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Sensory physiological investigation of carbon dioxide receptors in lepidoptera
0022-1910/90 $3.00 -I-0.00 Copyright 0 1990 Pergamon Press plc
J. insect Physiol.Vol. 36, No. 12, pp. 951-957, 1990 Printed in Great Britain. All rights resewed
SENSORY PHYSIOLOGICAL INVESTIGATION OF CARBON DIOXIDE RECEPTORS IN LEPIDOPTERA FRANZ Institut fiir Zoologie
II, UniversitCt
BOGNER*
Regensburg,
D-8400
(Received 25 April; revised 1 September
Regensburg,
F.R.G.
1990)
Abstract-This report indicates that CO,-receptors are present in labial palp pits of various species of Lepidoptera, suggesting this to be a common occurrence and not to be restricted to certain taxonomic groups. Stimulation of the carbon dioxide receptors revealed uniform response to increasing CO,-concentrations and showed that receptors were responsive to the normal COJevel in surrounding air (0.03%). Similar molecules such as CS2 did not elicit nearly as strong responses as carbon dioxide. The absolute levels of the dose-dependent responses in Lepidoptera showed great similarity to one another. Slight responses to stimulation by humidity or temperature were revealed to be actually dependent on trace CO2 . Therefore these receptors may be designated as olfactory unimodal cells. So called “natural complex odour”-receptors in Pieris were rigorously retested and are shown here to be simple CO,-receptors. The biological function of these CO,-receptors remains a matter of speculation. For the vast majority of Lepidoptera, small differences in CO,-content of the immediate environment (the “microclimate”) would be measurable. However, for the fraction of Lepidoptera occasionally exposed to high CO,-concentrations, the receptors could fulfill a more clear function. Key Word Index: CO,-receptors;
extracellular
sensitivity; specificity; olfaction; labial palp pit; Lepidoptera; single cell recordings
excitability of the respective Rhodogastria-receptors (cf. Bogner et al., 1986). Eltringham (1933) suggested that the pegs in the labial terminal cavity in Pieris were possibly involved in olfaction. Lee et al. (1985) documented such sensitivity and assigned their findings as “natural complex odour”-receptors; they omitted to include CO2 in the list of tested stimuli. In the light of previous findings of CO,-receptors in Rhodogastria (Bogner et al., 1986), Pieris brassicae was chosen as test animal in order to investigate if these receptors are in reality CO,-receptors. This study presents data from a rigorous retesting.
INTRODUCTION
Carbon dioxide is of importance in the life of various insects and behavioral CO,-sensitivity is well established in a large number of species. Physiological investigations of CO,-sensitive exteroreceptors in insects have been undertaken in various studies (Boistel, 1960; Lather, 1964; Kellogg, 1970; Stange, 1975; Bogner, 1986; Bogner et al., 1986; Bogner, 1989). Although there is still no well-founded idea on the biological function of CO,-sensitivity in Lepidoptera, single cell recordings in Bogner et al. (1986) clearly revealed CO,-receptors in an arctiid (Rhodogastria catinca) and a noctuid (Achaea lineradii) moth; these receptors show a high sensitivity and specificity even though various odourants elicit moderate responses. In an effort to build on these findings, receptors in labial palp pits were investigated in detail in various species of Lepidoptera (butterflies as well as moths) from different biotopes. In addition, Galleria (Pyralidae) was chosen as a test animal because it inhabits bee-hives, where CO,-levels can be relatively high (Hazelhoff, 1953; Seeley, 1974). In addition, this study replies to the question of the ‘multimodal
MATERIALS
*Present address: Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853-2702,U.S.A. 951
AND METHODS
Single cell recordings were taken from both sexes of several Rhodogastria catinca (Arctidii), as well as from the following Lepidoptera: (i) European species Autographa gamma L. (Noctuidae), Pieris brassicae L. (Pieridae), Argynnis paphia L. (Nymphalidae) and Galleria melonella L. (Pyralidae), (ii) African species Danaus chrysippus (L.) (Danainae), (iii) North American species Manduca sexta L. (Sphingidae) and (iv) Asiatic species Antherea perneyi BuerrinMeneville (Saturniidae). In this study quantitative data of responses to CO,-stimulation are shown from the respective receptors of Manduca sexta (n = 3),
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FRANZBOGNER
Galleria melonella (n = 6) and Pieris brassicae (n = 7). However, pilot tests in respective receptors
were made with the other species (stimuli were human breath and commercial CO,). All species were from laboratory cultures derived from field-caught specimens; the African species were made available by M. Boppre (Freiburg); Galleria by H. Bogenschiitz (Freiburg), and Manduca by H. Schweikel (Regensburg). The whole animals were fastened in a perplex holder with a sticky tape. Afterwards the labial palps were immobilized with wax and tungsten hooks and the scales were carefully removed with tiny pieces of sticky tape. For the single cell recordings I used standard electrophysiological techniques (Boeckh, 1962) as well as a conventional amplifying and recording system (cf. Sal3, 1976). Tungsten electrodes (tip 4 1 pm) were inserted into the pit cavity until cell responses could be recorded. This method provides an adequate sampling of the population of receptors, but does not allow to rule out the possibility that receptors other than the CO,-sensitive might exist in the labial palp pits [cf. Bogner et al. (1986), cf. Discussion]. Studies of the respective morphology of the labial palp pit (and their sensilla) are published in Lee et al. (1985) (Pieris) and Bogner et al. (1986) (Rhodogastria). The reference electrode was placed in the basis of palpi. The spikes were registered with a cassette tape recorder (SONY TC); selected playback of these recordings onto an oscilloscope were filmed with a “recordine” (Fa. Tiinnies, Freiburg). Cq-stimuli of variable (increasing) concentrations were provided from commercially available gas cylinders (mixture with pressurized air) and delivered via an electric valve through an empty syringe towards the antennae (flow rate of 10 ml/s). The CO,-content after the mixer-valve (Nigretti-Zamba) was checked spectroscopically with an IR-gas analyzer (BINOS-IR, Leybold-Haraeus). As control stimuli I applied commercial N,- and/or O,-gas from cylinders, dry ice, fresh chemically produced CO, (from sodium carbonate) as well as air led through a saturated aqueous solution of Ca(OH), (to remove COz). Olfactory stimuli were delivered as paraffin solutions from a syringe olfactometer (Kafka, 1970), the respective flow rates of all further stimuli was comparable to CO,-application. The odourants (at least 99% purity) were diluted in liquid paraffin (mostly 1: 100) before application. In each case 1 ml of these dilutions were filled into a 8 ml glass vial of a syringe. CS, was chosen as a test stimulus due to the similar molecule structure to CO*. For application of humid air stimuli, vial in syringes was loaded with wet filter paper (1 ml H,O per vial). Temperature stimuli were obtained with + 10°C changed air (thermistor checking at the organ); for these procedures syringes--containing brass cylinders (20 x 20 mm) instead of mentioned
vials-were heated or cooled at least 3 h before application. For retesting of respective Pieris-receptors, vial in syringes were used to house specimens of Pieris (both sexes) or Periplaneta (non-conspecific) as well as plant material of Brassica sp (foodplant) or Urtica (non-foodplant), to analyse their stimulatory effect. As controls, headspace air of freshly freeze-killed specimens as well as such one of living specimens after passage over humid KOH-pellets (removing COz) were employed. The stimulus duration was 1 s except in continuous or in sequential pulsed stimuli; the intervals between stimuli were at least 3 min. The preparation was kept under a stream of normal air (i.e. -0.03% COa) humified by passing through water in a gaswashing bottle, except during the stimulation. Spikes from an individual receptor cell could be recorded during many hours in general. The experimental room was maintained at 23°C. Spikes were counted over the entire stimulus time (1 s), i.e. imps/s-values given in the following are absolute, not only extrapolated from spike counts in the phasic parts of response (this is contrast to Lee et al., 1985). All data include the background level of the cell activity. Values in dose-dependent curves are means (k SD).
