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

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. 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. 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.

Cao X ，Jiang H 《BMC GENOMICS》

Integrated modeling of protein-coding genes in the Manduca sexta genome using RNA-Seq data from the biochemical model insect.

The genome sequence of Manduca sexta was recently determined using 454 technology. Cufflinks and MAKER2 were used to establish gene models in the genome assembly based on the RNA-Seq data and other species' sequences. Aided by the extensive RNA-Seq data from 50 tissue samples at various life stages, annotators over the world (including the present authors) have manually confirmed and improved a small percentage of the models after spending months of effort. While such collaborative efforts are highly commendable, many of the predicted genes still have problems which may hamper future research on this insect species. As a biochemical model representing lepidopteran pests, M. sexta has been used extensively to study insect physiological processes for over five decades. In this work, we assembled Manduca datasets Cufflinks 3.0, Trinity 4.0, and Oases 4.0 to assist the manual annotation efforts and development of Official Gene Set (OGS) 2.0. To further improve annotation quality, we developed methods to evaluate gene models in the MAKER2, Cufflinks, Oases and Trinity assemblies and selected the best ones to constitute MCOT 1.0 after thorough crosschecking. MCOT 1.0 has 18,089 genes encoding 31,666 proteins: 32.8% match OGS 2.0 models perfectly or near perfectly, 11,747 differ considerably, and 29.5% are absent in OGS 2.0. Future automation of this process is anticipated to greatly reduce human efforts in generating comprehensive, reliable models of structural genes in other genome projects where extensive RNA-Seq data are available.

Cao X ，Jiang H 《-》

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta.

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

Kanost MR ，Arrese EL ，Cao X ，Chen YR ，Chellapilla S ，Goldsmith MR ，Grosse-Wilde E ，Heckel DG ，Herndon N ，Jiang H ，Papanicolaou A ，Qu J ，Soulages JL ，Vogel H ，Walters J ，Waterhouse RM ，Ahn SJ ，Almeida FC ，An C ，Aqrawi P ，Bretschneider A ，Bryant WB ，Bucks S ，Chao H ，Chevignon G ，Christen JM ，Clarke DF ，Dittmer NT ，Ferguson LCF ，Garavelou S ，Gordon KHJ ，Gunaratna RT ，Han Y ，Hauser F ，He Y ，Heidel-Fischer H ，Hirsh A ，Hu Y ，Jiang H ，Kalra D ，Klinner C ，König C ，Kovar C ，Kroll AR ，Kuwar SS ，Lee SL ，Lehman R ，Li K ，Li Z ，Liang H ，Lovelace S ，Lu Z ，Mansfield JH ，McCulloch KJ ，Mathew T ，Morton B ，Muzny DM ，Neunemann D ，Ongeri F ，Pauchet Y ，Pu LL ，Pyrousis I ，Rao XJ ，Redding A ，Roesel C ，Sanchez-Gracia A ，Schaack S ，Shukla A ，Tetreau G ，Wang Y ，Xiong GH ，Traut W ，Walsh TK ，Worley KC ，Wu D ，Wu W ，Wu YQ ，Zhang X ，Zou Z ，Zucker H ，Briscoe AD ，Burmester T ，Clem RJ ，Feyereisen R ，Grimmelikhuijzen CJP ，Hamodrakas SJ ，Hansson BS ，Huguet E ，Jermiin LS ，Lan Q ，Lehman HK ，Lorenzen M ，Merzendorfer H ，Michalopoulos I ，Morton DB ，Muthukrishnan S ，Oakeshott JG ，Palmer W ，Park Y ，Passarelli AL ，Rozas J ，Schwartz LM ，Smith W ，Southgate A ，Vilcinskas A ，Vogt R ，Wang P ，Werren J ，Yu XQ ，Zhou JJ ，Brown SJ ，Scherer SE ，Richards S ，Blissard GW ... -  《-》

The immune signaling pathways of Manduca sexta.

Signal transduction pathways and their coordination are critically important for proper functioning of animal immune systems. Our knowledge of the constituents of the intracellular signaling network in insects mainly comes from genetic analyses in Drosophila melanogaster. To facilitate future studies of similar systems in the tobacco hornworm and other lepidopteran insects, we have identified and examined the homologous genes in the genome of Manduca sexta. Based on 1:1 orthologous relationships in most cases, we hypothesize that the Toll, Imd, MAPK-JNK-p38 and JAK-STAT pathways are intact and operative in this species, as are most of the regulatory mechanisms. Similarly, cellular processes such as autophagy, apoptosis and RNA interference probably function in similar ways, because their mediators and modulators are mostly conserved in this lepidopteran species. We have annotated a total of 186 genes encoding 199 proteins, studied their domain structures and evolution, and examined their mRNA levels in tissues at different life stages. Such information provides a genomic perspective of the intricate signaling system in a non-drosophiline insect.

Cao X ，He Y ，Hu Y ，Wang Y ，Chen YR ，Bryant B ，Clem RJ ，Schwartz LM ，Blissard G ，Jiang H ... -  《-》

A genome-wide analysis of antimicrobial effector genes and their transcription patterns in Manduca sexta.

Antimicrobial proteins/peptides (AMPs) are effectors of innate immune systems against pathogen infection in multicellular organisms. Over half of the AMPs reported so far come from insects, and these effectors act in concert to suppress or kill bacteria, fungi, viruses, and parasites. In this work, we have identified 86 AMP genes in the Manduca sexta genome, most of which seem likely to be functional. They encode 15 cecropins, 6 moricins, 6 defensins, 3 gallerimycins, 4 X-tox splicing variants, 14 diapausins, 15 whey acidic protein homologs, 11 attacins, 1 gloverin, 4 lebocins, 6 lysozyme-related proteins, and 4 transferrins. Some of these genes (e.g. attacins, cecropins) constitute large clusters, likely arising after rounds of gene duplication. We compared the amino acid sequences of M. sexta AMPs with their homologs in other insects to reveal conserved structural features and phylogenetic relationships. Expression data showed that many of them are synthesized in fat body and midgut during the larval-pupal molt. Certain genes contain one or more predicted κB binding sites and other regulatory elements in their promoter regions, which may account for the dramatic mRNA level increases in fat body and hemocytes after an immune challenge. Consistent with these strong mRNA increases, many AMPs become highly abundant in the larval plasma at 24 h after the challenge, as demonstrated in our previous peptidomic study. Taken together, these data suggest the existence of a large repertoire of AMPs in M. sexta, whose expression is up-regulated via immune signaling pathways to fight off invading pathogens in a coordinated manner.

He Y ，Cao X ，Li K ，Hu Y ，Chen YR ，Blissard G ，Kanost MR ，Jiang H ... -  《-》