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First published online September 19, 2006
Journal of Experimental Biology 209, 3729-3741 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02444
Receptor expression and sympatric speciation: unique olfactory receptor neuron responses in F1 hybrid Rhagoletis populations
1 Department of Entomology, Barton Lab, NYSAES, Cornell University, Geneva,
NY 14456, USA
2 Department of Biological Sciences, PO Box 369, Galvin Life Science Center,
University of Notre Dame, Notre Dame, IN 46556-0369, USA
3 Department of Entomology, University of Illinois, 320 Morrill Hall, 505
South Goodwin Avenue, Urbana, IL 61801, USA
* Author for correspondence at present address: Department of Ecology and Evolutionary Biology, University of California, Los Angeles, 618 Charles E. Young Drive South, PO Box 951786, Los Angeles, CA 90095-1786, USA (e-mail: shannonolsson{at}ucla.edu)
Accepted 13 July 2006
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Key words: host shift, hybridization, postzygotic isolation, single sensillum electrophysiology, discrimination, specificity
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)
because the strong divergent ecological selection pressures and genetic
architecture thought to be required for the process are more restrictive than
the precondition of geographic separation facilitating allopatric forms of
speciation. In order for speciation to occur, pre- and/or post-zygotic
barriers must be sufficient to reproductively isolate the populations while
the populations are at all times within `cruising range'
(Mayr, 1970) of each other.
Sympatric speciation is theoretically intriguing, however, because in the
absence of any physical barrier to gene flow the same traits differentially
adapting populations to alternative habitats pleiotropically act as important
pre- and/or postzygotic ecological barriers, reproductively isolating
specialist populations. Elucidating the morphological, physiological and/or
behavioral basis for these ecological barriers translates directly into
understanding involved in the underlying processes speciation.
The Rhagoletis pomonella sibling species complex is often cited as
an example of sympatric speciation in progress
(Berlocher, 1998). In 1867,
Benjamin Walsh first observed the shift of the apple maggot fly Rhagoletis
pomonella (Walsh), from its native host hawthorn (Crataegus
spp.) to introduced domestic apple (Malus pumila P. Mill) as the
formation of a new host race. Bush later postulated that members of the R.
pomonella group were established by sympatric speciation via
host plant shifts (Bush, 1969).
This complex contains a variety of host-specific frugivorous races and sibling
species (see Berlocher, 2000).
Electroantennographic, field trapping and flight-tunnel studies have shown
that flies from sympatric populations infesting hawthorn (Crataegus
mollis), domestic apple (Malus pumila) and flowering dogwood
(Cornus florida) fruit can distinguish among the unique volatile
blends identified from each host fruit. These populations not only
preferentially orient to their own natal host fruit volatiles
(Zhang et al., 1999;
Nojima et al., 2003a;
Nojima et al., 2003b;
Linn, Jr et al., 2003;
Linn, Jr et al., 2005b), but
avoid non-natal fruit blends (Forbes et
al., 2005) and display arrested flight when non-natal volatiles
are added to the host blend (Linn, Jr et
al., 2005a; Forbes et al.,
2005). Host fruit volatiles can thus exert both agonist and
antagonist effects on foraging behaviors. Because Rhagoletis flies
mate and oviposit directly on or near the fruit of their host plant
(Prokopy et al., 1971),
variation in olfactory host preference serves as a pre-mating barrier to gene
flow among flies infesting different host species
(Feder et al., 1994).
A recent study (Linn, Jr et al.,
2004) showed that olfactory cues can also serve as a form of
postzygotic isolation. F1 hybrids from crosses between apple-,
hawthorn- and dogwood-origin flies displayed significantly reduced orientation
to host volatiles in flight-tunnel assays. Hybrid flies failed to respond to
volatiles at concentrations generating maximum levels of upwind flight from
parent flies. Upwind orientation could generally be elicited (in 50-60% of the
flies tested) only when higher concentrations (ten times) of host volatiles
were used. These high concentrations normally antagonize the upwind flight of
parent flies (Dambroski et al.,
2005), suggesting a shift in olfactory response threshold for
hybrid flies. Furthermore, the few hybrid individuals that did orient at lower
concentrations (
10%) typically flew only when the two parental blends
were combined, indicating an alteration or relaxation in agonist/antagonist
pathways in the olfactory system of hybrids. This response to the combined
blends was also amplified at the higher concentrations. These observed changes
in olfactory preference could significantly reduce hybrid fitness in the field
by diminishing the ability of flies of mixed ancestry to locate hosts, and
successfully mate and oviposit on host fruit. Reduced hybrid fitness therefore
produces an additional barrier to gene flow among Rhagoletis
populations by generating a form of olfactory-based, postzygotic
isolation.
