Chromosome-level genome assembly of Bactrocera dorsalis reveals its adaptation and invasion mechanisms.

Bactrocera dorsalis is an invasive polyphagous pest causing considerable ecological and economic damage worldwide. We report a high-quality chromosome-level genome assembly and combine various transcriptome data to explore the molecular mechanisms of its rapid adaptation to new environments. The expansions of the DDE transposase superfamily and key gene families related to environmental adaptation and enrichment of the expanded and unique gene families in metabolism and defence response pathways explain its environmental adaptability. The relatively high but not significantly different expression of heat-shock proteins, regardless of the environmental conditions, suggests an intrinsic mechanism underlying its adaptation to high temperatures. The mitogen-activated protein kinase pathway plays a key role in adaptation to new environments. The prevalence of duplicated genes in its genome explains the diversity in the B. dorsalis complex. These findings provide insights into the genetic basis of the invasiveness and diversity of B. dorsalis, explaining its rapid adaptation and expansion.

Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control.

p53 gene family members in humans and other organisms encode a large number of protein isoforms whose functions are largely undefined. Using Drosophila as a model, we find that a p53B isoform is expressed predominantly in the germline where it colocalizes with p53A into subnuclear bodies. It is only p53A, however, that mediates the apoptotic response to ionizing radiation in the germline and soma. In contrast, p53A and p53B are both required for the normal repair of meiotic DNA breaks, an activity that is more crucial when meiotic recombination is defective. We find that in oocytes with persistent DNA breaks p53A is also required to activate a meiotic pachytene checkpoint. Our findings indicate that Drosophila p53 isoforms have DNA lesion and cell type-specific functions, with parallels to the functions of mammalian p53 family members in the genotoxic stress response and oocyte quality control.

Radiation with reticulation marks the origin of a major malaria vector.

Advances in genomics have led to an appreciation that introgression is common, but its evolutionary consequences are poorly understood. In recent species radiations the sharing of genetic variation across porous species boundaries can facilitate adaptation to new environments and generate novel phenotypes, which may contribute to further diversification. Most Anopheles mosquito species that are of major importance as human malaria vectors have evolved within recent and rapid radiations of largely nonvector species. Here, we focus on one of the most medically important yet understudied anopheline radiations, the Afrotropical Anopheles funestus complex (AFC), to investigate the role of introgression in its diversification and the possible link between introgression and vector potential. The AFC comprises at least seven morphologically similar species, yet only An. funestus sensu stricto is a highly efficient malaria vector with a pan-African distribution. Based on de novo genome assemblies and additional whole-genome resequencing, we use phylogenomic and population genomic analyses to establish species relationships. We show that extensive interspecific gene flow involving multiple species pairs has shaped the evolutionary history of the AFC since its diversification. The most recent introgression event involved a massive and asymmetrical movement of genes from a distantly related AFC lineage into An. funestus, an event that predated and plausibly facilitated its subsequent dramatic geographic range expansion across most of tropical Africa. We propose that introgression may be a common mechanism facilitating adaptation to new environments and enhancing vectorial capacity in Anopheles mosquitoes.

Whitefly genomes contain ribotoxin coding genes acquired from plants.

Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of 28S rRNA. These enzymes are widely distributed among plants and bacteria. Previously, we have described for the first time RIP genes in mosquitoes belonging to the Culicidae family. We showed that these genes are derived from a single event of horizontal gene transfer (HGT) from a prokaryotic donor. Mosquito RIP genes are evolving under purifying selection, strongly suggesting that these toxins have acquired a functional role. In this work, we show the existence of two RIP encoding genes in the genome of the whitefly Bemisia tabaci, a hemiptera species belonging to the Aleyrodidae family distantly related to mosquitoes. Contamination artifacts were ruled out analyzing three independent B. tabaci genome databases. In contrast to mosquito RIPs, whitefly genes harbor introns and according to transcriptomic evidence are transcribed and spliced. Phylogeny and the taxonomic distribution strongly support that whitefly RIP genes are derived from an independent HGT event from a plant source. These results, along with our previous description of RIPs in Diptera, suggest that the acquired genes are functional in these insects and confer some fitness advantage.

