Table 1.
Assigned tag sequences specific to big defensins in oyster DGE libraries.
Figure 1.
Oyster big defensin members are multi-domain defense polypeptides encoded by distinct genomic sequences.
a: Amino acid sequence alignment of big defensins from Crassostrea gigas and Tachypleus tridentatus (BDEF_TACTR, GenBank: P80957). The predicted signal peptides are in bold and underlined. The putative propeptides and the conserved cysteine residues are shadowed with grey and black backgrounds, respectively. I: hydrophobic N-terminal domain; II: C-terminal cysteine-rich domain (β-defensin-like). The putative furin-like cleavage motif (RXKR) is indicated by a dotted rectangle. Asterisks (*) indicate identical amino acid residues. b: A not-to-scale representation of oyster big defensin genomic sequences (Cg-bigdef1, Cg-bigdef2 and Cg-bigdef3). Black lines represent the untranslated regions of the exons. White boxes indicate the signal and the propeptide sequence, and the N-terminal domain of the mature peptide. Black boxes indicate the C-terminal domain of the mature peptides, and the grey box indicates a part of the 5′ untranslated region (37-bp) of the first exon found in Cg-bigdef3. Numbers indicate the length of exons and introns (in base pairs).
Figure 2.
Big defensins form a group predominantly present in mollusk species and closer to vertebrate defensins.
a: Multiple alignments of mature polypeptides of big defensins from bivalve mollusks, horseshoe crab and amphioxus. Identical amino acid residues are shaded with black backgrounds. b: Phylogenetic tree of different defensin groups, including big defensins, CSαβ-containing defensins, α-defensins, β-defensins and β-defensin-like peptides from fish. The tree was constructed using the Neighbour-Joining method in MEGA 4. Bootstrap sampling was reiterated 1,000 times. Sequences included in analyses of defensins were the following, where asterisks (*) indicate mollusk big defensin members newly identified in this study from GenBank: (i) big defensins: oysters Crassostrea gigas (Cg-BigDef1*: AEE92768, Cg-BigDef2*: AEE92775, Cg-BigDef3*: AEE92778) and C. virginica (BDEF_CRAVI*: CV133156), horseshoe crabs Tachypleus tridentatus (BDEF_TACTR: P80957) and Carcinoscorpius rotundicauda (BDEF_CARRO: CK086629), amphioxus Branchiostoma belcheri tsingtauense (BDEF_BRABE: AAO18674) and B. floridae (BDEF_BRAFL: ADH03419), scallops Argopecten irradians (AiBD: ABC61319) and Mizuhopecten yessoensis (MyBD: GH736001), clams Venerupis (Ruditapes) philippinarum (VpBD: ADM25826, VpBD-EST*: AM873974) and Mercenaria mercenaria (BDEF_MERME: GO915266), mussels Mytilus galloprovincialis (BDEF_MYTGA*: FL490131), M. californianus (BDEF1_MYTCA*: GE761911, BDEF2_MYTCA*: GE759807) and Bathymodiolus azoricus (BDEF_BATAZ: HM756150), abalone Haliotis diversicolor (BDEF_HALDV*: GT870909); (ii) CSαβ-containing defensins: oysters C. gigas (Cg-Defm: CAJ19280, Cg-Defh1: ABD66301, Cg-Defh2: ABD66302) and C. virginica (AOD: P85008), mussels M. edulis (DEFA_MYTED: P81610, DEFB_MYTED: P81611) and M. galloprovincialis (MGD-1: P80571, MGD-2: AAD52660), abalones Haliotis discus discus (DEF_HALDS: ACZ15982) and H. discus hannai (DEF_HALHA: ABF69125), scorpion Androctonus australis (DEF4_ANDAU: P56686), tick Dermacentor variabilis (VSNA1: AAO24323), fruit fly Drosophila melanogaster (DEFI_DROME: P36192), mosquitos Anopheles gambiae (DEFI_ANOGA: Q17027) and Aedes aegypi (DEFC_AEDAE: P81603), saprophytic fungus Pseudoplectania nigrella (PLECT_PSENR: