Marit Komendant

ANTENNAL CONTACT CHEMORECEPTION IN GROUND BEETLES (COLEOPTERA: CARABIDAE)

JOOKSIKLASTE (COLEOPTERA: CARABIDAE) ANTENNAALNE KONTAKTNE KEMORETSEPTSIOON

MARIT KOMENDANT

A Thesis submitted for the degree of Doctor of Philosophy in Entomology

Väitekiri ﬁlosooﬁadoktori kraadi taotlemiseks entomoloogia erialal

Tartu 2011 EESTI MAAÜLIKOOL ESTONIAN UNIVERSITY OF LIFE SCIENCES ANTENNAL CONTACT CHEMORECEPTION IN GROUND BEETLES COLEOPTERA: CARABIDAE

JOOKSIKLASTE (COLEOPTERA: CARABIDAE) ANTENNAALNE KONTAKTNE KEMORETSEPTSIOON

MARIT KOMENDANT

A Th esis submitted for the degree of Doctor of Philosophy in Entomology

Väitekiri ﬁ losooﬁ adoktori kraadi taotlemiseks entomoloogia erialal

Tartu 2011 Institute of Agricultural and Environmental Sciences Estonian University of Life Sciences

According to the verdict No 80 of March 21, 2011, the Doctoral Committee of the Agricultural and Natural Sciences of the Estonian University of Life Sciences has accepted this thesis for the defence of the degree of Doctor of Philosophy in Entomology.

Opponent: Prof. Habil. Dr. Vincas Būda Institute of Ecology, Vilnius University, Lithuania

Supervisors: Dr. Enno Merivee Estonian University of Life Sciences

Prof. Anne Luik Estonian University of Life Sciences

Defence of this thesis: Estonian University of Life Sciences, Karl Ernst von Baer House, Veski 4, Tartu, on May 6, 2011, at 10.00.

Th e English language was edited by Ingrid H. Williams and the Estonian by Tiina Halling.

Publication of this thesis is granted by the Estonian University of Life Sciences and by the Doctoral School of Earth Sciences and Ecology created under the auspices of European Social Fund.

ISBN 978-9949-484-00-3 CONTENTS

LIST OF ORIGINAL PUBLICATIONS ...... 7 1. INTRODUCTION ...... 8 2. AIMS OF THE STUDY ...... 12 3. MATERIAL AND METHODS ...... 13 3.1. Test beetles ...... 13 3.2. Electrophysiology ...... 13 3.3. Chemicals and preparation of stimulating solutions ...... 14 3.4. Feeding bioassays (V) ...... 15 3.5. Data analysis ...... 15 4. RESULTS ...... 17 4.1. Taste sensilla on the antennal fl agellum of P. oblongopunctatus and P. aethiops ...... 17 4.2. Spike shapes and amplitudes evoked by tested chemicals ...... 17 4.3. Eff ect of stimulus pH to the responses of the electrolyte-sensitive chemoreceptor neurons innervating antennal taste sensilla of P. aethiops and P. oblongopunctatus ...... 18 4.3.1. Response to 100 mmol l-1 acetate buff ers at pH 3–6 .....18 4.3.2. Response to 100 mmol l-1 phosphate buff ers at pH 5.8–8.1 (11.1) ...... 19 4.3.3. Response to 100 mmol l -1 Na +-salts with and without addition of 10 mmol l-1 NaOH...... 19 4.4. Plant sugar sensitivity in ground beetles ...... 20 4.4.1. Electrophysiological identifi cation of the sugar-sensitive neuron in antennal taste sensilla of P. aethiops ...... 20 4.4.2. Electrophysiological responses of the chemoreceptor neurons from antennal taste sensilla of P. oblongopunctatus to plant sugars and amino acids ...... 21 4.4.2.1. Response of antennal taste neurons to sugars ...21 4.4.2.2. Response of antennal taste neurons to amino acids ...... 22 4.5. Response of antennal taste neurons to plant alkaloids and glucosides ...... 22 4.5.1. Electrophysiological identifi cation of the alkaloid-sensitive neuron in antennal taste sensilla of P. oblongopunctatus .. 22

5 4.5.2. Stimulating eff ect of plant alkaloids and glucosides on the responses of the antennal chemoreceptor neurons ...23 4.5.3. Peripheral inhibition of the salt- and pH-sensitive neurons by some plant secondary compounds ...... 23 4.5.4. Eff ect of quinine hydrochloride on the feeding response of P. oblongopunctatus ...... 24 5. DISCUSSION ...... 25 5.1. pH sensitivity in ground beetles (I, II) ...... 25 5.1.1. Th e occurrence of two chemoreceptor neurons in insect contact chemoreceptors responding to salts ...... 25 5.1.2. Th e eff ect of stimulus pH to the salt- and pH-sensitive neurons in antennal taste sensilla of P. aethiops and P. oblongopunctatus ...... 26 5.1.3. pH preference in ground beetles ...... 27 5.2. Sensitivity of ground beetles to plant sugars and amino acids (III, IV) ...... 28 5.2.1. Response of the chemoreceptor neurons from antennal taste sensilla to sugars ...... 28 5.2.2. Response of the chemoreceptor neurons from antennal taste sensilla to amino acids ...... 30 5.3. Response of the chemoreceptor neurons in the antennal taste sensilla of P. oblongopunctatus to plant secondary compounds ...... 31 5.3.1. Stimulating eff ect of plant alkaloids and glucosides on the responses of the antennal chemoreceptor neurons ...... 31 5.3.2. Peripheral inhibition of the salt- and pH-sensitive neurons by some plant secondary compounds ...... 33 CONCLUSIONS ...... 35 REFERENCES ...... 37 SUMMARY IN ESTONIAN ...... 46 ACKNOWLEDGEMENTS ...... 51 PUBLICATIONS ...... 53 CURRICULUM VITAE ...... 129 ELULOOKIRJELDUS ...... 130 LIST OF PUBLICATIONS ...... 131

6 LIST OF ORIGINAL PUBLICATIONS

Th is thesis is a review of the following papers, which are referred to by Roman numerals in the text. Th e papers are reproduced by kind permission of the following journals: Physiological Entomology (I) and Journal of Insect Physiology (II, III, IV).

I Merivee, E., Ploomi, A., Milius, M., Luik, A., Heidemaa, M., 2005. Electrophysiological identifi cation of antennal pH receptors in the ground beetle Pterostichus oblongopunctatus. Physiological Entomology 30(2), 122–33. II Milius, M., Merivee, E., Williams, I., Luik, A., Mänd, M., Must, A., 2006. A new method for electrophysiological identifi cation of antennal pH receptor cells in ground beetles: the example of Pterostichus aethiops (Panzer, 1796) (Coleoptera, Carabidae). . Journal of Insect Physiology 52, 960–967. III Merivee, E., Must, A., Milius, M., Luik, A., 2007. Electrophysiological identifi cation of the sugar cell in antennal taste sensilla of the predatory ground beetle Pterostichus aethiops. Journal of Insect Physiology 53(4), 377–384. IV Merivee, E., Märtmann, H., Must, A., Milius, M., Williams, I., Mänd, M., 2008. Electrophysiological responses from neurons of antennal taste sensilla in the polyphagous predatory ground beetle Pterostichus oblongopunctatus (Fabricius 1787) to plant sugars and amino acids. Journal of Insect Physiology 54, 1213– 1219. V Komendant, M., Merivee, E., Must, A., Tooming, E., Williams, I., Luik, A. Electrophysiological response of the chemoreceptor neurons in the antennal taste sensilla to plant alkaloids and glucosides in the granivorous ground beetle Pterostichus oblongopunctatus. (in review) Table 1. Author’s contribution to each article (%) I II III IV V Idea and design 10% 80% 25% 0% 80% Data acquisition 50% 100% 50% 40% 100% Data analysis, statistics 50% 80% 50% 40% 80% Writing 10% 80% 15% 0% 90%

7 1. INTRODUCTION

Th e ground beetles (Coleoptera, Carabidae) with 40,000 species belong to the most numerous families of beetles, their distribution being cosmopolitan. Approximately 275 species have been found in Estonia (Haberman, 1968; Silfverberg, 2004). Binding of ground beetles to certain habitat types and microhabitats is determined by numerous biotic and abiotic factors such as food conditions, vegetation type, presence and distribution of competitors, life history and season, landscape characteristics, agricultural cultivation impacts, temperature and humidity conditions, light intensity, and others. Favourite wintering sites are well aerated. Habitat choice in many ground beetles is so speciﬁ c that they are often used to characterize habitats (Th iele, 1977; Lövei and Sunderland, 1996; Ings and Hartley, 1999; Cole et al., 2002; Blake et al., 2003; Eyre and Luﬀ , 2004; Purtauf et al., 2005).

Chemically mediated habitat selection has been shown to occur in several species of ground beetle by Evans (1982, 1983, 1984, 1988). By behavioural experiments, he demonstrated that olfactory neurons receptive to methyl esters of palmitic and oleic acid emitted by the mat-forming blue-green algae Oscillatoria animalis Agardh and Oscillatoria subbrevis Schmidle are present in sensillae on the antennae of those Bembidiini associated with the shores of saline lakes. Salt content and pH of the soil may also have importance in determining the distribution of ground beetles (Th iele, 1977; Paje and Mossakowski, 1984; Hoback et al., 2000; Irmler, 2001, 2003; Magura, 2002). Behavioural ablation experiments have shown that presumptive contact chemoreceptive sensilla are located on the ground beetles’ antennae (Paje and Mossakowski, 1984; Hoback et al., 2000).

Scanning electron microscope studies show that the antennae of ground beetles are well equipped with various chemoreceptors. In addition, to numerous small basiconic olfactory sensilla, approximately 56 to 70 larger bristle-like contact chemosensilla (35–200 μm in length) have been found on the antennal fl agellum of adults in several genera (Kim and Yamasaki, 1996; Merivee et al., 2000, 2001, 2002). In the ground beetle Nebria brevicollis these large sensilla are each innervated by one mechanoreceptive and four chemoreceptive neurons (Daly and Ryan, 1979). A similar set of neurons is typical for contact chemoreceptors in various insect groups (Morita and Shiraishi, 1985; Chapman, 1998; Jørgensen et al., 2006). 8 By electrophysiological single sensillum stimulations the occurrence of two chemoreceptor neurons responding to salts has been demonstrated in the antennal taste bristles of the ground beetle Pterostichus aethiops. One of them, producing spikes with negative polarity was highly sensitive to both ionic composition and concentration of the tested nine salt solutions showing a phasic-tonic type of reaction with a pronounced phasic component. Th e stimulating eff ect was dominated by the cations involved, and in most cases, monovalent cations were more eff ective stimuli than divalent cations. Th is neuron was classifi ed as the salt-sensitive neuron. Th e specifi c function for the second neuron (the “A-cell”) generating two- tipped spikes with positive polarity still remains to be explained. It was observed, however, that alkaline salts usually evoked substantially more spikes in the second neuron than acid salts, but the ionic composition of the salts also aff ected the responses of the second neuron (Merivee et al., 2004). Th us, the “A-cell” with positive spikes seems to be the most probable candidate for pH sensitivity in ground beetles, but no electrophysiogical evidence is available. Th e special function of the third and fourth chemoreceptor neurons of the sensillum also needs to be clarifi ed in ground beetles.

Data on food items are available for approximately 1100 species and subspecies of ground beetles of the world. A total of 73% are carnivorous, 19% are omnivorous and 8% are exclusively herbivorous (Th iele, 1977; Larochelle, 1990; Toft and Bilde, 2002). Of these, many species predate on seeds (Honek et al., 2003). Ground beetles use a variety of external stimuli in order to locate food. Behavioural tests and observations have indicated that visual, tactile, olfactory and gustatory cues may be involved (Wheater, 1989; Kielty et al., 1996; Toft and Bilde, 2002). Various sugars and amino acids function as strong phagostimulants in most phytophagous insects equipped with specialized receptor neurons for these chemicals (Chapman, 1998; Hansen-Delkeskamp, 1998; Schoonhoven and van Loon, 2002; Chapman, 2003) suggesting that these chemicals may have a phagostimulatory role in herbivorous and omnivorous ground beetles, too. Adults of the genus Pterostichus appear to be primarily carnivorous, feeding on small prey animals, but in many species, plant matter also forms part of their diet, and, in some the proportion of animal to plant matter eaten varies with the season (Th iele, 1977; Hengeveld, 1980; Lövei and Sunderland, 1996). Johnson and Cameron (1969) collected data on the extent of herbivory in more than 150 species of ground beetle. Some species in the genera Pterostichus, including Pterostichus oblongopunctatus,

9 Amara, Agonum and Harpalus consume conifer seeds and young seedlings immediately following germination. In Sweden, P. oblongopunctatus and Calathus micropterus are the most important predators of Pinus sylvestris seed. In the ﬁ eld, seed predation typically resulted in >20% seed mortality, reaching even higher levels (up to 60%) on some occasions and most predation (81% of all damaged seeds) was due to ground beetles (Heikkilä, 1977; Nystrand and Granström, 2000). Th erefore, as in other phytophagous insects, ground beetles feeding exclusively or partly on plant material, may also have plant sugar- and amino acid-sensitive neurons associated with their feeding habits although no experimental data is available.

In addition to detecting phagostimulants, most animals, including herbivorous insects, possess chemoreceptor neurons responding to a diverse range of feeding deterrent compounds. When stimulated, deterrent neurons reduce or fully stop feeding activity (Dethier, 1980; Schoonhoven and van Loon, 2002; Chapman, 2003; Wang et al., 2004). Natural deterrent stimuli recognized by chemoreceptor neurons of herbivorous insects constitute the largest and most structurally diverse class of gustatory stimuli. It has been shown that plant glucosides, for example, sinigrin and salicin, and various alkaloids like nicotine (pyrrolidine group), strychnine and quinine (quinoline group), caﬀ eine (purine group) and several others stimulate the deterrent neuron in many herbivorous insects. Th reshold concentrations for compounds that stimulate a deterrent neuron vary from 10 -7 to 10-2 mol l-1, but the majority of these are in the range of 10-4 to 10 -2 mol l-1. Th erefore, deterrent neurons fulﬁ l a central role in host-plant recognition and have, since their discovery (Ishikawa 1966), attracted much interest (Glendinning, 2002; Ryan, 2002; Schoonhoven and van Loon, 2002; Chapman, 2003; Jørgensen et al., 2007).

Herbivory, including pre- and post-dispersal plant seed predation, is extremely common in virtually all ecosystems. Seeds with their very high nutrient values per unit volume are extremely attractive to granivores as food (Janzen, 1971). To counterbalance the eff ects of herbivory, plants have developed a broad spectrum of secondary metabolites involved in plant defense, which are collectively known as antiherbivory compounds and can be classifi ed into three main sub-groups: nitrogen compounds (including alkaloids, cyanogenic glycosides and glucosinolates), terpenoids, and phenolics. Th ese compounds can act as feeding deterrents or toxins with various modes of action to herbivores, or reduce plant digestibility (Janzen, 1971; Jolivet, 1998; Ryan, 2002; Chapman, 2003; Hulme and 10 Benkman, 2004; Davis et al., 2008). Seeds are sources of some of the most toxic natural products known to humans and the secondary chemicals in seeds present formidable challenges to granivores. However, seed chemical defenses can only be assessed with reference to specifi c target organisms since a secondary chemical may not be equally toxic to all granivores. Some seeds are so toxic that all seed-predators avoid them (Harborne, 1993; Hulme and Benkman, 2004). Most toxins are usually in small concentrations (<5%) in the seed and some alkaloids, for example, can be lethal at concentrations as low as 0.1% (Hatzold et al., 1983; Harborne, 1993). Unfortunately, there is no evidence that plant secondary compounds cause feeding deterrency in ground beetles. However, due to selective seed predation by ground beetles (Toft and Bilde, 2002; Tooley and Brust, 2002), the presence of a deterrent neuron in their antennal contact chemoreceptors should be expected.

11 2. AIMS OF THE STUDY

Th e aims of this study were:

1) by electrophysiological experiments, to test the response of the chemoreceptor neurons in the antennal taste sensilla of the ground beetle P. oblongopunctatus to acetate and phosphate buff ers over a wide range of pH (3–11) in order to clarify the eff ect of stimulus pH on the neurons, and to clarify the possible existence of a pH-specifi c neuron in this species (I);

2) using various alkalized and non-alkalized salts as stimuli, to test the electrophysiological response of the antennal contact chemoreceptor neurons of the ground beetle P. aethiops to stimulus pH in order to collect additional data on functioning of a speciﬁ c pH-sensitive neuron in ground beetles (II);

3) by electrophysiological experiments, to determine whether sugar- and amino acid-sensitive receptor neurons are present in the antennal taste sensilla of P. aethiops (III) and P. oblongopunctatus (IV) by testing for stimulatory eﬀ ects of various sugars of live and decaying plant origin, and amino acids on these sensilla;

4) to test the stimulatory eﬀ ect of some toxic plant secondary compounds (alkaloids and glucosides) on the chemoreceptor neurons of the antennal taste sensilla of the granivorous P. oblongopunctatus in order to identify the speciﬁ c feeding deterrent neuron stimulated by these chemicals (V).

12 3. MATERIAL AND METHODS

3.1. Test beetles

Adult P. oblongopunctatus and P. aethiops were collected from a local population in southern Estonia. Th ey were kept in 30×20×10 cm plastic boxes ﬁ lled with moistened moss and pieces of brown-rotted wood in a refrigerator at 5–6 °C. Th ree to four days prior to the experiments, the beetles were transferred to room temperature (20–23 °C), fed with moistened cat food (Kitekat, Master Foods, Poland, I, II; Friskies Vitality+, Nestle´ Purina, Hungary, III, IV, V) and given clean water to drink every day.

3.2. Electrophysiology

Each beetle to be tested was restrained securely in a conical tube made of thin sheet-aluminium of a size that allowed the head and antennae to protrude from the narrower end. Th e wider rear end of the tube was blocked with plasticine to prevent the beetle from retreating out of the tube. Its antennae were ﬁ xed horizontally to the edge of an aluminium stand with special clamps and beeswax, so that the horizontally located large contact chemoreceptors (chaetoid taste sensilla) were visible from above under a light microscope, and easily accessible for micromanipulation from the side. Care was taken not to contaminate the taste sensilla by contact with instruments or beeswax. Each prepared test beetle was placed in a Faraday cage (15×10×5 cm) for electrophysiology.

Spikes from taste neurons of the taste sensilla were recorded by the conventional single sensillum tip-recording technique (Hodgson et al., 1955). To achieve a good signal-to-noise ratio, the indiff erent tungsten microelectrode was inserted into the base of the fl agellum free of muscular tissue. It was connected to an AC amplifi er via a calibration source, and earthed. Th e recording glass micropipette fi lled with stimulating solution was brought into contact with the tip of the sensillum by means of a micromanipulator under visual control through a light microscope at a magnifi cation of 300×. Immediately before each stimulation, 2–3 drops of solution were squeezed out of the micropipette tip by a syringe connected to the rear end of the micropipette by means of a silicone tube, and adsorbed onto a piece of fi lter paper attached to another micromanipulator, in order to

13 avoid concentration changes in the capillary tip due to evaporation. Spikes picked up by the recording electrode were fi ltered with a bandwidth set at 100–2000 Hz, amplifi ed (input impedance 10 GV, amplifi cation factor 2000×), monitored on an oscilloscope screen, and relayed to a computer via an analogue-to-digital input board DAS-1401 (Keithley, Taunton, MA, USA) for data acquisition, storage, and analysis using TestPoint software (Capital Equipment Corp., Billerica, MA, USA) at a sampling rate of 10 kHz. Responses were recorded from neurons of one (II) to fi fteen (I, III, IV, V) randomly selected sensilla on the fl agellomeres 1–9 of each test beetle. Each sensillum was stimulated once only with one solution (IV). In most cases, however, each sensillum was stimulated twice, with control (10 mmol l-1 electrolyte) and test stimulus solution, respectively. In each subsequent test beetle, the order of stimulation was reversed (I, II, III, V). During each 1 s (IV) or 5 s (I, II, III, V) stimulation period, spikes picked up by the recording electrode were counted and analysed. Responses of the sensilla to mechanical stimulation were also tested, especially in those rare cases when it was not clear whether a certain class of recorded spikes originated from the chemosensory or mechanosensory neuron. Th e tip of the sensillum was rapidly moved from its resting position toward the antennal shaft by 15–20°, held in this position for 2–3 s, and then rapidly moved back to its initial position using the recording electrode.

3.3. Chemicals and preparation of stimulating solutions

To explain the eff ect of stimulus pH to the responses of antennal chemoreceptor neurons, 100 mmol l-1 acetate and phosphate buff ers in the pH range 3–8.1 were prepared. For some experiments, the pH of the phosphate buff er was raised to 11.1 with NaOH. To produce stimulating buff ers of approximately the same pH but of diff erent concentrations of ions, the 100 mmol l-1 phosphate buff er (pH 7.6) was diluted in distilled water to 10 mmol l-1 with pH 7.8 (I). To test for the presence of the pH neuron in the antennal taste bristles of the ground beetle P. aethiops more precisely, three solutions of 100 mmol -1 + l Na -salts, Na2HPO4, NaCl and NaH2PO4 were prepared, acid, neutral and alkaline, respectively; these served as control stimuli. Test stimuli were prepared by adding small amounts of NaOH to the 100 mmol l-1 salts resulting in three electrolyte mixtures with 1 mmol l-1 NaOH and three mixtures with 10 mmol l-1 NaOH. Addition of NaOH to these salt solutions does not change their ionic composition and the Na+ concentration

14 increases only to a small extent (1% and 10%, respectively). At the same time, the pH level of these solutions rises substantially. Th e stimulating eﬀ ect of these sodium salt solutions with and without NaOH was expected to be approximately similar for the salt-sensitive neuron but not for the pH-sensitive neuron if present (Table 1, II). Th e ﬁ nal pH of the stimulating solutions was measured with a pH meter E6115 (Evikon, Estonia). Sugars (III; Table 1, IV), amino acids (Table 1, IV), alkaloids and glucosides (Table 1, V) were dissolved in 10 mmol l-1 electrolyte (KCl, NaCl, choline chloride) to give good electrical conductivity. Stimulating chemicals were kept at 5 °C for less than a week. Chemicals were obtained from AppliChem (Germany) and Sigma-Aldrich company.

3.4. Feeding bioassays (V)

Two-choice feeding experiments were carried out in 110-mm diameter glass Petri dishes. To ensure high air humidity inside the dishes, each was lined with a moistened Whatman ﬁ lter paper circle (Schleicher and Schuell, England). Two pine seeds (Pinus sibirica), treated for 20 min with distilled water (control) and 0.01 to 50 mmol l-1 quinine hydrochloride, respectively, were placed in the centre of each Petri dish, 50 mm from each other. Only seeds with seed coats removed were used in the experiments. Test beetles, one in each Petri dish, were allowed to choose between, and feed on the seeds for 24 h (20 °C, 14:10 light:dark). After 24 h, the beetles were removed and the percentage of predation on control seeds and seeds treated with alkaloid, was visually determined. At each concentration of quinine hydrochloride, the feeding preference of the beetles was tested in ﬁ ve replications (10 beetles in each).

3.5. Data analysis

Automated classiﬁ cation and counting of action potentials from taste sensilla was not an appropriate method for data analysis because two action potentials frequently coincided to produce an irregular waveform. Instead, spikes from several chemosensory neurons innervating antennal taste sensilla tested were visually distinguished by their polarity and amplitude (Figs. 1, III, IV, V), and counted, using TestPoint software. Firing rates (spikes/sec) were analysed by counting the number of spikes within 5 s after stimulus onset. Mean ﬁ ring rates and their standard errors

15 were calculated. t-test for paired samples (I, II, III,), t-test for independent samples (IV), Wilcoxon test (V) and ANOVA, Tukey test (IV) were used to determine the signifi cance of diff erences between means. Spike amplitude analysis was performed in order to explain which specifi c neuron was stimulated by the chemical classes tested (IV, V).

16 4. RESULTS

4.1. Taste sensilla on the antennal ﬂ agellum of P. oblongopunctatus and P. aethiops

Large blunt-tipped 100–200 μm long taste bristles are located in whorls in the distal part of fl agellomeres. Th ere are six on the fi rst fl agellomere and seven on the following fl agellomeres counting from the beetle’s head. In addition to those, there are six bristles symmetrically at the tip of the terminal fl agellomere. Hence, the total number of taste bristles on each antennal fl agellum is 68. All these bristles seemed to respond in a similar manner to the stimuli tested.

4.2. Spike shapes and amplitudes evoked by tested chemicals

Spike waveform analysis showed that spikes from four diff erent chemoreceptor neurons innervating antennal taste sensilla of the ground beetles P. oblongopunctatus and P. aethiops could be distinguished in the recordings obtained in response to tested chemicals. Spikes with negative polarity, 1–2.5 mV in amplitude, arose from the salt-sensitive neuron. Th is neuron generated spikes in a phasic-tonic manner and had an extremely short response latency lasting less than 10 ms (Fig. 1, III; Fig. 1, V). Two-tipped spikes with positive polarity, 2–8 mV in amplitude, were generated by the pH-sensitive neuron (Fig. 1, I, III; Figs. 1 and 2, IV). Compared to the spikes from the salt-sensitive neuron, these positive spikes appeared with a much longer delay (Fig. 1, II; Fig. 1A, I, V). Th e shape of these impulses is complex because they are composed of two separate electrical events in quick succession and most probably produced by two separate spike generation sites of the same neuron. Minor diff erences in the time interval between these two events occuring among antennal taste sensilla may cause considerable diff erences in the shape of the resulting double- spike. Usually, the double-spikes of the pH-sensitive neuron described are two-tipped (Fig. 2A and B, IV). In some sensilla, however, only a small characteristic notch or discontinuity in the rising phase of these impulses indicates their complex nature (Fig. 2C and D, IV). Th e sugar-sensitive neuron also produced spikes with negative polarity, but smaller in amplitude than those of the salt-sensitive neuron (Fig. 1B, III, IV; Fig. 1J, V). Th e fourth chemoreceptor neuron of the taste sensillum was stimulated by some alkaloids. Th e negative spikes it produced were variable in size

17 (Fig.1 B–E, V) but diﬀ erent from those originating from the salt- and sugar-sensitive neurons (Fig. 2, V).

Impulse activity of the mechanosensory neuron belonging to these taste sensilla and responding to bending of the bristle was recorded from some intact bristles as well as from bristles with a broken tip. Spikes produced by the mechanosensory neuron were small but variable in size, and negative in polarity (Fig. 1C and D, IV).

4.3. Eﬀ ect of stimulus pH to the responses of the electrolyte- sensitive chemoreceptor neurons innervating antennal taste sensilla of P. aethiops and P. oblongopunctatus

Two chemoreceptor neurons from fl agellar taste bristles of P. aethiops and P. oblongopunctatus may respond to stimulation with various electrolyte solutions in a phasic-tonic manner. According to their response characteristics these neurons were classifi ed as salt- (cation-) and pH-sensitive neuron distinguished by the amplitude, shape and polarity of spikes they generated (Fig. 1, II). Th e phasic component of the response was more pronounced in the salt-sensitive neuron compared to that of the pH-sensitive neuron (Figs. 2 and 3, II). Due to large variations in responsiveness of the pH-sensitive neuron from diff erent taste bristles and diff erent samples of beetles to the same stimulus (Fig. 4, A-cell, I), stimulating solutions were tested in pairs to allow a more precise comparison of the responses to the tested stimuli.

4.3.1. Response to 100 mmol l-1 acetate buﬀ ers at pH 3–6

Th e pH-sensitive neuron did not respond to acetate buff er at pH 3 and only a very weak response was observed at pH 4; fi rst second response below 1 spikes/s (Fig. 2A, A-cell, I). Th e number of spikes generated by the salt-sensitive neuron at pH 3 and 4 was much higher, fi rst second responses 5.7 and 9.5 spikes/s, respectively (Fig. 2A, B-cell, I). Accord- ing to how much the pH level of the stimulating buff ers increased further, more spikes by the pH- and salt-sensitive neurons were produced. However, even at pH 6, the mean fi ring rate of the pH-sensitive neuron remained below 5 spikes/s while that of the salt-sensitive neuron reached 23 spikes/s (Fig. 2B,C, I).

18 4.3.2. Response to 100 mmol l-1 phosphate buﬀ ers at pH 5.8–8.1 (11.1)

In most cases, phosphate buff ers were remarkably more stimulating for the pH-sensitive neuron compared to acetate buff ers; at pH 5.8, the neuron produced a mean of 8.1–10.5 spikes/s (Fig. 4A,B, A-cell, I). When buff er pH was increased from 5.8 to 7, the fi rst second fi ring rate of the pH-sensitive neuron increased by 50%, from 10.5 to 15.8 spikes/s. Even larger increases in the response, reaching up to 300–800%, were observed during next four seconds of the response after stimulus onset (Fig. 4A, A- cell, I). In contrast, the response of the salt-sensitive neuron to these two phosphate buff ers was approximately equal (Fig. 4A, B-cell, I). However, the salt-sensitive neuron responded to the larger jumps in stimulus pH. For example, when buff er pH was increased by three pH units, from 8.1 to 11.1, both of the neurons responded with signifi cant fi ring rate increase whereby relative change in the response of the pH-sensitive neuron was considerably larger compared to that of the salt-sensitive neuron, the fi r- ing rate increased during the fi rst second of the response by 1350% and 25%, respectively (Fig. 4C, A- and B-cell, respectively, I).

4.3.3. Response to 100 mmol l-1 Na+-salts with and without addition of 10 mmol l-1 NaOH

Th e limitation of the buff er series method used for testing the responses of the chemoreceptor neurons to stimulus pH is that buff ers with diff erent pH vary largely in both pH and ionic concentrations of the component salts. Th e method of alkalized salts reported here allows the preparation of stimulating salt solutions over a wide range of pH whereas changes in ionic concentrations of the component salts are minimal.

