Chem. Senses 28: 325-338,
2003
© Oxford University Press 2003
Functional Divergence of Spatially Conserved Olfactory Glomeruli in Two Related Moth Species
1 Department of Biology, University of Utah, Salt Lake City, UT 84112, USA 2 Arizona Research Laboratories—Division of Neurobiology, University of Arizona, Tucson, AZ 85721, USA
Correspondence to be sent to: Neil J. Vickers, Department of Biology, University of Utah, 257 S. 1400 E., Rm 201, Salt Lake City, UT 84112, USA. e-mail: vickers{at}biology.utah.edu
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Abstract |
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In different moth species, the number and spatial arrangement of olfactory glomeruli in the antennal lobe (AL) vary widely, but the spatial map within a species is thought to be invariant, making it possible to identify single glomeruli across individuals. We investigated the relationship between the physiological tuning of pheromone-selective interneurons and their association with specific, identified glomeruli in the macroglomerular complex (MGC) of the noctuid moth, Heliothis subflexa. Three odorants that are required for pheromone-source location in this species were tested individually and in blends. Recordings from 27 pheromone-specific projection neurons (PNs) indicated that the majority (48%) were selectively activated by the major pheromone component of this species, Z-11-hexadecenal (Z11–16:Ald), with 33% primarily tuned to Z-9-hexadecenal and 19% to Z-11-hexadecenol. Intracellular staining revealed that the dendrites of PNs tuned to Z11–16:Ald always branched within the largest glomerulus of the MGC, the cumulus. Similarly, each of the other two classes of PN was associated with a different `satellite' glomerulus in the MGC. The spatial configuration of the four-glomerulus H. subflexa MGC was indistinguishable from that previously reported in the closely related species, Heliothis virescens. Hence, as these two species diverged, changes in the association of satellite MGC glomeruli with particular odorants have occurred without a measurable change in the anatomical arrangement of the glomerular array.
Key words: antennal lobe, chemotopy, glomerulus, olfactory coding, pheromone
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Numerous moth species have an acute sense of smell, as exemplified by the ability of males to correctly identify and locate conspecific females releasing sex pheromones from long distances upwind (David and Birch, 1989
).
Previous investigations in two species of noctuid moth, Heliothis
virescens and Helicoverpa zea, have shown that a behavioral
preference for the conspecific pheromone blend is reflected in the
physiological responses of both olfactory receptor cells (ORCs) (Almaas and
Mustaparta, 1990,
1991;
Almaas et al., 1991;
Hansson et al., 1995;
Cossé et al.,
1998) and central projection neurons (PNs) (Christensen et
al., 1991,
1995;
Berg et al., 1998;
Vickers et al., 1998)
innervating the glomeruli of the male-specific macroglomerular complex (MGC).
It was shown that the number and spatial arrangement of the sexually dimorphic
glomeruli that constitute the MGC were different in the two species
(Christensen et al.,
1991,
1995;
Vickers et al.,
1998). Within both species, however, the position of each
glomerulus was invariant, thereby making it possible to identify all MGC
glomeruli reliably and repeatedly across individuals. Using single-unit
intracellular recording and staining methods, a precise chemotopic map was
revealed across the different MGC glomeruli in H. virescens by
showing that PNs with a similar physiological response profile always
innervated the same glomerulus (Vickers
et al., 1998). These results supported the hypothesis
that odor-blend discrimination in these species occurs through the brain's
recognition of different cross-glomerular (or combinatorial) activity patterns
in the AL (Christensen and White,
2000).
Phylogenetic studies using genetic markers have shown that Heliothis
subflexa is a close relative of H. virescens
(Cho et al., 1995;
Fang et al., 1997),
confirming earlier studies using morphological characters
(Mitter et al.,
1993). Nevertheless, the pheromone blends produced and released by
females of the two species are chemically distinct
(Roelofs et al.,
1974; Tumlinson et
al., 1975; Klun et al.,
1980,
1982; Teal et al.,
1981,
1986;
Vetter and Baker, 1983;
Heath et al., 1991).
