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.

Transcriptomic data recover a new superfamily-level phylogeny of Cucujiformia (Coleoptera, Polyphaga).

Cucujiformia, the largest taxon in the order Coleoptera, exhibits extraordinary morphological, ecological, and behavioral diversity. This infraorder is currently divided into seven superfamilies, but considerably incongruent relationships among superfamilies have been reported by recent phylogenomic studies. Here, we combined the 21 newly sequenced transcriptomes representing six superfamilies with nine previously published cucujiform genomes/transcriptomes to elucidate the phylogeny and evolution of Cucujiformia. The monophyly of each of five superfamilies were consistently supported by all phylogenetic analyses based on the twelve datasets (matrix occupancy, amino acid and nucleotide data) and the two analytical methods (maximum likelihood method and Bayesian inference). Both the amino acid datasets and the RY recoded nucleotide datasets recovered the monophyly of Cucujoidea. Topology test results statistically supported the following robust superfamily-level phylogeny in Cucujiformia: (Coccinelloidea, (Cleroidea, (Tenebrionoidea, (Cucujoidea, (Chrysomeloidea, Curculionoidea))))). Our divergence time analyses recovered a Permian origin of Cucujiformia and a Jurassic-Cretaceous origin of most superfamilies. The diversification of phytophagous beetles that occurred in the Cretaceous can be attributed to its co-evolution with angiosperms, supporting the hypothesis of a Cretaceous Terrestrial Revolution.

RNA-seq Analysis Reveals Mitochondrial and Cuticular Protein Genes Are Associated with Phosphine Resistance in the Rusty Grain Beetle (Coleoptera:Laemophloeidae).

The rusty grain beetle, Cryptolestes ferrugineus (Stephens), is a serious pest of stored grain, which has developed high levels of resistance to phosphine. In this study, five geographically distant populations of C. ferrugineus had been collected in China, specifically in granaries where phosphine fumigant is used for pest control, and they showed a high resistance ratio up to 1,907 (LC50 = 21.0 mg/liter). Then, a reference transcriptome was constructed to use as a basis for investigating the molecular mechanisms of phosphine resistance in this species, which consisted of 47,006 unigenes with a mean length of 1,090. Subsequently, the RNA-Seq analysis of individuals from the most susceptible and resistant populations led to the identification of 54 genes that are differentially expressed. GO and KEGG analysis demonstrated that genes associated with mitochondrial and respiration functions were significantly enriched. Also, the 'structural constituent of cuticle' term was annotated in the GO enrichment analysis and further qRT-PCR confirmed that the expression levels of nine cuticular protein genes were significantly increased in the resistant population. In conclusion, we present here a transcriptome-wide overview of gene expression changes between resistant and susceptible populations of C. ferrugineus, and this in turn documents that mitochondria and cuticular protein genes may play together a crucial role in phosphine resistance. Further gene function analysis should enable the provision of advice to expedite resistance management decisions.

Mitochondrial genomes of three Bostrichiformia species and phylogenetic analysis of Polyphaga (Insecta, Coleoptera).

Here we determined mitogenomes of three Bostrichiformia species. These data were combined with 51 previously sequenced Polyphaga mitogenomes to explore the higher-level relationships within Polyphaga by using four different mitogenomic datasets and three tree inference approaches. Among Polyphaga mitogenomes we observed heterogeneity in nucleotide composition and evolutionary rates, which may have affected phylogenetic inferences across the different mitogenomic datasets. Elateriformia, Cucujiformia, and Scarabaeiformia were each inferred to be monophyletic by all analyses, as was Bostrichiformia by most analyses based on two datasets with low heterogeneity. The large series Staphyliniformia was never recovered as monophyletic in our analyses. The Bayesian tree using a degenerated nucleotide dataset (P123_Degen) and a site-heterogeneous mixture model in PhyloBayes was supported as the best Polyphaga phylogeny: (Scirtiformia, (Elateriformia, ((Bostrichiformia, Cucujiformia), (Scarabaeiformia + Staphyliniformia)))). For Cucujiformia, the largest series, we inferred a superfamily-level phylogeny: ((Cleroidea, Coccinelloidea), (Tenebrionoidea, (Cucujoidea + Curculionoidea + Chrysomeloidea))).

Transcriptome-Wide Identification of MicroRNAs and Analysis of Their Potential Roles in Development of Indian Meal Moth (Lepidoptera:Pyralidae).