RESULTS CO,-receptors
of various Lepidoptera
Pilot tests for CO,-sensitivity of labial palp pit receptors were made with butterflies (Argynnis paphia, Danaus chrysippus) and moths (Antherea perneyi, Autographa gamma). All these species, though from very different biotopes, showed doubtless CO,-receptor sensitivities. Due to the insufficient availability no quantitative data are presented; however, all respective receptors of these species showed obviously a strong sensitivity to CO*stimuli. Figure 1 shows dose-dependencies of responses to CO* for Manduca sexta (Sphingidae). There is a strong increase in the responses up to a spike frequency of about 90 imps/s; 0.03% CO, (=air) elicited evident responses. Even a CO* concentration (0.015%) below the normal air-level was shown stimulative in one single recording. CO,-sensitivity was also found in Galleria melIonella (Pyralidae) (Fig. 2): CO+oncentrations higher than 1% evoked spikes of diminished amplitudes; to these stimuli the receptors remained excited after the cessation of the stimulus and took more time to return to previous unstimulated state. Details of dose-dependent characterization of six cells to stimulation with CO2 are shown in Fig. 3, the curve of the responses of these bee moths shows a strong increase to CO1 concentrations from 0.015% up to ranges of about 5%.
Carbon dioxide receptors in Lepidoptera
Fig. I. Dose-dependent responses of three labial palp pit receptor cells of Manduca Sexta (Sphingidae) to stimulation with CO,. Cell activity when preparation were kept in room air (A), or in 0,/N,-atmosphere (B). The cell response to 0.015% CO, corresponds with the reaction to stimulation with 1: 1 air/Or-mixtures. (Stimulus duration = 1 s.)
CO,-receptors
953
Fig. 3. Dose-dependent responses of labial palp pit receptor cells (+) of Galleria melonella (n = 6), (0) of Pieris brassicae(n = 7) and (0) of Rhodogastriacatinca* (n = 11) to stimulation with CO,. The cell response to 0.03% corresponds with stimulation by room air, and to 0.015%, with stimulation by 1:l air/Or-mixtures. Spontaneous activity of these cells kept in room air. *The values of fiodogastria (0) are from Bogner et al. (1986). Bars: standard deviation.
of Pieris brassicae
Pieris brassicae was chosen as test animal in order to investigate if the “complex odour”-receptors (as described by Lee et al., 1985) are in reality CO,-receptors. Figure 4A shows examples of the responses of a labial palp pit receptor to different CO,-concentrations. There is a strong dosedependency in the response (Fig. 3): 1% CO2 (also Fig. (A): D) triggers an increase in the number of spikes to about 110 imps/s, whereas 0.015% COr still elicits a response of about 35 imps/s, almost equivalent to half air-response. Stimulation with headspace air of confined male/female cabbage butterflies elicited similar responses to those evoked by headspace air of other insects, for example Periplaneta americana, or plant leaves [Fig. 4(B)]. If the headspace air of living Pieris is first passed through humid KOH-pellets or bleaching powder before reaching the receptors, the respective response was reduced drastically from about 100 imps/s to O-2 imps/s (in the case of the presented
recording to 0 imps/s). This lack of response corresponds to a receptor cell activity in CO,-free air suggesting that the cells are responding to CO, and not to odourants from conspecifics and foodplants (see Discussion). Aforementioned receptors perform a moderate sensitivity to odourants in Pieris (Table 1) and ergo seem to portray a comparable profile of the respective receptors in Rhodogastria (cf. Bogner et al., 1986), though not a whole variety of odourants was tested. Since responses to odourants always contain a reaction to the COP of the surrounding room air, in weak responses it is difficult to decide, whether CO, or the respective odourant is causing the stimulation. However, in pilot experiments these mentioned “weak” stimuli caused no responses when the respective headspace air was applied after passage over humid KOH-pellets (removing COr), whereas “strong” odourants still elicited responses after that procedure.
Fig. 2. Responses of a labial palp pit receptor cell of Galleriamelonella(Pyralidae) to room air (B), and to different CO,-concentrations (C-F): (C) 0.1% (D) l%, (E) lo%, (F) 5% (“unfiltered” recording, otherwise, as well as in Figs 4(A), 4(B) and 6 recording system according to Sa13, 1976). Unstimulated cell activity of this receptor cell (kept in room air) (A). Bars: stimulus duration (= I s), 2 mV.
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Fig. 4(A). Responses of a labial palp pit receptor cell of Pieris brussicue(Pieridae) to room air (B), and to different CO*-concentrations (C-F): (C) O.l%, (D) l%, (E) 5%, (F) 10% (receptor cell kept in room air except during stimulation). Cell activity of this receptor cell without stimulation (kept in room air) (A). Bars: stimulus duration (= 1 s), 2 mV.
