Comparative genomic analysis of C-type lectin-domain genes in seven holometabolous insect species.

C-type lectins (CTLs) recognize various glycoconjugates through carbohydrate recognition domains (CRDs) and they play important roles in immune responses. In this study, comparative genomic analysis of CTLs were performed in 7 holometabolous species. CTL-S1 to S8 and CTL-X1 to X4 orthologous groups existed in the 7 species, while CTL-X5 group with dual-CRD, CTL-S11 group with triple-CRD, CTL-S9 group with a long C-terminus and Lepidopteran specific CTL-S10 group were not conserved. SliCTL-S12 to S14 cluster was only present in Spodoptera litura, and CTL-S genes were expanded on chromosomes 2 L and 2 R in Drosophila melanogaster. Most IMLs were clustered into three groups and the numbers of IMLs vary among species due to gene duplications. D. melanogaster specific CTLs and Lepidopteran IMLs within each of the three groups evolved more rapidly with higher dN/dS ratios. Two CRDs in IMLs clustered into two clades, with conserved Cys4-Cys5 and Cys1-Cys2 bonds in the first and second CRDs, respectively. The CTL-S and CTL-X family members in S. litura were mainly expressed in the fat body of 5th but not 6th instar larvae, and responded differently to S. litura nucleopolyhedrovirus (SpltNPV) and Nomuraea rileyi infection. The transcription levels of SliCTLs that expressed in fat body but not highly expressed in hemocytes were decreased at the middle and late stages of SpltNPV infection, and the mRNA levels of SliCTLs highly or specifically expressed in hemocytes were mainly decreased by SpltlNPV, N. rileyi and Bacillus thuringiensis infection. These results provide valuable information for further exploration of CTL functions in host-pathogen interaction.

Lu Y ，Su F ，Zhu K ，Zhu M ，Li Q ，Hu Q ，Zhang J ，Zhang R ，Yu XQ ... -  《-》

Structural features, evolutionary relationships, and transcriptional regulation of C-type lectin-domain proteins in Manduca sexta.

C-type lectins (CTLs) are a large family of Ca(2+)-dependent carbohydrate-binding proteins recognizing various glycoconjugates and functioning primarily in immunity and cell adhesion. We have identified 34 CTLDP (for CTL-domain protein) genes in the Manduca sexta genome, which encode proteins with one to three CTL domains. CTL-S1 through S9 (S for simple) have one or three CTL domains; immulectin-1 through 19 have two CTL domains; CTL-X1 through X6 (X for complex) have one or two CTL domains along with other structural modules. Nine simple CTLs and seventeen immulectins have a signal peptide and are likely extracellular. Five complex CTLs have both an N-terminal signal peptide and a C-terminal transmembrane region, indicating that they are membrane anchored. Immulectins exist broadly in Lepidoptera and lineage-specific gene duplications have generated three clusters of fourteen genes in the M. sexta genome, thirteen of which have similar expression patterns. In contrast to the family expansion, CTL-S1∼S6, S8, and X1∼X6 have 1:1 orthologs in at least four lepidopteran/dipteran/coleopteran species, suggestive of conserved functions in a wide range of holometabolous insects. Structural modeling suggests the key residues for Ca(2+)-dependent or independent binding of certain carbohydrates by CTL domains. Promoter analysis identified putative κB motifs in eighteen of the CTL genes, which did not have a strong correlation with immune inducibility in the mRNA or protein levels. Together, the gene identification, sequence comparisons, structure modeling, phylogenetic analysis, and expression profiling establish a solid foundation for future studies of M. sexta CTL-domain proteins.

Rao XJ ，Cao X ，He Y ，Hu Y ，Zhang X ，Chen YR ，Blissard G ，Kanost MR ，Yu XQ ，Jiang H ... -  《-》

Genome-wide identification and characterization of basic helix-loop-helix transcription factors in Spodoptera litura upon pathogen infection.

