Chemoreception of Mouthparts: Sensilla Morphology and Discovery of Chemosensory Genes in Proboscis and Labial Palps of Adult Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract

Siphoning mouthparts, consisting of proboscis and labial palps, are the exclusive feeding organs and important chemosensory organs in most adult Lepidoptera. In this study, the general morphology of the mouthpart organs and precision architecture of the proboscis was described in adult Helicoverpa armigera. Three major sensilla types with nine subtypes including three novel subtypes were identified. The novel sensilla styloconica subtype 2 was the only one having a multiporous structure, which may play olfactory roles. For further understanding of the chemosensory functions of mouthpart organs, we conducted transcriptome analysis on labial palps and proboscises. A total of 84 chemosensory genes belonging to six different families including 4 odorant receptors (ORs), 6 ionotropic receptors (IRs), 7 gustatory receptors (GRs), 39 odorant binding proteins (OBPs), 26 chemosensory proteins (CSPs), and 2 sensory neuron membrane proteins (SNMPs) were identified. Furthermore, eight OBPs and six CSPs were identified as the novel genes. The expression level of candidate chemosensory genes in the proboscis and labial palps was evaluated by the differentially expressed gene (DEG) analysis, and the expression of candidate chemosensory receptor genes in different tissues was further investigated by quantitative real-time PCR (qRT-PCR). All the candidate receptors were detected by DEG analysis and qRT-PCR, but only a small part of the OR or IR genes was specifically or partially expressed in proboscis or labial palps, such as HarmOR58 and HarmIR75p.1, however, most of the GRs were abundantly expressed in proboscis or labial palps. The reported CO₂ receptors such as HarmGR1, GR2, and GR3 were mainly expressed in labial palps. HarmGR5, GR6, and GR8, belonging to the "sugar receptor" clade, were mainly expressed in proboscis or antenna and were therefore suggested to perceive saccharide. The results suggest that the mouthparts are mutually cooperative but functionally concentrated system. These works contribute to the understanding of chemical signal recognition in mouthpart organs and provide the foundation for further functional studies.

Keywords: Helicoverpa armigera; chemosensory genes; mouthparts; sensilla; transcriptome.

FIGURE 1

General morphology of the mouthpart organs and ultrastructure of the proboscis of adult Helicoverpa armigera. (A) Frontal view of the head shows the major siphoning mouthpart organs: proboscis (pr) and the pair of labial palps (lp). (B) The proboscis (pr) coiled up under resting state; one labial palp attached on the side (the other one was removed). (C–H) Scanning electron micrographs of proboscis. (C) Overall structure of proboscis: the rough distal section (dt) and the smooth proximal (px) and middle (md) sections were shown on the two elongated galeae (ge). (D) The distal section (dt) of two galeae shows many peg-shaped sensilla. (E) Dorsal (dl) and ventral ligulae (vl) on each galea. (F) Two major sensilla on the distal section: sensilla styloconica (ss), external sensilla basiconica subtype 2 (esb2) and sensilla chaetica subtype 2 (sch2). (G) Two types of sensilla on the proximal and middle sections: external sensilla basiconica subtype 1 (esb1) and sensilla chaetica subtype 1 (sch1). Plenty of cuticular processes (cp) arranged on the surface. (H) Triangular structure of cuticular processes.

FIGURE 2

Scanning electron micrographs of sensilla on the proboscis of adult H. armigera. (A) Sensilla styloconica subtype 1 (ss1) possessing a uniporous cone. (B) Sensilla styloconica subtype 2 (ss2) with a multiparous sphere. (C) The lotus-shaped pedestal of sensilla styloconica. (D) Long and short sensilla chaetica subtype 1 (sch1) on the proximal section. The cupped socket at the base of sch1 (white box). (E) Short sensilla chaetica subtype 1 (sch1) on the middle section. (F) Sensillum chaetica type 2 (sch 2) on the distal part of the proboscis. (G) External sensilla basiconica subtype 1 (esb1) with a basal socket and a top pore. (H) External sensilla basiconica subtype 2 (esb2) on a roof-shaped bulge and with a pore on the tip. (I) External sensilla basiconica type 3 (esb3) with an uniporous peak and a curving cone. (J) Internal sensilla basiconica type 1 (isb1) on the internal surface of the proboscis tube. (K) Internal sensilla basiconica type 2 (isb2).

FIGURE 3

The heat map of the expression level of ORs, IRs, and GRs based on the DEGs analysis and the relative expression level based on the qRT-PCR. The heat maps were generate based on the FPKM values. The column diagrams representing the relative expression level of each gene between four tissues of both sexes were generated based on 2^-ΔΔCt. The differences of expression among tissues and sexes were analyzed by one-way ANOVA and followed by Duncan’s test (P < 0.05). Different letters on the top of the columns represent significant difference at P < 0.05. (A) Expression level of ORs; (B) expression level of IRs; (C) expression level of GRs; An, antenna; Pr, proboscis; Lp, labial palps; Le, legs.

FIGURE 4

(A) The phylogenetic tree of OBPs from Lepidoptera species. The OBPs identified in our dataset are identified in bolding font. Eight novel OBPs are marked by orange circles. The “classic” OBP clade including PBP/GOBP, “plus-C” OBP, and “minus-C” is shown. Bootstrap values are shown by color gradation. The four species re H. armigera (Harm, red), H. assulta (Hass, black), S. littoralis (Slit, green), and B. mori (Bmor, blue). (B) Phylogenetic tree of 39 OBPs identified in our dataset. Their expression profiles were shown by a heat map based on the FPKM values. Eight novel OBPs are marked in blue font.

FIGURE 5

(A) The phylogenetic tree of CSPs from Lepidoptera species. The CSPs identified in our dataset are shown in bolding font. Six novel CSPs are marked by blue circles. Bootstrap values are shown by color gradation. The four species are H. armigera (Harm, red), H. assulta (Hass, black), S. littoralis (Slit, blue), and B. mori (Bmor, green). (B) Phylogenetic tree of 26 CSPs identified in our dataset. Their expression profiles were shown by a heat map based on the FPKM values. Six novel CSPs are marked in blue font.

FIGURE 6

(A) The phylogenetic tree of 21 reported SNMPs in 11 species revealed two separate clades of SNMP1 and SNMP2. (B) The expression profiles of two SNMPs identified in proboscis and labial palp were shown by a heat map based on the FPKM values.