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Pheromone receptor cells in the male moth Manduca sexta.

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Archives of Insect Biochemistry and Physiology 10:273-279 (1 989)
Pheromone Receptor Cells in the Male Moth
Manduca sexta
Karl-Ernst Kaissling, John G. Hildebrand, and James H. Tumlinson
Max-Planck-lnstitut fur Verhaltensphysiologie,Seewiesen, Federal Republic of Germany
K - E . K . ) ; Arizona Research Laboratories, Division of Neurobiology, University of Arizona,
Tucson (J.G.H.); lnsect Attractants, Behavior, and Basic Biology Research Laboratoy,
Agricultural Research Service, U.S. Department of Agriculture, Gainesville, Florida (1.H . T.)
Three types of pheromone receptor cells have been identified by electrophysiological recording from single antennal sensilla trichodea of the male sphinx
moth Manduca sexta. These cells responded best to the pheromone components (€,Z)-IO,I2-hexadecadienal(type A receptor cell), (€,€,Z)-10,12,14-hexadecatrienal (type B), and (€,€,€)-10,12,14-hexadecatrienal (type C). Cell type
B also responded to (€,Z)-ll,13-pentadecadienal,
which has been used experimentally as a pheromone substitute. In recordings from 20 trichoid hairs, 17
were found to be innervated by one cell of type A and one of type B; 3 trichoid hairs had cell types A and C.
Key words: olfaction, sex pheromone, electrophysiology, antenna
INTRODUCTION
Several aspects of the development and function of the olfactory system,
particularly with regard to perception of sex pheromone, have been studied
extensively in the moth Munducu sexta (for reviews see [1-3])/ while only one
component of the female’s sex-pheromone blend, (E,Z)-10,12-hexadecadienal
(ElO,Z12-16:AL or bombykal), has been identified [4].Because ultrastructural
observations had shown that the hairs of the sexually dimorphic sensillu tvichodeu
on the antennal flagellum of male M. sexta contain two receptor-cell dendrites
Acknowledgments: We thank Dr. R.E. Doolittle, Ms. A. Brabham, and Ms. M.M. Brennan of
the USDA (Gainesville, FL) for chemical synthesis and preparation of several compounds used
in this study; Drs. 1. Buckner and J. Svoboda of the USDA for generously supplying M. sexta
eggs; W. Knauf of Hoechst AC (Frankfurt/Main, FRC) for kindly providing M. sexta pupae for
some of these experiments; and Prof. H.J. Bestmann (Erlangen, FRC) for supplying EIO,Z12-16:AL
and Ell,Z13-15:AL used in some of these studies. We are also grateful to R. Montague, P. Randolph, and D. Sakiestewa for assistance with insect rearing and to Drs. T.A. Christensen, E.
Priesner, and T.R. Tobin for comments on the manuscript. This research was supported in part
by NIH grants (to J.C.H.) Al-17711, Al-23253, and NS-23405.
Received December 2,1988; accepted April 5,1989.
Address reprint requests to Dr. JohnG. Hildebrand, ARL Division of Neurobiology, 611 CouldSimpson Building, University of Arizona, Tucson, AZ 85721.
0 1989 Alan R. Liss, Inc.
274
Kaissling et al.
[5,6], it seemed likely that the female's pheromone blend would contain more
than one physiologically active component. Moreover, behavioral observations
also pointed to at least a second pheromone component in addition to E10,Z1216:AL [4,7].
Preliminary physiological experiments on trichoid hairs of the male antennal flagellum (Kaissling, unpublished) revealed one type of receptor cell that
responded to ElO,Z12-16:AL and a second type sensitive to (E,Z)-11,13-pentadecadienal (Ell,Z13-15:AL). Although this pentadecadienal was much less
effective than expected for an actual pheromone, both ElO,Z12-16:AL and
Ell,Z13-15:AL (previously called "C1.5" and regarded as a pheromone mimic
[8,9])have been used in studies of olfactory interneurons in the deutocerebrum
of adult male M.sexta [8-lo]. A reinvestigation of the mixture of volatile substances in solvent rinses of the intact pheromone gland of the female moth
revealed twelve CI6and CI8aldehydes (Table l), several or all of which might
have pheromonal functions [7].
