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Background Hymenoptera comprise extremely diverse insect species with extensive variation in their life histories. The Dryinidae, a family of solitary wasps of Hymenoptera, have evolved innovations that allow them to hunt using venom and a pair of chelae developed from the fore legs that can grasp prey. Dryinidae larvae are also parasitoids of Auchenorrhyncha, a group including common pests such as planthoppers and leafhoppers. Both of these traits make them effective and valuable for pest control, but little is yet known about the genetic basis of its dual adaptation to parasitism and predation. Results We sequenced and assembled a high-quality genome of the dryinid wasp Gonatopus flavifemur, which at 636.5 Mb is larger than most hymenopterans. The expansion of transposable elements, especially DNA transposons, is a major contributor to the genome size enlargement. Our genome-wide screens reveal a number of positively selected genes and rapidly evolving proteins involved in energy production and motor activity, which may contribute to the predatory adaptation of dryinid wasp. We further show that three female-biased, reproductive-associated yellow genes, in response to the prey feeding behavior, are significantly elevated in adult females, which may facilitate the egg production. Venom is a powerful weapon for dryinid wasp during parasitism and predation. We therefore analyze the transcriptomes of venom glands and describe specific expansions in venom Idgf-like genes and neprilysin-like genes. Furthermore, we find the LWS2-opsin gene is exclusively expressed in male G. flavifemur, which may contribute to partner searching and mating. Conclusions Our results provide new insights into the genome evolution, predatory adaptation, venom evolution, and sex-biased genes in G. flavifemur, and present genomic resources for future in-depth comparative analyses of hymenopterans that may benefit pest control.

Target‐site mutations and detoxification gene overexpression are two major mechanisms conferring insecticide resistance. Molecular assays applied to detect these resistance genetic markers are time‐consuming and with high false‐positive rates. RNA‐Seq data contains information on the variations within expressed genomic regions and expression of detoxification genes. However, there is no corresponding method to detect resistance markers at present. Here, we collected 66 reported resistance mutations of four insecticide targets (AChE, VGSC, RyR, and nAChR) from 82 insect species. Next, we obtained 403 sequences of the four target genes and 12,665 sequences of three kinds of detoxification genes including P450s, GSTs, and CCEs. Then, we developed a Perl program, FastD, to detect target‐site mutations and overexpressed detoxification genes from RNA‐Seq data and constructed a web server for FastD (http://www.insect-genome.com/fastd). The estimation of FastD on simulated RNA‐Seq data showed high sensitivity and specificity. We applied FastD to detect resistant markers in 15 populations of six insects, Plutella xylostella, Aphis gossypii, Anopheles arabiensis, Musca domestica, Leptinotarsa decemlineata and Apis mellifera. Results showed that 11 RyR mutations in P. xylostella, one nAChR mutation in A. gossypii, one VGSC mutation in A. arabiensis and five VGSC mutations in M. domestica were found to be with frequency difference >40% between resistant and susceptible populations including previously confirmed mutations G4946E in RyR, R81T in nAChR and L1014F in VGSC. And 49 detoxification genes were found to be overexpressed in resistant populations compared with susceptible populations including previously confirmed detoxification genes CYP6BG1, CYP6CY22, CYP6CY13, CYP6P3, CYP6M2, CYP6P4 and CYP4G16. The candidate target‐site mutations and detoxification genes were worth further validation. Resistance estimates according to confirmed markers were consistent with population phenotypes, confirming the reliability of this program in predicting population resistance at omics‐level., We developed a program called FastD to detect the insecticide target‐site mutations and overexpressed detoxification genes from RNA‐Seq data and constructed a corresponding web server for FastD (http://www.insect-genome.com/fastd) in this article.

The cotton mealybug, Phenacoccus solenopsis, is an invasive pest that can cause massive damage to many host plants of agricultural importance. P. solenopsis is highly polyphagous, and shows extreme sexual dimorphism between males and females. The functions of DNA methyltransferase (DNMT) enzymes in the cotton mealybug have not been well studied. Here, we carried out an investigation of DNMTs in cotton mealybug to study their roles in sexual dimorphism. We found that the cotton mealybug has two copies of PsDnmt1, but Dnmt3 is absent. We then amplified the full-length cDNAs of PsDnmt1A (2,225 bp) and PsDnmt1B (2,862 bp) using rapid amplification cDNA ends (RACE). Quantitative reverse transcriptase PCR shows that both PsDnmt1A and PsDnmt1B are highly expressed in adult males, while the expression of PsDnmt1B is 30-fold higher in gravid females than in virgin females. We knocked down PsDnmt1A and PsDnmt1B with small interfering RNAs (siRNAs), and both genes were successfully down-regulated after 24 h or 72 h in adult females and pupa (t-test, p &lt, 0.05). Down-regulating the expression of these two DNMT genes led to offspring lethality and abnormal body color in adult females. Furthermore, the silencing of PsDnmt1B induced abnormal wing development in emerged adult males. Our results provide evidence that PsDnmt1 plays a crucial role in regulating sexual dimorphism in the cotton mealybug.

