ins included in the shell d irec t th e crystal growth th a t is specific for the shell spec ies . C a 2+ a d so rb e d th rough specific t ranspor te rs and EHC032~ syn the­sized from C O , a re mixed u n d e r well regu la ted condit ions , th e reb y fo rm ing shell.

KEYW ORDS an ti l ipopolysaccharides; antimicrobria l pep tid e s ; biomineral; calcifica­tion; crustins; p enae id ins ; shell

222 A. T a s s a n a k a j o n e t al.

1. Shrimp Antimicrobial Peptides: Sequence Diversity and Functional

Characteristics of Different Isoforms

1.1. Introduction

Organisms lacking an adaptive immunity rely on antimicrobial peptides (AMPs) as the first line of defense against invading patho­gens (Hancock and Diamond 2000; Jenssen el al. 2006). AMPs are typically small cati­onic molecules (15 to 100 amino acids) which are considered to play an important role in the innate immune system. They are widely distributed in the whole living king­dom ranging from bacteria to plants, inver­tebrate and vertebrate species, including mammals. They display a broad spectrum of activity against bacteria, fungi, viruses, para­sites and even tumor cells. Although they differ in amino acid sequences, most AMPs adopt an amphipathic secondary structure that is believed to be essential for their anti­microbial action.

Numerous AMPs are found in inverte­brates, particularly in insects, where they play a major role in protection from invad­ing pathogens (Bulet and Stocklin 2005). In crustaceans, AMPs were first described in tlie hemocytes of the shore crab Carcinus maenas (Schnapp et al. 1996) as a 6.5 kDa proline-rich cationic protein displaying ac­tivity against both gram-positive and gram- negative bacteria. Not until 1997 was the first shrimp AMP family, named penaeidin, discovered in the Pacific white shrimp, Litopenaeus vannamei, by means of reverse- phase chromatography (Destoumieux el al. 1997). Subsequently, penaeidin sequences have been found in several penaeid shrimps by genomic approaches (Cuthbertson el al. 2002; Kang et al. 2004; Supungul el al. 2004). The other two shrimp AMP families, tlie cmstins and antilipopolysaccharide fac­tors (ALFs), have been identified from haemocyte cDNA libraries of several shrimp

species. The cmstins, which are homologues of the shore crab 11.5 kDa antibacterial pro­tein, were identified by expressed sequenced tag (EST) analysis from L. vannamei and L. setiferus (Bartlett et al. 2002; Vargas- Albores et al. 2004). Since then, cmstins and cmstin-like peptides have been identified from a variety of other species (Rattanachai et al. 2004; Hauton el al. 2006; Brockton el al. 2007; Jiravanichpaisal el al. 2007; Zhang el al. 2007; Supungul el al. 2008). Antilipo­polysaccharide factors (ALFs), first reported in horseshoe crabs (Morita et al. 1985; Aketagawa el al. 1986; Muta el al. 1987), have also been identified in shrimps (Supungul el al. 2004; Liu el al. 2005; Somboonwiwat el al. 2005). Moreover, an anionic antimi­crobial peptide, astacidin, which is derived from the limited proteolysis of hemocyanin, has been reported in L. vannamei (Destoumieux-Garzón et al. 2001). In this paper, we present a recent finding on the se­quence diversity of cationic AMPs identified from tlie black tiger shrimp Penaeus monodon database (http://pmonodon.biotec.or.th), their antimicrobial properties and potential application for disease control in aquaculture.

1.2. AMPs identified from the Penaeus monodon EST Database

As of December, 2007, tlie Penaeus monodon EST database (http://pmonodon.biotec.or.tli) contained a total of 40,001 EST sequences. Approximately 30,000 additional ESTs were obtained after the first establishment of tlie database according to the report by Tassanakajon el al. (2006). Clustering and sequence assembly identified 10,536 unique sequences represented by 3,227 contigs and 7,309 singletons. Each unique sequence was subjected to similarity searches against tlie GenBank database using BLASTx and BLASTn with an e-value cut off of <10 4. Of 10,536 unigenes, 5,648 (53.6%) showed a significant match whereas 4,888 (46.4%) did not match to any sequences in tlie database.

B io techno logy o f shrim p and shellfish 223

Among tlie known (annotated) genes, a total of 767 clones were identified as putative antimicrobial proteins. They were homo­logues of the three AMP families; penaeidins (284 clones), crustins (275 clones) and antilipopolysaccharide factors (208 clones). Sequence analysis revealed that each family of AMPs displays sequence diversity and constitutes multiple isofonns or subgroups (Table 1 ). The sequence and functional di­versity of the different isoform s of P. monodon AMPs are described below.

1.3. Penaeidins

The shrimp AMPs in tlie penaeidin family contain a unique structure comprised of an N-terminal proline-rich domain and a C- terminal cysteine-rich domain with six con­served cysteine residues engaged in three disulfide bonds. Penaeidins 1,2 and3 (PENI, -2 and -3) were first isolated from haemocytes of die white shrimp L. vannamei (Destoumieux el al. 1997). Subsequent phylogenetic analy­sis indicated that LitvanV\ IN I is a variant of LitvanPENl (Cuthbertson el al. 2002). To date, penaeidin sequences have been identi­fied from different penaeid shrimps by ge­nomic approaches and can be classified into four different subgroups (PEN2, -3, -4 and -5) based on their primary sequence diver­sity. The conserved key residues that appear to be a signature for PEN-2, -3 and -4 have been established in PenBase, http://www. penbase.immunaqua. com (Gueguen el al.2006) while tlie distinct sequence character­istics of PEN-5, recently identified in Fenneropenaeus chinensis, have also been described by Kang el al. (2007).

From the Penaeus monodon EST data­base, 284 ESTs representing penaeidins were identified and subjected to sequence analy­sis. Based on the sequence lengths and tlie conserved signature residues, they were clas­sified into two d ifferent subgroups: Pen?nonPEN3 and -5 (Fig. 1). The major penaeidin sequences (260 clones) found were classified as PEN3 whereas PEN5 sequences

T ab le 1. T he a b u n d a n c e o f p u ta tiv e an tim i­c ro b ia l p e p tid e s in th e Penaeus m o n o d o n EST d a ta b a s e (h t tp : / /p m o n o d o n .b io te c .o r .th ).

