The sense of smell in Odonata: An electrophysiological screening

Journal of Insect Physiology 70 (2014) 49–58

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The sense of smell in Odonata: An electrophysiological screening Silvana Piersanti a,1, Francesca Frati b,1, Eric Conti b, Manuela Rebora a,⇑,2, Gianandrea Salerno b,2 a b

Dipartimento di Chimica, Biologia e Biotecnologie, University of Perugia, Italy Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of Perugia, Italy

a r t i c l e

i n f o

Article history: Received 13 June 2014 Received in revised form 11 August 2014 Accepted 3 September 2014 Available online 16 September 2014 Keywords: Odonata Aquatic insects Electrophysiology Olfaction Libellula depressa Ischnura elegans

a b s t r a c t Volatile chemicals mediate a great range of intra- and interspecific signalling and information in insects. Olfaction has been widely investigated mostly in Neoptera while the knowledge of this sense in most basal insects such as Paleoptera (Odonata and Ephemeroptera) is still poor. In the present study we show the results of an electrophysiological screening on two model species, Libellula depressa (Libellulidae) and Ischnura elegans (Coenagrionidae), representatives of the two Odonata suborders Anisoptera and Zygoptera, with the aim to deep the knowledge on the sense of smell of this insect order. The antennal olfactory sensory neurons (OSNs) of these two species responded to the same 22 compounds (out of 48 chemicals belonging to different functional groups) encompassing mostly amines, carboxylic acids or aldehydes and belonging to green leaf volatiles, vertebrate related volatiles and volatiles emitted by standing waters bacteria. The properties of Odonata OSNs are very similar to those of ionotropic receptors (IRs) expressing OSNs in other insects. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The detection of chemical cues in the environment — which provide information on food, mates, danger, predators and pathogens — is essential for the survival of most animals. In particular insects possess a sophisticated olfactory system, a key physiological element for their survival and reproduction and one of the reasons of their extraordinary evolutionary success. Olfaction is organized at various levels, starting with reception of semiochemicals at the periphery, processing of signals in the antennal lobes, integration of olfactory and other sensory modalities in the higher processing centers of the brain, and ultimately translation of olfactory signals into behavior (Leal, 2013). The olfactory system and the chemicals that one insect is able to perceive are heavily dependent on the types of receptors expressed on the peripheral olfactory sensory neurons (OSNs), located mainly in insect antennae. These are insect-specific odorant receptors (ORs) (Vosshall et al., 1999) and more ancient ionotropic receptors (IRs) (Benton et al., 2009). The ORs vary greatly between species, while the IRs tend to be conserved across insect orders, possibly representing an ancestral insect chemodetection module for environmental cues ⇑ Corresponding author at: Dipartimento di Chimica, Biologia e Biotecnologie, University of Perugia, Via Elce di Sotto, 06123 Perugia, Italy. Tel.: +39 075 5855722; fax: +39 075 5855733. E-mail address: [email protected] (M. Rebora). 1 These authors contributed equally to this work. 2 Seniority of authorship is equally shared. http://dx.doi.org/10.1016/j.jinsphys.2014.09.003 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved.

of general interest (Croset et al., 2010). Olfaction has been widely investigated mostly in Neoptera while the knowledge of this sense in most basal insects such as Paleoptera (Odonata and Ephemeroptera) is still poor. Odonata adults have large eyes and reduced antennae and they are considered to be primarily visually oriented (Corbet, 1999). Ultrastructural investigations revealed the presence of singlewalled coeloconic sensilla located in pits on the antennal flagellum of some species belonging to Anisoptera and Zygoptera, with a morphology similar to that of olfactory sensilla (Rebora et al., 2008, 2009a; Piersanti et al., 2010). Together with these olfactory sensilla, deeply sunken sensilla located at the bottom of deep cavities and presenting some features of insect thermo-hygroreceptors are typical of Odonata adult antennae (Rebora et al., 2008, 2009a; Piersanti et al., 2010). An electrophysiological study stimulating the antennae of adults of the dragonfly Libellula depressa Linnaeus (Odonata: Libellulidae) with some generic odors showed that Odonata antennae possess functional OSNs (Rebora et al., 2012). Single cell recordings confirmed also the presence of thermo-hygroreceptive neurons in the antennal flagellum of L. depressa (Piersanti et al., 2011). In addition, a neuroanatomical investigation on the brain of the same species revealed that the antennal sensory neurons project to an aglomerular antennal lobe showing spherical knots that could allow for the perception of odor (Rebora et al., 2013). A first evidence of the use of olfaction in Odonata behavior has been given by a recent investigation (Piersanti et al., 2014) demonstrating by means of behavioral and

