Identification of Cuticular Protein Genes in the Colorado Potato Beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

Structural cuticular proteins (CPs) are the primary components of insect cuticle, linings of salivary gland, foregut, hindgut and tracheae, and midgut peritrophic membrane. Variation of CPs in insect cuticle can cause penetration resistance to insecticides. Moreover, depletion of specific CP by RNA interference may be a suitable way for the development of potential pest control traits. Leptinotarsa decemlineata (Say) CPs are poorly characterized at present, and therefore, we mined the genome and transcriptome data to better annotate and classify L. decemlineata CPs in this study, by comparison with the annotated CPs of Tribolium castaneum Browse (Coleoptera: Tenebrionidae). We identified 175 CP genes. Except one miscellaneous CP with an 18-amino acid motif, these CPs were classified into 7 families based on motifs and phylogenetic analyses (CPs with a Rebers and Riddiford motif, CPR; CPs analogous to peritrophins, CPAP3 and CPAP1; CPs with a tweedle motif, TWDL; CPs with a 44-amino acid motif, CPF; CPs that are CPF-like, CPFL; and CPs with two to three copies of C-X5-C motif, CPCFC). Leptinotarsa decemlineata CPRs could be categorized into three subfamilies: RR-1 (50), RR-2 (85), and RR-3 (2). The RR-1 proteins had an additional motif with a conserved YTADENGF sequence. The RR-2 members possessed a conserved RDGDVVKG region and three copes of G-x(3)-VV. Few genes were found in TWDL (9), CPAP1 (9), CPAP3 (8), CPF (5), CPFL (4), and CPCFC (2) families. The findings provide valuable information to explore molecular modes of penetration resistance to insecticides and to develop dsRNA-based control method in L. decemlineata.

Wang YW ，Li YZ ，Li GQ ，Wan PJ ，Li C ... -  《-》

Preliminary characterization of putative structural cuticular proteins in the malaria vector Anopheles sinensis.

The insect cuticle protects against environmental stresses such as insecticides, physical injury, dehydration and pathogenic microorganisms. Structural cuticular proteins (CPs) are the primary components of the cuticle, and are of interest for physiology-based pest management methods. Anopheles sinensis CPs are poorly characterised at present, and therefore we performed whole-genome sequence analysis and re-analysed available transcriptome data to identify potential insecticide resistance-associated CPs. Genome analysis revealed 238 putative CPs belonging to 11 different families; 136 proteins possessing a Rebers and Riddiford motif (CPR), 12 CPs analogous to peritrophins with a single chitin-binding domain (CPAP1), eight CPs analogous to peritrophins with three chitin-binding domains (CPAP3), four proteins with a 44 amino acid motif (CPF), five CPF-like (CPFL) proteins, nine Tweedle proteins, three proteins of low complexity with alanine residues (CPLCA), 26 proteins of low complexity with conserved glycine residues (CPLCG), seven proteins of low complexity with invariant W residues (CPLCW), 27 proteins of low complexity with proline residues (CPLCP), and one protein with two or three copies of a C-X -C motif (CPCFC). Two CPs belonging to the CPR and CPAP3 families were over-expressed in a deltamethrin-resistant strain. These results establish an information framework for An. sinensis CPs that provides a basis for further molecular research. © 2017 Society of Chemical Industry.

Zhou D ，Duan B ，Sun Y ，Ma L ，Zhu C ，Shen B ... -  《-》

Identification of cuticular protein genes and analysis of their roles in phosphine resistance of the rusty grain beetle Cryptolestes ferrugineus.