Basic helix-loop-helix (bHLH) transcription factors play an important role in a wide range of metabolic and developmental processes in eukaryotes, and bHLH proteins also participate in immune responses, especially in plants. However, their roles in insects upon entomopathogen infection are unknown. In this study, 54 bHLH genes in 41 families were identified in a polyphagous pest, Spodoptera litura, including a new bHLH gene in group B, which is specifically present in Lepidoptera and was thus named Lep. The conserved amino acids in the bHLH domain, structural architecture, and chromosomal distribution of bHLH genes in S. litura were analyzed. The bHLH genes in Plutella xylostella and Apis mellifera were also updated, and genome-wide comparison and phylogenetic analysis of bHLH members in 5 holometabolous insects were performed. The expression profiles of S. litura bHLH (SlbHLH) genes in 3 tissues at different developmental stages and their responses to S. litura nucleopolyhedrovirus (SpltNPV), Nomuraea rileyi (Nr), and Bacillus thuringiensis (Bt) infection were investigated. More SlbHLHs in group B were expressed and differentially expressed during pathogen infections, and SlbHLHs tended to be downregulated in the midgut of S. litura larvae after B. thuringiensis treatment. Our study provides an overview of bHLH family members in S. litura and their responses to different pathogens used for pest biocontrol. These findings on bHLH members may contribute to uncovering the mechanism of host-pathogen interaction.

Zhang J ，Zhu M ，Li Q ，Tang T ，Wen L ，Zhong J ，Zhang R ，Yu XQ ，Lu Y ... -  《-》

A single-CRD C-type lectin is important for bacterial clearance in the silkworm.

C-type lectins (CTLs) depend on the carbohydrate-recognition domain (CRD) to recognize carbohydrates by a Ca(2+)-dependent mechanism. In animals, CTLs play critical roles in pathogen recognition, activation of the complement system and signaling pathways. Immulectins (Dual-CRD CTLs) in lepidopteran are involved in recognizing pathogens. However, little is known about the immune-related functions of insect single-CRD CTLs. Here, we reported the characterization of C-type lectin-S3 (CTL-S3), a single-CRD CTL from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S3 gene is 672 bp, which encodes a putative protein of 223 amino acids. CTL-S3 gene was expressed in a variety of tissues. Levels of CTL-S3 mRNA in fertilized eggs and whole larvae were elevated upon bacterial challenges. CTL-S3 was secreted to larval hemolymph. The recombinant protein (rCTL-S3) binds to bacterial cell wall components and bacteria. CTL-S3 inhibited the growth of Bacillus subtilis and caused agglutination of Staphylococcus aureus. More importantly, CTL-S3 facilitated the rapid clearance of Escherichia coli and Staphylococcus aureus from the body cavity of larvae. Taken together, our results suggested that CTL-S3 may function as an opsonin in larval hemolymph to enhance the clearance of pathogens.

Zhan MY ，Shahzad T ，Yang PJ ，Liu S ，Yu XQ ，Rao XJ ... -  《-》

Thioredoxin peroxidase gene is involved in resistance to biocontrol fungus Nomuraea rileyi in Spodoptera litura: gene cloning, expression, localization and function.

Thioredoxin peroxidases (Tpxs) are a ubiquitous family of antioxidant enzymes that play important roles in protecting organisms against oxidative stress. Here, one Tpx was cloned from Spodoptera litura named as SlTpx. The full-length cDNA consists of 1165 bp with 588 bp open reading frame, encoding 195 amino acids. The putative amino acid sequence shared >70% identity with Tpxs from other insects. Phylogenetic analysis revealed that SlTpx is closely related to other available lepidopteran Tpxs. Real-time PCR analysis showed that SlTpx can be induced by Nomuraea rileyi infection in some detected tissues at the mRNA level. The strongest expression was found in hemocytes of unchallenged and N. rileyi-challenged S. litura. Western blotting showed SlTpx protein in the hemocytes, head and cuticle from normal S. litura. However, when N. rileyi was inoculated into the body cavity of S. litura larvae, SlTpx protein was detected in head, hemocytes, fatbody, midgut, malpighian tubule, but not in the hemolymph and cuticle. Moreover, time-course analysis showed that SlTpx mRNA/protein expression levels were up-regulated in the hemocytes, when S. litura were infected by N. rileyi or injected with H2O2. The levels of N. rileyi-induced reactive oxygen species (ROS) in hemocytes were evaluated, and revealed that N. rileyi infection caused generation of ROS, and induced changes in expression of SlTpx. In addition, the heterologously expressed protein of this gene in Escherichia coli showed antioxidant activity; it removed H2O2 and protected DNA. Knocking down SlTpx transcripts by dsRNA interference resulted in accelerated insect death with N. rileyi infection. This is believed to be the first report showing that SlTpx has a significant role in resisting oxidative stress caused by N. rileyi infection.

Chen H ，Yin Y ，Feng E ，Li Y ，Xie X ，Wang Z ... -  《-》