J Comp Physiol (1983) 152:219-229

Journal of Comparative Physiology. A

9 Springer-Verlag 1983

Response characteristics and specificity of chemoreceptors in Hemilepistus reaumuri (Crustacea, Isopoda)

Giinter Seelinger
Institut fiir Zoologie, Universit/itsstrasse 31, D-8400 Regensburg, Federal Republic of Germany Accepted February 17, 1983

Summary. 1. Chemoreceptor units were investigated electrophysiologically in the apical sensory cone on the antenna of the desert isopod, Hemilepistus reaumuri. Activity of 141 single units was re: corded from fibres in the antennal nerve. 2. The receptors responded to a variety of chemical substances with excitation up to 300 imp/s or with inhibition. They were classified into distinct physiological response types with non-overlapping response spectra (Table 1). 3. Two physiological response types were excited by airborne odour stimuli: (a) butyric acid cells were sensitive to short-chain fatty acids and aldehydes (Table 3); (b) amine cells responded to short-chain mono- and diamines (Table 5). 4. Four groups of gustatory cells ,were found: (c) sugar cells responded to several sugars and eglucosides (Table 6); (d) calcium cells were excited by CaC12 which was inhibitory in most other cells; (e) amino acid cells were found in only two cases, responding to 1-asparagine and 1-methionine; (f) 29 cells responded only to an aqueous rinse of desert woodlice, and not to other stimuli tested. 5. Olfactory cells responded to n-fatty acids (namines, respectively) with phasic-tonic excitation at low concentrations, but with phasic excitation followed by an off-response at higher concentrations (Figs. 1, 3). Complete inhibition could be caused by applying still higher concentrations of the same substances. 6. Transition from phasic-tonic to phasic responses occurred at low initial impulse frequencies with some stimulus substances, but at high initial frequencies with other substances in the same receptor cell (Figs. 1, 3). Chain length of the stimulus molecule influenced phasic-tonic behaviour in a different manner in both olfactory cell groups (Tables 2, 4). 7. No influence of stimulus concentration on

time course was found in sugar cells (Figs. 4a, b, 5). There were, however, remarkable differences in the poststimulatory frequency decay for different sugars (Fig. 4 a, b).

Introduction
The function of arthropod chemoreceptors has been primarily studied in terrestrial insects and in comparatively few Crustacea, mainly in aquatic decapods. Both groups of animals live in different environments where different classes of substances are available for communication and food detection. A general comparison of insect and crustacean chemoreceptors must take these differences into account. Furthermore, it seems desirable to postpone such a comparison until more different Crustacea will be investigated and some general features can be delineated for this group. Some crustaceans are especially interesting in this context because they have conquered a terrestrial environment. It may be asked whether their chemosensory system has evolved in parallel to the insects', and if a distinction between olfactory and gustatory organs can be made which is difficult in aquatic organisms (Atema 1977). The desert isopod Hernilepistus reaumuri is certainly among the most successful terrestrial crustaceans. Electrophysiological studies showed that mechanical and chemical stimuli are perceived by a sensory organ at the tip of the second antenna (Seelinger 1977). This cone-shaped organ plays an important role in social contexts (Linsenmair and Linsenmair 1971 ; Linsenmair 1972) and probably also in food detection. The present paper describes responses of such apical cone receptors to chemical stimuli with em-

220

G. Seelinger: Chemoreceptors of Hemilepistus 1 s, air stream velocity 3 m/s. Initiation and termination of the stimulus was recorded by a thermistor circuit. The tip opening of the stimulus syringe was 1.8 mm wide and located 3-4 mm from the receptor organ. A vacuum pump removed air from the preparation during the experiments. Syringes were refilled twice a week, or at the beginning of an experiment. Both taste and odour stimuli were presented at 2 min intervals. After 5-10test stimuli a control stimulus was given: contact with distilled water as a control for taste stimuli, a syringe with paraffin oil for odour stimuli. Intensity of the cell response was measured as the number of impulses in the first 100 ms period of the response, beginning with the first impulse after stimulus initiation. Control values were subtracted from the test values. Experiments were performed at room temperature from 20-26 ~

phasis on three different aspects. Firstly, it is asked whether response types of different specificity can be defined. Secondly, the classification of apical cone receptors as olfactory or gustatory cells is discussed. Finally, emphasis is laid on the description of response parameters, since examples from other crustacean chemoreceptors show a variety of response patterns. In the crayfish, Orconectes, different receptor types are characterised by their specific time course of excitation (Bauer and Hatt 1980). Phasic and tonic responses were reported for antennular receptors of the spiny lobster (Fuzessery 1978). In Hernilepistus, chemical nature and concentration of the stimuli affected the time course of chemoreceptor responses.
Materials and methods
Hemilepistus reaumuri (Audouin and Savigny) males and
females from Gabes, Tunesia, were kept in the laboratory at Regensburg, F R G , for several months at 28 ~ and less than 40% rel. humidity. Experiments were performed on adult animals and on juveniles not younger than nine months old.

