Specific Molecular Signatures for Type II Crustins in Penaeid Shrimp Uncovered by the Identification of Crustin-Like Antimicrobial Peptides in Litopenaeus vannamei

Abstract

Crustins form a large family of antimicrobial peptides (AMPs) in crustaceans composed of four sub-groups (Types I-IV). Type II crustins (Type IIa or "Crustins" and Type IIb or "Crustin-like") possess a typical hydrophobic N-terminal region and are by far the most representative sub-group found in penaeid shrimp. To gain insight into the molecular diversity of Type II crustins in penaeids, we identified and characterized a Type IIb crustin in Litopenaeus vannamei (Crustin-like Lv) and compared Type II crustins at both molecular and transcriptional levels. Although L. vannamei Type II crustins (Crustin Lv and Crustin-like Lv) are encoded by separate genes, they showed a similar tissue distribution (hemocytes and gills) and transcriptional response to the shrimp pathogens Vibrio harveyi and White spot syndrome virus (WSSV). As Crustin Lv, Crustin-like Lv transcripts were found to be present early in development, suggesting a maternal contribution to shrimp progeny. Altogether, our in silico and transcriptional data allowed to conclude that (1) each sub-type displays a specific amino acid signature at the C-terminal end holding both the cysteine-rich region and the whey acidic protein (WAP) domain, and that (2) shrimp Type II crustins evolved from a common ancestral gene that conserved a similar pattern of transcriptional regulation.

Keywords: WAP domain; crustacean; host defense peptide; host-pathogen interaction; invertebrate immunity; molecular diversity.

Figure 1

Litopenaeus vannamei Type IIb crustins. Amino acid sequences alignment of Type IIb crustins from L. vannamei (Crustin-like Lv) and the crustinPm7 from Penaeus monodon. Identical residues are highlighted in black. Triangles (▼) indicate the 12 conserved cysteine residues found in crustins. The whey acidic protein (WAP) domain is underlined by a solid red line.

Figure 2

Litopenaeus vannamei Type II crustins are encoded by distinct genomic sequences. A not-to-scale representation of L. vannamei Type II crustins, Type IIa (Crustin Lv) and Type IIb (Crustin-like Lv). White boxes indicate the exons and the black line indicates the intron. The numbers show the size (in base pairs) of the exons and introns.

Figure 3

Gene expression distribution of Type II crustins in shrimp tissues. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of Crustin Lv (Type IIa) and Crustin-like Lv (Type IIb) transcript levels in different tissues from naïve (N) and Vibrio-stimulated (S) shrimp. The expression of the β-actin gene was used as an endogenous control. HE: hemocytes, GL: gills, ML: muscle, NC: nerve cord, FG: foregut, HP: hepatopancreas, MG: midgut, HG: hindgut.

Figure 4

Relative expression profile of Crustin Lv (Type IIa) and Crustin-like Lv (Type IIb) genes in (A) circulating hemocytes and (B) midgut of shrimp at 48 h after experimental infections with the Gram-negative Vibrio harveyi ATCC 14126 (white bars) or the White spot syndrome virus (black bars). Results are presented as mean ± standard deviation of relative expressions (three biological replicates). WSSV: White spot syndrome virus. W-free: tissue homogenate inoculum prepared from WSSV-free shrimp.

Figure 5

Relative abundance of Crustin-like Lv (Type IIb) transcripts during shrimp development. Results are present as mean ± standard deviation (three biological replicates). The red dotted line indicates the basal expression level of the Crustin-like Lv gene in hemocytes from juvenile shrimp. EI: fertilized eggs at 0–4 h post-spawning; EII: fertilized eggs at 7–11 h post-spawning; NI: nauplius I; NV: nauplius V; ZI: protozoea I; ZIII: protozoea III; MI: mysis I; MIII: mysis III; PL2: postlarva 2; PL9: postlarva 9; PL17: postlarva 17. Different letters indicate significant differences among the developmental stages from EII to PL17 (one-way ANOVA/Tukey, p < 0.05). Asterisks (*) shows significant differences between each developmental stage and hemocytes from juveniles (one-way ANOVA/Tukey, p < 0.05).