The aim of our study was to test ElO,Z12-16:AL and two of the newly identified pheromone components, as well as Ell,Z13-15:AL, by a single-sensillum
recording method in order to begin to characterize pheromone receptor cells
in the antennae. Such information, together with knowledge about the roles
of pheromone components in controlling the behavior of the male moth, is
needed for ongoing studies of central processing of olfactory information in
M.sexta [8-111.
MATERIALS AND METHODS
Manduca sexta (Lepidoptera, Sphingidae) were raised at the University of
Arizona as described previously [8] or obtained as pupae from W. Knauf,
Hoechst AG (Frankfurt/Main).
A male moth (1-3 days posteclosion) was mounted with one antenna fixed
TABLE 1. Compounds Identified in Solvent Rinses of Intact Pheromone Glands of Calling
Virgin-FemaleMoths 171
Full name
Blend component"
Abbreviation
(Z)-9-hexadecenal
*(Z)-1I-hexadecenal
(E)-11-hexadecenal
(S)-16-hexadecanal
*( E, Z)-lO,l2-hexadecadienal
(E,E)-IO,12-hexadecadienal
*(E, E, Z)-10,12,14-hexadecatrienal
*( E,E,E)-10,12,14-hexadecatrienal
(Z)-11-octadecenal
*(Z)-13-octadecenal
(S)-18-octadecanal
(Z,Z)-ll,13-octadecadienal
Z9-16:AL
Zll-16:AL
E11-16:AL
S-16AL
ElO,Z12-16:AL
ElO,E12-16:AL
ElO,El2,Z14-16:AL
ElO,El2,E14-16:AL
Z11-18:AL
Z13-18:AL
S-18:AL
Zll,Z13-18:AL
Approx. nglgland
0.8
13.4
6.8
15.7
23.8
3.9
ca.llb
ca. 1 . 2 ~
6.2
2.2
4.8
1.4
'Compounds tested in this study are indicated with an asterisk (").
bAlthough clearly present in the gland rinses, the trienals could not be reliably quantified owing
to their instability during gas-liquid chromatography. The values given here are rough estimates [7].
Pheromone Receptors in Manduca sexta
275
to a wire by bridges of paraffin wax. The tips of several trichoid hairs on the
anterior half of one or more antennal annuli (i.e., closer to the leading edge of
the annulus than to its trailing, scale-covered side as the antenna is held during flight) were clipped off. Electrical recording from an individual opened
hair was accomplished with a glass capillary filled with sensillum lymph saline
solution [ 121.
The stimulus compounds (Table 1) were loaded on 1 cm2 pieces of filter
paper in glass cartridges (7 mm diameter). The odor stimulus was delivered
by passing an air current of 100 ml/s through the cartridge, which was positioned 5 cm from the antennal flagellum.
To compare the effectiveness of the compounds, we used stimulus loads
necessary to produce a standard response of 10-20 action potentials within
the second following the first nerve impulse. The stimulus loads were prepared in decadic steps between lop4pg and 1pg, with intermediate steps with
factors of 2or 3 in some cases. The stimulus compounds, summarized in Table
1, were prepared by J.H.T., R.E. Doolittle, A. Brabham, and M.M. Brennan
(USDA, Gainesville, FL) [7,13]. ElO,Z12-16:AL and Ell,Z13-15:AL used in early
experiments were kindly provided by H. Bestmann (Erlangen).
RESULTS
In each of 50 sensiZla trichodea tested, one receptor cell responded to E10,Z1216:AL at cartridge loads of 1ng. In one out of a total of eight animals, this cell
produced action potentials with a much larger recorded amplitude than those
of the second cell in the sensillum, which responded to Ell,Z13-15:AL (Fig. 1).