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electrophysiological assays that adults of the damselfly Ischnura elegans Vander Linden (Odonata: Coenagrionidae) are attracted by olfactory cues emitted by prey, that they perceive through olfactory antennal sensilla. In the present investigation we show the results of an electrophysiological screening on two model species, L. depressa and I. elegans, representatives of the two Odonata suborders Anisoptera and Zygoptera, with the aim to deep the knowledge on the sense of smell of dragonflies and damselflies and describe possible differences between them. In consideration that electroantennography (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), we used this technique to test the odor specificity in L. depressa. Considering that Zygoptera antennae show a lower number of coeloconic sensilla (Rebora et al., 2009a; Piersanti et al., 2010) in comparison with Anisoptera, we could not use EAG in I. elegans and we performed single cell recording (SCR), particularly useful when the number of stimulated neurons is small and gives only a minute EAG (Schiestl and Marion-Poll, 2002). The antennae were stimulated with 48 compounds carrying various functional groups and active on coeloconic olfactory sensilla of other insects and/or potentially involved in some aspects of Odonata biology. Ultrastructural investigations are reported to show the morphology of the antennal olfactory sensilla in I. elegans. 2. Materials and methods 2.1. Insects L. depressa was chosen as model species for Anisoptera, as in previous investigations (Piersanti et al., 2011; Rebora et al., 2008, 2012), because it is easy to collect, very common in Italy, and the larvae can be easily maintained in laboratory condition until the adult emergence. Larvae of L. depressa, attributed to the last instar (F-0) were collected in natural ponds in Central Italy (Perugia, Umbria) in the period March–April 2012. The specimens were kept outdoor, under natural conditions (Perugia from April to July 2012) of temperature (mean monthly temperature from 12 to 26 °C) and light, inside plastic containers (60  40  40 cm) with water, detritus, flora and fauna from the collecting site. The larvae were fed ad libitum with plankton (Daphnia spp. and Cyclops spp.) until adult emergence. EAG was conducted on male and female adults of L. depressa 2 days after their emergence. I. elegans was chosen as model species for Zygoptera as in previous investigations (Piersanti et al., 2014), because it is one of the few odonatan species that can be reared in the laboratory (Van Gossum et al., 2003). I. elegans was maintained at 25 ± 2 °C, a LD 16:8 h photoperiod and 60–80% RH conditions. The larval stages were reared in aquaria and fed ad libitum with Artemia salina nauplii and freshwater plankton (Daphnia sp., Cyclops sp.). Males and females were held separately after the emergence and were reared in small insectaries (50  50  50 cm wooden cages covered with bee netting). Adults were fed ad libitum with Drosophila melanogaster flies. Insectaries and aquaria were provided via artificial solar illumination (36W/94 Philips TLD, Roosendaal, The Netherlands). SCR were carried out on antennae of male and female adults 2– 14 days after their emergence. 2.2. Ultrastructural investigations Observations on scanning (SEM) and transmission (TEM) electron microscopy were performed on the antennal flagellum of

males and females of I. elegans to describe the external and internal morphology of the sensilla previously described in L. depressa (Rebora et al., 2008) and other Odonata (Rebora et al., 2009a; Piersanti et al., 2010). Specimens were prepared as described by Rebora et al. (2008). 2.3. Chemicals To perform the electrophysiological screening we selected 48 chemicals (Table 1) belonging to different functional groups (sulfur compounds, primary alcohols, phenols, ketones, carboxylic esters, carboxylic acids, amines, aldehydes, acetate esters). Odorants were from Sigma Aldrich (St. Louis, MO, USA) and were of the highest grade available (P98%); before use they were dissolved in paraffin oil, diethyl ether, ethanol or distilled water (Table 1), to obtain 10% (v/v) solutions. Because very few data are available on the physiology and the biological role of olfactory sensilla in Odonata (Rebora et al., 2012; Piersanti et al., 2014), to increase the probability of testing chemicals able to elicit responses, we selected odorants perceived by coeloconic olfactory sensilla in other insects and /or potentially involved in some aspects of Odonata biology. In detail, our odorants can be grouped into the following categories (Table 1): GLV = green leaf volatiles or leaf extracts; very common compounds produced by different damaged plants as antimicrobial and perceived by olfactory sensilla (often coeloconic sensilla) in many insects (Ruther, 2000; Shields and Hildebrand, 2001); VR = vertebrate related volatiles. Odors emitted by vertebrates and perceived by many insects through sensilla coeloconica and grooved pegs (Hill et al., 2010). In particular we selected volatiles emitted by the preen gland of birds (Soini et al., 2007) that represent the main Odonata predators; CDV = volatiles active on ionotropic receptors (IR) located in the coeloconic sensilla of D. melanogaster antennae (Yao et al., 2005; Benton et al., 2009). Some of these compounds are also active on coeloconic sensilla and/or grooved pegs of other insects, such as Orthoptera (Altner et al., 1981), Blattoidea (Altner et al., 1977), Lepidoptera (Hill et al., 2010) and Hemiptera (Diehl et al., 2003), where IR sequences have been identified (Croset et al., 2010). WBR = water bacteria related volatiles. Odors emitted by bacteria of stagnant water (habitat where many dragonflies and damselflies oviposit) and used by mosquitoes to positively select oviposition sites (Du and Millar, 1999; Ponnusamy et al., 2008). Some of them are amines and carboxylic acids which are functional groups reported as active on IRs (Rytz et al., 2013). Many chemicals belong to more than one category (Table 1). When comparing the activity of the different compounds on receptor neurons, no compensation was made for differences in volatility. The 10% solutions were kept in a freezer at 18 °C until used. In the present investigation, 6 odorants already tested by EAG and SCR in L. depressa (Rebora et al., 2012) were tested only on I. elegans, while the remaining 42 were tested in both species (Table 1). 2.4. Electroantennography 2.4.1. Recordings The antenna of L. depressa was carefully excised from the head. Scape and pedicel were removed in order to avoid any noise due to the numerous mechanoreceptors located on these structures. The antennal flagellum bearing the olfactory sensilla was mounted between two glass capillary electrodes (1.5 mm o.d, 1.2 mm i.d.) filled with Ringer solution (Beadle and Ephrussi, 1936), containing 5 g/l of polyvinylpyrrolidone (Fluka), in contact with a silver wire.