Here, we investigate a potential source for the changes in the hybrid
olfactory system by examining peripheral chemoreception in F1
hybrid flies to behaviorally relevant volatiles from the parent host fruit.
Few studies have examined peripheral chemoreception in F1 insect
hybrids, and those that have were concerned only with pheromonal reception.
These previous studies revealed four different characteristics of hybrid
olfactory receptor neurons (ORNs): (1) hybrids can possess identical ORNs to
parental populations, but in different proportions [Ips pini
(Mustaparta et al., 1985)];
(2) hybrids can respond more similarly to one parent over the other
[Agrotis ipsilon x A. segetum
(Gadenne et al., 1997)]; (3)
hybrid ORNs can generate intermediate spike amplitudes
(Roelofs et al., 1987) and
frequencies (Cossé et al.,
1995); [two pheromone races of Ostrinia nubilalis]; and
(4) hybrid ORNs can exhibit a variety of responses, some similar to the
parents, and some `atypical' responders [Ctenopseustis obliquana
x Ctenopseustis sp.
(Hansson et al., 1989);
Heliothis subflexa x H. virescens
(Baker et al., 2006)]. In this
study, we test the hypothesis that Rhagoletis hybrid ORNs also
exhibit unique characteristics from parent populations, and that these
peripheral alterations can impact olfactory host volatile preference.
Previously, we found that ORNs from various parental Rhagoletis
host populations demonstrated inter-population variation in ORN sensitivities
and temporal firing patterns to an array of key host fruit volatiles
(Olsson et al., 2006b).
However, all populations tested possessed similar classes of ORNs responding
with similar odor specificities (Olsson et
al., 2006a). In the present study, we report a unique finding:
crosses between apple, hawthorn and dogwood host populations of R.
pomonella generated F1 hybrids with distinct and diverse
alterations in ORN response profiles absent from parent population ORNs. We
discuss possible causes of these alterations in ORN specificity as well as
their potential effect on oriented flight to host volatiles.
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).
Chemical stimuli and neurophysiological analyses were similar to those
previously published (Olsson et al.,
2006a; Olsson et al.,
2006b). We briefly overview these methods below.
Rhagoletis origins and rearing conditions
Apple and hawthorn Rhagoletis pomonella (Walsh) flies were
collected as larvae from infested fruit at Grant and Fennville, Michigan, and
Urbana, Illinois, USA during the 1999-2002 field seasons and reared to
adulthood in the laboratory using standard Rhagoletis protocols
(Feder at al., 1989). Dogwood
flies were collected from Granger and Raccoon Lake, Indiana, USA from
2000-2002 and were treated in the same manner as the host races. After
overwintering as pupae at 5°C for 4-7 months, eclosing adults were placed
in a controlled environmental chamber (24°C, 15 h:9 h light:dark
photoperiod, 60-70% relative humidity) and fed a diet of brewers yeast, brown
sugar and molasses (Neilson and McAllen,
1965). Adults were mass crossed in mating cages containing at
least 20 females and 20 males and supplied with water, food and four Red
Delicious variety apples for female oviposition. Puparia were placed in small,
clear plastic Solo cupsTM containing moist vermiculite and held in a
constant temperature chamber to allow flies to develop into adults.
Olfactory stimuli
Synthetic blends and sources of chemicals were the same as reported
previously (see Olsson et al.,
2006a). Stock solutions (1 µg µl-1) of individual
key fruit volatiles and specific fruit blends in hexane were prepared
according to the key volatiles determined for each fruit through behavioral
and electrophysiological analyses (see
Table 1)
(Olsson et al., 2006a).
Dilutions of each compound (1, 10 and 100 ng µl-1, and 1 µg
µl-1) were prepared for dose response trials. 10 µl of each
diluted compound or blend was pipetted onto filter paper (5 mmx15
mm) in disposable Pasteur pipettes. Blank cartridges, containing only filter
paper plus solvent, were also prepared.
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Electrophysiological recording
Adult Rhagoletis were confined in the tapered, cut end of a 100
µl pipette tip with only their heads protruding and immobilized with dental
wax. A sharpened tungsten wire was inserted into the right eye, serving as a
ground electrode. Electrolytically sharpened tungsten microelectrodes were
used to establish contact with the ORNs. The recording electrode was
positioned using a preparation microscope with up to 200x magnification
and an electrophysiological recording unit with combined joystick
micromanipulators and amplifier (Syntech INR-5, Hilversum, The Netherlands).
In most cases, the ventral portion of the left antenna was used for
recording.