Results

Electrophysiology. Action potentials of single sense cells were

recorded from the axons in the distal part of the antennal nerve. The recording electrode was a platinum-iridium wire which had been electrolytically sharpened to 2-3 gm tip diameter. It was covered with a glass mantle for electric insulation except the very tip. A platinum-coat was applied to the tip by electrolysis immediately before the experiment. The recording electrode was inserted through the joint membrane of the distal antennal segment, the reference electrode (an uninsulated platinum-iridium wire) through a sensory pit on this segment. Standard equipment was used for amplification and registration of the action potentials. Stimuli and stimulus application: Various chemicals were selected as stimuli which were known to elicit responses in other arthropod chemoreceptors: fatty acids, aldehydes, alcohols, amines, esters, sugars, glucosides, inorganic salts, amino acids, and other organic substances (a detailed list is given by Seelinger 1977). Complex stimuli were fresh and rotten grass extract and a distilled water rinse of desert isopods which contained pheromones of unknown chemical structure (Linsenmair 1972). The term 'taste stimuli' refers to water-soluble substances of very low vapor pressure which can only reach the receptors after contact of the receptor organ with the test solution. 'Odour stimuli' are stimuli of higher vapor pressure which evaporate from the solvent and are transmitted by the air. Taste stimuli were dissolved in distilled water and applied to the apical cone in glass capillaries of 100 gm tip diameter. A syringe connected to the capillary by a tubing was used to renew the fluid at the capillary tip immediately before stimulation, in order to prevent concentration changes due to evaporation. Sugars and amino acids were freshly dissolved before each experiment. Stimulus onset was characterised by a weak electrical artifact, Stimulus end (withdrawal of the capillary) was recorded with 50 ms accuracy by a photocell. Odour substances were diluted in paraffin oil (Merck DAB 7). For quantitative studies, a stimulus airstream was blown over the antennal tip from a syringe olfactometer described by Kafka (1970) which provided steep rise and decay of the stimulus (about 10 ms). Stimulus duration was approx.

Basic morphological features of the apical cone on the Hemilepistus antenna were described in a previous paper (Seelinger 1977) and may be briefly summarised here. The apical cone is up to 150 gm long and consists of about 55 hair sensilla which are fused except for their distal portions. The somata of about 450 receptor cells are located in the last antennal segment. Their unbranched dendrites reach the very tip of the sensory hairs where an apical pore of 0.2-0.3 gm diameter in each hair provides access for chemical stimuli. The number of dendrites per hair varies from 3 to 11. Two central hairs show modifications indicative of a mechanoreceptive function (Mead et al. 1976). Chemoreceptors usually had little or no spontaneous activity during the experiments. Units firing spontaneously at more than 5 imp/s were not investigated. Peak frequencies of up to 300 imp/s were obtained during stimulation with some chemicals. Size and shape of the impulses did not or only slightly change during stimulation; sometimes it was possible to evaluate the activity of more than one receptor unit in the same recording. Three criteria were used to discriminate between apical cone receptors and other neuronal activities: (a) basal ablation of the antenna did not eliminate cell activity; this criterion could only be checked if the preparation was still vital after the experimental program; (b) taste receptors responded only when the stimulant was brought into contact with the cone tip, and not with other sensilla; (c) responses to olfactory stimuli were eliminated or considerably delayed when the cone-tip was covered with a water-filled glass capillary.

Physiological response types

Preparations were usually viable for 1-2 h. Consequently, 30-60 stimuli were presented to most

G. Seelinger: Chemoreceptors of Hemilepistus

221

Table 1. Responses of 38 chemoreceptor cells to 10 -2 diluted olfactory and 0.1 mol/1 gustatory stimuli. Numbers indicate sequence of recordings; receptor units from the same recording have identical numbers, but different letters. Horizontal bar." inhibition; open circle: no response; smaller full circle: weak response; larger full circle: strong response (for definition see text). No symbol." not tested