Both species share the same primary pheromone component,
(Z)-11-hexadecenal (Z11–16:Ald), but each requires the presence
of a different set of secondary components to communicate with conspecifics.
In a wind-tunnel bioassay, H. virescens males flew upwind when a
small amount of (Z)-9-tetradecenal (Z9–14:Ald) was combined
with Z11–16:Ald (0.05:1 ratio), but their flight was arrested when a
chemically similar odorant, (Z)-11-hexadecenyl acetate
(Z11–16:Ac), was added to this binary blend
(Vickers and Baker, 1997).
Recent behavioral studies have revealed that (Z)-9-hexadecenal
(Z9–16:Ald) and (Z)-11-hexadecenol (Z11–16:OH) are
required in addition to Z11–16:Ald by H. subflexa males, but
that upwind flight was unaffected by the addition of Z11–16:Ac to this
blend (Vickers, 2002). All
four of these odorants have been isolated from female H. subflexa
pheromone glands (Teal et al.,
1986; Heath et al.,
1991; Teal and Tumlinson,
1997).
The MGC in H. virescens receives input from at least four
different populations of ORCs on the antenna
(Hansson et al.,
1995; Berg et al.,
1998). Two of these pathways represent the conspecific pheromone
blend (Z11–16:Ald and Z9–14:Ald) and are mapped to two glomeruli
(the cumulus and a dorso-medial glomerulus, DM,
Figure 1A). Z11–16:Ac is
not a component of the H. virescens pheromone blend, but it is
produced and emitted by H. subflexa females
(Teal et al., 1986;
Heath et al., 1991)
and it inhibits upwind flight in H. virescens
(Vickers and Baker, 1997).
This interspecific antagonist is represented by a third pathway that is mapped
to a separate glomerulus situated in an anteromedial position relative to the
cumulus (AM) (Vickers et al.,
1998). These studies confirmed that the MGC in this insect,
besides being involved in intraspecific communication, also plays a role in
processing and discriminating chemically similar signals released by
interspecific females. This chemotopic organization could serve as an
important aid in maintaining reproductive isolation.
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60 µm from the AL
surface), the four glomeruli of the MGC were clearly visible. (B) Confocal
photomicrograph at approximately the same depth below the AL surface in H.
subflexa revealed an MGC comprised of four glomeruli in a spatial
configuration that was virtually indistinguishable from that of H.
virescens. In both species a large, multi-lobed glomerulus (the cumulus)
occupied the area closest to the entrance of the antennal nerve (AN). The
three remaining `satellite' glomeruli were arranged around the cumulus.
Because of their anatomical similarity the same nomenclature was used to
describe the satellite glomeruli in both species: AM = antero-medial; DM =
dorso-medial; VM = ventro-medial. Scale bar = 100 µm. Dorsal, d; medial,
m.In this study, we test the hypothesis that glomeruli in the MGC of H. subflexa males (like their H. virescens congeners) are also organized chemotopically. We used intracellular recording and staining to match the odor-tuning properties of glomerular PNs with the spatial assignment of their dendritic arborizations in the MGC glomeruli. Based on results from other heliothine species, we predicted that neurons with similar physiological response profiles would show consistent patterns of dendritic arborization within specific identifiable glomeruli across individual males. In addition, by comparing the results of these studies with others on heliothine moths (particularly H. virescens), we gained insight into the divergence of male olfactory characteristics that have accompanied the evolution of these species.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Insects
A series of H. subflexa colonies were established between 1997 and
1999 from pupae and eggs sent to the University of Arizona and the University
of Utah from North Carolina State University. Neonate larvae were reared on
either a pinto-bean diet (Shorey and Hale,
1965) in large ice-cream cups or a corn-soy blend diet (A. Sheck,
personal communication) in individual 30 ml medicine cups fitted with
cardboard lids (WLM Inc., Newark, NJ). Once the larvae had pupated they were
removed from the cups and pupae were segregated by sex. Male pupae were placed
in an environmental chamber (Percival Scientific, Boone, IA) at 25°C, 60%
RH, 14:10 L:D reversed light cycle and allowed to eclose. Males were aged
daily and cohorts of aged males were kept in plastic containers. Males between
the ages of 3 and 10 days were utilized in neurophysiological experiments.