A constant flow of charcoal-filtered and humidified air passed over the
antenna from a stimulus air controller at approximately 2.6 l min-1
(Syntech, CS-5, Hilversum, The Netherlands) through a metal tube protruding
approximately 10 mm from the antenna. Stimulation was performed by inserting
the tip of the test pipette into a hole in the metal tube, approximately 10 cm
before the outlet. The test pipette was connected to the stimulus air
controller, which generated air puffs (1.3 l min-1 during 0.5
s) through the pipette and replaced a complimentary air stream during that
time period.
The analog signal originating from the ORNs was amplified (10x) (Syntech INR-05, Hilversum, The Netherlands), sampled (31746.0 samples s-1) and filtered (200 Hz-3000 Hz with 50/60 Hz suppression) via USB-IDAC connection to a computer (Syntech, Hilversum, The Netherlands). Action potentials were extracted as digital spikes from the analog signal according to top-top amplitudes using Syntech Auto Spike 32 v. 1.1b-3.2 software. When co-located in the same sensilla, individual neurons were sorted manually for each recorded trace based on differences in the amplitude of their action potentials (spikes). Changes in amplitude due to excessive firing (i.e. `pinching') were taken into account.
In the event of a contact, ORNs were screened with the three fruit blends at 10 µg, and the blank (hexane). These stimuli were tested at least once at the beginning and, in nearly all cases, end of each recording period. All stimuli were presented in 0.5 s air puffs at approximately 1 min. intervals to allow the ORNs to return to baseline firing rate. If the neuron(s) responded to one or more of the blends (see below for definition of response), then all 11 components were tested individually at a concentration of 10 µg. Those compounds eliciting responses were subsequently tested in dose-response trials (10 and 100 ng, 1 µg and 10 µg) to determine each cell's sensitivity to those chemicals.
Data analysis
For each ORN testing period, spike frequencies of the blank (hexane) were
calculated every 500 ms for the 10.5 s recording period (including 1 s pre-
and 9.5 s post-stimulus onset). In the majority of the cases, more than one
blank trial was presented. Spike counts per 500 ms were then averaged across
all blank trials. An increase in spike frequency for the 500 ms following
stimulus presentation was considered a response if it rose >3 standard
deviations (s.d.) above the blank mean. ORN spike increases below this level
were not considered further. A response threshold was calculated as the lowest
concentration eliciting a spike frequency increase >3 s.d. over the mean of
the blank trials. In the few cases where threshold could not be determined
(owing to incomplete dose-response trials or cell death), threshold was given
as 75% of the lowest concentration eliciting a response <4 s.d. of the
blank mean or 50% of a response >4 s.d. of the mean. Sensitivities were
assigned as reciprocals of the threshold values (e.g. 10 ng
threshold=104, 100 ng=103, 1 µg=102, and
10 µg threshold=101).
Each ORN response pattern (i.e. the array of biologically relevant host
volatiles to which each ORN responded) to 10 µg of the 11 host compounds
(Table 1) was first compared to
the ORN response patterns to 10 µg of the same compounds in Olsson et al.
(Olsson et al., 2006a). If an
ORN responded to the same array of host compounds as ORNs found in the parent
population study (Olsson et al.,
2006a), it was considered `parent-like' and labeled with the
appropriate ORN class (A-E) determined by cluster analysis
(Olsson et al., 2006a) (see
Table 2). If the ORN responded
to unique combinations of compounds not found in any parent classification,
then it was designated as `hybrid-like' and labeled with the lettered class or
classes it most closely resembled. Cells in classes D and E were difficult to
classify as `parent-' or `hybrid-like' because of the diversity and number of
compounds to which they responded. To maintain the most conservative and
accurate depiction of `hybrid' cells, these cells were not classified as
either category. This is also true for a small number of cells (N=6)
in the first three classes (A-C) as well.
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For statistical comparison of ORN responses, parsimony networks depicting
the interrelationship of single cell sensillum response patterns for parents
(Olsson et al., 2006a;
Olsson et al., 2006b) and
F1 hybrids to the 11 volatile compounds tested in the study were
constructed using the program TCS v. 1.13
(Clement et al., 2000). These
networks provided a graphic overview of ORN response relationships for the
entire sample population, allowing for multi-dimensional characterization of
similarities and differences in the patterns of variation for parents and
hybrids. Each of the compounds, except for pentyl hexanoate and butyl
hexanoate, was considered to represent the equivalent of a different
nucleotide base position in a DNA sequence, with + and 0 neuron responses
coded as if they were alternate base pairs for a genetic polymorphism (e.g. A
and T). Pentyl hexanoate and butyl hexanoate were recoded as a single volatile
for network construction and branch length calculation because of the high
positive correlation in neuron response observed between the two compounds
(parent population r=0.697, P<0.0001, 76 d.f.;
F1 hybrid population r=0.812, P<0.0001, 117
d.f.). Significant connections between different response patterns based on
parsimony were limited to one step because of the low number of sites
(compounds). In order to connect all response patterns, the maximum number of
connections was set to four and networks are shown without breaking
reticulations.