Selective sweeps on novel and introgressed variation shape mimicry loci in a butterfly adaptive radiation.

Natural selection leaves distinct signatures in the genome that can reveal the targets and history of adaptive evolution. By analysing high-coverage genome sequence data from 4 major colour pattern loci sampled from nearly 600 individuals in 53 populations, we show pervasive selection on wing patterns in the Heliconius adaptive radiation. The strongest signatures correspond to loci with the greatest phenotypic effects, consistent with visual selection by predators, and are found in colour patterns with geographically restricted distributions. These recent sweeps are similar between co-mimics and indicate colour pattern turn-over events despite strong stabilising selection. Using simulations, we compare sweep signatures expected under classic hard sweeps with those resulting from adaptive introgression, an important aspect of mimicry evolution in Heliconius butterflies. Simulated recipient populations show a distinct 'volcano' pattern with peaks of increased genetic diversity around the selected target, characteristic of sweeps of introgressed variation and consistent with diversity patterns found in some populations. Our genomic data reveal a surprisingly dynamic history of colour pattern selection and co-evolution in this adaptive radiation.

Structure of Drosophila melanogaster ARC1 reveals a repurposed molecule with characteristics of retroviral Gag.

The tetrapod neuronal protein ARC and its Drosophila melanogaster homolog, dARC1, have important but differing roles in neuronal development. Both are thought to originate through exaptation of ancient Ty3/Gypsy retrotransposon Gag, with their novel function relying on an original capacity for self-assembly and encapsidation of nucleic acids. Here, we present the crystal structure of dARC1 CA and examine the relationship between dARC1, mammalian ARC, and the CA protein of circulating retroviruses. We show that while the overall architecture is highly related to that of orthoretroviral and spumaretroviral CA, there are substantial deviations in both amino- and carboxyl-terminal domains, potentially affecting recruitment of partner proteins and particle assembly. The degree of sequence and structural divergence suggests that Ty3/Gypsy Gag has been exapted on two separate occasions and that, although mammalian ARC and dARC1 share functional similarity, the structures have undergone different adaptations after appropriation into the tetrapod and insect genomes.

Genome editing retraces the evolution of toxin resistance in the monarch butterfly.

Identifying the genetic mechanisms of adaptation requires the elucidation of links between the evolution of DNA sequence, phenotype, and fitness1. Convergent evolution can be used as a guide to identify candidate mutations that underlie adaptive traits2-4, and new genome editing technology is facilitating functional validation of these mutations in whole organisms1,5. We combined these approaches to study a classic case of convergence in insects from six orders, including the monarch butterfly (Danaus plexippus), that have independently evolved to colonize plants that produce cardiac glycoside toxins6-11. Many of these insects evolved parallel amino acid substitutions in the α-subunit (ATPα) of the sodium pump (Na+/K+-ATPase)7-11, the physiological target of cardiac glycosides12. Here we describe mutational paths involving three repeatedly changing amino acid sites (111, 119 and 122) in ATPα that are associated with cardiac glycoside specialization13,14. We then performed CRISPR-Cas9 base editing on the native Atpα gene in Drosophila melanogaster flies and retraced the mutational path taken across the monarch lineage11,15. We show in vivo, in vitro and in silico that the path conferred resistance and target-site insensitivity to cardiac glycosides16, culminating in triple mutant 'monarch flies' that were as insensitive to cardiac glycosides as monarch butterflies. 'Monarch flies' retained small amounts of cardiac glycosides through metamorphosis, a trait that has been optimized in monarch butterflies to deter predators17-19. The order in which the substitutions evolved was explained by amelioration of antagonistic pleiotropy through epistasis13,14,20-22. Our study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities2,6-11,15,17-19.