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Table 1 Chemicals belonging to different functional groups used in the electrophysiological screening on Libellula depressa (EAG) and Ischnura elegans (SCR). In the last two columns the comparison between the electrophysiological responses of the two species is reported. Test compounds

Dimethyl trisulfide 1-Hexadecanol 1-Undecanol (E)-3-hexen-1-ol (E)-2-hexen-1-ol Decanol Nonanol Octanol Hexanol Pentanol Isoamyl alcohol Isobutyl alcohol Z3-hexen-1-ol Phenol 2-pentadecanone Nonanone Cyclohexanone Butanone Methyl tetradecanoate Methyl salicylate (Z)-3-hexenyl butyrate Hexyl butyrate Benzyl acetate Tetradecanoic acid Nonanoic acid Heptanoic acid Hexanoic acid Pentanoic acid Butyric acid Propionic acid Lactic acid Indole 3-Methylindole Isoamylamine Ammonia (28% in H2O) 1,4 diaminobutane Phenylacetaldehyde Decanal Nonanal Octanal Heptanal Hexanal Butanal Propanal (E)-2-hexenal (E)-2-hexenyl acetate Hexyl acetate (Z)-3-hexenyl acetate *

Solvent

Diethyl ether Diethyl ether Diethyl ether Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Diethyl ether Ethanol Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Ethanol Paraffin oil Paraffin oil Paraffin oil Paraffin oil Water Water Paraffin oil Diethyl ether Diethyl ether Water Water Water Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil Paraffin oil

Odorant categories

WBR VR VR GLV GLV VR, CDV CDV CDV GLV, CDV GLV, CDV GLV, CDV GLV, CDV GLV WBR VR CDV CDV CDV WBR GLV GLV GLV CDV WBR GLV, WBR GLV, VR GLV, VR GLV, CDV VR VR VR WBR WBR CDV VR, CDV CDV CDV VR GLV, VR GLV, VR GLV, VR, CDV GLV, CDV GLV, CDV GLV, CDV GLV GLV GLV GLV

Functional group

Sulfur compounds Primary alcohols Primary alcohols Primary alcohols Primary alcohols Primary alcohols Primary alcohols Primary alcohols Primary alcohols Primary alcohols Primary alcohols Primary alcohols Primary alcohols Phenols Ketones Ketones Ketones Ketones Carboxylic esters Carboxylic esters Carboxylic esters Carboxylic esters Carboxylic esters Carboxylic acids Carboxylic acids Carboxylic acids Carboxylic acids Carboxylic acids Carboxylic acids Carboxylic acids Carboxylic acids Amines Amines Amines Amines Amines Aldehydes Aldehydes Aldehydes Aldehydes Aldehydes Aldehydes Aldehydes Aldehydes Aldehydes Acetate esters Acetate esters Acetate esters

Electrophysiological responses Libellula depressa

Ischnura elegans

X X X

X X X

X* X

X X

X

X X

X

X X X X X* X*

X X X X X X

X X X*

X X X

X* X X X X X

X X X X X X

X*

X

From Rebora et al. (2012).