A n tim ic ro b ia l p e p t id e F req u e n c y

A n til ip o p o ly s a c c h a r id e fa c to rs 2 0 8

ALF Pm 1 1

ALF Pm2 2

AFF Pm 3 18 4

AFF Pm 4 1

AFF Pm 5 2

AFF Pm 6 16

AFF Pm 7 2

AFF Pm 8 1

C ru s tin s 2 7 5

c ru s t in P m l 55

c ru s tin P m 2 * 2

c ru s tin P m 3 1

c ru s tin P m 4 15 8

c ru s tin P m 5 1

c ru s tin P m ó 2

c ru s t in P m 7 (c ru s-lik eP m ) 56

P e n a e id in s 2 8 4

P e n m o n Pen3 2 6 0

P e n m o n Pen5 2 4

" n o t full len g th

(24 clones) were found much less frequently. PenmonV\ i \3 and -5 sequences encode pre­dicted peptides of 74 and 79 amino acid residues, respectively, including a signal peptide of 19 amino acids. All PenmonV\ 1X3 predicted peptide sequences possess the fol­lowing signature: Gin1, Gly5, Arg13 Gly18, Ser35, His37, Gin43 and Ala46, similar to the conserved key residues of PEN3 described by Gueguen el al. (2006). However, PenmonVEN5 sequences do not contain all the reported conserved key residues (Gin1, Ser5, Arg13, Ser18, Arg37, Gin43 and Ala46) pro­posed by Kang el al. (2007), but show

224 A. Tassanakajon et ai

1 .................. 1 0 ......................... 2 0 ........................ 3 0 ...................... 4 0 ...................... 5 0 .............

_____________ t t t t ,i234567' ▼* t tPenmon P E N 3 -1 HRLWCLVFLASFALVCQA QGYCGGYXRPFPRPTÏGGG-----------YKgVPV CTSCHRLST1 jAPACCRQLGRCCL-AK2TYG '4Penmon PE N 3 -3 MRLWCLVFLASFALVCQA QGY'.GGYTRPFr RPPYG '1 -----------YHlVPV CISCH FL7F: jAFACCRQ: GRCCDAKQTyI 74Penmon P E N 3-2 MRLWCLVFLASFALVCQA QGYcGÖfflRPFI RPPYG G -----------YHgVPV C ÏSC B R LSFL Q A R ä O C I^ L R ||Ä d |k q tY* 14Penmon P E N 3-4 MRLWCLVFLASFALVCQA QGYcGGWRPFPKPPYG ------------- YHBVPV CTSCHRLSFL'JARÄOCROLRROCI>AKQTYQ 74Penmon P E N 3 -5 MRLWCLVFLASFALVCQA QGYcGGYTh i F ZgPPYQGG-----------YH.fVPV CT SCH M I L j A ACCRQ LGRCC D§K J T Y Q 74

Penmon P E N 5-1 jlRLW CLVFL . SFALVCQ" QGYKGGYT RPPYG R P IN T R P IS R l Penmon P E N 5-2 MRLWCLVFL-SFALVCQ QGYKG6YTGSYÍ RPPYG R P IST R P ISR « Pen/non P E N 5 -3 MRLWCLVFL'/SFALVCQ* QGYK GGYTC S Y S RPPYG SP PI S TR PI S R |

iTG C T SC H TIT FD K jlveC R Q FG R O C "A l.K --| 7 9 iTG ÇTSCHTITFDKAl .CCRQF GRCC :A L K -- | 7 9 WG CTSCHT’ TTFnKATACCRQFGROC-A’ 7 9

Signal peptide Prolina-rich domain Cyataina-rich domain

Fig. 1. P re d ic te d a m in o a c id s e q u e n c e c o m p a r is o n o f PEN3, -5 a n d th e ir v a rian ts from Penaeus m o n o d o n . T he full s e q u e n c e s o f th e p e n a e id in s w e re a lig n ed u sin g C lu sta lW a n d g a p s w e re in tro d u c e d to o p tim iz e th e a lig n m en t. C y ste in e s a re in b o ld fa c e , id en tica l re s id u e s a n d c o n s e rv a ­tive re p la c e m e n ts a re s h a d e d . T he aste risk (*) in d ic a te s a m in o ac id id e n tify a n d (.) a n d (:) in d ic a te a m in o a c id sim ilarity . T he a rro w s in d ic a te c o n s e rv e d re s id u e s o f p e n a e id in s u b g ro u p s .

variations at Gly5 and His37 which are iden­tical to tlie residues in PEN3, and at Ser18 which is a specific residue in the subgroup 5 and was found in all PenmonPEN5 se­quences. Variants of Penm onPEN3 (PenmonPEN3-l to PenmonPEN3-5) and PenmonPEN5 (Penm onPEN 5-l to PenmonPEN5-3) show polymorphisms at four and two amino acid residues, respec­tively (Fig. 1 ). It should also be noted that tlie penaeidin-5 cDNA from P. monodon re­ported previously by Hu el al. (2006) is iden­tical to PenmonPEN3-3 reported here. Ac­cording to the conserved key residues for PEN3 proposed in PenBase by Gueguen el al. (2006) and those proposed for PEN5 by Kang el al. (2007), this cDNA sequence should be classified as a PEN3 isoform and not PEN5. Analysis of penaeidin sequences through PenBase, http://www. penbase. immunaqua.com, would allow a systematic nomenclature and classification of the penaeidin family and prevent confusion with tlie increasing number of independently de­rived penaeidin sequences from a variety of shrimp species.

In L. vannamei and L. setiferus, genes from each of the three penaeidin subgroups (PEN2, -3, and -4) have been found and shown to be expressed in a single individual

(Cuthbertson et al. 2008), whilst hi P. monodon only two subgroups (PEN3 and -5) were found with tlie expression level of tlie later being much less abundant. This probably indicates that PenmonPEN5 is not constitu- tively expressed but rather may be regulated in response to certain physiological stimuli. Further expression studies are needed to test tins notion.

In general, penaeidins possess antibac­terial activity predominantly directed against gram-positive bacteria and antifungal activ­ity against filamentous fungi. PEN2 and -3 from L. vannamei both exhibit similar anti­microbial properties although PEN3 is more effective at low concentrations (Destoumieux el al. 1999). In L. setiferus, the antimicro­bial properties of PEN3 and -4 were com­pared and it was found that LitsetP\ i \3 has a broader range of microbial targets whilst LrisetPENT is generally more effective against fungi (Cuthbertson et al. 2004). FenchiPEN 5 displayed activities against gram-positive and gram-negative bacteria and fungi (Kang et al. 2007). Since antimi­crobial properties of the four different sub­groups of penaeidins have been well char­acterized by several research groups, we do not further characterize PenmonPEN3 and -5. The structure and function of penaeidins

B io techno logy o f shrim p and shellfish 225

have been subjected to extensive review elsewhere (Bachère el al. 2000; Cuthbertson et al. 2008).