MicroRNAs (miRNAs) have been reported to play indispensable roles in regulating various developmental processes via the posttranscriptional repression of target genes in insect species. In the present paper, we studied the miRNAs in Indian meal moth (Plodia interpunctella (Hübener)), one of the most economically important stored grains pests around the world. In total, 12 small RNA libraries from four developmental stages of P. interpunctella were constructed, and 178 known and 23 novel miRNAs were identified. In addition, the expression profiles of these miRNAs were assessed across different developmental stages and miRNAs that were highly expressed in eggs, larvae, pupae, and adults were identified. Specifically, 100, 61, and 52 miRNAs were differentially expressed between eggs and larvae, larvae and pupae, and pupae and adults, respectively. The KEGG and GO analysis of the predicted target genes suggested the essential roles of miRNAs in the regulation of complex development of P. interpunctella. Importantly, we also found a set of miRNAs might be involved in the larval metamorphic molting process, with their expressions increasing and then decreasing during the larva-pupa-adult stages of P. interpunctella. In conclusion, the current paper has discovered numerous miRNAs, and some key miRNAs that might be responsible for regulating development in P. interpunctella. To our knowledge, this is the first study to document miRNAs and their expression patterns in interpunctella, and those findings would lay an important molecular foundation for future functional analysis of these miRNAs in P. interpunctella.

Mitogenome evolution in ladybirds: Potential association with dietary adaptation.

Dietary shifts can alter the relative availability of different nutrients and are therefore associated with metabolic adaptation in animals. The Coccinellidae (ladybirds) exhibits three major types of feeding habits and provides a useful model to study the effects of dietary changes on the evolution of mitogenomes, which encode proteins directly involved in energy metabolism. Here, mitogenomes of three coccinellid species were newly sequenced. These data were combined with other ten previously sequenced coccinellid mitogenomes to explore the relationship between mitogenome evolution and diets. Our results indicate that mitogenomic data can be effectively used to resolve phylogenetic relationships of Coccinellidae. Strong codon usage bias in coccinellid mitogenomes was predominantly determined by nucleotide composition. The 13 mitochondrial protein-coding genes (PCGs) globally evolved under negative constraints, with some PCGs showing a stronger purifying selection. Six PCGs (nad3, nad4L, and nad5 from Complex I; cox1 and cox3 from Complex IV; and atp6 from Complex V) displayed signs of positive selection. Of these, adaptive changes in cox3 were potentially associated with metabolic differences resulting from dietary shifts in Coccinellidae. Our results provide insights into the adaptive evolution of coccinellid mitogenomes in response to both dietary shifts and other life history traits.

Reference gene selection to determine differences in mitochondrial gene expressions in phosphine-susceptible and phosphine-resistant strains of Cryptolestes ferrugineus, using qRT-PCR.

Cryptolestes ferrugineus is a serious pest of stored grain and has developed high levels of resistance to phosphine fumigants in many countries. Measuring differences in expression levels of certain 'resistant' genes by quantitative real-time PCR (qRT-PCR) may provide insights into molecular mechanisms underlying resistance to phosphine in C. ferrugineus, but reliable qRT-PCR results depend on suitable reference genes (RGs). We evaluated the stability of nine candidate RGs across different developmental stages and phosphine strains of C. ferrugineus, using four softwares. The results showed that RPS13 and EF1α were the most stable RGs, whereas α-TUB was the least under developmental stages. Across the different strains, RPS13 and γ-TUB were the most stable RGs, whereas CycA and GAPDH were the least. We confirmed the reliability of the selected RGs by qRT-PCR analyses of the mitochondrial cox1 gene. Expression of cox1 was not significantly different in the phosphine-resistant strain compared with the phosphine-susceptible strain, but three mitochondrial genes (nad3, atp6 and cob) were significantly down-regulated. These results suggest that alterations in the expressions of these three genes may be associated with phosphine resistance in C. ferrugineus. The findings will facilitate future functional genomics studies on the development and phosphine resistance in C. ferrugineus.

Characterization of transcriptome in the Indian meal moth Plodia interpunctella (Lepidoptera: Pyralidae) and gene expression analysis during developmental stages.

The Indian meal moth Plodia interpunctella (Lepidoptera: Pyralidae) is a worldwide pest that causes serious damage to stored foods. Although many efforts have been conducted on this species due to its economic importance, the study of genetic basis of development, behavior and insecticide resistance has been greatly hampered due to lack of genomic information. In this study, we used high throughput sequencing platform to perform a de novo transcriptome assembly and tag-based digital gene expression profiling (DGE) analyses across four different developmental stages of P. interpunctella (egg, third-instar larvae, pupae and adult). We obtained approximate 9gigabyte (GB) of clean data and recovered 84,938 unigenes, including 37,602 clusters and 47,336 singletons. These unigenes were annotated using BLAST against the non-redundant protein databases and then functionally classified based on Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes databases (KEGG). A large number of differentially expressed genes were identified by pairwise comparisons among different developmental stages. Gene expression profiles dramatically changed between developmental stage transitions. Some of these differentially expressed genes were related to digestion and cuticularization. Quantitative real-time PCR results of six randomly selected genes conformed the findings in the DGEs. Furthermore, we identified over 8000 microsatellite markers and 97,648 single nucleotide polymorphisms which will be useful for population genetics studies of P. interpunctella. This transcriptomic information provided insight into the developmental basis of P. interpunctella and will be helpful for establishing integrated management strategies and developing new targets of insecticides for this serious pest.

