Article

Defining behavioral and molecular differences between summer and migratory monarch butterflies

Department of Neurobiology, University of Massachusetts Medical School, Plantation Street, Worcester, MA 01605, USA.
BMC Biology (Impact Factor: 7.98). 04/2009; 7(1):14. DOI: 10.1186/1741-7007-7-14
Source: PubMed

ABSTRACT

In the fall, Eastern North American monarch butterflies (Danaus plexippus) undergo a magnificent long-range migration. In contrast to spring and summer butterflies, fall migrants are juvenile hormone deficient, which leads to reproductive arrest and increased longevity. Migrants also use a time-compensated sun compass to help them navigate in the south/southwesterly direction en route for Mexico. Central issues in this area are defining the relationship between juvenile hormone status and oriented flight, critical features that differentiate summer monarchs from fall migrants, and identifying molecular correlates of behavioral state.
Here we show that increasing juvenile hormone activity to induce summer-like reproductive development in fall migrants does not alter directional flight behavior or its time-compensated orientation, as monitored in a flight simulator. Reproductive summer butterflies, in contrast, uniformly fail to exhibit directional, oriented flight. To define molecular correlates of behavioral state, we used microarray analysis of 9417 unique cDNA sequences. Gene expression profiles reveal a suite of 40 genes whose differential expression in brain correlates with oriented flight behavior in individual migrants, independent of juvenile hormone activity, thereby molecularly separating fall migrants from summer butterflies. Intriguing genes that are differentially regulated include the clock gene vrille and the locomotion-relevant tyramine beta hydroxylase gene. In addition, several differentially regulated genes (37.5% of total) are not annotated. We also identified 23 juvenile hormone-dependent genes in brain, which separate reproductive from non-reproductive monarchs; genes involved in longevity, fatty acid metabolism, and innate immunity are upregulated in non-reproductive (juvenile-hormone deficient) migrants.
The results link key behavioral traits with gene expression profiles in brain that differentiate migratory from summer butterflies and thus show that seasonal changes in genomic function help define the migratory state.

