Olfaction in dragonflies: Electrophysiological evidence

https://doi.org/10.1016/j.jinsphys.2011.11.018Get rights and content

Abstract

The problem of olfaction in Paleoptera (Odonata, Ephemeroptera) cannot be considered fully elucidated until now. These insects have been traditionally considered anosmic, because their brain lacks glomerular antennal lobes, typically involved in Neoptera odor perception. In order to understand if the presumed coeloconic olfactory receptors described on the antennal flagellum of adult Odonata are really functioning, we performed an electrophysiological investigation with electroantennogram (EAG) and single cell recordings (SCR), using Libellula depressa L. (Odonata, Libellulidae) as a model species. Odors representing different chemical classes such as (Z)-3-hexenyl acetate (acetate ester), (E)-2-hexenal, octanal (aldehydes), (Z)-3-hexen-1-ol (alcohol), propionic acid, butyric acid (carboxylic acids), and 1,4-diaminobutane (amine) were tested. Most of the tested chemicals elicited depolarizing EAG responses in both male and female antennae; SCR show unambiguously for the first time the presence of olfactory neurons in the antennae of L. depressa and strongly support the olfactory function of the coeloconic sensilla located on the antennal flagellum of this species.

Electrophysiological activity may not necessarily indicate behavioral activity, and the biological role of olfactory responses in Odonata must be determined in behavioral bioassays. This study represents a starting point for further behavioral, electrophysiological, neuroanatomical and molecular investigation on Odonata olfaction, a research field particularly interesting owing to the basal position of Paleoptera, also for tracing evolutionary trends in insect olfaction.

Highlights

► Olfaction in Paleoptera (Odonata/Ephemeroptera) is still an open question. ► Libellula depressa bears presumed olfactory sensilla on its antennae. ► We performed an electrophysiological investigation (EAG, SCR) on these antennae. ► Depolarizing EAG and SCR responses to chemicals were recorded in males and females. ► This is the first clear identification of olfactory receptor neurons in Odonata.

Introduction

In insects, the sense of smell is a complex and highly sensitive modality, governing essential decisions such as choice of mates, food, and oviposition sites. A pair of antennae bearing olfactory sensilla represents the main olfactory organ of hexapods and the olfactory sensory system is organized in a very similar fashion in most insects.

The problem of olfaction in Paleoptera (Odonata, Ephemeroptera) is still an open question. These insects have been traditionally considered anosmic, because their brain lacks glomerular antennal lobes and mushroom body calyces, which in Neoptera are involved in odor perception (Strausfeld et al., 1998, Farris, 2005).

Slifer and Sekhon (1972), in an ultrastructural overview on adult antennal flagellum of some Odonata species, identified coeloconic sensilla located in simple and compound cavities. On the basis of their apparent porous cuticle, the authors hypothesized a chemosensory function for these sensilla.

A recent ultrastructural investigation (SEM, TEM) on the adult of the dragonfly Libellula depressa (Odonata: Libellulidae) revealed sensilla located in pits on the lateral–ventral side of the antennal flagellum (Rebora et al., 2008). These sensilla are represented by sensilla coeloconica and by deeply sunken sensilla styloconica. The sensilla coeloconica (Fig. 1) are innervated by three unbranched dendrites entering the peg and they show a dendritic sheath ending at the base of the peg. The peg shows no socket and its cuticle is irregular with wide pore-like structures at the base of which actual pores with pore tubules are evident. The structure of these sensilla is in agreement with that reported for single-walled insect olfactory receptors. The deeply sunken sensilla are represented by two kinds of sensilla styloconica located at the bottom of deep cavities evident on the antennal surface as simple openings (Fig. 1). These sensilla are no-pore sensilla with inflexible socket and unbranched dendrites and, notwithstanding their structural differences, they share common features typical of thermo-hygroreceptors. The presence of putative olfactory and thermo-hygroreceptors has been confirmed in ultrastructural investigations on several Odonata families belonging to the suborders Anisoptera and Zygoptera (Rebora et al., 2009a, Piersanti et al., 2010).

Single cell electrophysiological recordings from adult males and females of L. depressa, stimulating the antenna by rapid changes in temperature and humidity, showed the occurrence of a dry, a moist and a cold receptor neurons on the antennal flagellum, probably located in the deeply sunken sensilla styloconica (Piersanti et al., 2011).

Odonata are believed to have secondarily invaded freshwater environments. Therefore, the putative olfactory coeloconic sensilla identified on their antennae could be “silent receptors”, in which olfactory receptor genes are pseudogenes, as it occurs in secondarily aquatic mammals (Freitag et al., 1998).

On the basis of these data, in order to understand if the coeloconic olfactory receptors located on Odonata antennae represent vestigial organs or are really functioning, we performed an electrophysiological investigation with electroantennogram (EAG) and single cell recordings (SCR), using L. depressa as a model species. EAG measures the total amount of electrophysiological responses in the insect antennae, thus providing a general measure of odorant reception at the peripheral level (Schneider, 1957, Roelofs, 1984, Park et al., 2002). When recording the frequency of action potentials in single olfactory receptor neurons using the SCR technique, the specificity in each neuron is revealed (Masson and Mustaparta, 1990). SCR is particularly useful when the number of stimulated neurons is small and gives only a minute EAG (Schiestl and Marion-Poll, 2002).

Section snippets

Insects

Larvae of L. depressa, attributed to the ultimate (F-0) stage were collected in ponds in Central Italy (Perugia, Umbria) in the periods March–April 2010 and 2011. The specimens were kept outdoor in plastic containers (60 × 40 × 40 cm) with water, detritus, flora and fauna from the collecting site, in natural conditions of temperature, humidity and light. The larvae were fed ad libitum with plankton (Daphnia spp. and Cyclops spp.) up to the emergence of the adults. In the experiments male and female

Electroantennography

Depolarizing EAG responses were recorded in both males and females (Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6).

The magnitude of the response to the standard stimulus (octanal at 10% v/v) was similar in the two sexes (males, −0.014 mV ± 0.002 SE; females, −0.011 mV ± 0.001 SE (t = 0.90; d.f. = 29; P = 0.374).

Discussion

Our results show that some chemicals, belonging to different classes, elicit EAG activity in the antennae of L. depressa. In particular, in females all the tested chemicals elicited response except (Z)-3-hexen-1-ol, while in males the tested chemicals elicited response except (Z)-3-hexenyl acetate, (Z)-3-hexen-1-ol and butyric acid. Differences between male and female antennae have been highlighted also in the EAG dose–response curves to octanal. In both sexes EAG responses generally increased

Acknowledgments

We are grateful to Bill Hansson for his suggestions about chemicals and to Tor Jorgen Almaas for his support in the collection of some preliminary data. Many thanks to Jonas M. Bengtsson for his helpful support in single cell recordings. Thank you very much to the anonymous referees for their suggestions.

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