The complete mitochondrial genome of Sympiezomias velatus (Coleoptera: Curculionidae).

Here, we determined the complete mitogenome sequence of Sympiezomias velatus (Coleoptera: Curculionidae: Entiminae). This mitogenome was 15,592 bp long with an A + T content of 74.1% and contains 13 protein-coding genes (PCGs), 21 transfer RNA genes (tRNAs), 2 ribosomal RNA unit genes and a large non-coding region (putative control region). The trnI gene was not found in the S. velatus mitogenome. The order and orientation of the mitochondrial genes were identical to the inferred ancestral arrangement of insects except for trnR which was changed to be adjacent the nad3 gene. All tRNAs had the typical cloverleaf structure, except for trnS1 which lacked the dihydrouridine arm. The Bayesian phylogenetic tree of 10 Entiminae species based on the concatenated nucleotide sequences of 13 PCGs strong supported a sister relationship of S. velatus and Barynotus obscures.

Transcription and induction profiles of three novel P450 genes in Liposcelis bostrychophila (Psocoptera: Liposcelididae).

The cDNAs of three novel P450 genes, CYP4CB1, CYP4CC1, and CYP4CD1 (GenBank accessions EU979550, EU979549, and EU979551, respectively), were sequenced and characterized from the psocid Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae). CYP4CB1, CYP4CC1, and CYP4CD1 contain open reading frames of 1533, 1512, and 1536 nucleotides that encode 511,504, and 512 amino acid residues, respectively. The putative proteins of CYP4CB1, CYP4CC1, and CYP4CD1 show predicted molecular mass of 59.65, 58.87, and 59.71 kDa, with a theoretical isoelectric point of 6.59, 8.80, and 8.84, respectively. The N-terminal transmembrane domain was only found in CYP4CB1 suggested it is a typical microsomal P450. Phylogenetic analysis showed a close relationship of CYP4CB1, CYP4CC1, and CYP4CD1 with CYP4AW1, CYP4L4, and CYP4E2. Quantitative real-time reverse transcriptase-polymerase chain reaction indicated that these three genes were expressed at all tested developmental stages. In addition, the highest expression occurred in the adult stage, which suggested that these three P450 genes may play important roles in adulthood. The transcripts of CYP4CB1 and CYP4CC1 in adult psocids could be induced to the highest level at 36 and 24 h after the exposure to deltamethrin and paraoxon-methyl (50% lethal concentration [LC50]), respectively, whereas CYP4CD1 remained unchanged. CYP4CD1 transcripts, however, increased rapidly at 8 h after aldicarb (LC50) induction and reached the peak at 36 h. The induction profiles of the three P450 genes suggested that CYP4CB1 and CYP4CC1 are possibly associated with deltamethrin and paraoxonmethyl metabolism in psocids, whereas CYP4CD1 is probably involved in aldicarb metabolism. However, our assumption needs to be further verified by recombinant protein expression of these proteins as well as RNA interference of these genes.

Cloning and characterization of acetylcholinesterase 1 genes from insecticide-resistant field populations of Liposcelis paeta Pearman (Psocoptera: Liposcelididae).

The psocid, Liposcelis paeta Pearman, is an increasingly important polyphagous pest of stored products worldwide. Intensive use of organophosphorous insecticides for pest control has facilitated resistance development in psocids in China. Three insecticide-resistant field populations of L. paeta were collected from Nanyang city of Henan Province (NY), and Wuzhou (WZ) and Hezhou (HZ) cities of Guangxi Province, China. Previous studies have shown that psocids have different susceptibilities to insecticides. In addition, their AChE susceptibilities to paraoxon-ethyl and demeton-S-methyl also differed from each other. Acetylcholinesterase 1, which is one of the major targets for organophosphate insecticides, has been fully cloned and sequenced from these populations of L. paeta. Comparison of both nucleotide and deduced amino acid sequences revealed nucleotide polymorphisms among L. paeta ace 1 genes from different populations, but none of these polymorphisms correspond to the active sites in AChE 1 from other insects. The results of comparative quantitative real-time PCR indicated that the relative expression level of HZ ace 1 gene was the highest among three populations, which was 1.20 and 1.02-fold higher than those of NY and WZ populations, respectively. This may due to an epigenetic inheritance phenomenon, which allows organisms to respond to a particular environment through changes in gene expression.