Remarkable diff erences between the pH- and salt-sensitive neurons regarding their responsiveness to the pH of the stimulating salt solutions were demonstrated by electrophysiological experiments with alkalized and non-alkalized salts. In the pH-sensitive neuron, rates of fi ring evoked by the mixture of 100 mmol l-1 Na+-salts and 10 mmol l-1 NaOH were signifi cantly higher compared to those caused by the salts alone (Fig. 3, II). Th e stimulating eff ect of NaOH to the pH neuron was highest in the mixture with NaCl (pH 9.6), fi ring rate during the fi rst second of the

19 response was 31 spikes/s and it did not fall below 17 spikes/s also during the next several seconds recorded. Th e diff erence compared to the response caused by 100 mmol l-1 NaCl alone (pH 7.6) was 10–22 times depending on the response time. Th e mixtures of 10 mmol l-1 NaOH with 100 -1 mmol l NaH2PO4 (pH 5.8) and Na2HPO4 (pH 10.6) were 2–7 times more eff ective stimuli for the pH-sensitive neuron compared to these non- alkalized salts (pH 4.6 and 8.6, respectively). In contrast, the presence or absence of 10 mmol l-1 NaOH in the stimulating salt solution had only little or no eff ect on the spike production of the salt-sensitive neuron.

4.4. Plant sugar sensitivity in ground beetles

4.4.1. Electrophysiological identiﬁ cation of the sugar-sensitive neuron in antennal taste sensilla of P. aethiops

In the ground beetle P. aethiops, by single sensillum tip recording technique, in addition to the salt- and pH-sensitive neurons, the third chemoreceptor neuron of the antennal taste sensilla was electrophysiologically identifi ed as a sugar-sensitive neuron. Th is neuron generated spikes of considerably smaller amplitude than those of the salt- and pH-sensitive neurons (Fig. 1B, III), and phasic-tonically responded to sucrose and glucose over the range of 1–1000 mmol l-1 tested. Th e responses of the sugar- sensitive neuron to stimulating solutions containing glucose or sucrose were signifi cantly stronger compared to the control solution (p<0.05, t-test for paired samples; Figs. 2A and 3A, III). Responses were concentration dependent, with sucrose evoking more spikes than glucose. During the fi rst second of the response, mean rates of fi ring of the sugar-sensitive neuron reached up to 19 and 37 spikes/s when stimulated with 1000 mmol l-1 glucose and sucrose, respectively. Th ereafter, the rates of fi ring quickly fell below 5 spikes/s (sucrose), or close to zero (glucose).

In contrast, the responses of the salt-sensitive neuron were not signifi cantly aff ected by the glucose content of the stimulating solutions (p>0.05, t-test for paired samples; Fig. 2B, III). It was observed, that sucrose had a little stimulating eff ect to the fi ring rate of the neuron, however (p<0.05, t-test for paired samples; Fig. 3B, III). Th e responses of the pH-sensitive neuron were also aff ected to a small degree by the content of sucrose and glucose in the stimulating solutions, but in a more complicated manner. Firing rate of the neuron increased to some degree at low concentrations (1 and

20 10 mmol 11) of the sugars (p<0.05, t-test for paired samples). At higher concentrations (100 and 1000 mmol l-1) of the sugars the opposite was observed, however: ﬁ ring rates of the pH-sensitive neuron were slightly suppressed by both glucose and sucrose compared to the responses evoked by 10 mmol l-1 KCl (control) alone (p<0.05, t-test for paired samples; Figs. 2 and 3, III).

4.4.2. Electrophysiological responses of the chemoreceptor neurons from antennal taste sensilla of P. oblongopunctatus to plant sugars and amino acids

Choline chloride at a concentration of 10 mmol l-1 served as the control stimulus, and as the electrolyte in stimulating mixtures with sugars and amino acids to ensure a good signal-to-noise ratio. Typically, choline chloride evoked only the salt-sensitive neuron to discharge with a relatively low rate of ﬁ ring (approximately 15 spikes/s) during the ﬁ rst second after stimulus onset (Fig. 3A, IV). Only in rare cases were a few spikes from other sensory neurons of the sensillum observed (Fig. 1A; Table 1, IV). Th erefore, the stimulating properties of choline chloride were suitable for classifying and counting action potentials of the responses evoked by the mixtures of this electrolyte with sugars and amino acids.

4.4.2.1. Response of antennal taste neurons to sugars

Responses of antennal taste neurons to stimulation by various 100 mmol l-1 sugars in mixtures with 10 mmol l-1 choline chloride, varied with the sugar tested. Only three out of twelve sugars, maltose, sucrose and glucose, evoked strong phasic-tonic responses from the sugar-sensitive neuron (Fig. 3B–E; Table 1, IV). Th e mean rates of fi ring of the neuron during the fi rst second after 100 mmol l-1 stimulation reached 49.9, 46.5 and 20.9 spikes/s, respectively. Dose/response curves of these three active sugars are demonstrated in Fig. 4 (IV). In the range of 1–1000 mmol l-1, the response to glucose was approximately two times lower than that of maltose and sucrose (p<0.05, ANOVA, Tukey test). In contrast, no differences were found between responses to maltose and sucrose (p>0.05, ANOVA, Tukey test). Th e remaining nine 100 mmol l-1 sugars had only a small or no stimulatory eff ect on the neuron. To test for possible seasonal variation in the functioning of the sugar-sensitive neuron, its responses

21 to 100 mmol l-1 sucrose were compared in active and hibernating beetles in July (46.5 spikes/s) and February (48.8 spikes/s), respectively but no diﬀ erences between their responses were observed (t = -0.57; d.f. = 18; p= 0.57; t-test for independent samples).

4.4.2.2. Response of antennal taste neurons to amino acids

Responses of antennal taste neurons to the seven 10 and 100 mmol l-1 L-amino acids in a mixture with 10 mmol l-1 choline chloride varied with the type and concentration of amino acid tested. At 10 mmol l-1, none of the amino acids stimulated the neurons. At 100 mmol l-1, these chemicals evoked only a few spikes (Table 1, IV) with amplitudes smaller than those generated by the salt-sensitive neuron (Fig. 3G–I, IV). No initial phasic component in the sequence of these spikes was observed. Frequently, they appeared with some delay in the responses, sometimes 5–10 s after stimulus onset (Fig. 3J, IV). Spike amplitude analysis showed that these small impulses were generated by the 4th chemosensory neuron rather than by the sugar-sensitive neuron. Th ough amplitudes of spikes induced by sugars (sucrose) and amino acids varied and overlapped to a large extent their histograms had diﬀ erent maxima (Fig. 5, IV), indicating that two neurons may be involved. Since the mean numbers of these small spikes during the ﬁ rst second after stimulus onset did not exceed 3 spikes/s, this neuron has probably no specialized receptor sites for amino acids, at least for those which were tested.

4.5. Response of antennal taste neurons to plant alkaloids and glucosides

4.5.1. Electrophysiological identiﬁ cation of the alkaloid-sensitive neuron in antennal taste sensilla of P. oblongopunctatus

Some tested alkaloids (quinine, caﬀ eine) and an alkaloid salt (quinine hydrochloride) induced a neuron of the taste sensilla to produce spikes with negative polarity, but with a considerably longer period of latency compared to the spikes of the salt- and sugar-sensitive neurons (Fig. 1B–E, V). Spike amplitude histograms show two maxima, indicating that spikes evoked by salt (NaCl), sugar (sucrose) and alkaloid (quinine hydrochloride) were generated by three diﬀ erent neurons, however, the salt-, sugar- and

22 alkaloid-sensitive neuron, respectively (Fig. 2, V). Spike amplitudes of the chemoreceptor neurons from diﬀ erent taste sensilla varied to a some extent, from 0.7 to 2 mV (Fig. 1C,E, V). As a result, in most cases, spike amplitudes generated by the alkaloid-sensitive neuron were shorter than those of the salt-sensitive neuron (Fig. 1B,C, V). In some cases, however, opposite spike amplitude ratios of the neurons were observed (Fig. 1D, V).

4.5.2. Stimulating eﬀ ect of plant alkaloids and glucosides on the responses of the antennal chemoreceptor neurons

Only two of the tested chemicals (Table 1, V), 1 mmol l-1 quinine and 10 mmol l-1quinine hydrochloride, were highly stimulating for the antennal alkaloid-sensitive neuron, evoking 8.9 and 22.8 spikes/s, respectively. Th e stimulatory eff ect of 10 mmol l-1 caff eine was very weak, 2.4 spikes/s. In contrast, a slight inhibiting eff ect of strychnine and sinigrin to the activity of the neuron was observed. Respective fi ring rates were very low, however, below 2 spikes/s (Table 1, V). Other plant chemicals tested had little eff ect on spike production of the alkaloid-sensitive neuron. Phasic-tonic response of the alkaloid-sensitive neuron to quinine hydrochloride was concentration dependent over the range of 0.001 to 50 mmol l-1 with the response threshold at 0.01 mmol l-1, and maximum rate of fi ring equal to 67 spikes/s at 50 mmol l-1 (Figs. 3 and 4, V). However, compared to the spike trains of the salt-sensitive neuron (Figs. 1 and 5A–F, V), the phasic component of the response produced by the alkaloid-sensitive neuron was less pronounced (Fig. 1B–E; Fig. 4; Fig. 5C–F, V).

4.5.3. Peripheral inhibition of the salt- and pH-sensitive neurons by some plant secondary compounds

23 close to zero during the 5 s adaptation period (Fig. 4, V). In response to 10 mmol l-1 NaCl alone, the mean number of spikes produced by the pH- sensitive neuron was relatively low compared to that of the salt-sensitive neuron, and varied to some extent in diff erent sensilla (Table 1, V). At quinine hydrochloride concentrations above 0.01 mmol l-1, the mean fi ring rate of the pH-sensitive neuron dropped to zero (Fig. 3, V). It was also observed that nicotine and salicin, which did not aff ect the fi ring rate of the alkaloid-sensitive neuron, greatly reduced the response of the pH-sensitive neuron (Table 1, V). Sinigrin and caff eine suppressed neural activity of the salt- and pH-sensitive neuron, respectively. Th is suggested that the antennal taste sensilla of P. oblongopunctatus may detect plant defensive compounds not only through the activation of specifi c alkaloid-sensitive neuron but also through inhibition of the salt- and pH-sensitive neurons.

4.5.4. Eﬀ ect of quinine hydrochloride on the feeding response of P. oblongopunctatus

Feeding choice experiments showed that one of the most stimulating chemicals for the alkaloid-sensitive neuron, quinine hydrochloride, had a concentration dependent deterrent eﬀ ect on seed predation in the ground beetle P. oblongopunctatus, with the threshold concentration at 1 mmol l-1. Th us, the threshold concentration of this alkaloid to evoke a behavioural response was a hundred times higher than that for an electrophysiological response of the alkaloid-sensitive neuron. At higher concentrations, the deterrent eﬀ ect of quinine hydrochloride was very strong. It was observed that pine seeds treated with 50 mmol l-1 quinine hydrochloride were never predated by the beetles (Fig. 6, V).

24 5. DISCUSSION

5.1. pH sensitivity in ground beetles (I, II)

5.1.1. Th e occurrence of two chemoreceptor neurons in insect contact chemoreceptors responding to salts

In a number of insects, including the ground beetles P. oblongopunctatus (I) and P. aethiops (Merivee et al., 2004; II) frequently two neurons of the taste sensilla respond to salt solutions (Dethier and Hanson, 1968; Haskell and Schoonhoven, 1969; Den Otter, 1972; Mitchell and Schoon- hoven, 1973; Mitchell and Seabrook, 1973; Blaney, 1974; Hanson, 1987; Bernays and Chapman, 2001a; Schoonhoven and van Loon, 2002). It is suggested that they function as a cation and an anion neuron (Ishikawa, 1963; Elizarov, 1978; Hanson, 1987; Schoonhoven and van Loon, 2002). Temporal patterns of fi ring and the concentration/response curves are quite diff erent for the two neurons. In the cation neuron with a phasic- tonic type of reaction, the magnitude of the response is proportional to the logarithm of salt concentration over the dynamic range (Evans and Mellon, 1962; Hanson, 1987). It is believed that the stimulating eff ect of salts to taste receptors is dominated by the cations involved, and that monovalent cations are more eff ective stimuli than divalent cations (Evans and Mellon, 1962; Schoonhoven and van Loon, 2002). Th is neuron type corresponds to the salt-sensitive neuron of the antennal taste bristles in Pterostichus (Merivee et al., 2004).

Specifi c stimuli for the second salt neuron are yet to be clarifi ed: it has been named the ‘anion neuron’ on the basis of a supposed sensitivity to salt anions or the ‘fi fth neuron’ (Ishikawa, 1963; Dethier and Hanson, 1968; Hanson, 1987; Liscia et al., 1997; Liscia and Solari, 2000). Th is neuron is characteristically less responsive to diff erences in salt concentration (Dethier, 1977; Liscia et al., 1997), similar to that observed in the pH-sensitive neuron of P. oblongopunctatus. In the strict sense of the term, it is not correct to give the name ‘salt neuron’ to a neuron that does not respond to changes in salt concentration. On the other hand, a study in Grammia geneura shows increasing activity in two neurons in response to potassium and sodium chlorides at concentrations ranging from 10 to 1000 mmol l-1 (Bernays and Chapman, 2001a). Dethier (1977) was the fi rst to realize that it is not at all certain that the best stimulus for the second salt neuron is indeed salt. Electrophysiological evidence is provided that

25 the so-called ‘ﬁ fth’ neuron in taste chemosensilla of blowﬂ ies responds to deterrent compounds, such as quinine, amiloride, nicotine and caffeine (Liscia and Solari, 2000). Very little attention has been paid to this neuron. Th is is unfortunate because this lack of information handicaps any attempt to construct an overview of the control of food detection and habitat selection in insects on the basis of their chemosensory input.

5.1.2. Th e eﬀ ect of stimulus pH to the salt- and pH-sensitive neurons in antennal taste sensilla of P. aethiops and P. oblongopunctatus

Results of the electrophysiological experiments conducted with acid, neutral and alkaline salts (Merivee et al., 2004), acetate and phosphate buff ers (I), and alkalized and non-alkalized salts (II) demonstrated that, in addition to the salt-sensitive neuron, the existence of a pH-sensitive neuron in antennal chaetoid taste sensilla of the ground beetles P. aethiops and P. oblongopunctatus is possible. A strong relationship between stimulus pH and spike production by the antennal pH-sensitive neuron was found: signifi cantly higher rates of fi ring were recorded at higher pH values compared to those observed at lower pH of the electrolyte solutions tested at pH ranged from 3 to 11. However, the methods of various salts and buff ers used to test the eff ect of pH on contact chemoreceptor neurons in ground beetles have limitations in that these stimuli with diff erent pH vary largely in both pH and ionic concentration of the component salts leaving the exact stimulating role of these two factors for the neurons open (Merivee et al., 2004; I). Th erefore, a method of alkalized (with NaOH) Na+ salts was developed allowing preparation of stimulating solutions over a wide range of pH with no diff erences in their ionic composition and minimal diff erences in their salt concentration. Using this method for stimulation of antennal contact chemoreceptors in P. aethiops (II), 2.5–10-fold increase in the fi rst second fi ring rate of the pH-sensitive neuron in response to the mixture of various 100 mmol l-1 salts and 10 mmol l-1 NaOH (pH was observed (4–30 spikes/s) compared to that of 100 mmol l-1 NaCl alone (1–8 spikes/s). Th us, the high stimulating eff ect of alkalized salts to the pH-sensitive neuron could be attributed to pH alone as it was increased by 1.3 to 2.0 pH units depending on the salt. In comparison, fi ring rate of the salt-sensitive neuron changed by only 16–27%.

26 If the response of the pH-sensitive neuron depends on solution pH level only, there should be equivalent responses to buff ers at diff erent concentrations at the same pH. However, the results do not confi rm this: 10 mmol l-1 phosphate buff er had a greater stimulating eff ect on the pH-sensitive neuron of P. oblongopunctatus than 100 mmol l-1 buff er (I). Th is phenomenon can be explained by interaction of diff erent chemical stimuli to chemosensory neurons. Other chemicals, as well as the pH of the stimulating solution, may infl uence the responses of insect taste receptors to one or another stimulating compound (Hanson, 1987; Liscia et al., 1997; Bernays et al., 1998; Liscia and Solari, 2000; Bernays and Chapman, 2001b; Schoonhoven and van Loon, 2002). Presumably, salts of stimulating buff er solutions at high concentration suppressed the neural activity of the pH-sensitive neuron when stimulated with 100 mmol l-1 phosphate buff er in 10 and 100 mmol l-1 phosphate buff er experiments in P. oblongopunctatus.

5.1.3. pH preference in ground beetles

Preferences for particular soil pH, varying between 3 and 9, in ground beetles were discovered using laboratory choice experiments some decades ago (Krogerus, 1960; Paje and Mossakowski, 1984). More recently, in ecological fi eld experiments, a correlation between the abundance of some ground beetles and soil pH has been found (Irmler, 2001, 2003). Our results suggest that in P. aethiops and P. oblongopunctus in their preferred acid forest habitats and overwintering sites in brown-rotted wood at pH 3–5, the antennal pH-sensitive neuron does not discharge or discharges at very low frequency with the fi rst s fi ring rate close to 1 spike/s or lower. Areas with a higher pH seem to be unfavourable to this insect and when contacted the pH-sensitive neuron signals with a stronger response. Th is may occur, for example, in places with decaying plant material on the soil surface. Many of the materials composted contain signifi cant amounts of protein, which is converted to ammonia toxic to many organisms and serving as the primary substrate in the nitrifi cation processes. Nitrifi cation processes have been observed at pH levels ranging from 6.6 to 9.7 (Odell et al., 1996). Obviously, these ground beetles avoid places with a high pH.

Adults of P. aethiops and P. oblongopunctus may spend seven to eight months, from September to April, in their preferred overwintering sites in

27 brown-rotted wood in Estonia. Fungi often lower the pH of the extracellular medium through secretion of organic acids and thus establish a pH gradient around the mycelium. Th e organic acid oxalate is produced by most, if not all, wood-degrading fungi. Usually, a pH of 3–6 is observed in both white- and brown-rotted wood. Stable moisture content is quaranteed as a result of metabolic water production during the wood-degrading process. For cultures of the white-rot fungus Merulius tremellosus incubated in air, the optimum water content was 2 g/1 g of wood. Moister conditions were less favourable to delignifi cation, possibly because the water impeded aeration (Reid, 1985; Varela and Tien, 2003). Similar prolonged stability of water content and aeration is probably also quaranteed in brown-rotting wood which seems to be a favourable microenvironment of ground beetles for their prolonged hibernation. Brown-rotted wood may off er good protection for hibernating ground beetles against entomopathogenic fungi and parasites which play an important role in their population dynamic. Up to 41% parasitism by nematodes and ectoparasitic fungi was found on 14 species of Bembidion in Norway depending on habitat selection of the host (Andersen and Skorping, 1991). Eggs of P. oblongopunctatus suff ered 83% mortality in fresh litter but only 7% in sterilized soil (Hees- sen, 1981). Although predators, parasites, and pathogens aff ect all ground beetle developmental stages, quantitative data remain scarce. So, antennal pH receptors of the ground beetles seem to be a powerful tool in their selection for favourable acid habitats and microhabitats crucial to survival.

5.2. Sensitivity of ground beetles to plant sugars and amino acids (III, IV)

5.2.1. Response of the chemoreceptor neurons from antennal taste sensilla to sugars

Th ree out of the twelve sugars tested in P. aethiops and P. oblongopunctatus, maltose, sucrose and glucose, evoked strong responses from the sugar- sensitive neurons while the others had little or no eﬀ ect. Most stimulating were the two disaccharides, maltose and sucrose (III, IV), and this is in agreement with results for a number of other insect species (Mitchell and Gregory, 1979; Schoonhoven and van Loon, 2002; Albert, 2003; Sandoval and Albert, 2007). Sucrose is a disaccharide consisting of two monosaccharides, α-glucose and β-fructose, joined by a glycosidic bond between carbon atom 1 of the glucose unit and carbon atom 2 of the fructose unit.

28 Maltose is composed of two molecules of glucose joined together through α-1.4-glycoside linkage. Th us, the presence of a terminal α-glucose unit seems to be a common and important feature of the molecular structure determining the stimulatory properties of these disaccharides in Pteros- tichus. Other monosaccharide units in the molecular structure of sucrose and maltose were of no importance in this respect as the sugar-sensitive neuron responses to these two disaccharides were similar. In contrast, the trisaccharide raffi nose has a non-terminal α-glucose unit and does not evoke a response from the antennal sugar-sensitive neuron in this species (IV). In the red turnip beetle, Entomoscelis americana, maltose (α-glucose-1.4-glucose) was at least an order of magnitude more eff ective in activating the maxillary sugar-sensitive neuron than trehalose (α-glucose- 1.1-glucose) illustrating the importance of the 1.4 linkage in these disaccharide molecules (Mitchell and Gregory, 1979). Compared to maltose and sucrose, the third active sugar glucose was approximately two times less eff ective at stimulating the antennal sugar-sensitive neuron of both P. oblongopunctatus and P. aethiops (III, IV). Th is can be partly explained by the fact that glucose exists in at least two anomeric forms, α and β. Th ese two forms interconvert over a timescale of hours in aqueous solution, to a fi nal stable ratio of α:β 36:64, in a process called mutarotation. So the physiologically active α-anomere forms only 36% of the glucose solution which is refl ected in its relatively low dose/response curve. Th e order of stimulatory eff ectiveness of sugars may vary in diff erent insect species or even be diff erent for receptors on various parts of the body (Blaney, 1974; Rüth, 1976; Bernays and Chapman, 2001; Schoonhoven and van Loon, 2002).

No insect so far investigated responds to all sugars, and no single sugar is a major phagostimulant for all insects. For a given insect species, only some sugars elicit a response to a greater or lesser extent while other sugars elicit no response. Plant sugars which were found to evoke responses from the antennal contact chemoreceptors in P. oblongopunctatus, i.e., maltose, sucrose and glucose (IV), function as strong phagostimulants for most phytophagous insects, ﬂ ies, cockroaches and others, equipped with specialized receptors to detect sugars (Dethier et al., 1956; Blaney, 1974; Rüth, 1976; Amakawa, 2001; Liscia et al., 2002; Schoonhoven and van Loon, 2002; Chapman, 2003; Sandoval and Albert, 2007). Th is suggests that these sugars may have the same function in P. oblongopunctatus, probably due to its partial herbivory. Although, in contrast to sucrose and glucose, maltose is uncommon in nature, it can be found in considerable

29 amounts in germinating seeds as a product of the enzymatic degradation of starch. Obviously, high sensitivity of P. oblongopunctatus to maltose is related to predation by the species on conifer seeds (Heikkilä, 1977; Nystrand and Granström, 2000).

Several carbohydrates, in addition to glucose, such as cellobiose, arabinose, xylose, mannose, rhamnose and galactose are known as components of cellulose and hemicelluloses. Th ey are released by brown-rot fungi during enzymatic wood decay. All water-soluble carbohydrates are ultimately consumed, so that brown-rotted wood consists almost entirely of modiﬁ ed lignin. Brown-rot fungi appear to depolymerize wood in the early stages of decay much more rapidly than the decay products can be metabolized. Th e occurrence of excess wood decomposition products may help explain the frequent presence of other wood scavengers in brown-rotted wood (Eriksson et al., 1990; Zabel and Morrell, 1992; Valášková and Baldrian, 2006). As wood decomposes, it passes through a range of decay stages, each colonized by a succession of diverse saproxylic insect assemblages (Grove, 2002). Wood-decay products may serve as chemical cues for these saproxylic insects and be involved in this colonization process although no electrophysiological or behavioural evidence is yet available. Some ground beetles, including P. oblongopunctatus, prefer brown-rotted wood for their prolonged hibernation (Haberman, 1968; Lindroth, 1986). Our results showed, however, that P. oblongopunctatus was not able to sense water-soluble wood sugars released by brown-rot fungi, except for glucose (IV), indicating that these chemicals are not involved in the search for hibernation sites by this species, although others may be.

5.2.2. Response of the chemoreceptor neurons from antennal taste sensilla to amino acids

Since amino acids, in addition to sugars, stimulate feeding behaviour in various phytophagous insects these animals have amino acid-speciﬁ c neurons in their taste sensilla. Amino acids and sugars may stimulate the same neuron. Such a versatile receptor neuron is present in the polyphagous caterpillar of Grammia geneura. Th ese insects have, in addition to an amino acid sensitive neuron in their lateral sensillum styloconicum, a neuron in another sensillum which responds to seven (out of twenty) amino acids. Th is latter neuron can also be stimulated by sucrose, glucose, and trehalose (Schoonhoven and van Loon, 2002; Chapman, 2003). Because

30 of its multiple specifi city this neuron was named a ‘‘phagostimulatory neuron’’, rather than a ‘‘sugar’’ or ‘‘amino acid’’ neuron (Bernays and Chapman, 2001). However, in P. oblongopunctatus, the seven amino acids tested (IV) had only little or no stimulatory eff ect on the neurons of the antennal taste sensilla suggesting that these neurons have no ability to perceive these amino acids. Th e weakly stimulatory eff ect (below 3 spikes/s) of some 100 mmol l-1 amino acids on the 4th chemosensory neuron of these sensilla can be characterized as non-specifi c, or modulating to the responses of non-target chemosensory neurons, the phenomenon well known in insect chemoreceptor functioning (Hanson, 1987; Liscia et al., 1997; Liscia and Solari, 2000; Bernays and Chapman, 2001; Chapman, 2003; Schoonhoven and van Loon, 2002; I; III).

5.3. Response of the chemoreceptor neurons in the antennal taste sensilla of P. oblongopunctatus to plant secondary compounds

5.3.1 Stimulating eﬀ ect of plant alkaloids and glucosides on the responses of the antennal chemoreceptor neurons

Th e experimental data presented show that the “fourth” chemoreceptor neuron in the antennal taste sensilla of the ground beetle P. oblongopunctatus (IV) may respond to some plant alkaloids and alkaloid salts such as quinine and quinine hydrochloride (V). Th is ﬁ nding seems to be in agreement with literature data on the feeding habits of this species. P. oblongopunctatus is one of the most common and widespread ground beetles in the forests of northern Europe (Lindroth, 1985; Niemelä et al., 1994). In most stands of Pinus sylvestris, seed predation results in 20% seed mortality, although occasionally it reaches 60%. Post-dispersal pine seed predation in the Swedish boreal forests studied was due mainly to the two ground beetle species P. oblongopunctatus and Calathus micropterus. Both are able to consume conifer seeds. Th e larger species, P. oblongopunctatus, is considered to be the more important pine seed predator of the two (Heikkilä, 1977; Nystrand and Granström, 2000).

31 of defensive compounds has favoured specialization in seed-predators and helps to explain why a large proportion of them are specialist feeders (Hulme and Benkman, 2004). Th e omnivorous ground beetle P. oblongopunctatus predates on many small animals, most frequently on Coleoptera, Diptera, Collembola, Acari etc. (Larochelle, 1990), as well as on conifer seeds (Heikkilä, 1977; Nystrand and Granström, 2000). Probably, the species feeds on seeds of various plants but respective data are not available. Selective seed predation has been observed in many other ground beetles, however (Toft and Bilde, 2002; Tooley and Brust, 2002).

To discriminate between toxic and nontoxic seeds, P. oblongopunctatus possesses a specifi c feeding deterrent neuron innervating its antennal chaetoid taste sensilla (V). A strong stimulatory eff ect of quinine (quinoline group) and quinine hydrochloride to this neuron was observed. Th e threshold concentration for the latter at 0.01 mmol l-1 was close to the upper limit of the threshold concentrations for the plant secondary compounds that stimulate a deterrent neuron in other herbivorous insects (Schoonhoven and van Loon, 2002; Chapman, 2003; Schoonhoven et al., 2005), probably due to the fact that toxin concentration in seeds is usually much higher than in other parts of the plant (Janzen, 1971; Hulme and Benkman, 2004; Davis et al., 2008). Th e response of the neuron to quinine hydrochloride was concentration dependent with maximum rates of fi ring reaching up to 70 spikes/s at 50 mmol l-1. In two-choice behavioural experiments, quinine hydrochloride had a strong concentration dependent deterrent eff ect on feeding of P. oblongopunctatus beetles with the response threshold at 1 mmol l-1. Th us, the threshold concentration of this alkaloid to evoke a behavioural response was a hundred times higher than that for an electrophysiological response of the alkaloid-sensitive neuron. In contrast to quinine hydrochloride, the response of the alkaloid-sensitive neuron to caff eine (purine group) was very weak, only 2.4 spikes/s at 10 mmol l-1 concentration. Th e eff ect of another quinoline alkaloid, strychnine, and sinigrin, to the neuron was rather inhibitory. No response of the neuron to tested pyrrolidine alkaloid, nicotine, and salicin (glucoside) was observed suggesting that the antennal alkaloid-sensitive neuron of P. oblongopunctatus is not equally sensitive to all plant defensive chemicals. Nevertheless, the list of toxic compounds capable of stimulating the antennal alkaloid-sensitive neuron of P. oblongopunctatus is likely to grow longer as more plant defence chemicals are tested (V).