Summary Rice stem borer (RSB, Chilo suppressalis) is an insect pest that causes huge economic losses every year. Control efforts rely heavily on chemical insecticides, which leads to serious problems such as insecticide resistance, environment pollution, and food safety issues. Therefore, developing alternative pest control methods is an important task. Here, we identified an insect‐specific microRNA, miR‐14, in RSB, which was predicted to target Spook (Spo) and Ecdysone receptor (EcR) in the ecdysone signalling network. In‐vitro dual luciferase assays using HEK293T cells confirmed the interactions of Csu‐miR‐14 with CsSpo and with CsEcR. Csu‐miR‐14 exhibited high levels of expression at the end of each larval instar stage, and its expression was negatively correlated with the expression of its two target genes. Overexpression of Csu‐miR‐14 at the third day of the fifth instar stage led to high mortality and developmental defects in RSB individuals. We produced 35 rice transformants to express miR‐14 and found that three lines had a single copy with highly abundant miR‐14 mature transcripts. Feeding bioassays using both T0 and T1 generations of transgenic miR‐14 rice indicated that at least one line (C#24) showed high resistance to RSB. These results indicated that the approach of miRNAs as targets has potential for improving pest control methods. Moreover, using insect‐specific miRNAs rather than protein‐encoding genes for pest control may prove benign to non‐insect species, and thus is worthy of further exploration.

The accurate rise and fall of active hormones is important for insect development. The ecdysteroids must be cleared in a timely manner. However, the mechanism of suppressing the ecdysteroid biosynthesis at the right time remains unclear. Here, we sequenced a small RNA library of Chilo suppressalis and identified 300 miRNAs in this notorious rice insect pest. Microarray analysis yielded 54 differentially expressed miRNAs during metamorphosis development. Target prediction and in vitro dual-luciferase assays confirmed that seven miRNAs (two conserved and five novel miRNAs) jointly targeted three Halloween genes in the ecdysteroid biosynthesis pathway. Overexpression of these seven miRNAs reduced the titer of 20-hydroxyecdysone (20E), induced mortality, and retarded development, which could be rescued by treatment with 20E. Comparative analysis indicated that the miRNA regulation of metamorphosis development is a conserved process but that the miRNAs involved are highly divergent. In all, we present evidence that both conserved and lineage-specific miRNAs have crucial roles in regulating development in insects by controlling ecdysteroid biosynthesis, which is important for ensuring developmental convergence and evolutionary diversity.

Background Insecticide resistance is a substantial problem in controlling agricultural and medical pests. Detecting target site mutations is crucial to manage insecticide resistance. Though PCR-based methods have been widely used in this field, they are time-consuming and inefficient, and typically have a high false positive rate. Acetylcholinesterases (Ace) is the neural target of the widely used organophosphate (OP) and carbamate insecticides. However, there is not any software available to detect insecticide resistance associated mutations in RNA-Seq data at present. Results A computational pipeline ACE was developed to detect resistance mutations of ace in insect RNA-Seq data. Known ace resistance mutations were collected and used as a reference. We constructed a Web server for ACE, and the standalone software in both Linux and Windows versions is available for download. ACE was used to analyse 971 RNA-Seq data from 136 studies in 7 insect pests. The mutation frequency of each RNA-Seq dataset was calculated. The results indicated that the resistance frequency was 30%–44% in an eastern Ugandan Anopheles population, thus suggesting this resistance-conferring mutation has reached high frequency in these mosquitoes in Uganda. Analyses of RNA-Seq data from the diamondback moth Plutella xylostella indicated that the G227A mutation was positively related with resistance levels to organophosphate or carbamate insecticides. The wasp Nasonia vitripennis had a low frequency of resistant reads (