Fig. 4(B). Responses of a labial palp pit receptor cell of Pieris brassicuemale to stimulation with headspaee air of living specimens of a conspecific male (A) or female (B, C), of a dead conspecific female (D), of a living specimen of Periplunetuumericann (F), of fresh leaves of Urtica (G) and of Brussicu sp. (H, I), of a living conspecific male, after passage over humid KOH-pellets (K). For comparison: stimulation with room air (E), with COP-free air (L). Except for (L) (CO,-free air) the preparate was kept in normal air (0.03% CO,), only in (K) stimulus air stream passed over KOH. The confinement was 30 min (A, B, D, F-H, K) or only 1 min (C, I) before stimulation. The selection of the stimuli was in consequence after Lee et al. (1985). Bars: stimulus duration (= 1 s), 2 mV.
Investigations of CO,-sensitive receptors on labial pulp pits of Rhodogastria
Stimulation with CS, which has a similar molecular structure to CO1, elicited dose-dependent responses (Fig. 5), but even unphysiologically high concentrations (e.g. undiluted CS2) caused no more than 70-80 imps/s. For comparison, in the same cells 1% CO, elicited spike frequencies in the range of 90 imps/s [Fig. 5(D)], responses to air pulses (0.03% COa) levels about 20 imps/s. To decide whether the weak responses to stimuli like humidity and temperature were actually caused by those stimuli, these stimuli were applied under CO,-free conditions. Without CO, the mentioned stimuli revealed no response at all (see Fig. 6 for temperature stimuli).
DISCUSSION
The investigated receptors of labial palpi are described as CO,-receptors, (i) because the CO,-stimuli are by far the most effective stimuli within natural concentrations and they elicit a response which is dependent on the concentration, and (ii) because the activity of the receptors is reduced by removal of CO* from room air (cf. Bogner et al., 1986). It is not ruled out that other receptors than CO,-sensitive may exist in the labial palp pits (cf. methods); however, there were never any indications for such an assumption, neither in this study nor in the previous one (Bogner et al., 1986). If one takes into account the molecule numbers in the headspace air of stimuli, CO, stimuli fall within the following range: headspace air (= 0.03% CO*) of
Carbon dioxide receptors in Lepidoptera
955
Table 1. Odourants tested at IO-’ concerttration on labial palp pit receptors of Pieris
Test compound
Response
Hexane Ethylalcohol Hexanol Pentanol Butanal Hexanal Butyl amin Geraniol Benzylalkohol Toluol Cyclohexenol Butylacetate Propylacetate Octanic acid Terpineol Cyclopentanone Cum01 Acetic acid Propionic acid Butyric acid
0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + -
Fig. 5. Dose-dependence of labial palp pit receptor cells (n =4) of Rhodogastria catinca (Arctiidae) to stimulation with headspace air of parathn dilutions of CS, (preparate kept in room air). (A) Cell activity in CO,-free air or N,-atmosphere, (J3) in room air (=0.03%); (C) cell activity to stimulation with room air, and (D) to 1% CO, stimuli.
of the various species. For that reason the higher “spontaneous” cell activity in Pieris in comparison to the one in other species is not discussed. In addition, the opening of the pit is narrowed by the electrode during the recordings. Moreover, in the living animal, stimuli are bound to be affected by the scales around the pit opening (which were removed always for the measurement). As far as ecological statements are concerned, an absolute comparison of values is even risky, because the investigated species of the various taxonomic groups stem from many different biotopes. The receptors in the labial palp pit of Pieris show a high CO,-sensitivity. The responses to headspace air of conspecifics or of foodplants reveal that the results for Pieris by Lee et al. (1985) should not be attributed to a response to “natural complex odours”, but rather to CO2 alone. Thus pheromones are not important in this context [as Lee et al. (1985) assumed] because headspace air of Pieris-wings of both sexes (Pieris pheromone will only be emitted from special wing-pouches; cf. Bergstrom and Lundgren, 1973) never elicited a response different from that to ordinary air. My own experiments show that carbon dioxide is the component of these “natural complex odours” which is mainly responsible for stimulation [cf. Fig. 4(B)], supporting the view that these receptors are attuned to co,
+ : 325% increase; - : <25% decrease, 0: no effect on the cell activity, compared to air control. a 10m3 paraffine dilution contains about 10” CO, molecules, of paralhne dilutions of pentanol lOI7 and hexanol lOI6 (cf. Kakfa, 1970; Sal3, 1976). The dose-dependent reaction to CS2 lies within the scope of responses to other odourants, but only if (certainly unphysiological) maximal concentrations of CS2 are used. One should also keep in mind that cell responses to an odourous stimulus always contain a response to the CO2 of the room air as well (about 30 imps/s), so that reactions to odourants are in reality weaker. Temperature and humidity stimuli which trigger a weak increase in cell activity in room-air (Bogner et al., 1986) show no response at all without C02. Therefore, it is now possible to ascribe the slight response to temperature or humidity changes (described in Bogner et al., 1986) to the presence of CO, in air. For that reason these receptors could be considered as unimodal. The middle ranges (0.03-l% C02) of the given dose-dependent curves show conspicuous differences in Pieris and Galleria. This result should not be overemphasized since the stimuli parameters cannot be considered strictly equivalent for the sensilla in the pit I
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A
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C D Fig. 6. Activity of a palp pit receptor cell of Rhodogastriu catinca (Arctiidae) kept in room air (A), N,/O,-atmosphere (B) showing the “real spontaneous” activity. Spike frequencies after application of warm CO,-free air (C), or cold CO,-free air (D) apparently not evoking any response. C, D bars: stimulus duration (= 1 s); amplitude 2 mV.