Nearest neighbor distances (NNDs) were calculated as a metric to describe the degree of similarity/difference in neuron response patterns observed among and between parental and F1 hybrid flies. NND measures the dispersion of a population sample. As estimated in the current study, NNDs represented a summary statistic quantifying in a single value the overall similarity or difference in response patterns of ORNs depicted in the parsimony networks within and between parent and hybrid populations. To determine the NND for a neuron, the neuron's response to the suite of compounds tested was coded as a series of 1s and 0s depending upon whether the volatiles did (1) or did not (0) induce a statistically significant neuronal response, with pentyl hexanoate and butyl hexanoate again considered a single volatile (see above for statistical determination of a response). The resulting response vector for the reference neuron was then compared with each of the ORN responses in a chosen comparison population (parent or F1 hybrid), with pairwise response distances calculated as the absolute value of the total difference between the reference and comparison response vectors. The neuron in the comparison population displaying the fewest number of differences to the reference neuron was considered the nearest neighbor and the difference the NND for the reference neuron. In cases in which the reference neuron population and comparison population were the same, the reference neuron was excluded from the comparison population when NND values were calculated. In the results section, we present the mean and histograms of the distributions of NNDs for comparisons of parent (reference) vs parent (comparison) populations, parent vs F1 hybrid, and F1 hybrid vs parent.
Mean NND values were assessed for statistical significance by parametric bootstrapping based on Monte Carlo generated probability distributions. For the parent vs F1 hybrid and F1 hybrid vs parent comparisons, this involved randomly drawing computer generated parent (N=77) and F1 hybrid (N=118) simulated data sets with replacement from a combined neuron response sampling pool of parent and hybrid patterns. Mean NND values were then calculated between the simulated hybrid and parent data sets and a probability value (P value) estimated from the simulated distribution of NND values as the number of times in 10 000 trials that a simulated NND value as great or greater than the observed value was obtained. For the parent to parent analysis, the P value instead represents the proportion of randomly drawn data sets (N=77) sampled with replacement from the F1 hybrid population that had a mean NND to the actual parent population the same or less than the observed parent to parent mean NND value in 10 000 trials. This latter analysis determined whether any observed difference between parent and hybrid response patterns could be caused by biased sampling of parent neurons due to smaller sample sizes in parent (N=77) vs hybrid (N=118) populations.
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;
Olsson et al., 2006b).
Fig. 1 illustrates typical
parent and hybrid ORN response profiles for three basic classes of
Rhagoletis ORNs, as described by Olsson et al.
(Olsson et al., 2006a) (see
Table 2). As
Fig. 1 shows, although hybrid
ORNs still responded to the same compounds as the parent ORNs, they also
responded to other, sometimes structurally unrelated, compounds.
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) into the five major ORN classes representing increasing
levels of complexity with respect to the number of compounds eliciting a
response (Table 2). An
additional 55 cells were categorized as Class D or E. Owing to the diverse
response profiles of cells in classes D and E (i.e. response to at least two
long-chain esters and/or several host volatiles) these cells were not labeled
as either parent- or hybrid-like and are not graphically represented. The y
axis depicts sensitivities [i.e. reciprocal thresholds displayed as
log(sensitivity)] to each host volatile as determined by dose-response trials.
The lowest threshold responses (i.e. 10 ng) are shown as the highest
sensitivity bars [log(10 000)=4]. The highest thresholds (i.e. 10 µg or 10
000 ng) are the lowest bars [log(10)=1]. Each vertical line for the 11
compounds represents a single ORN, and the response profile is preserved from
top to bottom for each contacted cell.