The capillary tubes were drawn to a fine point using the microelectrode puller PC-10 (Narishige, Tokyo, Japan) to get an inner diameter wide enough to enable insertion of the excised antenna. The base of the flagellum was inserted into the reference glass electrode. The recording electrode was connected to the antennal tip (with the apex of the flagellum cut off). The analog signal was detected through a probe with a high-input impedance preamplifier (10) (EAG Kombi-probe, Syntech, Germany), and was captured and processed with a data acquisition controller (IDAC-4, Syntech, Germany) and analyzed using EAG 2000 software (Syntech, Germany). 2.4.2. Stimulations Test compounds diluted as 10% solution were delivered as 20 ll samples placed on a filter paper (15 mm  15 mm). We used high doses of compounds to stimulate OSNs in consideration of previous investigations where we had fairly weak EAG responses in L. depressa (Rebora et al., 2012). The impregnated filter paper was

placed into a glass Pasteur pipette (150 mm in length) constituting an odor cartridge. The control stimulus consisted of a similar pipette containing a filter paper impregnated with 20 ll aliquot of corresponding solvent. Fresh stimulus pipettes were prepared every day. The tip of the glass pipette was placed about 3 mm into a small hole in the wall of a L-shaped glass tube (130 mm long, 12 mm diameter) oriented towards the antennal preparation (5 mm away from the preparation). The stimuli were provided as 1 s puffs of purified, charcoal-filtered air into a continuous humidified main air stream (up to 60% RH obtained by bubbling air through a washing glass bottle) at 2500 ml min1, that was flowing over the antennal preparation at a velocity of 50 cm/s generated by an air stimulus controller (CS-55, Syntech, Germany). At least 1 min interval was allowed between successive stimulations for antenna recovery. Preliminary tests showed that at room temperature dragonfly antenna preparations lasted up to 15 min, with no noticeable decreases in EAG responses over this time period. For this reason it was not possible to perform complete series (more

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than 40 stimulations) on each antenna. Each compound was tested on at least 8 antennae per sex. A total of 70 insects, 34 males and 36 females, were used in the experiments. On the basis of previous recordings (Rebora et al., 2012) (E)-2-hexenal was chosen as reference standard stimulus and presented to each antenna at the beginning, in the middle and at the end of the recording series to confirm activity of the antennal preparation. For each recording series, a pipette with only filter paper was also used to check any contamination of filter paper and to evaluate the air effect in the EAG response. Test compounds were presented in a random sequence. 2.4.3. Statistical analyses For evaluation of EAG responses, the maximum deflection of the recorded EAG signal after stimulation with an odor was used. L. depressa sensitivity to the different chemical compounds was recorded as a percentage of all recorded EAG responses to the reference standard stimulus ((E)-2-hexenal at 10% v/v). This allows compensation for changes of the sensitivity of an antenna during the course of an experiment and the comparison between experiments performed with antennae of different sensitivities. EAG responses for each gender, and separately for those compounds dissolved in different solvents, were compared by 1-way analysis of variance (ANOVA). As post hoc comparisons, Dunnett test was used to compare the response to each compounds to those of the respective control (paraffin oil, diethyl ether, ethanol or distilled water) (Statistica 6.0, Statsoft Inc., 2001). Before the analysis, Box–Cox transformations were used to reduce data heteroscedasticity (Sokal and Rohlf, 1998). 2.5. Single cell recordings 2.5.1. Recordings Before testing, each I. elegans adult was placed inside a channel (diameter 4 mm) drilled through a Plexiglas cube (side 22 mm) and immobilized by Patafix (UHU Bostik, Milano, Italy) and adhesive tape. The antennae, exposed at the top of the holder, were fastened to the Patafix by tungsten hooks. The pits containing the sensilla on the latero-ventral side of the antenna were well exposed to the stimulation, and easily accessible for microelectrode penetration from a side. Nerve impulses from single receptor neurons were recorded extracellularly using tungsten microelectrodes sharpened in a highly concentrated solution of KNO2 to a near tip diameter of 0.2 lm. The recording electrode penetrated the cuticle inside one of the pits located on the latero-ventral side of the flagellum, using a micromanipulator (MMO-203 Narishige) under visual control with a light stereomicroscope (WILD M420 connected with a Wild zoom 1:5) at 200. Under the stereomicroscope, while inserting the recording electrode into the pit, it was not possible to distinguish between the different sensilla located in the cavities. Moreover, in consideration of the incomplete electrical isolation of receptor neurons from neighboring sensilla (Lee and Baker, 2008), each electrode could be picking up spikes from a number of receptor neurons not necessary from the same sensillum. These aspects were considered when analyzing the spikes (see below). The indifferent electrode was inserted into the pedicellum making contact with the hemolymph. A conventional electrophysiological set-up for extra cellular single cell recording was used. The ring was mounted on anti-vibration table and shielded with a Faraday cage. For data acquisition a 10 gain probe (Universal Single Ended Probe, Type PRS-1, Synthec) was used. The amplified analogue signal was monitored on an audio monitor and captured and processed with a data acquisition controller (IDAC-4, Syntech, Germany). Spike activity was recorded by the computer software Autospike (Syntech, Germany).