The cotton mealybug, Phenacoccus solenopsis, is an invasive pest that can cause massive damage to many host plants of agricultural importance. P. solenopsis is highly polyphagous, and shows extreme sexual dimorphism between males and females. The functions of DNA methyltransferase (DNMT) enzymes in the cotton mealybug have not been well studied. Here, we carried out an investigation of DNMTs in cotton mealybug to study their roles in sexual dimorphism. We found that the cotton mealybug has two copies of PsDnmt1, but Dnmt3 is absent. We then amplified the full-length cDNAs of PsDnmt1A (2225 bp) and PsDnmt1B (2862 bp) using rapid amplification cDNA ends (RACE). Quantitative reverse transcriptase PCR shows that both PsDnmt1A and PsDnmt1B are highly expressed in adult males, while the expression of PsDnmt1B is 30-fold higher in gravid females than in virgin females. We knocked down PsDnmt1A and PsDnmt1B with small interfering RNAs (siRNAs), and both genes were successfully down-regulated after 24 h or 72 h in adult females and pupa (t-test, p < 0.05). Down-regulating the expression of these two DNMT genes led to offspring lethality and abnormal body color in adult females. Furthermore, the silencing of PsDnmt1B induced abnormal wing development in emerged adult males. Our results provide evidence that PsDnmt1 plays a crucial role in regulating sexual dimorphism in the cotton mealybug.

The precise increase and decrease of hormone ecdysone are critical for accurate development in insects. Most previous works focus on transcriptional activation of ecdysone production; however, little is known about the mechanism of switching off ecdysone biosynthesis after ecdysis. Here, we showed that the precursor microRNA-14 (pre-miR-14) encodes two mature miRNAs in silkworm; both of these two mature miRNAs regulate various genes in the ecdysone-signalling pathway. Bmo-miR-14-5p targets on nine genes whereas Bmo-miR-14-3p targets on two genes in the same pathway. These two mature miRNAs increased immediately after the ecdysis, efficiently suppressing the 20-hydroxyecdysone (20E) biosynthesis, the upstream regulation, and the downstream response genes. Knocking down either of two mature miRNAs or both of them delays moult development, impairing development synchrony in antagomir-treated groups. In addition, overexpressing Bmo-miR-14-5p but not Bmo-miR-14-3p significantly affected the 20E titer and increased the moulting time variation, suggesting that Bmo-miR-14-5p, though it is less abundant, has more potent effects in development regulation than Bmo-miR-14-3p. In summary, we present evidence that a pre-miRNA encodes two mature miRNAs targeting on the same pathway, which significantly improves miRNA regulation efficiencies to programmatically switch off ecdysone biosynthesis.

The genomes and transcriptomes of hundreds of insects have been sequenced. However, insect community lacks an integrated, up-to-date collection of insect gene data. Here, we introduce the first release of InsectBase, available online at http://www.insect-genome.com. The database encompasses 138 insect genomes, 116 insect transcriptomes, 61 insect gene sets, 36 gene families of 60 insects, 7544 miRNAs of 69 insects, 96,925 piRNAs of Drosophila melanogaster and Chilo suppressalis, 2439 lncRNA of Nilaparvata lugens, 22,536 pathways of 78 insects, 678,881 untranslated regions (UTR) of 84 insects and 160,905 coding sequences (CDS) of 70 insects. This release contains over 12 million sequences and provides search functionality, a BLAST server, GBrowse, insect pathway construction, a Facebook-like network for the insect community (iFacebook), and phylogenetic analysis of selected genes.

Long non-coding RNA (lncRNA) is a class of noncoding RNA >200 bp in length that has essential roles in regulating a variety of biological processes. Here, we constructed a computational pipeline to identify lncRNA genes in the diamondback moth (Plutella xylostella), a major insect pest of cruciferous vegetables. In total, 3,324 lncRNAs corresponding to 2,475 loci were identified from 13 RNA-Seq datasets, including samples from parasitized, insecticide-resistant strains and different developmental stages. The identified P. xylostella lncRNAs had shorter transcripts and fewer exons than protein-coding genes. Seven out of nine randomly selected lncRNAs were validated by strand-specific RT-PCR. In total, 54–172 lncRNAs were specifically expressed in the insecticide resistant strains, among which one lncRNA was located adjacent to the sodium channel gene. In addition, 63–135 lncRNAs were specifically expressed in different developmental stages, among which three lncRNAs overlapped or were located adjacent to the metamorphosis-associated genes. These lncRNAs were either strongly or weakly co-expressed with their overlapping or neighboring mRNA genes. In summary, we identified thousands of lncRNAs and presented evidence that lncRNAs might have key roles in conferring insecticide resistance and regulating the metamorphosis development in P. xylostella.