1.4. Crustins

Crustins are homologues of ‘carciniiT, an 11.5 kDa antibacterial protein from the shore crab Carcinus maenus. The cDNA of crustins have been reported from a variety of crusta­ceans including L. vannamei (Bartlett el al. 2002; Vargas-Albores et al. 2004), L. setiferus (Bartlett et al. 2002), P. monodon (Supungul et al. 2004), Marsupenaeus japon icus (R attanachai et al. 2004), Fenneropenaeus chinensis (Zhang et al.2007), Panulirus argus (Stoss et al. 2004), Homarus gammarus (Hauton et al. 2006), C. maenas (Brockton et al. 2007) and Pacifastacus leniusculus (Jiravanichpaisal et al. 2007). Those crustins that have been described to date have diverse amino acid sequences but with a relatively conserved C-terminus of 12 cysteine residues includ­ing a single whey acidic protein (WAP) do­main. The WAP domain generally consists of 50 amino acid residues with eight cysteine residues at defined positions. They form four intracellular disulfide bonds creating a tightly packed structure (Griitter el al. 1988). The WAP domain-containing proteins are wide­spread throughout vertebrates and inverte­brates and have diverse biological functions including antibacterial activity and protease inhibitory activity (Sallenave 2000; Hagiwara et al. 2003).

From the Penaeus monodon EST data­base, 275 ESTs representing crustins were identified. Clustering analysis revealed tlie presence of five isoforms including the four isoforms reported previously (Crus/’/n 1—I) (Supungul el al. 2004). The amino acid se­quence alignment of the full length se­quences of all isoforms except crustin/7n2 suggested that isoform 3 is a subset of isoform 4. Each isoform was found at a dif­ferent frequency (Table 1 ), and sequence variation was also observed within each

isoform suggesting both differential expres­sion and potential functional polymorphisms. The crus tin isoforms are very different in length and primary sequence and they are tlie most diverged representatives of tlie AMP families in the species. Alignment of the crus tin sequences revealed very diverse amino-terminal signal peptide and glycine- rich regions whilst tlie carboxyl-terminal re­gion, where the WAP domain resided, is more conserved (Fig. 2). CmstinP/nT, one of the major isoforms, contained a very long glycine-rich repeat and this isoform is 92% identical at the amino acid sequence level to the P. monodon eras tin reported by Chen el al. (2004). All isofonns contain 12 conserved cysteine residues in the C-tenninus that par­ticipate in the formation of six disulfide bonds. The phylogenetic analysis of WAP domains from various invertebrate WAP do- main-containing proteins showed a high di­versity and could be divided into three dis­tinct groups (Fig. 3). The first group com­posed of crustacean single WAP proteins. The second group contained crab carcinins, lobster and crayfish crustins. The last group contained all the shrimp crustins including all isoforms of P. monodon crustins. How­ever, CrastinPw77, later named the crus­like/bn, is quite distinct from the other isoforms and formed a subgroup with a eras tin-like protein Fc 1 from F. chinensis (Zhang el al. 2007). The difference of tlie WAP domains may indicate different biological activity of the proteins.

Expression analysis in various shrimp tissues revealed different patterns of expres­sion of different crastin isofonns. For exam­ple, crustin/bnl was expressed in all tissues examined including haemocytes, lymphoid organs, gili, intestine, eyestalk, hepatopan- creas, epipodite and antemial gland whereas the cmtinP«75 transcript was observed only in epipodite and eyestalks (data not shown). The difference in tissue distribution of the crastin transcripts probably indicates diverse functions of crustins as immune effectors,

226 A. Tassanakajon et ai

c r u s t i n ñ n l c ru s tin F m 6 c ru s t in F m 3 , 4 c ru s tin F m 5 c r u s - l ik e P m

c ru s t in F ra l c ru s tin P m 6 c ru s t in F m 3 , 4 c ru s tin P m 5 c ru s - l ik e P m

M K G L G V IL F C -V L A M A S A - M K G V SV A IL C C V L A A A S A - M K G V K A V IL C S L L A T A L A - M RV A G Y LW A V A SV A V TD G M L K F W L S W A V A V V H A - - *

G K FRG SGTYG GGG SYG GG GSY GG GLG GG VSG GG LGG GV NGG

----------------------------------------------------------------------------------------------------------------------------------------------------- 1 7 1 8

GLGG GVNGGGLGGGLGGGVNGGGLGGGGAYGGGLGGGLGGGGVNGGGLGGGLGGGVQGGG 1 1 9------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Q Y 2 1

----------------------------------------------------------------------------------------------------------------------------------------------------- 1 7

c ru s t in P m l c ru s tin F m 6 crustin iR m S, 4 c ru s tin F m 5 c r u s - l ik e P m

-------------------------- QSW H G G RPG ----------- G F P G ---- G G R P --------G G F PG G G — R P G G R P — G G F P - N E R Q G SN R F F N G G F G G G V N S G F G G G F G G G F P G -A G G F Q G G G — F P G G F G G IG G F P GLG G G L G G G V H G G G LG G G LG G G V H G G G LG G G LG G G LS-G G L G G G LG — RP G G G L G R PG G G LIG F G V P G Q G L V D S L N G L IS G G G F P G G H FP G Q G G H F P G Q G G H F P G Q G G N F P G Q G --------------- -Q D K G N A D T R F L G G ---------L G V P G ---- G G V PG V G G G FLPG V --------PG HGG VV PGG GG

517 1

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c ru s t in P m l crustinjR m ß c ru s tin F m S , 4 c ru s tin P m S c r u s - l ik e P m

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F Y — E F F A 1 4 5 F F G 1 6 1FYGRNVKG 2 6 9YYWGR 1 6 9 A G SQ G 1 3 6

Fig. 2. M ultip le a lig n m e n t o f d e d u c e d a m in o a c id s e q u e n c e s o f Penaeus m o n o d o n c ru s tin s. T h e a s te risk (*) in d ic a te s a m in o a c id id en tify a n d (.) a n d (:) in d ic a te a m in o a c id sim ilarity. T he signal p e p tid e s , g lycine rich reg io n s , a n d W A P d o m a in s a re in d ic a te d b y w h ite , gray, a n d b lack b o x es , re sp ec tiv e ly . T h e b o ld s le tte rs c o r re s p o n d to th e c y s te in e rich reg io n s .

although multifunctional roles in develop­ment or differentiation cannot be excluded.

