Olfaction with legs-Spiders use wall-pore sensilla for pheromone detection

Abstract

The sense of smell is a central sensory modality of most terrestrial species. However, our knowledge of olfaction is based on vertebrates and insects. In contrast, little is known about the chemosensory world of spiders and nothing about how they perform olfaction despite their important ecological role. The orb-weaving spider Argiope bruennichi lends itself to an in-depth study on olfaction as it is one of the few spider species whose volatile sex pheromone, emitted by females to attract males, is known. We combined ultrastructural and electrophysiological analyses and found that previously overlooked sensilla with wall pores are abundant on all walking legs of A. bruennichi males. We compared the ultrastructure of these wall-pore sensilla with those known to perform olfaction in insects, exploring similarities and differences. Electrophysiological single sensillum recordings demonstrated that the wall-pore sensilla in A. bruennichi respond highly sensitive and in a concentration-dependent manner to the sex pheromone. Our study demonstrates male-specific sensilla for detecting signaling females, whereas females and subadult males are devoid of wall pore sensilla. In a preliminary comparative morphological analysis using 19 species from 16 spider families, we found that wall-pore sensilla occur in male spiders from most, but not in basally branching clades or in Salticids, suggesting that wall-pore sensilla evolved at least once within spiders and were lost at least once. This research significantly expands our knowledge of the sensory ecology of spiders, will stimulate studies on the diversity and function of sensilla, as well as studies on the evolution of olfaction in arthropods.

Keywords: Araneae; chemosensing; electrophysiology; mate attraction; ultrastructure.

Fig. 1.

External appearance and cross-sections of wall-pore sensilla in A. bruennichi males. (A) SEM-overview of a wall-pore sensillum (wps) with socket (so) and a blunt tip. Note the smooth nonporous base of the sensillum shaft (sh). (B) Sensillum shaft showing wall pores (some with arrowheads) in the grooved region. (C) TEM cross-section of the median region of the sensillum shaft shows that wall pores (wp) are connected to the longitudinal shaft canals (lca), as well as to the outer sensillum lymph space (osl) inside the sensillum. (D) Wall pores are absent at the basal region of the sensillum, but the longitudinal shaft canals (lca) are present throughout the shaft. Dendrites (de) are confined to the inner sensillum lymph space (isl) surrounded by the dendritic sheath (ds). (E) Close-up showing three dendrites in the inner sensillum lymph space, surrounded by the continuous dendritic sheath. Further labels; pasc processes of accessory sheath cells, sca spoke canal, swc shaft-wall cuticle.

Fig. 2.

Semischematic (A–C) and schematic (D) reconstruction of a wall-pore sensillum on a walking leg of an A. bruennichi male and of a wp-sensillum of an insect (E). (A) Overview of cellular anatomy in longitudinal view. Sensilla are innervated by 1 to 4 (here 3) receptor cells. Mind that the distal processes of the accessory sheath cells are not depicted in (A) but in (B) and (C). (B) Cross-section through the mid-region of the sensillum shaft showing numerous wall pores. The shaft wall cuticle is perforated, giving it a double-walled appearance. (C) Cross-section through the basal region of the sensillum shaft which lacks wall pores. Section levels for (B) and (C) are indicated by lines in A. (D) Schematic representation of a wp-sensillum featuring the most important similarities and differences compared with (E) insect wp-sensilla. (E) Representative wall-pore sensillum of insects adopted from Wehner & Gehring: Zoology, Thieme 2013. Labels: asc accessory sheath cells, cu leg cuticle, de dendritic processes, ds dendritic sheath, ecm extracellular matrix, epc epidermal cell, gc glial cell, isc inner (glandular) sheath cell, isl inner sensillum lymph space, lca longitudinal shaft wall canals, msc median sheath cell, osc outer sheath cell, osl outer sensillum lymph space, pasc digitiform distal processes of accessory sheath cells, rc chemoreceptive cell, sca spoke canals, so socket, swc shaft wall cuticle, wp wall pores.

Fig. 3.

Distribution of wall-pore sensilla (wp-sensilla) and tip-pore sensilla (tp-sensilla) on the first walking leg of an A. bruennichi male. (A) Representative area of the tibia showing 16 wp-sensilla (light blue arrows) and one s-shaped and longer tp-sensillum (red arrow). (B) Distribution map of chemosensilla, viewed from dorsal, prolateral, ventral, and retrolateral (Top to Bottom). Labels: light blue dots: wp-sensilla, red dots: tp-sensilla; tr: trochanter. See supplement for distribution maps of 2nd, 3rd, and 4th walking legs (SI Appendix, Fig. S2).

Fig. 4.

Mate attraction via volatile odor and electrophysiological response of single sensillum recording. (A) Scenario of female-produced volatile TMMC captured by wall-pore sensilla of the male A. bruennichi. (B) Responses of a wall-pore sensillum (femur of the first walking leg of a male A. bruennichi) to the synthetic pheromone. TMMC was dissolved in dichloromethane (DCM) and loaded as stimulus to the filter paper in the test tube. Responses to different dosages are labeled in each panel. Bar at bottom shows the 0.5 s puffing period after initial recording of the spontaneous potentials for 2 s. The amplitudes of the potentials are in mV. (C) Responses of a wall-pore sensillum (tibia of the first walking leg of a male A. bruennichi) to a DCM solvent control, a silk extract of 0.1 female equivalent (fe) that contained about 490 ng of the pheromone, and the synthetic pheromone TMMC in a loading dose of 400 ng. (D) Responses of a wall-pore sensillum (tibia of the first walking leg of a male A. bruennichi) to a DCM solvent control, a female-silk-contained minor component octanamide in a loading dose of 12 μg, and a silk extract of 0.02 female equivalent (fe) that contained approximately 100 ng of the pheromone. (E) Representative pattern of spontaneous action potentials recorded from cohosted neurons in the tested wp-sensilla of A. bruennichi males. In total, four physiologically active neurons, named as “Cell_a” to “Cell_d” were observed. These neurons are organized in a binary or ternary cohosting pattern, i.e., a+b, a+c, and a+b+d. The neurons are presented in overlays or separated forms along with the color coding. The pheromone-mediated action potentials were recorded from the neuron Cell_a labeled in red.

Fig. 5.

Taxon sampling for FE-SEM-based screening for wall-pore sensilla in the spider (Araneae) tree of life. Adapted from refs. –.