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). These
cells were classified as parent-like in the first columns of classes A, B and
C. Of the 118 recorded ORNs, 6.7%, 9.3% and 12.7% of the cells could be
categorized as parent-like class A, B or C cells, respectively. Several ORNs
from hybrid individuals responded exclusively to 1-octen-3-ol (class A: one
ORN from an apple x hawthorn hybrid, two from hawthorn x apple,
three from apple x dogwood, and two from dogwood x apple),
dihydro-ß-ionone (class B: two ORNs from apple x hawthorn hybrids,
three from hawthorn x apple, and one ORN from a dogwood x apple
hybrid) or hexyl butanoate (Class B: one ORN each from apple x hawthorn,
hawthorn x apple, and dogwood x apple hybrids), as in parent class
A and B ORNs. Two of the class B apple x hawthorn hexyl butanoate
responders also responded to propyl hexanoate as was found in one instance of
the parent population recordings. Several cells also responded with class C
response profiles (response to 4,8-dimethyl-1,3(E),7-nonatriene
and/or 3-methylbutan-1-ol, either exclusively or with one or more other
compounds): five ORNs from apple x hawthorn hybrids, six ORNs from
hawthorn x apple, two from apple x dogwood, and two from dogwood
x apple. Other ORNs from hybrid individuals showed response patterns that were not observed in any of the parent flies. Because these response patterns were never found for any parent population ORNs, these cells were classified as `hybrid-like' and are shown in Fig. 2 in the right columns of classes A, B and C. For example, hybrid-like class A ORNs recorded from apple x hawthorn individuals (black bars) responded to 1-octen-3-ol (an alcohol) as did parent population ORNs, but also responded to dihydro-ß-ionone (a ketone), 4,8-dimethyl-1,3(E),7-nonatriene (a hydrocarbon), propyl hexanoate, pentyl hexanoate, butyl hexanoate (all esters), isoamyl acetate (an acetate) and/or ethyl acetate (acetate). These response profiles were never recorded from any parent population class A cells. Likewise, class B hybrid-like hawthorn x apple ORNs (white bars) responded to dihydro-ß-ionone or hexyl butanoate as in parent population ORNs, but also responded to 4,8-dimethyl-1,3(E),7-nonatriene, and/or ethyl acetate, 3-methylbutan-1-ol or isoamyl acetate. Several other examples are listed with their corresponding response profiles in Fig. 2, classes A-C. Note that cells with profiles resembling multiple classes are repeated for each class they resembled and indicated by asterisks on the x axis. In total, 29/63 (46%) of the cells exhibiting characteristics of parent class A, B, or C cells responded to unique combinations of volatiles absent from parent population recordings. Furthermore, both hybrid- and parent-like ORN profiles were observed in the same individual in 14 of 39 hybrid individuals tested (36%; six apple x hawthorn individuals, four hawthorn x apple, two apple x dogwood, and two dogwood x apple individuals), and twice in the same sensillum (one each from an apple x hawthorn and hawthorn x apple individual).
Fig. 2 also provides
response threshold sensitivities (i.e. reciprocal dose-response thresholds)
for each contacted ORN. The figures show a great deal of variation in ORN
sensitivity both within and between individuals. Mann-Whitney tests found very
few significant differences (P>0.05) in host volatile sensitivity
between hybrid populations or between hybrids and parents for any tested
volatile. Only the dogwood parent population
(Olsson et al., 2006b)
vs its reciprocal hybrid crosses was significantly different
(P<0.05) with respect to sensitivity to
4,8-dimethyl-1,3(E),7-nonatriene.
Parsimony networks were constructed for parent and F1 hybrid ORN
responses depicting the relationships of neuron response to the 11 fruit
volatile compounds tested in the study
(Fig. 3). Unlike the graphs
above, which group general classes (A-E) of response patterns based upon
cluster analysis (Table 2)
(Olsson et al., 2006a),
parsimony networks statistically compare ORN response profiles for exact
matches, with each node representing a different profile. For example, a
neuron responding to hexyl butanoate, propyl hexanoate and butyl hexanoate
(compounds 3, 5 and 8 from Fig.
2) would be pooled only with other neurons having that same
profile. Thus, each node in the network represents a unique response profile
and each single straight line between nodes reveals a one step difference in
response (i.e. response to one different volatile) between the two profiles.
Each subsequent line reveals another single compound difference in response
profile up to a maximum of four differences. The TCS network method allows for
more direct statistical comparison of the breadth in response pattern
variation between parent and hybrid populations. The networks highlighted some
similarities and several major differences in the patterns of neuron response
between parent and hybrid populations. For example, a number of parent neurons
(41/77=53.2%) fell into clusters A-D (see dark nodes at the top of
Fig. 3A). These neurons
characteristically responded to just one or a few of the 11 compounds tested,
and are similar to the classes previously determined by cluster analysis
(Table 2). Clusters A-D were
also evident in the hybrid population. However, the proportion of hybrid
neurons constituting these five categories was significantly lower
(28/118=23.7%) than the proportions observed for the parents (53.2%;
P<0.0001, two-tailed Fisher exact test). Another major difference
was that the hybrid population contained many neurons that responded to one
compound in addition to those characteristic of clusters A-D
(Fig. 3B).
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Parent neurons not belonging to the five clusters responded to varying combinations of multiple compounds; apart from the five clusters, no parental neurons responded to only one compound (notice lack of labeled white nodes, Fig. 3A). Several of the multiple response neurons had shared counterparts in the F1 hybrid population (10/30=33.3%, black nodes Fig. 3A,B). By contrast, the F1 hybrid population contained a higher proportion of unique, multi-compound response patterns not seen in the parent population (56/67=83.6% unique to F1 hybrids vs 66.7% unique to parents, unlabelled white nodes, Fig. 3A,B).