Full-text

Available from: Sriramana Kanginakudru, Nov 11, 2014
7.43
Abstract
Background
In the fall, Eastern North American monarch butterflies (Danaus plexippus) undergo a magnificent long-range migration. In contrast to spring and summer butterflies, fall
migrants are juvenile hormone deficient, which leads to reproductive arrest and increased longevity. Migrants also use a time-compensated sun compass to help them
navigate in the south/southwesterly direction en route for Mexico. Central issues in this area are defining the relationship between juvenile hormone status and oriented
flight, critical features that differentiate summer monarchs from fall migrants, and identifying molecular correlates of behavioral state.
Results
Here we show that increasing juvenile hormone activity to induce summer-like reproductive development in fall migrants does not alter directional flight behavior or its
time-compensated orientation, as monitored in a flight simulator. Reproductive summer butterflies, in contrast, uniformly fail to exhibit directional, oriented flight. To
define molecular correlates of behavioral state, we used microarray analysis of 9417 unique cDNA sequences. Gene expression profiles reveal a suite of 40 genes whose
differential expression in brain correlates with oriented flight behavior in individual migrants, independent of juvenile hormone activity, thereby molecularly separating fall
migrants from summer butterflies. Intriguing genes that are differentially regulated include the clock gene vrille and the locomotion-relevant tyramine beta hydroxylase
gene. In addition, several differentially regulated genes (37.5% of total) are not annotated. We also identified 23 juvenile hormone-dependent genes in brain, which
separate reproductive from non-reproductive monarchs; genes involved in longevity, fatty acid metabolism, and innate immunity are upregulated in non-reproductive
(juvenile-hormone deficient) migrants.
Conclusion
The results link key behavioral traits with gene expression profiles in brain that differentiate migratory from summer butterflies and thus show that seasonal changes in
genomic function help define the migratory state.
Background
Eastern North American monarch butterflies (Danaus plexippus) undergo a spectacular fall migration during which they travel distances up to ~4000 km to reach their
overwintering grounds in central Mexico [1]. In contrast to spring and summer butterflies, fall migrants are juvenile hormone (JH) deficient, which leads to reproductive
arrest (diapause), increased longevity, and increased abdominal fat stores [2,3]. Fall migrants also use a time-compensated sun compass to help them navigate in the
south/southwesterly direction [4-6]. Reproductive quiescence persists at the overwintering areas in Mexico until spring, when the butterflies break diapause, become
reproductively competent, mate, and fly northward to lay fertilized eggs on newly emerged milkweed plants in the southern United States [7,8].
The migrant offspring give rise to three to four successive generations of reproductively active butterflies that repopulate the northern range of their habitat. It is unclear
whether the successive generations of spring and summer butterflies have oriented flight activity to the north and/or whether they are following the progressive northerly
increase in milkweed abundance, while avoiding undue heat stress that would occur if they remained in the southern United States throughout the summer [7]. The late-
July/early-August generations of summer butterflies, some of whose offspring become fall migrants, appear to be the best example of butterflies that do not exhibit
oriented flight behavior [9,10]. However, the precise type of flight behavior that the summer monarchs actually manifest has not been rigorously examined. It is also
unclear whether JH deficiency and the accompanying reproductive quiescence are required for ongoing time-compensated sun compass orientation in fall migrants.
We recently developed a brain expressed sequence tag (EST) resource for monarch butterflies that likely represents ~50% of genes in the monarch genome [11]. Using
high-density microarrays of the 9417 unique cDNA sequences in the EST resource, a blueprint of gene expression patterns can be compared and contrasted between
different conditions that may help define the molecular substrates that characterize the summer and migratory states.
Research article
Defining behavioral and molecular differences between summer and migratory monarch butterflies
Haisun Zhu
, Robert J Gegear
, Amy Casselman, Sriramana Kanginakudru and Steven M Reppert
*
BMC Biology 2009, 7:14 doi:10.1186/1741-7007-7-14
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1741-7007/7/14
Received: 15 December 2008
Accepted: 31 March 2009
Published: 31 March 2009
© 2009 Zhu et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Corresponding author: Steven M Reppert
† Equal contributors
Department of Neurobiology, University of Massachusetts Medical School, Plantation Street, Worcester, MA 01605, USA
For all author emails, please log on.
*
Steven.Reppert@umassmed.edu
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BMC Biology
Open Access
Research article
Defining behavioral and molecular differences between summer
and migratory monarch butterflies
Haisun Zhu
, Robert J Gegear
, Amy Casselman, Sriramana Kanginakudru
and Steven M Reppert*
Address: Department of Neurobiology, University of Massachusetts Medical School, Plantation Street, Worcester, MA 01605, USA
Email: Haisun Zhu - Haisun.Zhu@gmail.com; Robert J Gegear - Robert.Gegear@umassmed.edu;
Amy Casselman - Amy.Casselman@umassmed.edu; Sriramana Kanginakudru - Sriramana.Kanginakudra@umassmed.edu;
Steven M Reppert* - Steven.Reppert@umassmed.edu
* Corresponding author †Equal contributors
Abstract
Background: In the fall, Eastern North American monarch butterflies (Danaus plexippus) undergo
a magnificent long-range migration. In contrast to spring and summer butterflies, fall migrants are
juvenile hormone deficient, which leads to reproductive arrest and increased longevity. Migrants
also use a time-compensated sun compass to help them navigate in the south/southwesterly
direction en route for Mexico. Central issues in this area are defining the relationship between
juvenile hormone status and oriented flight, critical features that differentiate summer monarchs
from fall migrants, and identifying molecular correlates of behavioral state.
Results: Here we show that increasing juvenile hormone activity to induce summer-like
reproductive development in fall migrants does not alter directional flight behavior or its time-
compensated orientation, as monitored in a flight simulator. Reproductive summer butterflies, in
contrast, uniformly fail to exhibit directional, oriented flight. To define molecular correlates of