2.5.2. Stimulations Test compounds and controls were delivered as 5 ll samples using the same procedure described for EAG. Also in this case, we used a quite high doses of compounds on the basis of the previous investigations on Odonata OSN (Rebora et al., 2012). The tip of the glass pipette constituting the odor cartridge was placed about 3 mm into a small hole in the wall of a plastic pipette (50 mm long, 6 mm inner diameter and 2 mm inner diameter in the tip) oriented towards the antenna (5 mm away from the preparation). The stimuli were provided as described for EAG. Test compounds were presented in a random sequence at intervals of at least 1 min. Each contact was used until it was lost but rarely they lasted enough to complete the series (up to 48 stimulations). Occasionally, on OSNs or on neurons which did not respond to chemicals, we performed very basic experiments with humidity changes (from R = 30% to D = 20% RH and from R = 30% to H = 60% RH) (obtained turning on or off the passage of the continuous main air stream through the washing glass bottle and/or through a silica gel cartridge able to retain humidity – see EAG Section). A total of 75 insects, 36 males and 39 females, were used in the experiments. The spike trains were analyzed offline by computer software Autospike (Syntech, Germany). To distinguish responses from colocated neurons, the different RNs were sorted into spike populations on the basis of their amplitude. Only spikes clearly separated from noise were evaluated. Responses of individual RNs, identified in each spike trains, were calculated as increase or decrease in the firing rate, relatively to the pre-stimulus rate. The response to a stimulus was obtained by counting action potentials (spikes) during 1 s, starting from the time after the stimulation period at which the earliest response for the neuron was found, and deducting the number of action potentials during 1 s immediately prior to the response. To achieve net responses (N) we subtracted the value of the response elicited by the control. Since, as explained above, it was not possible to see the sensilla under the stereomicroscope before inserting the electrode, each electrode could be picking up spikes from a number of receptor neurons, not necessarily from the same sensillum, at different distances. For this reason, and considering that in most part of recordings it was not possible to test the whole series of compounds, in the present investigation we had to consider each OSN in each contact separately and we could not group them classifying them by virtue of their distinguishable amplitude or their specificity and attribute them to the different olfactory sensilla, as the SCR technique normally allows (Masson and Mustaparta, 1990). For the same reason to compare the data we normalized net responses in absolute value (|N|) to the pre-stimulus firing rate (R), to obtain the factor (F) of increase (or decrease) of the discharge rate respect to the pre-stimulus condition:

F ¼ ðjNj þ RÞ=R We considered excitatory or inhibitory responses if the factor of increase (or decrease) F respect to the pre-stimulus firing rate exceeded the value of 1.5.

3. Results 3.1. L. depressa EAG responses were recorded in both males and females of L. depressa (Fig. 1). In females (Fig. 2) 1-undecanol, (E)-3-hexen-1ol, phenol, (Z)-3-hexenyl butyrate, nonanoic acid, heptanoic acid, hexanoic acid, pentanoic acid, isoamylamine, ammonia, heptanal, butanal, propanal and (E)-2-hexenal elicited EAG activity significantly higher in comparison with the EAG activity recorded in

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Fig. 1. Examples of excitatory (heptanoic acid, ammonia, (E)-2-hexanal) and inhibitory ((E)-2-hexen-1-ol, phenol, isoamylamine,) EAG response waveforms of L. depressa. Dotted line corresponds to 1 s stimulus.

response to the relative solvent, while no significant difference was present between the EAG activity recorded in response to the other tested compounds (see Table 1) and the EAG activity recorded in response to the relative solvent (diethyl ether: F = 14.3; d.f. = 6, 66; P < 0.001; paraffin oil: F = 19.91; d.f. = 32, 438; P < 0.001; ethanol: F = 0.40; d.f. = 2, 26; P = 0.678; water: F = 22.4; d.f. = 2, 29; P < 0.001). Among the chemicals that elicited a response in females, (E)-3-hexen-1-ol, phenol and isoamylamine elicited an inhibitory response while all the others elicited an excitatory response (Fig. 2). In males (Fig. 3) we obtained the same responses obtained in females except for 1-undecanol, (E)-3-hexen-1-ol and butanal, that did not elicit any significant response, and (E)-2-hexen-1-ol, hexyl butyrate and hexanal that elicited significant response compared to the response to the relative solvent (diethyl ether: F = 7.85; d.f. = 6, 52; P < 0.001; paraffin oil: F = 15.95; d.f. = 32, 368; P < 0.001; ethanol: F = 0.53; d.f. = 2, 13; P = 0.600; water: F = 22.06; d.f. = 2, 26; P < 0.001). Among the chemicals that elicited a response in males, (E)-2-hexen-1-ol, phenol and isoamylamine elicited an inhibitory response while all the others elicited an excitatory response. In both females and males the magnitude of the response to phenol, pentanoic acid, hexanoic acid heptanoic acid and (E)-2hexenal was particularly high (P0.008 mV and 60.015 mV) in comparison with that of the other chemicals. 3.2. I. elegans The adult of I. elegans shows 3–4 (occasionally up to 7) pits located on small bulges on the lateral-ventral side of each antenna (Fig. 4a and b). Each pit contains 1–3 olfactory sensilla (Fig. 4c), visible on the antennal surface, each innervated by two olfactory sensory neurons (Fig. 4d and e). The same pits represent the openings of deep cavities hosting other sensilla (Fig. 4f and g). Some of these have features similar to the type-1 sensilla of L. depressa, showing a grooved peg with cuticular fingers in its distal portion (Fig. 4f). Successful contacts (Fig. 5) were obtained from ORNs in 24 insects (10 males and 14 females), but due to a low quality of the signal (low signal to noise ratio) only the responses to odors from 13 damselflies (20 different contacts) are presented here. In this regard, it is worth to remember the technical difficulty of obtaining high-quality electrophysiological recordings from coeloconic sensilla. In addition to their small size, they are more susceptible to damage by the electrode and yield recordings with lower signal to noise ratios than the other types of sensilla (Yao et al., 2005). This is particularly true for Odonata (Rebora et al., 2012) where coeloconic antennal sensilla are small and located inside pits.