Nearest neighbor distances (NNDs) were calculated as summary statistics to quantify the difference between parent and F1 hybrid population ORN responses relative to the variation present within parent flies (see Fig. 4A-C for histograms of NND values for parent vs parent, parent vs F1 hybrid, and F1 hybrid vs parent comparisons). The mean NND calculated for the F1 hybrid vs parent comparison (0.831) was highly significant (P<0.0003) and much larger than the mean NND values estimated for the parent vs parent or parent vs F1 hybrid analyses (mean NND=0.493 and 0.338, respectively). The high NDD value for the F1 hybrid vs parent comparison reflects the large proportion of unique neuron response patterns present in the F1 hybrid population that were not seen in parent flies. By contrast, the relatively low and non-significant mean NND for the reciprocal parent vs F1 hybrid comparison (0.338; P=0.326) was due to the majority of neuron response patterns measured in the parent population having counterparts or close companions in the hybrid population. Finally, the low mean NND for the parent vs parent comparison reflects the high proportion of parental neurons with shared response patterns and the relatively few neurons having unique response patterns.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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).
Although a small proportion of parents responded to multiple blends
(Linn, Jr et al., 2005a), most
flew only to their own natal host blend
(Zhang et al., 1999;
Nojima et al., 2003a;
Nojima et al., 2003b;
Linn, Jr et al., 2005b), and
were antagonized by the presence of non-natal host volatiles
(Linn, Jr et al., 2005a).
Hybrids, on the other hand, did not respond to host volatile blends at
concentrations eliciting maximum response in the parental studies. The small
proportion that did respond did so only to combined blends from both parent
hosts. Additionally, if the concentrations of the volatiles were increased
tenfold, approximately 50-60% of the flies flew upwind to both single and/or
combined blends of the parents. These high concentrations arrested the upwind
flight of parents.
The unexpected results of our study indicate that peripheral chemoreception
is also significantly altered and potentially compromised in F1
hybrids between apple, hawthorn and dogwood host populations of R.
pomonella. In a previous study of the three parent populations, all
recorded ORNs could be grouped into five distinct classes with distinguishing
characteristics (Olsson et al.,
2006a) (Table 2).
Although 54% of the 63 Class A-C ORNs from hybrids could also be classified
into one of the first three parent ORN classes, 46% possessed unique and
highly divergent response profiles not observed in any parent population (Figs
1 and
2). In fact, some of the hybrid
ORN response profiles appeared to be combinations of profiles found in various
parental ORN classes. Still others possessed completely unique profiles.
Parsimony networks and nearest neighbor distances (Figs
3 and
4) were used to statistically
summarize differences in the response profiles within and between parent and
F1 hybrid neurons. These analyses reflect the diversity of ORN
response profiles in the F1 hybrid population as compared to the
more homogeneous parent ORNs. Monte Carlo parametric bootstrapping shows that
these unique ORN response profiles are an inherent characteristic of hybrid
neurons, and are not simply due to experimental sampling bias in the parent
population.
Contrary to previous studies in which F1 hybrid ORN responses
generally resembled one parent or a mixture of both parents, hybrid
Rhagoletis ORNs in hybrid individuals appeared to have unique
response profiles completely absent in any parent population. Abnormalities in
the peripheral system of F1 hybrids, therefore, cannot simply be
the result of the inheritance of traits from both parents. Rather, the unique
profiles indicate some form of breakdown in the development and function of
the ORNs. Although it is not surprising that each host-related taxon responds
to its own set of unique host volatile cues
(Zhang et al., 1999;
Nojima et al., 2003a;
Nojima et al., 2003b), it is
surprising that these hybrid abnormalities occur in spite of the fact that the
different host-related taxa are morphologically indistinguishable and lack any
fixed allozyme or nuclear and mtDNA sequence differences
(Berlocher, 2000;
Feder et al., 1988;
Feder et al., 2003;
Feder et al., 2005).
There are several potential explanations for the altered neurophysiology in
Rhagoletis F1 hybrids. It is possible that genomic
incompatibilities between parent Rhagoletis populations result in
physical deformities in the structure of olfactory sensilla and alter the
response properties of the ORNs they house. In Drosophila, the
dominant mutation Sco causes specific alterations in chemoreception
(Dubin et al., 1995), as well
as a severe reduction in expression of specific antennal odor receptors and
malformations in certain basiconic sensilla that may result in the loss of
responsiveness to certain chemicals (A. Ray, personal communication). However,
in Rhagoletis hybrids, any obvious deformities in sensilla were never
observed. We also did not discern any significant difficulties in contacting
ORNs, which might be assumed if those sensilla were malformed.
