Autofluorescence-Based Identification and Functional Validation of Antennal Gustatory Sensilla in a Specialist Leaf Beetle

Abstract

Herbivorous insects mainly rely on their sense of taste to decode the chemical composition of potential hosts in close range. Beetles for example contact and scan leaves with their tarsi, mouthparts and antennal tips, i.e., appendages equipped with gustatory sensilla, among other sensillum types. Gustatory neurons residing in such uniporous sensilla detect mainly non-volatile compounds that contribute to the behavioral distinction between edible and toxic plants. However, the identification of gustatory sensilla is challenging, because an appendage often possesses many sensilla of distinct morphological and physiological types. Using the specialized poplar leaf beetle (Chrysomela populi, Chrysomelidae), here we show that cuticular autofluorescence scanning combined with electron microscopy facilitates the identification of antennal gustatory sensilla and their differentiation into two subtypes. The gustatory function of sensilla chaetica was confirmed by single sensillum tip-recordings using sucrose, salicin and salt. Sucrose and salicin were found at higher concentrations in methanolic leaf extracts of poplar (Populus nigra) as host plant compared to willow (Salix viminalis) as control, and were found to stimulate feeding in feeding choice assays. These compounds may thus contribute to the observed preference for poplar over willow leaves. Moreover, these gustatory cues benefited the beetle's performance since weight gain was significantly higher when C. populi were reared on leaves of poplar compared to willow. Overall, our approach facilitates the identification of insect gustatory sensilla by taking advantage of their distinct fluorescent properties. This study also shows that a specialist beetle selects the plant species that provides optimal development, which is partly by sensing some of its characteristic non-volatile metabolites via antennal gustatory sensilla.

Keywords: Chrysomela populi; antenna; contact chemosensation; cuticular autofluorescence; gustation; herbivory; leaf beetle; sensilla chaetica.

FIGURE 1

Distinct autofluorescent properties among different antennal sensilla types facilitate identification of gustatory sensilla chaetica. (A) The antenna of adult C. populi consists of scapus (sc), pedicellus (pe) and nine flagellomeres (fl) of which the apical flagellomere was the focus of this study (fl9, dotted frame); mxp - maxillary palp; dorsal view by scanning electron microscopy. (B) Cuticular autofluorescence scanning by confocal laser microscopy showing one stack (consisting of 300 scans at 0.5 μm thickness) from the (i) dorsal and (ii) ventral side; (iii) single scan from i) as indicated by dashed frame. (C) Lambda scan indicates two subtypes of s. chaetica due to their distinct fluorescence (subtype 1 in blue; subtype 2 in green with arrowheads). (D) Scanning electron microscopy reveals a blunt end of both s. chaetica subtypes, but different terminal pore types between subtype 1 and 2 (arrowheads).

FIGURE 2

Gustatory receptor neurons in antennal sensilla chaetica of subtype 2 respond to salicin, sucrose and salt. Mean spike frequency toward different tastants (dissolved in 30 mM KCl) at different concentrations; 30 mM KCl as control; n ≥ 3. Subtype 1 s. chaetica did not respond to any of the tastants. Dashed line indicates background activity of gustatory neurons due to 30 mM KCl in tastant solution.

FIGURE 3

Sucrose and salicin are major compounds in leaves of Populus nigra, but less concentrated in Salix viminalis. Mean concentration of salicin and different saccharides in leaves of P. nigra (n = 6) or S. viminalis (n = 3) as measured by liquid chromatography or gas chromatography coupled to mass spectrometry after extracting fresh leaves in methanol. Authentic standards were used for identification and quantification.

FIGURE 4

Weight gain of C. populi depends on host plant species. Using no-choice diet with leaves from P. nigra or S. viminalis species shows that individual poplar leaf beetles gain significantly more weight over time on P. nigra (n = 7) compared to S. viminalis (n = 7); Two Way Repeated Measurement ANOVA with pairwise multiple comparison (Holm–Sidak method). ^∗P < 0.05, ^∗∗P < 0.01.