Zelda and the maternal-to-zygotic transition in cockroaches.

In the endopterygote Drosophila melanogaster, Zelda is an activator of the zygotic genome during the maternal-to-zygotic transition (MZT). Zelda binds cis-regulatory elements (TAGteam heptamers), making chromatin accessible for gene transcription. Zelda has been studied in other endopterygotes: Apis mellifera and Tribolium castaneum, and the paraneopteran Rhodnius prolixus. We studied Zelda in the cockroach Blattella germanica, a hemimetabolan, short germ-band, and polyneopteran species. B. germanica Zelda has the complete set of functional domains, which is typical of species displaying ancestral features concerning embryogenesis. Interestingly, we found D. melanogaster TAGteam heptamers in the B. germanica genome. The canonical one, CAGGTAG, is present at a similar proportion in the genome of these two species and in the genome of other insects, suggesting that the genome admits as many CAGGTAG motifs as its length allows. Zelda-depleted embryos of B. germanica show defects involving blastoderm formation and abdomen development, and genes contributing to these processes are down-regulated. We conclude that in B. germanica, Zelda strictly activates the zygotic genome, within the MZT, a role conserved in more derived endopterygote insects. In B. germanica, zelda is expressed during MZT, whereas in D. melanogaster and T. castaneum it is expressed beyond this transition. In these species and A. mellifera, Zelda has functions even in postembryonic development. The expansion of zelda expression beyond the MZT in endopterygotes might be related with the evolutionary innovation of holometabolan metamorphosis. DATABASES: The RNA-seq datasets of B. germanica, D. melanogaster, and T. castaneum are accessible at the GEO databases GSE99785, GSE18068, GSE63770, and GSE84253. In addition, the RNA-seq library from T. castaneum adult females is available at SRA: SRX021963. The B. germanica reference genome is available as BioProject PRJNA203136.

A Gene Expression Screen in Drosophila melanogaster Identifies Novel JAK/STAT and EGFR Targets During Oogenesis.

The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) and epidermal growth factor receptor (EGFR) signaling pathways are conserved regulators of tissue patterning, morphogenesis, and other cell biological processes. During Drosophila oogenesis, these pathways determine the fates of epithelial follicle cells (FCs). JAK/STAT and EGFR together specify a population of cells called the posterior follicle cells (PFCs), which signal to the oocyte to establish the embryonic axes. In this study, whole genome expression analysis was performed to identify genes activated by JAK/STAT and/or EGFR. We observed that 317 genes were transcriptionally upregulated in egg chambers with ectopic JAK/STAT and EGFR activity in the FCs. The list was enriched for genes encoding extracellular matrix (ECM) components and ECM-associated proteins. We tested 69 candidates for a role in axis establishment using RNAi knockdown in the FCs. We report that the signaling protein Semaphorin 1b becomes enriched in the PFCs in response to JAK/STAT and EGFR. We also identified ADAM metallopeptidase with thrombospondin type 1 motif A (AdamTS-A) as a novel target of JAK/STAT in the FCs that regulates egg chamber shape. AdamTS-A mRNA becomes enriched at the anterior and posterior poles of the egg chamber at stages 6 to 7 and is regulated by JAK/STAT. Altering AdamTS-A expression in the poles or middle of the egg chamber produces rounder egg chambers. We propose that AdamTS-A regulates egg shape by remodeling the basement membrane.

Unravelling relationships among the shared stripes of sailors: Mitogenomic phylogeny of Limenitidini butterflies (Lepidoptera, Nymphalidae, Limenitidinae), focusing on the genera Athyma and Limenitis.