To further investigate the biological function of P. monodon crustins, three isofonns (crustin/V/1, -5 and crus-like/’/n) were selected for the production of recombinant proteins in the E. coli system. The recombinant proteins expressed mainly in insoluble inclusion bodies were solubi­lized, purified and characterized for their antibacterial activity against several strains of gram-positive and gram-negative bacte­ria. Recombinant crus-like/’/)? showed the broadest spectrum of activity against vari­ous strains of bacteria including all five tested gram-positive bacteria (Staphylococcus

aureus, S. haemolyticus, Aerococcus viridans, Bacillus megaterium and Micrococcus luteus) with MIC values ranging from 0.312 to 10 u \ I, and five of the six gram-negative bacteria (Table 2). However, potency against gram negative bacteria ranged from strong (Escherichia coli 363 and Vibrio harveyi with MIC values ranging from to 2.5 to 5 u.\ I), intermediate (Klebsiella pneumoniae) to weak or no activity (E. cloacae and S. thyphimurium). In contrast, the other two isoforms were far less active (Table 2). Recombinant crustin/’/n I was active against gram-positive bacteria with the highest ac­tivity against S. aureus (MIC of 3.13 to 6.25 u.M) and no detectable activity against

B io te ch n o lo g y o f sh rim p and shellfish 227

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c•Ccc >:cc ?idcC r i : C CFG: c

,KHH,DIHKHHt<,GEHf<,QEHGEH'■GEH',QEH,GQH.KEH’,GEHt<,NDTf<,NDT-G R-G R Ù-GKj .<

•Cf<GPI- 51 K-PAH 4 9 K-TAE 5 0 K -P P S 4 9 K -P P S 5 0 C -PPS 4 9 C -PPS 4 9 C -PPS 51 K -P P S 5 3 K -P P S 5 2 C-PAT 5 3 K -P IV 4 9 C -P IV 4 9 L-AVG 4 7 j -A W 47 '-V PQ 4 6

B.elafinHs P 19957

.SWDPm AY464465

.SWDLv AY465833

.carcinin Cm AJ237947729

.crustinP/ CF5423041000549

.crustinHg AJ786653

crus likoPm EF654658

921 .crustinFc DQ097703

cmstinPm3,4 BI018072, C1415873

■crustini.s2 AF430077108592 crustinLcl AF430076

155•crvJStinA4J2 AB121741

•cmstinPm6306

•crustinP/n1 CD766060

765 ■cmstinPm2 BI784444

Fig. 3 . S e q u e n c e a lig n m e n t o f th e W A P d o m a in s in Penaeus m o n o d o n c ru s tin s a n d o th e r c ru s ­ta c e a n W A P d o m a in -c o n ta in in g p ro te in s . SW D , sin g le -w h ey a c id ic d o m a in ; Hs, H o m o sapiens; Pm, Penaeus m o n o d o n ; Lv, L itopenaeus vannam ei; Cm, Carcinus maenas; PI, Pacifastacus len iusculus; H g, H om arus gam m arus; Pc, Fenneropenaeus ch inensis; Ls, L itopenaeus setiferus; M j, M arsupenaeus ja p o n ic u s . (A) C lu sta lW m e th o d . An a s te risk in d ic a tes a m in o a c id id en tity . C o n se rv e d C ys a re s h a d o w e d a n d b o x e d . (B) R o o te d p h y lo g e n e tic tre e c o n s tru c te d b y N e ig h b o r-Jo in in g t re e m e th o d (b o o ts tra p = 1 0 0 0 ). W A P d o m a in o f H o m o sapiens e lafin (e lafinH s) is d e f in e d a s th e o u t g ro u p .

five of the six gram-negative bacteria tested and a weak activity against E. coli 363 (MIC of 50 to 100 ti\ I). Recombinant crustin/’/nô was only active against three of the six strains of gram-positive bacteria (S. aureus, S. haemolyticus and M. latens) with MIC

values ranging from 1.56 to 50 u \ I. Fur­ther studies on tlie mechanism of inhibition of crustin/’/n I (Supungul et al. 2008) and crustin-like Pm (Amparyup 2008) against bacteria has revealed crustins to have bac­tericidal effects. Besides the antimicrobial

228 A. T a s s a n a k a j o n e t al.

T ab le 2 . A n tim icrob ial activ ities o f crus-likePm , c ru s tin P m l a n d c ru s tin P m 5 p e p tid e s ag a in s t g ram -p o sitiv e a n d g ram -n eg a tiv e b a c te r ia in a liquid g ro w th in h ib ition assay

M ic r o o rg a n is m M IC v a lu e (m M )

c ru s t in P m l c ru s -lik e P m c ru s tin P m 5

G ra m (+) b a c te r ia

A e ro c o c c u s v ir id a n s 50-100 0 .3 1 2 -0 .625 >100

S ta p h y lo co cus a u re u s 3 .13 -6 .25 5 -10 0 .7 8 -1 .5 6

S ta p h y lo co cus h a e m o ly t ic u s 50-100 2 .5 -5 12 .5 -25

M ic ro c o c c u s lu te u s 2 5 -5 0 2 .5 -5 2 5 -5 0

Bacillus m e g a te r iu m 6 .2 5 -1 2 .5 0 1.25 -2 .5 >100

G ra m ( - ) b a c te r ia

E n te ro b a c te r c lo a c a e >100 >100 >100

K lebsie lla p n e u m o n ia e >100 1 0-20 >100

S a lm o n e lla th y p h im u r iu m >100 >100 >100

Escherich ia c o li 363 50-100 2 .5 -5 >100

Erw in ia c a ro to v o ra >100 ND >100

V ib rio h a rvey i 639 >100 2 .5 -5 >100

M IC a re e x p re ss e d a s th e in terval a - b , w h e re a is th e h ig h e s t c o n c e n tr a tio n te s te d a t w h ic h m ic ro o rg a n ism s a re g row ing , a n d b th e lo w e st c o n c e n tr a tio n th a t c a u s e d 1 0 0 % g ro w th inh ib i­tio n . ND = n o t d e te rm in e d

activity, the WAP domain is also recognized as a signature motif for a serine protease inhibitor. However, the recom binant crustin/’/n I, -5 and crus-like/’/)? do not pos­ses a detectable protease inhibitory activity (data not shown).

