Biotechnology of Marine Invertebrates —Recent Advances in … · Biotechnology of Marine...
Transcript of Biotechnology of Marine Invertebrates —Recent Advances in … · Biotechnology of Marine...
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
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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
Q P V P C S S D Y K C G G -L D K C C F D R C L G Q H V C K P P S R P IT C S N D Y K C G G -V D K C C Y D R C L Q E H V C K P P S P P V T C S H D F K C A G -L D K C C F D R C L G E H V C K P P S R P IQ C A H D G Q C Y Ä S N D K C C F D R C L G E H V C K PA T IC R Q D T D C F G -S D K C C F D T C L N D T V C K P IV
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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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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