Xiang-Jun Rao

Identification of candidate chemosensory genes in the antennal transcriptome of Tenebrio molitor (Coleoptera: Tenebrionidae).

We present the first antennal transcriptome sequencing information for the yellow mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae). Analysis of the transcriptome dataset obtained 52,216,616 clean reads, from which 35,363 unigenes were assembled. Of these, 18,820 unigenes showed significant similarity (E-value <10(-5)) to known proteins in the NCBI non-redundant protein database. Gene ontology (GO) and Cluster of Orthologous Groups (COG) analyses were used for functional classification of these unigenes. We identified 19 putative odorant-binding protein (OBP) genes, 12 chemosensory protein (CSP) genes, 20 olfactory receptor (OR) genes, 6 ionotropic receptor (IR) genes and 2 sensory neuron membrane protein (SNMP) genes. BLASTX best hit results indicated that these chemosensory genes were most identical to their respective orthologs from Tribolium castaneum. Phylogenetic analyses also revealed that the T. molitor OBPs and CSPs are closely related to those of T. castaneum. Real-time quantitative PCR assays showed that eight TmolOBP genes were antennae-specific. Of these, TmolOBP5, TmolOBP7 and TmolOBP16 were found to be predominantly expressed in male antennae, while TmolOBP17 was expressed mainly in the legs of males. Several other genes were identified that were neither tissue-specific nor sex-specific. These results establish a firm foundation for future studies of the chemosensory genes in T. molitor.

Identification of Putative Carboxylesterase and Glutathione S -transferase Genes from the Antennae of the Chilo suppressalis (Lepidoptera: Pyralidae)

In insects, rapid degradation of odorants in antennae is extremely important for the sensitivity of olfactory receptor neurons. Odorant degradation in insect antennae is mediated by multiple enzymes, especially the carboxylesterases (CXEs) and glutathione S-transferases (GSTs). The Asiatic rice borer, Chilo suppressalis, is an economically important lepidopteran pest which causes great economic damage to cultivated rice crops in many Asian countries. In this study, we identified 19 putative CXE and 16 GST genes by analyzing previously constructed antennal transcriptomes of C. suppressalis. BLASTX best hit results showed that these genes are most homologous to their respective orthologs in other lepidopteran species. Phylogenetic analyses revealed that these CXE and GST genes were clustered into various clades. Reverse-transcription quantitative polymerase chain reaction assays showed that three CXE genes (CsupCXE8, CsupCXE13, and CsupCXE18) are antennae-enriched. These genes are candidates for involvement in odorant degradation. Unexpectedly, none of the GST genes were found to be antennae-specific. Our results pave the way for future researches of the odorant degradation mechanism of C. suppressalis at the molecular level.

Molecular cloning and analysis of PGRP-L1 and IMD from silkworm Bombyx mori

Peptidoglycan is one of the major components of bacterial cell wall. The innate immune system of insects utilizes a group of peptidoglycan recognition proteins (PGRPs) for the recognition of specific peptidoglycans and activating immune signaling pathways. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are two important signaling molecules of the IMD pathway. Here we cloned and characterized PGRP-L1 and IMD from the domesticated silkworm Bombyx mori (BmPGRP-L1 and BmIMD). BmPGRP-L1 gene consists of five exons that encodes a polypeptide of 304 amino acids with a transmembrane region and an extracellular PGRP domain. The PGRP domain lacks key residues for the amidase activity. BmIMD cDNA encodes a polypeptide of 250 amino acids with a death domain. BmPGRP-L1 and BmIMD were expressed in various tissues and induced by bacterial challenges. In addition, in vivo blocking of the PGRP domain by the antiserum or purified antibody significantly reduced the expression of some antimicrobial peptide genes. The extracellular region of BmPGRP-L1 bound to diaminopimelic acid-type and lysine-type peptidoglycans. Overexpression of full-length BmIMD in Drosophila Schneider 2 cells significantly induced three antimicrobial peptide genes. These results suggest that BmPGRP-L1 and BmIMD may be players in the IMD pathway of B. mori. This study provides a foundation for further studies on the functions of silkworm IMD pathway.