The phylogenetic relationships of the nymphalid butterfly tribe Limenitidini are best known for the genera Limenitis and Adelpha, model taxa for evolutionary processes such as Batesian mimicry and rapid adaptive radiations. Whereas these American limenitidines have received the most attention, phylogenetic relationships of their Asian relatives are still controversial and largely unexplored. Even one of the largest genera in Asia, Athyma, is polyphyletic. To clarify the phylogenetic relationships of these Asian Limenitidini, a total of 53 representatives were sampled; 37 have their mitogenomes sequenced for the first time. Our phylogenetic results confirm that mitogenomic data provides well-resolved relationships at most major levels of the phylogeny, even using different partition schemes or different inference methods. Interestingly, our results show that some Athyma taxa are embedded within the genus Limenitis, whereas the genus Tacola, previously considered to be a synonym of Athyma, needs to be recognized as a valid clade. Additionally, the other Limenitidini genera in Asia (namely Tarattia, Litinga, Sumalia, Pandita and Patsuia) are now grouped either within Athyma or Limenitis, so these genera need to be sunk. Importantly, we also show that the mainly Old World Limenitis and entirely New World Adelpha are sister groups, confirming the relevance of Asian lineages to global studies of Limenitis evolution.

Spatiotemporally controlled genetic perturbation for efficient large-scale studies of cell non-autonomous effects.

Studies in genetic model organisms have revealed much about the development and pathology of complex tissues. Most have focused on cell-intrinsic gene functions and mechanisms. Much less is known about how transformed, or otherwise functionally disrupted, cells interact with healthy ones toward a favorable or pathological outcome. This is largely due to technical limitations. We developed new genetic tools in Drosophila melanogaster that permit efficient multiplexed gain- and loss-of-function genetic perturbations with separable spatial and temporal control. Importantly, our novel tool-set is independent of the commonly used GAL4/UAS system, freeing the latter for additional, non-autonomous, genetic manipulations; and is built into a single strain, allowing one-generation interrogation of non-autonomous effects. Altogether, our design opens up efficient genome-wide screens on any deleterious phenotype, once plasmid or genome engineering is used to place the desired miRNA(s) or ORF(s) into our genotype. Specifically, we developed tools to study extrinsic effects on neural tumor growth but the strategy presented has endless applications within and beyond neurobiology, and in other model organisms.

Genome-based identification and analysis of ionotropic receptors in Spodoptera litura.

The ability to sense and recognize various classes of compounds is of particular importance for survival and reproduction of insects. Ionotropic receptor (IR), a sub-family of the ionotropic glutamate receptor family, has been identified as one of crucial chemoreceptor super-families, which mediates the sensing of odors and/or tastants, and serves as non-chemosensory functions. Yet, little is known about IR characteristics, evolution, and functions in Lepidoptera. Here, we identify the IR gene repertoire from a destructive polyphagous pest, Spodoptera litura. The exhaustive analyses with genome and transcriptome data lead to the identification of 45 IR genes, comprising 17 antennal IRs (A-IRs), 8 Lepidoptera-specific IRs (LS-IRs), and 20 divergent IRs (D-IRs). Phylogenetic analysis reveals that S. litura A-IRs generally retain a strict single copy within each orthologous group, and two lineage expansions are observed in the D-IR sub-family including IR100d-h and 100i-o, likely attributed to gene duplications. Results of gene structure analysis classify the SlitIRs into four types: I (intronless), II (1-3 introns), III (5-9 introns), and IV (10-18 introns). Extensive expression profiles demonstrate that the majority of SlitIRs (28/43) are enriched in adult antennae, and some are detected in gustatory-associated tissues like proboscises and legs as well as non-chemosensory organs like abdomens and reproductive tissues of both sexes. These results indicate that SlitIRs have diverse functional roles in olfaction, taste, and reproduction. Together, our study has complemented the information on chemoreceptor genes in S. litura, and meanwhile allows for target experiments to identify potential IR candidates for the control of this pest.

Simultaneous radiation of bird and mammal lice following the K-Pg boundary.