Molecular characterization of two novel deltamethrin-inducible P450 genes from Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae).

Two novel P450 genes, CYP6CE1 and CYP6CE2 (GenBank accession number: EF421245 and EF421246), were cloned and characterized from psocid, Liposcelis bostrychophila. CYP6CE1 and CYP6CE2 contain open reading frames of 1,581 and 1,563 nucleotides that encode 527 and 521 amino acid residues, respectively. The putative proteins of CYP6CE1 and CYP6CE2 show predicted molecular weights of 60.76 and 59.83 kDa with a theoretical pI of 8.58 and 8.78, respectively. CYP6CE1 and CYP6CE2 share 74% identity with each other, and the deduced proteins are typical microsomal P450s sharing signature sequences with other insect CYP6 P450s. Both CYP6CE1 and CYP6CE2 share the closest identities with Hodotermopsis sjoestedti CYP6AM1 at 48% among the published sequences. Phylogenetic analysis showed a closer relationship of CYP6CE1 and CYP6CE2 with CYP6 members of other insects than with those from other families. Quantitative real-time RT-PCR showed that both CYP6CE1 and CYP6CE2 are expressed at all developmental stages tested. Interestingly, CYP6CE2 transcripts decreased from the highest in 1st nymph to the lowest in adults, which seemed to suggest developmental regulation. However, neither CYP6CE1 nor CYP6CE2 were stage specific. The CYP6CE1 and CYP6CE2 transcripts in adults increased significantly after deltamethrin exposure. Recombinant protein expression studies are needed to determine the real functions of these proteins.

Validation of endogenous reference genes for insecticide-induced and developmental expression profiling of Liposcelis bostsrychophila (Psocoptera: Liposcelididae).

Four housekeeping genes named Lbbeta-Actin1, Lbbeta-Actin2, Lbalpha-Tubulin, and LbGapdh were cloned from Liposcelis bostrychophila using the combined techniques of reverse transcriptase-PCR (RT-PCR) with rapid amplification of cDNA ends (RACE). The GenBank accession numbers were FJ196622, FJ447483, FJ595242, and FJ595241, respectively. The full-length cDNA of Lbbeta-Actin1 was a 1,772 bp sequence with an open reading frame (ORF) encoded 376 amino acids, while Lbbeta-Actin2 was 1,350 bp in length containing an ORF encoded 376 amino acids. Furthermore, the 1,565 bp cDNA of Lbalpha-Tubulin had an ORF of 1,350 bp encoding 450 amino acids. And LbGapdh possessed an ORF of 333 amino acids. Sequences analysis and phylogenetic trees generated from the nucleotide sequences of their coding regions revealed a relationship that was closer to other insects than to mammals. The four genes together with 18S rRNA were quantified for transcription stability in L. bostrychophila, and the geNorm software ranked from the most to least were Lbbeta-Actin1 > LbGapdh > Lbalpha-Tubulin > Lbbeta-Actin2 > Lb18S rRNA for deltamethrin induction, while ranked Lbbeta-Actin1 > Lbalpha-Tubulin > Lbbeta-Actin2 > LbGapdh > Lb18S rRNA for the different developmental stages.

Molecular characterization of two nicotinic acetylcholine receptor subunits from Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae).

Two nicotinic acetylcholine receptor (nAChR) subunit genes, Lbalpha1 and Lbalpha8, were isolated and characterized from psocid, Liposcelis bostrychophila Badonnel, using the rapid amplification of cDNA ends (RACE) technique. They are the first two nAChR family members isolated from the insect order of Psocoptera. The full-length cDNAs of Lbalpha1 (GenBank accession number: EU871527) and Lbalpha8 (EU871526) consist of 2,025 and 1,763 nucleotides, respectively, and an open reading frame of 1,644 and 1,608 bp encoding 547 and 535 amino acid proteins, respectively. Both genes have typical features of nAChR family members, though they share only 56% identity in amino acid sequence. The dendrogram generated by the MEGA 3.1 program shows that the protein deduced by Lbalpha1 had the closest phylogenetic relationship to Agamalpha1 from Anopheles gambiae and Amelalpha1 from Apis mellifera, and Lbalpha8 shares the highest identity with Agamalpha8 from An. gambiae and Amelalpha8 from A. mellifera. Quantitative real-time PCR analysis showed that Lbalpha1 was expressed 2.03-6.54-fold higher than Lbalpha8 at the different developmental stages of L. bostrychophila. The highest expression levels of Lbalpha1 and Lbalpha8 were both detected at adult stage and the lowest were at the third and fourth nymphal stages, respectively. There was a stable and relatively low expression level for Lbalpha1, whereas there was a descending expression pattern for Lbalpha8 in the 1st through the 4th nymphal stadia.