FRANZBOGNER
956 Biological
SigniJicance
Behaviour investigations have proven CO,-sensitivity (Nicolas and Sillans, 1989) for many different species of arthropods: for example in blood-sucking arthropods (e.g. ticks, Garcia, 1962; insects, Friend and Smith, 1977; mosquitoes, Riissler, 1961; Gillies, 1980) including tsetseflies (Bursell, 1987), as well as in wood-eating beetles (Paim and Beckel, 1964), root-eating insects (Klingler, 1966) and honeybees (Lather, 1967; Seeley, 1974). Variable CO1concentrations (of 0.03-7%) in the nest of fig-wasps are essential in triggering sex-specific hatching of offspring (Gailil et al., 1973). Ant workers tend to move up CO,-gradients outside the nest, and this gas therefore has been called the simplest pheromone in an animal communication systems (Wilson, 1976). The biological significance of the CO,-receptors in Lepidoptera is uncertain. However, we can speculate in certain cases: it seems conceivable that CO*sensitivity is an indicator for lacriphagous moths (Banzinger, 1972) because these species suck the tear glands of mammals in complete darkness. Almost the same holds true for lepidopterans that live from overripe or rotten fruit (e.g. Charaxes). COZreceptors have been found in the labial palp pits of bee moths (Galleria melonella, Pyralidae), whose way of living in the bee-hives, where CO2 can reach levels of 9.8% (Walla, 1948) may depend upon the measurement of CO,-levels. This ability could enable those moths to locate sites of low CO,-levels for oviposition. CO,-enrichments in atmosphere can have such effect as slowing larval growth, increasing mortality [Junonia coenia (Lep.: Nymphalidae), Fajer et al., 19891, or enhancing larval growth [Phaseolus lunata (Lep.: Noctuidae), Osbrink et al., 19871; it can also fail to effect larval growth [Pectinophora gossypieZZa (Lep.: Gelechiidae), Akey et al., 19881. Local CO,-differences, may be of potential importance to CO, sensing arthropods. The microclimate within a plant culture shows, besides an annual change, a significant daily change in their COZconcentration levels (e.g. Atkins and Pate, 1977). CO,-minimal levels in vegetation layers may drop to 0.014% (Reinau, 1954) or increase during the night to a maximum of 1.2% (Nakayama and Kimball, 1988). Besides these rather big differences as far as area is concerned, there are significant CO,-changes dependent on the respective biotope, the species of plant, and physiological condition of the plant and/or even the leaves (e.g. Inoue et al., 1968; Field, 1987). CO,-levels are also affected by photosynthesis rates, which need to be specified, as well as by other physiological parameters (for example Cd-plant; Bazzaz and Carlson, 1984; Wray and Strain, 1987). It still remains an open question whether Lepidoptera react behaviourally to these CO,-differences (in time or space) or whether they merely measure these differences. The CO, receptors that were investigated here would respond to differences of
0.015-0.3% with an increase of cell activity from about 20 to 70 imps/s (in the case of Pieris). According to the dose-response curve the animal could distinguish between varying COz-concentrations of up to 3% (see above). Thus as yet unmeasured microclimatological CO,-differences in plant structures could have an additional function for Lepidoptera, for example in selection of leaves for oviposition. Certainly imaginable, but doubtful, is the possibility that these receptors are only relics reflecting an attunement to the higher air-CO, concentrations in the historical past (Gammon et al., 1985). Despite these qualifications, it is now for the first time possible to make a definitive statement about the physiology of lepidopteran palps, structures which according to Kirstensen (1984) are of fundamental taxonomic importance in Lepidoptera. Judging for their numbers, CO,-receptors must play a basic role in the palps. One is not dealing here with an adaptive feature of restricted distribution. Lepidoptera of many different biotopes as well as of different geographical areas possess these organs and share the CO,-sensitivity. In addition, the existence of a defined neuroanatomical “CO,-glomerulus” (cf. Bogner et al., 1986) suggests a still unknown but basic function in the life of Lepidoptera. Acknowledgements-Basic
results of this study were a portion of mv dissertation (Universitv of Regensbura, 1987). I thank J.-Boeckh for his steady interest in the process of the study, C. Albers for providing the mixer valve and the gas analyzer, also people named in the Materials and Methods section for providing the various species of butterflies and moths. I am indebted to M. Boppr& H. Sal3 and U. Waldow as well as to T. Eisner and B. Johnson for stimulating criticism. This study was financially supported by the Deutsche Forschungsgemeinschaft (DFG). REFERENCES Akey D. H., Kimball B. A. and Mauney J. R. (1988) Growth and develooment of the pink bollwotm Pectinophora gossypieia (Lep: Gelechiidae) on bolls of cotton grown in enriched carbon dioxide atmospheres. Envir. Ent. 17, 452-45.5.
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Carbon dioxide receptors in Lepidoptera Bogner F., Boppre M., Ernst K. E. and Boeckh J. (1986) CO, sensitive receptors in labial palps of Rhodogastria moths (Lep.: Arctiidae): physiology, fine structure and central projection. J. Comp. Physiol. A 158, 741-749. Boistel J. (1960) Caracteristiques fonctionnelles des fibres nerveuses et des recepteurs tactiles et olfactifs des insectes. PhD Thesis, Librairie Amette, Paris. Bursell J. (1987) The effect of wind-borne odours on the direction of tlight in tsetse flies Glossina ssp. Physiol. Em. 12, 149-156. Eltringham H. (1933) Thesenses of insects. Methuen, London. Faier E. D.. Bowers M. D. and Bazzaz F. A. (1989) The effects of enriched carbon dioxide atmospheres on plant-insect herbivore interactions. Science 243, 1198-1200. Field C. B. (1987) Leaf-age effects on stomata1 conductance. in Stomata1 Function (Edited by Zeiger E., Farguhar G. D. and Cowan I. R.), pp. 367-384. Stanford University Press, Stanford, California. Friend W.G. andSmithJ. J. B. (1977)Factorsaffectingfeeding by blood-sucking insects. A. Rev. Ent. 22, 309-331. GaililJ.,ZeroniM.andBar-ShalomD.(1973)Carbondioxide and ethylene effects in the coordination between the pollinatorBlastophagaquadricepsand thesyconiuminFicus religiosa. New Phytol. 72, 1113-1127. GammonR.H.,SundquistE.T.andFraserP.J.(1985)History ofcarbondioxideintheatmosphere.InAtmosphericCarbon Dioxide and the Global Carbon Cycle (Edited by Trabalka J. R.), pp. 2563. Oakridge. Garcia R. (1962) Carbon dioxide as an attractant for certain ticks. Ann. Ent. Sot. Am 55, 6055606. Gillis M. T. (1980) The role of carbon dioxide in host-finding by mosquitoes (Diptera: Culicidae): areview. BUN.Em. Res. 70, 525-532. HazelhoffE.F.(1953)Floculationinabeehiveduringsummer. Physiol. Comp. Ecol. 3, 229. Inoue E., Uchijima Z., Udagawa T., Horie T. and Kobayashi K. (1968) Studies of energy and gas exchange within crop canopies: CO,-flux within and above a corn plant canopy. J. Agr. Meteor. (Tokyo) 23, 165-176. Kafka W. A. (1970) Molekulare Wechselwirkung bei der Erregungeinzelner Riechsinneszellen. 2. Vergl. Physiol. 70, 105-143. Kellogg F. E. (1970) Water vapour and carbon dioxide receptors in Aedes aegyti. J. Insect Physiol. 16, 99-108. Kirstensen N. P. (1984) Studies on the morphology and systematic of primitive Lepidoptera. Steenstrupia (Copenhagen) 10, 141-191. Klingler J. (1966) Uber den Sitz der CO,-Rezeptoren bei der Larve von Othiorrhychus sulcatus. Ent. exp. Appl. 9, 271-277.