Acetic acid Butyric acid* Hexanoic acid * Octanoic acid Decanoic acid Ethanol Butanol Hexanal Octanal Decana[ Benzatdehyde Hex.- ac.- ally[es ter But.-mc.-geranytest Hex.-ac.-nitrile Ethanol Butanol Hexano[ Octanot DecaDgl Citronella[ Geraniot Citrorleltal Ethylamine Butylamine * Hexytamine* Octylamine Decylamine D,L-Vatine L-Nethionine L- G[utamine L-A sparaqine * Glucose* Sucro se NaCl KCL CaCt 2 * Hemilepistus r i n s e *

O--O--Of@CO OOOOoOo0@O OQO@O000~O 0000000000 0000000000 0000000@00 @ 00@0@0000 000@@ 00000 0000000000 0000000000 000000000@ 000@000000 0000000000 - 0000000000 0000000000 0000000000 0000000000 0000000000 - 0000000000 0000000000 0000000000 000000--000 ooo0oooo00 ooooo-oo0o oooo ooo0 oooo-ooooo -oooo-ooooo 00000000--0 00000000--0 oooooooo0o ooooo00oo0 oooooooo-o -oooooooooo ooooooo@oo oeooooeooo o---ooo oooeoeoooo

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000 00000

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(75%) of the cells investigated. Recordings from the same cell were obtained on two consecutive days in a few cases, and several hundred stimuli were applied. The investigation comprised two distinct phases. In the first phase, 38 cells were tested with a program of 37 different stimuli at standard concentrations. Taste substances were given at 0.1 mol/1 concentration, odour substances at 10 -2 dilutions in paraffin oil. The olfactometer was not used for stimulus delivery in the first phase; instead, olfactory stimulus solutions were taken up in a capillary of 100 ~tm tip diameter which then was brought within 0.5 cm distance from the antennal tip. Response intensity was defined as the maximal impulse frequency of any 300 ms period during stimulation. Responses were classified as 'inhibition' (suppression of spontaneous activity), 'no response' (frequency lower or about as high as control value), 'weak excitation' (20-50 imp/s

above control level), or 'strong excitation' (more than 50 imp/s above control level). Responses to controls were usually weak or non-detectable. Some cells, however, were excited or inhibited by water after previous treatment with effective stimuli. Rinsing with distilled water was repeated in these cases until the water response was normal again. Moderate changes in impulse frequency were observed upon contact with salt solutions, e.g. weak excitation by KC1 and, frequently, inhibition by CaC12. Strong responses, however, were only elicited in any given cell by a small selection of chemically related substances. Consequently, the receptors could be classified into distinct physiological response types. Table 1 summarises the responses of all cells which showed a strong response to at least one test substance. In the second phase of investigation, receptors were checked for their physiological response type with a short program of the most effective stimuli

222

G. Seelinger: Chemoreceptors of Hemilepistus

Fig. 1. Responses of a butyric acid cell to different concentrations of some n-fatty acids (original recordings). Concentration means dilution of the stimulus substance in paraffin oil contained in the olfactometer. Second trace of oscilloscope shows record of stimulus airstream with a thermistor circuit. Stimulus shape is almost rectangular (see Kafka 1970), slower changes in the recording trace are mainly due to inertia of the thermistor. Time marks: 100 ms

which are marked with an asterisk in Table 1. Then a more detailed program including the stimulus found to be effective and chemically related substances at various concentrations was tested. Odour stimuli were delivered from the olfactometer. Physiological response types were named after the most effective stimuli. The following numbers of cells were found: (a) 37 butyric acid cells. Receptors of this type responded to short-chain fatty acids and sometimes to aldehydes. Fresh and rotten grass extracts were also effective olfactory stimuli. (b) 49 amine cells. They responded to shortchain mono- and diamines and also to fresh and rotten grass extract. (c) 18 sugar cells. They responded to various sugars and glucosides. (d) 6 calcium cells responded to CaC12, KC1, and some other inorganic salts. (e) 29 'Hemilepistus rinse' cells responded exclusively to the distilled water rinse obtained from desert woodlice. (f) 2 amino acid cells responded to 1-asparagine, 1-methionine, weakly to 1-histidine and Meucine, but not to d,l-valine, glycine, 1-alanine, and 1-glutamine. Types a and b responded to airborne stimuli,

types c - f responded only to contact with stimulus solutions. Response types a-d are described in more detail below. This is not possible for type e and f, since amino acid cells were not found during the second phase of investigation and Hemilepistus rinse cells did not respond to any other stimulus than the chemically undefined rinse solution. Butyric acid cells Time course. Figure I shows responses of a butyric acid cell to various concentrations of n-fatty acids. For any given substance, the time course changed with concentration although this effect is not always visible at first glance. The responses to butyric acid may serve to demonstrate the influence of concentration on time course. Almost tonic firing was obtained near threshold (defined as 10% of the maximal response frequency measured in a cell). The frequency dropped to the resting level at the end of the stimulus. Application of higher concentrations resulted in a high initial frequency followed by a more or less rapid decrease. Activity eventually ceased before stimulus termination; after stimulation impulses appeared again and their frequency rose to a second maximum. Application of high concentrations could fail to elicit