In the other moths in this study, the receptor cells of a trichoid hair could
not be distinguished on the basis of impulse amplitude or shape.
b.
E 10,E 12 , E 14- 16:A L , 2x 10 -3 y g
----LA-c.
- ,
d.
E10, E12,Zlh-l6:AL. Z X ~ O - ~ J J ~
I
I,,
-4-
E l l , Z13-15:AL, lO-’pg
I
1 mvl
1s
Fig. 1. DC recordings from two receptor cellsof onesensillurn trichodeurn. Cell type A (larger
nerve impulses) responds best to EIO,Z12-16:AL (a). Cell type B (smaller nerve impulses) responds
best to E10,E12,Z14-16:AL (c) and i s also sensitive to Ell,Z13-15:AL (d). Neither receptor
cell responds to E10,E12,E14-16:AL (b). Loads of stimulant substances on the filter papers are
given in pg.
Kaissling et al.
276
Experiments intended to discriminate between the two cells innervating the
hair under study used the method of selective adaptation of one of the receptor cells within the sensillum [14]. In this procedure, one of the two receptor
cells was adapted in order to determine whether the other cell innervating
the hair responded to a test compound. Such an experiment, involving pairs
of successive stimuli each lasting 1 s, is illustrated in Figure 2. Records a, b,
and c demonstrate that the nerve-impulse response of a cell adapted within
about 1 s to a low level or to zero. Stimulation during the ensuing second
with another pulse of the same stimulant evoked little or no response. A
a . (1) EIO.Zl2-16:AL
( 2 ) same
b. (1) ElO,E12,Z14-16:AL
(21 same
.-p----<*/-
C.
(1) Ell.Zl3-15:AL
d. (1) ElO.Zl2-16:AL
(2) ElO,E12,Z14-16:AL
e. (1)
f.
(1) Ell, Z13-15:AL
( 2 ) E 10,E12,ZlL-l6:AL
( 2 ) Ell,Z13-15.AL
1. s t i m u l u s
1s
2. s t i m u l u s
1s
Fig. 2. DC recordings from two receptor cells of o n e sensillum trichodeum demonstrating
selective adaptation. The nerve impulses of both cells have the same amplitude. Two stimuli,
each lasting 1 s, were presented in immediate succession (as indicated by the markers below
the records). As described in Materials and Methods, the stimulus load for each compound
was selected to produce a standard response, so that all stimuli were comparably effective.
a-c: Double stimulation with t h e same compound reveals a reduced (adapted) response (i.e.,
fewer action potentials) during the second stimulus. The stimuli were a) EIO,Z12-16:AL, 1 0 ng,
twice; b) E10,E12,Z14-16:AL, 1 0 ng, twice; c) Ell,Zl3-15:AL, 100 ng, twice. d,e: Unadapted
response to the second stimulus suggests that each of the 2 compounds excites a separate
receptor cell. The stimuli were d ) EIO,Z12-16:AL followed by E10,E12,Z14-16:AL; e) EIO,E12,Z1416:AL followed by EIO,Z12-16:AL. f,g: Reduced response to the second stimulus suggests that
both compounds act o n t h e same receptor cell. The stimuli were f ) Ell,Z13-15:AL followed by
E1O,E12,Z14-16: AL; g) E10,E12,Z14-16:AL followed by E l 1 ,Z13-I5 :AL.
Pheromone Receptors in Manduca sexta
277
response evoked by the second stimulus of a pair (Fig. 2, records d and e)
must have been generated by a separate cell that was not excited (and not
adapted) by the first stimulus. Therefore, ElO,Z12-16:AL and ElO,E12,Z14-16:AL
acted on different receptor cells. The second stimulus elicited no response if
the two stimuli were ElO,E12,Z14-16:AL and EllrZ13-15:AL (Fig. 2, records f
and g). Both of these compounds therefore must have acted on the same receptor cell. The theoretical possibility that we were recording from one sensory
cell with two types of receptor sites seems to us most unlikely in view of the
cited morphological findings and our observation of different impulse amplitudes in sensilla of one moth.