Most crustins reported so far are active against only gram-positive bacteria, except­ing tlie crus-like/’/)? winch also kills some gram-negative bacteria. Although it is quite clear that crustins are antimicrobial proteins, some isofonns winch showed low killing activity may be engaged in other biological functions, or else require different physi­ological conditions or partners to be active. Variations in the response of crastin expres­sion to various pathogens have been ob­served in several shrimps (Supungul et al. 2004; Vargas-Albores et al. 2004; Amparyup et al. 2008) as well as in H. gammarus (Hauton et al. 2006) and P. leniusculus (Jiravanichpaisal et al. 2007). Moreover, we found that the expression of crustin/7n5,

which is a novel crastin recently isolated from a gill-epipodite cDNA library of P. monodon was significantly up-regulated under heat stress when the animals were maintained at 35°C, compared to those at 30°C (unpub­lished data). Inconsistent patterns of corre­lation between expression of crastin isofonns in response to microorganisms and the re­sponse of some crastin isofonns to other physiological stress may indicate potential other functions for these proteins outside of immunity. Further investigation is required to elucidate the trae functions of various crustins from crastacean species.

1.5. Antilipopolysaccharide factors

Antilipopolysaccharide factor (ALF) is a small basic protein originally isolated from hem ocytes of the horseshoe crab, L.polyphem us, (LALF) and Tachypleus tridentatus (TALF) (Tanaka et al. 1982; Aketagawa et al. 1986). LALF binds to and

B io techno logy o f shrim p and shellfish 229

ALFPm6 MRVSVFS-MILWWAASFAPQCQASGWEALVPAIANKLTSLWESGEFELLGHYCSFNVT 59 ALF P m l MRVSVLT-MALTVALAVALPSQCSAAGWGAFMPSIATRLTGLWETGELELLGHYCTYSVK 59 ALF Pm 3 MRVSVLVSLVLWSLVALFAPQCQAQGWEAVAAAVASKIVGLWRNEKTELLGHECKFTVK 60 ALF Pm2 MR--VLVSFLMALSLIALMP-RCQGQGVQDLLPALVEKIAGLWHSDEVEFLGHSCRYSQR 57 ALF Pm8 MTSSTARGMFSLVCFLLIASASARPQLGDILGSLVETFVENAIKTTEITILDNYCLLSRS 60

ALF Pmè PKFKRWQLYFRGRMWCPGWTTIRGQAETRS-RSGWGRTTQDFVRKAFRAGIITESEAQA 118 ALFPm7 PT FQQWQLY FI GSMWCPGWTPIRGVAETRS - RSGVVGKMTQDFVRKALRADLLSKEEAET 118 ALF Pm3 PYLKRFQVYYKGRMWCPGWTAIRG2ASTRS-QSGVAGKTAKDFVRKAFQKGLISQQEANQ 119 ALF Pm2 PSFYRWELYFNGRMWCPGWAPFTGRSRTRS-PSGAIEHATRDFVQKALQSNLITEEDARI 116 ALFPm8 PYLKKFEVHYRAELKCPGWTTIVGKGRDHTNPTNSELAAIKDFIGQALKKGLVTDEEAAQ 120

• # * * * » • * • * ■ *

ALFPm6 WLNN 122 ALF Pm7 WLSH 122ALF Pm3 WLSS 123ALF Pm2 WLEH 120ALF Pm8 YL— 122

• *

Fig. 4. C o m p a ris o n o f th e d e d u c e d a m in o a c id s e q u e n c e s o f ALF cD N A s. T he aste risk (*) indi­c a te s a m in o a c id id e n tity a n d (.) a n d (:) in d ic a te a m in o a c id sim ilarity . T he b o ld le tte rs re p re s e n t th e p u ta tiv e signal s e q u e n c e s . T h e gray b o x in d ic a te s th e p u ta tiv e LPS-binding site.

neutralizes LPS and exhibits an anti-bacterial effect on the growth of the grain-negative bacteria Salmonella minnesota but not on the grain-positive S. aureus (Morita el al. 1985). Since, the discovery of horseshoe crab ALFs, various lipopolysaccharide-binding proteins and derivatives have attracted great interest as candidate therapeutic agents for the man­agement of septic shocks (Vallespi el al. 2000).

In penaeid shrimps, tlie cDNAs of ALF were identified and characterized in Penaeus monodon (Supungul et al. 2002, 2004), L. setiferus (Gross et al. 2001), F. chinensis (Liu et al. 2005) and M. japonicus (Nagoshi et al. 2006). Five different ALF sequences (ALF Pm I-A LF Pm 5) previously reported in P. monodon could be divided into two groups: the Aid 'Pm I and 2 as group A and the ALF P m3, 4 and 5 as group B. Each group has a unique LPS binding site (C R Y SQ R PSFY RW ELY FN G R M W C fo r g roup A andCKFTVKPYLKRFQVYYKGRMWC for group B). Recently, we showed that tlie two groups of P. monodon ALFs are encoded by different genomic loci (Tharntada et al.

2008) and that Aid '¡’m l and3 were the ma­jor or authentic ALFs in the haemocytes whereas the other isofonns may result from aberrant RNA splicing. In addition, sequence variation of the major isolorm, Aid 7V/3, has also been reported (Somboonwiwat el al. 2006).

From tlie Penaeus monodon database, 208 ESTs representing candidate ALF se­quences were identified. Clustering analy­sis indicated the presence of five isofonns, and three of them (Aid77n6, -7 and -8) are novel ALF Pm isofonns rather than abenant RNA splices. Sequence alignment showed that all ALF sequences contained a signal peptide at the N-tenninus and a predicted LPS binding site with clustered positive charges between two conserved cysteine residues (Fig. 4). Interestingly, eachisofonn contained quite a distinct putative LPS-bind­ing site which suggested that they each might have a different ability to bind the microbial cell wall components or to different LPS isoforms.