Fig. 2. EAG responses (mean ± s.e.m.) of female antennae of L. depressa to synthetic compounds. For the responses to squared compounds see Rebora et al. (2012). Bars of the same colors show responses of compounds dissolved in the same solvent. The responses are compared with those of the respective solvent (⁄P < 0.05; ANOVA, Dunnett test). EAG responses have been normalized to the E-2-hexenal responses.

Co-located ORNs were effectively sorted on the basis of their spike amplitude. The average pre-stimulus firing frequency was 8.6 ± 1.7 spike/s with the lowest value of 0.5 spike/s and the highest of 21.4 spike/s recorded only one time. The recordings showed the presence of ORNs responding to some of the tested odors (Figs. 5 and 6). In particular 1-undecanol,

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hexanal, butanal, propanal, (E)-2-hexenal, (Z)-3-hexenyl acetate elicited response from at least one ORN, while the other tested compounds (see Table 1) elicited no response (Fig. 6). Among the chemicals that elicited responses, all of them elicited excitatory response but, in some contacts, phenol, cyclohexanone, pentanoic acid, butyric acid, propionic acid, propanal and (E)-2-hexenal induced an inhibitory response (Fig. 6). Hexanoic acid, pentanoic acid, butyric acid, propionic acid, isoamylamine, ammonia, heptanal, hexanal, butanal, (E)-2-hexenal and (Z)-3-hexenyl acetate represent the chemicals eliciting the strongest response (10 6 F 6 65) (Fig. 6). The chemicals that induced a higher percentage of response in consideration of the total number of contacts stimulated are phenol (37.5%), ammonia (52.9%), butyric acid (40%), propionic acid (46.7%) and (E)-2-hexenal (46.7%) (Fig. 6). Many OSNs responded to more than 5 chemicals with a maximum of 13 chemicals. We identified the presence of one OSN responding to 1-undecanol, (E)-3-hexen-1-ol, phenol, nonanoic acid, heptanoic acid, butyric acid, propionic acid, 1,4 diaminobutane, octanal, heptanal, hexanal, butanal, (E)-2-hexenal, also responding as a typical moist cell i.e. to humidity increase with an increase in the firing rate (+55 spikes/s) and to humidity decrease with a decrease in the firing rate (37 spikes/s) (Fig. 7). A comparison between the results of the electrophysiological olfactory screening in L. depressa and I. elegans (Table 1) shows that, among the 48 tested chemicals, L. depressa and I. elegans responded to 23 chemicals with a difference of only one chemical each, as hexyl butyrate elicited a response only in L. depressa while cyclohexanone elicited a response only in I. elegans. Moreover, (E)2-hexen-1-ol, (E)-3-hexen-1-ol and isoamylamine, show an opposite response in the two species. In particular, considering that some compounds can be attributed to more than one category, the compounds that elicited a response in both species are 14 GLV, 7 VR, 2 WBR and 8 CDV on a total of 24 GLV, 13 VR, 7 WBR and 20 CDV. Considering the functional groups, the active compounds were mainly carboxylic acids followed by amines, alcohols and aldehydes.

4. Discussion

Fig. 3. EAG responses (mean ± s.e.m.) of male antennae of L. depressa to synthetic compounds. For the responses to squared compounds see Rebora et al. (2012). Bars of the same colors show responses of compounds dissolved in the same solvent. The responses are compared with those of the respective solvent (⁄P < 0.05; ANOVA, Dunnett test). EAG responses have been normalized to the E-2-hexenal responses.