Alternatively, each recorded antennal sensillum in Rhagoletis housed between one and three neurons discriminated by differences in amplitude. It is possible that genomic incompatibilities governing neuron-to-sensilla mapping caused parent ORNs to become co-located in hybrid sensilla. These multiple ORNs then possessed identical amplitudes that could not be distinguished by the methods in this study, and appeared as single ORNs with unique response profiles. However, during the diminished amplitude (pinching) that often occurs with a high frequency response to an odorant (see Fig. 1C parent), one might be able to distinguish an unaffected ORN. Additionally, 15 of the 29 hybrid-like cells were already found co-located with ORNs easily discriminated by amplitude. It is unlikely that all of these hybrid-like ORNs were the result of co-located ORNs that were never distinguished.
The most parsimonious explanation for the observed changes in hybrid
Rhagoletis neurophysiology involves the ORNs themselves. Studies of
Drosophila have shown that odorant receptors confer the entire
odorant response spectrum of an olfactory neuron
(Elmore et al., 2003;
Dobrista et al., 2003;
Hallem et al., 2004).
Therefore, any changes in response profile seen in hybrid ORNs are most likely
the result of alterations in the olfactory receptors themselves. Olfactory
receptor neurons have traditionally been thought to express only one type of
olfactory receptor conferring the entire response profile of that neuron
(Jacquin-Joly and Merlin,
2004). However, a recent study
(Goldman et al., 2005) found
multiple, naturally expressed and completely functional odor receptors
co-expressed within a single neuron.
A recent study of chemoreception in hybrids between two closely related
moth species, Heliothis virescens and H. subflexa, found
that some hybrid ORNs responded to unique combinations of pheromone components
absent from either parent species (Baker et
al., 2006). Combined with both flight tunnel assays
(Vickers, 2006a) and antennal
lobe recordings (Vickers,
2006b), Baker et al. (Baker et
al., 2006) postulated that the "broad yet specific
tuning of certain ORN types" suggests that multiple olfactory
receptors were expressed in the dendritic membranes of the olfactory neurons.
In congruence with the hypothesis by Baker et al. for Heliothine
hybrids (Baker et al., 2006),
it is also possible that Rhagoletis hybrid ORNs express multiple
receptors, in combinations absent from parent ORNs. This could produce the
diverse response profiles witnessed in hybrid ORN responses. For instance,
some hybrid ORNs responded to combinations of 1-octen-3-ol,
4,8-dimethyl-1,3(E),7-nonatriene, hexyl butanoate and/or
dihydro-ß-ionone concurrently (Figs
1 and
2). In the parent populations
we found separate ORNs specific to these single components at the
concentrations tested in the current study
(Olsson et al., 2006a)
(Fig. 1,
Table 2). If parent populations
possessed separate receptor types for each of these compounds, then it is
possible that hybrid ORNs expressed multiple receptors for these compounds in
a single neuron.
Unfortunately, "remarkably little is known" about how
neurons determine which receptors to express
(Hallem and Carlson, 2004). In
Drosophila, Acj6, a POU-domain protein transcription factor, has been
suggested to impact receptor gene choice
(Clyne et al., 1999). In Acj6
mutant flies, a percentage of ORNs responded normally, a percentage were
unresponsive, and another subset had unique response profiles that were absent
from wild-type flies. This pattern is similar to what occurred in
Rhagoletis hybrids. It is possible that crosses between parent
Rhagoletis populations result in genomic incompatibilities that
affect POU-domain transcription factors and produce receptor misexpression at
the periphery. This, in turn, could leave some ORN response profiles intact
while others become distorted, gaining or losing response to certain odorants
by altering the type of receptor proteins they express. Many (36%) of the
tested individuals possessed both parent- and hybrid-like ORNs concurrently.
In fact, two individuals possessed both parent- and hybrid-like cells in the
same sensillum. The relationship between transcription factors and OR genes
has been suggested as a pathway for the unique responses found in
Heliothine hybrids (Baker et al.,
2006).
However, unlike the Heliothine study where the hybrid ORNs
displayed a stereotypical, though unique, set of responses from the parents,
the Rhagoletis hybrid ORN response profiles were much more diverse,
and almost random in appearance. We observed ORN response profiles with
virtually every combination of the 11 host volatiles tested (note hybrid A, B,
and C columns of Fig. 2). This
reshuffling and randomization of receptor expression indicates a fundamental,
and likely early, breakdown in odor receptor gene choice. Recent work in
Drosophila has identified both positive and negative
cis-regulatory elements that act in combinatorial code to dictate
organ- and neuron-specific expression of individual receptor genes
(Ray et al., 2005). It is
possible that a breakdown in this combinatorial code early in development
caused by genomic incompatibilities in parent host populations could cause the
changes in receptor expression proposed for the Rhagoletis
hybrids.