Figure 6

The molecular signature of shrimp Type II crustins. (A) Amino acid sequence alignments of the C-terminal region holding the 12 conserved cysteine residues (the crustin signature [39]) of Type IIa and Type IIb crustins from penaeid shrimp: Litopenaeus vannamei (Crustin Lv1, Crustin Lv3, LvCrustin I, LvCrustin P, Crustin-like Lv), Litopenaeus setiferus (Crustin Ls1, Crustin Ls2, Crustin Ls3), Litopenaeus schmitti (CrusLsch), Farfantepenaeus paulensis (CrusFpau), Farfantepenaeus brasiliensis (CrusFbra), Farfantepenaeus subtilis (CrusFsub), Fenneropenaeus chinensis (Fc-crus 2, CruFc), Fenneropenaeus indicus (Fi-crustin), Penaeus monodon (crustinPm1, crustinPm4, crustinPm5, crustinPm6, crustinPm7, Crustin-like Pm), and Marsupenaeus japonicus (Crustin Mj, Crustin-like Mj). (B) Consensus amino acid sequence of shrimp Type IIa and Type IIb crustins. X indicates any amino acid. Identical residues are highlighted in black. Specific amino acid residues conserved in shrimp Type IIa and Type IIb crustins are highlighted in blue and yellow, respectively. Triangles (▼) indicate the 12 conserved cysteine residues found in crustins. The whey acidic protein (WAP) domain is underlined by a solid red line. (C) A not-to-scale representation of shrimp Type IIa and Type IIb crustins indicating the N-terminal glycine-rich region and the C-terminal crustin signature (cysteine-rich region + WAP domain). MW: molecular weight. pI: theoretical isoelectric point. * Sequence obtained from [16] (not deposited in any database).

Figure 7

Penaeid shrimp Type II crustins form two distinct phylogenetic clades. The phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 6. Bootstrap sampling was reiterated 1000 times. Sequences included in analyses were the following: (i) Type I crustins (“Carcinins”): the Pp-crustin from Portunus pelagicus (GenBank: JQ965930), the Sc-crustin from Scylla serrata (GenBank: HQ638025), the CrusSp from Scylla paramamosain (GenBank: EU161287), the CruHa1 from Hyas araneus (GenBank: EU921641), PtCrustin from Portunus trituberculatus (GenBank: FJ612106), the carcinin (11.5-kDa peptide) from Carcinus maenas (GenBank: AJ427538), the PlCrustin2 from Pacifastacus leniusculus (GenBank: EF523613), the Pc-crustin 2 from Procambarus clarkii (GenBank: GQ301202), the MjCruI-1 and MjCruI-2 from Marsupenaeus japonicus [11], and the carcininPm2 from Penaeus monodon [10]; (ii) Type IIa crustins (“Crustins”): the LvCrustin I (GenBank: AY488492), LvCrustin P (GenBank: AY488494), Crustin Lv1 (GenBank: AF430071) and Crustin Lv3 (GenBank: AF430073) from Litopenaeus vannamei (indicated by black circles, ●), the Crustin Ls1 (GenBank: AF430078), Crustin Ls2 (GenBank: AF430077) and Crustin Ls3 (GenBank: AF430079) from Litopenaeus setiferus, the CrusLsch (GenBank: EF182748) from Litopenaeus schmitti, the CrusFpau (GenBank: EF182747) from Farfantepenaeus paulensis, the CrusFbra (GenBank: EF601055) from Farfantepenaeus brasiliensis, the CrusFsub (GenBank: EF450744) from Farfantepenaeus subtilis, the Crustin Mj (GenBank: AB121741) from M. japonicus, the crustinPm1 (GenBank: FJ686014), crustinPm4 (GenBank: FJ686015), crustinPm5 (GenBank: FJ380049) and crustinPm6 (GenBank: GH717910) from P. monodon, the Fc-crus 2 (GenBank: FJ853147) from Fenneropenaeus chinensis and the PJC1 (GenBank: FJ797417), PJC2 (GenBank: FJ797418), PJC3 (GenBank: FJ797419), and PJC4 (GenBank: FJ797420) from Panulirus japonicus; (iii) Type IIb crustins (“Crustin-like”): the Crustin-like Lv (GenBank: JQ824114, FE049920 and FE049921) from L. vannamei (indicated by black squares, ■), the Crustin-like Mj from M. japonicus [16], the CruFc (GenBank: DQ097703) from F. chinensis, the Fi-crustin (GenBank: GQ469987) from Fenneropenaeus indicus and the Crustin-like Pm (GenBank: GQ334395, JX912161 and GU299808) and crustinPm7 (GenBank: EF654658) from P. monodon; (iv) Type III crustins (“Single WAP domain-containing proteins or SWD”): the LvSWD (GenBank: AY464465) and LvSWDi [40] from L. vannamei, the SWDPm1 (GenBank: EU623979) and SWDPm2 (GenBank: EU623980) from P. monodon, the FcSWD (GenBank: EF216349) from F. chinensis and the MjSWD (GenBank: AU176270) from M. japonicus; (v) Type IV crustins (“Double WAP domain-containing proteins or DWD”): the LvSLPI (GenBank: EF467169) from L. vannamei, the PmDWD (GenBank: BI784457) from P. monodon, the FcDWD (GenBank: GQ303571) from F. chinensis, and the MjDWD (GenBank: EU095018) from M. japonicus.