G. Seelinger: Chemoreceptors of Hemilep&tus

Table 2. Effect of chain-length and stimulus concentration on phasic-tonic behaviour of butyric acid cells. F10/F1 : ratio for quantification of phasic-tonic behaviour (see text). Concentration means dilution of stimulus substance in paraffin oil. Each value represents a mean from 8-18 individual cells

223

F1 vs conc.

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Concentration Propionic acid Butyric acid Valeric acid Hexanoic acid

10-4 0.38 + 0.28 0.56___ 0.16 0.57+0.15 0.62 + 0.14

10- a 0.08 + 0,13 0.23 + 0,20 0.30+0.16 0.36 + 0.09

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any response or suppress spontaneous activity completely although the cell responded to lower concentrations of the same substance. Some stimulus substances caused drastic changes in time course with increasing concentrations while others did so to a lower degree. Two or three n-fatty acids (C3-C6 in varying combinations for individual cells, e.g. C3 + C4, C4-C6) were highly effective stimulants for a given butyric acid cell in that they were able to elicit high frequency responses of rather tonic character at certain concentrations. Homologues of lower chain length (C2C3) usually yielded phasic responses of high initial frequency in a narrow concentration range above threshold and suppressed cell activity completely at higher concentrations. Homologues of higher than optimal chain length (C6-C7) caused tonic responses of low frequency near threshold. High concentrations often elicited phasic responses of moderate peak frequency and sometimes suppression of cell activity, too. A quantitative estimation of the phasic-tonic character was obtained by measuring the instantaneous response frequency in 100 ms intervals as FI, F2, etc. and calculating the ratio F10/F1. Table 2 gives the mean FI0/Fa values for all cells tested with 10 .4 and 10 .3 dilutions of the most effective n-fatty acids. A decrease of the tonic component with increasing concentration is obvious for all substances.

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Fig. 2. Upper part." Dose-response curves of two butyric acid cells for the effective n-fatty acids. Abscissa: stimulus intensity in molecules per crn a air. Ordinate: impulse frequency of the first 100 ms of the response ( = F1). Cell A is recognisable by open circles and broken lines, cell B by full circles and solid lines. Letters at top end of dose-response curves specify stimulus substances: P propionic acid, B butyric acid, V valeric acid, H hexanoic acid. Standard deviations from 9-10 individual responses are given for some concentrations in cell B in order to demonstrate response variability. Lower part: Correlation of initial response frequency F1 and the ratio F10/F1 for the same pair of cells. Abscissa: F1 (imp/s). Ordinate: F10/F1

Odour specificity
Butyric acid was an effective stimulus for all cells of this group while the responses to other homologues differed from cell to cell. Other effective substances were butanal, pentanal, heptanal, benzaldehyde, t-2-hexanal, acrylic acid, iso-butyric acid, iso-valeric acid, and 3-butenoic acid. All these substances, however, were only effective in some of the cells tested. Some other aldehydes were always ineffective: ethanal, propanal, heptanal, octanal, nonanal, decanal, cumaldehyde, salicyl aldehyde, anis aldehyde (tested in 10 -3 concentration).

Relative specificity of chemoreceptors is usually described by dose-response curves for the effective stimuli. Butyric acid cells, as has been shown above, respond to different concentrations of the same substance with different time courses. Doseresponse curves are therefore changed in shape when different time intervals of the responses are evaluated. This problem cannot be avoided by elimination of all' phasic' responses, since' phasic' and 'tonic' are only two extremes of a continuous range. Moreover, dose-response curves for a standard time interval would lead to misinterpretations of the relative stimulating capacities, because the time course is more phasi c for the short-chain homologues. A more comprehensive description of the cells' responses can be provided by a combination of dose-response curves for the initial response frequency F1 and a plot correlating this initial frequency with the ratio F10/F1. Figure 2 compares