32 Quinoline alkaloids are among the most potent insect feeding deterrents occurring mainly in Rutaceous and Rubiaceous plants. Th ey can be present in virtually every part of the plant or just a speciﬁ c part like the bark, rhizome, leaf or seed (Openshaw, 1967; Michael, 2003; Aniszewski, 2007). Th ese alkaloids represent a large and structurally diverse group of plant defensive compounds. When contacted, some of them may be detected by stimulation of a speciﬁ c antennal alkaloid-sensitive neuron of the ground beetles predating on seeds, similarly to the quinine-sensitivity of P. oblongopunctatus. Th e ability of granivores to perceive plant defensive compounds allows them to avoid toxic seeds, and survive. High sensitivity of P. oblongopunctatus to plant sugars, commonly known as strong phagostimulants for most phytophagous insects, seems to be also related to its partially granivorous feeding habits. Th e disaccharide maltose was one of the most stimulatory sugars for the sugar-sensitive neuron also innervating antennal taste sensilla of P. oblongopunctatus (IV).

5.3.2. Peripheral inhibition of the salt- and pH-sensitive neurons by some plant secondary compounds

While stimulating the alkaloid-sensitive neuron, quinine hydrochloride strongly reduced spike production by the salt- and pH-sensitive neurons which also innervate these sensilla in Pterostichus spp. (Merivee et al., 2004; I, II). Increasing the quinine hydrochloride concentration, while keeping the concentration of NaCl constant, caused a dose-dependent decrease in spike generation by the salt-sensitive neuron. At 10 mmol l-1 and higher concentrations of quinine hydrochloride, predominant fi ring of the alkaloid-sensitive neuron was observed while the fi ring rate of other neurons dropped almost to zero. It was also observed that several tested plant secondary compounds, salicin, sinigrin, caff eine and nicotine, which had only little or no eff ect on the fi ring rate of the alkaloid-sensitive neuron, would greatly reduce the responses of the salt- and pH-sensitive neurons of the antennal taste sensillum of P. oblongopunctatus. Th ese results suggest that the antennal taste sensilla in P. oblongopunctatus may detect toxic and deterrent plant secondary compounds not only through activation of the specifi c alkaloid-sensitive neuron but also through inhibition of taste neurons activated by salts and stimulus pH; this is similar to deterrent inhibition of taste neurons activated by sugars and water in Drosophila (Meunier et al., 2003). Since foodplant acceptability to herbivores depends on the balance between stimulation and deterrency, similar interactions

33 between stimulating chemicals at peripheral taste neurons of herbivorous insects are of widespread occurrence. Plant toxins and deterrents may inhibit sugar neurons (Frazier, 1986; van Loon, 1990; Messchendorp et al., 1996; Bernays and Chapman, 2000; Schoonhoven and van Loon, 2002; Meunier et al., 2003; Schoonhoven et al., 2005), water neurons (Meunier et al., 2003), and sugars and salts may inhibit deterrent neurons (Simmonds and Blaney, 1983; Shields and Mitchell, 1995; Glendinning et al., 2000; Schoonhoven and van Loon, 2002; Schoonhoven et al., 2005). Deterrent compounds that on their own do not stimulate any neuron within a sensillum may also decrease the responsiveness of a neuron responding to a nutrient, as exempliﬁ ed by sinigrin inhibiting the inositol neuron in Heliothis virescens and H. subﬂ exa (Bernays and Chapman, 2000; Schoonhoven and van Loon, 2002).

Survival of most terrestrial animals requires ability to detect environmental NaCl, a nutrient essential for fl uid and electrolyte homeostasis and for a multitude of other physiological processes (Lindemann, 1996; Contreras and Lundy, 2000). Th e gustatory system allows insects to detect and ingest salt, to discriminate between diff erent salts, and to avoid high salt concentrations. However, there are only few data that relate input from inorganic salts to feeding behaviour in phytophagous insects. In some insects, salts have a deterrent eff ect on feeding (Chapman, 2003; Schoonhoven and van Loon, 2002). In contrast, larvae of Drosophila show dose-dependent responses to NaCl: the appetitive responses to low concentration gradually turn into aversion as concentration is increased up to 200 mmol l-1 and higher (Miyakawa, 1982; Liu et al., 2003; Niewalda et al., 2008). Some other insects also respond positively to dilute salts. Cockroaches and housefl ies prefer dilute sodium and potassium chlorides and refuse distilled water. In the two-choice situation, many animals give a bimodal response to sodium chloride. Acceptance at low concentrations switches to rejection at high concentrations (Dethier, 1977). In these cases, inorganic salt at low concentration may act as a phagostimulant. Th erefore, NaCl at 10 mmol l-1 may also have a phagostimulatory eff ect in P. oblongopunctatus although no behavioural data is available. Th is assumption could explain the inhibition of spike production of the salt-sensitive neuron by some toxic compounds in P. oblongopunctatus (V) as being similar to how plant deterrents inhibit activity of other phagostimulatory neurons in other phytophagous insects.

34 6. CONCLUSIONS

1. For the fi rst time, a specifi c pH-sensitive chemoreceptor neuron was identifi ed in insect taste sensilla, most probably related to habitat and microhabitat selection of the species (P. aethiops and P. oblongopunctatus; I, II);

2. For the ﬁ rst time, a sugar-sensitive chemoreceptor neuron responding to 1–1000 mmol l-1 sucrose and glucose was identiﬁ ed in the taste sensilla of ground beetles. Th e phasic-tonic response of the neuron to these sugars was concentration dependent (P. aethiops, III);

3. Th e disacharides with an a-1.4-glycoside linkage, sucrose and maltose, were the two most stimulatory sugars out of 12 tested for the antennal sugar-sensitive neuron of P. oblongopunctatus evoking 70 spikes/s at 1000 mmol l-1. Th e stimulatory eﬀ ect of glucose was approximately two times lower. Due to the partial herbivory of P. oblongopunctatus these plant sugars are probably involved in its search for food, for example, for conifer seeds (IV);

4. None of the water-soluble sugars released by brown-rot fungi during enzymatic wood decay (cellobiose, arabinose, xylose, mannose, rhamnose and galactose) stimulated the antennal sugar-sensitive neuron in P. oblongopunctatus (IV);

5. Th e weak stimulating eﬀ ect (below 3 spikes/s) of some 100 mmol l-1 amino acids (methionine, serine, alanine, glutamine) to the 4th chemosensory neuron of the antennal taste sensilla of P. oblongopunctatus was characterized as non-speciﬁ c, or modulating the responses of non-target chemosensory neurons (IV);

6. For the ﬁ rst time, the occurrence of an alkaloid-sensitive feeding deterrent neuron in the antennal taste sensilla of ground beetles (P. oblongopunctatus) was documented (V);

7. Some tested compounds (quinine and quinine hydrochloride) that stimulate the alkaloid-sensitive neuron in P. oblongopunctatus may strongly inhibit activity of other neurons (salt- and pH-sensitive neuron) of the sensillum (V);

35 8. Several tested plant secondary compounds, salicin, sinigrin, caff eine and nicotine, which had only little or no eff ect on the fi ring rate of the alkaloid-sensitive neuron, through peripheral inhibition, would greatly reduce the responses of other chemoreceptor neurons of the sensillum in P. oblongopunctatus (V).

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45 SUMMARY IN ESTONIAN

JOOKSIKLASTE (COLEOPTERA: CARABIDAE) ANTENNAALNE KONTAKTNE KEMORETSEPTSIOON

Rööv- ja segatoidulised jooksiklased oma suure liigirikkuse ja arvukuse tõttu omavad arvestatavat majanduslikku tähtsust kui põllu- ja aiakahjurite arvukuse olulised looduslikud reguleerijad.

Mitme- ja segatoiduliste jooksiklaste jaoks leidub põldudel alati piisavalt toitu. Jooksiklaste peamisteks toiduobjektideks on lehetäid, hooghänna- lised, liblikate ja mardikate munad, vastsed ning valmikud, kahetiivaliste munad ja vastsed ning vaablaste ebaröövikud. Suuremate liikide toiduks sobivad ka vihmaussid ja teod. Suure osa sega- ja taimtoiduliste liikide toi- dumenüüsse kuuluvad taimede, sealhulgas umbrohtude seemned. Just selle tõttu võivad jooksiklased erinevalt kitsalt spetsialiseerunud röövtoidulistest putukatest ja parasitoididest migreeruda põldudele varakevadel, enne kui sinna ilmuvad kultuurtaimede kahjurid. Hävitades kahjureid arvukuse tõusu algfaasis hoitakse ära nende hilisem massesinemine ja majanduslik kahju. Seepärast on mahetootmise ja integreeritud kahjuritõrje tingimustes manipulatsioonid jooksiklaste liigirikkuse ja arvukusega tähtsal kohal.

Jooksiklaste seotus teatud elupaigatüüpide ning varje- ja talvituskohtadega on määratud mitmesuguste biootiliste ja abiootiliste teguritega nagu toitu- mistingimused, taimkattetüüp, konkurentide olemasolu ja levik, aastaaeg, maastiku iseärasused, maaharimise mõju, temperatuuri, valgus- ja niiskus- tingimused jne. Pinnase sooladesisaldus ja pH võivad olla samuti tähtsad tegurid jooksiklaste levikul. Käitumiskatsetega on näidatud, et vastavad kontaktsed kemoretseptoosed sensillid ehk maitseharjased paiknevad nende mardikate tundlatel. Paljude jooksiklaste elupaigavalik on nii spetsiiﬁ line, et sageli kasutatakse neid elupaikade iseloomustamiseks. Välisstiimulid ja otsingulise käitumise mehhanismid, mille abil jooksiklased oma toidu asukoha kindlaks teevad ja saagirikastele aladele peatuma jäävad, pole täpselt teada. Samal ajal, kui paljud liigid leiavad oma toidu nähtavasti juhusliku otsingu teel, kütivad mitmed teised, päevase aktiivsusega liigid, saaki nägemise abil. Paljud liigid kasutavad aga toiduga seotud lõhna- signaale. Keemilise informatsiooni kasutamine on jooksiklastel ilmselt tunduvalt rohkem levinud kui väheste avaldatud andmete põhjal võiks arvata. Jooksiklaste käitumist reguleerivaid keemilisi välisstiimuleid ning neid vastuvõtvate kemoretseptorite talitlust on seni üllatavalt vähe uuritud. 46 Elektronmikroskoopilised uuringud on näidanud, et jooksiklaste tundlatel esineb tuhandeid erinevaid kemoretseptoorseid sensille. Lisaks arvukatele pisikestele pulkjatele haistesensillidele, paikneb nende ﬂ agellumil 56 kuni 70 suurt 35–200 μm pikkust harjasjat kontaktset kemosensilli. Kivijook- sikul Nebria brevicollis on need sensillid varustatud nelja kemoretseptoorse ja ühe mehhanoretseptoorse neuroniga. See on tüüpiline neuronite komp- lekt enamiku putukarühmade kontaktsetes kemosensillides. Nendest on jooksiklastel seni elektrofüsioloogiliselt kindlaks tehtud ainult ühe, soolaneuroni, funktsioon. Ülejäänud kolme kemoretseptoorse neuroni täpsem funktsioon pole teada. Kontaktset kemoretseptsiooni on põhjali- kumalt uuritud paljudel taimtoidulistel putukatel, kellel need neuronid võivad reageerida väga mitmesugustele taimsetele keemilistele stiimulitele, enamasti aga toitumise stimulantidele (taimsed suhkrud, aminohapped, suhkuralkoholid jt.) ja deterrentsetele ühenditele (alkaloidid, glükosiidid jt.). Kuna stimulantsetel ja deterrentsetel ühenditel on toidu- ja peremees- taime äratundmisel keskne roll, on nad viimastel aastakümnetel pälvinud üha suuremat tähelepanu.

Käesoleva töö eesmärgiks on elektrofüsioloogilise meetodi abil selgitada antennaalseid kontaktseid kemoretseptoreid (maitseharjaseid) innerveerivate neuronite funktsiooni ümarselg-süsijooksikul (Pterostichus aethiops) ja metsa-süsijooksikul (P. oblongopunctatus). Erinevate meetoditega (puhver- lahuste seeriad, leelistatud ja leelistamata soolad) testiti ärrituslahuste pH mõju nende neuronite reaktsioonile ning spetsiiﬁ lise pH-tundliku neuroni võimalikku esinemist nende liikide antennaalsetes maitseharjastes. Kuna mõlemad uuritavad liigid on osaliselt taimtoidulised (granivoorid), testiti ka antennaalsete maitseneuronite reaktsiooni mitmesugustele taimsetele suhkrutele ja aminohapetele, et selgitada nende ainete vastuvõtmisele spetsialiseerunud neuronite olemasolu ja talitluse iseärasusi jooksiklaste tundlatel. Olulise osa tööst moodustavad elektrofüsioloogilised ja etoloogi- lised eksperimendid, mille käigus selgitati taimeseemnetes leiduvate toksi- liste ja deterrentsete ainete (alkaloidid ja glükosiidid) stimuleerivat toimet nimetatud jooksiklaste antennaalsete maitseharjaste kemosensoorsetele neuronitele. Katsete eesmärgiks oli selgitada spetsiiﬁ lise deterrendineuroni olemasolu ja nende ainete võimalikku inhibeerivat toimet maitseharjase teistele neuronitele. Nii ümarselg-süsijooksik kui ka metsa-süsijooksik on videvikuaktiivsusega omnivoorsed metsaliigid, kelle toidumenüüs on tähtsal kohal okaspuude seemned. Katsemardikad koguti Lõuna-Eesti metsadest.

47 Elektrofüsioloogilistes katsetes mõõdeti spetsiaalse aparatuuri abil mardikate antennaalseid maitseharjaseid innerveerivate neuronite reaktsioone ärrituslahustele, kasutades sensilli tipust neuronite impulss-aktiivsuse rakuvälise registreerimise meetodit. Selleks, et suurendada ärrituslahuste elektrijuhtivust, lahustati suhkrud, aminohapped, alkaloidid ja glükosiidid vajalikus kontsentratsioonis 10 mmol l-1 elektrolüüdi (NaCl, koliinklo- riid) lahuses. Puhtad ained hangiti ﬁ rmadelt AppliChem (Saksamaa) ja Sigma-Aldrich. Lahuste pH taset mõõdeti portatiivse pH-meetri E6115 (Evikon, Estonia) abil. Neuronite reaktsiooni mõõdeti toimepotentsiaa- lide (närviimpulsside) arvuna sekundis. Et testida elektrofüsioloogiliselt aktiivsete alkaloidide deterrentset toimet mardikate toitumise aktiivsusele, viidi läbi vajalikud käitumuslikud valikukatsed alkaloidiga töödeldud ja töötlemata männiseemnetega. Katseandmete analüüsimisel kasutati erinevaid statistilisi teste, kasutades arvutitarkvara Statistica (StatSoft, Inc., USA).

Elektrofüsioloogilised katsed atsetaat- ja fosfaatpuhvrite seeriatega ning leelistatud ja leelistamata sooladega pH vahemikus 3 kuni 11 näitasid, et ümarselg-süsijooksiku ja metsa-süsijooksiku antennaalsetes maitseharjastes paikneb kaks neuronit, soola- ja pH-tundlik neuron, mis reageerivad nende elektrolüütide lahustele. pH-tundliku neuroni keskmine impulss-aktiivsus tõusis alati koos ärrituslahuse pH-taseme tõusuga ulatudes aluselise reaktsiooniga ärrituslahuste puhul (100 mmol l-1 NaCl + 10 mmol l-1 NaOH; pH 9,6) maksimaalselt kuni 30 imp/s, samal ajal kui soola-tundliku neuroni aktiivsus ei muutunud või muutus ainult vähesel määral. Samas täheldati, et ka ärrituslahustes olevate soolade iooniline koostis mõjutas märgatavalt pH-tundliku neuroni närviimpulsside sagedust. Saadud tulemus on huvipakkuv, kuna varem pole putukate kontaktsetes kemosensoorsetes sensillides spetsiiﬁ lise pH-tundliku neuroni olemasolu täheldatud. Uuritud jooksiklaste antennaalne pH-tundlik neuron näib olevat seotud mardikate elupaiga ja talvituskohtade valikuga. pH-tundliku neuroni impulssaktiivsus puudub või on madalseisus (alla 5 imp/s) pH väärtustel 3 kuni 6. See happelisuse piirkond vastab pinnase pH tasemele uuritud jooksiklaste metsabiotoopides ja eelistatud talvituskohtades pruunmädaniku poolt lagundatud puidus, kus mardikad veedavad 7–8 kuud, septembrist aprillini.

Käesoleva uurimuse raames identiﬁ tseeriti esmakordselt granivoorsetel lülijalgsetel (ümarselg-süsijooksikul ja metsa-süsijooksikul) elektrofüsio-

48 loogiliselt suhkrutundliku neuroni olemasolu. Katsed näitasid, et 12-st testitud taimsest suhkrust 3 (maltoos, sahharoos ja glükoos) osutusid füsioloogiliselt aktiivseteks, stimuleerides antennaalsete maitseharjaste suhkrutundliku neuroni impulssaktiivsust. Tüübilt oli neuroni reakt- sioon 1–1000 mmol l-1 suhkrulahusele faasilis-tooniline, sõltudes lahuse kontsentratsioonist. Metsa-süsijooksikul osutusid 1,4-glükosiidsidemega disahhariidid maltoos ja sahharoos füsioloogiliselt aktiivseimateks, tõstes 1000 mmol l-1 kontsentratsioonil suhkrutundliku neuroni impulss-aktiivsuse tasemele 70 imp/s. Glükoosi stimuleeriv toime oli ligi kaks korda madalam. Kuna maltoosi leidub looduses peamiselt idanevates seemnetes ja noortes idantites, võib oletada, et uuritud jooksiklaste suhkrutundlikkus on seotud nende osalise taimtoidulisusega (mardikad toituvad okaspuude seemnetest ja noortest idanditest). Ükski puidu ensümaatilise lagunemise käigus tekkivast vees lahustuvast suhkrust (tsellobioos, arabinoos, ksüloos, mannoos, ramnoos ja galaktoos) ei osutunud füsioloogiliselt aktiivseks, mistõttu nad ei osale nähtavasti metsa-süsijooksiku otsingulises käitu- mises. Mõned testitud seitsmest 100 mmol l-1 aminohappest (metioniin, seriin, alaniin, glutamiin) avaldasid nõrka stimuleerivat toimet (alla 3 imp/s) metsa-süsijooksiku maitseharjase kemosensoorsetele neuronitele, mida võib aga iseloomustada kui maitseharjase neuronite reaktsiooni mittespetsiiﬁ list modulatsiooni.

Antennaalsete maitseharjaste kemosensoorsete neuronite reaktsiooni taimsetele alkaloididele ja glükosiididele testiti metsa-süsijooksikul. Katsete tulemusena identiﬁ tseeriti granivoorsetel lülijalgsetel esmakordselt alka- loiditundlik neuron. Ilmnes, et testitud viiest alkaloidist avaldasid sellele neuronile tugevat stimuleerivat toimet vaid kiniin ja selle sool kiniinhüd- rokloriid. Alkaloiditundliku neuroni faasilis-toonilised reaktsioonid 0,001 kuni 50 mmol l-1 olid võrdelises sõltuvuses lahuse kontsentratsioonist. Neuroni erutusläveks kiniinhüdrokloriidi suhtes osutus kontsentratsioon 0,01 mmol l-1, maksimaalseid reaktsioone (67 imp/s) täheldati kontsentratsioonil 50 mmol l-1. Käitumiskatsetes osutus kiniinhüdrokloriid tugevaks toitumise deterrendiks. Kofeiini stimuleeriv efekt sellele neuronile oli väga nõrk (alla 2 imp/s), nikotiin aktiivsust ei mõjutanud, strühniin aga pärssis nõrgalt selle neuroni impulssaktiivsust.

Lisaks kiniini ja kiniinhüdrokloriidi alkaloidineuronit stimuleerivale toimele võivad nad tugevalt pärssida metsa-süsijooksiku maitseharjase soola- ja pH-tundliku neuroni impulssaktiivsust. Samuti ilmnes, et

49 glükosiidid sinigriin ja salitsiin ning alkaloidid kofeiin ja nikotiin, mille mõju alkaloiditundlikule neuronile oli nõrk või puudus üldse, võivad samuti tugevalt pärssida soola- ja pH-tundliku neuroni impulssaktiivsust. Saadud tulemused lubavad oletada, et jooksiklase antennaalsed maitseharjased võivad taimseid toksilisi ja deterrentse toimega aineid avastada nii alkaloiditundliku neuroni stimuleerimise kui ka maitseharjase teiste kemosensoorsete neuronite periferaalse inhibitsiooni teel.

50 ACKNOWLEDGEMENTS

Th is study was carried out at the Institute of Agricultural and Environ- mental Sciences of the Estonian University of Life Sciences. I would like to express my gratitude to my supervisors Enno Merivee and Prof. Anne Luik who has been supporting and helping me completing in this thesis.

I am grateful to Kaljo Voolma and Heino Õunap for their review of my thesis and good comments on how to improve it.

I sincerely thank my colleagues in the Institute of Agricultural and En- vironmental Sciences for their support.

Th e Estonian Science foundation (Grants No 5423 and 6958) ﬁ nancially supported this study.

51 I

PUBLICATIONS Merivee, E., Ploomi, A., Milius, M., Luik, A., Heidemaa, M., 2005.

ELECTROPHYSIOLOGICAL IDENTIFICATION OF ANTENNAL PH RECEPTORS IN THE GROUND BEETLE PTEROSTICHUS OBLONGOPUNCTATUS.

Physiological Entomology 30(2), 122–33. Physiological Entomology (2005) 30, 122–133

Electrophysiological identification of antennal pH receptors in the ground beetle Pterostichus oblongopunctatus

ENNO MERIVEE1 , ANGELA PLOOMI1 ,MARITMILIUS1 ,ANNE LUIK1 and M I K K H E I D E M A A 2 1Estonian Agricultural University, Institute of Plant Protection, Tartu and 2University of Tartu, Institute of Zoology and Hydrobiology, Tartu, Estonia

Abstract. Electrophysiological responses of antennal taste bristles to 100 mM acetate and phosphate buffers were tested at pH 3–11 in the ground beetle Pterostichus oblongopunctatus (F.) (Coleoptera, Carabidae). Additionally, responses of these sensilla to 10 and 100 mM phosphate buffers were compared with each other. Generally, in response to these stimulating solutions, two sensory cells, classified as a salt cell (cation cell) and a pH cell, respectively, showed action potentials distinguished by differences in their amplitudes and polarity of spikes. The firing rate of the cation cell increased with increasing buffer concentration, and was influenced by buffer pH in a complicated way. The best stimulus for the second cell (pH cell) was pH of the stimulating buffer solution. As the pH of the stimulus solution increased, higher rates of firing were produced by the pH cell. For example, the number of action potentials elicited by 100 mM phosphate buffer at pH 11.1 was approximately 16-fold higher compared with that at pH 8.1, and firing rates during the first second of the response were 27.9 and 1.7 imp/s, respectively. The pH cell did not fire or fired at very low frequency (first second response below 5 imp/s) at pH 3–6. This level of acidity probably represents the pH preferences of this ground beetle in its forest habitat and hibernating sites. By contrast to the cation cell, the pH cell responded to increases in buffer concentration by decreasing its firing rate. Key words. Action potentials, firing rate, taste sensilla, tip recording.

Introduction behavioural experiments concerning the ground beetles’ ability to perceive salt content and pH of the ground The life of carabids from egg to death of the adult proceeds are contradictory (Thiele, 1977). Nevertheless, Paje & in direct physical contact with the ground (from which the Mossakowski (1984) demonstrated convincingly that name of the beetles is derived in English: ground beetles). behaviourally ground beetles have certain pH preferences Due to scarce data, we can only assume that, in searching concerning their habitat; ablation experiments conducted and selection of their habitat, refugia and hibernating sites, by these authors suggested that pH receptors are located besides other stimuli, ground beetles perceive and analyse on the beetles’ antennae. external chemical information, such as soil pH, salt content Scanning electron microscope studies demonstrate that and concentration. However, some data obtained from the most probable candidates for pH receptors (i.e. large blunt-tipped sensilla) can be found abundantly on the antennae of all ground beetles. These antennae consist of Correspondence: Dr Enno Merivee, Estonian Agricultural a scapus, pedicel and nine flagellomeres. The taste receptors University, Institute of Plant Protection, 64 Kreutzwaldi Street, stand in a whorl of six to seven bristles, in distal parts of the 51014 Tartu, Estonia. E-mail: [email protected] flagellomeres. In addition, six bristles are located in pairs

122 # 2005 The Royal Entomological Society

55 pH receptors in P. oblongopunctatus 123 symmetrically at the antennal tip (Kim & Yamasaki, 1996; Chemicals and preparation of buffer solutions Merivee et al., 2000, 2001, 2002). According to Daly & Ryan (1979), these taste bristles in the ground beetle Before experiments, 100 mM acetate buffers in the pH Nebria brevicollis are innerved with five neurones: four range 3–6 were prepared by dissolving glacial acetic acetate chemoreceptor cells and one mechanoreceptor cell. In and anhydrous sodium acetate in distilled water. Stimulat- electrophysiological experiments with acid, neutral and ing 100 mM buffers with higher levels of pH (5.8–8.1) were alkaline salts, it was demonstrated that two chemoreceptor made with combinations of Na2HPO4 and NaH2PO4. For cells of the antennal taste bristles respond to these salt some experiments, the pH of phosphate buffer was raised to solutions in ground beetles. One of the cells, the salt cell, 11.1 with NaOH. The pH of the 100 mM borax used in some was sensitive to ionic (cationic) content and concentration test series with acetate buffers was 9.3. All chemicals of the stimulating solution. Firing rates of the second cell were obtained from AppliChem (Germany). To produce were proportional to the pH level of the stimulating stimulating buffers of approximately the same pH but of salt solutions tested (Merivee et al., unpublished data). different concentrations of ions, the 100 mM phosphate Unfortunately, the tested salts were dissolved in distilled buffer (pH 7.6) was diluted in distilled water to 10 mM water and not in buffer solutions. It is likely that the pH solution (pH 7.8). The final pH of the stimulating solutions level of the tested salt solutions was unstable and changed was measured with a pH meter E6115 (Evikon, Estonia). during experiments. Thus, the function of the second salt cell (pH cell) requires further testing using buffer-controlled pH solutions. To date, electrophysiological evidence con- Stimulation and recording of action potentials cerning the occurrence of pH receptors in insects is lacking, but it has been shown repeatedly that the pH of stimulating Electrophysiological recordings were made from large solutions may affect neural responses of insect taste sensilla chaetoid taste sensilla on the antennal flagellum of the (Shiraishi & Morita, 1969; Bernays et al., 1998). beetle with a tip-recording technique (Hodgson et al., The aim of the present study is to test responses of 1955). To achieve a good signal-to-noise ratio and avoid antennal taste bristles to acetate and phosphate buffers in registration of muscular activities from two basal antenno- a wide range of pH values (3–11) in the ground beetle meres, the scape and pedicel and from other parts of insect’s Pterostichus oblongopunctatus F. (Coleoptera, Carabidae) body, an indifferent tungsten microelectrode was inserted to investigate the functioning of pH receptors in ground into the base of the flagellum. The recording microelec- beetles. trode, a glass micropipette (diameter at the tip approximately 20 mm) filled with stimulating buffer solution was placed over the sensillum tip by means of a micromanipulator under visual control with a light microscope at Materials and methods magnification 300. Immediately before each stimulation, two to three drops of the stimulating solution were squeezed Test beetles out of the micropipette tip, and absorbed onto a piece of filter paper attached to an additional micromanipulator to Test beetles were collected in April 2003 from their reduce concentration increases due to evaporation. A hibernation sites in forest margins in southern Estonia. copper wire made contact with high-impedance preampli- The beetles were kept in plastic boxes filled with moistened fier input. The signal picked up by the recording electrode moss in a refrigerator at þ5 C. Three or four days before was amplified and filtered with a band width set at the electrophysiological tests, the beetles were kept in 100–2000 Hz and monitored on an oscilloscope. Recordings similar boxes at room temperature (þ 22 C). The beetles of the first 5 s of the response were transferred directly to a were given clean water to drink and fed with moistened cat computer via an analogue-to-digital input board DAS-1401 food (Kitekat, Master Foods, Poland) every day. (Keithley, Taunton, Massachusetts) for data acquisition, Test beetles were immobilized by placing them tightly storage and further analysis using TestPoint (Capital into a special conical tube made of thin sheet-aluminium Equipment Corp., Billerica, Massachusetts) software. The of a size that allowed their head and antennae to protrude sampling data rate was 10 kHz. only a little from the narrower end. The wider rear end of Due to variations in responsiveness of different taste the conical tube was blocked with a piece of plasticine to bristles to the same stimuli, on the antennae of one and prevent the beetle from retreating out of the tube. The the same as well as different beetles, stimulating buffers antennae of immobilized intact beetles were fastened hori- were tested in pairs to allow a more precise comparison of zontally on the edge of a special aluminium stand with tiny the responses to the tested stimuli. Thus, each taste sensil- amounts of beeswax, such that some horizontally located lum was tested twice. Up to 15 sensilla on one antenna of a antennal taste bristles were visible from above under a light test beetle were in a suitable position for stimulations and microscope, and easily accessible for micromanipulations recordings. Primarily, these sensilla were tested with the first from the side. Contamination of tested taste bristles and stimulus. Then, after approximately 30 min, the stimulat- their contact with preparation instruments and beeswax was ing/recording micropipette was rinsed with distilled water avoided. several times and filled with the second stimulus to test the

