The immune signaling pathways of Manduca sexta

Abstract

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.

Keywords: Expression profiling; Gene annotation; Insect immunity; RNA-Seq; Transcriptome.

Fig. 1

Phylogenetic relationships of Spätzles in M. sexta, B. mori, T. castaneum, and D. melanogaster. (A) Tree. Based on the sequence alignment of 29 full-length Spätzles, a tree was generated with branches shown in colors representing closely related groups. (B) Aligned sequences of the cystine-knot cytokine domains in M. sexta Spätzles-1 through 7. Cys residues are indicated in a red font. Some Cys residues may form intra- (1-1, 2-2, 3-3) and inter- (4) chain disulfide bonds. Proteolytic activation sites, known for Spätzle-1, are predicted to be next to the Arg (red) in Spätzle-2 through 6. The putative processing site (RXXR) is underlined in Spätzle-3 and 5.

Fig. 2

Transcript profiles of the putative signaling protein genes in the 52 tissue samples. The mRNA levels, as represented by log₂(FPKM+1) values, are shown in the gradient heat map from blue (0) to red (≥10). The values of 0–0.49, 0.50–1.49, 1.50–2.49 ··· 8.50–9.49, 9.50–10.49 10.50–11.49, and 11.50–12.49 are labeled 0, 1, 2 … 9, A, B and C, respectively. The cDNA libraries are constructed from the following tissues and stages: head [2^nd (instar) L (larvae), d1 (day 1); 3^rd L, d1; 4^th L, d0.5; 4^th L, late; 5^th L, d0.5; 5^th L, d2; 5^th L, pre-W (pre-wandering); P (pupae), late; A (adults), d1; A, d3; A, d7], fat body (4^th L, late; 5^th L, d1; 5^th L, pre-W; 5^th L, W; P, d1-3; P, d15-18; A, d1-3; A, d7-9), whole animals [E (embryos), 3h; E, late; 1^st L; 2^nd L; 3^rd L), midgut (2^nd L; 3^rd L; 4^th L, 12h; 4^th L, late; 5^th L, 1–3h; 5^th L, 24h; 5^th L, pre-W; 5^th L, W; P, d1; P, d15-18; A, d3-5; 4^th L, 0h), Malpighian tubules (MT) (5^th L, pre-W; A, d1; A, d3), muscle (4^th L, late; 5^th L, 12h; 5^th L, pre-W; 5^th L, W), testis (P, d3; P, d15-18; A, d1-3), and ovary (P, d15-18; A, d1). Some libraries (underlined) are from single-end sequencing; the others are from paired-end sequencing. Note that some synonymous libraries exhibit different FPKMs due to method differences. Panel A, Toll; B, Imd with JNK branch; C, MAPK-JNK-p38; D, JAK-STAT; E, pi- si- and mi-RNA pathways, F, autophagy; G, apoptosis.

Fig. 2

Transcript profiles of the putative signaling protein genes in the 52 tissue samples. The mRNA levels, as represented by log₂(FPKM+1) values, are shown in the gradient heat map from blue (0) to red (≥10). The values of 0–0.49, 0.50–1.49, 1.50–2.49 ··· 8.50–9.49, 9.50–10.49 10.50–11.49, and 11.50–12.49 are labeled 0, 1, 2 … 9, A, B and C, respectively. The cDNA libraries are constructed from the following tissues and stages: head [2^nd (instar) L (larvae), d1 (day 1); 3^rd L, d1; 4^th L, d0.5; 4^th L, late; 5^th L, d0.5; 5^th L, d2; 5^th L, pre-W (pre-wandering); P (pupae), late; A (adults), d1; A, d3; A, d7], fat body (4^th L, late; 5^th L, d1; 5^th L, pre-W; 5^th L, W; P, d1-3; P, d15-18; A, d1-3; A, d7-9), whole animals [E (embryos), 3h; E, late; 1^st L; 2^nd L; 3^rd L), midgut (2^nd L; 3^rd L; 4^th L, 12h; 4^th L, late; 5^th L, 1–3h; 5^th L, 24h; 5^th L, pre-W; 5^th L, W; P, d1; P, d15-18; A, d3-5; 4^th L, 0h), Malpighian tubules (MT) (5^th L, pre-W; A, d1; A, d3), muscle (4^th L, late; 5^th L, 12h; 5^th L, pre-W; 5^th L, W), testis (P, d3; P, d15-18; A, d1-3), and ovary (P, d15-18; A, d1). Some libraries (underlined) are from single-end sequencing; the others are from paired-end sequencing. Note that some synonymous libraries exhibit different FPKMs due to method differences. Panel A, Toll; B, Imd with JNK branch; C, MAPK-JNK-p38; D, JAK-STAT; E, pi- si- and mi-RNA pathways, F, autophagy; G, apoptosis.

Fig. 3

Domain structures (A), phylogenetic relationships (B), and gene orders (C) of Tolls in M. sexta. (A) Signal peptide (SP), Leu-rich repeat (LRR), amino- and carboxyl-terminal (NT & CT) LRRs, low complexity (LC) region, transmembrane (TM) segment, and TIR (Toll/interleukin-1 receptor) domain are shown in different colors and shapes as indicated. (B) Amino acid sequences of the 58 full-length Toll proteins from M. sexta, B. mori, T. castaneum, A. gambiae, and D. melanogaster are aligned to generate the tree with its branches in different colors for closely related groups. (C) Orientations and orders of the Toll genes in the five insects are schematically shown as arrows in the same colors as in panel B. Arrows for the single exon genes are in black frame.

Fig. 4

Putative signaling pathways and regulators for antimicrobial immune responses in M. sexta. Panels A, Toll; B, Imd with JNK branch; C, MAPK-JNK-p38; D, JAK-STAT; E, pi- si- and mi-RNA pathways, F, autophagy; G, apoptosis. Panels A through G are described in the text.