AI d 7V/2 and -3 isofonns were expressed as recombinant proteins in the yeast Pichia pastoris for further characterization of their

230 A. T a s s a n a k a j o n e t al.

T ab le 3 . A n tim ic rob ia l ac tiv itie s o f ALFPm3, tiVanPEN-3 a n d crus-likePm a g a in s t g ram -p o sitiv e a n d g ram -n eg a tiv e b a c te r ia in a liquid g ro w th in h ib ition a ssay

M ic ro o rg a n ism MIC [|lM]

ALFPm3 L/vanPEN3

(D esto u m ieu x e ta /. 1999)

crus-likePm

G ram -positive b a c te r ia

A e roco ccu s v ir id a ns 1 .5 6 -3 .1 2 0 .3 -0 .6 0 .3 1 2 -0 .6 2 5

Bacillus m e g a te r iu m 0 .1 9 -0 .3 9 2 .5 -5 1 .2 5 -2 .5

M ic ro co ccu s lu teu s 1 .5 6 -3 .1 2 1 .2 5 -2 .5 2 .5 -5

S taphy lococcus aureus 5 0 -1 0 0 >40 5 -1 0

S taphylococus h a e m o ly tic u s ND ND 2 .5 -5

G ram -negative b a c te r ia

E n te robacte r c loacae 3 .1 2 -6 .2 5 >40 >100

Erw inia c a ro to vo ra 1 .5 6 -3 .1 2 >40 ND

Escherichia c o li 3 6 3 0 .0 9 5 -0 .1 9 5 -1 0 2 .5 -5

Klebsie lla p n e u m o n ia e 3 .1 2 -6 .2 5 >40 1 0 -2 0

Salm one lla th y p h im u r iu m 6 .2 5 -1 2 .5 >40 >100

V ib rio a lg in o ly tic u s 0 .3 9 -0 .7 8 >40 ND

V ib rio harveyi 0 .7 8 -1 .5 6 >40 2 .5 -5

V ib rio p e n a e ic id a 2 5 -5 0 >40 ND

Fungi

Botrytis c ine rea 3 .1 2 -6 .2 5 5 -1 0 ND

Fusarium o x y s p o ru m 1 .5 6 -3 .1 2 5 -1 0 ND

M IC a re e x p re ss e d a s th e in terval a - b , w h e re a = th e h ig h e s t c o n c e n tr a tio n te s te d a t w h ich m ic ro o r­g an ism s a re g ro w in g , a n d b = th e lo w e st c o n c e n tr a tio n th a t c a u s e d 1 0 0 % g ro w th in h ib itio n . N D = n o t d e te rm in e d

antimicrobial properties. Overexpression of rALFF/«3 but not rALI 7V/2 was attained. The purified rALFF/«3 and the crude pro­tein of rALI 7V/2 were then used for further characterization. The rALFPm3 has a broad activity spectrum with anti-fungal properties against both tested filamentous fungi, and anti-bacterial activities against both gram- positive (4/4 tested species) and gram-nega­tive (8/8 tested species) bacteria, with high potency against the natural shrimp patho­gens, V. harveyi (MIC of 0.78 to 1.56 u.\ I) and V. alginolyticus (MIC of 0.39 to 0.78 pM) (Table 3). The bactericidal activity against E. coli 363 and B. megaterium was also evi­denced (Somboonwiwat el ai. 2005). The synthetic peptide corresponding to a part of tlie LPS binding site of ALI / ’/i/3 was shown

to only exhibit activity against gram-posi­tive bacteria suggesting tlie involvement of tlie full molecule for activity against gram- negative bacteria. The crude protein of rALI :/ :m2 was also active against the gram- positive bacteria, B. megaterium, and the gram-negative bacteria, E. coli 363 (data not shown). However, it would be interesting to compare the antimicrobial properties be­tween the two isoforms once sufficient rAI.I /7)?2 is available. ALF cDNAs have been identified in other shrimps but their antimicrobial properties remain to be char­acterized. Nevertheless, amongst tlie three families of shrimp AMPs, tlie ALF displayed tlie broadest spectrum of antimicrobial ac­tivities including against natural shrimp pathogens. The other two shrimp AMP

B io techno logy o f shrim p and shellfish 231

families, crus-like/V; and LivanPEN-3 re­vealed lower activities (LîvôhPEN-3) or a reduced target spectrum (crus-like/V/) un­der these conditions compared to ALF/V/3 (Table 3). Moreover, it has recently been shown by RNA interference (RNAi) in both whole animals and in cell cultures that tlie crayfish ALF can interfere with the replica­tion of white spot syndrome virus (Liu et al. 2006). The exciting potential antiviral prop­erty of rALFPm3 is now under investigation, but clearly signals the need for a more ex­tensive screening of other host proteins in­cluding ALF proteins for anti-viral activity against tlie suite of shrimp viral pathogens.

1.6. The potential use of antimicrobial peptides for disease control

in aquaculture

Aquatic animals including shrimps live in an environment enriched with pathogenic organisms and, therefore, antibiotics are commonly used in aquaculture to prevent or treat disease outbreaks. Most fanners use antibiotics prophylactically, some on a daily basis. In addition to the elevated costs, tlie continuous use of antibiotics has resulted in residues of varying concentrations of anti­biotics in the water and mud, and subse­quently, the development or selection for antibiotic resistance in bacteria in the envi­ronment. Moreover, there could be an ad­verse effect on species biodiversity which ultimately may affect the cultureed shrimps, humans and environment.

Antimicrobial peptides provide a good therapeutic alternative for tlie treatment of diseases in aquaculture. They are natural molecules with a broad spectrum of activity against a wide range of microorganisms, are easy to produce, and are less prone to induce resistance. Some antimicrobial peptides are already in clinical and commercial use (Reddy et al. 2004) but their use for disease control in aquaculture has yet to be demon­strated. However, it is quite promising that the shrimp AMPs reported here could be potential candidates as an alternative to an­

tibiotics in shrimp farming. As shown here, rALF/V/3 possess broad spectrum of anti­microbial activity and effectively kills the shrimp pathogen, V. harveyi, at a MIC of 0.78 to 1.56 u.\ I whereas tlie commonly used an­tibiotic in aquaculture, oxytetracyline kills the bacteria at a MIC of 72.1 to 90.3 u.\ I (Vaseeharan el al. 2005). More recently, the shrimp AMPs LitvanPen3 and ALF/V/3 were tested for their antiviral activity against her­pes simplex vims type 1, human adenovirus respiratory strain, and rotavirus S A l l (Carriel-Gomes et al. 2007). Both shrimp AMPs exhibited significant activity against HSV-1 although they were active at near cytotoxic concentrations. The activity of shrimp AMPs against shrimp virases has not yet been clearly demonstrated due to tlie lack of shrimp cell lines. Nevertheless, their po­tential use in shrimp fanning to overcome severe disease outbreaks is quite promising. Besides their antimicrobial function, AMPs are also known to act as mediators of inflam­mation influencing diverse processes such as cell proliferation, wound healing, cytokine release and immune induction (Zaiou 2007). The application of AMPs as a food additive to enhance shrimp immunity is another strat­egy to combat microbial infections. Evi­dently, the genes encoding these AMPs rep­resent good candidates for the genetic im­provement of shrimps for resistance to se­vere pathogens.