# 2005 The Royal Entomological Society, Physiological Entomology, 30, 122–133 124 E. Merivee et al. same sensilla again. The space of time between the two extremely large, approximately 8 mV peak-to-peak. By con- successive stimulations ensured complete disadaptation of trast, action potentials with negative spikes originating the receptor cells. After the test series, test beetles remained from the B-cell are three- to four-fold smaller in amplitude in good condition, and no differences were observed in the compared with the action potentials generated by the A-cell responses of their taste bristles recorded at the beginning (Fig. 1A). The responses to tested stimuli were phasic–tonic and the end of the test series. Antennal sensilla from four to in type, with a more pronounced phasic component in six beetles were tested in each pair of stimuli. B-cells. On the recordings, action potentials from two cells frequently interacted to produce an irregular waveform. In these cases, the regularity of firing as the principal para- Data management and analysis meter for visual separating and identifying action potentials was used (Fig. 1A). In some cases, responses of the A-cells Action potentials from several sensory cells of taste sen- were of a bursting nature (Figs 1C,D). Usually action silla were distinguished by differences in their amplitude, potentials generated by the same cell had similar amplitude. duration, shape and polarity of spikes. In some cases, the However, in a number of cases, especially at the beginning regularity of firing as the principal parameter for separating of the B-cell’s response, a few action potentials could be and identifying action potentials was used. Automatic observed whose amplitude was remarkably greater than the classifying and counting of action potentials from taste others (Fig. 1B). sensilla was clearly not an appropriate method for data analysis because spike amplitude of a certain cell often changed in time or with concentration, and two action Responses to acetate buffers at pH 3 and 4 potentials frequently interacted to produce an irregular waveform. Instead, the analysis used was visual, employing The A-cell did not respond to 100 mM acetate buffer at TestPoint software (CEC Capital Equipment, Bedford, pH 3 and only a very weak response was observed at pH 4: New Hampshire). Firing rates of receptor cells were the average number of impulses during the first and follow- expressed as a number of spikes per second. All data ing seconds of the response was less than 1. At the same were analysed using Microsoft Excel (Microsoft Corp., time, the A-cell of the same bristles strongly responded to Redmond, Washington) and Statistica (StatSoft, Inc., borax: on average, 23 impulses per first second of the Tulsa, Oklohoma) software. response were recorded (Fig. 2A, A-cell), demonstrating that the tested cells were in good functional condition. The B-cell responded to 100 mM acetate buffers at pH 3 Results and 4 also relatively weakly: 5.7 and 9.5 impulses per first second of the response, respectively (P < 0.05). The Taste sensilla on the antennal flagellum in response of the same B-cells to borax was 24 imp/s. Dur- P. oblongopunctatus ing the following seconds of the response (in the tonic part), the firing rate of the B-cell stabilized at the level of m Large blunt-tipped 100–200- m long flagellar taste 1–3 imp/s; compared with the first second of the response, P. oblongopunctatu bristles of the ground beetle s can be the opposite trend was noted. The firing rate of the cell at quantified at low magnification under the light microscope. pH 4 appeared to be lower than that at pH 3, although The largest bristles occur on the first flagellomere counting the difference was not statistically significant (P > 0.05; from the beetle’s head. The length of the bristles towards the Fig. 2A, B-cell). Some typical 5-s responses of antennal tip of the antenna decreases, and the shortest of the bristles taste sensilla to stimulating solutions at pH 3 and 4 are can be found at the tip of the terminal flagellomere. Bristles shown in Fig. 3. are located in whorls in the distal part of flagellomeres. There are six on the first flagellomere and seven on the following flagellomeres. In addition to those, there are six bristles symmetrically at the tip of the terminal flagellomere. Responses to acetate buffers at pH 4 and 5 Hence, the total number of taste bristles on each antennal flagellum is 68. All these bristles seemed to respond in a In response to stimulation with acetate buffer at pH 5, the similar manner to the stimuli tested. A-cells generated significantly more action potentials (first second mean response 3.6 impulses) compared with the response at pH 4 (0.9 impulses per first second of the Characterization of action potentials recorded from antennal response) (P < 0.05; Fig. 2B, A-cell). taste sensilla During the first two seconds of the response, B-cells fired at a significantly higher frequency at pH 5 than at Two sensory cells innervating flagellar taste bristles in pH 4 (P < 0.05; first second responses 15.9 and 9.7 imp/s, P. oblongopunctatus responded to stimulation with acetate respectively). The third second response was approxi- buffers, phosphate buffers and borax: A- and B-cell. Action mately equal (P > 0.05). As observed in the test series potentials with positive spikes generated by the A-cell are with stimulating solutions at pH 3 and 4, during the last

# 2005 The Royal Entomological Society, Physiological Entomology, 30, 122–133

57 pH receptors in P. oblongopunctatus 125

Fig. 1. Action potentials recorded from two cells of antennal taste bristles in response to 100 mM phosphate buffer at pH 8.1 in the ground beetle Pterostichus oblongopunctatus. (A) The beginning of the electrophysiogical response. Arrow- head indicates the beginning of stimulation. AC and BC, action potentials from A- and B-cells, respectively, distinguished by differences in their amplitudes and polarity of spikes. Action potentials from two cells interacted frequently to produce an irregular waveform (arrows). In the phasic part of the response, the B-cell fired in a highly regular manner (black dots), which facilitated differentiation and counting of impulses of two cells. Cali- bration bar ¼ 5 mV. (B) The phasic beginning of the response to phosphate buffer at pH 8.1. Only one cell responded. Often, especially at the beginning of the B-cell’s response, a few action potentials could be observed whose amplitude was remarkably greater than others (indicated by arrows). (C) The A-cell frequently fired in bursts. Impulses of one of the bursts (marked with black bar) are shown at a shorter timescale (D).

2 s of stimulation, B-cells generated significantly less ences in mean firing rates of A-cells were not statistically action potentials at pH 5 compared with the response at significant during the first second of the response pH 4 (P < 0.05; Fig. 2B, B-cell). (P > 0.05), these differences were significant during the following seconds of the response (P < 0.05; Fig. 2C, A-cell). Responses to acetate buffers at pH 5 and 6 First second mean firing rates of the B-cells in response to stimulating solutions at pH 5 and 6 were 17 and As the pH of the 100 mM acetate buffers tested 22.7 imp/s, respectively. The difference between the increased, higher rates of firing were generated by the means was statistically significant (P < 0.05). During the A-cells, although these remained at a relatively low level, next 4 s of the response, no differences were observed in below 5 imp/s. When acetate buffers at pH 5 and pH 6 relatively low firing rates caused by the two buffer solu- were used for stimulation, in every pair of stimuli tested, tions (P > 0.05; Fig. 2C, B-cell). Thus, comparing B-cell the less acid buffer solutions caused A-cells to produce responses to acetate buffers with pH 3–6, it became evi- more action potentials per second: first second firing dent that solutions of higher pH caused B-cells to fire at rates of 3.7 and 4.6 imp/s, respectively. Although differ- higher frequencies during the first second of the response.

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Fig. 2. Responses of antennal taste bristles to 100 mM acetate buffers at pH 3–6. Because the test insects and their taste sensilla varied greatlyintheirresponsestothesamestimulus,stimuliweretestedinpairswithoneandthesamebristle.(A)TheA-celldidnotfire, or fired at very low frequency, when stimulated by acetate buffers at pH 3 and 4. However, strong responses to borax showed that the same cells were in a good functional condition. (B and C) According to how the pH level of stimulus solution increased, higher rates of firing were observed in A-cells. The increase in pH caused B-cells to fire more frequently, especially during the first second of the response, but to a remarkably lesser extent than in A-cells. Vertical bars indicate SE of the means; asterisk (*) and n.s. indicate whether the difference between the mean firing rates is significant or not, respectively (paired t-test, P < 0.05). n,Numberoftaste bristles tested.

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Fig. 3. Some typical recordings of the responses of an antennal taste sensillum to 100 mM acetate buffers and borax. (A and B) Responses to acetate buffers at pH 3 and 4. Only one cell responded. Stimulating the same taste bristle with borax solution caused two cells to fire, demonstrating that both cells of the bristle were in a good condition and responded strongly when an adequate stimulus was provided (C). Some small impulses from B-cells among large impulses originating from A-cells are indicated by arrows. (D) Beginning of the same recording (C) at a shorter timescale.

During the next 4 s of the response, in most cases, similar ively. In addition, during the following seconds of the firing rates were observed in response to the buffers tested response, firing rate (4–8.3 imp/s) generated by the neutral in pairs. However, in some cases (stimulations at pH 4 buffer (pH 7) was remarkably higher compared with that and 5), stimulating solution at lower pH elicited a signifi- (0.4–2.4 imp/s) generated by the slightly acid buffer (pH 5.8) cantly higher firing rate in the tonic part of the response (P < 0.05; Fig. 4A, A-cell). The responses of B-cells to these of the B-cell (Fig. 2B). two phosphate buffers were approximately equal (Fig. 4A, B-cell).

Responses to phosphate buffers at pH 5.8 and 7.0 Responses to phosphate buffers at pH 5.8 and 8.1 The frequency of action potentials, generated by 100 mM phosphate buffers both in A- and B-cells, was remarkably Responses of the A-cell to stimulation with phosphate higher than with acetate buffers. In A-cells, the number of buffers at pH 5.8 and 8.1 were similar to these recorded in a spikes per first second of the response was 10.5 and 15.8, test series with buffers at pH 5.8 and 7. However, differences caused by stimulating solutions at pH 5.8 and 7, respect- between respective firing rates were considerably greater in

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Fig. 4. Responses of antennal taste sensilla to 100 mM phosphate buffers at pH 5.8–11.1. Stimuli were tested in pairs with the same sensilla to overcome the different sensitivity of different taste bristles. In A-cells, as a rule, stimulating solutions of high pH caused remarkably higher rates of firing compared with those caused by solutions at lower pH (A–C). The pH of the stimulus solution also affected the responses of the B-cells but in a much more complicated way. For example, the B-cell’s responses to buffers at pH 5.8 and 7 did not differ very much (A). In a test series with pH 5.8 and 8.1, the firing rate of the B-cells was affected by an increase in pH in the opposite way at the beginning and end of stimulation, positively and negatively, respectively (B). When the responses of the B-cells to phosphate buffers at pH 8.1 and 11.1 were compared with each other, it became evident that the solution with pH 11.1 was a more effective stimulus for this cell. However, compared with the responses of the A-cell, B-cell’s firing rate depended remarkably less on the pH value of the stimulating solution (C). Vertical bars indicate standard errors (SE) of the means; asterisk (*) and n.s. indicate a significant and not significant difference between the mean firing rates, respectively (paired t-test, P < 0.05). n, Number of bristles tested.

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61 pH receptors in P. oblongopunctatus 129 the test series with stimulating solutions at pH 5.8 and 8.1 Responses to phosphate buffers at pH 8.1 and 11.1 (first second responses 8.0 and 23.1 imp/s, respectively; Fig. 4B, A-cell). Recordings obtained in test series with phosphate buffers In B-cells, differences between firing rates elicited by demonstrate that different sets of taste sensilla, belonging to stimulating buffers at pH 5.8 and 8.1 were relatively small different beetles, may vary greatly in their responses to the but statistically significant (P < 0.05). During the first sec- same stimulus. For example, 1.7 and 23.1 spikes per first ond of the responses, mean firing rate at pH 5.8 was a little second of the response to buffer solution with pH 8.1 were lower compared with that caused at pH 8.1, 25 and recorded from A-cells in test series where responses to 28.2 imp/s, respectively. The opposite was observed in the buffers at pH 5.8 and 8.1 and 8.1 and 11.1 were compared, tonic part of the responses; firing rates caused by buffer respectively (Fig. 4B,C). At the same time, the same A-cells, solution at pH 5.8 were slightly higher compared with those which responded very weakly to buffer at pH 8.1, responded at pH 8.1 (Fig. 4B, B-cell). Some typical original recordings strongly to buffer at pH 11.1 (P < 0.05), with first second of the responses to stimulating buffers at pH 5.8 and 8.1 are responses of 1.7 and 27.9 imp/s, respectively (Fig. 4C, shown in Fig. 5. A-cell).

Fig. 5. Typical responses recorded from one and the same antennal taste sensillum stimulated by 100 mM phosphate buffers at pH 5.8 (A) and 8.1 (C). (B) and (D) show the same responses on a shorter time scale, respectively. Two cells are firing. (A,C) and (B,C) show action potentials from A- and B-cells, respectively. Some impulses generated by the B-cell are marked with arrows. Compared with the B-cell, the neural activity level of the A-cell depended much more on the pH of the stimulating solution.

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Although the B-cells responded to buffer at pH 11.1 more concentration. Differences were significant during the first strongly than at pH 8.1 (P < 0.05), these cell’s responses to 4 s of the response (P < 0.05), but not during the final fifth those buffers during the first second differed to a much less second (P > 0.05; Fig. 7, A-cell). The opposite was noticed extent compared with those observed in A-cells, 34.4 and when responses of the B-cell were compared with each 27.9 imp/s, respectively. Furthermore, in the tonic part of other; higher firing rates occurred when stimulating with the response, the number of spikes at pH 11.1 was remark- buffer solution at higher concentration, first second ably higher than at pH 8.1 (Fig. 4C, B-cell). Some examples responses 31.2 and 21.9 imp/s, elicited by 100 and 10 mM of the responses of the antennal taste sensilla to phosphate phosphate buffers, respectively (P < 0.05). In the tonic buffers at pH 8.1 and 11.1 are demonstrated in Fig. 6. part of the B-cell’s response, firing rates decreased rapidly, and the cell responded equally to 10 and 100 mM buffers (Fig. 7, B-cell).

Responses to 10 and 100 mM phosphate buffers (pH 7.8 and 7.6, respectively) Discussion When responses of A-cells to 10 and 100 mM phosphate buffers were compared, it became evident that remarkably In a number of insects, including the ground beetle higher firing rates were elicited by the solution at lower P. oblongopunctatus, frequently two cells of the taste sensilla

Fig. 6. Recordings of some typical responses of an antennal chaetoid taste sensillum to phosphate buffers at pH 8.1 (A) and pH 11.1 (C). (B) and (D) show the same responses on a shorter time scale, respectively. Some small neural impulses generated by the B-cell among large impulses originated by the A-cell are marked with arrows. Responses of both A-cell, and B-cells were of a phasic–tonic type.

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Fig. 7. Responses of antennal taste sensilla to 100 and 10 mM phosphate buffers (pH measured 7.6 and 7.8, respectively). A- and B-cells responded to changes in buffer solution concentration in opposite manner. By contrast to A-cells, in B-cells, buffer solution at higher concentration was a more effective stimulus, especially during the first 2 s of the response. Asterisk (*) and n.s. inidcate whether differences between mean firing rates were statistically significant or not, respectively (paired t-test, P < 0.05). respond to salt solutions (Dethier & Hanson, 1968; Haskell quinine, amiloride, nicotine and caffeine (Liscia & Solari, & Schoonhoven, 1969; Den Otter, 1972; Mitchell 2000). Very little attention has been paid to this cell. This is & Schoonhoven, 1973; Mitchell & Seabrook, 1973; Blaney, unfortunate because this lack of information handicaps any 1974; Hanson, 1987; Bernays & Chapman, 2001a; attempt to construct an overview of the control of food Schoonhoven & van Loon, 2002). It is suggested that they detection and habitat selection in insects on the basis of function as an anion and a cation cell (Ishikawa, 1963; their chemosensory input. Elizarov, 1978; Hanson, 1987; Schoonhoven & van Loon, In the ground beetle P. oblongopunctatus, the firing rate 2002). Temporal patterns of firing and the concentration– of the second cell, the A-cell, innervating antennal taste response curves are quite different for the two cells. In the sensilla, was correlated positively with pH values of the cation cell with a phasic–tonic type of reaction, the magnitude 100 mM acetate and phosphate buffers tested at pH 3–11 of the response is proportional to the logarithm of salt over the range of the 5-s stimulation period. Differences in concentration over the dynamic range (Evans & Mellon, firing rates of the A-cell elicited by the different pH of the 1962; Hanson, 1987). It is believed that the stimulating effect stimulating solutions may be very large. For example, the of salts to taste receptors is dominated by the cations number of impulses elicited by 100 mM phosphate buffer at involved, and that monovalent cations are more effective pH 11.1 was approximately 16-fold higher compared with stimuli than divalent cations (Evans & Mellon, 1962; Schoon- that elicited by 100 mM phosphate buffer at pH 8.1. When hoven & van Loon, 2002). This cell type corresponds to the responses to 10 and 100 mM phosphate buffers with nearly B-cell of the antennal taste bristles in P. oblongopunctatus. the same pH (pH 7.8 and 7.6, respectively) were compared Specific stimuli for the second salt cell are yet to be with each other, it became evident that a solution at lower identified: it has been named the ‘anion’ cell on the basis concentration had a stronger stimulating effect on the of a supposed sensitivity to salt anions or the ‘fifth’ cell A-cell than a solution at higher concentration. At the (Ishikawa, 1963; Dethier & Hanson, 1968; Hanson, 1987; same time, as expected, the response of B-cells to 10 and Liscia et al., 1997; Liscia & Solari, 2000). This cell is char- 100 mM phosphate buffer was the opposite: a solution at a acteristically less responsive to differences in salt concentra- higher concentration caused higher rates of firing. tion and often responds in bursts (Dethier, 1977; Liscia Therefore, it can be concluded, that the best stimulus for et al., 1997), similar to that observed in P. oblongopunctatus. the antennal taste bristles’ A-cell in the ground beetle In the strict sense of the term, it is not correct to give the P. oblongopunctatus is not salt but the pH level of the name ‘salt cell’ to a cell that does not respond to changes in stimulus solution. The present results confirm the salt concentration. On the other hand, a study in Grammia suggestion made by Paje & Mossakowski (1984) that, in geneura shows increasing activity in two salt cells in ground beetles, pH receptors are located on the antennae. response to KCl at concentrations ranging from 10 to To date, pH receptors have not been described in other 1000 mM. NaCl also produced responses in the two insects, and it is possible that the pH of the stimulating cells with slightly higher firing rates than KCl (Bernays & solution may prove to be an important stimulus for other Chapman, 2001a). Dethier (1977) was the first to realize chemoreceptors. that it is not at all certain that the best stimulus for the If the response of the A-cell depends on solution pH level second salt cell is indeed salt. Electrophysiological evidence only, there should be equivalent responses to buffers at is provided that the so-called ‘fifth’ cell in taste chemosen- different concentrations at the same pH. However, the silla of blowflies responds to deterrent compounds, such as results do not confirm this: 10 mM buffer had a greater

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64 132 E. Merivee et al. stimulating effect on the A-cell than 100 mM buffer. This Blaney, W.M. (1974) Electrophysiological responses of the terminal phenomenon can be explained by interaction of different sensilla on the maxillary palps of Locusta migratoria (L.) to some chemical stimuli to chemosensory cells. Other chemicals, as electrolytes and non-electrolytes. Journal of Experimental well as the pH of stimulating solution, may influence the Biology, 60, 275–293. responses of insect taste receptors to one or another stimu- Daly, P.J. & Ryan, M.F. (1979) Ultrastructure of antennal sensilla of Nebria brevicollis (Fab.) (Coleoptera: Carabidae). Interna- lating compound (Hanson, 1987; Liscia et al., 1997; Bernays tional Journal of Insect Morphology and Embryology, 8, 169–181. et al., 1998; Liscia & Solari, 2000; Bernays & Chapman, den Otter, C.J. (1972) Differential sensitivity of insect chemo- 2001b; Schoonhoven & van Loon, 2002). Presumably, receptors to alkali cations. Journal of Insect Physiology, 18, salts of stimulating buffer solutions at high concentration 109–131. suppressed the neural activity of pH cell when stimulated Dethier, V.G. (1977) The taste of salts. American Scientist, 65, with 100 mM phosphate buffer in 10 and 100 mM phosphate 744–751. buffer experiments in P. oblongopunctatus. Dethier, V.G. & Hanson, F.E. (1965) Taste papillae of the blowfly. The pH cell of antennal taste sensilla in P. oblongopunc- Journal of Cellular and Comparative Physiology, 65, 93–100. tatus does not fire, or fires at very low frequency (first Dethier, V.G. & Hanson, F.E. (1968) Electrophysiological second response below 5 imp/s) at pH 3–6. This acid range responses of the blowfly to sodium salts of fatty acids. Proceedings of the National Academy of Sciences, U.S.A., 60, of pH values most likely represents the pH values in the 1269–1303. forest habitat and hibernating sites of this ground beetle Elizarov, Y.A. (1978) Insect Chemoreception. Moscow University (Thiele, 1977; Paje & Mossakowski, 1984). Press, Russia. It is noteworthy that the action potentials registered by Elizarov, Y.A. & Sinitzina, E.E. (1974) Contact chemoreceptors in antennal taste bristles’ cation and pH cells in the ground Aedes aegypti L. Zoological Journal, 53, 577–584 (in Russian). beetle P. oblongopuctatus are negative and positive in their Evans, D.R. & Mellon, D. (1962) Stimulation of a primary taste polarity, respectively. Furthermore, other electrophysiolo- receptor by salts. Journal of General Physiology, 45, 651–661. gists studying insects’ chemoreceptors have noticed that Frazier, J.L. & Hanson, F.E. (1986) Electrophysiological recording spikes in sensillum tip-recording technique (Hodgson and analysis of insect chemosensory responses. Insect–Plant et al., 1955) may have positive as well as negative polarity Interactions (ed. by T. A. Miller and J. Miller), pp. 285–330. Springer, Germany. (Hanson & Wolbarsht, 1962; Dethier & Hanson, Hansen-Delkeskamp, E. (2001) Responsiveness of antennal taste 1965; Elizarov & Sinitzina, 1974; Elizarov, 1978; Hansen- hairs of the apterygotan insect, Thermobia domestica (Zygento- Delkeskamp, 2001; Albert, 2003). Because the sensillum is ma); an electrophysiological investigation. Journal of Insect an integral part of the recording circuit, any cellular Physiology, 47, 689–697. properties and changes may contribute to uncontrolled Hanson, F.E. (1987) Chemoreception in the fly: the search for variability in the shape of the recorded action potentials the liverwurst receptor. Perspectives in Chemoreception (Frazier & Hanson, 1986). At present, there appears to be and Behaviour (ed. by R. F. Chapman, E. A. Bernays and no satisfactory explanation for this phenomenon. J. G. Stoffolano), pp. 99–122. Springer, Germany. Hanson, F.E. & Wolbarsht, K.L. (1962) Dendritic action potentials in insect chemoreceptors. American Zoologist, 2, 528–534. Haskell, P.T. & Schoonhoven, L.M. (1969) The function of certain mouth part receptors in relation to feeding in Schistocerca Acknowledgements gregaria and Locusta migratoria migratorioides. Entomologia Experimentalis et Applicata, 12, 429–440. Contract grant sponsor: Estonian Science Foundation; Hodgson, E.S., Lettvin, J.Y. & Roeder, K.D. (1955) Physiology of Contract grant numbers: 5423 and 4105. a primary chemoreceptor unit. Science, 122, 417–418. Ishikawa, S. (1963) Responses of maxillary chemoreceptors in the larvae of the silkworm, Bombyx mori,tostimulationby carbohydrates. Journal of Cellular and Comparative Physiology, References 61, 99–107. Kim, J.L. & Yamasaki, T. (1996) Sensilla of Carabus (Isiocarabus) Albert, P.J. (2003) Electrophysiological responses to sucrose from a fiduciarius saishutoicus Csiki (Coleoptera: Carabidae). Interna- gustatory sensillum on the larval maxillary palp of the spruce tional Journal of Insect Morphology and Embryology, 25, budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: 153–172. Tortricidae). Journal of Insect Physiology, 49, 733–738. Liscia, A. & Solari, P. (2000) Bitter taste recognition in the blowfly: Bernays, E.A. & Chapman, R.F. (2001a) Taste cell responses in the Electrophysiological and behavioral evidence. Physiology and polyphagous arctiid, Grammia geneura: towards a general pattern Behaviour, 70, 61–65. for caterpillars. Journal of Insect Physiology, 47, 1029–1043. Liscia, A., Solari, P., Majone, R., Tomassini Barbarossa, I. & Bernays, E.A. & Chapman, R.F. (2001b) Electrophysiological Crnjar, R. (1997) Taste receptor mechanisms in the blowfly: responses of taste cells to nutrient mixtures in the polyphagous evidence of amiloride-sensitive and insensitive receptor sites. caterpillar of Grammia geneura. Journal of Comparative Physiol- Physiology and Behavior, 62, 875–879.7. ogy A, 187, 205–213. Merivee, E., Ploomi, A., Rahi, M., Luik, A. & Sammelselg, V. Bernays, E.A., Glendinning, J.I. & Chapman, R.F. (1998) Plant (2000) Antennal sensilla of the ground beetle Bembidion lampros acids modulate chemosensory responses in Manduca sexta Hbst (Coleoptera, Carabidae). Acta Zoologica (Stockholm), 81, larvae. Physiological Entomology, 23, 193–201. 339–350.

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Merivee, E., Ploomi, A., Luik, A., Rahi, M. & Sammelselg, V. Paje, F. & Mossakowski, D. (1984) pH-preferences and habitat (2001) Antennal sensilla of the ground beetle Platynus dorsalis selection in carabid beetles. Oecologia (Berlin), 64, 41–46. (Pontoppidan, 1763) (Coleoptera, Carabidae). Microscopy Schoonhoven, L.M. & van Loon, J.J.A. (2002) An inventory of Research and Technique, 55, 339–349. taste in caterpillars: each species its own key. Acta Zoologica Merivee, E., Ploomi, A., Rahi, M., Bresciani, J., Ravn, H.P., Luik, A. & Academiae Scientiarum Hungarica, 48, 215–263. Sammelselg, V. (2002) Antennal sensilla of the ground beetle Bembidion Shiraishi, A. & Morita, H. (1969) The effects of pH on the labellar properans Steph. (Coleoptera, Carabidae). Micron, 33, 429–440. sugar receptor of the fleshfly. Journal of General Physiology, 53, Mitchell, B.K. & Schoonhoven, L.M. (1973) Taste receptors 450–470. in Colorado beetle larvae. Journal of Insect Physiology, 20, Thiele, H.-U. (1977) Carabid beetles in their environment. 1787–1793. Zoophysiology and Ecology 10. Springer, Germany. Mitchell, B.K. & Seabrook, W.D. (1973) Electrophysiological investigations on tarsal chemoreceptors of the spruce budworm, Choristoneura fumiferana (Lepidoptera). Journal of Insect Physiology, 20, 1209–1218. Accepted 20 October 2004

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66 II Milius, M., Merivee, E., Williams, I., Luik, A., Mänd, M., Must, A., 2006.

A NEW METHOD FOR ELECTROPHYSIOLOGICAL IDENTIFICATION OF ANTENNAL PH RECEPTOR CELLS IN GROUND BEETLES: THE EXAMPLE OF PTEROSTICHUS AETHIOPS (PANZER, 1796) (COLEOPTERA, CARABIDAE).

Journal of Insect Physiology 52, 960–967. ARTICLE IN PRESS

Journal of Insect Physiology 52 (2006) 960–967 www.elsevier.com/locate/jinsphys

A new method for electrophysiological identiﬁcation of antennal pH receptor cells in ground beetles: The example of Pterostichus aethiops (Panzer, 1796) (Coleoptera, Carabidae) Ã Marit Miliusa, Enno Meriveea, , Ingrid Williamsb, Anne Luika, Marika Ma¨nda, Anne Musta

aEstonian University of Life Sciences, Institute of Agricultural and Environmental Sciences, 64 Kreutzwaldi Street, 51014 Tartu, Estonia bRothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK

Received 20 March 2006; received in revised form 2 June 2006; accepted 7 June 2006

Abstract

The responses of antennal taste sensilla of the ground beetle Pterostichus aethiops to 100 mM Na+-salts and their mixtures with 1 and 10 mM NaOH were compared. An increase in pH by 0.3–0.6 units in 100 mM Na+-salt solutions, caused by the content of 1 mM NaOH, was too small, except for alkaline Na2HPO4, to influence the firing rate of the cation cell and pH cell significantly. However, different sensitivity of the two cells to increased pH was clearly demonstrated when the concentration of NaOH in 100 mM stimulating salt solutions was increased to 10 mM. Increasing pH by 1.2–2 units caused the 1st s firing rate to increase by 140–1050% and 0–26% in the pH cell and cation cell, respectively. Compared to the buffer series method used for identification of the pH receptors in ground beetles earlier, considerably stronger responses of the pH cell to a similar increase in pH were observed when the NaOH method was used for testing. At the same time, undesirable changes in salt ions concentration that occur when stimulating solutions differing by 1–2 pH units are prepared were much smaller using the latter method. Behavioural and ecological relevance of the results is discussed. r 2006 Elsevier Ltd. All rights reserved.