Stimulation
Details of the preparation of animals for recording and the experimental
set-up have been described in detail previously
(Christensen et al.,
1995; Vickers et al.,
1998). All olfactory stimuli were delivered as a series of five 40
ms pulses (interpulse interval 300 ms) from an odor cartridge directed at one
antenna. The rapid presentation of brief odor pulses allows an assessment of
the ability of each neuron to follow the temporal pattern of the stimulus. The
odorants included the following single compounds that have been identified as
pheromone components in H. subflexa or other closely related
heliothine moths, including H. virescens and H. zea:
(Z)-11-hexadecenal (Z11–16:Ald), (Z)-9-tetradecenal
(Z9–14:Ald), (Z)-9-hexadecenal (Z9–16:Ald),
(Z)-11-hexadecenyl acetate (Z11–16:Ac),
(Z)-11-hexadecenol (Z11–16:OH). In addition, several synthetic
mixtures were tested: H. subflexa 4-mix, H. subflexa 3-mix;
H. virescens 1:0.5 blend, H. virescens 2-mix; and H.
virescens 6-mix (ratios detailed in
Table 1). As eight positions on
the odor cartridge-holder assembly were available only two blends (along with
the five single odorants and a blank control) were tested against any given
neuron. Typically, one H. subflexa and one H. virescens
mixture were used. In earlier experiments, an H. subflexa female
pheromone gland extract was used as a stimulus. Gland extracts were prepared
by removing the female pheromone gland during the fourth to sixth hour of
scotophase and placing it in 50 µl hexane for 1 min. Single odorants were
loaded onto filter paper strips (0.7 cm x 3.5 cm, Whatman No. 1,
Maidstone, UK) as 10 µl aliquots to give a final dosage of 10 ng. For
blends, individual compounds were loaded in the stated ratio with respect to
10 ng of Z11–16:Ald. Thus, a 1:0.05 mixture of
Z11–16:Ald:Z9–14:Ald had a dosage of 10 ng Z11–16:Ald and
0.5 ng Z9–14:Ald loaded onto the filter paper substrate. A blank
cartridge containing only a filter paper strip was used as a control.
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Morphology
Intracellular neurophysiological recordings were obtained using
borosilicate glass microelectrodes filled at the tip with Lucifer yellow CH
(LY, 4–6% in 0.2 M LiCl) and backfilled with 2 M LiCl. Resistance of the
microelectrodes varied between 150 and 400 M
. Following physiological
characterization, LY was injected into the neuron by passing negative direct
current (up to 1 nA for 10 min). Details of histological procedures to prepare
brains for microscopic examination have been provided in detail previously
(Vickers et al.,
1998). Wholemount and sectioned material were examined on either a
Bio-Rad MRC 600 (equipped with: Nikon Optiphot-2 microscope, 100 mW Argon
laser light source, filter cube: 457 nm excitation) or Zeiss LSM 510 (25 mW
Argon laser light source, excitation: 458/514 nm dichroic, emission: LP 475
nm) laser scanning confocal microscopes (LSCM). Serial optical images were
collected at intervals of either 1 or 2 µm.