The rusty grain beetle, Cryptolestes ferrugineus (Stephens) is one of the most economically important stored grain pests, and it has evolved the high resistance to phosphine. Cuticular proteins (CPs) are the major structural components of insect cuticle, and previous studies have confirmed that CPs were involved in insecticide resistance. However, the CPs of C. ferrugineus are still poorly characterized, and thus we conducted transcriptome-wide identification of CP genes and analyze their possible relationships with phosphine resistance in this pest. In this study, a total of 122 putative CPs were annotated in the C. ferrugineus transcriptome data by blasting with the known CPs of Tribolium castaneum. The analysis of conserved motifs revealed these CPs of C. ferrugineus belonging to 9 different families, including 87 CPR, 13 CPAP1, 7 CPAP3, 3 Tweedle, 1 CPLCA, 1 CPLCG, 5 CPLCP, 2 CPCFC, and 3 CPFL proteins. The further phylogenetic analysis showed the different evolutionary patterns of CPs. Namely, we found some CPs (CPR family) formed species-specific protein clusters, indicating these CPs might occur independently among insect taxa, and while some other CPs (CPAP1 and CPAP3 family) shared a closer correlation based on the architecture of protein domains. Subsequently, the previous RNA-seq data were applied to establish the expression profiles of CPs in a phosphine susceptible and resistant populations of C. ferrugineus, and a large amount of CP genes were found to be over-expressed in resistant insects. Lastly, an up-regulated CP gene (CPR family) was selected for the further functional analysis, and after this gene was silenced via RNA interference (RNAi), the sensitivity to phosphine was significantly enhanced in C. ferrugineus. In conclusion, the present results provided us an overview of C. ferrugineus CPs, and which suggested that the CPs might play the critical roles in phosphine resistance.

Tang PA ，Hu HY ，Du WW ，Jian FJ ，Chen EH ... -  《-》

Downregulation of imidacloprid resistant genes alters the biological parameters in Colorado potato beetle, Leptinotarsa decemlineata Say (chrysomelidae: Coleoptera).

Colorado potato beetle, Leptinotarsa decemlineata Say (coleoptera: chrysomelidae), is the important pest of potato all over the world. This insect pest is resistant to more than 50 active compounds belonging to various chemical groups. Potential of RNA interference (RNAi) was explored to knock down transcript levels of imidacloprid resistant genes in Colorado potato beetle (CPB) under laboratory conditions. Three important genes belonging to cuticular protein (CP), cytochrome P450 monoxygenases (P450) and glutathione synthetase (GSS) families encoding imidacloprid resistance were targeted. Feeding bio-assays were conducted on various stages of imidacloprid resistant CPB lab population by applying HT115 expressing dsRNA on potato leaflets. Survival rate of insects exposed to CP-dsRNA decreased to 4.23%, 15.32% and 47.35% in 2nd, 3rd and 4th instar larvae respectively. Larval weight and pre-adult duration were also affected due to dsRNAs feeding. Synergism of RNAi with imidacloprid conducted on the 2nd instar larvae, exhibited 100% mortality of larvae when subjected to reduced doses of GSS and CP dsRNAs along with imidacloprid. Utilization of three different dsRNAs against imidacloprid resistant CPB population reveal that dsRNAs targeting CP, P450 and GSS enzymes could be useful tool in management of imidacloprid resistant CPB populations.

Naqqash MN ，Gökçe A ，Aksoy E ，Bakhsh A ... -  《-》

Identification and expression analysis of cuticular protein genes in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

Structural cuticular proteins (CPs) are major components of the insect cuticle, and they play critical roles in insect development and insecticide resistance. Here, a total of 196 CP genes were successfully annotated in the Plutella xylostella genome. On the basis of motif analysis, these CPs were classified into 10 different families, including 122 CPR, 12 CPAP1, 8 CPAP3, 9 CPLCP, 2 Tweedle, 1 CPF, 1 CPFL, 1 CPCFC, 17 CPG and 2 18 aa proteins, and the remaining 21 unclassified CPs were classed as cuticular proteins hypothetical (CPH). A phylogenetic analysis of CPs from different insects revealed species-specific clades of RR-1 and RR-2 genes, suggesting that CP gene duplication might occur independently among insect taxa, while we also found that some other CPs (such as CPAP1 and CPAP3) had a closer relationship based on their conserved domain architecture. Using available RNAseq libraries, the expression profiles of the CPs were analyzed over the four developmental stages of the insect (i.e., egg, larva, pupa, and adult), revealing stage-specific expression patterns for the CPs. In a chlorpyrifos resistant strain, 18 CP genes were found to be more than two-fold upregulated compared to the susceptible control strain, and qRT-PCR analysis showed that these CP genes were overexpressed after exposure to chlorpyrifos, suggesting a potential role in the molecular mechanism of insecticide resistance in P. xylostella. This study provides the tools and molecular basis to study the role of CPs in the post-embryonal development and the mechanisms of insecticide resistance of P. xylostella.

Chen EH ，Hou QL 《-》