2. Biomineralization of Marine Organisms

2.1. Biomineralization

A variety of animals including bacteria, fun­gus, plants and animals have the ability to synthesize m ineral crystals called biominerals. Calcium phosphate in verte­brate bone and teeth, calcium carbonate in molhisk shell and some sea algae, silica in sponges and diatoms, calcium sulfate in jelly­fish, small magnetic particles in chitons and magnetic bacteria are well-known examples of biominerals.

232 A. T a s s a n a k a j o n e t al.

oyster s Prism atic layer rn

C fbí¿{arnellaFoliate löyer

Fig. 5 . S tru c tu re o f o y s te r shell. O y s te r shell is c o m p o s e d o f p rism a tic layer, c ro s s lam ella r a n d fo lia te layer from o u ts id e to in s ide th a t a re all m a d e o f C a C 0 3. Small a m o u n t o f o rg a n ic m atrix c o m p o n e n ts p o ssib ly p lay im p o r ta n t ro le s to p ro d u c e th e s tru c tu re o f c rystal s tru c tu re s sp ec ific fo r e a c h layer.

These biominerals function to maintain tlie framework of the body, besides regula­tion of tlie mineral balance in the body to function as a reservoir, recognition of the direction by detecting the earth magnet (bio­logical magnets in birds), sense of hearing and body balance (statolith of fish and jelly­fish), teeth of vertebrates and chitons.

A variety of biominerals have been uti­lized in human life: Coins, dishes and music apparatus, jewelries such as pearl, cameo and corals, industrial materials such as chalk, cement and marble, functional food additives such as cliitin. Biominerals of fish statolith and fossil are biomarkers to evaluate the age of fish and era of tlie fossil.

In the 17th century, biominerals espe­cially having economical value were stud­ied by microscopic observation to judge tlie truth or fake. In tlie late 20th century, elec­tron microscopy has been used for the ob­servation, followed by the biochemical analysis of the components of the shell.

Synthesis of the shell proceeds under physiological condition, moderate tempera­

ture and pH, suggesting that there are so many merits in this system to be learned in tlie utilization for tlie implant of the bone and teeth in the medical field, production of light and tough materials in the industrial field.

2.2. Structure of shell

In this section, the author would like to ex­plain tlie structure of the shell by taking oys­ter (Crassostrea gigas) as the example. Molluscan body consists of periostracum and ostracium The structure of oyster shell is shown in Fi g. 5. The periostracum that is not calcified and organic matrix covers tlie whole shell body. In the case of oyster, most of the periostracum is lost except for the edge of tlie shell.

The ostracum is calcified and separated into three parts, prismatic layer, cross lamel­lar and foliate layer from outside to inside. The prismatic layer is composed of column structure made of calcite. The cross lamel­lar, also called chalk, demonstrates the rough matrix structure formed with thin plate-like

B io techno logy o f shrim p and shellfish 233

Fig. 6. N a tu ra lly o b ta in e d c a lc ite (left) a n d o y s te r shell (right). B oth h ave s a m e crysta l s tru c tu re , b u t th e a p p a r e n t fe a tu re s a re c lear ly d istinc tive .

crystal. Foliate layer is composed with dis­tinct plate-like crystals accumulating each other.

Naturally, CaC03 forms calcite or arago­nite crystals or otherwise amorphous struc­ture. In the case of shell, most CaC03 crys­tal is calcite (Fig. 6), but the inner side of pearl oyster is aragonite, which produces rarely a pearl. Pearl is composed of thou­sands of CaC03 layer having a 0.4 pm thick­ness. When light shines into pearl layers, beautiful pink interfering color occurs by the mechanism of multiple layer interference. Pigments among CaC03 layers sometimes causes specific colors such as gold and black. The color of the bottom layer derived from organic materials sometimes causes a beau­tiful blue color.

After hatching, oyster larvae float 2-3 weeks and settle on the bottom of the sea with their left side shell down. Since most oysters live on the mud of estuaries under natural condition, they suffer from the change in mud condition according to tlie tidal change. They have selected two living strategy. One is that they settle on the other adult oyster of the prior generation to maintain the gili opened to the seawater. Another strategy is to lower tlie shell weight not to be buried in the mud. Chalk structure of the shell is helpful for them to lower tlie body weight. Due to their living strategy, the

morphology of oyster is quite different from other bivalves.

2.3. Function of organic substances for biomineralization

Although CaC03 is a main constituent of the shell, morphology of tlie shell is highly spe­cies-specific (Fig. 7). In addition, the struc­ture of the shell is different even in the same species as mentioned above. A possible as­sumption so far proposed is an implication of organic matrix compounds contained in the shell. Although the amount of the organic matrix component is very small (0.1-10%), these components possibly lead die shell struc­ture specific for tlie shell species and portion specific of the shell even in the one species. The organic matrix components are roughly classified into soluble and insoluble fractions into water or EDTA solution. Generally, water or EDTA soluble fraction contains as­partate rich proteins and insoluble fraction contains glycine, serine and alanine rich pro­teins (Crenshaw 1996, Sudo 1997).

Calcification in the shell proceeds ac­cording to the following reaction.

Ca2+ + 2HC032- -» H20 + C 0 2 + CaC03

Ca2+ is adsorbed in the body through spe­cific transporters expressed in the membrane of the epithelial cells of mantle, gili and

234 A. T a s s a n a k a j o n e t al.

oyster

t rmtV » *

scallop

pearl oyster top shellFig. 7 . S p e c ie s sp ec ific s tru c tu re o f m o llu sk shells. All o f shells a re c o m p o s e d o f C a C 0 3 crys­tals, b u t th e a p p a r e n t fe a tu re s a re c learly d iffe ren t.