MOLECULAR CHARACTERIZATION OF TWO ACETYLCHOLINESTERASE GENES FROM THE RICE LEAFFOLDER, Cnaphalocrocis medinalis (LEPIDOPTERA: PYRALIDAE)

In this study, two full-length cDNA sequences (Cmace1 and Cmace2) encoding putative acetylcholinesterases (AChEs) were cloned and characterized from the rice leaffolder, Cnaphalocrocis medinalis, an important lepidopteran rice pest in Asia. Cmace1 encodes a CmAChE1 consisting of 689 amino acid residues, while Cmace2 encodes a 639 amino acids CmAChE2. The two CmAChEs both have N-terminal signal peptides and conserved motifs including the catalytic triad, choline-binding sites, oxianion hole, acyl pocket, peripheral anionic subsite, and the characteristic FGESAG motif and conserved 14 aromatic amino acids. Phylogenetic analysis showed that Cmace1 and Cmace2 are clustered into distinct clusters that are completely diverged from each other. Reverse-transcription quantitative PCR analysis revealed that Cmace1 and Cmace2 were predominately expressed in the larval brain and at the fifth-instar larvae stage, and the transcription levels of Cmace1 were significantly higher than those of Cmace2 in all the tested samples. Recombinant CmAChE1 and CmAChE2 were heterologously expressed in baculovirus system. Using acetylthiocholine iodide (ATChI) as substrate, the Michaelis constant (Km ) values of rCmAChE1 and rCmAChE2 were 39.81 ± 6.49 and 68.29 ± 6.72 μmol/l, respectively; and the maximum velocity (Vmax ) values of the two rCmAChEs were 0.60 ± 0.02 and 0.31 ± 0.06 μmol/min/mg protein, respectively. Inhibition assay indicated that rCmAChE1 was more sensitive to the organophosphate insecticides chlorpyrifos and triazophos than rCmAChE2. This study is the first report of molecular cloning and biochemical characterization of two acetylcholinesterase genes/enzymes in C. medinalis.

Identification and expression profiles of putative cytochrome P450 monooxygenase genes from Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

In insects, cytochrome P450 monooxygenases (P450s) have received considerable attention for their roles in the detoxification of harmful chemicals and insecticide resistance. The rice leaf‐folder, Cnaphalocrocis medinalis, is a severe lepidopteran rice pest. In this research, 36 putative P450 genes were identified from the transcriptome dataset of C. medinalis. Phylogenetic analysis showed that these genes fell into four clans, including the mitochondrial clan (6 genes), CYP2 clan (5 genes), CYP3 clan (16 genes) and CYP4 clan (9 genes), and were classified into 19 families and 30 subfamilies. Since the CYP6 P450 family in insects is the major family related to the detoxification of xenobiotics, the expression profiles of five genes in the CYP6 family were determined by real‐time quantitative PCR. These genes were differentially expressed in various larval tissues, including the midgut, Malpighian tubules and fat body. Of these, CYP6AW1 and CYP6CV1 were mainly expressed in the midgut, while CYP6AE76 was highly expressed in the fat body. CYP6AB62 and CYP6AE28 were ubiquitously expressed. The transcription of CYP6AE28 was significantly induced by abamectin at LD20 dose. Taken together, the results provided useful information for the characteristics of P450s in C. medinalis and for future functional studies.

Immune functions of insect βGRPs and their potential application

Abstract Insects rely completely on the innate immune system to sense the foreign bodies and to mount the immune responses. Germ‐line encoded pattern recognition receptors play crucial roles in recognizing pathogen‐associated molecular patterns. Among them, &bgr;‐1,3‐glucan recognition proteins (&bgr;GRPs) and gram‐negative bacteria‐binding proteins (GNBPs) belong to the same pattern recognition receptor family, which can recognize &bgr;‐1,3‐glucans. Typical insect &bgr;GRPs are comprised of a tandem carbohydrate‐binding module in the N‐terminal and a glucanase‐like domain in the C‐terminal. The former can recognize triple‐helical &bgr;‐1,3‐glucans, whereas the latter, which normally lacks the enzymatic activity, can recruit adapter proteins to initiate the protease cascade. According to studies, insect &bgr;GRPs possess at least three types of functions. Firstly, some &bgr;GRPs cooperate with peptidoglycan recognition proteins to recognize the lysine‐type peptidoglycans upstream of the Toll pathway. Secondly, some directly recognize fungal &bgr;‐1,3‐glucans to activate the Toll pathway and melanization. Thirdly, some form the ‘attack complexes’ with other immune effectors to promote the antifungal defenses. The current review will focus on the discovery of insect &bgr;GRPs, functions of some well‐characterized members, structure‐function studies and their potential application. Highlights&bgr;GRPs and GNBPs belong to the same PRR family that mainly exist in insects.The N‐terminal region specifically binds to &bgr;‐1,3‐glucans.&bgr;GRPs/GNBPs activate the Toll pathway and phenoloxidase system.