(E)-3-hexen-1-ol, (E)-2-hexen-1-ol, (Z)-3-hexen-1-ol, phenol, cyclohexanone, (Z)-3-hexenyl butyrate, nonanoic acid, heptanoic acid, hexanoic acid, pentanoic acid, butyric acid, propionic acid, isoamylamine, ammonia, 1,4 diaminobutane, octanal, heptanal,

The present electrophysiological screening on the two model species, L. depressa and I. elegans, representatives of the two Odonata suborders Anisoptera and Zygoptera, shows that the antennal OSNs respond nearly to the same chemicals. Indeed, we noted a response to the same 22 compounds in both species, and in addition a response to cyclohexanone in I. elegans and to hexyl butyrate in L. depressa. These differences are negligible considering that EAG and SCR techniques can give different results in the same insect, both quantitatively and qualitatively (Wibe, 2004). Such similar response profile by two odonatan species, phylogenetically distant and with a very different biology, suggests that the sense of smell in dragonflies and damselflies is not specialized, probably remaining in the entire order quite similar to what it was in the common ancestor. Our data confirm that Odonata OSNs respond to odorants belonging to all the categories we selected, because perceived by coeloconic olfactory sensilla in other insects and /or potentially involved in some aspects of Odonata biology. In detail, we obtained responses to 16 of the 24 green leaf volatiles (GLV) tested. These very common compounds, produced as traces by un-stressed plants and rapidly formed within a few seconds upon stress, have several behavior-modifying properties in phytophagous arthropods, such as attraction to the host plants, enhancing or inhibition of the attractiveness of pheromones, direct location of sexual mates and selection of the oviposition site (Scala

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Fig. 4. Antennal flagellum of the adult of Ischnura elegans under SEM (a–c, f) and TEM (d–e, g). (a) Antenna showing scape (S), pedicel (P) and flagellum (F) with sensilla in pits (arrows) on its lateral-ventral side; (b) detail of the flagellum showing the pits located on small bulges (arrows); (c) detail of a pit with a single-walled coeloconic olfactory sensillum showing pores (P); (d,e) cross sections showing the outer (d) and the inner (e) dendritic segments (D) of the two neurons innervating each single-walled coeloconic olfactory sensillum; (f) detail of one cavity showing two coeloconic porous olfactory sensilla (arrows) and one sensillum with a grooved peg with cuticular fingers (double arrow), located at the bottom of the cavity; (g) longitudinal section of the flagellum showing one of the deep convoluted cavities hosting on the bottom sensilla with a grooved peg and cuticular fingers (arrow). C, cuticle, D, dendrites.

Fig. 5. Examples of raw spike trains showing excitatory (propionic acid and ammonia) and inhibitory (phenol and butyric acid) responses of I. elegans ORNs to odors. On the right side, cell templates from analyzed recordings. Bars represent 1 s stimulations.

et al., 2013). In addition, these compounds have been reported to play a synomonal role in tritrophic interactions in the recruitment of predators and parasitoids of herbivores (Scala et al., 2013). On this account, many studies showed that predators and parasitoids are attracted to single GLVs or to a set of GLVs (James, 2005; Kessler and Baldwin, 2001). Odonata have a well-earned reputation for being generalist predators mainly catching their prey in flight; otherwise it is well known that they can also capture sessile

alive preys, such as aphids and other phytophagous arthropods, by gleaning (Corbet, 2008). On this account the perception of GLV could be useful for these huge predators to orient in their ‘‘green environment’’ and improve prey location. We recorded responses to 2 of the 7 WBR tested (one of them is also a GLV) and responses to 8 of the 14 VR tested (four of them also belong to the GLV category). Volatiles emitted by bacteria naturally occurring in waters, in particular associated with bacterial digestion of organic material such as plant debris, could be a useful signal for many dragonflies and damselflies to locate suitable sites for oviposition and larval growth. Indeed, notwithstanding larvae of different species are adapted to different habitat (Corbet, 2008), the presence of submerged aquatic plants and plant debris are generally positively selected by ovipositing females, because they provides larval refuges from predators (Corbet, 2008). As far as the volatiles emitted by vertebrates are concerned, sensitivity to these compounds could be useful for dragonflies and damselflies to escape predators, in particular birds that virtually are the only effective vertebrate predators on mature dragonflies and an important threat for damselflies too (Corbet, 2008). On this account, it is very interesting that the response spectra of olfactory sensilla coeloconica and grooved pegs to vertebrate related compounds (such as ammonia, carboxylic acids and aldehydes that we tested with positive responses on dragonflies and damselflies) appear to be under some degree of conservation among insects as diverse as moths, mosquitoes, fruit flies and locusts (Hill et al., 2010). This putative conservation suggests a significant role for these sensilla in eliciting or modulating a vertebrate-related behavior that in the most part of cases is a repulsion but can be an attraction in haematophagous insects (Hill et al., 2010). The sense of smell of Odonata is surely poor in comparison with that of many other insects, also in consideration of the simple organization of the antennal lobe (Rebora et al., 2013). However, recent behavioral and electrophysiological investigation on the damselfly

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Fig. 6. SCR responses of I. elegans antennae to synthetic compounds. Each bar represents the factor of increase or decrease (F) of discharge rate of different cells (as reported in material and methods, we could not group OSNs classifying them by virtue of their distinguishable amplitude or their specificity). The number of bars for each compound represents the number of cells in which the compound has been tested. Bars with a white spot are considered responses (F > 1.5). The percentage of responses on the total number of cells in which each compound has been tested is reported on the right.