The diversification of parasite groups often occurs at the same time as the diversification of their hosts. However, most studies demonstrating this concordance only examine single host-parasite groups. Multiple diverse lineages of ectoparasitic lice occur across both birds and mammals. Here, we describe the evolutionary history of lice based on analyses of 1107 single-copy orthologous genes from sequenced genomes of 46 species of lice. We identify three major diverse groups of lice: one exclusively on mammals, one almost exclusively on birds and one on both birds and mammals. Each of these groups radiated just after the Cretaceous-Paleogene (K-Pg) boundary, the time of the mass extinction event of the dinosaurs and rapid diversification of most of the modern lineages of birds and mammals.

Draft genomes of two blister beetles Hycleus cichorii and Hycleus phaleratus.

Background: Commonly known as blister beetles or Spanish fly, there are more than 1500 species in the Meloidae family (Hexapoda: Coleoptera: Tenebrionoidea) that produce the potent defensive blistering agent cantharidin. Cantharidin and its derivatives have been used to treat cancers such as liver, stomach, lung, and esophageal cancers. Hycleus cichorii and Hycleus phaleratus are the most commercially important blister beetles in China due to their ability to biosynthesize this potent vesicant. However, there is a lack of genome reference, which has hindered development of studies on the biosynthesis of cantharidin and a better understanding of its biology and pharmacology. Results: We report 2 draft genomes and quantified gene sets for the blister beetles H. cichorii and H. phaleratus, 2 complex genomes with >72% repeats and approximately 1% heterozygosity, using Illumina sequencing data. An integrated assembly pipeline was performed for assembly, and most of the coding regions were obtained. Benchmarking universal single-copy orthologs (BUSCO) assessment showed that our assembly obtained more than 98% of the Endopterygota universal single-copy orthologs. Comparison analysis showed that the completeness of coding genes in our assembly was comparable to other beetle genomes such as Dendroctonus ponderosae and Agrilus planipennis. Gene annotation yielded 13 813 and 13 725 protein-coding genes in H. cichorii and H. phaleratus, of which approximately 89% were functionally annotated. BUSCO assessment showed that approximately 86% and 84% of the Endopterygota universal single-copy orthologs were annotated completely in these 2 gene sets, whose completeness is comparable to that of D. ponderosae and A. planipennis. Conclusions: Assembly of both blister beetle genomes provides a valuable resource for future biosynthesis of cantharidin and comparative genomic studies of blister beetles and other beetles.

The Z chromosome is enriched for sperm proteins in two divergent species of Lepidoptera.

Genes that promote sexual conflict, such as those with a sex-limited fitness benefit, are expected to accumulate differentially on sex chromosomes relative to autosomes. Few tests of this hypothesis exist for male homogametic (ZZ) taxa, however, and most use RNA expression data to identify such genes. Here, we employ a different identification method by using proteomic analysis of sperm cells to identify genes with a sex-limited benefit. We tested for a bias in genomic location of sperm protein genes in two species of Lepidoptera. An excess of sperm protein genes was identified on the Z chromosomes of both the Carolina sphinx moth (Manduca sexta) and the monarch butterfly (Danaus plexippus). Taking into consideration a Z-autosome fusion in monarchs, we discover that the ancestrally sex-linked portion of the genome is the source of this enrichment, while the newly sex-linked portion still appears similar to autosomes in relative abundance of sperm protein genes. Together, these results point to an enrichment of male-beneficial genes on the Z chromosome and demonstrate the usefulness of proteomic datasets in sexual conflict research.

An analysis of 67 RNA-seq datasets from various tissues at different stages of a model insect, Manduca sexta.