Neurophysiological analysis
Intracellular responses were monitored continuously by oscilloscope and recorded on either FM tape (Vetter model D instrumentation tape recorder) or VHS tape (Vetter PCM recorder). Analysis of action potential trains was performed off-line using either Axon Instruments' Axoscope or Run Technologies' Datapac2000. Acquisition was triggered on the rising phase of the first stimulus pulse and 2 s of pre-trigger and 4 s of post-trigger data were recorded. Acquired records were then analyzed for neuronal spiking activity by setting user-defined thresholds that varied according to the quality of the recording. Spike data were then converted into instantaneous frequency–time histograms (IFTs) so that responses could be compared across stimuli within the same neuron or across different neurons.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Morphology of the MGC in H. subflexa males
Detailed morphological examination of the AL in male H. subflexa
moths revealed that the MGC is dominated by a large glomerulus situated near
the entrance of the antennal nerve into the AL
(Figure 1B) as demonstrated in
other noctuid species (Christensen et al.,
1991,
1995;
Hansson et al., 1994;
Anton and Hansson, 1995; Berg
et al., 1998,
2002;
Vickers et al.,
1998). In the sphingid moth, Manduca sexta as well as two
other heliothine moths, this glomerulus was named the `cumulus' because of its
complex, multi-lobed structure (Hansson
et al., 1992; Vickers
et al., 1998; Berg
et al., 2002). We therefore use the same term for this
large glomerulus in H. subflexa. Three additional satellite glomeruli
are positioned near the cumulus in a precise spatial arrangement that is
indistinguishable from the H. virescens MGC
(Figure 1A). We previously
devised a scheme for naming each satellite glomerulus according to its spatial
position relative to the cumulus. In H. virescens these are the AM
(antero-medial), VM (ventro-medial) and DM (dorso-medial) glomeruli
(Vickers et al.,
1998). Given the close anatomical similarities between the two
species, we have now adopted this nomenclature for the satellite glomeruli in
H. subflexa (Figure
1B).
Functional characterization of antennal lobe neurons in H. subflexa
Successful recordings were obtained from 31 out of 48 H. subflexa
males. In 18 of these, a total of 27 neurons responded to one or more of the
test odorants or blends. Neurons were categorized according to the observed
differences in their responses to the stimuli tested. Type 1 PNs
showed excitatory responses to only a single odorant and any blend containing
that odorant. Type 2 PNs also showed excitatory or inhibitory
responses to one or more of the other test odorants
(Christensen et al.,
1995; Vickers et al.,
1998). Included in the Type 2 category were PNs that were
primarily excited by a single odorant but showed enhanced responses (either
elevated peak rate of firing or improved temporal resolution in response to
pulsatile stimuli) to a blend containing that odorant. Inhibitory responses
were characterized by a reproducible hyperpolarization of the membrane
potential with each odor pulse. Typically, these inhibitory responses were
phase-locked to stimulus delivery or were observed following the offset of the
last stimulus pulse. Spontaneous activity of neurons was variable but
instantaneous frequencies were typically 50 Hz or less.
Z11–16:Ald-responsive PNs
About half of the odor-responsive PNs (13 out of 27 or 48%) were primarily responsive to one odorant: Z11–16:Ald (Figures 2 and 5). Nine of these PNs were exclusively responsive to Z11–16:Ald (Type 1), whereas four displayed secondary responses (either excitatory, inhibitory, or enhanced) to other pheromonal odorants (Type 2).
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Type 1: Physiology
These PNs were strongly depolarized by the presence of Z11–16:Ald and
showed neither excitatory nor inhibitory responses to other odorants. No
quantitative or qualitative difference in response was noted between
presentation of Z11–16:Ald alone, the synthetic pheromone mixtures
(H. subflexa 3- or 4-mix and H. virescens 2- or 6-mix; see
Table 1), or a female gland
extract (FE; see Materials and methods). All nine PNs in this category were
able to track the temporal dynamics of the stimulus, and responded with a
discrete burst of action potentials to each of the five pulses in the stimulus
train (Figure 2).
