RESULTS AND DISCUSSION

This is the first study to demonstrate an electrophysiological and behavioral response to ecologically relevant volatile compounds and CO₂ by olfactory sensilla present on an ovipositor. We amplified the signal-to-noise ratio by anchoring the ovipositor at three-fourths its length from the base on the recording electrode such that the sensory tip remains free to respond to stimuli. Another method of arranging small antennae in series during an EAG improves the signal-to-noise ratio (Park and Baker, 2002) but such a method fails with sensory organs such as ovipositors, where the tip is of critical importance as the tip is immersed in conducting gel in such an arrangement and the sensilla are not exposed to the stimulus.

The ovipositor of S. fusca responded strongly to pollen-receptive phase volatiles (12–18 mV) (Fig. 1D). Electrophysiologically active compounds of this volatile profile, (E)-β-ocimene and decanal, were also tested individually, to which the ovipositor also responded [(E)-β-ocimene: 0.4±0.34 mV (mean±s.d.), n=3; decanal: 0.183±0.076 mV, n=3; Fig. 1E,F]. (E)-β-ocimene is a well-known HIPV (Dicke and Baldwin, 2010) and is a good candidate for a context-dependent function when combined with CO₂. The ovipositor of the parasitoid A. westwoodi that arrives post-pollination did not respond to volatiles of the pollen-receptive phase because this stimulus was ecologically inappropriate as the volatile profile of the syconium changes dynamically with the ontogeny of the syconium (Borges et al., 2013). The parasitoid's ovipositor, however, responded strongly (16.2±4.02 mV, n=14) to 0.5% CO₂ (Fig. 1G); this response disappeared when the tip was excised indicative of the presence of the CO₂ sensillum at the tip.

To characterize the ovipositor's electrophysiological response to CO₂ in an organismal context, we performed behavioral assays. In the first assay, 27 out of 40 experimental wasps that had silicone-occluded antennae landed on the tip within 2 min of CO₂ release. With control air flows, these wasps were not attracted to the tip, although a few chance landings did occur. The number of wasps landing at the micropipette tip with blank air and with CO₂ were taken as paired data in each trial; we found that wasps landed significantly more often on the micropipette tip when CO₂ was present (Wilcoxon matched pairs signed rank test, v=0, n=8 trials, P=0.013; Fig. 1H). Because CO₂ sensors may be present elsewhere on the insect body and these may have been also involved in the above response, to demonstrate the ability of the ovipositor to react to CO₂, we performed another assay. In this assay, only the ovipositor of a tethered wasp was exposed to a 5 s stimulus of 0.5% CO₂ injected into the background flow of blank air, in response to which the ovipositor deflected towards the CO₂ source until the CO₂ puff terminated (Movie 2).

CO₂ plays an important role in insect–plant and host–insect interactions when perceived at particular concentrations against the background (Guerenstein and Hildebrand, 2008). Olfactory receptor neurons located on the antennae and maxillary palp mediate the response to CO₂ in Drosophila and Anopheles gambiae (Suh et al., 2004; Wasserman et al., 2013; Lu. et al., 2007). Context specificity may explain the effectiveness and reliability of this non-specific cue as a short-range oviposition attractant or repellent (Stange, 1999; Goyret et al., 2008). For insect larvae that do not exhibit host-seeking behavior (Brodeur and Boivin, 2004), such as fig wasp larvae that are restricted to their individual galls, it is crucial for ovipositing females to precisely find oviposition sites with the help of their ovipositors. A spatially variable concentration of CO₂ around different galls, owing to the respiration of developing host wasp larvae at different developmental stages and limited permeability of gall tissue, could possibly be used to assess the suitability of the oviposition site. Given that the respiration rate is high in figs (Galil et al., 1973) and that A. westwoodi parasitizes pre-pupal stages of the early-arriving gallers Sycophaga testacea and Sycophaga stratheni (P.Y. and R.M.B., unpublished data) that develop within large galls, clustered towards the cavity of the syconium (Fig. 1A), we expected its ovipositor to encounter a relatively high CO₂ concentration deep inside the syconium. We therefore tested the ovipositor response to 0.5% CO₂, which is 1/20th of the highest concentration recorded from figs (Galil et al., 1973). Wasps also frequently expose the sensilla-rich ovipositor tip to chemical cues by an in–out scanning motion (Movie 3) in which the tip is exerted beyond the sheath and then retracted; this motion could help the sensilla-rich tip to perceive volatiles even when the ovipositor continues to remain covered by the sheath prior to oviposition [during oviposition, the sheath remains outside the fig (Fig. 1A); furthermore, the ovipositor sheath has only mechanosensory structures and no chemosensilla (Fig. 2A)]. The previously reported (Ghara et al., 2011) unidentified and sparsely occurring sensillum in the ovipositor of parasitoids (Fig. 2B) resembles the auricillic CO₂-sensillum reported in the antennae of other insects (Stange and Stowe, 1999).

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Fig. 2.

Demonstration of the porous olfactory nature of sensilla on the ovipositor. SEM images of (A) ovipositor sheath and (B) putative CO₂ sensillum. Silver nitrate staining: (C) silver impregnation inside the ovipositor, (D) unstained control. X-ray tomography images of (E) silver nitrate-stained ovipositor and (F) control. Scale bars (unless specified), 10 µm.

We used silver nitrate staining followed by X-ray tomography (XRT) to investigate the porous, olfactory nature of ovipositor sensilla. Upon penetration through the pores, if any, silver turns black upon exposure to light or organic material. We observed impregnation of silver inside the ovipositor under bright field (Fig. 2C) which was absent in the control (Fig. 2D). XRT indicated the presence of silver along the mid-section of the ovipositor in addition to the tip, reflecting pores in ovipositor sensilla (Fig. 2E). A faint electron-dense patch at the tip and another region in the control (Fig. 2F) is due to zinc in the parasitoid ovipositor (Kundanati and Gundiah, 2014). TEM imaging to explore the internal structure of sensilla was unsuccessful because of the micro-dimensions of the ovipositor (thinner than a human hair) and a strongly sclerotized tip (Ghara et al., 2011), resulting in insurmountable difficulties with fixation and sectioning.

The ability of the antennal olfactory system to evolve rapidly and adapt to changing conditions contributes to the success of insects (Hansson and Stensmyr, 2011). Our study demonstrates olfactory perception of ecologically relevant volatiles in an ovipositor, but the evolution of olfaction in the ovipositor and its role in host-finding across insect taxa remain to be investigated. We predict that olfactory sensilla will occur on ovipositors of insects that are completely dependent on their ovipositor to locate hidden hosts and where a concentration build-up of informative volatiles can occur. For example, the wasp pollinators of F. racemosa have only three sensilla on their ovipositors (Ghara et al., 2011) and these are likely to be mechano-chemosensory, but not olfactory, in nature. This is because, unlike NPFWs, pollinating wasps enter into fig syconia, have an abundance of relatively easily accessible oviposition sites, and may not need precise chemosensors that can detect volatile gradients. Further explorations into the sensory nature of ovipositors in such systems and into measuring these volatile gradients can provide designs for ovipositor-inspired micro-chemosensors.