Antimicrobial activities of a proline-rich proprotein from Spodoptera litura

ABSTRACT Antimicrobial peptides (AMPs) are produced by the stimulated humoral immune system. Most mature AMPs contain less than 50 amino acid residues. Some of them are generated from proproteins upon microbial challenges. Here, we report the antimicrobial activities of a proline‐rich proprotein, named SlLebocin1 (SlLeb1), from the tobacco cutworm Spodoptera litura. SlLebocin1 cDNA contains a 477‐bp open reading frame (ORF). It is mainly expressed in hemocytes and the midgut in naïve larvae. The transcript level was significantly induced in hemocytes but repressed in the midgut and fat body by bacterial challenges. The proprotein contains 158 amino acids with 3 RXXR motifs that are characteristic of some Lepidopteral lebocin proproteins. Four peptides corresponding to the predicted processed fragments were synthesized chemically, and their antimicrobial activities against two Gram‐negative and two Gram‐positive bacterial strains were analyzed. The peptides showed differential antimicrobial activities. For Escherichia coli and Bacillus subtilis, only the C‐terminal fragment (124–158) showed strong inhibitory effects. For Staphylococcus aureus, all peptides showed partial inhibitions. None of them inhibited Serratia marcescens. Bacterial morphologies were examined by the scanning electron microscopy and confocal laser scanning microscopy. The antimicrobial peptides either disrupted cellular membrane or inhibited cell division and caused elongated/enlarged morphologies. The results may provide ideas for designing novel antibiotics. HighlightsA lebocin proprotein from S. litura was characterized.Some synthetic peptides affected bacterial growth.Peptide‐treated bacteria showed different morphologies.

Cloning and analysis of peptidoglycan recognition protein-LC and immune deficiency from the diamondback moth, Plutella xylostella

Peptidoglycan (PGN) exists in both Gram-negative and Gram-positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP-LC and IMD (PxPGRP-LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP-LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP-LC and IMD homologs. PxPGRP-LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP-LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full-length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP-LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella.

Transcriptome sequencing reveals abundant olfactory genes in the antennae of the rice leaffolder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

The rice leaffolder, Cnaphalocrocis medinalis Guenée, is an important lepidopteran pest of rice in Asia. Insect olfactory proteins, including olfactory receptors (ORs), ionotropic receptors (IRs), odorant‐binding proteins (OBPs), chemosensory proteins (CSPs) and sensory neuron membrane proteins (SNMPs), are responsible for perception of sex pheromones and host plant volatiles, and thus regulate insect behavior. In the present study, transcriptome sequencing was conducted for C. medinalis antennae to identify genes involved in olfaction. A total of 45800 unigenes were assembled from the transcriptome dataset. Of these, 19696 (43.0%) unigenes were annotated by searching against the NCBI non‐redundant database. Functional classification of these unigenes were also conducted by using the Gene Ontology (GO) and Cluster of Orthologous Groups (COG) databases. We identified 90 putative olfactory genes (including 37 novel ones): 46 ORs, 15 IRs, 12 OBPs, 15 CSPs and two SNMPs. BLASTX best hit results indicated that these genes were most identical to their respective orthologs from other lepidopteran insects. Quantitative reverse transcription‐PCR (qRT‐PCR) assays were performed to investigate the expression profiles of newly identified OR genes. All of the OR genes were antennae‐specific or antennae‐enriched. Of these, CmedOR28 and CmedOR31 were mainly expressed in male antennae, while CmedOR27 and CmedOR32 were enriched in female antennae. Our results establish a solid foundation for future functional studies of these genes.

Molecular cloning and analysis of a C-type lectin from silkworm Bombyx mori

C-type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non-catalytic carbohydrate-recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single-CRD CTL, named C-type lectin-S2 (BmCTL-S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL-S2 is expressed in a variety of immune-related tissues, including hemocytes and fat body among others. BmCTL-S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL-S2) bound different bacterial cell wall components and bacterial cells. rBmCTL-S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL-S2 is a pattern-recognition receptor with antibacterial activities.