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Lather V. (1964) Elektrophysiologische Untersuchungen an einzelnen Rezeptoren fiir Geruch, Kohlendioxyd, Luftfeuchtigkeit und Temperatur aufden Antennen der Arbeitsbiene und Drohne (Apis mellifera L.). Z. Vergl. Physiol. 48, 587-623. Lather V. (1967) Verhaltensreaktionen der Bienenarbeiterin bei Dressur auf Kohlendioxid. Z. Vergl. Physiol. 54, 75-84. Lee J. K., Seizer R. and Altner H. (1985) Lamellated outer dendritic segments of a chemoreceptor within wall-pore sensilla in the labial palp-pit organ of the butterfly, Pieris ravae. CeZZTiss. Res. 240. 333-342. Nakayama F. S. and KimballB. A. (1988) Soilcarbon dioxide distribution and flux within the open-top chamber. Agric. J. 80, 394-398. Nicolas G. and Sillans D. (1989) Immediate and latent effects of carbon dioxide on insects. A. Rev. Ent. 34, 97-l 16. OsbrinkW. L.A.,TrumbleJ.T. andWagnerR. E.(1987)Host suitability of Phaseolus Iunata for Trichoplusia ni (Lep: Noctuidae) in controlled carbon dioxide atmospheres. Envir. Ent. 16, 639. Paim U. and Beckel W. E. (1964) The behaviour of the larvae Orthosoma brunneum (Coleoptera: Cerambycidae) in relation to gases in the logs inhabited by the larvae. Can. J. Zool. 42, 327-353. Reinau E. H. (1954) Der Schwellenwert des CO, in Luft der Griingrenze eines Zuckerrtibenschlages bei Gewitterregen. Ber. Dt. Bot. Ges. 67, 44-47. Rijssler H. P. (1961) Versuche zur geruchlichen Anlockung weiblicher Stechmiicken (Aedes aegyptii, Culicidae). Z. Vergl. Physiol. 44, 184-23 1. Sal3 H. (1976) Zur nerviisen Codierung von Geruchsreizen bei Periplaneta americana. J. Comp. Physiol. A 107, 49-65. Seeley T. D. (1974) Atmospheric carbon dioxide regulation in honey bee (Apis mellifera) Colonies. J. Insect Physiol. 20, 2301-2305. Stange G. (1975) Linear relation between stimulus concentration and primary transduction process in insect CO,-receptors. In Olfaction and Taste V (Edited by Denton D. A. and Coghlan J. P.), pp. 205-209. Academic Press, New York. Walla F. (1948) Der Kohlensiiure-Gehalt in verschlossenen Bienenstijcken wiihrend des Winters. Bienenvater 73,331. Wilson E. 0. (1976) The Insect Societies. Harvard University Press, Cambridge. Wray S. M. and Strain B. R. (1987) Interaction of age and competition under CO, enrichment. Funct. Ecol. 1, 145-149.
J. insect Physiol.Vol. 36, No. 12, pp. 951-957, 1990 Printed in Great Britain. All rights resewed
SENSORY PHYSIOLOGICAL INVESTIGATION OF CARBON DIOXIDE RECEPTORS IN LEPIDOPTERA FRANZ Institut fiir Zoologie
II, UniversitCt
BOGNER*
Regensburg,
D-8400
(Received 25 April; revised 1 September
Regensburg,
F.R.G.
1990)
Abstract-This report indicates that CO,-receptors are present in labial palp pits of various species of Lepidoptera, suggesting this to be a common occurrence and not to be restricted to certain taxonomic groups. Stimulation of the carbon dioxide receptors revealed uniform response to increasing CO,-concentrations and showed that receptors were responsive to the normal COJevel in surrounding air (0.03%). Similar molecules such as CS2 did not elicit nearly as strong responses as carbon dioxide. The absolute levels of the dose-dependent responses in Lepidoptera showed great similarity to one another. Slight responses to stimulation by humidity or temperature were revealed to be actually dependent on trace CO2 . Therefore these receptors may be designated as olfactory unimodal cells. So called “natural complex odour”-receptors in Pieris were rigorously retested and are shown here to be simple CO,-receptors. The biological function of these CO,-receptors remains a matter of speculation. For the vast majority of Lepidoptera, small differences in CO,-content of the immediate environment (the “microclimate”) would be measurable. However, for the fraction of Lepidoptera occasionally exposed to high CO,-concentrations, the receptors could fulfill a more clear function. Key Word Index: CO,-receptors;
extracellular
sensitivity; specificity; olfaction; labial palp pit; Lepidoptera; single cell recordings
excitability of the respective Rhodogastria-receptors (cf. Bogner et al., 1986). Eltringham (1933) suggested that the pegs in the labial terminal cavity in Pieris were possibly involved in olfaction. Lee et al. (1985) documented such sensitivity and assigned their findings as “natural complex odour”-receptors; they omitted to include CO2 in the list of tested stimuli. In the light of previous findings of CO,-receptors in Rhodogastria (Bogner et al., 1986), Pieris brassicae was chosen as test animal in order to investigate if these receptors are in reality CO,-receptors. This study presents data from a rigorous retesting.