In recordings from 20 trichoid hairs, three types of receptor cells could be
distinguished, two per hair (Table 2). Each hair was innervated by a receptor
cell that responded to E10rZ12-16:AL (cell type A). The second cell in 17 trichoid hairs responded to Ell,Z13-15:AL and, with much higher sensitivity,
to ElO,El2,Z14-16:AL; the (E,E,E)-isomer(ElO,E12,E14-16:AL) was less effective (cell type B). In three of the 20 trichoid sensilla, the second receptor cell
did not respond to Ell1Z13-15:AL and clearly responded better to E10,E12,
E14-16:AL than to the (E,E,Z)-isomer (cell type C). Cells of types B and C
appeared to be somewhat less sensitive than those of type A, as judged from
the stimulus loads necessary to elicit the standard responses.
DISCUSSION
Each of the long olfactory hairs (sensilla trichodea) on the antenna1 flagella of
adult male M . sexta contains the distal dendritic segments of two receptor cells
[5,6]. In this study, we found that about 50% of these receptor cells are sensitive to ElO,Z12-16:AL (type A cells), and a majority of the rest are tuned to
ElO,El2,Z14-16:AL (type B cells). Both of these substances are major components of the blend found in solvent rinses of the female pheromone gland,
and they are the most important components for eliciting characteristic pheromone-dependent behavior in male moths [7]. In fact, in wind-tunnel experiments a mixture of E10jZ12-16:AL and E10,E12,Z14-16:ALr at a concentration
of 0.02 female gland equivalents, had the same effects as the complete synthetic blend or the gland rinse [7]. Because receptors tuned to E1OTE12,El4-16:AL
are present in the antenna, however, and in view of the fact that intracellular
TABLE 2. Types of Receptors Cells in Trichoid Sensilla
Stimulus compounds
ElO,Z12-16:AL
ElO,E12,Zl4-16:AL
ElO,El2,E14-16:AL
Ell,Z13-15:AL
A
(n = 20)
0.001
nr
nr
nrc
Receptor cell types"
B
(n = 17)
nrb
o.003
0.03
0.1
C
(n = 3)
nr
0.03
o.003
nrc
"Stimulus loads (pg) in cartridges eliciting a standard response of 10-20 nerve impulses within
1 s following the first impulse. The figures for the most effective compounds for these cell types
are underlined.
bNo response was observed up to cartridge loads of 0.1 pg (nr) or 1 pg (nrc).
278
Kaisslinget al.
recordings from central olfactory interneurons reveal responses to that substance [lo], it seems likely that the (E,E,E) isomer has subtle effects on behavior that have not yet been recognized.
The most effective stimulants for the central neurons were ElO,Z12-16:AL,
ElO,E12,Z14-16:AL, and E10rE12,E14-16:AL. It remains to be shown whether
other components of the female’s pheromone blend are perceived by the male
moth. Preliminary tests showed no effects of (Z)-11-hexadecenaland (2)-13octadecenal on the three types of receptor cells described in this paper. A recent
study of central processing of pheromonal information in M. sexta using all 12
components of the blend indicated that only eight of those substances elicited recognizable responses in neurons in the antennal lobe of the brain [lo].
The findings reported here raise challenging questions about the development of sensilla, as have similar observations in other species of moths. One
wonders how the established pattern of differentiative cell divisions that give
rise to the trichoid sensillum [5] generates different sets of receptor cells in
different trichoid sensilla. Thus, it would be interesting to learn what developmental mechanism accounts for the formation of the relatively few sensilla
in the male antenna that contain a receptor cell (type C cell) tuned to the minor
pheromone ElO,E12,E14-16:AL.