O 3 CaCOj I C a C 0 3 C a C 0 3 C a C 0 3

C a C 0 3 C a C 0 3 0aC O 3 C a 0 0 3 Ca

0 3 C a C 0 31 C a C 0 3 C a C 0 3 C a C 0 3

C a C 0 3 C a C 0 3 0 a 0 0 3 C a C 0 3 Oa

Fig. 8 . P u ta tiv e fu n c tio n s o f o rg a n ic m atrix p ro te in s . T h e se p ro te in s a s s u m e to p lay th e fu n c ­tio n to c o m b in e th e C a C 0 3 c ry s ta ls a n d le a d th e fo rm o f th e s e u n its by su p p re ss in g th e g ro w th o f th e c ry sta l fo rm atio n .

intestine. I ICO ,2 is partly adsorbed from the surrounding water, but most of them are syn­thesized from C 0 2 produced through TCA cycle.

Ca2+ and I ICO ,2 are mixed and concen­trated in the specific space around tlie spe­cific area surrounded by tlie shell and the epithelia of tlie mantle and crystallized there. The more the core materials exist, crystalli­zation starts under the lower concentration of both compounds. Some kind of organic materials are possibly involved in the initia­

tion of crystallization. After crystallization begins, sheets composed of other organic materials surround tlie crystals and lead tlie form of crystals to the expected form.

Some matrix protein fractionated in the soluble fraction exhibits an enzyme activity, Nacrein isolated from pearl oyster (Pinctada fiicata) in 1996 showed carbonic anliydrolase activity that catalyzed the formation of CO ,2 from I ICO ,2 (Miyamoto 1996).

Figure 8 demonstrates the putative function of organic matrix proteins. Direct

B io techno logy o f shrim p and shellfish 235

evidences to show tlie function of organic matrix proteins are not available at present, but these proteins assume to play the func­tion to combine the CaC03 crystals and lead tlie form of these units by suppressing tlie development of tlie crystal formation.

2.4. Common proteins involved in biomineralization am ong

animals

Among studies so far reported, evolutional relationship of shell matrix proteins are few. Dennatopontin gene has been detected in mammals, arthropods and sponge, and Nacrein and N66 found in pearl oyster has been also found among various animals (Sarasliina 2006).

Perlucin, perlustrin and perlwapin re­ported from abalone demonstrates homology with known proteins. Perlucin is a kind of C-type lectin having carbohydrate binding domain and shares a variety of function involved in biomineral formation of mam­mal, bird, fish and sea urchin (Maini el al. 2000). Perlustrin shows a homology with N-tenninal domain of insulin-like growth factor binding protein (Weiss el al. 2001). Perlwapin exhibits a homology with mam­malian whey acidic protein (Treccani 2006).

As described above, there have been only limited reports suggesting tlie evolu­tional relationship of shell matrix proteins. Thus, most proteins involved in the synthe­sis of biominerals have evolved independ­ently. Acidic amino acids such as aspartate and glutamate implicating in the chelating Ca2+ may play an important function in the biomineralization and the proteins having these amino acids apparently showed homol­ogy among different animals.

2.5. Transportation of Ca2+ for biomineralization

Metal ions are essential for a variety of bio­logical systems in animals. Metal ions are transported into cells by membrane associ­

ated proteins including divalent metal trans­porters (DMTs), which have been identified from various species (Andrews el al. 1999). DMT1 (also called DCT1 or Nramp2) was reported as a homologous protein of Nramp which was implicated in natural resistance to infection by intracellular parasites in mouse (Vidal el al. 1993; Groenheid el al. 1995; Govoni and Gros 1998). DMT1 was identified in rat as DCT1 through expression cloning to search for an mRNA that pro­moted Fe2+ uptake activity (Gunshin el al. 1997). Mutations in tlie DMT1 gene caused a defect in intestinal iron absorption and red cell iron utilization in rat and mouse, sug­gesting that DMT1 plays an important role in iron absorption by intestinal cells (Fleming el al. 1997, 1998; Su el al. 1998). The pres­ence of an iron regulatory element (IRE) in m ammalian DMTs suggests that DMT mRNA is implicated in post-transcriptional regulation by intracellular Fe2+ concentration (Lee el al. 1998; Andrews 1999). Accord­ingly, iron deficiency in rat induced a remark­able accumulation in the mRNA level of DMT 1 due to the function of IRE (Gunshin el al. 2001).

We cloned a DMT gene from scallop Mizuhopecten yessoensis as a transporter possibly involved in the extraordinary accu­mulation of cadmium (Toyohara el al. 2005). Scallop DMT (scDMT) mRNA was strongly expressed in the gili and intestine. As a re­sult of the functional analysis using electrophysiological method using Xenopus laevis oocytes, scDMT transported Fe2+ and Cd2+, the same as mammalian DMT. Inter­estingly, scDMT differs from mammalian DMT in possessing an ability to transport Ca2+.

It must be stressed that scDMT most ef­fectively transports Ca2+ at 10 mM (Takagi et al. 2007). Considering that the Ca2+ con­centration of sea water is approximately 10 mM, this result indicates that scallop pos­sibly absorbs Ca2+ directly from sea water by scDMT. For further characterization of

236 A. T a s s a n a k a j o n e t al.

scDMT, it is necessary to examine the Ca2+ transport activity in a wide range of Ca2+ concentration.

Ca2+ transport activity of DMT, specifi­cally detected for the mollusks, suggests the implication of mollusks DMT in the shell fonnation process. Recently, it was reported that tlie crystal of CaC03 is formed in the hemocytes, not in tlie mantle, and released CaCO, crystals are used for the shell syn­thesis (Mount et al. 2004). Together with the fact that the higher level of mRNA expres­sion of scDMT was detected in tlie gili and intestine, Ca2+ in sea water is transported by scDMT localized in the epithelium of these tissues and presumably transferred into tlie hemocytes to make CaC03 crystals.

2.6. Conclusions

In this paper, we described the recent ad­vance in shrimp antimicrobial peptide and

shell formation as examples of tlie studies of marine invertebrates. Marine invertebrates include a variety of animals and some of them are very important for human life. Shrimp and shell are the most useful animals among marine invertebrates, but there remain so many animals to be studied. We hope tlie studies of marine invertebrates will serve the development of biotechnology possibly promising the prosperous human life in tlie future.

Acknowledgements

The part of this work is supported by Thailand National Center for Genetic Engineering and Biotechnology (BIOTEC) and the Commission on Higher Education. Exchange of scientists under the JSPS-NRCT Program is acknowledged. The authors also acknowledge the support given to the Ph.D students from the Royal Golden Jubilee Ph.D Program, Thailand Research Fund.

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