Keywords: Taste sensilla; Tip recording; Action potential rate; Predatory insects; Habitat preferences

1. Introduction External stimuli crucial in habitat choice of ground beetles have been purely studied on the receptor level, however. The ground beetles (Coleoptera, Carabidae) with 40,000 Preferences for particular soil pH, varying between 3 and species belong to the most numerous families of beetles, 9, in ground beetles were discovered using laboratory their distribution being cosmopolitan. Approximately 430 choice experiments some decades ago (Krogerus, 1960; species have been found in Fennoscandia, Denmark and Paje and Mossakowski, 1984). More recently, in ecological Baltic countries, 530 species occur in Czech Republic field experiments, a strong correlation between the (Lo¨vei and Sunderland, 1996; Kula and Purchart, 2004; abundance of some ground beetles and soil pH has been Silferberg, 2004). Their bio-indication importance is found (Irmler, 2001, 2003). Electrophysiological studies derived from their dependence on specific biotic and with alkaline, neutral and acid salts have indicated that in abiotic conditions of the site: food availability, competi- addition to the salt (cation) cell, the pH cell also probably tors, vegetation type, humidity, light and temperature innervates the antennal taste bristles in the ground beetle conditions, soil pH and salt content etc. Habitat choice is Pterostichus aethiops (Panzer, 1796) (Merivee et al., 2004). so specific that carabids are often used to characterize Experiments with acetate and phosphate buffer series in the habitats (Thiele, 1977; Lo¨vei and Sunderland, 1996). pH range of 3–11 confirmed this assumption in the ground beetle Pterostichus oblongopunctatus (Fabricius, 1787) (Merivee et al., 2005). The pH cell discovered seems to be Ã Corresponding author. specific to ground beetles, as it is not found in other insects. E-mail address: [email protected] (E. Merivee). In order to explain the role of this cell in the beetles’

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M. Milius et al. / Journal of Insect Physiology 52 (2006) 960–967 961 behaviour more precisely, further electrophysiological Addition of NaOH to these salt solutions does not change studies on the distribution and functioning of the pH- their ionic composition and the Na+ concentration receptive cell in ground beetles are justified. increases only to a small extent (1% and 10%, respec- P. aethiops is a European species found from the British tively). At the same time, the pH level of these solutions Isles east to Perm in Russia, south to Serbia and north into rises substantially. The stimulating effect of these sodium southern Fennoscandia and Finland. In mountains of salt solutions with and without NaOH was expected to be Central and Eastern Europe their abundance considerably approximately similar for the salt cell but not for the pH increases with increasing altitude up to subalpine zones. cell if present. Results of these experiments are reported in This spring-breeding, wingless, eurytopic ground beetle this paper. The size, number and location of antennal taste populates wet coniferous, deciduous as well as mixed bristles in the ground beetle P. aethiops have been forests with acid soil pH. Also, its preferred overwintering described earlier (Merivee et al., 2004). sites in brown-rotted wood laying on the ground where, in autumn, the beetles concentrate in great numbers are 2. Material and methods strongly acid (pH 3–5; Merivee, unpublished data). Frequently, hibernating beetles have been found in moss 2.1. Test beetles and litter. In its preferred habitats, it is one of the most numerous ground beetles (Haberman, 1968; Thiele, 1977; The specimens of P. aethiops used in the experiments Lindroth, 1985, 1986; Wachmann et al., 1995; Ermakov, were collected in May 2005 in southern Estonia. They were 2004; Kula and Purchart, 2004; Silferberg, 2004; Sk"o- stored in plastic boxes filled with moistened moss and sand dowski, 2005). in a refrigerator at +5 1C. Two or three days before In P. oblongopunctatus, it was demonstrated that the pH electrophysiological tests the beetles were transferred to cell did not fire or fired at very low frequency at pH 3–6, similar boxes at room temperature (+22 1C). They were first s response below 5 imp/s declining during the next four given clean water to drink and fed with moistened cat food s of the stimulation close to zero. This level of acidity (Kitekat, Master Foods, Poland) every day. probably represents the pH preferences of this ground For the electrophysiological tests, each test beetle was beetle in its forest habitat and hibernating sites (Merivee restrained by placing it into a conical tube made of thin et al., 2005). In this study we show that similar correlations sheet of aluminium of a size that allowed its head and between electrophysiological responses of the pH cell and antennae to protrude a little from the narrower end. The pH of the preferred habitats may occur also in the ground wider rear end of the conical tube was closed with a piece of beetle P. aethiops. plasticine to prevent the beetle from retreating out of the The limitation of the buffer series method used for tube. The antennae of the restrained beetle were fastened testing the responses of pH receptor cells to pH in ground horizontally on the edge of a special aluminium stand with beetles earlier (Merivee et al., 2005) is that buffers with tiny amounts of beeswax, so that horizontally located different pH vary largely in both pH and electrolyte antennal taste bristles were visible from above under a light concentration. The NaOH method reported here allows to microscope, and easily accessible for micromanipulations prepare stimulating salt solutions over a wide range of pH from the side. Contamination of tested taste bristles and whereas changes in electrolyte concentrations are minimal. their contact with preparation instruments and beeswax Over the dynamic range of the stimulus/response curve, the was carefully avoided. action potential rate of a taste cell is a logarithmic function of the stimulus concentration. The maximum firing rate of 2.2. Chemicals and preparation of stimulating solutions a phasic-tonically responding taste cell evoked by strong stimuli does usually not exceed 40–50 imp/s (Evans and Chemicals of analytical grade of purity used in the Mellon, 1962; Hanson, 1987; Merivee et al., 2004). experiments were obtained from AppliChem (Germany). Consequently, a 1–10% increase in concentration of Stimulating test solutions prepared are shown in Table 1. the stimulating salt solution causes a change of less than pH of the solutions ranging from 4.6 to 10.6 was measured 1 imp/s in the firing rate of the taste cell, which is hardly with the pH meter E6115 (Evikon, Estonia). detectable in electrophysiological experiments. These cal- culations were taken into account when designing the 2.3. Stimulation and recording of action potentials current experiments. To test for the presence of the pH cell in the antennal Electrophysiological recordings were made from the taste bristles of the ground beetle P. aethiops more large chaetoid taste sensilla on the antennal flagellum of the + precisely, three solutions of 100 mM Na -salts, Na2HPO4, restrained beetle using a tip-recording technique (Hodgson NaCl and NaH2PO4 were prepared, acid, neutral and et al., 1955). To achieve a good signal-to-noise ratio and alkaline, respectively; these served as control stimuli. Test avoid registration of muscular activities from two basal stimuli were prepared by adding small amounts of NaOH antennomeres, the scape and pedicel and from other parts to the 100 mM salts resulting in three electrolyte mixtures of insect’s body, an indifferent tungsten microelectrode was with 1 mM NaOH and three mixtures with 10 mM NaOH. inserted into the base of the flagellum. The recording

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Table 1 the two successive stimulations ensured complete disadap- Stimulating solutions prepared for the experiments tation of the receptor cells allowing similar responses in our preliminary experiments to several successive stimulations Stimulating solution Concentration (mM) pH with the same stimulus. Taste bristles from 30 to 69 beetles

1. NaH2PO4 100 4.6 were tested in each pair of stimuli. 2. NaH2PO4 100 4.9 NaOH 1 2.4. Data management and analysis 3. NaH2PO4 100 5.8 NaOH 10 4. NaCl 100 7.6 Action potentials from several sensory cells of taste 5. NaCl 100 8.2 sensilla were distinguished by differences in their ampli- NaOH 1 tude, shape and polarity of spikes. Automated classiﬁca- 6. NaCl 100 9.6 NaOH 10 tion and counting of action potentials was clearly not an

7. Na2HPO4 100 8.6 appropriate method for data analysis because action 8. Na2HPO4 100 9.0 potentials from two or three cells frequently interacted to NaOH 1 produce an irregular waveform. Instead, the analysis used 9. Na2HPO4 100 10.6 was visual, employing TestPoint software. Firing rates of NaOH 10 receptor cells were expressed as a number of spikes per second. All data were analysed using Microsoft Excel (Microsoft Corp., Redmond, Washington) and Statistica microelectrode, a glass micropipette with a tip diameter of (StatSoft, Inc., Tulsa, Oklahoma) software. approximately 20 mm filled with stimulating solution, was placed over the sensillum tip by means of a micromanipu- 3. Results lator under visual control with a light microscope at Â magnification 300. Immediately before each stimulation, Two sensory cells from the antennal taste bristles two to three drops of the stimulating solution were responded to the tested electrolytes in a phasic-tonic squeezed out of the micropipette tip, and adsorbed onto manner. These two cells were the cation and the pH cells a piece of filter paper attached to an additional micro- distinguished by the amplitude and polarity of action manipulator to reduce concentration increases due to evaporation. A copper wire made contact with a high- impedance preamplifier input. The signal picked up by the recording electrode was amplified and filtered with a band width set at 100–2000 Hz and monitored on an oscilloscope screen. Recordings of the first 5 s of the response were transferred directly to a computer hard disc via an analogue-to-digital input board DAS-1401 (Keithley, Taunton, Massachusets) for data acquisition, storage and further analysis using TestPoint (Capital Equipment Corporation, Bedford, New Hampshire) software. The rate of sampling data was 10 kHz. The first s firing rate includes initial phasic burst of action potentials. Four next s characterize the following low decline in the rate of firing of the sensory cells tested. Due to variations in responsiveness of different taste bristles to the same stimuli, stimulating solutions were tested in pairs to allow more precise comparison of the responses to salt solutions with and without NaOH. Thus, each taste bristle was tested twice. Responses of one taste bristle of each test beetle were recorded. To eliminate the possible effect of stimulation order to the responses, 100 mM Na+-salt alone and that containing NaOH alternatively were chosen for the first stimulus. No deteriorating or disturbing effect on the receptor cells Fig. 1. Example recordings from antennal taste sensilla of the ground caused by used low content of NaOH in stimulating salt beetle Pterostichus aethiops in response to stimulating electrolyte solutions solutions was observed. Then, after approximately 10 min, with different pH. CC and pHC show action potentials from the cation the stimulating/recording micropipette was rinsed with cell and pH cell, respectively. Arrows indicate the beginning of the stimulation. Calibration bar: 5 mV. (A) Response to 100 mM NaCl distilled water several times and filled with the second (pH 7.6). (B) Response to the mixture of 100 mM NaCl and 10 mM NaOH stimulus to test the same sensillum again. The time between (pH 9.6).

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M. Milius et al. / Journal of Insect Physiology 52 (2006) 960–967 963 potentials they generated. The phasic component of the the cation cell. Usually NaCl was the most stimulating salt response was more pronounced in the cation cell compared for this cell. When the responses to the three Na+-salts to the pH cell. The action potentials with positive polarity with and without NaOH were compared with each other, it from the pH cell were very large, 7–8 mV peak-to-peak, became evident that the presence of 1 mM NaOH in the whereas those with negative polarity generated by the 100 mM salts had no effect on the ﬁring rate of the cation cation cell were 3–4 times smaller (Fig. 1). cell, except for the 1st s response in the test series with NaCl, where a few more action potentials were generated 3.1. Responses to 100 mM Na+-salts with and without by the mixture of this salt and NaOH compared to NaCl addition of 1 mM NaOH alone (Fig. 2).

The stimulating effect of the 100 mM salts tested on the 3.2. Responses to 100 mM Na+-salts with and without of pH cell, depended on their pH. Salt solutions with higher 10 mM NaOH pH evoked stronger responses. Most test beetles did not respond to the acid 100 mM NaH2PO4 (pH 4.6) nor to the Differences between the pH and cation cells regarding mixture (pH 4.9) of this 100 mM salt and 1 mM NaOH; their responsiveness to the pH of the stimulating solutions respective mean firing rates during the 1st s of the response became much more obvious when the concentration of were below 1 imp/s. The stimulating effects of 100 mM NaOH in the alkalized test salt was raised from 1 to NaCl (pH 7.6) and Na2HPO4 (pH 8.6) solutions were 10 mM. In the pH cell, rates of firing evoked by the mixture + considerably higher compared to NaH2PO4, with 1st s of 100 mM Na -salts and 10 mM NaOH were significantly firing rates of 9 and 13 imp/s, respectively. During the higher compared to the responses caused by the salts alone following seconds these rates of firing fell 2–3 times. No (Fig. 3). The stimulating effect of NaOH to the pH cell was significant differences were observed between the firing greatest in the mixture with NaCl (pH 9.6), firing rate rates of the pH cell caused by 100 mM NaCl and the during the 1st s of the response was 31 imp/s and it did not mixture (pH 8.2) of 100 mM NaCl and 1 mM NaOH. fall below 17 imp/s also during the next several seconds Significantly more action potentials were generated by the recorded. The difference compared to the response caused pH cell in response to the solution of 100 mM Na2HPO4 by 100 mM NaCl alone was 10–22 times depending on the and 1 mM NaOH (pH 9.0), however, compared to the response time. The mixtures of 10 mM NaOH with response evoked by 100 mM Na2HPO4 alone (Fig. 2). 100 mM NaH2PO4 (pH 5.8) and Na2HPO4 (pH 10.6) were By contrast, the relationship between the salt pH and the 2–7 times more effective stimuli for the pH cell compared number of action potentials generated was not observed in to these non-alkalized salts (pH 4.6 and 8.6, respectively).

Fig. 2. Responses of the pH cell and cation cell of the antennal taste sensilla of the ground beetle P. aethiops to various 100 mM Na+-salts and their mixtures with 1 mM NaOH. Vertical bars denote 7SE of the means. Asterisks indicate signiﬁcantly different means: paired t-test, Po0.05.

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Fig. 3. Responses of the pH cell and cation cell of the antennal taste sensilla of the ground beetle P. aethiops to various 100 mM Na+-salts and their mixtures with 10 mM NaOH. Vertical bars denote 7SE of the means. Asterisks indicate signiﬁcantly different means: paired t-test, Po0.05. Note the different y-scales.

The presence or absence of 10 mM NaOH in the cell, the other a pH cell. The salt as well as the pH receptors stimulating salt solution had little or no effect on the rate seem to be common in ground beetles and are probably of ﬁring of the cation cell. However, a 10–40% increase in related to habitat choice by these ground-dwelling insects the ﬁring rate was observed in response to the mixture of (Thiele, 1977; Paje and Mossakowski, 1984; Irmler, 2001, 10 mM NaOH with 100 mM NaH2PO4 and NaCl at the 2003). Surprisingly, no pH receptors have been found in beginning of the 5 s stimulation period (Fig. 3). other insects so far. This is probably due to the fact that insect’ gustation has been less studied than their olfaction 4. Discussion (Chapman, 1998; Hansson, 1999; Blomquist and Vogt, 2003; Christensen, 2005). Instead, in some other insects, two The results of the electrophysiological experiments sensory cells of a taste sensillum respond to stimulating salt conducted with Na+-salts and Na+-alkali solutions in the solutions (Dethier and Hanson, 1968; Haskell and Schoon- ground beetle P. aethiops agree well with earlier data on hoven, 1969; Den Otter, 1972; Mitchell and Schoonhoven, contact chemoreception of ground beetles, obtained from 1973; Mitchell and Seabrook, 1973; Blaney, 1974; Hanson, experiments with various salts (Merivee et al., 2004) and 1987; Bernays and Chapman, 2001a; Schoonhoven and van buffers (Merivee et al., 2005). Accordingly, two sensory Loon, 2002) but it is believed that they function as an anion cells of the taste bristles located on the antennae of ground and a cation cell (Ishikawa, 1963; Elizarov, 1978; Hanson, beetles respond to electrolytes: one of them is a salt (cation) 1987; Schoonhoven and van Loon, 2002).

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In addition to the specific stimulus, also frequently material on the soil surface. Many of the materials named the best stimulus, other chemical factors such as composted contain significant amounts of protein, which ionic composition and concentration of electrolytes, the is converted to ammonia toxic to many organisms and presence of non-electrolytes, and pH of the stimulating serving as the primary substrate in the nitrification solution, may all influence the response of different kinds processes. Nitrification processes have been observed at of insect contact chemoreceptive cells to some extent pH levels ranging from 6.6 to 9.7 (Odell et al., 1996). (Hanson, 1987; Liscia et al., 1997; Bernays et al., 1998; Obviously, the ground beetle P. aethiops avoids these Liscia and Solari, 2000; Bernays and Chapman, 2001b; places with a high pH. Schoonhoven and van Loon, 2002). In this context, it is not Six to seven months, from September to April, adults of surprising that the response of the antennal cation cell of P. aethiops may spend in its preferred overwintering sites in P. aethiops, in some cases, was also increased by 17–40% brown-rotted wood in Estonia. Fungi often lower the pH by pH increase up to two pH units of the stimulating of the extracellular medium through secretion of organic solution, which is some thirty times smaller increase in acids and thus establish a pH gradient around the firing rate than was observed in the pH cell responses under mycelium. The organic acid oxalate is produced by most, the same stimulating conditions. Similar stimulatory effect if not all, wood-degrading fungi. Usually, pH 3–6 is of pH to the cation cell responses was observed in observed in both white- and brown-rotted wood. Stable electrophysiological experiments with acetate and phos- moisture content is quaranteed as a result of metabolic phate buffers also in the ground beetle P. oblongopunctatus water production during the wood-degrading process. For (Merivee et al., 2005). cultures of white-rot fungus Merulius tremellosus incubated Our tests demonstrated that an increase in pH by 0.3–0.6 in air, the optimum water content was 2 g/1 g of wood. units in 100 mM Na+-salt solutions, caused by the 1 mM Moister conditions were less favourable to delignification, NaOH content, was too small, except for Na2HPO4,to possibly because the water impeded aeration (Reid, 1985; influence the firing rate of the pH cell or cation cell Varela and Tien, 2003). Similar prolonged stability of significantly. However, different sensitivity of the two cells water content and aeration is probably also quaranteed in to increased pH was clearly demonstrated when the brown-rotting wood which seems to be a favourable concentration of NaOH in 100 mM stimulating salt microenvironment to ground beetles for their prolonged solutions was increased to 10 mM resulting in pH increase hibernation. by 1.2–2 units depending on the salt. Compared to the Brown-rotted wood may offer good protection for acetate and phosphate buffers method (Merivee et al., hibernating ground beetles against entomopathogenic 2005), a comparable increase in pH by 1–2 units of the fungi and parasites which play an important role in their stimulatory salt solution using the method of adding small population dynamic. Up to 41% parasitism by nematodes amounts of NaOH to 100 mM Na+-salts evoked a and ectoparasitic fungi was found on 14 species of considerably larger increase in the rate of firing of the pH Bembidion in Norway depending on habitat selection cell: respective rates of firing during the 1st s of the of the host (Andersen and Skorping, 1991). Eggs of response increased by 30–400% and 140–1050%, respec- P. oblongopunctatus suffered 83% mortality in fresh litter tively, depending on buffers and salts used. but only 7% in sterilized soil (Heessen, 1981). Although In the stimulating solutions differing by 1.2–2 pH units, predators, parasites, and pathogens affect all ground beetle and using the NaOH method, the difference in Na+ developmental stages, quantitative data remain scarce. So, concentrations is only 10% and the salt anion concentra- antennal pH receptors of the ground beetles P. aethiops tion does not change. By contrast, using the method of seem to be a powerful tool in their selection for favourable buffers, respective concentrations of content ions may acid habitats and microhabitats crucial to survive. differ up to 30 times, depending on the pH range and salts Chemically mediated habitat selection has been shown to involved. Taking into consideration that both salt cations occur in several species of ground beetles by Evans (1982, and anions of the stimulating solution may affect the pH 1983, 1984, 1988). He demonstrated that olfactory cells cell firing rate, large differences in the concentration of salt receptive to methyl esters of palmitic and oleic acid emitted ions are undesirable when testing pH cell responses to pH. by the mat-forming blue-green algae Oscillatoria animalis Thus, it seems that the NaOH method is, besides the Agardh and Oscillatoria subbrevis Schmidle are present in buffers method, a reliable tool for studying pH receptor sensillae on the antennae of those Bembidiini associated functioning in insects. with the shores of saline lakes. Low concentrations of Our results suggest that in P. aethiops, in its preferred formic acid, a defence secretion in the ground beetle acid forest habitats and overwintering sites in brown-rotted Harpalus rufipes, was shown by Luff (1986) to cause wood at pH 3–5, the antennal pH sensitive cell does not aggregation in this species. Formic acid is also produced by discharge or discharges at very low frequency with the first some wood-decaying fungi as a result of enzymatic s firing rate close to 1 imp/s or lower. Areas with a higher decarboxylation of oxalic acid accumulating in large pH seem to be unfavourable to this insect and when quantities in the cell sap of various plants and as a major contacted the pH cell signals with a stronger response. This product of carbohydrate metabolism by many molds may occur, for example, in places with decaying plant (Shimazono and Hayaishi, 1957). Probably, formic acid

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966 M. Milius et al. / Journal of Insect Physiology 52 (2006) 960–967 could be perceived and used for long-distance orientation Hansson, B.S. (Ed.), 1999. Insect Olfaction. Springer, Berlin, Heidelberg, and aggregation in brown-rotted wood by P. aethiops as New York, Barcelona, Hong Kong, London, Milan, Paris, Singapore, well. Tokyo, p. 457. Haskell, P.T., Schoonhoven, L.M., 1969. The function of certain mouth part receptors in relation to feeding in Schistocerca gregaria and Acknowledgements Locusta migratoria migratorioides. Entomologia Experimentalis et Applicata 12, 429–440. Contract grant sponsor: Estonian Science Foundation; Heessen, H.J.L., 1981. Egg mortality in P. oblongopunctatus (Coleoptera, Contract Grant No. 5423 and 5736. Carabidae). Oecologia 50, 233–235. Hodgson, E.S., Lettvin, J.Y., Roeder, K.D., 1955. Physiology of a primary chemoreceptor unit. Science 122, 417–418. References Irmler, U., 2001. Characterisierung der Laufka¨fergemeinschaften schleswig-holsteinischer Wa¨lder und Mo¨glichkeiten ihrer o¨kologischen Andersen, J., Skorping, A., 1991. Parasites of carabid beetles: prevalence Bewertung. Angewandte Carabidologie. Laufka¨fer im Wald depends on habitat selection of the host. Canadian Journal of Zoology (Suppl. 2), 21–32. 69, 1216–1220. Irmler, U., 2003. The spatial and temporal pattern of carabid beetles on Bernays, E.A., Chapman, R.F., 2001a. Taste cell responses in the arable fields in northern Germany (Schleswig–Holstein) and their value polyphagous arctiid, Grammia geneura: towards a general pattern for as ecological indicators. Agriculture, Ecosystems & Environment 98, caterpillars. Journal Insect Physiology 47, 1029–1043. 141–151. Bernays, E.A., Chapman, R.F., 2001b. Electrophysiological responses Ishikawa, S., 1963. Responses of maxillary chemoreceptors in the of taste cells to nutrient mixtures in the polyphagous caterpillar larvae of the silkworm, Bombyx mori, to stimulation by carbohy- of Grammia geneura. Journal of Comparative Physiology A 187, drates. Journal of Cellular and Comparative Physiology 61, 205–213. 99–107. Bernays, E.A., Glendinning, J.I., Chapman, R.F., 1998. Plant acids Krogerus, R., 1960. O¨kologische Studien u¨ber nordische Moorarthropo- modulate chemosensory responses in Manduca sexta larvae. Physio- den. Societas Scientiarum. Fennica, Commentationes Biologicae 21 logical Entomology 23, 193–201. (3), 1–238. Blaney, W.M., 1974. Electrophysiological responses of the terminal Kula, E., Purchart, L., 2004. The ground beetles (Coleoptera: Carabidae) sensilla on the maxillary palps of Locusta migratoria (L.) to some of forest altitudinal zones of the eastern part of the Krusˇne´hory Mts. electrolytes and non-electrolytes. 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Methods in Insect Sensory Neuroscience. 1997. Taste receptor mechanisms in the blowfly: evidence of amiloride- CRC Press, Boca Raton, London, New York, Washington, p. 435. sensitive and insensitive receptor sites. Physiology & Behavior 62, Den Otter, C.J., 1972. Differential sensitivity of insect chemoreceptors to 875–879. alkali cations. Journal of Insect Physiology 18, 109–131. Lo¨vei, G.L., Sunderland, K.D., 1996. Ecology and behavior of ground Dethier, V.G., Hanson, F.E., 1968. Electrophysiological responses of the beetles (Coleoptera:Carabidae). Annual Review of Entomology 41, blowfly to sodium salts of fatty acids. Proceedings of the National 231–256. Academy of Sciences, USA 60, 1269–1303. Luff, M.L., 1986. Aggregation of some Carabidae in pitfall traps. In: den Elizarov, Yu.A., 1978. Insect Chemoreception. Moscow University Press, Boer, P.J. (Ed.), Carabid Beetles. Gustav Fisher, Stuttgart, Moscow, p. 232. (in Russian). pp. 385–397. Ermakov, A.I., 2004. Structural changes in the carabid fauna of forest Merivee, E., Renou, M., Ma¨nd, M., Luik, A., Heidemaa, M., Ploomi, A., ecosystems under a toxic impact. Russian Journal of Ecology 35, 2004. Electrophysiological responses to salts from antennal chaetoid 403–408. taste sensilla of the ground beetle Pterostichus aethiops. Journal of Evans, D.R., Mellon, D., 1962. Stimulation of a primary taste receptor by Insect Physiology 50, 1001–1013. salts. Journal of General Physiology 45, 651–661. Merivee, E., Ploomi, A., Milius, M., Luik, A., Heidemaa, M., 2005. Evans, W.G., 1982. Oscillatoria sp. (Cyanophyta) mat metabolites Electrophysiological identification of antennal pH-receptors in the implicated in habitat selection in Bembidion obtusidens (Coleopt.: ground beetle Pterostichus oblongopunctatus (Coleoptera, Carabidae). Carabidae). Journal of Chemical Ecology 8, 671–678. Physiological Entomology 30, 122–133. Evans, W.G., 1983. Habitat selection in the Carabidae. 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76 III Merivee, E., Must, A., Milius, M., Luik, A., 2007.

ELECTROPHYSIOLOGICAL IDENTIFICATION OF THE SUGAR CELL IN ANTENNAL TASTE SENSILLA OF THE PREDATORY GROUND BEETLE PTEROSTICHUS AETHIOPS.

Journal of Insect Physiology 53(4), 377–384. ARTICLE IN PRESS

Journal of Insect Physiology 53 (2007) 377–384 www.elsevier.com/locate/jinsphys

Electrophysiological identiﬁcation of the sugar cell in antennal taste sensilla of the predatory ground beetle Pterostichus aethiops Ã Enno Merivee , Anne Must, Marit Milius, Anne Luik

Estonian University of Life Sciences, Institute of Agricultural and Environmental Sciences, 64 Kreutzwaldi Street, 51014 Tartu, Estonia

Received 6 October 2006; received in revised form 14 December 2006; accepted 21 December 2006

Abstract

By single sensillum tip recording technique, in addition to the salt and pH cells found in antennal taste sensilla of some ground beetles earlier, the third chemosensory cell of four innervating these large sensilla was electrophysiologically identified as a sugar cell in the ground beetle Pterostichus aethiops. This cell generated action potentials of considerably smaller amplitude than those of the salt and pH cells, and phasic-tonically responded to sucrose and glucose over the range of 1–1000 mM tested. Responses were concentration dependent, with sucrose generating more spikes than glucose. During the first second of the response, maximum rates of firing of the sugar cell reached up to 19 and 37 imp/s when stimulated with 1000 mM glucose and sucrose, respectively. Three to four seconds later, the responses decreased close to zero. Both sugars are important in plant carbohydrate metabolism. These ground dwelling insects may come into contact with live and decayed plant material everywhere in their habitat including their preferred overwintering sites in brown- rot decayed wood. In conclusion, we hypothesize that high content of soluble sugars in their overwintering sites and refugia is unfavourable for these ground beetles, most probably to avoid contact with dangerous fungi. r 2007 Elsevier Ltd. All rights reserved.