INTRODUCTION
Carbon dioxide is of importance in the life of various insects and behavioral CO,-sensitivity is well established in a large number of species. Physiological investigations of CO,-sensitive exteroreceptors in insects have been undertaken in various studies (Boistel, 1960; Lather, 1964; Kellogg, 1970; Stange, 1975; Bogner, 1986; Bogner et al., 1986; Bogner, 1989). Although there is still no well-founded idea on the biological function of CO,-sensitivity in Lepidoptera, single cell recordings in Bogner et al. (1986) clearly revealed CO,-receptors in an arctiid (Rhodogastria catinca) and a noctuid (Achaea lineradii) moth; these receptors show a high sensitivity and specificity even though various odourants elicit moderate responses. In an effort to build on these findings, receptors in labial palp pits were investigated in detail in various species of Lepidoptera (butterflies as well as moths) from different biotopes. In addition, Galleria (Pyralidae) was chosen as a test animal because it inhabits bee-hives, where CO,-levels can be relatively high (Hazelhoff, 1953; Seeley, 1974). In addition, this study replies to the question of the ‘multimodal
MATERIALS
*Present address: Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853-2702,U.S.A. 951
AND METHODS
Single cell recordings were taken from both sexes of several Rhodogastria catinca (Arctidii), as well as from the following Lepidoptera: (i) European species Autographa gamma L. (Noctuidae), Pieris brassicae L. (Pieridae), Argynnis paphia L. (Nymphalidae) and Galleria melonella L. (Pyralidae), (ii) African species Danaus chrysippus (L.) (Danainae), (iii) North American species Manduca sexta L. (Sphingidae) and (iv) Asiatic species Antherea perneyi BuerrinMeneville (Saturniidae). In this study quantitative data of responses to CO,-stimulation are shown from the respective receptors of Manduca sexta (n = 3),
952
FRANZBOGNER
Galleria melonella (n = 6) and Pieris brassicae (n = 7). However, pilot tests in respective receptors
were made with the other species (stimuli were human breath and commercial CO,). All species were from laboratory cultures derived from field-caught specimens; the African species were made available by M. Boppre (Freiburg); Galleria by H. Bogenschiitz (Freiburg), and Manduca by H. Schweikel (Regensburg). The whole animals were fastened in a perplex holder with a sticky tape. Afterwards the labial palps were immobilized with wax and tungsten hooks and the scales were carefully removed with tiny pieces of sticky tape. For the single cell recordings I used standard electrophysiological techniques (Boeckh, 1962) as well as a conventional amplifying and recording system (cf. Sal3, 1976). Tungsten electrodes (tip 4 1 pm) were inserted into the pit cavity until cell responses could be recorded. This method provides an adequate sampling of the population of receptors, but does not allow to rule out the possibility that receptors other than the CO,-sensitive might exist in the labial palp pits [cf. Bogner et al. (1986), cf. Discussion]. Studies of the respective morphology of the labial palp pit (and their sensilla) are published in Lee et al. (1985) (Pieris) and Bogner et al. (1986) (Rhodogastria). The reference electrode was placed in the basis of palpi. The spikes were registered with a cassette tape recorder (SONY TC); selected playback of these recordings onto an oscilloscope were filmed with a “recordine” (Fa. Tiinnies, Freiburg). Cq-stimuli of variable (increasing) concentrations were provided from commercially available gas cylinders (mixture with pressurized air) and delivered via an electric valve through an empty syringe towards the antennae (flow rate of 10 ml/s). The CO,-content after the mixer-valve (Nigretti-Zamba) was checked spectroscopically with an IR-gas analyzer (BINOS-IR, Leybold-Haraeus). As control stimuli I applied commercial N,- and/or O,-gas from cylinders, dry ice, fresh chemically produced CO, (from sodium carbonate) as well as air led through a saturated aqueous solution of Ca(OH), (to remove COz). Olfactory stimuli were delivered as paraffin solutions from a syringe olfactometer (Kafka, 1970), the respective flow rates of all further stimuli was comparable to CO,-application. The odourants (at least 99% purity) were diluted in liquid paraffin (mostly 1: 100) before application. In each case 1 ml of these dilutions were filled into a 8 ml glass vial of a syringe. CS, was chosen as a test stimulus due to the similar molecule structure to CO*. For application of humid air stimuli, vial in syringes was loaded with wet filter paper (1 ml H,O per vial). Temperature stimuli were obtained with + 10°C changed air (thermistor checking at the organ); for these procedures syringes--containing brass cylinders (20 x 20 mm) instead of mentioned
vials-were heated or cooled at least 3 h before application. For retesting of respective Pieris-receptors, vial in syringes were used to house specimens of Pieris (both sexes) or Periplaneta (non-conspecific) as well as plant material of Brassica sp (foodplant) or Urtica (non-foodplant), to analyse their stimulatory effect. As controls, headspace air of freshly freeze-killed specimens as well as such one of living specimens after passage over humid KOH-pellets (removing COz) were employed. The stimulus duration was 1 s except in continuous or in sequential pulsed stimuli; the intervals between stimuli were at least 3 min. The preparation was kept under a stream of normal air (i.e. -0.03% COa) humified by passing through water in a gaswashing bottle, except during the stimulation. Spikes from an individual receptor cell could be recorded during many hours in general. The experimental room was maintained at 23°C. Spikes were counted over the entire stimulus time (1 s), i.e. imps/s-values given in the following are absolute, not only extrapolated from spike counts in the phasic parts of response (this is contrast to Lee et al., 1985). All data include the background level of the cell activity. Values in dose-dependent curves are means (k SD).
RESULTS CO,-receptors
of various Lepidoptera
Pilot tests for CO,-sensitivity of labial palp pit receptors were made with butterflies (Argynnis paphia, Danaus chrysippus) and moths (Antherea perneyi, Autographa gamma). All these species, though from very different biotopes, showed doubtless CO,-receptor sensitivities. Due to the insufficient availability no quantitative data are presented; however, all respective receptors of these species showed obviously a strong sensitivity to CO*stimuli. Figure 1 shows dose-dependencies of responses to CO* for Manduca sexta (Sphingidae). There is a strong increase in the responses up to a spike frequency of about 90 imps/s; 0.03% CO, (=air) elicited evident responses. Even a CO* concentration (0.015%) below the normal air-level was shown stimulative in one single recording. CO,-sensitivity was also found in Galleria melIonella (Pyralidae) (Fig. 2): CO+oncentrations higher than 1% evoked spikes of diminished amplitudes; to these stimuli the receptors remained excited after the cessation of the stimulus and took more time to return to previous unstimulated state. Details of dose-dependent characterization of six cells to stimulation with CO2 are shown in Fig. 3, the curve of the responses of these bee moths shows a strong increase to CO1 concentrations from 0.015% up to ranges of about 5%.
Carbon dioxide receptors in Lepidoptera
Fig. I. Dose-dependent responses of three labial palp pit receptor cells of Manduca Sexta (Sphingidae) to stimulation with CO,. Cell activity when preparation were kept in room air (A), or in 0,/N,-atmosphere (B). The cell response to 0.015% CO, corresponds with the reaction to stimulation with 1: 1 air/Or-mixtures. (Stimulus duration = 1 s.)
