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

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Highlights

  • The genome of Manduca sexta was sequenced and assembled, with a total genome size of 419.4 Mbp.

  • 15,451 protein-coding genes were annotated, and 2498 of these were manually curated.

  • RNA-seq data from many tissues and developmental stages informed the gene models and provide insights into gene expression patterns.

  • Gene families for a broad set of topics in M. sexta biology were analyzed.

Abstract

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.

Introduction

Insects in the order Lepidoptera, moths and butterflies, include more than 150,000 species with enormous diversity. They include some of the most striking and beautiful of insect species, as well as many of the world's most serious agricultural pests (Powell, 2003). Lepidopteran insects have been the subjects of extensive experimental studies in genetics, molecular biology, and biochemistry of a wide array of physiological processes, and they include model systems and species that have unique ecological or economic importance (Goldsmith and Marek, 2010). Investigation of lepidopteran biology is beginning to benefit from advances in genomic sequencing, with published draft genomes available for the commercial silkworm, Bombyx mori (International Silkworm Genome, Consortium, 2008, Mita et al., 2004, Xia et al., 2004), the first lepidopteran genome sequenced, and several additional species including butterflies Danaus plexippus (Zhan et al., 2011), Heliconius melpomene (Dasmahapatra et al., 2012), Melitaea cinxia (Ahola et al., 2014), Papilio glaucus (Cong et al., 2015), and moths Plutella xylostella (You et al., 2013) and Spodoptera frugiperda (Kakumani et al., 2014). We report here a draft sequence for the genome of Manduca sexta, known as the tobacco hornworm or the Carolina sphinx moth, the first genome from the family Sphingidae. M. sexta is in the same superfamily, Bombycoidea, as B. mori but their biology differs dramatically. While B. mori has been domesticated for silk production and feeds exclusively on mulberry leaves, M. sexta is a wild species that feeds on solanaceous plants as larvae, including the crops tobacco and tomato.

M. sexta has been used extensively as a classic biochemical and physiological model for laboratory research on a wide array of topics over the last 40 years. It is an important model species for investigations of development and metamorphosis (Gilbert et al., 2002, Hiruma and Riddiford, 2010, Nijhout et al., 2014, Truman et al., 2006, Truman and Riddiford, 2007), neurobiology and olfaction (Heinbockel et al., 2013, Martin et al., 2011), lipid metabolism (Canavoso et al., 2001), immunity (Kanost and Nardi, 2010), parasitoid- and pathogen-host interactions (Amaya et al., 2005, Chevignon et al., 2015), mechanisms of Bacillus thuringiensis Cry toxins (Soberon et al., 2010), insect-plant interactions (Schuman et al., 2015), midgut physiology (Wieczorek et al., 2003) and many other aspects of insect biochemistry, physiology, and behavior. Annotation and expression analysis of gene families in the M. sexta genome described here provide new insight into a diversity of important topics in insect biology.

Section snippets

DNA sequencing and assembly

A M. sexta colony started from eggs obtained from Carolina Biological Supply and maintained at Kansas State University for more than 15 years was the source of genomic DNA for the sequencing project. We carried out four generations of single-pair sibling inbreeding to reduce heterozygosity. We isolated DNA from a single male pupa by proteinase K and RNAse A treatment of homogenized tissues, followed by phenol-chloroform extraction, and ethanol precipitation (Bradfield and Wyatt, 1983). We

Sequencing, assembly, and annotation

We sequenced DNA from a single male pupa, using 454 sequencing technology. We used a male for sequencing to avoid complications in assembly from the highly repetitive W chromosome present only in females (Sahara et al., 2012). Our Msex_1.0 genome assembly (see Methods for details) had a final size of 419.4 Mbp consistent with a prior measurement of 422 ± 12 Mbp for the M. sexta genome (Hanrahan and Johnston, 2011). The Msex 1.0 assembly has excellent contiguity with contig and scaffold N50s of

Conclusions

M. sexta is and has been a powerful and important model system for studies of many areas of insect biology for many decades. Through the reported draft genome sequence, representative transcriptome data, and the detailed studies of M. sexta genes and gene families that are associated with selected genomic, biochemical, and physiological systems, we make a major step on the long journey toward understanding both the specific details and the larger context of the complex biology of this

Funding

Primary funding for DNA sequencing and assembly and RNA-seq was from grants from NIH(GM041247) to M.R. Kanost and from DARPA to G.W. Blissard. Funding to support annotation of specific gene families or analysis of genome features is listed in Supplemental Table S22 (Author Contacts, Topic Areas, and Funding).

Acknowledgments

We thank Sandy Youngeberg, Janice Beal, and Marjolein Schat for insect rearing. We also thank Julie Poulain and Corinne Da Silva from the Genoscope (Centre National de Séquençage, Evry, France) for raw transcript sequence data from fat body and hemocyte libraries, which were used to refine annotation of certain M. sexta genes.

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