The NCBI accession numbers of insect ace-1 and ace-2 genes

The SRA accession numbers of 971 RNA-Seq data used for detecting mutations

Insects undergo metamorphosis, involving an abrupt change in body structure through cell growth and differentiation. Rice stem stripped borer (SSB), Chilo suppressalis, is one of the most destructive rice pests. However, little is known about the regulation mechanism of metamorphosis development in this notorious insect pest. Here, we studied the expression of 22,197 SSB genes at seven time points during pupa development with a customized microarray, identifying 622 differentially expressed genes (DEG) during pupa development. Gene ontology (GO) analysis of these DEGs indicated that the genes related to substance metabolism were highly expressed in the early pupa, which participate in the physiological processes of larval tissue disintegration at these stages. In comparison, highly expressed genes in the late pupal stages were mainly associated with substance biosynthesis, consistent with adult organ formation at these stages. There were 27 solute carrier (SLC) genes that were highly expressed during pupa development. We knocked down SLC22A3 at the prepupal stage, demonstrating that silencing SLC22A3 induced a deficiency in pupa stiffness and pigmentation. The RNAi-treated individuals had white and soft pupa, suggesting that this gene has an essential role in pupal development.

Primers used for RT-PCR validation of three lncRNA genes and their overlapping protein coding genes. (DOCX 12Â kb)

The number of specifically-expressed and differentially-expressed lncRNAs in various populations (DOCX 14Â kb)

Background Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNA primarily expressed in germ cells that can silence transposons at the post-transcriptional level. Accurate prediction of piRNAs remains a significant challenge. Results We developed a program for piRNA annotation (Piano) using piRNA-transposon interaction information. We downloaded 13,848 Drosophila piRNAs and 261,500 Drosophila transposons. The piRNAs were aligned to transposons with a maximum of three mismatches. Then, piRNA-transposon interactions were predicted by RNAplex. Triplet elements combining structure and sequence information were extracted from piRNA-transposon matching/pairing duplexes. A support vector machine (SVM) was used on these triplet elements to classify real and pseudo piRNAs, achieving 95.3 ± 0.33% accuracy and 96.0 ± 0.5% sensitivity. The SVM classifier can be used to correctly predict human, mouse and rat piRNAs, with overall accuracy of 90.6%. We used Piano to predict piRNAs for the rice stem borer, Chilo suppressalis, an important rice insect pest that causes huge yield loss. As a result, 82,639 piRNAs were predicted in C. suppressalis. Conclusions Piano demonstrates excellent piRNA prediction performance by using both structure and sequence features of transposon-piRNAs interactions. Piano is freely available to the academic community at http://ento.njau.edu.cn/Piano.html. Electronic supplementary material The online version of this article (doi:10.1186/s12859-014-0419-6) contains supplementary material, which is available to authorized users.

ChiloDB is an integrated resource that will be of use to the rice stem borer research community. The rice striped stem borer (SSB), Chilo suppressalis Walker, is a major rice pest that causes severe yield losses in most rice-producing countries. A draft genome of this insect is available. The aims of ChiloDB are (i) to store recently acquired genomic sequence and transcriptome data and integrate them with protein-coding genes, microRNAs, piwi-interacting RNAs (piRNAs) and RNA sequencing (RNA-Seq) data and (ii) to provide comprehensive search tools and downloadable data sets for comparative genomics and gene annotation of this important rice pest. ChiloDB contains the first version of the official SSB gene set, comprising 80 479 scaffolds and 10 221 annotated protein-coding genes. Additionally, 262 SSB microRNA genes predicted from a small RNA library, 82 639 piRNAs identified using the piRNApredictor software, 37 040 transcripts from a midgut transcriptome and 69 977 transcripts from a mixed sample have all been integrated into ChiloDB. ChiloDB was constructed using a data structure that is compatible with data resources, which will be incorporated into the database in the future. This resource will serve as a long-term and open-access database for research on the biology, evolution and pest control of SSB. To the best of our knowledge, ChiloDB is one of the first genomic and transcriptome database for rice insect pests. Database URL: http://ento.njau.edu.cn/ChiloDB.