CO,-receptors
953
Fig. 3. Dose-dependent responses of labial palp pit receptor cells (+) of Galleria melonella (n = 6), (0) of Pieris brassicae(n = 7) and (0) of Rhodogastriacatinca* (n = 11) to stimulation with CO,. The cell response to 0.03% corresponds with stimulation by room air, and to 0.015%, with stimulation by 1:l air/Or-mixtures. Spontaneous activity of these cells kept in room air. *The values of fiodogastria (0) are from Bogner et al. (1986). Bars: standard deviation.
of Pieris brassicae
Pieris brassicae was chosen as test animal in order to investigate if the “complex odour”-receptors (as described by Lee et al., 1985) are in reality CO,-receptors. Figure 4A shows examples of the responses of a labial palp pit receptor to different CO,-concentrations. There is a strong dosedependency in the response (Fig. 3): 1% CO2 (also Fig. (A): D) triggers an increase in the number of spikes to about 110 imps/s, whereas 0.015% COr still elicits a response of about 35 imps/s, almost equivalent to half air-response. Stimulation with headspace air of confined male/female cabbage butterflies elicited similar responses to those evoked by headspace air of other insects, for example Periplaneta americana, or plant leaves [Fig. 4(B)]. If the headspace air of living Pieris is first passed through humid KOH-pellets or bleaching powder before reaching the receptors, the respective response was reduced drastically from about 100 imps/s to O-2 imps/s (in the case of the presented
recording to 0 imps/s). This lack of response corresponds to a receptor cell activity in CO,-free air suggesting that the cells are responding to CO, and not to odourants from conspecifics and foodplants (see Discussion). Aforementioned receptors perform a moderate sensitivity to odourants in Pieris (Table 1) and ergo seem to portray a comparable profile of the respective receptors in Rhodogastria (cf. Bogner et al., 1986), though not a whole variety of odourants was tested. Since responses to odourants always contain a reaction to the COP of the surrounding room air, in weak responses it is difficult to decide, whether CO, or the respective odourant is causing the stimulation. However, in pilot experiments these mentioned “weak” stimuli caused no responses when the respective headspace air was applied after passage over humid KOH-pellets (removing COr), whereas “strong” odourants still elicited responses after that procedure.
Fig. 2. Responses of a labial palp pit receptor cell of Galleriamelonella(Pyralidae) to room air (B), and to different CO,-concentrations (C-F): (C) 0.1% (D) l%, (E) lo%, (F) 5% (“unfiltered” recording, otherwise, as well as in Figs 4(A), 4(B) and 6 recording system according to Sa13, 1976). Unstimulated cell activity of this receptor cell (kept in room air) (A). Bars: stimulus duration (= I s), 2 mV.
954
FRANZBOGNER
Fig. 4(A). Responses of a labial palp pit receptor cell of Pieris brussicue(Pieridae) to room air (B), and to different CO*-concentrations (C-F): (C) O.l%, (D) l%, (E) 5%, (F) 10% (receptor cell kept in room air except during stimulation). Cell activity of this receptor cell without stimulation (kept in room air) (A). Bars: stimulus duration (= 1 s), 2 mV.
Fig. 4(B). Responses of a labial palp pit receptor cell of Pieris brassicuemale to stimulation with headspaee air of living specimens of a conspecific male (A) or female (B, C), of a dead conspecific female (D), of a living specimen of Periplunetuumericann (F), of fresh leaves of Urtica (G) and of Brussicu sp. (H, I), of a living conspecific male, after passage over humid KOH-pellets (K). For comparison: stimulation with room air (E), with COP-free air (L). Except for (L) (CO,-free air) the preparate was kept in normal air (0.03% CO,), only in (K) stimulus air stream passed over KOH. The confinement was 30 min (A, B, D, F-H, K) or only 1 min (C, I) before stimulation. The selection of the stimuli was in consequence after Lee et al. (1985). Bars: stimulus duration (= 1 s), 2 mV.
Investigations of CO,-sensitive receptors on labial pulp pits of Rhodogastria
Stimulation with CS, which has a similar molecular structure to CO1, elicited dose-dependent responses (Fig. 5), but even unphysiologically high concentrations (e.g. undiluted CS2) caused no more than 70-80 imps/s. For comparison, in the same cells 1% CO, elicited spike frequencies in the range of 90 imps/s [Fig. 5(D)], responses to air pulses (0.03% COa) levels about 20 imps/s. To decide whether the weak responses to stimuli like humidity and temperature were actually caused by those stimuli, these stimuli were applied under CO,-free conditions. Without CO, the mentioned stimuli revealed no response at all (see Fig. 6 for temperature stimuli).
DISCUSSION
The investigated receptors of labial palpi are described as CO,-receptors, (i) because the CO,-stimuli are by far the most effective stimuli within natural concentrations and they elicit a response which is dependent on the concentration, and (ii) because the activity of the receptors is reduced by removal of CO* from room air (cf. Bogner et al., 1986). It is not ruled out that other receptors than CO,-sensitive may exist in the labial palp pits (cf. methods); however, there were never any indications for such an assumption, neither in this study nor in the previous one (Bogner et al., 1986). If one takes into account the molecule numbers in the headspace air of stimuli, CO, stimuli fall within the following range: headspace air (= 0.03% CO*) of
Carbon dioxide receptors in Lepidoptera
955
Table 1. Odourants tested at IO-’ concerttration on labial palp pit receptors of Pieris
Test compound
Response
Hexane Ethylalcohol Hexanol Pentanol Butanal Hexanal Butyl amin Geraniol Benzylalkohol Toluol Cyclohexenol Butylacetate Propylacetate Octanic acid Terpineol Cyclopentanone Cum01 Acetic acid Propionic acid Butyric acid
0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + -
Fig. 5. Dose-dependence of labial palp pit receptor cells (n =4) of Rhodogastria catinca (Arctiidae) to stimulation with headspace air of parathn dilutions of CS, (preparate kept in room air). (A) Cell activity in CO,-free air or N,-atmosphere, (J3) in room air (=0.03%); (C) cell activity to stimulation with room air, and (D) to 1% CO, stimuli.