BACKGROUND: Manduca sexta is a large lepidopteran insect widely used as a model to study biochemistry of insect physiological processes. As a part of its genome project, over 50 cDNA libraries have been analyzed to profile gene expression in different tissues and life stages. While the RNA-seq data were used to study genes related to cuticle structure, chitin metabolism and immunity, a vast amount of the information has not yet been mined for understanding the basic molecular biology of this model insect. In fact, the basic features of these data, such as composition of the RNA-seq reads and lists of library-correlated genes, are unclear. From an extended view of all insects, clear-cut tempospatial expression data are rarely seen in the largest group of animals including Drosophila and mosquitoes, mainly due to their small sizes. RESULTS: We obtained the transcriptome data, analyzed the raw reads in relation to the assembled genome, and generated heatmaps for clustered genes. Library characteristics (tissues, stages), number of mapped bases, and sequencing methods affected the observed percentages of genome transcription. While up to 40% of the reads were not mapped to the genome in the initial Cufflinks gene modeling, we identified the causes for the mapping failure and reduced the number of non-mappable reads to <8%. Similarities between libraries, measured based on library-correlated genes, clearly identified differences among tissues or life stages. We calculated gene expression levels, analyzed the most abundantly expressed genes in the libraries. Furthermore, we analyzed tissue-specific gene expression and identified 18 groups of genes with distinct expression patterns. CONCLUSION: We performed a thorough analysis of the 67 RNA-seq datasets to characterize new genomic features of M. sexta. Integrated knowledge of gene functions and expression features will facilitate future functional studies in this biochemical model insect.

The draft genome of whitefly Bemisia tabaci MEAM1, a global crop pest, provides novel insights into virus transmission, host adaptation, and insecticide resistance.

BACKGROUND: The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is among the 100 worst invasive species in the world. As one of the most important crop pests and virus vectors, B. tabaci causes substantial crop losses and poses a serious threat to global food security. RESULTS: We report the 615-Mb high-quality genome sequence of B. tabaci Middle East-Asia Minor 1 (MEAM1), the first genome sequence in the Aleyrodidae family, which contains 15,664 protein-coding genes. The B. tabaci genome is highly divergent from other sequenced hemipteran genomes, sharing no detectable synteny. A number of known detoxification gene families, including cytochrome P450s and UDP-glucuronosyltransferases, are significantly expanded in B. tabaci. Other expanded gene families, including cathepsins, large clusters of tandemly duplicated B. tabaci-specific genes, and phosphatidylethanolamine-binding proteins (PEBPs), were found to be associated with virus acquisition and transmission and/or insecticide resistance, likely contributing to the global invasiveness and efficient virus transmission capacity of B. tabaci. The presence of 142 horizontally transferred genes from bacteria or fungi in the B. tabaci genome, including genes encoding hopanoid/sterol synthesis and xenobiotic detoxification enzymes that are not present in other insects, offers novel insights into the unique biological adaptations of this insect such as polyphagy and insecticide resistance. Interestingly, two adjacent bacterial pantothenate biosynthesis genes, panB and panC, have been co-transferred into B. tabaci and fused into a single gene that has acquired introns during its evolution. CONCLUSIONS: The B. tabaci genome contains numerous genetic novelties, including expansions in gene families associated with insecticide resistance, detoxification and virus transmission, as well as numerous horizontally transferred genes from bacteria and fungi. We believe these novelties likely have shaped B. tabaci as a highly invasive polyphagous crop pest and efficient vector of plant viruses. The genome serves as a reference for resolving the B. tabaci cryptic species complex, understanding fundamental biological novelties, and providing valuable genetic information to assist the development of novel strategies for controlling whiteflies and the viruses they transmit.

The Complete Mitochondrial Genome of Delia antiqua and Its Implications in Dipteran Phylogenetics.