224

G. Seelinger: Chemoreceptors of

Hemilepistus

two butyric acid cells which differ in their responses to valeric acid. This stimulus elicits strong and rather tonic responses in cell B while cell A responds with a low frequency phasic response. Differences like this one also occurred in simultaneous recordings from pairs of cells in the same preparation. Therefore, they cannot be due to variations in stimulus presentation. An overview on variability of the butyric acid cell is given in Table 3 by the response profiles for two different dilution steps of some fatty acids. The initial response frequency F1 was selected as a measure of the response intensity, since this value is less affected by changes in time course than longer time intervals. It appears that the response profiles for n-fatty acids vary gradually from cell to cell without tendencies for subgroup formation. No correlation is apparent between optimal chain length of n-fatty acids and the effects of other fatty acids. Receptor units from one preparation may either be similar to each other or as different as cells from different preparations. Amine cells Figure 3 shows responses of an amine cell to different concentrations of some n-amines. Time course changes from tonic to phasic-tonic or to purely phasic with increasing concentration, as was described for the butyric acid cells. Complete inhibition by high concentrations was also observed. Influence of chain-length, however, was not like in the former cell group. Long-chain amines often yielded high frequency phasic responses (Table 4). Individual amine cells responded to two, three, four or even five of the n-amines with strong excitation at suitable concentrations. Other effective amines were allyl amine, iso-butylamine, cadaverin 9 and putrescine, while ethylene diamine, diethylamine, diallylamine a n d trimethylamine never elicited a response. Response profiles for 10 -4 and 10 -3 diluted amines are shown in Table 5. Although no clear separation of subgroups occurs, we can recognise a rather uniform group most sensitive to n-amines C~-C 7, to cadaverine and to putrescine. Cells more sensitive to short-chain namines do not respond to the diamines. Sugar cells Sugar cells responded to an effective stimulus with a high initial frequency followed by a lower plateau. A second maximum occurred after stimulus removal, then the frequency dropped to the resting level (Fig. 4A). Concentration effects on time course were investigated with dilution series

Table 3. Response profiles of butyric acid cells for fatty acids in 10 -~ (a) and 10 -3 (b) dilutions. Numbers indicate sequence of recordings, n-fatty acids are separated from unsaturated or branched fatty acids by a blank field. Sizes of full circles stand for categories of relative response intensity as follows (from small to big): less than 26% of maximal response measured in this cell; 26-50% ; 51-75% ; 76-100%. Response intensity measured as mean frequency of the first 100 ms of the response

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Fig. 3. Responses of an amine cell to different concentrations of some n-amines (original recordings). For explanations see legend of Fig. 1

Table 4. Effect of chain length on stimulus concentration on phasic-tonic behaviour of amine cells. See also legend of Table 2 and text
Concentration 10 - 4 10 - 3

Propyl amine Butyl amine Pentyl amine Hexyl amine Heptyl amine

0.52 _ 0.14 0.52 _+0.23 0.50 _+0.23 0.44 _+0.22 0.40 _+0.24

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quency decay. A fast decay was found for a-methylglucoside ( - 135_+ 50 imp/s 2) and fructose (-ll0 slower decay for maltose ( - 70 + 30 imp/s 2) and p-nitrophenyl-~-glucoside ( - 3 5 + 3 0 imp/s 2, see also Figs. 4A, B). Specificity for different sugars varied from cell to cell (Table6). Maltose, fructose, glucose, lactose, ~-methylglucoside, and p-nitrophenyl-0~glucoside were effective stimuli in some cells, while iso-maltose, sorbose and fl-methylglucoside were always ineffective. The lowest threshold found for glucose was 2 mmol/1, the highest one 200 mmol/1. Calcium cells Very few cells of this response type were found, but they were easily recognisable by their strong response to 0.1 tool/1 CaC12. This stimulus inhibited most other receptors completely. Each point of the following description is based on 2-3 cells only and no statistical analysis can be presented. Calcium cells responded to concentrations of CaC12 from 10mmol/1 up. Excitations of 60-90 imp/s were obtained at 0.5-1 tool/1 concentrations. Responses were tonic or phasic-tonic with a somewhat more pronounced initial maximum at higher concentrations. Poststimulatory excitation was very long-lasting, sometimes up to 5 s. In contrast, stimulation with KC1 yielded fast-fading excitations (Fig. 4C). This difference was obvious at all concentrations and excitation levels, and the experimenter could easily decide which of both substances was applied by listening to the response of a single cell coming from a loudspeaker.