Keywords: Ground beetles; Antennal taste sensilla; Sugar cell; Tip recording technique; Habitat selection; Brown-rotted wood; Hibernation

1. Introduction insects suggesting that it is characteristic for other ground beetles too. In the ground beetles Pterostichus aethiops and Most electrophysiologists studying insect contact che- Pterostichus oblongopunctatus, two chemoreceptive cells moreception have focused on taste sensilla of herbivores have been electrophysiologically identified: they are the salt (Chapman, 1998,2003; Schoonhoven and van Loon, 2002) (cation) cell and pH cell probably involved in choice of and some other insect groups (van der Starre, 1972; Ru¨th, habitat and overwintering sites of these insects (Merivee 1976; Hanson, 1987; Hansen-Delkeskamp, 1992; Murata et al., 2004, 2005; Milius et al., 2006). The occurrence of the et al., 2002), related to their feeding and oviposition. pH receptive cell in the sensillar apparatus seems to be Usually, these sensilla are located on mouthparts, anten- unique for ground beetles, as it is not found in other nae, ovipositor and tarsi. Very little is known about insects. Stimulating chemical compounds, specific for other functioning of taste sensilla in predatory insects such as two chemoreceptive cells innervating antennal taste sensilla ground beetles (Carabidae). In the tribes Platynini, of ground beetles, are not known. Pterostichini and Bembidiini, approximately 70 taste Besides other chemosensory cells, phagostimulatory bristles are located on the antennae of adults (Merivee sugar cells are present in all phytophagous insects that have et al., 2000, 2001, 2002). Four chemoreceptive and one been studied and are almost certainly present universally mechanoreceptive cell innervate these sensilla in the ground (Schoonhoven and van Loon, 2002; Chapman, 2003). Sugar beetle Nebria brevicollis (Daly and Ryan, 1979). Similar set cells have been also found in various flies (Shiraishi and of sensory cells is widespread in contact chemoreceptors of Morita, 1969; van der Starre, 1972; Hanson, 1987; Liscia et al., 1998, 2002; Furuyama et al., 1999; Amakawa, 2001; Ã Corresponding author. Sadakata et al., 2002; Murata et al., 2004), cockroaches E-mail address: [email protected] (E. Merivee). (Ru¨th, 1976; Becker and Peters, 1989; Hansen-Delkeskamp

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378 E. Merivee et al. / Journal of Insect Physiology 53 (2007) 377–384 and Hansen, 1995; Hansen-Delkeskamp, 1998)and tip by means of a micromanipulator under visual control apterygotan insects (Zygentoma) (Hansen-Delkeskamp, through a light microscope at a magnification of 300 Â . 2001). These results suggest that also in ground beetles, Immediately before each stimulation, 2–3 drops of solution taste sensilla may house the sugar cell. The aim of this study were squeezed out of the micropipette tip, and adsorbed onto is to test stimulatory effect of glucose and sucrose, important a piece of filter paper attached to an additional micromani- in plant carbohydrate metabolism (Kruger, 1993), to pulator, in order to avoid concentration changes in the antennal taste bristles in the ground beetle P. aethiops capillary tip due to evaporation. Due to response, variability (Panzer, 1796). This ground dwelling forest insect of different taste bristles to the same stimulus stimulating (Thiele, 1977) may come into contact with live and decayed solutions were tested in pairs to allow a more precise plant material everywhere in its habitat including its comparison of the responses to the tested stimuli. Thus, each preferred overwintering sites in brown-rot decayed wood. taste sensillum was tested twice. Primarily, three to five sensilla Results of these experiments are reported in this paper. of each test beetle were tested with the control stimulus. Then, approximately after 30 min, the stimulating/recording micro- 2. Material and methods pipette was rinsed with distilled water several times and filled with the second stimulating solution containing sugar to test 2.1. Test beetles the same sensilla again. In each next test beetle the order of presentation of stimulating solutions was alternated and then Test beetles were collected in their preferred overwintering filtered with a band width set at 100–2000 Hz and amplified sites in brown-rot decayed wood at forest margins in southern (input impedance 10 GO) signals were monitored on an Estonia in September and October 2005. Beetles were kept in oscilloscope screen, and relayed to a computer via an plastic boxes filled with moistened moss and pieces of brown- analogue-to-digital input board DAS-1401 (Keithley, Taun- rot decayed wood in refrigerator at 5–6 1C for a couple of ton, MA, USA) for data acquisition, storage, and analysis weeks. Three to four days before experiments, the beetles using TestPoint software (Capital Equipment Corporation, were exposed to room temperature (20 1C), fed with Billerica, MA, USA) at a sampling rate of 10 kHz. Recordings moistened cat food (Kitecat, Master Foods, Poland) and were made during 5-sec stimulation period in order to given clean water to drink every day. characterize both rapid phasic and relatively slow stabilization Intact test beetles were restrained by placing them tightly period of the responses of the chemosensory cells involved. into a special conical tube made of thin sheet aluminium of a size that allowed their head and antennae to protrude 2.3. Data analysis only a little from the narrower end. The wider rear end of the conical tube was blocked with a piece of plasticine to Automatic classifying and counting of action potentials prevent the beetle from retreating out of the tube. from taste sensilla was clearly not an appropriate method Thereafter, antennae of the beetles were fixed horizontally for data analysis because two action potentials frequently on the edge of an aluminium stand with special clamps and interacted to produce an irregular waveform. Instead, spikes tiny amounts of beeswax, so that horizontally located large from several chemosensory cells innervating antennal taste chaetoid taste sensilla were well visible from above under a sensilla tested were visually distinguished by their amplitude light microscope, and easily accessible for micromanipula- and polarity of spikes, and counted, using TestPoint tions from the side. Contamination of tested taste bristles software. In some cases, the regularity of firing as the and their contact with preparation instruments and principal parameter for separating and identifying action beeswax was carefully avoided. potentials was used, whereas contact artefact lasting up to 25 ms seen at onset of stimulus was not corrected by our 2.2. Stimulation and recording of action potentials recording amplifier, 3–4 spikes may have lost. So as the contact artefact of the same duration occurs in both cases, Chemicals of analytical grade of purity used in the when stimulated with control as well as with test solution experiments were obtained from AppliChem (Germany). containing sugar, it does not essentially affect main results Ten millimolar KCl was used as an electrolyte in the mixtures and conclusions of the work. Firing rate (imp/s) was used to with sugars, and as a control solution. pH of stimulating measure the response. t-test for paired samples was used to solutions varying between 6.1 and 6.2 was measured with the determine different means significantly. Responses from pH meter E6115 (Evikon, Estonia). To minimize the possible three to five taste sensilla of each test beetle were analysed. microbial contamination, stimulating sugar solutions were The number of beetles (N) in each test series was 7–16. prepared no longer than 24 h before experiments. Action potentials from taste bristles were recorded by 3. Results conventional single sensillum tip-recording technique. To reach good signal-to-noise ratio, a grounded tungsten 3.1. Responses of antennal taste sensilla to 10 mM KCl microelectrode was inserted into the base of the flagellum. The recording glass micropipette with tip diameter of 20 mm Stimulation of antennal taste bristles of the ground and filled with stimulating solution was placed over the bristle beetle P. aethiops by 10 mM KCl only, serving as an ARTICLE IN PRESS

E. Merivee et al. / Journal of Insect Physiology 53 (2007) 377–384 379 electrolyte and control solution in the experiments, caused numerous spikes of the third cell were observed in the two chemosensory cells to respond phasic-tonically. These recordings (Fig. 1B). The responses of the third cell to cells were the salt (cation) cell and the pH cell. Compared stimulation solutions containing glucose were significantly to the action potentials with negative polarity generated by stronger compared to control solution (po0.05), except for the salt cell those of the pH cell were positive and the solutions with 1 and 1000 mM glucose at the end of the considerably larger in amplitude (Fig. 1A). In various 5-s stimulation period, when the responses fell much below samples of test insects, the first second response of the pH 1 imp/s and they did not differ from control solution cell to this electrolyte varied to a great degree, from 4 (p40.05). During the first second of stimulation, over the to15 imp/s. During the next 4 s of stimulation, however, the range of 1–1000 mM glucose tested, firing rate of the third rate of firing of the pH cell fell to the level of 0–2 imp/s cell drastically increased with concentration increase from (Figs. 2C and 3C). In contrast to the pH cell, firing rate of 2 to 19 imp/s (po0.05). During the next 4 s of stimulation, the salt cell was highly stable in different samples of test firing rate of the third cell fell below 1–2 imp/s, and no beetles with first second responses varying between 11 and dependence of the response on glucose concentration was 13 imp/s (Figs. 2B and 3B). In some recordings, a few small observed any longer. This cell was classified as a phasic- action potentials with negative polarity of the third cell tonic sugar cell (Fig. 2A). were observed. Their mean rate of firing usually did not In contrast, the responses of the salt cell were not exceed 1 imp/s at the beginning of stimulation. Thereafter, significantly affected by the glucose content of the this low rate of firing fell to the level close to zero. stimulating solutions (p40.05). Its first second responses varied between 11 and 14 imp/s. The responses fell below 3.2. Responses of antennal taste sensilla to the mixtures of 1 imp/s during the next s of stimulation, and close to zero 10 mM KCl and glucose thereafter (Fig. 2B). The responses of the pH cell were also affected by the content of glucose in the stimulating In response to the mixtures of 10 mM KCl and sugars, in solutions, but in a more complicated manner. Firing rate of addition to the action potentials from the salt and pH cells, this cell increased to some degree at low concentrations of

Fig. 1. Typical responses from antennal taste bristles of the ground beetle Pterostichus aethiops: (A) 10 mM KCl (control solution), (B) the mixture of 10 mM KCl and 100 mM sucrose. Arrows indicate the beginning of stimulation. Action potentials of the sugar cell (SC), salt cell (CC) and pH cell (pHC) were distinguished.

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Fig. 2. Firing rates of antennal taste sensilla in response to glucose in the ground beetle P. aethiops. To compare stimulating effects of glucose more precisely, the control and stimulus solutions were tested in pairs. Asterisks show signiﬁcantly different means (po.05). Vertical bars indicate SE. Note the different y-scales.

glucose (1 and 10 mM solutions). At the beginning of the Our results demonstrated that the presence of sucrose responses (first two seconds), stimulating effect of glucose in stimulating solutions caused the sugar cell to respond to the pH cell was statistically significant (po0.05). At strongly being nearly silent when stimulated by 10 mM KCl higher concentrations (100 and 1000 mM solutions) the alone. Difference was statistically significant (po0.05). opposite was observed, however: firing rates of the pH cell Stimulatory effect of sucrose to the sugar cell was nearly were slightly suppressed by glucose compared to the two times stronger, however, compared to that of glucose. responses evoked by 10 mM KCl alone (Fig. 2C). First second responses varied from 3 to 37 imp/s depending on the sugar concentration. A strong dependence of the 3.3. Responses of antennal taste sensilla to the mixtures of sugar cell firing rate on the sucrose concentration was 10 mM KCl and sucrose noted throughout the 5-s period of stimulation. In contrast, 1–1000 mM sucrose had only little or no Generally, responses of antennal taste bristles to sucrose stimulatory effect to the salt cell (Fig. 3B). It was also were similar to those obtained in tests with glucose (Fig. 3A). observed that 1 and 10 mM solutions of sucrose were

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Fig. 3. Responses of antennal taste sensilla to sucrose in the ground beetle P. aethiops. Details as in Fig. 2. slightly stimulatory for the pH cell. At higher concentra- they are often used to characterize habitats (Thiele, 1977; tions, however, this sugar caused the pH cell to ﬁre at lower Lo¨vei and Sunderland, 1996; Ings and Hartley, 1999; Cole frequency of action potentials compared to that evoked by et al., 2002; Blake et al., 2003; Eyre and Luff, 2004; Purtauf 10 mM control solution (Fig. 3C). et al., 2005). Chemically mediated habitat selection has been shown to occur in several species of ground beetles by 4. Discussion Evans (1982, 1983, 1984, 1988). He demonstrated that olfactory cells receptive to methyl esters of palmitic and Binding of ground beetles to certain habitat types and oleic acid emitted by the mat-forming blue-green algae microhabitats is determined by numerous biotic and Oscillatoria animalis Agardh and Oscillatoria subbrevis abiotic factors such as food conditions, vegetation type, Schmidle are present in sensillae on the antennae of those presence and distribution of competitors, life history and Bembidiini associated with the shores of saline lakes. Salt season, landscape characteristics, agricultural cultivation content and pH of the soil are also important factors in impacts, temperature and humidity conditions, light distribution of ground beetles (Thiele, 1977; Paje and intensity, and others. Favourite wintering sites are well Mossakowski, 1984; Hoback et al., 2000; Irmler, aerated. Habitat choice in ground beetles is so speciﬁc that 2001,2003; Magura, 2002). Behavioural experiments have

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382 E. Merivee et al. / Journal of Insect Physiology 53 (2007) 377–384 shown that presumptive contact chemoreceptive sensillae insects, whereas in other species this receptor responds are located on the ground beetles’ antennae (Paje and exclusively to sucrose or glucose or fructose or other Mossakowski, 1984; Hoback et al., 2000). carbohydrate. These compounds function as strong pha- By electrophysiological experiments it is convincingly gostimulants and important stimuli for host recognition to demonstrated that the salt and pH cells innervate most herbivorous insects (Mitchell and Gregory, 1979; numerous antennal taste bristles in ground beetles indeed. Schoonhoven, 1987; Bernays and Chapman, 2001; Bernays The used conventional single sensillum tip-recording et al., 2002; Schoonhoven and van Loon, 2002; Chapman, technique (Hodgson et al., 1955), and the location of the 2003) suggesting that the sugar cell found in antennal taste grounded microelectrode at the base of antennal flagellum bristles of the predatory ground beetle P. aethiops may also free of muscle tissues guaranteed that action potentials be related to detection for food. recorded originated from the sensory cells of the taste On the other hand, gustatory apparatuses are usually sensillum contacted and stimulated by the recording located in the labella, legs, wings, palpi and less frequently in microelectrode. The unusual positive polarity and extre- the antennae. Further, there exist strong evidences that the mely long duration of the potentials generated by the pH pH cell at least, and probably also the salt cell innervating cell have been repeatedly pointed out and discussed earlier these antennal taste bristles are responsible for habitat in various ground beetles. Unfortunately, we have no selection in some ground beetles (Thiele, 1977; Paje and satisfactory explanation for this phenomenon today Mossakowski, 1984; Hoback et al., 2000). So, participation (Merivee et al., 2004, 2005). Detailed transmission electron of the sugar cell in searching for suitable habitat is also microscope studies are needed to get new detailed possible in ground beetles. The predatory ground beetle information on the inner structure of these sensilla. P. aethiops may come into contact with live and decayed Intracellular recordings of action potentials from the pH plant material everywhere in its habitat including its cell may probably finally solve the problem of the real preferred overwintering sites in brown-rot decayed wood polarity of their unusual positive potentials recorded by which does not contain soluble sugars and cellulose, and extracellular single sensillum tip-recording technique. Due which is composed of pure lignin. Stable moisture content to the technical complicacy of intracellular recording of and aeration are quaranteed as a result of metabolic water action potentials from insect antennal taste sensilla covered production during the wood-degrading process. The content with a thick and hard cuticular wall, no such attempts have of sugars and pH depend on the assembly of wood-decaying been made so far, however. With regard to the pH fungi involved, and change during this process (Reid, 1985; receptive cell, it was suggested that in P. aethiops and Enoki et al., 1988; Zabel and Morrell, 1992; Varela and Tien, P. oblongopunctatus, in their preferred acid forest habitats 2003) reflecting the quality of decaying wood as a favourable and overwintering sites in brown-rotted wood at pH 3–5 microenvironment to ground beetles for their prolonged the antennal pH sensitive cell does not discharge or hibernation. In addition, brown-rotted wood may offer good discharges at very low frequency with the first second protection for hibernating ground beetles against entomo- firing rate close to 1 imp/s or lower. Areas with higher pH pathogenic fungi and parasites which play an important role seems to be unfavourable for these insects and when in their population dynamic. Up to 41% parasitism by contacted the pH cell signals with a stronger response nematodes and ectoparasitic fungi was found on 14 species (Merivee et al., 2004, 2005; Milius et al., 2006). Therefore, of Bembidion in Norway depending on habitat selection we hypothesize that the function of the third chemorecep- of the host (Andersen and Skorping, 1991). Eggs of tive cell of the taste bristles identified as a sugar cell in the P. oblongopunctatus suffered 83% mortality in fresh litter forest ground beetle P. aethiops, is also related to habitat but only 7% in sterilized soil (Heessen, 1981). Although and/or microhabitat choice. This cell generated action predators, parasites, and pathogens affect all developmental potentials of considerably smaller amplitude than those of stages of ground beetles, quantitative data remain scarce. the salt and pH cells, and responded phasic-tonically to Hence, antennal pH, salt and sugar receptors of the ground sucrose and glucose over the all range of 1–1000 mM beetle P. aethiops seem to be a powerful tool in their selection stimulating solutions tested, a span that covers the range of of suitable habitats and microhabitats crucial to survive. soluble sugar levels generally present in green plants, i.e. 1–50 mM/l (Schoonhoven and van Loon, 2002; Chapman, 2003). Significantly, higher rates of firing were recorded in Acknowledgements response to sucrose compared to that evoked by glucose. During the first second of the response, maximum rates of Contract grant sponsor: Estonian Science Foundation; firing of the sugar cell reached up to 19 and 37 imp/s when Contract grant numbers 5423 and 5736. stimulated with glucose and sucrose, respectively. Both sugars are important in plant carbohydrate metabolism. References These results are in a good consistency with literature data obtained by similar experiments with phytophagous Amakawa, T., 2001. Effects of age and blood sugar levels on the proboscis insects. Various mono-, di-, and trisaccharides may extension of the blow fly Phormia regina. Journal of Insect Physiology stimulate the sugar cell with different effectiveness in some 47, 195–203.

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Paje, F., Mossakowski, D., 1984. pH-preferences and habitat selection in Perspectives in Chemoreception and Behavior. Springer, New York, carabid beetles. Oecologia (Berlin) 64, 41–46. pp. 69–97. Purtauf, T., Roschewitz, I., Dauber, J., Thies, C., Tscharntke, T., Wolters, Schoonhoven, L.M., van Loon, J.J.A., 2002. An inventory of taste in V., 2005. Landscape context of organic and conventional farms: caterpillars: each species its own key. Acta Zoologica Academiae influences on carabid beetle diversity. Agriculture, Ecosystems and Scientiarum Hungaricae 48, 215–263. Environment 108, 165–174. Shiraishi, A., Morita, H., 1969. The effects of pH on the labellar sugar Reid, I.D., 1985. Biological delignification of aspen wood by solid-state receptor of the fleshfly. The Journal of General Physiology 53, 450–470. fermentation with the white-rot fungus Merulius tremellosus. Applied Thiele, H.-U., 1977. Carabid Beetles in Their Environment. Zoophysiology and Environmental Microbiology 50, 133–139. and Ecology, vol.10. Springer, Berlin, p. 369. Ru¨th, E., 1976. Elektrophysiologie der Sensilla Chaetica auf den Antennen Van der Starre, H., 1972. Tarsal taste discrimination in the blowfly, von Periplaneta americana. Journal of comparative Physiology 105, Calliphora vicina Robineau-Desvoidy. Netherlands Journal of Zool- 55–64. ogy 22, 227–282. Sadakata, T., Hatano, H., Koseki, T., Koganezawa, M., Shimada, I., Varela, E., Tien, M., 2003. Effect of pH and oxalate on hydroquinone- 2002. The effect of amiloride on the labellar taste receptor cells of the derived hydroxyl radical formation during brown rot wood degrada- fleshfly Boettcherisca peregrina. Journal of Insect Physiology 48, tion. Applied and Environmental Microbiology 69, 6025–6031. 565–570. Zabel, R.A., Morrell, J.J., 1992. Chemical changes in wood caused by Schoonhoven, L.M., 1987. What makes a caterpillar eat? The sensory decay fungi. In: Wood Microbiology Decay and its Prevention. code underlying feeding behavior. In: Chapman, R.F.et al. (Ed.), Academic, San Diego, pp. 195–224.

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ELECTROPHYSIOLOGICAL RESPONSES FROM NEURONS OF ANTENNAL TASTE SENSILLA IN THE POLYPHAGOUS PREDATORY GROUND BEETLE PTEROSTICHUS OBLONGOPUNCTATUS (fABRICIUS 1787) TO PLANT SUGARS AND AMINO ACIDS.

Journal of Insect Physiology 54, 1213–1219. Journal of Insect Physiology 54 (2008) 1213–1219

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Journal of Insect Physiology

journal homepage: www.elsevier.com/locate/jinsphys

Electrophysiological responses from neurons of antennal taste sensilla in the polyphagous predatory ground beetle Pterostichus oblongopunctatus (Fabricius 1787) to plant sugars and amino acids

Enno Merivee *, Helina Ma¨rtmann, Anne Must, Marit Milius, Ingrid Williams, Marika Ma¨nd

Estonian University of Life Sciences, 1 Kreutzwaldi Street, 51014 Tartu, Estonia

ARTICLE INFO ABSTRACT

Article history: The responses of antennal contact chemoreceptors, in the polyphagous predatory ground beetle Received 10 March 2008 Pterostichus oblongopunctatus, to twelve 1–1000 mmol l1 plant sugars and seven 10–100 mmol l1 Received in revised form 13 May 2008 amino acids were tested. The disaccharides with an a-1.4-glycoside linkage, sucrose and maltose, were Accepted 19 May 2008 the two most stimulatory sugars for the sugar-sensitive neuron innervating these contact chemosensilla. The firing rates they evoked were concentration dependent and reached up to 70 impulses/s at Keywords: 1000 mmol l1. The stimulatory effect of glucose on this neuron was approximately two times lower. This Contact chemoreceptors can be partly explained by the fact that glucose exists in at least two anomeric forms, a and b. These two Electrophysiology Firing rate forms interconvert over a timescale of hours in aqueous solution, to a final stable ratio of a:b 36:64, in a Feeding process called mutarotation. So the physiologically active a-anomere forms only 36% of the glucose Phytophagy solution which was reflected in its relatively low dose/response curve. Due to the partial herbivory of P. Polyphagy oblongopunctatus these plant sugars are probably involved in its search for food, for example, for conifer seeds. Several carbohydrates, in addition to glucose, such as cellobiose, arabinose, xylose, mannose, rhamnose and galactose are known as components of cellulose and hemicelluloses. They are released by brown-rot fungi during enzymatic wood decay. None of them stimulated the antennal sugar-sensitive neuron. They are therefore not implicated in the search for hibernation sites, which include rotting wood, by this beetle. The weak stimulating effect (below 3 impulses/s) of some 100 mmol l1 amino acids (methionine, serine, alanine, glutamine) to the 4th chemosensory neuron of these sensilla was characterized as non-specific, or modulating the responses of non-target chemosensory neurons. ß 2008 Elsevier Ltd. All rights reserved.

1. Introduction in the spring; newly emerged adults appear in the autumn and hibernate in decaying wood, under the bark of tree trunks, in forest Many species of ground beetles (Coleoptera, Carabidae) are litter and under stones (Haberman, 1968; Lindroth, 1986). important as natural enemies of phytophagous pest insects Scanning electron microscope studies show that the antennae (Kromp, 1999; Symondson et al., 2002), but the chemical cues of ground beetles are well equipped with various chemoreceptors. that they use in habitat selection and food recognition have been In addition to numerous small basiconic olfactory sensilla, larger little studied. More knowledge of these cues and their sensory bristle-like contact chemosensilla have been found on the antennal detection may aid the development of strategies to manipulate flagellum of adults in several genera: approximately 70 such carabid assemblages for plant protection in the future. The sensilla in Bembidion, Platynus and Pterostichus (Merivee et al., common Euro-Siberian ground beetle, Pterostichus oblongopuncta- 2000, 2001, 2002, 2005) and 56 in Carabus (Kim and Yamasaki, tus, the subject of this study, is a eurytopic woodland species, 1996). In Nebria brevicollis these large sensilla are each innervated occuring in both deciduous and coniferous forests. It reproduces by one mechanoreceptive and four chemoreceptive neurons (Daly and Ryan, 1979). In Pterostichus aethiops and P. oblongopunctatus, specific chemical stimuli have been identified by electrophysiology for three of the four chemosensory neurons. One responds to * Corresponding author at: Estonian University of Life Sciences, Institute of Agricultural and Environmental Sciences, 1 Kreutzwaldi Street, 51014 Tartu, various salts and another to the pH of the stimulating solution Estonia. (Merivee et al., 2004, 2005; Milius et al., 2006). In P. aethiops, the E-mail address: [email protected] (E. Merivee). third neuron is sensitive to sucrose and glucose, important in plant

89 1214 E. Merivee et al. / Journal of Insect Physiology 54 (2008) 1213–1219 carbohydrate metabolism (Merivee et al., 2007). As habitat water to drink every day. Electrophysiological experiments with selection in ground beetles appears to be influenced by soil pH hibernating beetles were conducted in February and March 2006 and salinity (Krogerus, 1960; Thiele, 1977; Paje and Mossakowski, and with active beetles from June to August 2007. 1984; Hoback et al., 2000; Irmler, 2001), the antennal pH- and salt- sensitive neuron may be involved in this process. Electrophysio- 2.2. Electrophysiology logical experiments with the pH-sensitive neuron in P. oblongopunctatus and P. aethiops demonstrated that this neuron did not fire Each beetle to be tested was restrained securely in a conical tube or fired at very low frequency (first second response below made of thin sheet-aluminium of a size that allowed the head and 5 impulses/s) when stimulated at pH 3–6. This level of acidity antennae to protrude from the narrower end. The wider rear end of reflects the pH preferences of these species in their forest habitat the tube was blocked with plasticine to prevent the beetle from and hibernation sites in brown-rotted wood. Firing rates of retreating out of the tube. Its antennae were fixed horizontally to the the neuron increased with increase in pH, up to 20–30 impulses/s edge of an aluminium stand with special clamps and beeswax, so at pH 8–11 (Merivee et al., 2005; Milius et al., 2006). These that the horizontally located large chaetoid taste sensilla were electrophysiological results are in agreement with the acid soil visible from above under a light microscope, and easily accessible for pH preferences of P. oblongopunctatus obtained in behavioural micromanipulation from the side. Care was taken not to contam- and ecological experiments (Paje and Mossakowski, 1984; Irmler, inate the taste sensilla by contact with instruments or beeswax. 2001). Each prepared test beetle was placed in a Faraday cage (15 cm In addition to the salt- and pH-sensitive neuron, the sugar- 10 cm 5 cm) for electrophysiology. sensitive neuron present in the same antennal taste sensillum may Action potentials from taste neurons of the sensilla were also be involved in habitat/microhabitat selection by these species. recorded by the conventional single sensillum tip-recording Various sugars and amino acids function as strong phagostimu- technique. To achieve a good signal-to-noise ratio, the indifferent lants in most phytophagous insects equipped with specialized tungsten microelectrode was inserted into the base of the flage- receptor neurons for these chemicals (Schoonhoven and van Loon, llum free of muscular tissue. It was connected to an AC amplifier 2002; Chapman, 2003). Adults of the genus Pterostichus appear to via a calibration source, and earthed. The recording glass micro- be primarily carnivorous, feeding on small prey animals, but in pipette filled with stimulating solution was brought into contact many species, plant matter also forms part of their diet, and, in with the tip of the sensillum by means of a micromanipulator some the proportion of animal to plant matter eaten varies with under visual control through a light microscope at a magnification season (Thiele, 1977; Hengeveld, 1980; Lo¨vei and Sunderland, of 300. Immediately before each stimulation, 2–3 drops of 1996). Johnson and Cameron (1969) collected data on the extent of solution were squeezed out of the micropipette tip, and adsorbed herbivory in more than 150 species of ground beetles. Some onto a piece of filter paper attached to another micromanipulator, species in the genera Pterostichus, including P. oblongopunctatus, in order to avoid concentration changes in the capillary tip due Amara, Agonum and Harpalus consume conifer seeds and young to evaporation. Action potentials picked up by the recording seedlings immediately following germination. In Sweden, P. electrode were filtered with a bandwidth set at 100–2000 Hz, oblongopunctatus and Calathus micropterus are the most important amplified (input impedance 10 GV), monitored on an oscillo- predators of Pinus sylvestris seed. In the field, seed predation scope screen, and relayed to a computer via an analogue-to-digital typically resulted in >20% seed mortality, reaching even higher input board DAS-1401 (Keithley, Taunton, MA, USA) for data levels (up to 60%) on some occasions and most predation (81% of all acquisition, storage, and analysis using TestPoint software damaged seeds) was due to ground beetles (Heikkila¨, 1977; (Capital Equipment Corp., Billerica, MA, USA) at a sampling rate Nystrand and Granstro¨m, 2000). Therefore, as in phytophagous of 10 kHz. Responses were recorded from neurons of three to five insects, polyphagous predatory ground beetles, including P. randomly selected taste sensilla on the flagellomeres 1–9 of each oblongopunctatus, may also have plant sugar- and amino acid- test beetle. Each sensillum was stimulated once only with one sensitive neurons associated with their feeding habits. solution. Action potentials generated during the first second after The aim of this study was to determine whether sugar- and the beginning of stimulus application were counted and analysed. amino acid-sensitive receptor neurons are present in the antennal Responses of the sensilla to mechanical stimulation were also taste sensilla of adult P. oblongopunctatus by testing for stimulatory tested, especially in those rare cases when it was not clear effects of various sugars, mostly of plant origin, and amino acids on whether a certain class of recorded action potentials originated these sensilla by electrophysiology. As the composition and content from the chemosensory or mechanosensory neuron. The tip of the of water-soluble sugars differ substantially in live and decaying sensillum was rapidly moved from its resting position toward the plant matter (Eriksson et al., 1990; Zabel and Morrell, 1992; antennal shaft by 15–208, held in this position for 2–3 s, and then Vala´sˇkova´ and Baldrian, 2006) the reaction spectrum of any sugar- rapidly moved back to its initial position using the recording sensitive antennal neurons, if present, should give some information electrode. on the functioning of antennal contact chemoreceptors and how they might be involved in habitat selection and food recognition by 2.3. Chemical stimuli this species. The main tests were carried out using twelve 1–1000 mmol l1 2. Materials and methods sugars and seven 10–100 mmol l1 amino acids (Table 1)dissolved in 10 mmol l1 choline chloride to give good conductivity. 2.1. Insects Compared to KCl and NaCl, which are frequently used as electrolytes in solutions of stimulating non-electrolytes, choline Adult P. oblongopunctatus were collected from a local popula- chloride elicits only a few action potentials of large amplitude tion in southern Estonia. They were kept in plastic boxes filled with generated by the salt- and pH-sensitive neuron innervating the moistened moss and pieces of brown-rotted wood in a refrigerator antennal taste sensilla in ground beetles. As a result, discrimina- at 5–6 8C. Three to four days prior to the experiments, the beetles tion and counting of small spikes generated by the sugar-sensitive were transferred to room temperature (23 8C), fed with moistened neuron is easier, especially at the beginning of the response. cat food (Friskies Vitality+, Nestle´ Purina, Hungary) and given clean Chemicals of analytical grade purity were obtained from

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Table 1 Responses of neurons of antennal chaetoid taste sensilla of P. oblongopunctatus for 1 s to stimulation with various carbohydrates and amino acids presented in 10 mmol l1 choline chloride

Stimulating solution Concentration Mean impulses/s Neuron responding Beetles/bristles Monosaccharide units (mmol l1) (S.E.) and glycosidic bonds

Choline chloride (control) 10 0.15 0.04 SNa and/or 4thN 26/130

D(+)-Pentoses Arabinose 100 0.23 0.83 SN and/or 4thN 6/30 Xylose 100 0.62 0.15** SN and/or 4thN 7/34

D(+)-Hexoses Glucose 100 20.87 1.41** SC 15/72 a-glu, 36%; b-glu, 64% Galactose 100 0.16 0.06 SN and/or 4thN 6/30 Mannose 100 0.46 0.19* SN and/or 4thN 9/45

D()-Hexoses Fructose 100 0.00* 6/30

L-Hexoses Rhamnose 100 0.26 0.18 SN and/or 4thN 6/30

D(+)-Disaccharides Cellobiose 100 0.18 0.11* SN and/or 4thN 9/43 b-glu, glu; 1 ! 4 Lactose 100 0.51 0.15* SC and/or 4thC 8/40 b-gal, glu; 1 ! 4 Maltose 100 49.9 2.65** SN 10/48 a-glu, glu; 1 ! 4 Sucrose 100 46.5 2.01** SN 23/111 a-glu, b-fru; 1 ! 2

D(+)-Trisaccharides Rafﬁnose 100 0.13 0.13 SN and/or 4thN 5/25 a-gal, a-glu, b-fru; 1 ! 6, 1 ! 2

L-Amino acids Methionine 100 2.32 0.42** SN and/or 4thN 12/60 Proline 100 0.68 0.23* SN and/or 4thN 7/35 Serine 100 2.76 0.51** SN and/or 4thN 6/30 Alanine 100 2.78 0.46** SN and/or 4thN 9/42 Asparagine 100 0.91 0.2** SN and/or 4thN 10/50 Glutamine 2.49 0.44** SN and/or 4thN 12/60 g-Aminobutyric acid 100 0.35 0.09 SN and/or 4thN 10/47

The number of spikes from the salt-sensitive neuron is not indicated. a At very low rate of firing firm classification of the spiking neuron was not possible. * Responses significantly different from choline chloride alone at P < 0.01. ** Responses significantly different from choline chloride alone at P < 0.001 (t-test for independent samples).