In the Heliothine study, Baker et al. suggested that because the
regulation of agonist/antagonist behaviors are believed to occur primarily in
the antennal lobe and higher brain centers, a simple change in receptor
expression at the periphery could allow a shift in the agonistic/antagonist
properties of a compound (Baker et al.,
2006). Olfactory receptor neurons expressing one type of olfactory
receptor target a single glomerulus in the antennal lobe of the brain
(Vosshall et al., 2000;
Gao et al., 2000), and ORNs
will target the same glomeruli regardless of normal receptor expression
(Dobrista et al., 2003). If an
ORN targeting a glomerulus that is part of an agonistic pathway now expresses
receptors for antagonistic compounds, it could drastically alter the
behavioral relevance of those compounds. It is this type of shift in receptor
expression that could have allowed hawthorn-origin flies to shift to apples
and develop antagonistic responses to former host compounds. A subtle shift in
receptor expression from neurons targeting agonist pathways to neurons
targeting antagonist pathways could promote a shift from one host to another.
A plasticity in receptor expression could also promote the variation in
behavior witnessed in parent populations
(Linn, Jr et al., 2005a).
If host populations with contrasting behavioral responses to host volatiles
differ as to their expression of olfactory receptors, then hybridization could
introduce the genomic incompatibilities discussed above. Formerly attractant
compounds whose receptors are now expressed in multiple neurons could
stimulate multiple glomeruli, sending conflicting input to the CNS and causing
hybrids to respond to a combination of host blends as observed in behavioral
trials (Linn, Jr et al.,
2004). Furthermore, although hybrid ORN sensitivities were not
significantly lower to host volatiles than parent ORNs, hybrids could still
require higher concentrations in order to respond behaviorally because the
hybrid antennal lobe requires a much higher concentration to process the
confused input from the periphery. It is known that antennal lobe neurons are
much less sensitive than receptor neurons [Hansson and Christensen
(Hansson and Christensen,
1999) and references therein]. Alternatively, there are still some
`parental type' ORNs present in F1 hybrids (Figs
2 and
3). These parent-like ORNs were
often found in conjunction with hybrid-like ORNs on the same individual.
Perhaps F1 hybrid individuals can only detect host blends at
significantly higher concentrations because there are fewer ORNs that still
respond `normally' to the host components.
Fig. 1 shows that in several
crosses roughly half of the cells tested possessed these altered,
`hybrid-like' response profiles.
These observed changes in hybrid ORN response profiles could have
significant impacts on hybrid olfactory behavior. By all other accounts, the
hybrid crosses are viable and fertile
(Linn, Jr et al., 2004).
However, olfaction has been shown to be an important factor in host selection
(Zhang et al., 1999;
Nojima et al., 2003a;
Nojima et al., 2003b;
Linn, Jr et al., 2003;
Linn, Jr et al., 2005a).
Chemosensory dysfunction in hybrids could significantly hinder their ability
to locate hosts (Linn, Jr et al.,
2004). This, in turn, would reduce their prospect of finding a
mate and ovipositing onto host fruit, given that Rhagoletis flies
mate and oviposit on or near the fruit of their host
(Prokopy et al., 1971).
Furthermore, the lack of sensitivity to high concentrations of volatiles in
F1 hybrids could reduce their ability to avoid overripe,
substandard fruit for oviposition preference
(Linn, Jr et al., 2004).
Together, these factors could significantly reduce hybrid reproductive fitness
in the field, and prevent further gene flow between hybrids and other
individuals. Thus, reduced chemosensory ability in Rhagoletis
F1 hybrids can serve as an effective postzygotic barrier to gene
flow between parent populations. More testing, including
mark-release-recapture field tests with F1 hybrids, are required to
confirm the impact of reduced chemosensory ability on hybrid host location
(Linn, Jr et al., 2004).
The results of the present study indicate that the reduced ability of
Rhagoletis hybrids to orient to host volatiles may be due, in part,
to significantly altered specificity in hybrid ORNs. The results also suggest
that subtle differences in receptor-neuron targeting in the parents could
account for differences in agonist/antagonist response to host volatiles and
could lead to genetic incompatibilities and receptor misexpression in the
hybrid offspring. Further testing involving the analysis of second generation
F2 and backcross hybrids is required to determine whether these
incompatibilities have direct impacts on olfactory behavior in the hybrid
populations. Alternatively, differences in olfactory behavior may be due to
alterations at higher brain centers, and the changes in ORN response seen here
may instead be the pleiotropic consequences of other phenotypes under
host-associated selection, such as developmental timing
(Feder and Filchak, 1999).
Nevertheless, the abnormalities found in Rhagoletis raise several new
questions regarding the development of peripheral olfactory pathways and their
impacts on behavior and speciation, which can only lead to exciting new
discoveries in the study of chemical senses and evolutionary biology.
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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