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

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.

Keywords: Innate immunity; Insect; Insect biochemistry; Lepidoptera; Moth; Synteny; Tobacco hornworm.

Fig. 1

The Manduca sexta gene repertoire and molecular species phylogeny. Approximately half of the 15,451 M. sexta genes have identifiable orthologs in the representative genomes of mammals, human and mouse (pie chart, blue), suggesting that these are ancient genes likely to have been present in the metazoan ancestor. A further 32% of M. sexta genes exhibit orthology to genes from the other seven representative insect species (green, 22% or 3427 genes), or only to genes from the other five lepidopteran species (pink, 10% or 1588 genes). Of the remaining genes, some have orthologs (orange) or homologs (red) in other animal species (other metazoan species from OrthoDB), leaving 417 M. sexta genes (app. 3%) without any recognizable homologs (yellow, e-value cutoff 1e-3). Employing aligned protein sequences of universal single-copy orthologs to estimate the molecular species phylogeny rooted with the starlet sea anemone, Nematostella vectensis, shows that the Lepidoptera and Diptera exhibit the fastest rates of molecular divergence. All nodes have 100% bootstrap support. The boxplots show the distributions of percent amino acid identities between M. sexta proteins and their best-reciprocal-hits from mammal species (MAM; median 34.9%), insect species (INS; median 40.1%), and lepidopteran species (LEP; median 60.2%). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Remarkably conserved synteny among the Lepidoptera. A. Predicted contiguous ancestral regions (CARs) for common ancestors at the major nodes of the insect species phylogeny. Pie charts show distributions of CAR lengths (number of orthologous anchor genes) with diameters proportional to the total number of anchor genes in the CARs. The molecular species phylogeny was built from single-copy orthologs from four representative species from four major groups – Lepidoptera, Drosophila, Culicidae, and Hymenoptera – and rooted with the body louse, Pediculus humanus. B. Examining the fates of the 1329 Holometabola CAR gene neighbor pairs in the sixteen extant species classifies them as kept (dark green, maintained neighbors), likely kept (light green, inferred maintained neighbors), lost (red, no longer neighbors), or ambiguous (orange, missing orthologs). C. Evolutionary distances between species pairs, in terms of branch lengths from the phylogeny in panel A, are plotted against the percentage of orthologous gene anchors maintained in synteny (top) and the syntenic pair to gene ratio (bottom, number of neighboring gene pairs/number of genes maintained in synteny), with linear regressions of all species pairs (solid lines) and all non-lepidopteran species pairs (dashed lines). s.s., substitutions per site; HYM, Hymenoptera; LEP, Lepidoptera; DRO, Drosophila; CUL, Culicidae; Aaeg, Aedes aegypti; Aatr, Anopheles atroparvus; Agam, Anopheles gambiae; Amel, Apis mellifera; Bmor, Bombyx mori; Cqui, Culex quinquefasciatus; Dmel, Drosophila melanogaster; Dmoj, Drosophila mojavensis; Dple, Danaus plexippus; Dpse, Drosophila pseudoobscura; Dvir, Drosophila virilis; Hmel, Heliconius melpomene; Hsal, Harpegnathos saltator; Msex, Manduca sexta; Nvit, Nasonia vitripennis; Pbar, Pogonomyrmex barbatus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Overview of gene transcripts and their relative levels in the 52 cDNA libraries. (a) Distribution of genes and their transcripts based on splicing variants per gene; (b) Percentages of OGS2.0 genes (left) and sums of their FPKM values (right) in the five FPKM categories. The 52 libraries are in the same order as described in He et al. (2015).

Fig. 4

Gene expression of 68 gut serine proteases and their close homologs in various tissue samples. (a) The mRNA levels, as represented by log₂(FPKM +1) values, are shown in the gradient heat map from blue (0) to red (≥10) (Cao et al., 2015c); (b) Average FPKM values in whole body, Malpighian tubules (MT), and other tissues; (c) stage-dependent transcription in midgut tissues from 2nd instar larvae (L2), late 3rd instar (L3L), early (L4E) and late (L4L) 4th instar, 1–3 h, day 1, pre-wandering (PW) and wandering (W) 5th instar larvae, day 1 and days 15–18 pupae, and days 3–5 adults. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Manduca Hox gene cluster. (A) A phylogenetic tree was constructed using translations of the Hox and Shx gene homeodomains from Manduca sexta (Ms), Heliconius melpomene (Hm), Bombyx mori (Bm) and Danaus plexippus (Dp). Homeodomains were extracted from genomic annotations and aligned using ClustalW. A maximum likelihood tree was generated with 100 bootstrap replicates using PhyML with a LG + G model with parameters sampled from the data. Manduca orthologs are highlighted. (B) A single Manduca scaffold contained the majority of the Hox cluster, including four Shx genes. The orientation of ShxD is reversed relative to the other Lepidoptera. An Apis mellifera (bee) Hox cluster is displayed as a representative ancestral insect cluster.