of glucose in seven cells. The ratio F10/FI was calculated for 62 responses to 0.02-1 mol/1 concentrations of glucose and the mean for each cell was set to 1. The resulting relative values were plotted vs. FI (Fig. 5) 1. The regression line indicates a 7% decrease of F10/F1 when F1 was changed from 50 to 170 imp/s, which is not significantly different from a horizontal line. Hence, initial response frequency and plateau increased almost proportionally to each other. The slope of the poststimulatory frequency decay remained fairly constant in the range from 0.02-0.5 mol/1 glucose at - 7 0 15 imp/s 2 (0.5 s after stimulus termination). Different sugars and glucosides were applied to 10 sugar cells at 0.1 mol/1 concentration. Similar response patterns were obtained with all effective stimuli, except for the poststimulatory fre1 Plotting F I 0 / F I vs. concentration would have separated cells of different sensitivity on different positions on the abscissa, while initial response frequency covered at least 2/3 of the range of Fig. 5 in each single cell

226 Table 5. Response profiles of amine cells for amines in 10 -+ (a) and 10 -3 (b) dilutions. For explanation of symbols see legend of Table 3 100

G. Seelinger: Chemoreceptors of Hemilepistus

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50 100 150 Imp/s Fig. 5. Phasic-tonic character of sugar cell responses to glucose at different excitation levels. Values from 7 sugar cells are pooled. Each point represents a single response. Abscissa: initial response frequency F1. Ordinate: phasic-tonic ratio F10/F1 divided by the mean F10/Fl-value of the respective cell. All responses with F1 g.e. 50 imp/s are included and the resulting regression line is plotted Table 6. Response profiles of 10 sugar cells for some sugars and c~-glucosides. For explanation of symbols see legend of Table 3

9 6~B
61A
~'

9 .oil. 9 9 9 9149149
" 9
9

gig" ill"
O0"

" O OO:o 9 o O O 0 "

" 9 O O o

" " 9 "O0"

O " g o " 9 9

48A
61.

9 * 9
9 9

OOOo 9
9 l g O

79A
79B 89B

91 "
"

9 c O * o * 0

" O g i g

112 +3
8s 64 s4 10s s7 is ss

9 9 9 9 OOOO 9 O O O O O 9
9 9 9 9 O! 9 9 l O g O 9 9 9 1 4O 9 9 i 9 9

9
9 9

R e s p o n s e s t o K C 1 , SrC12 a n d M g C 1 2 w e r e a b o u t o n e o r d e r o f m a g n i t u d e less s e n s i t i v e , BaC12 w a s still less e f f e c t i v e ( t e s t e d i n 2 cells). I n h i b i t i o n o f s p o n t a n e o u s a c t i v i t y w a s c a u s e d b y 0.1 m o l / 1 N a C 1 . M i x t u r e s o f N a C 1 a n d CaC12 w e r e less effect i v e w i t h i n c r e a s i n g N a C l - c o n c e n t r a t i o n (1 cell).

9 O O O O O O 9 . O o o o O O 9 9 9

9 9 g O O D 9 OOo 9

G. Seelinger:Chemoreceptorsof Hemilepistus
Discussion

227 ander 1977), Titanethes (Abraham and Wolsky 1929), Oniscus and Porcellio (Henke 1960), Metoponorthus (Mead etal. 1976) and Hemilepistus (Schneider 1973 ; Seelinger 1977). Behavioural evidence for a chemoreceptive function was given in several cases, indicating contact perception of food and social stimuli (Henke 1960; Gupta 1962; Mead 1970; Schneider 1971; Linsenmair and Linsenmair 1971; Linsenmair 1972). Fischbach (1954) suggested an additional olfactory role. Alexander (1977) recorded responses upon contact with glutamic acid. The present paper shows that both olfactory and gustatory stimuli are perceived in the Hemilepistus apical cone by sPecialised receptors. Sugar cells, amino acid cells and calcium cells are probably concerned with food recognition, Hemilepistus rinse cells may be the receptors specialised for social recognition (Linsenmair 1972). Fatty acids and amines are also present in the natural food of desert isopods (green plants, human and other faeces, rotten organic materials; Schneider 1971). It remains unclear whether the apical cone plays a role in orientation to food sources or merely serves to check food for edibility. Distance responses to olfactory stimuli have not yet been demonstrated. Both the nature of the effective stimuli and its presumed biological functions would suggest the apical cone of Hemilepistus is a mixed olfactory-gustatory organ. A comparison of threshold concentrations found for Hemilepistus with those reported for decapods (see above) reveals that taste receptors of the isopod are much less sensitive. This may be explained by the fact that compounds of 'prey odour' are diluted rapidly in water and can guide lobster and crayfish to the source as soon as they are detected. Sugar and salt stimuli in food sources of the desert, however, wilt be concentrated by sun and dry air within short time and may even have to be dissolved in secretions before they can be tasted.