Type 1: Morphology
In four individuals, a single Z11–16:Ald/Type 1 PN was stained after
physiological characterization. LSCM images revealed that in each case, the
dendritic arborizations of these neurons were restricted to the largest MGC
glomerulus, the cumulus (Figure
3). In a fifth individual, recordings were made separately from
two neurons. One responded primarily to Z9–16:Ald (see below) while the
other responded selectively to Z11–16:Ald. LSCM images confirmed
staining of two PNs, each with a cell body in the medial cell cluster
(Figure 4). In one PN,
dendritic arborizations were restricted to DM, while in the other, branches
were found exclusively in the cumulus
(Figure 4). In all five
preparations, the axons of stained PNs projected through the inner
antenno-cerebral tract (IACT) to the protocerebrum.
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Type 2: Physiology
Two PNs responded to antennal stimulation with either Z11–16:Ald or Z11–16:OH, but the responses to the two odorants were quantitatively different (Figure 5, cells 1, 2). Both PNs responded to each pulse of Z11–16:Ald with a discrete burst of action potentials. In contrast, the threshold for activation to Z11–16:OH was higher, but the responses were nonetheless phase-locked to the stimulus pulses. Two additional neurons responded to Z11–16:Ald, but the response was greatly enhanced when this odorant was presented as part of a blend (Figure 5, cells 3 and 4). None of the neurons of this type were stained successfully.
Z9–16:Ald-responsive PNs
Another third of the odor-responsive PNs (9 out of 27 or 33%) were primarily responsive to Z9–16:Ald (Figure 6). Five of these PNs were exclusively responsive to Z9–16:Ald (Type 1), whereas four displayed secondary responses (either excitatory, inhibitory, or enhanced) to other pheromonal odorants (Type 2).
Type 1: physiology
Five PNs showed low-threshold excitatory responses to antennal stimulation
with Z9–16:Ald, and neither excitatory nor inhibitory responses to other
test odorants. In two of these neurons
(Figure 6A, cells 3–5),
every odor pulse evoked a burst of spikes that was phase-locked to the
stimulus train. None of the neurons in this category were stained.
Type 2: physiology
Four neurons were classified as Z9–16:Ald Type 2
(Figure 6B). Three of these had
similar response profiles, showing strong depolarizing responses to
Z9–16:Ald alone or to the H. subflexa FE-blend
(Figure 6B, cells 2–4).
Unlike the other PN types discussed thus far, these three neurons were also
specifically inhibited by other test odorants or blends. Interestingly, even
though it contained a small amount of the excitatory stimulus Z9–16:Ald,
stimulation with the H. virescens 6-mix
(Table 1) resulted in a
pronounced hyperpolarization and complete inhibition of all background
activity in two PNs (Figure 6B,
cells 2 and 3). Therefore, the interspecific blend of six odorants had a
greater effect on these neurons than did any of the individual components of
the blend. This suggests that the strongly inhibitory response to the blend
emerged as a result of the integration of signals converging from multiple MGC
glomeruli that responded to this specific combination of odorants. The fourth
Type-2 PN had a response profile that was distinctly different from others in
this category. Stimulation with either Z9–16:Ald or Z9–14:Ald
evoked robust excitatory responses in this neuron
(Figure 6B, cell 1), while
other odorants evoked neither excitatory nor inhibitory responses.
Type 2: morphology
AL neurons were stained in two of the above-mentioned preparations. In the
first, two different PNs were characterized (as discussed above;
Figure 4). In a second
preparation, recordings were obtained from two neurons with similar
physiological properties (Figure
6B, cells 2 and 3) but only one neuron (a PN) was stained
sufficiently after dye injection. This PN had branches that were restricted to
the MGC, with particularly intense staining in DM
(Figure 7). The cell body was
in the lateral cluster and the axon projected through the MACT directly to the
inferior lateral protocerebrum (ILP).