AppliChem (Germany). To minimize microbial contamination, usually only the salt-sensitive neuron fired, producing spikes with stimulating solutions were prepared within 48 h of electrophy- negative polarity, 1–2 mV in amplitude. However, in some siology. recordings, a few spikes from other sensory neurons were also observed. The pH-sensitive neuron generated very large action 2.4. Data analysis potentials, 2–4 mV peak-to-peak, with initial positive polarity (Fig. 1A). The shape of these impulses is complex because they are Automated classification and counting of action potentials from composed of two separate electrical events in quick succession and taste sensilla was not an appropriate method for data analysis most probably produced by two separate spike generation sites of because two action potentials frequently interacted to produce an the same neuron. Minor differences in the time interval between irregular waveform. Instead, spikes from several chemosensory these two events occuring among antennal taste sensilla may neurons innervating antennal taste sensilla tested were visually cause considerable differences in the shape of the resulting double- distinguished by their polarity and amplitude (Fig. 1), and counted, impulse. Usually, the double-impulses of the pH-sensitive neuron using TestPoint software. In some cases, the regularity of firing was described are two-tipped (Fig. 2A and B). In some sensilla, used as the principal parameter for separating and identifying however, only a small characteristic notch or discontinuity in action potentials. Firing rates (impulses/s) were analysed by the rising phase of these impulses indicates their complex nature counting the number of action potentials within 1 s after stimulus (Fig. 2C and D). Typically the mixtures of 10 mmol l1 choline onset. Mean responses and their standard errors were calculated. chloride and some sugars evoked a sugar-sensitive neuron, in addition to the salt-sensitive neuron, to discharge. The sugar- 3. Results sensitive neuron generated negative spikes smaller than those of the salt-sensitive neuron. In a few recordings, irrespective of the 3.1. Spike waveforms generated by neurons of the stimulating solution, rare, very small action potentials with antennal chaetoid taste sensilla negative polarity from the 4th chemosensory neuron from these sensilla were also observed (Fig. 1B). So far, chemical stimuli Spike waveform analysis of electrophysiological recordings eliciting a response specific to the 4th chemosensory neuron have obtained in chemical and mechanical stimulation experiments not been identified. Impulse activity of the mechanosensory showed that at least four chemoreceptive and one mechanor- neuron belonging to these taste sensilla and responding to bending eceptive neurons innervate each antennal chaetoid taste sensillum of the bristle was recorded from some intact bristles as well as from in P. oblongopunctatus. In response to 10 mmol l1 choline chloride, bristles with a broken tip. Action potentials produced by the

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Fig. 1. Action potential waveforms recorded from the tip of antennal chaetoid taste sensilla in the ground beetle P. oblongopunctatus. CN, SN, pHN, 4thN, and MN Fig. 2. Variation of the complex action potential shape produced by the pH-sensitive represent action potentials produced by the salt-, sugar-, pH-, 4th chemo- and neuron from different antennal taste sensilla. A, B: Two-tipped impulses with mechano-sensitive neuron, respectively. Downward deflection from the baseline equal and unequal positive peaks, respectively. In other cases, a notch (C) or a 1 represents negative polarity. (A) Response of a taste sensillum to 10 mmol l discontinuity (D, arrowhead) in the rising phase of these impulses indicates their choline chloride (control) 0.2–0.4 s after the beginning of stimulation. Arrowhead complex nature. These double-impulses are caused by two separate action potential shows a characteristic notch in the rising phase of the spike from the pH-sensitive generation sites of the same neuron. Note the shortening of time interval betveen neuron indicating the presence of two spike generation sites in the neuron the two electrical events from A to D. Lower traces show upper traces in a shorter membrane. (B) A recording of an atypical response from a taste sensillum to the timescale. mixture of 10 mmol l1 choline chloride and 100 mmol l1 sucrose 0.5 s after the beginning of stimulation demonstrating negative action potentials with different amplitudes generated by three different chemosensory neurons. (C) Response of responses evoked by the mixtures of this electrolyte with sugars the taste bristle to bending. Arrows show 1 mV calibration pulses from a and amino acids. calibration source. Horizontal doubled black line indicates period of the rapid movement of the sensillum tip towards antennal shaft and holding it in this position (single black line) approximately 1.4 s which evoked strong firing of the 3.3. Responses of antennal taste neurons to sugars MN. The movement of the sensillum back to its initial resting position (dotted line) caused only a weak response. (D) The same recording as (C) but in a shorter Responses of antennal taste neurons to stimulation by various timescale. 100 mmol l1 sugars in mixtures with 10 mmol l1 choline chloride, varied with the sugar tested. Only three out of twelve mechanosensory neuron were small but variable in size, and sugars, maltose, sucrose and glucose, evoked strong phasic-tonic negative in polarity (Fig. 1C and D). responses from the sugar-sensitive neuron (Table 1; Fig. 3B–E). The mean rates of firing of the neuron during the first second after 3.2. Responses of neurons from antennal taste sensilla to 10 mmol l1 100 mmol l1 stimulation reached 49.9, 46.5 and 20.9 impulses/s, choline chloride alone respectively. Dose/response curves of these three active sugars are demonstrated in Fig. 4. In the range of 1–1000 mmol l1, the Choline chloride at a concentration of 10 mmol l1 served as the response to glucose was approximately two times lower than that control stimulus, and as the electrolyte in stimulating mixtures of maltose and sucrose. In contrast, no differences were found with sugars and amino acids to ensure a good signal-to-noise ratio. between responses to maltose and sucrose. The remaining nine Typically, choline chloride evoked only the salt-sensitive neuron 100 mmol l1 sugars had only a small or no stimulatory effect on to discharge with a relatively low rate of firing (approximately the neuron. Action potentials generated by the sugar-sensitive and 15 impulses/s) during the first second after stimulus onset the 4th chemosensory neuron were small, varied in size, and their (Fig. 3A). Only in rare cases were a few action potentials from amplitudes appeared to overlap. Therefore, at a very low rate of other sensory neurons of the sensillum observed (Fig. 1A; Table 1). firing, firm classification of the spiking neuron was not always Therefore, the stimulating properties of choline chloride were possible with these non-stimulatory or slightly stimulatory sugars. suitable for classifying and counting action potentials of the The mean response they evoked was much lower than 1 impulses/s

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Fig. 3. Example recordings from antennal taste sensilla to some carbohydrates and amino acids in P. oblongopunctatus. CN and SN represent action potentials from the salt- and sugar-sensitive neuron, respectively. Arrowheads show small action potentials most probably generated by the 4th chemosensory neuron.

(Table 1; Fig. 3F). To test for possible seasonal variation in the functioning of the sugar-sensitive neuron, its responses to 100 mmol l1 sucrose were compared in active and hibernating beetles in July (46.5 impulses/s) and February (48.8 impulses/s), respectively but no differences between their responses were observed (t = 0.57; d.f. = 18; P = 0.57; t-test for independent samples).

3.4. Responses of antennal taste neurons to amino acids

Responses of antennal taste neurons to the seven 10 and 1 1 100 mmol l L-amino acids in a mixture with 10 mmol l choline chloride varied with the type and concentration of amino acid tested. At 10 mmol l1, none of the amino acids stimulated the neurons. At 100 mmol l1, these chemicals evoked only a few action potentials (Table 1) with amplitudes smaller than those generated by the salt-sensitive neuron (Fig. 3G–I). No initial phasic component in the sequence of these spikes was observed. Fig. 4. Concentration/response curves of the sugar-sensitive neuron from antennal Frequently, they appeared with some delay in the responses, taste sensilla obtained in experiments with three physiologically active plant sugars in P. oblongopunctatus. Action potentials were counted for 1 s after the onset of the sometimes 5–10 s after stimulus onset (Fig. 3J). Spike amplitude stimulus. Vertical bars show S.E. of the means; means denoted with different letters analysis showed that these small impulses were generated by the grouped by the horizontal bar are signiﬁcantly different at P < 0.05 (ANOVA, Tukey 4th chemosensory neuron rather than by the sugar-sensitive test, 6 N 23).

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36:64, in a process called mutarotation. So the physiologically active a-anomere forms only 36% of the glucose solution which is reflected in its relatively low dose/response curve. The order of stimulatory effectiveness of sugars may vary in different insect species or even be different for receptors on various parts of the body (Blaney, 1974; Ru¨ th, 1976; Bernays and Chapman, 2001; Schoonhoven and van Loon, 2002). Since amino acids, in addition to sugars, stimulate feeding behaviour in various phytophagous insects these animals have amino acid-specific neurons in their taste sensilla. Amino acids and sugars may stimulate the same neuron. Such a versatile receptor neuron is present in the polyphagous caterpillar of Grammia geneura. These insects have, in addition to an amino acid sensitive neuron in their lateral sensillum styloconicum, a neuron in another sensillum which responds to seven (out of twenty) amino acids. This latter neuron can also be stimulated by sucrose, glucose, and trehalose (Schoonhoven and van Loon, 2002; Chapman, 2003). Because of its multiple specificity this neuron was named a Fig. 5. Histograms of relative spike amplitudes from neurons stimulated by sugars ‘‘phagostimulatory cell’’, rather than a ‘‘sugar’’ or ‘‘amino acid’’ cell and amino acids in antennal taste sensilla of P. oblongopunctatus. aSN, a4thN and (Bernays and Chapman, 2001). However, in P. oblongopunctatus, aCN represent spike amplitudes of the sugar-, 4th chemo-, and salt-sensitive the seven amino acids tested had only little or no stimulatory effect neuron, respectively. Normal distribution curves of respective samples are indicated. on the neurons of the antennal taste sensilla suggesting that these neurons have no ability to perceive these amino acids. The weakly stimulatory effect (below 3 impulses/s) of some 100 mmol l1 neuron. Though amplitudes of spikes induced by sugars (sucrose) amino acids on the 4th chemosensory neuron of these sensilla can and amino acids varied and overlapped to a large extent their be characterized as non-specific, or modulating to the responses of histograms had different maxima (Fig. 5). Since the mean numbers non-target chemosensory neurons, the phenomenon well known of these small spikes during the first second after stimulus in insect chemoreceptor functioning (Hanson, 1987; Liscia et al., onset did not exceed 3 impulses/s, this neuron has probably no 1997; Liscia and Solari, 2000; Bernays and Chapman, 2001; specialized receptor sites for amino acids, at least for those which Chapman, 2003; Schoonhoven and van Loon, 2002; Merivee et al., were tested. 2005; Merivee et al., 2007). No insect so far investigated responds to all sugars, and no 4. Discussion single sugar is a major phagostimulant for all insects. For a given insect species, only some sugars elicit a response to a greater or Three out of the twelve sugars tested, maltose, sucrose and lesser extent while other sugars elicit no response. Plant sugars glucose, evoked strong responses from the sugar-sensitive neurons which were found to evoke responses from the antennal contact while other had little or no effect. Most stimulating were the two chemoreceptors in P. oblongopunctatus, i.e., maltose, sucrose and disaccharides, maltose and sucrose, and this is in agreement with glucose, function as strong phagostimulants for most phytopha- results for a number of other insect species (Mitchell and Gregory, gous insects, flies, cockroaches and others, equipped with 1979; Schoonhoven and van Loon, 2002; Albert, 2003; Merivee specialized receptors to detect sugars (Dethier et al., 1956; Blaney, et al., 2007; Sandoval and Albert, 2007). Sucrose is a disaccharide 1974; Ru¨ th, 1976; Amakawa, 2001; Liscia et al., 2002; Schoon- consisting of two monosaccharides, a-glucose and b-fructose, hoven and van Loon, 2002; Chapman, 2003; Sandoval and Albert, joined by a glycosidic bond between carbon atom 1 of the glucose 2007). This suggests that these sugars may have the same function unit and carbon atom 2 of the fructose unit. Maltose is composed of in P. oblongopunctatus, probably due to its partial herbivory. two molecules of glucose joined together through a-1.4-glycoside Although, in contrast to sucrose and glucose, maltose is uncommon linkage. Thus, the presence of a terminal a-glucose unit seems to in nature, it can be found in considerable amounts in germinating be a common and important feature of the molecular structure seeds as a product of the enzymatic degradation of starch. determining the stimulatory properties of these disaccharides in P. Several carbohydrates, in addition to glucose, such as cello- oblongopunctatus. Other monosaccharide units in the molecular biose, arabinose, xylose, mannose, rhamnose and galactose are structure of sucrose and maltose were of no importance in this known as components of cellulose and hemicelluloses. They are respect as the sugar-sensitive neuron responses to these two released by brown-rot fungi during enzymatic wood decay. All disaccharides were similar. In contrast, the trisaccharide raffinose water-soluble carbohydrates are ultimately consumed, so that has a non-terminal a-glucose unit and does not evoke a response extensively brown-rotted wood consists almost entirely of from the antennal sugar-sensitive neuron in this species. In the red modified lignin. Brown-rot fungi appear to depolymerize wood turnip beetle, Entomoscelis americana, maltose (a-glucose-1.4- in the early stages of decay much more rapidly than the decay glucose) was at least an order of magnitude more effective in products can be metabolized. The occurrence of excess wood- activating the maxillary sugar-sensitive neuron than trehalose (a- decomposition products may help explain the frequent presence of glucose-1.1-glucose) illustrating the importance of the 1.4 linkage other wood scavengers in brown-rotted wood (Eriksson et al., in these disaccharide molecules (Mitchell and Gregory, 1979). 1990; Zabel and Morrell, 1992; Vala´sˇkova´ and Baldrian, 2006). As Compared to maltose and sucrose, the third active sugar glucose wood decomposes, it passes through a range of decay stages, each was approximately two times less effective at stimulating the colonized by a succession of diverse saproxylic insect assemblages antennal sugar-sensitive neuron of P. oblongopunctatus. This can be (Grove, 2002). Wood-decay products may serve as chemical cues partly explained by the fact that glucose exists in at least two for these saproxylic insects and be involved in this colonization anomeric forms, a and b. These two forms interconvert over a process although no electrophysiological or behavioural evidence timescale of hours in aqueous solution, to a final stable ratio of a:b is yet available. Some ground beetles, including P. oblongopuncta-

94 E. Merivee et al. / Journal of Insect Physiology 54 (2008) 1213–1219 1219 tus, prefer brown-rotted wood for their prolonged hibernation Kromp, B., 1999. Carabid beetles in sustainable agriculture: a review on pest control efficacy, cultivation impacts and enhancement. Agric. Ecosyst. Environ. 74, (Haberman, 1968; Lindroth, 1986). Our results showed, however, 187–228. that P. oblongopunctatus was not able to sense water-soluble wood Lindroth, C.H., 1986. Fauna Entomologica Scandinavica, Vol. 15: The Carabidae sugars released by brown-rot fungi, except for glucose, indicating (Coleoptera) of Fennoscandia and Denmark, II. E.J. Brill, Leiden, pp. 230–497. With an appendix on the family Rhysodidae. that these chemicals are not involved in the search for hibernation Liscia, A., Solari, P., 2000. Bitter taste recognition in the blowfly: electrophysiolo- sites by this species, although others may be. gical and behavioral evidence. Physiol. Behav. 70, 61–65. 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Ultrastructure of antennal sensilla of Nebria brevicollis ostichus aethiops. J. Insect Physiol. 53, 377–384. (Fab) (Coleoptera: Carabidae). Int. J. Insect Morphol. Embryol. 8, 169–181. Milius, M., Merivee, E., Williams, I., Luik, A., Ma¨nd, M., Must, A., 2006. A new method Dethier, V.G., Evans, D.R., Rhoades, M.V., 1956. Some factors controlling the inges- for electrophysiological identification of antennal pH receptor cells in ground tion of carbohydrates by the blowfly. Biol. Bull. 111, 204–222. beetles: the example of Pterostichus aethiops (Panzer, 1796) (Coleoptera, Car- Eriksson, K.-E.L., Blanchette, R.A., Ander, P., 1990. Microbial and Enzymatic Degra- abidae). J. Insect Physiol. 52, 960–967. dation of Wood Components. Springer-Verlag, New York. Mitchell, B.K., Gregory, P., 1979. Physiology of the maxillary sugar sensitive cell in Grove, S.J., 2002. Saproxylic insect ecology and the sustainable management of the red turnip beetle, Entomoscelis americana. J. Comp. 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95 V Komendant, M., Merivee, E., Must, A., Tooming, E., Williams, I., Luik, A.

ELECTROPHYSIOLOGICAL RESPONSE OF THE CHEMO- RECEPTOR NEURONS IN THE ANTENNAL TASTE SEN- SILLA TO PLANT ALKALOIDS AND GLUCOSIDES IN THE GRANIVOROUS GROUND BEETLE PTEROSTICHUS OBLON- GOPUNCTATUS.

Journal of Chemical Ecology (in review) ELECTROPHYSIOLOGICAL RESPONSE OF THE CHEMORECEPTOR NEURONS IN THE ANTENNAL TASTE SENSILLA TO PLANT ALKALOIDS AND GLUCOSIDES IN THE GRANIVOROUS GROUND BEETLE Pterostichus oblongopunctatus

MARIT KOMENDANT, ENNO MERIVEE*, ANNE MUST, ENE TOOMING, INGRID WILLIAMS, ANNE LUIK

Estonian University of Life Sciences 1 Kreutzwaldi Street, 51014 Tartu, Estonia Abstract Electrophysiological responses of chemoreceptor neurons from the antennal chaetoid taste sensilla to several plant alkaloids and glucosides were tested in the ground beetle Pterostichus oblongopunctatus. For the fi rst time, a specifi c alkaloid-sensitive neuron phasic-tonically responding to quinine and quinine hydrochloride was found in these insects, most probably related to their granivorous feeding habit. Th e response to quinine hydrochloride was concentration-dependent at 0.001 to 50 mmol l-1 with the response threshold at 0.01 mmol l-1, and a maximum rate of fi ring of 67 spikes/sec at 50 mmol l-1. Th e stimulatory eff ect of caff eine was very weak. In addition, both quinine and quinine hydrochloride strongly inhibited spike production by the salt- and pH-sensitive neurons when presented in mixtures with 10 mmol l-1 NaCl. It was also observed that several tested plant secondary compounds, salicin, sinigrin, caff eine and nicotine, which had only little or no eff ect on the fi ring rate of the alkaloid-sensitive neuron, would greatly reduce the responses of the salt- and pH-sensitive neurons, suggesting that the antennal taste sensilla of P. oblongopunctatus may detect plant defensive compounds both through the activation of a specifi c alkaloid-sensitive neuron and through peripheral inhibition of other chemoreceptor neurons of the taste sensillum.

Key Words Granivory, Contact chemoreception, Plant secondary compounds, Alkaloid-sensitive neuron, Peripheral inhibition, Feeding deterrent INTRODUCTION

Ground beetles (Coleoptera, Carabidae) are the dominant ground-living invertebrate predators in many ecosystems. Because of their potential role in foodweb dynamics and pest control, the food and feeding habits of ground beetles have been investigated in more detail than of any other group of predatory arthropods. Data on food items are available for approximately 1100 species and subspecies of ground beetles of the world. A total of 73% are carnivorous, 19% are omnivorous and 8% are exclusively herbivorous (Th iele 1977; Larochelle 1990; Toft and Bilde 2002). Of these, many species predate on seeds (Honek et al. 2003). Ground beetles use a variety of external stimuli in order to locate food. Behavioural tests and observations have indicated that visual, tactile, olfactory and gustatory cues may be involved (Wheater 1989; Kielty et al. 1996; Toft and Bilde 2002). Scanning electron microscopic studies have demonstrated that thousands of various olfactory and gustatory sensilla are located on the ground beetles’ antennae (Daly and Ryan 1979; Merivee et al. 2000, 2001, 2002). Very little is known, however, about the chemical compounds to which they respond.

Electrophysiological investigations of the chemoreception capabilities of ground beetles started only recently with experiments on antennal chaetoid contact chemoreceptors. Approximately 70 of these large sensilla (35- 200 μm in length) have been found on the antennal fl agellum of various ground beetles including the ground beetle Pterostichus oblongopunctatus (Merivee et al. 2000, 2001, 2002, 2005). Typically, four chemoreceptive and one mechanoreceptive neuron innervate these sensilla in various insect groups (Morita and Shiraishi 1985; Chapman 1998; Jørgensen et al. 2006). A similar set of sensory neurons has been found for the antennal chaetoid contact chemoreceptors of the ground beetle Nebria brevicollis (Daly and Ryan 1979) suggesting that it may be characteristic for these sensilla in P. oblongopunctatus, too, though, histological data for this species are not available. In the ground beetles Pterostichus aethiops and P. oblongopunctatus, specifi c chemical stimuli have been identifi ed by electrophysiology for three of the four chemosensory neurons. Two respond to various electrolyte solutions. By their specifi c response characteristics these neurons were classifi ed as salt- (cation-) and pH-sensitive neurons (Merivee et al. 2004, 2005; Milius et al. 2006). Th e third chemoreceptor neuron is sensitive to sucrose, maltose and glucose, important in plant

101 carbohydrate metabolism (Merivee et al. 2007, 2008), most probably related to granivory of these two forest species (Heikkilä 1977; Nystrand and Granström 2000). Th e action potentials (spikes) produced by the salt-, pH-, and sugar-sensitive neurons can be distinguished by their waveform, polarity and amplitude. Th e pH-sensitive neuron generates large two-tipped spikes with positive polarity. In contrast, the polarity of the one-tipped spikes from the salt- and sugar-sensitive neurons is negative. It has been also observed that the spikes generated by the sugar-sensitive neuron are shorter in amplitude compared to those generated by the salt-sensitive neuron (Merivee et al. 2004, 2005, 2007). Carbohydrates function as strong phagostimulants to most herbivorous insects, equipped with specialized receptors to detect these compounds (Chapman 1998; Schoonhoven and van Loon 2002; Chapman 2003) suggesting that these chemicals may have phagostimulatory role in herbivorous and omnivorous ground beetles, too. Unfortunately, no data exist on the function of the 4th chemoreceptor neuron probably also located in these antennal sensilla of ground beetles, including P. oblongopunctatus (Daly and Ryan 1979; Merivee et al. 2008).

In addition to detecting phagostimulants, most animals, including herbivorous insects, possess chemoreceptor neurons responding to a diverse range of feeding deterrent compounds. When stimulated, deterrent neurons reduce or fully stop feeding activity (Dethier 1980; Schoonhoven and van Loon 2002; Chapman 2003; Wang et al. 2004). Natural deterrent stimuli recognized by chemoreceptor neurons of herbivorous insects constitute the largest and most structurally diverse class of gustatory stimuli. It has been shown that plant glucosides, for example, sinigrin and salicin, and various alkaloids like nicotine (pyrrolidine group), strychnine and quinine (quinoline group), caﬀ eine (purine group) and several others stimulate the deterrent neuron in many herbivorous insects. Th reshold concentrations for compounds that stimulate a deterrent neuron vary from 10-7 to 10-2 mol l-1, but the majority of these are in the range of 10-4 to 10-2 mol l-1. Th erefore, deterrent neurons fulﬁ l a central role in host-plant recognition and have, since their discovery (Ishikawa 1966), attracted much interest (Glendinning 2002; Ryan 2002; Schoonhoven and van Loon 2002; Chapman 2003; Jørgensen et al. 2007).

Herbivory, including pre- and post-dispersal plant seed predation, is extremely common in virtually all ecosystems. Seeds with their very high

102 nutrient values per unit volume are extremely attractive to granivores as food (Janzen 1971). To counterbalance the eff ects of herbivory, plants have developed a broad spectrum of secondary metabolites involved in plant defense, which are collectively known as antiherbivory compounds and can be classifi ed into three main sub-groups: nitrogen compounds (including alkaloids, cyanogenic glycosides and glucosinolates), terpenoids, and phenolics. Th ese compounds can act as feeding deterrents or toxins with various modes of action to herbivores, or reduce plant digestibility (Janzen 1971; Jolivet 1998; Ryan 2002; Chapman 2003; Hulme and Benkman 2004; Davis et al. 2008). Seeds are sources of some of the most toxic natural products known to humans and the secondary chemicals in seeds present formidable challenges to granivores. However, seed chemical defenses can only be assessed with reference to specifi c target organisms since a secondary chemical may not be equally toxic to all granivores. Some seeds are so toxic that all seed-predators avoid them (Harborne 1993; Hulme and Benkman 2004). Most toxins are usually in small concentrations (<5%) in the seed and some alkaloids, for example, can be lethal at concentrations as low as 0.1% (Hatzold et al. 1983; Harborne 1993). Unfortunately, there is no evidence that plant secondary compounds cause feeding deterrency in ground beetles.

However, due to selective seed predation by ground beetles (Toft and Bilde, 2002; Tooley and Brust, 2002), the presence of a deterrent neuron in their antennal contact chemoreceptors should be expected. We tested the stimulatory eﬀ ect of some plant alkaloids and glucosides on the chemoreceptor neurons innervating the antennal chaetoid taste sensilla in the omnivorous P. oblongopunctatus, known as an important predator on conifer seeds (Heikkilä 1977; Nystrand and Granström 2000). Th ese experiments are reported in this paper.

103 METHODS AND MATERIALS

Insects Adult test beetles of P. oblongopunctatus were collected from a local population in southern Estonia in September and October 2008. Th ey were kept in 30×20×10 cm plastic boxes ﬁ lled with moistened moss and pieces of brown-rotted wood in a refrigerator at 5-6 ºC. A week prior to the experiments, the beetles were transferred to room temperature (22 ºC), fed with moistened cat food (Friskies Vitality+, Nestlé Purina, Hungary) and given clean water to drink every day. Electrophysiological experiments were conducted from November 2009 to February 2010.

Electrophysiology Th e beetle to be tested was intact and restrained securely in a conical tube made of thin sheet-aluminium of a size that allowed the head and antennae to protrude from the narrower end. Th e wider rear end of the tube was blocked with plasticine to prevent the beetle from retreating out of the tube. Its antennae were ﬁ xed horizontally to the edge of an aluminium stand with special clamps and beeswax, so that the horizontally-located large chaetoid taste sensilla were visible from above under a light microscope, and easily accessible for micromanipulation from the side. Care was taken not to contaminate the taste sensilla by contact with instruments or beeswax. Each prepared test beetle was placed in a Faraday cage (15x10x5 cm) for electrophysiology.

Spikes from chemoreceptor neurons of the sensilla were recorded by the conventional single sensillum tip-recording technique (Hodgson et al. 1955). To achieve a good signal to noise ratio, the indiff erent tungsten microelectrode was inserted into the base of the fl agellum free of muscular tissue. It was connected to an AC amplifi er via a calibration source, and earthed. Th e recording glass micropipette fi lled with stimulating solution was brought into contact with the tip of the sensillum by means of a micromanipulator under visual control through a light microscope at a magnifi cation of 300X. Approximately, two sec before each stimulation, 2-3 drops of solution were squeezed out of the micropipette tip by a syringe connected to the rear end of the micropipette by means of a silicone tube, and adsorbed onto a piece of fi lter paper attached to another micromanipulator, in order to avoid concentration changes in the capillary tip due to evaporation. During each 5 sec stimulation period, spikes picked up by the recording electrode were fi ltered with a bandwidth set at 100–2000 Hz, amplifi ed (input impedance 10 GΩ; amplifi cation factor

104 2000X), monitored on an oscilloscope screen, and relayed to a computer via an analogue-to-digital input board DAS-1401 (Keithley, Taunton, MA, USA) for data acquisition, storage, and analysis using TestPoint software (Capital Equipment Corp., Billerica, MA, USA) at a sampling rate of 10 kHz. Responses were recorded from neurons of two to three randomly-selected taste sensilla on the ﬂ agellomeres 1 to 9 of each test beetle. Each sensillum was stimulated twice, with control (10 mmol l-1 NaCl) and test stimulus solution, respectively. In each subsequent test beetle, the order of stimulation was reversed.

Chemicals Sodium chloride, sucrose, sinigrin, salicin and caﬀ eine were purchased from AppliChem (Germany). Quinine, quinine hydrochloride dihydrate, nicotine and L-strychnine were obtained from Sigma-Aldrich company. Except for quinine and strychnine, stimulating chemicals, at concentration of 10 mmol l-1, and were diluted in 10 mmol l-1 sodium chloride. Due to their poor solubility in water, 1 mmol l-1 quinine and 0.1 mmol l-1 strychnine in 10 mmol l-1 sodium chloride were used in the experiments. Additionally, to assess the threshold concentration and concentration/response curve for one of the most active compounds, quinine hydrochloride dihydrate, a water soluble salt of quinine, was tested at concentrations ranging from 0.001 to 50 mmol l-1. All solutions were kept at 5°C for less than one week.

Feeding bioassays Two-choice feeding experiments were carried out in 110-mm diameter glass Petri dishes. To ensure high air humidity inside the dishes, each was lined with a moistened Whatman ﬁ lter paper circle (Schleicher & Schuell, England). Two pine seeds (Pinus sibirica), treated for 20 min with distilled water (control) and 0.01 to 50 mmol l-1 quinine hydrochloride, respectively, were placed in the centre of each Petri dish, 50 mm from each other. Only seeds with seed coats removed were used in the experiments. Test beetles, one in each Petri dish, were allowed to choose between, and feed on the seeds for 24 h (20 °C, 14:10 light:dark). After 24 h, the beetles were removed and the percentage of predation on control seeds and seeds treated with alkaloid, was visually determined. At each concentration of quinine hydrochloride, the feeding preference of the beetles was tested in ﬁ ve replications (10 beetles in each).