Olfaction or taste
Biological role, morphology and physiology of crustacean chemoreceptors are best understood in aquatic decapods. Two receptor systems are especially interesting: the thin-walled aesthetascs on the first antennae and sensory pegs on the dactylopodites. The classical criteria for discrimination between olfactory and gustatory organs would not be applicable to these structures: high thresholds, watery medium and contact perception for taste organs, low thresholds and distance perception in air medium for olfactory organs. Both systems respond in water to amino acids and some other components of prey extracts with high sensitivity. Thresholds of 10-5-10 - l ~ mol/1 were reported for the antennular system (Shepheard 1974; Fuzessery etal. 1978), 1 0 - 5 - 1 0 - 9 tool/1 for the dactyl receptors (Bauer et al. 1981; Derby and Atema 1982a, b). Behavioural responses to prey can be elicited over considerable distances via both systems, although the nature of these responses is quite different: complicated searching behaviour upon aesthetasc stimulation, grasping reflexes upon stimulation of the dactyls (Atema 1977). Atema proposes a definition of smell and taste on the basis of the biological survival function which would be applicable to both land and water arthropods. 'Smell' is understood as perception of stimuli requiring complicated central processing, e.g. orientation and social recognition cues, and is located on the antennae. 'Taste' would be defined as perception of stimuli controlling food uptake via simple reflex mechanisms and would be found on legs and mouthparts. However, many examples hardly fit into this general scheme, e.g. the antennal sugar receptors of the honeybee which can reflexively release proboscis extension. Moreover, it is often difficult to determine the biological role of an organ in all its complexity. It seems more appropriate, in the case of terrestrial arthropods, to refer to the classical distinction between airborne odour stimuli and taste stimuli dissolved in water, although transitions may occur. The same organ may be involved in olfaction or gustation in different situations. Water beetles can follow an odour trail in air and under water using the same receptors (Schaller 1926; Behrend 1971). Insect sensilla with a single terminal pore are usually regarded as 'taste hairs'. In some cases, however, sensitivity to typical odour substances has been demonstrated (St~idler and Hanson 1975; Riith 1976). Apical sensory hairs with a tip pore are present on the second antenna of many terrestrial isopods, probably as homologous structures: in Ligia (Alex-

Quality discrimination
The apical cone of Hemilepistus reaumuri contains at least six groups of receptor cells which respond to different chemical stimuli without an overlapping of their response spectra. Two groups of olfactory cells are present, responding to short-chain fatty acids and to amines, respectively. This is similar to findings in coeloconic sensilla of the locust (Kafka 1970) and in pore plates of Dytiscus (Behrend /971). A cell response depends on the presence of a certain functional group within the molecule and is modified by other features, e.g. chain length. Discrimination between amines on

228

G. Seelinger: Chemoreceptors of Hemilepistus

one hand and fatty acids on the other hand is certainly possible in Hemilepistus. Discrimination between substances stimulating the same receptor group requires cells of different specificity within the group. Different response spectra are present in both olfactory cell groups. While butyric acid cells show individual variation, subgroups may be present in the amine cell group. Olfactory as well as sugar cells seem able to discriminate between many substances. Time patterns may also play a role in quality discrimination. Chemical stimuli usually don't have sharply defined temporal or spatial distributions, but more or less regular pulses of high concentration at the receptors may result from the behaviour of certain animals. Examples are the sniffing of mammals, the antennal flicking of decapod crustacea and the zigzagging flight of a male moth across a pheromone plume. Both time pattern of the stimulus and dynamic properties of the receptors will influence the response pattern in these cases. This also applies to Hemilepistus. While walking around the isopod rapidly waves its antennae. The flicking movements result in short (40-80ms) contacts with the substratum at 100-300 ms intervals (determined with a videorecorder). Consequently, gustatory and close range olfactory stimuli should occur in short pulses. Phasic-tonic response characteristics of olfactory cells and differences in the post-stimulatory frequency decline of gustatory cells should cause ' smooth' a n d ' pulsed' response patterns for different stimuli. A different function of flicking was shown in the spiny lobster Panulirus (Price and Ache 1977; Schmitt and Ache 1979). Flicking lowered the threshold of aesthetasc receptors for amino acids and delayed adaptation effects, probably by improving exposure of the sensilla to the surrounding medium.