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Z11–16:OH-responsive PNs
Five neurons out of 27 (19%) responded primarily to stimulation with Z11–16:OH (Figure 8). Two of these neurons were exclusively responsive to Z11–16:OH (Type 1), whereas another three displayed secondary responses (either excitatory, inhibitory, or enhanced) to other pheromonal odorants (Type 2).
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Type 1: physiology
Two neurons exhibited rather high-threshold (but nevertheless selective)
responses to Z11–16:OH (Figure
8A). For one neuron (Figure
8A, cell 1), bursts of action potentials were phase-locked to each
stimulus pulse and the instantaneous frequency of each burst exceeded firing
rates for any other stimulus. A second neuron
(Figure 8A, cell 2) was weakly
excited by Z11–16:OH when presented alone but responded much more
vigorously to the FE possibly due to a greater concentration of
Z11–16:OH in the gland extract. Neither neuron was stained.
Type 2: physiology
Three neurons responded to Z11–16:OH and to the H. subflexa
FE. Responses of these neurons (Figure
8B, cells 1–3) were not quantitatively or qualitatively
different for these two stimuli. However, secondary responses to other
odorants took on different forms in these neurons. One cell
(Figure 8B, cell 1) was
specifically inhibited by stimulation with Z9–14:Ald. Two neurons
(Figure 8B, cells 2 and 3) in
this category were also excited to some extent by antennal stimulation with
Z9–14:Ald.
Type 2: morphology
In one preparation LSCM images revealed a single PN with a cell body in the
medial cell cluster. Arborizations were restricted to the AM glomerulus of the
MGC (Figure 9).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In H. subflexa a blend of three female-produced odorants (Z11–16:Ald, Z9–16:Ald, and Z11–16:OH) was necessary and sufficient to stimulate upwind flight and source location by conspecific males (Vickers, 2002
). Based upon
this finding, as well as previous observations from other heliothine species,
we were able to make several predictions about the functional and anatomical
organization of the MGC glomeruli in H. subflexa. First, we
postulated that some olfactory PNs would show odorant-specific responses and
would remain segregated in different MGC glomeruli. Second, we expected to
find a functionally and anatomically distinct population of PNs that would
integrate information from more than one of these input pathways (Christensen
et al., 1991,
1995;
Vickers et al.,
1998). In the present study, we found one population of PNs that
was selectively responsive to either Z11–16:Ald, Z9–16:Ald or
Z11–16:OH (type 1 PNs) as well as another set of PNs with firing
patterns that were modulated by more than one test odorant (type 2 PNs). As in
other moths, therefore, this organization indicates that pheromone processing
is not simply governed by labeled lines, but involves a complex,
cross-glomerular and combinatorial code that represents the attractive
pheromonal blend in H. subflexa. Evolution of MGC structure and function
Processing of olfactory information in the AL glomeruli has been studied in
a number of Lepidopteran species, including representatives of the families
Bombycidae (Bombyx mori), Pyralidae (Ostrinia nubilalis),
Saturniidae (Antheraea polyphemus), Sphingidae (Manduca
sexta), and other Noctuidae (Agrotis segetum, Spodoptera littoralis,
Trichoplusia ni) (Koontz and
Schneider, 1987; Hansson et al.,
1992,
1994;
Anton and Hansson, 1995;
Ochieng et al., 1995;
Todd et al., 1995;
Anton et al., 1997;
Christensen, 1997;
Hansson, 1997;
Kanzaki et al.,
2003). These species all utilize pheromonal blends that differ in
their chemical composition, either in the specific components present, or in
the relative amount of each odorant present in the blend. The number and size
of glomeruli in the male MGC also vary substantially for these different
species, perhaps reflecting the need to process and discriminate between
different conspecific and interspecific pheromone blends
(Christensen, 1997;
Hansson, 1997;
Anton and Homberg, 1999). These
data show that the spatial arrangement of glomeruli in the MGC is
evolutionarily labile, but they do not explain how the evolution of
pheromone-receptor specificity, or changes in the structure and function of
central olfactory pathways in the MGC, might come about. What changes in
olfactory processing circuitry have occurred in H. virescens and
H. subflexa to accompany their semiochemical divergence and the
males' behavioral requirement for distinct pheromone blends? Morphologically,
the H. subflexa MGC is essentially indistinguishable from that of
H. virescens (Figure
1) but the odorant-response profiles of central olfactory PNs (and
ORCs; T.C. Baker et al., submitted for publication) have clearly
changed over the course of evolution
(Figure 10). The data
presented here show that as H. virescens and H. subflexa
have diverged, satellite MGC glomeruli (those adjacent to the cumulus) have
undergone changes in their odorant tuning. In some cases, these shifts have
been accompanied by a divergence in the specific association with attractive
or antagonistic odorants. Below we outline some of the basic principles of
organization that are emerging from studies across several related
species.