Delia antiqua is a major underground agricultural pest widely distributed in Asia, Europe and North America. In this study, we sequenced and annotated the complete mitochondrial genome of this species, which is the first report of complete mitochondrial genome in the family Anthomyiidae. This genome is a double-stranded circular molecule with a length of 16,141 bp and an A+T content of 78.5%. It contains 37 genes (13 protein-coding genes, 22 tRNAs and 2 rRNAs) and a non-coding A+T rich region or control region. The mitochondrial genome of Delia antiqua presents a clear bias in nucleotide composition with a positive AT-skew and a negative GC-skew. All of the 13 protein-coding genes use ATN as an initiation codon except for the COI gene that starts with ATCA. Most protein-coding genes have complete termination codons but COII and ND5 that have the incomplete termination codon T. This bias is reflected in both codon usage and amino acid composition. The protein-coding genes in the D. antiqua mitochondrial genome prefer to use the codon UUA (Leu). All of the tRNAs have the typical clover-leaf structure, except for tRNASer(AGN) that does not contain the dihydrouridine (DHU) arm like in many other insects. There are 7 mismatches with U-U in the tRNAs. The location and structure of the two rRNAs are conservative and stable when compared with other insects. The control region between 12S rRNA and tRNAIle has the highest A+T content of 93.7% in the D. antiqua mitochondrial genome. The control region includes three kinds of special regions, two highly conserved poly-T stretches, a (TA)n stretch and several G(A)nT structures considered important elements related to replication and transcription. The nucleotide sequences of 13 protein-coding genes are used to construct the phylogenetics of 26 representative Dipteran species. Both maximum likelihood and Bayesian inference analyses suggest a closer relationship of D. antiqua in Anthomyiidae with Calliphoridae, Calliphoridae is a paraphyly, and both Oestroidea and Muscoidea are polyphyletic.

Unique synteny and alternate splicing of the chitin synthases in closely related heliothine moths.

Chitin is an extracellular biopolymer that contributes to the cuticular structural matrix in arthropods. As a consequence of its rigid structure, the chitinous cuticle must be shed and replaced to accommodate growth. Two chitin synthase genes that encode for chitin synthase A (ChSA), which produces cuticular exoskeleton, and chitin synthase B (ChSB), which produces peritrophic membrane, were characterized in the genomes of two heliothine moths: the corn earworm/cotton bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) and the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). In both moths, the two genes were arranged in tandem with the same orientation on the same strand with ChSB located 5' of ChSA. Sequence comparisons showed that the coding sequences were highly conserved with homologues from other species but that the tandem juxtaposed genomic arrangement of the two genes was unique in these insects. The mechanism that has led to this arrangement is unclear but is most likely a recent recombinational event. Transcript mapping of HzChSB and HzChSA in H. zea demonstrated that both transcripts were differentially spliced in various tissues and larval stages. The identification of the HzChSB-E12b alternate spliced transcript is the first report of alternate splicing for the ChSB group. The importance of this splice form is not clear because the protein produced would lack any enzymatic activity but retain the membrane insertion motifs. As for other insects, these genes provide an important target for potential control through RNAi but also provide a subject for broad scale genomic recombinational events.

Large-scale detection and analysis of adenosine-to-inosine RNA editing during development in Plutella xylostella.

Adenosine-to-inosine (A-to-I) RNA editing is site-specific modification of RNAs that increases the diversity of the transcriptome and proteome. Most insects undergo complete metamorphosis, including four life cycle stages: egg, larva, pupa and adult. Many previous studies have confirmed that RNA-editing events occur in a development-specific manner; in other words, RNA-editing levels change during metamorphosis. Here, we describe an effort to identify the developmental specificity of RNA-editing events using a large-scale computational analysis of RNA-seq data derived from four developmental stages of the diamondback moth, Plutella xylostella. One thousand one hundred and eighty-seven A-to-I RNA-editing sites were predicted to be developmental stage specific (false-discovery rate <0.01) and 1,094 of these sites were located in protein-coding regions. Editing of 152 sites resulted in an altered amino acid residue. A putative adult-specific A-to-I RNA-editing site was verified by comparing cDNA sequences with its corresponding genomic locus at different stages of the P. xylostella life cycle. Our findings will help elucidate the role of A-to-I RNA editing in the regulation of metamorphosis. Further studies detailing changes in the extent of editing are needed to establish how as yet unknown regulatory factors are involved in the editing mechanism and what biological functions' editing serves.