Sensilla Morphology and Complex Expression Pattern of Odorant Binding Proteins in the Vetch Aphid Megoura viciae (Hemiptera: Aphididae)

Abstract

Chemoreception in insects is mediated by several components interacting at different levels and including odorant-binding proteins (OBPs). Although recent studies demonstrate that the function of OBPs cannot be restricted to an exclusively olfactory role, and that OBPs have been found also in organs generally not related to chemoreception, their feature of binding molecules remains undisputed. Studying the vetch aphid Megoura viciae (Buckton), we used a transcriptomic approach to identify ten OBPs in the antennae and we examined the ultrastructural morphology of sensilla and their distribution on the antennae, legs, mouthparts and cauda of wingless and winged adults by scanning electron microscopy (SEM). Three types of sensilla, trichoid, coeloconic and placoid, differently localized and distributed on antennae, mouthparts, legs and cauda, were described. The expression analysis of the ten OBPs was performed by RT-qPCR in the antennae and other body parts of the wingless adults and at different developmental stages and morphs. Five of the ten OBPs (MvicOBP1, MvicOBP3, MvicOBP6, MvicOBP7, and MvicOBP8), whose antibodies were already available, were selected for experiments of whole-mount immunolocalization on antennae, mouthparts, cornicles and cauda of adult aphids. Most of the ten OBPs were more expressed in antennae than in other body parts; MvicOBP1, MvicOBP3, MvicOBP6, MvicOBP7 were also immunolocalized in the sensilla on the antennae, suggesting a possible involvement of these proteins in chemoreception. MvicOBP6, MvicOBP7, MvicOBP8, MvicOBP9 were highly expressed in the heads and three of them (MvicOBP6, MvicOBP7, MvicOBP8) were immunolocalized in the sensilla on the mouthparts, supporting the hypothesis that also mouthparts may be involved in chemoreception. MvicOBP2, MvicOBP3, MvicOBP5, MvicOBP8 were highly expressed in the cornicles-cauda and two of them (MvicOBP3, MvicOBP8) were immunolocalized in cornicles and in cauda, suggesting a possible new function not related to chemoreception. Moreover, the response of M. viciae to different components of the alarm pheromone was assessed by behavioral assays on wingless adult morph; (-)-α-pinene and (+)-limonene were found to be the components mainly eliciting an alarm response. Taken together, our results represent a road map for subsequent in-depth analyses of the OBPs involved in several physiological functions in M. viciae, including chemoreception.

Keywords: RT-qPCR; behavioral assays; chemoreception; immunolocalization; odorant-binding proteins; vetch aphid.

FIGURE 1

SEM images showing the distribution and morphology of different sensilla on wingless M. viciae antennae. (A–C) Type II trichoid sensilla located on the terminal part of the antenna (arrowheads in (A)), and on processus terminalis (C) showing a blunt tip with a grooved surface (B,C’). (D) Global view of primary rhinaria on 5 and 6th segments (arrowheads) with a type I trichoid sensilla (arrow). (E) Details of the primary rhinaria on the 6th segment composed of 1 large placoid sensillum (LP) with porous structures (white arrowheads), 2 small placoid sensilla (SP), and 4 coeloconic pegs surrounded by cuticular fringes (black arrowheads). (G,H) Detail of type I (CI in (G)) and type II (CII in (H)) coeloconic sensilla in the 6th segment surrounded by cuticular fringes (arrowheads). (I) Detail of placoid sensillum of 5th segment. Porous structures were visible on the flat surface (arrowhead in (K)). (L) Placoid sensilla forming the secondary rhinaria of the 3rd segment (arrowhead) and trichoid sensilla (arrow). (F,J,M) Details of type I trichoid sensilla showing a groove surface and porous structures on the tip. (N) Detail of a placoid sensillum with a smooth surface not surrounded by cuticular fringes and small pores on the flat surface. Bars in (A,E,I,M), 10 μm; bars in (B,F–H,K), 1 μm; bar in (C), 5 μm; bars in (C’,J,N), 500 nm; bars in (D,L), 50 μm.

FIGURE 2

SEM images showing sensilla on M. viciae mouthparts, cauda and cornicles. (A) Long sensilla symmetrically distributed (arrowhead) and short sensilla (encircled) situated on the labium tip. (B) Detail of long sensilla tip with pre-apical expansion (black arrow in (A)) or in the shape of a cup (arrow in (A,B)). (C) Detail of long sensilla branched tip (arrowhead). (D–F) Detail of porous (arrow in (E)) or fissure like structures (arrowheads in (E,F)) on long sensilla and finger-like extensions (arrowhead in (D)) on M. viciae cauda. Finally, SEM observation highlights the presence of cuticular finger-like structures (arrowheads in (G,H)) on cornicle surface. Moreover, hole-like structures are evident among cuticular tufts (arrows in (I)) of cornicle terminal region. Bar in (A), 50 μm; bars in (B,E,F), 1 μm; bar in (C), 2 μm; bar in (D), 25 μm; bar in (G), 100 μm; bars in (H,I), 5 μm.

FIGURE 3

Relative expression level of M. viciae OBPs in different body parts. OBP expression levels were quantified by RT-qPCR. Bars represent the standard deviation of the mean for 3 independent experiments. Significant differences are denoted by asterisks (Tukey’s test, (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001)). Lg, legs; Cd, cornicles-cauda; Hd, head; Bd, body; An, antennae. Reference genes: RPL32, RPS9. Calibrator sample: antennae.

FIGURE 4

(A–Y) Whole-mount immunolocalization experiments showing the OBP expression in type II trichoid sensilla located on the antennal tip (A–D), in type II trichoid sensilla on the 6th antennal segment (F–I), in primary rhinaria on the 5th (K–N) and 6th segments (P–S) and in secondary placoid sensilla on the 3rd segment (U–X). (E,J,O,T,Y) Negative controls. Bars in (A–T), 10 μm; bars in (U–Y), 25 μm. (A’–N’) Whole-mount immunolocalization experiments showing the OBP localization in the mouthparts (arrowhead in (A’)), in the cauda (arrowhead in (B’)) and in cornicles (arrow in (B’)).(E–G,C’–E’) Immunolocalization of OBPs in the long sensilla on the labium sides. (G’–L’) OBPs detection in hair-like structures and finger-like projections in cauda and in cornicles. (F’,M’,N’) Negative controls. Bars in (A’,B’), 250 μm; bars in (C’–F’,H’,K’), 10 μm; bars in (M’), 50 μm; bars in (G’,I’,J’,L’,N’), 20 μm

FIGURE 5

Relative expression level of M. viciae OBPs in different nymphal instars. OBP expression levels were quantified by RT-qPCR. Bars represent the standard deviation of the mean for 3 independent experiments. Significant differences are denoted by asterisks (Tukey’s test, (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001)). I, 1st nymphal instar; II, 2nd nymphal instar; III, 3rd nymphal instar; IV, 4th nymphal instar; Ap, winged adults; Al, winged adults. Reference genes: RPL32, RPS9. Calibrator sample: 1st nymphal instar

FIGURE 6

Behavioral responses of M. viciae to the main compounds identified in the insect’s cornicle secretions and to the mixture containing (E)-β-farnesene 14.2%, (–)-α-pinene 11.8%, β-pinene 74%. The repellency index R was calculated by the formula R = (C–T)/(C+T), where T indicates the number of aphids in the arm with the compound to be tested and C, those in the control arm. Asterisks indicate that the repellence observed is significantly different from the control (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, Student’s t-test)