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Principle 1. The cumulus is the site for processing the main pheromone component, Z11–16:Ald
Many heliothine species utilize Z11–16:Ald as the most abundant
component of their respective pheromone blends
(Arn et al., 1992). In
both H. virescens and H. zea, PNs that responded to
Z11–16:Ald were the most frequently encountered type of MGC neuron (58%
and 56%, respectively) and Z11–16:Ald PNs that were stained always had
dendritic arborizations within the cumulus (Christensen et al.,
1991,
1995;
Vickers et al.,
1998). Similarly, in H. subflexa, Z11–16:Ald PNs
were the most frequently encountered (48%), and all PNs of this type that were
stained had dendritic arborizations confined to the cumulus (Figures
3 and
4). In both H.
virescens and H. zea, multiglomerular PNs were also identified
(Christensen et al.,
1991; Vickers et al.,
1998). These neurons always had a primary response to
Z11–16:Ald, or were specifically activated by any blend containing
Z11–16:Ald. Multiglomerular neurons with a primary sensitivity to
Z11–16:Ald were not stained in the present study, but they also likely
exist in H. subflexa. Nevertheless, the results obtained from stained
uniglomerular PNs provide compelling evidence that the cumulus is the common
site for processing the odorant Z11–16:Ald, the most abundant component
of the pheromone blend in H. subflexa and almost all other heliothine
moths studied to date.
Principle 2. Other MGC glomeruli are specific for secondary pheromone components
Additional pheromone components, specifically Z9–16:Ald and
Z11–16:OH, are also required to elicit significant levels of upwind
flight and source location by H. subflexa males
(Vickers, 2002), and neurons
specifically tuned to each of these odorants have been documented in this
study (Figures 6 and
8). While more PNs must be
stained, we now have evidence that at least a subset of these narrowly tuned
PNs also have dendritic arborizations restricted to particular MGC glomeruli
other than the cumulus. For example, a single PN sensitive to Z9–16:Ald
was found to arborize in DM (Figure
4A). In addition, a multiglomerular PN arborizing in all MGC
glomeruli, but with noticeably more branching in DM, also responded to
Z9–16:Ald (Figure 7).
This PN had a soma in the lateral cell cluster, and its axon projected from
the AL directly to the ILP. In H. virescens and H. zea, PNs
showing selectivity for a secondary component or antagonist were always
uniglomerular (Vickers et al.,
1998), which makes this type of PN in H. subflexa
unusual. This finding may, however, be indicative of a general undersampling
of PNs with somata in the lateral cell cluster. The existence of similar
multiglomerular neurons was also recently reported in B. mori, but
their function in olfactory processing remains to be clarified in this species
(Kanzaki et al.,
2003).
Principle 3. Divergence of the odorant–glomerulus association
The odorants processed in the DM glomerulus of H. virescens and
H. subflexa are different (Z9–14:Ald and Z9–16:Ald,
respectively, Figure 10), but
they carry a similar importance as a necessary component of the pheromone
blends in these two species. For this central shift in chemical representation
in the DM glomerulus of these two species to have appeared, a similar change
in the specificity of the sensory input must also have occurred.