Q53I06); (iii) α-defensins: rhesus monkey Macaca mulatta (DEF1_MACMU: P60030), house mouse Mus musculus (CRYP-1: NP_034161), Norway rat Rattus norvegicus (DEF2_RAT: Q62715), rabbit Oryctolagus cuniculus (DEF3_RABIT: P01376), domestic guinea pig Cavia porcellus (DEF1A_CAVPO: P11478), Homo sapiens (HNP-1: NP_004075, HNP-3: AAA35753, HNP-4: NP_001916); (iv) β-defensins: spiny lobster Panulirus japonicus (PJD1: ACM62357, PJD2: ACM62358), chicken Gallus gallus (GAL4: NP_001001610), wild turkey Meleagris gallopavo (GPV-1: AAG09213), R. norvegicus (RBD-4: NP_071989), water buffalo Bubalus bubalis (DEFB_BUBBU: ABI36600); cattle Bos taurus (DEFB_BOSTA: CAC15400), goat Capra hircus (DEFB_CAPHI: ABF71365), H. sapiens (HBD-2: NP_004933, HBD-3: NP_061131); (v) fish defensins: rainbow trout Oncorhynchus mykiss (DEF_ONCMY: ABR68250), zebrafish Danio rerio (DEF_DANRE: AM181358), fugu pufferfish Takifugu rubripes (DEF_TATRU: CAJ57646), spotted green pufferfish Tetraodon nigroviridis (DEF_TATNI: CAJ57644), Japanese flounder Paralichthys olivaceus (DEF_PAROL: ADA84138).
Figure 3.
Relative expression of Cg-BigDef transcripts in oyster hemocytes by real-time quantitative PCR.
a: Expression analysis of the three Cg-BigDef forms at 12 h post-stimulation with sterile sea water (white bars, SSW) and heat-killed bacteria (black bars, stimulation). Asterisks (*) indicate significant differences between conditions according to the Student's t-test (p<0.05). b: Time-course expression levels of transcripts of the inducible Cg-BigDef1 at 0, 3, 8, 24, 48, 72 and 120 h after Vibrio splendidus LGP32 and V. tasmaniensis LMG 20012T challenge or SSW injection. Data analysis was performed with the Pfaffl method. Significant differences (Student's t-test, p<0.05) are indicated by different lowercase letters (a, b, and c). The absence of significant difference is indicated by the use of identical lowercase letters.
Figure 4.
Localization of Cg-BigDef mRNA expression in unchallenged and Vibrio-infected oyster tissues by in situ hybridization (ISH).
Histological sections of C. gigas were analyzed by ISH using Cg-BigDef -DIG-UTP antisense (a-e) and sense (f) riboprobes. In unchallenged oysters, Cg-BigDef1 (a) and Cg-BigDef3 (b) labeling appeared in hemocytes (arrows) located in blood vessels and infiltrating oyster tissues. In Vibrio-challenged oysters, strong hybridization signals were detected with Cg-BigDef1 antisense riboprobe in hemocytes located in blood vessel (c) and invading massively connective tissues of the oyster organs such as the digestive gland (d) and the gills (e). Control sections with the sense Cg-BigDef1 riboprobe were devoid of labeling (f). (Scale bars: 100 µm).
Figure 5.
Reversed-phase HPLC of gill acid extracts from unchallenged (black line) and immune-challenged oysters analyzed 12 h (green line), 24 h (blue line), and 48 h (red line) after an injection of heat-killed bacteria.
Elution was performed with a linear gradient of 0-60% of acetonitrile in acidified water over 40 minutes at a flow rate of 0.6 ml/min (grey line). Absorbance was monitored at 214 nm. One fraction eluted with 34% acetonitrile (arrow) showed an increased absorbance over the time course. Ions compatible with the mass of a double-charged Cg-BigDef1 (asterisks) were identified by MALDI-TOF MS in this fraction only. They were found in challenged oysters at 12, 24 and 48 h (see m/z values) but not in unchallenged oysters.
Table 2.
Nucleotide sequence of primers used in this study.