Fig. 6

Summary of the families of genes coding for chitin metabolism enzymes and chitin binding proteins (CBPs) in the M. sexta genome. Dotted lines indicate the binding of CBPs to chitin by their chitin binding domains (CBD – orange), and lines with an arrow indicate that chitin metabolism enzymes and their functions in chitin synthesis (chitin synthases, CHS – green), deacetylation (chitin deacetylases, CDA – blue) and degradation (chitinases, CHT – red). For CDA and CHT, a phylogenetic tree has been constructed using the neighbor-joining method, with 1000 replications of bootstrap analyses, implemented in MEGA 6.06 (Tamura et al., 2011). CPAP: cuticular proteins analogous to peritrophin; PMP: peritrophic matrix protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Location, structure, and phytogeny of the chorion gene cluster and chorion genes of M. sexta. A. Possible configurations of the chorion locus. Represented are the four different relative orientations between chorion gene containing scaffolds, scaffold00032 and scaffold00064. The two annotated contiguous chorion gene clusters and the non-chorion orthologs adjacent to the scaffold00064 cluster are shown. The reverse complementary strands of the scaffolds are represented in red. scaffold04803 may be adjacent to scaf-fold00032 or to scaffold00064. B. Phylogenetic tree of all chorion protein sequences, based on maximum likelihood. Each class is divided into two subclasses which are clustered together in the genome (early A in green, middle A in red, middle B in blue, and early B in magenta). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Pathways for the synthesis and secretion of diacylglycerol (DG) in fat body. Fatty acids (FA) entering the fat body, produced de novo, or released from triacylglycerol (TG) by the action of lipases (TGL, ATGL and HSL) on the lipid droplets are reused in part to form DG for export. The acyl-CoA formed by ACS could enter the synthesis of DG through the PA-pathway or through the MG-pathway, which would use monoacylglycerol (MG) produced by the hydrolysis of stored TG. Export of DG to circulating lipophorin is expected to involve the lipid transfer protein (LTP) and other membrane proteins, such as the lipophorin receptor. A similar scheme could be envisaged for the synthesis and export of DG from midgut tissue.

Fig. 9

An overview of the immune system in M. sexta. Pathogens and their surface molecules (in blue font) are recognized by pattern recognition receptors in the plasma or on the immune cells. A serine protease/serine protease homolog system (shown as pink pacmans) is activated by sequential proteolytic cleavage to generate active phenoloxidases and Spätzle-1. Serpins (colored triangles) modulate melanization and cytokine effects by inhibiting immune SPs. The putative intracellular pathways (Toll, Imd, MAPK-JNK-p38, JAK-STAT) are activated by cytokines (e.g., Spätzle-1) and microbial compounds (e.g., DAP-PG) through receptors, adaptors, kinases (red spheres), and transcription factors (colored ovals), which transactivate the expression of immunity-related genes (e.g., AMPs). Newly synthesized proteins either replenish the defense molecules used up in the initial reaction or serve as effectors to kill the survived pathogens. Autophagy, apoptosis, and RNA interference are involved in insect antiviral responses. The stimulatory and inhibitory steps are depicted as red arrows and blue bars, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 10

Domain architecture, structural model, phylogenetic relationships, and expression profiles for some of the nondigestive serine proteases and serine protease homologs (SPs/ SPHs) in M. sexta. a) SP50, representing the 52 multidomain SPs/SPHs, has the domains organized in the same way as those in its ortholog Drosophila Nudel; PAP3, one of the 42 clipdomain SPs/SPHs, activates proPOs in the presence of SPH1 and SPH2. b) 3D model of the clip domain-1 in PAP3 is highly similar to the known structure of PAP2 clip domain-1 (Huang et al., 2007). α helix, red; β strand, yellow; coil, green; Cys, pink. c) Phylogenetic analysis of the entire clip-domain SP/SPH sequences in groups A (black, SPH, group-3 clip domain), B (red, SP, group-2 clip domain), C (green, SP, group-1a clip domain), and D (blue, SP, group-1b or-1c clip domain). d) PAP3 and SP50 mRNA levels in M. sexta tissues from various life stages. X-axis, RNA-seq library number; Y-axes, FPKM values of SP50 (black dotted line) and PAP3 (red solid line). PAP3 transcripts are abundant in fat body of wandering larvae and early pupae; SP50 mRNA levels are high in fat body and ovary of late pupae and adults. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)