Electrophysiological studies have in fact revealed evidence that some of the
ORC pathways in these moths are considerably more broadly tuned than others.
Sensillum recordings from the antennae of H. subflexa males, for
example, indicate that some ORCs respond not only to Z9–16:Ald as
expected, but also to Z9–14:Ald. These ORCs however, are about two
orders of magnitude more sensitive to Z9–16:Ald than to Z9–14:Ald
(T.C. Baker et al., submitted for publication). In our data set we
found one Z9–16:Ald Type 2 PN (Figure
6B, cell 1) that also responded to Z9–14:Ald. A similar
situation in which an olfactory pathway was found to be more broadly tuned to
these same two odorants was reported in H. zea
(Christensen et al.,
1991; Cossé et
al., 1998; Vickers et
al., 1998). This pathway may explain why H. zea
males were attracted to pheromone blends in which Z9–16:Ald was replaced
with a small amount of Z9–14:Ald
(Vickers et al.,
1991). This substitution test, however, failed to attract H.
subflexa (Vickers, 2002),
reflecting perhaps the much lower affinity of Z9–16:Ald-sensitive ORCs
in this species for Z9–14:Ald, as compared with H. zea (T.C.
Baker et al., submitted for publication).
The capacity of some ORCs and PNs to respond to Z9–14:Ald in addition
to other odorants is an increasingly common theme amongst heliothine moths
(Cossé et al.,
1998; Vickers et al.,
1998) and could be a major factor in speciation. In the current
study, one Z9–16:Ald Type 2 PN
(Figure 6B, cell 1) and two
Z11–16:OH Type 2 PNs (Figure
8B, cells 2, 3) also responded to Z9–14:Ald. The occurrence
of these more broadly tuned sensory and central neurons suggests that the
divergence of a single olfactory pathway into different sub-pathways with
differing degrees of odorant selectivity may eventually lead to functional
shifts in the odorant specificity of a given glomerulus.
Principle 4. Divergence of functional significance in MGC glomeruli
Finally, our results also indicate that the functional significance of at
least one of the satellite MGC glomeruli can shift through evolution. For
example, in H. virescens, it was discovered that PNs responsive to
Z11–16:Ac (a potent behavioral antagonist) innervated only the AM
glomerulus (Vickers et al.,
1998). In contrast, studies of ORCs
(Berg et al., 1998),
synaptic activity associated with ORCs
(Galizia et al.,
2000), and PNs (Vickers, unpublished observations) in H.
virescens indicated that Z11–16:OH/Z9–14:Ald responses were
mediated through the VM glomerulus. Surprisingly, in the current study of
H. subflexa, the only stained PN that was activated by both of these
attractive odorants was localized not to VM, but to the AM glomerulus, which
serves the antagonist function in H. virescens
(Figure 9A). We speculate that
Z11–16:OH-specific PNs (Figure
8) also arborize in this glomerulus and that activity in AM would
be required in combination with that of the cumulus and DM for H.
subflexa males to fly upwind (Figure
10). If this result can be confirmed by additional PN recording
and staining, it would suggest that the odorants associated with the AM
glomerulus, as well as the specific behavioral significance of activating this
particular glomerulus, have diverged in these two heliothine species.
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Acknowledgments |
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Supported by the National Science Foundation, IBN-9905683 to NJV. Thanks to Dr John Hildebrand for helpful discussion and encouragement during the early stages of this project. Many thanks to Dr Fred Gould, Dr Amy Sheck, Mark Sisterson, and Sara Oppenheim of North Carolina State University for their help in establishing a series of H. subflexa colonies and for their suggestions for colony maintenance. Thanks to the following for help with rearing and maintenance of moth colonies and data analysis: Keri Swearingen, Dave Kelly, Matt Pond.
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References |
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Accepted April 2, 2003
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