Origin of temporalpatterns in olfactory cells

Responses to olfactory stimuli from the syringe olfactometer usually were phasic-tonic at lower concentrations and phasic or inhibited at high concentrations. Inhibition could also be obtained with more gradually rising stimuli when a capillary with stimulus solution was slowly moved towards the receptor organ. An initial increase of activity was followed by a decrease, and frequently by complete inhibition. Impulses would not be observed until withdrawal of the capillary. These observations suggest, that time courses of olfactory cell re-

sponses may be a result of excitatory and inhibitory influences of the same stimulus substance. Both effects are characterised by a threshold concentration and their rate of increase with concentration. These values vary individually from cell to cell and from substance to substance. High frequency tonic responses can only be elicited if the excitatory effect is strong and has a much lower threshold than the inhibitory effect. Phasic excitation and off-responses may reflect the changes of stimulus concentrations at the receptor from non-effective through excitatory to inhibitory, and back. The term inhibition here refers to the partial or total suppression of impulses in the receptor axon. It has no implications for the underlying mechanism. A possible mechanism of inhibition by short-chain fatty acids is a change in acidity of the receptor medium. Kafka (1970) found strong excitation followed suddenly by inhibition when locust olfactory cells were stimulated by formic acid. Inhibition of butyric acid cells by butyric acid in Hemilepistus occurred at concentrations from 1014 to 1016 molecules/cm3 air. This may be sufficient to influence the pH value of the medium surrounding the receptors under certain conditions. However, it is not an extremely high concentration. The water beetle Dytiscus shows positive behavioural responses to still higher concentrations, and reproducibility of responses in Hemilepistus (up to several hundred times over several days) indicated that no damage of receptor cells occurred. Inhibition of amine cells cannot be explained by changes in the pH value since no correlation exists between dissociation constants and inhibitory effect of the n-amines. It is also different from the inhibitory effect of long-chain amines on the Bombyx pheromone receptor which is inhibited at low concentrations and excited at higher concentrations (Kaissling 1977). Octylamine, a potent inhibitor, has no structural similarity to the key compound Bombycol and probably interacts with the lipid membrane. The excitatory effect of amines in Hemilepistus, however, is probably due to specific receptor mechanisms and usually has a lower threshold than the inhibitory effect. Acknowledgements. I am grateful to Prof. J. Boeckh, Dr. U.
Watdow and to all other colleagues who helped with discussion and criticism of the manuscript.

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G. Seelinger: Chemoreceptors of Hemilepistus Alexander CG (1977) Antennal sense organs in the isopod Ligia oceanica. Mar Behav Physiol 5:61-77 Atema J (1977) Functional separation of smell and taste in fish and crustacea. In: LeMagnen J, MacLeod P (eds) Olfaction and taste, vol VI. IRL Press, London Washington, DC, pp 165-174 Behrend K (1971) Riechen in Wasser und Luft bei Dytiscus marginalis L. Z Vergl Physiol 75:108-122 Bauer U, Hatt H (1980) Demonstration of three different types of chemosensifive units in the crayfish claw using a computerized evaluation. Neurosci Lett 17:209-214 Bauer U, Dudel J, Hatt H (1981) Characteristics of single chemoreceptive units sensitive to amino acids and related substances in the crayfish leg. J Comp Physiol 144:67-74 Derby CD, Atema J (1982a) Narrow-spectrum chemoreceptor cells in the walking legs of the lobster Homarus americanus: taste specialists. J Comp Physiol 146:181-189 Derby CD, Atema J (1982b) Chemosensitivity of walking legs of the lobster Homarus americanus: neurophysiological response spectrum and thresholds. J Exp Biol 98:303-315 Fischbach E (1954) Licht-, Schwere- und Geruchssinn bei Isopoden. Zool Jahrb Abt Allg Zool Physiol 65:141 170 Fuzessery ZM (1978) Quantitative stimulation of antennular chemoreceptors of the spiny lobster, Panulirus argus. Comp Biochem Physiol 60:303-308 Fuzessery ZM, Carr WES, Ache BW (1978) Antennular chemosensitivity in the spiny lobster, Panulirus argus: Studies of taurine sensitive receptors. Biol Bull 154:226-240 Gupta M (1962) Contact chemoreception in Oniscus asellus and Porcellio scaber. J Zool Soc Ind 14:145-149 Henke G (1960) Sinnesphysiologische Untersuchungen bei Landisopoden, insbesondere bei Poreellio scaber. Verh Dtsch Zool Ges 54:167-171 Kafka WA (1970) Molekulare Wechselwirkungen bei der Erregung einzelner Riechzellen. Z Vergl Physiol 70:105-143 Kaissling KE (1977) Control of insect behaviour via chemoreceptor organs. In: Shorey HH, McKelvey JJ jr (eds) Chemical control of insect behaviour: theory and applications. Wiley, London New York, pp 45-65

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