Data analysis Automated classiﬁ cation and counting of action potentials from taste sensilla was not an appropriate method for data analysis be-

105 cause two action potentials frequently coincided to produce an irregular waveform. Instead, spikes from several chemosensory neurons innervating antennal taste sensilla tested were visually distinguished by their polarity and amplitude (Fig. 1), and counted, using TestPoint software. Firing rates (spikes/sec) were analysed by counting the number of action potentials within 5 sec after stimulus onset. Mean fi ring rates and their standard errors were calculated. Paired sample Wilcoxon signed rank was used to determine the signifi cance of diff erences between means. Spike amplitude analysis was performed in order to explain which specifi c neuron was stimulated by the chemical classes tested.

106 RESULTS

Spike shapes and amplitudes evoked by tested chemicals Spike waveform analysis showed that spikes from four diff erent chemoreceptor neurons innervating antennal taste sensilla of the ground beetle Pterostichus oblongopunctatus could be distinguished in the recordings obtained in response to tested chemicals. Earlier, three of the neurons, the salt- (Merivee et al. 2004), pH- (Merivee et al. 2005; Milius et al. 2006) and sugar-sensitive neurons (Merivee et al. 2007, 2008), were identifi ed in this species. Spikes with negative polarity, 1–2 mV in amplitude, arose from the salt-sensitive neuron. Th is neuron generated spikes in a phasic-tonic manner and had an extremely short response latency lasting less than 10 msec (Fig. 1A–J). Two-tipped spikes with positive polarity, 2–4 mV in amplitude, were generated by the pH-sensitive neuron (Merivee et al. 2005; Milius et al. 2006). Compared to the spikes from the salt-sensitive neuron, these positive spikes appeared with a much longer delay (Fig. 1A,F). Th e sugar- sensitive neuron also produced spikes with negative polarity, but smaller in amplitude than those of the salt-sensitive neuron (Fig. 1J). Some tested alkaloids (quinine, caff eine) and an alkaloid salt (quinine hydrochloride) induced a neuron of the taste sensilla to produce spikes with negative polarity, but with a considerably longer period of latency compared to the spikes of the salt- and sugar-sensitive neurons (Fig. 1B–E). Spike amplitude histograms show two maxima, indicating that spikes evoked by salt (NaCl), sugar (sucrose) and quinine hydrochloride were generated by three diff erent neurons, the salt-, sugar- and alkaloid-sensitive neuron, respectively (Fig. 2). Spike amplitudes of the chemoreceptor neurons from diff erent taste sensilla varied to a some extent, from 0.7 to 2 mV (Fig. 1C,E). As a result, in most cases, spike amplitudes generated by the alkaloid-sensitive neuron were shorter than those of the salt-sensitive neuron (Fig. 1B,C). In some cases, however, opposite spike amplitude ratios of the neurons were observed (Fig. 1D).

Eff ect of plant alkaloids and glucosides on the responses of the antennal chemoreceptor neurons Only two of the tested chemicals (Table 1), 1 mmol l-1 quinine and 10 mmol l-1quinine hydrochloride, were highly stimulating for the antennal alkaloid-sensitive neuron, evoking 8.9 and 22.8 spikes/sec, respectively. Th e stimulatory eff ect of 10 mmol l-1 caff eine was very weak, 2.4 spikes/sec. In contrast, some inhibiting eff ect of strychnine and sinigrin to the activity of the neuron was observed. Respective fi ring rates were

107 very low, however (Table 1). Other plant chemicals did not considerably aﬀ ect the spike production of the alkaloid-sensitive neuron. Phasic-tonic response of the alkaloid-sensitive neuron to quinine hydrochloride was concentration dependent over the range of 0.001 to 50 mmol l-1 with the response threshold at 0.01 mmol l-1, and maximum rate of ﬁ ring equal to 67 spikes/sec at 50 mmol l-1 (Figs. 3 and 4). However, compared to the spike trains of the salt-sensitive neuron (Fig. 1; Fig. 5A–F), the phasic component of the response produced by the alkaloid-sensitive neuron was less pronounced (Fig. 1B–E; Fig. 4; Fig. 5C–F).

In addition to stimulation of the alkaloid-sensitive neuron, both quinine and quinine hydrochloride strongly inhibited spike generation by the salt- and pH-sensitive neurons when presented in mixtures with 10 mmol l-1 NaCl (Table 1). Increasing the quinine hydrochloride concentration while keeping the concentration of NaCl constant at 10 mmol l-1 caused a dose- dependent decrease in spike generation by the salt-sensitive neuron (Figs. 3–5). In most tested sensilla, at concentrations above 10 mmol l-1 quinine hydrochloride, only the alkaloid-sensitive neuron fi red while the fi ring rate of the salt-sensitive neuron of the taste sensillum decreased close to zero during the 5 sec adaptation period (Fig. 4). In response to 10 mmol l-1 NaCl alone, the mean number of spikes produced by the pH-sensitive neuron was relatively low compared to that of the salt-sensitive neuron, and varied to some extent in diff erent sensilla (Table 1). At quinine hydrochloride concentrations above 0.01 mmol l-1, the mean fi ring rate of the pH-sensitive neuron dropped to zero (Fig. 3). It was also observed that nicotine and salicin, which did not aff ect the fi ring rate of the alkaloid- sensitive neuron, greatly reduced the response of the pH-sensitive neuron (Table 1). Sinigrin and caff eine suppressed neural activity of the salt- and pH-sensitive neuron, respectively. Th is suggested that the antennal taste sensilla of P. oblongopunctatus may detect plant defensive compounds not only through the activation of specifi c alkaloid-sensitive neuron but also through inhibition of the salt- and pH-sensitive neurons.

Eﬀ ect of quinine hydrochloride on the feeding response of P. oblongopunctatus Feeding choice experiments showed that one of the most stimulating chemicals for the alkaloid-sensitive neuron, quinine hydrochloride, had a concentration dependent deterrent eﬀ ect on seed predation in the ground beetle P. oblongopunctatus, with the threshold concentration at 1 mmol l-1. Th us, the threshold concentration of this alkaloid to evoke a behavioural

108 response was a hundred times higher than that for an electrophysiological response of the alkaloid-sensitive neuron. At higher concentrations, the deterrent eﬀ ect of quinine hydrochloride was very strong. It was observed that pine seeds treated with 50 mmol l-1 quinine hydrochloride were never predated by the beetles (Fig. 6).

109 DISCUSSION

Th e experimental data presented show that the “fourth” chemoreceptor neuron in the antennal taste sensilla of the ground beetle Pterostichus oblongopunctatus may respond to some plant alkaloids such as quinine. Th is ﬁ nding is in agreement with literature data on the feeding habits of this species. P. oblongopunctatus is one of the most common and widespread ground beetles in the forests of northern Europe (Lindroth 1985; Niemelä et al. 1994). In most stands of Pinus sylvestris, seed predation results in 20% seed mortality, although occasionally it reaches 60%. Post-dispersal pine seed predation in the Swedish boreal forests studied was due mainly to the two ground beetle species P. oblongopunctatus and Calathus micropterus. Both are able to consume conifer seeds. Th e larger species, P. oblongopunctatus, is considered to be the more important pine seed predator of the two (Heikkilä 1977; Nystrand and Granström 2000).

Like many herbivores, numerous insect seed-predators have biochemical adaptations to deal with toxic plant secondary compounds, including various detoxifi cation and sequestration mechanisms (Jolivet 1998; Hulme and Benkman 2004; Schoonhoven et al. 2005; Després et al. 2007). However, the diffi culties of dealing with more than a few diff erent kinds of defensive compounds has favoured specialization in seed-predators and helps to explain why a large proportion of them are specialist feeders (Hulme and Benkman 2004). Th e omnivorous ground beetle P. oblongopunctatus predates on many small animals, most frequently on Coleoptera, Diptera, Collembola, Acari etc. (Larochelle 1990), as well as on conifer seeds (Heikkilä 1977; Nystrand and Granström 2000). Probably, the species feeds on seeds of various plants but respective data are not available. Se- lective seed predation has been observed in many other ground beetles, however (Toft and Bilde, 2002; Tooley and Brust, 2002).

To discriminate between toxic and nontoxic seeds, P. oblongopunctatus possesses a speciﬁ c feeding deterrent neuron innervating its antennal chaetoid taste sensilla. A strong stimulatory eﬀ ect of quinine (quinoline group) and quinine hydrochloride to this neuron was observed. Th e threshold concentration for the latter at 0.01 mmol l-1 was close to the upper limit of the threshold concentrations for the plant secondary compounds that stimulate a deterrent neuron in other herbivorous insects (Schoonhoven and van Loon 2002; Chapman 2003; Schoonhoven et al. 2005), probably

110 due to the fact that toxin concentration in seeds is usually much higher than in other parts of the plant (Janzen 1971; Hulme and Benkman 2004; Davis et al. 2008). Th e response of the neuron to quinine hydrochloride was concentration dependent with maximum rates of fi ring reaching up to 70 spikes/secat 50 mmol l-1. In two-choice behavioural experiments, quinine hydrochloride had a strong concentration dependent deterrent eff ect on feeding of P. oblongopunctatus beetles with the response threshold at 1 mmol l-1. Th us, the threshold concentration of this alkaloid to evoke a behavioural response was a hundred times higher than that for an electrophysiological response of the alkaloid-sensitive neuron. In contrast to quinine hydrochloride, the response of the alkaloid-sensitive neuron to caff eine (purine group) was very weak, only 2.4 spikes/sec at 10 mmol l-1 concentration. Th e eff ect of another quinoline alkaloid, strychnine, and sinigrin, to the neuron was rather inhibitory. No response of the neuron to tested pyrrolidine alkaloid, nicotine, and salicin (glucoside) was observed suggesting that the antennal alkaloid-sensitive neuron of P. oblongopunctatus is not equally sensitive to all plant defensive chemicals. Nevertheless, the list of toxic compounds capable of stimulating the antennal alkaloid-sensitive neuron of P. oblongopunctatus is likely to grow longer as more plant defence chemicals are tested.

Quinoline alkaloids are among the most potent insect feeding deterrents occurring mainly in Rutaceous and Rubiaceous plants. Th ey can be present in virtually every part of the plant or just a speciﬁ c part like the bark, rhizome, leaf or seed (Openshaw 1967; Michael 2003; Aniszewski 2007). Th ese alkaloids represent a large and structurally diverse group of plant defensive compounds. When contacted, some of them may be detected by stimulation of a speciﬁ c antennal alkaloid-sensitive neuron of the ground beetles predating on seeds, similarly to quinoline alkaloid-sensitivity of P. oblongopunctatus. Th e ability of granivores to perceive plant defensive compounds allows them to avoid toxic seeds, and survive. High sensitivity of P. oblongopunctatus to plant sugars, commonly known as strong phagostimulants for most phytophagous insects, seems to be also related to its partially granivorous feeding habits. Th e disaccharide maltose was one of the most stimulatory sugars for the sugar-sensitive neuron also innervating antennal taste sensilla of P. oblongopunctatus (Merivee et al. 2008). Maltose is uncommon in nature, it can be found in considerable amounts in germinating seeds as a product of the enzymatic degradation of starch.

111 While stimulating the alkaloid-sensitive neuron, quinine hydrochloride strongly reduced spike production by the salt- and pH-sensitive neurons which also innervate these sensilla in Pterostichus spp. (Merivee et al. 2004, 2005; Milius et al. 2006). Increasing the quinine hydrochloride concentration, while keeping the concentration of NaCl constant, caused a dose-dependent decrease in spike generation by the salt-sensitive neuron. At 10 mmol l-1 and higher concentrations of quinine hydrochloride, predominant fi ring of the alkaloid-sensitive neuron was observed while the fi ring rate of other neurons dropped almost to zero. It was also observed that several tested plant secondary compounds, salicin, sinigrin, caff eine and nicotine, which had only little or no eff ect on the fi ring rate of the alkaloid-sensitive neuron, would greatly reduce the responses of the salt- and pH-sensitive neurons of the antennal taste sensillum of P. oblongopunctatus. Th ese results suggest that the antennal taste sensilla in P. oblongopunctatus may detect toxic and deterrent plant secondary compounds not only through activation of the specifi c alkaloid-sensitive neuron but also through inhibition of taste neurons activated by salts and stimulus pH; this is similar to deterrent inhibition of taste neurons activated by sugars and water in Drosophila (Meunier et al. 2003). Since foodplant acceptability to herbivores depends on the balance between stimulation and deterrency, similar interactions between stimulating chemicals at peripheral taste neurons of herbivorous insects are of widespread occurrence. Plant toxins and deterrents may inhibit sugar neurons (Frazier 1986; van Loon 1990; Messchendorp et al. 1996; Bernays and Chapman 2000; Schoonhoven and van Loon 2002; Meunier et al. 2003; Schoonhoven et al. 2005), water neurons (Meunier et al. 2003), and sugars and salts may inhibit deterrent neurons (Simmonds and Blaney 1983; Shields and Mitchell 1995; Glendinning et al. 2000; Schoonhoven and van Loon 2002; Schoonhoven et al. 2005). Deterrent compounds that on their own do not stimulate any neuron within a sensillum may also decrease the responsiveness of a neuron responding to a nutrient, as exemplifi ed by sinigrin inhibiting the inositol neuron in Heliothis virescens and H. subfl exa (Bernays and Chapman 2000; Schoonhoven and van Loon 2002).

112 trations. However, there are only few data that relate input from inorganic salts to feeding behaviour in phytophagous insects. In some insects, salts have a deterrent eff ect on feeding (Chapman 2002; Schoonhoven and van Loon 2003). In contrast, larvae of Drosophila show dose-dependent responses to NaCl: the appetitive responses to low concentration gradually turn into aversion as concentration is increased up to 200 mmol l-1 and higher (Miyakawa 1982; Liu et al. 2003; Niewalda et al. 2008). Some other insects also respond positively to dilute salts. Cockroaches and housefl ies prefer dilute sodium and potassium chlorides and refuse distilled water. In the two-choice situation, many animals give a bimodal response to sodium chloride. Acceptance at low concentrations switches to rejection at high concentrations (Dethier 1977). In these cases, inorganic salt at low concentration may act as a phagostimulant. Th erefore, NaCl at 10 mmol l-1 may also have a phagostimulatory eff ect in P. oblongopunctatus although no behavioural data is available. Th is assumption could explain the inhibition of spike production of the salt-sensitive neuron by some toxic compounds in P. oblongopunctatus as being similar to how plant deterrents inhibit activity of other phagostimulatory neurons in other phytophagous insects.

In conclusion we state that • the occurrence of an alkaloid-sensitive neuron in the antennal taste sensilla of the ground beetle P. oblongopunctatus was documented; • the alkaloid-sensitive neuron found is most probably related to the granivorous feeding habit of the species; • some tested compounds (quinine and quinine hydrochloride) that stimulate the alkaloid-sensitive neuron may inhibit activity of other neurons (salt- and pH-sensitive neuron) of the sensillum; • several tested plant secondary compounds, salicin, sinigrin, caff eine and nicotine, which had only little or no eff ect on the fi ring rate of the alkaloid-sensitive neuron, through peripheral inhibition, would greatly reduce the responses of other chemoreceptor neurons of the sensillum.

113 Acknowledgements Th e study was supported by Estonian target ﬁ nancing project SF0170057s09 and Estonian Science Foundation grant no. 6958.

114 REFERENCES

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119 Legends for ﬁ gures

Figure 1. Spike waveforms and response types of the chemoreceptor neurons from the antennal chaetoid taste sensilla in P. oblongopunctatus. CN, AN, pHN and SN represent spikes produced by the salt-, alkaloid-, pH- and sugar-sensitive neuron, respectively. Downward defl ection from the baseline represents negative polarity. Recordings A–C, D, and E were obtained from three diff erent sensilla from the same animal, respectively. F–J, recordings were made from the antennal taste sensilla of diff erent beetles. B, C, E, F, I, note the long response latency of the AN; fi rst spike in the spike train of the AN is shown by an arrow. Usually, spike amplitude of the AN was smaller compared to that of CN (see B and C), but in some sensilla they were larger than CN spikes (demonstrated in D). Note the pronounced phasic component of the response from CN (see A–J) and SN (see J), and response with no clear phasic component of the AN (see B, C, E) in these 0.5 sec fragments of the original 5 sec recordings.

Figure 2. (A) Histogram of relative spike amplitudes from neurons stimulated by sucrose and quinine hydrochloride in antennal taste sensillum of P. oblongopunctatus. aAN, aCN and aSN represent spike amplitudes of the alkaloid-, salt- and sugar-sensitive neuron, respectively. Normal distribution curves of respective samples are indicated. Two successive stimulations of the same sensillum with 10 mmol l-1 sucrose and 10 mmol l-1 quinine hydrochloride in the mixture with 10 mmol l-1 NaCl, respectively, were performed. Spikes amplitudes from the two 30 sec recordings were measured and analysed. (B,C) Sample 1 sec fragments of the respective original recordings.

Figure 3. Spike frequencies of the salt- and alkaloid-sensitive neurons in the antennal chaetoid taste sensilla of P. oblongopunctatus upon stimulation with 10 mmol l-1 NaCl mixed with diﬀ erent concentrations of quinine hydrochloride (QHC). CN, AN, pHN, salt-, alkaloid and pH-sensitive neuron, respectively. N, the number of tested beetles. Responses from two to three taste sensilla were tested from each test beetle. Vertical bars denote ± SE of the means.

Figure 4. Responses of the salt- and alkaloid-sensitive neurons from antennal taste sensilla of P. oblongopunctatus during 5 sec stimulation with 10 mmol l-1 NaCl mixed with diﬀ erent concentrations of quinine

120 hydrochloride. CN and AN show spike numbers produced by the salt- and alkaloid-sensitive neuron, respectively; N, the number of tested beetles. Responses from two to three taste sensilla were tested from each test beetle. Vertical bars denote ± SE of the means.

Figure 5. Sample responses of an antennal taste sensillum of P. oblongopunctatus to diﬀ erent concentrations of quinine hydrochloride. Note that spike production of the alkaloid-sensitive neuron increased and that of the salt-sensitive neuron decreased with quinine hydrochloride (QHC) concentration increase. CN and AN show spikes from the salt- and alkaloid-sensitive neurons, respectively.

Figure 6. Deterrent eﬀ ect of quinine hydrochloride (QHC) to the feeding response of P. oblongopunctatus in two-choice experiments. At each variant of seed treatment the feeding preference of the beetles between treated (QHC) and control (distilled water) seeds was tested in ﬁ ve replications (10 beetles in each).

Table 1. Eff ect of tested plant alkaloids and glucosides to the spike production of the chemoreceptor neurons from antennal chaetoid taste sensilla of P. oblongopunctatus. All test chemicals were diluted in 10 mmol l-1 sodium chloride. Due to low solubility of quinine and strychnine in water these chemicals were tested at a lower concentration than others. pHN, CN, AN indicate spikes from pH-, salt- and alkaloid-sensitive neurons, respectively. Asterisks indicate signifi cantly diff erent means, P<0.05 (paired t-test).

121 Figure 1.

A 1 mV CN pHN NaCl 10 mmol l-1

B AN CN AN NaCl 10 mmol l-1 quinine hydrochloride dihydrate 1 mmol l-1

C CN AN -1 AN NaCl 10 mmol l quinine hydrochloride dihydrate 50 mmol l-1

D AN CN AN NaCl 10 mmol l-1 quinine hydrochloride dihydrate 1 mmol l-1

AN E CN AN NaCl 10 mmol l-1 caffeine 10 mmol l-1 F

CN pHN NaCl 10 mmol l-1 nicotine 10 mmol l-1 G

CN NaCl 10 mmol l-1 strychnine 0.1 mmol l-1 AN H CN NaCl 10 mmol l-1 salicin 10 mmol l-1 I CN NaCl 10 mmol l-1 sinigrin 10 mmol l-1 J CN SN NaCl 10 mmol l-1 sucrose 10 mmol l-1

0 0.1 0.2 0.3 0.4 0.5 Time (sec)

122 Figure 2.

35 A aAN/aCN 30 aSN/aCN

10 Number of observations

0 0,45 0,50 0,55 0,60 0,65 0,70 0,75 0,80 0,85 0,90 0,95 1,00 Ratio of spike amplitudes

10 mmol l-1 quinine hydrochloride in 10 mmol l -1 NaCl

B salt-sensitive neuron alkaloid-sensitive neuron

10 mmol l-1 sucrose in 10 mmol l -1 NaCl

C salt-sensitive neuron sugar-sensitive neuron

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (sec)

123 Figure 3.

70 N=7

30 CN

First second response (spikes/sec) 10 pHN 0 AN

0 0.001 0.01 0.1 1 10 50 Concentration of QHC (mmol l-1 )

124 Figure 4.

A 70 60 QHC 0.1 mmol l ¹ 50 N=7 40 CN 30 20

10 AN

Mean firing rate (spikes/sec) 0 pHN

B 70 60 QHC 1 mmol l ¹ N=23 50 40 30 CN 20 AN 10 pHN

Mean firing rate (spikes/sec) 0 C 70 60 QHC 10 mmol l ¹ AN N=23 50 40 30 20 10 CN

Mean firing rate (spikes/sec) 0 pHN

D 70 AN 60 QHC 50 mmol l ¹ N=26 50 40 30 20 10 CN pHN Mean firing rate (spikes/sec) 0 1 2 3 4 5 Time (sec)

125 Figure 5.

-1 CN 10 mmol l NaCl (control) A

-1 -1 CN 0.01 mmol l QHC in 10 mmol l NaCl B

0.1 mmol l-1 QHC in 10 mmol l-1 NaCl CN AN AN C

-1 -1 CN 1 mmol l QHC in 10 mmol l NaCl AN AN D

CN 10 mmol l-1 QHC in 10 mmol l-1 NaCl AN E

CN 50 mmol l-1 QHC in 10 mmol l-1 NaCl 8 AN F

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (sec)

126 Figure 6.

120 * 100 *

80 * n.s n.s 60

Feeding preference (%) 40

0 -1 1 -1 -1 -1 - o o ontro ontro ont o Contr l Cl 0 ml Cl Crl0 Contr l ml HC 1 m Q ml HC 0 QHC 50 m QHC 1 m QHC .1 mml Q .01 mml

127 (bristles) Tested beetles N Tested (spikes/s±SE) to test solution Mean 5 s response 5 s response Mean

-1 (spikes/s±SE) Mean 5 s response 5 s response Mean to NaCl 10 mmol l to NaCl ANAN 1.1 ± 0.37 0.7± 0.21 8.9 ± 1.19** 0.3 ± 0.08* AN 0.8 ± 0.20 0.9 ± 0.24 AN 1.7 ± 0.46 0.8 ± 0.22** AN 0.1 ± 0.11 0.1 ± 0.11 AN 1.0 ± 0.27 22.8 ± 2.91** AN 1.0 ± 0.20 2.4 ± 0.44** CNCN 11.7 ± 0.82 11.1 ± 0.86 10.1 ± 0.81* 10.2 ± 0.84 CN 10.9 ± 0.63 12.1 ± 0.82 CN 11.8 ± 0.61 5.2 ± 0.35** CN 14.2 ± 0.67 10.9 ± 2.5 CN 12.4 ± 0.94 11.7 ± 0.85 neuron Responding ) -1 10 pHN 8.2 ± 2.3 0.0 ± 0.00** 15(45) (mmol l Concentration eine 10 pHN 3.2 ± 0.88 1.7 ± 0.73* 11(34) Salicin 10 pHN 1.2 ± 0.64 0.7 ± 0.10** 15(30) Sinigrin 10 pHN 0.6 ± 0.22 0.5 ± 0.19 16(48) Caﬀ Quinine 1 pHN 0.4 ± 0.21 0.0 ± 0.00* 9(27) Quinine Quinine Nicotine 10 pHN 12.3 ± 2.97 0.4 ± 0.17* 25(50) Strychnine 0.1 pHN 0.2 ± 0.08 1.6 ± 0.67* 8(24) Compound hydrochloride (salt of quinine) CN 16.2 ± 1.88 1.6 ± 0.60** Quinoline alkaloids Quinoline Glucosides Purine alkaloids Purine Pyrrolidine alkaloids Pyrrolidine Table 1.

128 CURRICULUM VITAE

First name: Marit Last name: Komendant Date birth: May 10th 1980 Address: Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia E-mail: [email protected]

Institution and position held: 2006–... Selteret OÜ oﬃ ce manager

Education: 2006–2010 PhD studies in Entomology, Estonian University of Life Sciences 2004–2006 MSc studies in Plant Protection, Estonian University of Life Sciences 2000–2004 BSc studies in Horticulture, Estonian Agricultural University 1998–2000 Räpina Higher Gardening School 1986–1998 Gymnasium of Forseliuse

Academic degree: Master’s Degree (2006), Th esis: Antennal pH receptors in the ground beetles Pterostichus aethiops and P. oblongopunctatus (Coleoptera, Carabidae), Estonian University of Life Sciences.

Research interests: Biosciences and Environment, Ecology, Biosystematics and -physiology (Insect behaviour and sensor physiology)

Languages spoken: Estonian, English, German, Russian

129 ELULOOKIRJELDUS

Eesnimi: Marit Perekonnanimi: Milius Sünniaeg: 10. mai 1980 Aadress: Põllumajandus- ja keskkonnainstituut, Eesti Maaülikool, Kreutzwaldi 1, Tartu 51014 E-mail: [email protected]

Töökoht ja amet: 2006–... Selteret OÜ büroojuht

Haridus: 2006–2010 doktoriõpe entomoloogia erialal, Eesti Maaülikool 2004–2006 magistriõpe taimekaitse erialal, Eesti Maaülikool 2000–2004 bakalaureuseõpe aianduse erialal, Eesti Põllumajandusülikool 1998–2000 Räpina Kõrgem Aianduskool 1986–1998 Forseliuse Gümnaasium

Teaduskraad: Teadusmagister 2006, väitekiri: “Antennaalsed pH-retseptorid süsijooksikutel Pterostichus aethiops ja P. oblongopunctatus (Coleoptera, Carabidae)”, Eesti Maaülikool

Teadustöö põhisuunad: Bio- ja keskkonnateadused, Ökoloogia, biosüstemaatika ja -füsioloogia (Putukate käitumine ja sensoorne füsioloogia)

130 LIST OF PUBLICATIONS

1.1. Publications indexed in the ISI Web of Science database:

Merivee, E., Märtmann, H., Must, A., Milius, M., Williams, I., Mänd, M., 2008. Electrophysiological responses from neurons of antennal taste sensilla in the polyphagous predatory ground beetle Pterostichus oblongopunctatus (Fabricius 1787) to plant sugars and amino acids. Journal of Insect Physiology 54, 1213–1219.

Merivee, E., Must, A., Milius, M., Luik, A., 2007. Electrophysiological identiﬁ cation of the sugar cell in antennal taste sensilla of the predatory ground beetle Pterostichus aethiops. Journal of Insect Physiology 53, 377–384.

Milius, M., Merivee, E., Williams, I., Luik, A., Mänd, M., Must, A., 2006. A new method for Electrophysiological identiﬁ cation of antennal pH receptor cells in ground beetles: the example of Pterostichus aethiops (Panzer, 1796) (Coleoptera, Carabidae). Journal of Insect Physiology 52, 960–967.

Merivee, E., Ploomi, A., Milius, M., Luik, A., Heidemaa, M., 2005. Electrophysiological identiﬁ cation of antennal pH receptors in the ground beetle Pterostichus oblongopunctatus. Physiological Entomology 30(2), 122–33.

1.2. Papers published in other peer-reviewed international journals with a registered code:

Merivee, E., Must, A., Milius, M., Luik, A., 2006. External stimuli in searching for favourable habitat, overwintering sites and refugia of ground beetles: a short review. Agronomy Research 4, 299–302.

3.5. Papers in Estonian and in other peer-reviewed research journals with a local editorial board:

Milius, M., Merivee, E., Mänd, M., Ploomi, A., 2005. Metsa- süsijooksiku antennaalsete maitseharjaste reaktsioonid 10 ja 100 mM fosfaatpuhvritele. EPMÜ Teadustööde kogumik 220, 228–230.

131 VIIS VIIMAST KAITSMIST

THEA KULL REPRODUCTION ECOLOGY AND GENETIC DIVERSITY OF DECLINING SEDGE (CAREX) SPECIES Prof. Tiiu Kull Dr. Tatjana Oja November 23, 2011

MARGIT HEINLAAN ECOTOXICOLOGICAL EVALUATION OF SYNTHETIC NANOPARTICLES AND PARTICULATE ENVIRONMENTAL SAMPLES. Prof. Henri-Charles Dubourguier Prof. Kalev Sepp Dr. Anne Kahru December 14, 2011

REIN DRENKHAN EPIDEMIOLOGICAL INVESTIGATION OF PINE FOLIAGE DISEASES BY THE USE OF THE NEEDLE TRACE METHOD Dr. Märt Hanso January 13, 2011

MIHKEL KIVISTE CONDITION AND RESIDUAL BEARING CAPACITY OF EXISTING REINFORCED CONCRETE STRUCTURES Prof. Jaan Miljan January 21, 2011

TATJANA KUZNETSOVA PLANTATIONS OF NATIVE AND INTRODUCED TREE SPECIES IN THE RECLAMATION OF OIL SHALE POST-MINING AREAS Dr. Malle Mandre Dr. Henn Pärn January 25, 2011

ISBN 978-9949-484-00-3