Approximate correspondence between the proportion of a pheromone in
the female’s blend and the frequency of occurrence in the male’s antenna of
receptor cells specialized to detect that substance has been described in other
species, for example for the third pheromone component of Antherueu pernyi
[15]. Meng et al. [15] discussed the idea that the relative rate of release of a
pheromone compound from the female gland, the relative number of corresponding receptor cells, and the sensitivity of those cells determine the potential distance from the female over which the compound can be detected by
the male. It can be expected from our results that this potential threshold volume [161 differs for the various pheromone components of M. sextu as well.
LITERATURE CITED
1. Hildebrand JG: Metamorphosis of the insect nervous system: Influences of the periphery
on the postembryonic development of the antennal sensory pathway in the brain of Manduca
sexta. In: Model Neural Networks and Behavior. Selverston A, ed. Plenum, New York, pp
129-148 (1985).
2. Christensen TA, Hildebrand JG: Functions, organization, and physiology of the olfactory
pathways in the lepidopteran brain. In: Arthropod Brain: Its Evolution, Development, Structure and Functions. Gupta AP, ed. John Wiley, New York, pp 457-484 (1987).
3. Homberg U, Christensen TA, Hildebrand JG: Structure and function of the deutocerebrum
in insects. Annu Rev Entomol34,477 (1989).
4. Starratt AM, Dahm KH, Allen N, Hildebrand JG, Payne TL, Roller H: Bombykal, a sex pheromone of the sphinx moth Manduca sexta. Z Naturforsch 34C, 9 (1979).
5. Sanes JR, Hildebrand JG: Origin and morphogenesis of sensory neurons in an insect antenna.
Dev Biol51,300 (1976).
6. Keil T: Fine structure of the pheromone-sensitive sensilla on the antenna of the hawkmoth,
Manduca sexta. Tissue Cell, 21,139 (1989).
7. Tumlinson JH, Brennan MM, Doolittle RE, Mitchell ER, Brabham A, Mazomenos BE,
Baumhover AH, Jackson DM: Identification of a pheromone blend attractive to Manduca
sexta (L.) males in a wind tunnel. Arch Insect Biochem Physiol10,255 (1989).
Pheromone Receptors in Manduca sexta
279
8. Christensen TA, Hildebrand JG: Male-specific, sex pheromone-selective projection neurons
in the antennal lobes of the moth Munducu sextu. J Comp Physiol [A] 160,553 (1987).
9. Christensen TA, Hildebrand JG: Frequency coding by central olfactory neurons in the sphinx
moth Munducu sexta. Chem Senses 13, 123 (1988).
10. Christensen TA, Hildebrand JG, Tumlinson JH, Doolittle RE: Sex pheromone blend of Mundum
sextu: Responses of central olfactory interneurons to antennal stimulation in male moths.
Arch Insect Biochem Physiol 10,281 (1989).
11. Waldrop B, Christensen TA, Hildebrand JG: GABA-mediated synaptic inhibition of projection neurons in the antennal lobes of the sphinx moth Munducu sextu. J Comp Physiol [A]
261,23 (1987).
12. Kaissling K-E, Thorson J: Insect olfactory sensilla: Structural, chemical and electrical aspects
of the functional organization. In: Receptors for Neurotransmitters, Hormones and Pheromones in Insects. Sattelle DB, Hall LM, Hildebrand JG, eds. ElsevieriNorth Holland,
Amsterdam, pp 261-282 (1980).
13. Doolittle RE, Brabham A, Tumlinson JH: Sex pheromone of Munducu sextu (L.): Stereoselective
synthesis of (E,E,Z)-10,12,14-hexadecatrienal
and isomers. J Chem Ecol in press, (1989).
14. Kaissling K-E, Zack Strausfeld C, Rumbo ER: Adaptation processes in insect olfactory receptors: Mechanisms and behavioral significance. Ann NY Acad Sci 510,104 (1987).
15. Meng LZ, Wu CH, Wicklein M, Kaissling K-E, Bestmann HJ: Number and sensitivity of
three types of pheromone receptor cells in Antherueu pernyi and A. polyphemus. J Comp Physiol
in press, (1989).
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Biol5,443 (1963).
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