I. elegans showed the involvement of olfaction in the perception of the prey (Piersanti et al., 2014) and the differences in the response profile between males and females of L. depressa observed in the present investigation and in the previous one (Rebora et al., 2012) could be linked to a possible involvement of olfaction in the reproductive behavior. Further behavioral investigations are therefore necessary to better clarify the function of olfaction in

Odonata and to test the hypothesis suggested by the present electrophysiological screening. Considering the last category of compounds (CDV), that we decided to test because they were active on ionotropic receptors (IR) located in the coeloconic sensilla of D. melanogaster (Yao et al., 2005; Benton et al., 2009), and in many cases also active on coeloconic sensilla and/or grooved pegs of other insects where

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combination of OR and IR, as in all the investigated Neoptera, or an intermediate condition, such as Zygentoma, were the presence of the coreceptor Orco but no OR has been demonstrated (Missbach et al., 2014). These data on Odonata, together with similar investigations on Ephemeroptera where coeloconic olfactory antennal sensilla have been described (Rebora et al., 2009b), can be very important for understanding the evolution of invertebrate senses and olfaction in particular.

Acknowledgements

Fig. 7. Analyzed recordings (cell templates in the right side) from I. elegans hygrosensitive receptor neuron (moist cell) responding to changes in air relative humidity (RH). R, 30% RH; D, 20% RH; M, 60% RH; (T 22 °C). The same cell responds also to different odors.

IR sequences have been identified (Croset et al., 2010), we recorded responses to 9 of the 20 volatiles tested. Drosophila’s IR expressing neurons show typical odor-response properties, listed below, which are different from those observed for OR (Rytz et al., 2013): (1) IRs and ORs detect distinct classes of odors, with the strongest IR ligands (amines, carboxylic acids or aldehydes) recognized only weakly or not at all by ORs, and the strongest OR ligands (predominantly esters, alcohols and ketones) not stimulating any IR neurons (Hallem and Carlson, 2006); (2) IR neurons are less sensitive than OR neurons (Yao et al., 2005; Silbering et al., 2011; Getahun et al., 2012) and are generally more broadly tuned, because multiple IRs can be coexpressed per neuron while OR expression generally follows a one neuron-one receptor rule (Benton et al., 2009); (3) IR neurons show a low base line activity and robust agonistic-evoked neuronal responses are extremely sparse (they require longer stimulation times to key odorants and respond with lower response intensities) (Getahun et al., 2012); (4) in some OSNs expressing IR the neuronal firing rate increases and decreases with increasing and decreasing humidity, respectively (Yao et al., 2005; Benton et al., 2009). These properties have been observed also in Odonata OSNs, in particular: (1) L. depressa and I. elegans antennae are sensitive to carboxylic acids followed by amines, alcohols and aldehydes. (2) in I. elegans, where SCR recordings were performed, many cells responded to numerous odors leading to the conclusion that OSNs are quite broadly tuned; (3) damselfly OSNs showed a low baseline activity and a low increase of firing rate (only in three cases more than 100 spikes/s); (4) In I. elegans we recorded from an OSN responding not only to odors but also to variation in humidity gradient. In a recent paper Missbach et al. (2014) demonstrated that only IRs are employed in Archaeognatha olfaction. In the same investigation the electrophysiological recordings show the same typical properties of the IRs expressing olfactory neurons reported above but the response spectrum is much larger, encompassing not only to acids, aldehydes and amines but also alcohols, esters and chetones. Authors concluded that if IRs are the only olfactory receptors type in basal insects, they should exhibit a broader spectrum of possible ligands (Missbach et al., 2014), and this assumption could be extended also to Odonata where, notwithstanding the best ligands are mostly acids, aldehydes and amines, we also recorded responses to alcohols and some esters. The molecular basis of olfaction in Paleoptera is so far unknown but all these features suggest the possibility that OSNs of Odonata mainly, or exclusively, express IR. Molecular investigations are needed to clarify if Odonata OSNs express only IR, as it has been demonstrated in Archaeognatha (Missbach et al., 2014), a

We are grateful to A. Luchetti and A. Sotgiu for insect rearing and to L. Farabi and F. Alberti for helping in data collection. We thank R. Dolciami, Director of Centro Ittiogenico Provinciale del Trasimeno (Perugia, Italy), for the supply of plankton used to rear larval stages of I. elegans. Funding was provided by the Italian Ministry for University and Research (MIUR-Fondo Integrativo per la Ricerca di Base – F.I.R.B., 2010, RBFR10Z196).

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