Molecular Characterization and Expression Analysis of the C-Type Lectin Domain Family 4 Member F in Litopenaeus vannamei against White Spot Syndrome Virus

Abstract

White spot disease (WSD) outbreaks pose a significant threat to the Pacific white shrimp (Litopenaeus vannamei) farming industry. The causative agent is the white spot syndrome virus (WSSV). There are no effective treatments for WSD so far. Therefore, understanding the resistance mechanisms of L. vannamei against the WSSV is crucial. C-type lectins (CTLs) are important pattern recognition receptors (PRRs) that promote agglutination, phagocytosis, encapsulation, bacteriostasis, and antiviral infections. This study cloned the C-type lectin domain family 4 member F (LvCLEC4F) from L. vannamei. LvCLEC4F contains a 492 bp open reading frame (ORF) encoding a protein of 163 amino acids, including a carbohydrate recognition domain (CRD). Following a challenge with the WSSV, the expression profile of LvCLEC4F was significantly altered. Using RNA interference (RNAi) technology, it was found that LvCLEC4F promotes WSSV replication and affects the expression levels of genes related to the regulation of apoptosis, signaling and cellular stress response, and immune defense. Meanwhile, the hemolymph agglutination phenomenon in vivo was weakened when LvCLEC4F was knocked down. These results indicated that LvCLEC4F may play an important role in the interaction between L. vannamei and WSSV.

Keywords: LvCLEC4F; RNAi; WSSV; gene expression; innate immunity; viral disease control.

Figure 1

cDNA and amino acid sequence of LvCLEC4F. The start and stop codons are indicated by boxes. The predicted carbohydrate recognition domain (CRD) is indicated by an underline. The predicted phosphorylation sites are indicated in bold font. The predicted signal peptide structure is indicated by a gray shadow.

Figure 2

The amino acid sequence alignment of CLEC4F. One hundred percent identical residues are indicated by a black shadow. Seventy-five percent identical residues are indicated by a dark gray shadow. Fifty percent identical residues are indicated by a light gray shadow. The GenBank accession numbers of CLEC4F are as follows: L. vannamei (XP_027212325.1), Rattus norvegicus (NP_446205.1), Canis lupus dingo (XP_025326743.1), Gorilla gorilla gorilla (XP_055234888.1), Danio rerio (XP_009299422.1), Fenneropenaeus chinensis (XP_047498904.1), Homo sapiens (KAI4034935.1), Bos taurus (XP_027410747.1), Xenopus tropicalis (XP_031750858.1), Homarus americanus (XP_042233235.1), Toxorhynchites rutilus septentrionalis (XP_055620181.1), Mus musculus (NP_058031.2), Ophiophagus hannah (ETE57245.1), Nibea albiflora (KAG8004890.1), Ictalurus punctatus (NP_001187725.1), Biomphalaria glabrata (XP_055887399.1).

Figure 3

Phylogenetic tree analysis of CLEC4F. The GenBank accession numbers of CLEC4F sequences are identical to those listed in Figure 2. The LvCLEC4F marker of L. vannamei is ▲.

Figure 4

Expression profiles of LvCLEC4F in the hepatopancreas, gill, muscle, and eyestalk of healthy L. vannamei. *: p < 0.05.

Figure 5

Expression profiles of LvCLEC4F in the hepatopancreas, gill, muscle, and eyestalk of L. vannamei post-WSSV infection. (A) The expression level of LvCLEC4F in the hepatopancreas at different time points post-WSSV infection. (B) The expression level of LvCLEC4F in the gill at different time points post-WSSV infection. (C) The expression level of LvCLEC4F in the muscle at different time points post-WSSV infection. (D) The expression level of LvCLEC4F in the eyestalk at different time points post-WSSV infection. *: p < 0.05, **: p < 0.01.

Figure 6

WSSV replication was suppressed after knocking down LvCLEC4F. (A) The knockdown efficiency of LvCLEC4F in the hepatopancreas at 36 and 48 h post-WSSV infection. (B) The WSSV viral load in the dsGFP+WSSV group (as control), the WSSV group (as control), and the dsLvCLEC4F+WSSV group after knocking down LvCLEC4F. (C) The survival curve of L. vannamei post-WSSV infection. *: p < 0.05, **: p < 0.01.

Figure 7

The observation of hemolymph agglutination in L. vannamei following WSSV infection after knocking down LvCLEC4F. (A) PBS group (scale bar = 100 μm). (B) WSSV group (scale bar = 100 μm). (C) dsGFP+WSSV group (scale bar = 100 μm). (D) dsLvCLEC4F+WSSV group (scale bar = 100 μm). (E) PBS group (scale bar = 50 μm). (F) WSSV group (scale bar = 50 μm). (G) dsGFP+WSSV group (scale bar = 50 μm). (H) dsLvCLEC4F+WSSV group (scale bar = 50 μm).

Figure 8

Expression profiles of immune-related genes after knocking down LvCLEC4F. (A) The expression level of Bcl-2 at different time points post-WSSV infection after knocking down LvCLEC4F. (B) The expression level of caspase 3 at different time points post-WSSV infection after knocking down LvCLEC4F. (C) The expression level of caspase 8 at different time points post-WSSV infection after knocking down LvCLEC4F. (D) The expression level of p38MAPK at different time points post-WSSV infection after knocking down LvCLEC4F. (E) The expression level of Lyz at different time points post-WSSV infection after knocking down LvCLEC4F. *: p < 0.05, **: p < 0.01.