of the various species. For that reason the higher “spontaneous” cell activity in Pieris in comparison to the one in other species is not discussed. In addition, the opening of the pit is narrowed by the electrode during the recordings. Moreover, in the living animal, stimuli are bound to be affected by the scales around the pit opening (which were removed always for the measurement). As far as ecological statements are concerned, an absolute comparison of values is even risky, because the investigated species of the various taxonomic groups stem from many different biotopes. The receptors in the labial palp pit of Pieris show a high CO,-sensitivity. The responses to headspace air of conspecifics or of foodplants reveal that the results for Pieris by Lee et al. (1985) should not be attributed to a response to “natural complex odours”, but rather to CO2 alone. Thus pheromones are not important in this context [as Lee et al. (1985) assumed] because headspace air of Pieris-wings of both sexes (Pieris pheromone will only be emitted from special wing-pouches; cf. Bergstrom and Lundgren, 1973) never elicited a response different from that to ordinary air. My own experiments show that carbon dioxide is the component of these “natural complex odours” which is mainly responsible for stimulation [cf. Fig. 4(B)], supporting the view that these receptors are attuned to co,
+ : 325% increase; - : <25% decrease, 0: no effect on the cell activity, compared to air control. a 10m3 paraffine dilution contains about 10” CO, molecules, of paralhne dilutions of pentanol lOI7 and hexanol lOI6 (cf. Kakfa, 1970; Sal3, 1976). The dose-dependent reaction to CS2 lies within the scope of responses to other odourants, but only if (certainly unphysiological) maximal concentrations of CS2 are used. One should also keep in mind that cell responses to an odourous stimulus always contain a response to the CO2 of the room air as well (about 30 imps/s), so that reactions to odourants are in reality weaker. Temperature and humidity stimuli which trigger a weak increase in cell activity in room-air (Bogner et al., 1986) show no response at all without C02. Therefore, it is now possible to ascribe the slight response to temperature or humidity changes (described in Bogner et al., 1986) to the presence of CO, in air. For that reason these receptors could be considered as unimodal. The middle ranges (0.03-l% C02) of the given dose-dependent curves show conspicuous differences in Pieris and Galleria. This result should not be overemphasized since the stimuli parameters cannot be considered strictly equivalent for the sensilla in the pit I
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A
B
C D Fig. 6. Activity of a palp pit receptor cell of Rhodogastriu catinca (Arctiidae) kept in room air (A), N,/O,-atmosphere (B) showing the “real spontaneous” activity. Spike frequencies after application of warm CO,-free air (C), or cold CO,-free air (D) apparently not evoking any response. C, D bars: stimulus duration (= 1 s); amplitude 2 mV.
FRANZBOGNER
956 Biological
SigniJicance
Behaviour investigations have proven CO,-sensitivity (Nicolas and Sillans, 1989) for many different species of arthropods: for example in blood-sucking arthropods (e.g. ticks, Garcia, 1962; insects, Friend and Smith, 1977; mosquitoes, Riissler, 1961; Gillies, 1980) including tsetseflies (Bursell, 1987), as well as in wood-eating beetles (Paim and Beckel, 1964), root-eating insects (Klingler, 1966) and honeybees (Lather, 1967; Seeley, 1974). Variable CO1concentrations (of 0.03-7%) in the nest of fig-wasps are essential in triggering sex-specific hatching of offspring (Gailil et al., 1973). Ant workers tend to move up CO,-gradients outside the nest, and this gas therefore has been called the simplest pheromone in an animal communication systems (Wilson, 1976). The biological significance of the CO,-receptors in Lepidoptera is uncertain. However, we can speculate in certain cases: it seems conceivable that CO*sensitivity is an indicator for lacriphagous moths (Banzinger, 1972) because these species suck the tear glands of mammals in complete darkness. Almost the same holds true for lepidopterans that live from overripe or rotten fruit (e.g. Charaxes). COZreceptors have been found in the labial palp pits of bee moths (Galleria melonella, Pyralidae), whose way of living in the bee-hives, where CO2 can reach levels of 9.8% (Walla, 1948) may depend upon the measurement of CO,-levels. This ability could enable those moths to locate sites of low CO,-levels for oviposition. CO,-enrichments in atmosphere can have such effect as slowing larval growth, increasing mortality [Junonia coenia (Lep.: Nymphalidae), Fajer et al., 19891, or enhancing larval growth [Phaseolus lunata (Lep.: Noctuidae), Osbrink et al., 19871; it can also fail to effect larval growth [Pectinophora gossypieZZa (Lep.: Gelechiidae), Akey et al., 19881. Local CO,-differences, may be of potential importance to CO, sensing arthropods. The microclimate within a plant culture shows, besides an annual change, a significant daily change in their COZconcentration levels (e.g. Atkins and Pate, 1977). CO,-minimal levels in vegetation layers may drop to 0.014% (Reinau, 1954) or increase during the night to a maximum of 1.2% (Nakayama and Kimball, 1988). Besides these rather big differences as far as area is concerned, there are significant CO,-changes dependent on the respective biotope, the species of plant, and physiological condition of the plant and/or even the leaves (e.g. Inoue et al., 1968; Field, 1987). CO,-levels are also affected by photosynthesis rates, which need to be specified, as well as by other physiological parameters (for example Cd-plant; Bazzaz and Carlson, 1984; Wray and Strain, 1987). It still remains an open question whether Lepidoptera react behaviourally to these CO,-differences (in time or space) or whether they merely measure these differences. The CO, receptors that were investigated here would respond to differences of
0.015-0.3% with an increase of cell activity from about 20 to 70 imps/s (in the case of Pieris). According to the dose-response curve the animal could distinguish between varying COz-concentrations of up to 3% (see above). Thus as yet unmeasured microclimatological CO,-differences in plant structures could have an additional function for Lepidoptera, for example in selection of leaves for oviposition. Certainly imaginable, but doubtful, is the possibility that these receptors are only relics reflecting an attunement to the higher air-CO, concentrations in the historical past (Gammon et al., 1985). Despite these qualifications, it is now for the first time possible to make a definitive statement about the physiology of lepidopteran palps, structures which according to Kirstensen (1984) are of fundamental taxonomic importance in Lepidoptera. Judging for their numbers, CO,-receptors must play a basic role in the palps. One is not dealing here with an adaptive feature of restricted distribution. Lepidoptera of many different biotopes as well as of different geographical areas possess these organs and share the CO,-sensitivity. In addition, the existence of a defined neuroanatomical “CO,-glomerulus” (cf. Bogner et al., 1986) suggests a still unknown but basic function in the life of Lepidoptera. Acknowledgements-Basic
results of this study were a portion of mv dissertation (Universitv of Regensbura, 1987). I thank J.-Boeckh for his steady interest in the process of the study, C. Albers for providing the mixer valve and the gas analyzer, also people named in the Materials and Methods section for providing the various species of butterflies and moths. I am indebted to M. Boppr& H. Sal3 and U. Waldow as well as to T. Eisner and B. Johnson for stimulating criticism. This study was financially supported by the Deutsche Forschungsgemeinschaft (DFG). REFERENCES Akey D. H., Kimball B. A. and Mauney J. R. (1988) Growth and develooment of the pink bollwotm Pectinophora gossypieia (Lep: Gelechiidae) on bolls of cotton grown in enriched carbon dioxide atmospheres. Envir. Ent. 17, 452-45.5.
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