behavioral state, we used microarray analysis of 9417 unique cDNA sequences. Gene expression
profiles reveal a suite of 40 genes whose differential expression in brain correlates with oriented
flight behavior in individual migrants, independent of juvenile hormone activity, thereby molecularly
separating fall migrants from summer butterflies. Intriguing genes that are differentially regulated
include the clock gene vrille and the locomotion-relevant tyramine beta hydroxylase gene. In addition,
several differentially regulated genes (37.5% of total) are not annotated. We also identified 23
juvenile hormone-dependent genes in brain, which separate reproductive from non-reproductive
monarchs; genes involved in longevity, fatty acid metabolism, and innate immunity are upregulated
in non-reproductive (juvenile-hormone deficient) migrants.
Conclusion: The results link key behavioral traits with gene expression profiles in brain that
differentiate migratory from summer butterflies and thus show that seasonal changes in genomic
function help define the migratory state.
Published: 31 March 2009
BMC Biology 2009, 7:14 doi:10.1186/1741-7007-7-14
Received: 15 December 2008
Accepted: 31 March 2009
This article is available from: http://www.biomedcentral.com/1741-7007/7/14
© 2009 Zhu et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Page 2
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Page 2 of 14
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Background
Eastern North American monarch butterflies (Danaus plex-
ippus) undergo a spectacular fall migration during which
they travel distances up to ~4000 km to reach their over-
wintering grounds in central Mexico [1]. In contrast to
spring and summer butterflies, fall migrants are juvenile
hormone (JH) deficient, which leads to reproductive
arrest (diapause), increased longevity, and increased
abdominal fat stores [2,3]. Fall migrants also use a time-
compensated sun compass to help them navigate in the
south/southwesterly direction [4-6]. Reproductive quies-
cence persists at the overwintering areas in Mexico until
spring, when the butterflies break diapause, become
reproductively competent, mate, and fly northward to lay
fertilized eggs on newly emerged milkweed plants in the
southern United States [7,8].
The migrant offspring give rise to three to four successive
generations of reproductively active butterflies that repop-
ulate the northern range of their habitat. It is unclear
whether the successive generations of spring and summer
butterflies have oriented flight activity to the north and/or
whether they are following the progressive northerly
increase in milkweed abundance, while avoiding undue
heat stress that would occur if they remained in the south-
ern United States throughout the summer [7]. The late-
July/early-August generations of summer butterflies, some
of whose offspring become fall migrants, appear to be the
best example of butterflies that do not exhibit oriented
flight behavior [9,10]. However, the precise type of flight
behavior that the summer monarchs actually manifest has
not been rigorously examined. It is also unclear whether
JH deficiency and the accompanying reproductive quies-
cence are required for ongoing time-compensated sun
compass orientation in fall migrants.
We recently developed a brain expressed sequence tag
(EST) resource for monarch butterflies that likely repre-
sents ~50% of genes in the monarch genome [11]. Using
high-density microarrays of the 9417 unique cDNA
sequences in the EST resource, a blueprint of gene expres-
sion patterns can be compared and contrasted between
different conditions that may help define the molecular
substrates that characterize the summer and migratory
states.
Here we show that increasing JH activity to induce sum-
mer-like reproductive development in fall migrants did
not alter directional flight behavior or its time-compen-
sated orientation, as monitored in a flight simulator.
Summer butterflies, on the other hand, uniformly failed
to exhibit directional, oriented flight. Microarray analysis
revealed 40 JH-independent genes whose differential
expression in brain correlated with directional flight
behavior in fall migrants. Moreover, we have identified 23
JH-dependent genes in brain, which separate reproductive
from non-reproductive butterflies. These data provide an
unprecedented foray into the genomic regulation of
migratory behaviors in monarch butterflies.
Results and discussion
Increased juvenile hormone activity in migrants does not
disrupt directed flight or time-compensated orientation
Because several aspects of migratory behavior are a conse-
quence of continued JH deficiency, for example, repro-
ductive quiescence and increased longevity [2,3], we
examined whether the oriented flight behavior character-
istic of fall migrants also depends on persistent JH insuffi-
ciency. This was evaluated by increasing JH activity with
the potent JH analog methoprene [12] and then monitor-
ing the effect on reproductive state and time-compensated
flight orientation. Preliminary studies showed that the
topical treatment of migrants with 200 g of methoprene
on day 1 and day 3 consistently led to summer-like repro-
ductive development in both sexes by day 14, while vehi-
cle (control) applications of acetone did not (data not
shown; see below).
Both methoprene- and vehicle-treatment groups were
housed indoors in either a 12 hr light-12 hr dark (LD)
cycle that was timed to coincide with the prevailing light-
ing conditions or a 6 hr-delayed LD cycle. These two light-
ing cycles, which differed in their timing relative to each
other, were used to test whether flight orientation was
time compensated, because altering the timing of the
daily light-dark cycle should cause predictable changes in
the direction the butterflies fly, if time compensation is
operable. For example, the 6-hr delay in LD should cause
a clockwise shift in orientation of 72° to 120°, relative to
the non-shifted LD group, if flight direction is fully time
compensated; the degree of the shift expected depends on
how rapidly the sun's azimuth varies during the time of
day the studies were performed, which was 12° to 20° per
hour for the current studies.
Fourteen days after the first methoprene treatment, butter-
flies housed in either LD or the 6-hr delayed LD cycle were
tethered, and over the next 5 days individual flight direc-
tion and group orientation were examined in butterflies
flown outdoors in a flight simulator. Of 62 migrants that
flew continuously for 5 to 10 min in the simulator, 48
individuals (77% of total) flew directionally, which was
defined as flying with a Z-score 500 (Figure 1); these
directional migrants comprised the four groups that were
evaluated for the time-compensated orientation analysis
(Figure 2).
Regardless of treatment (methoprene or vehicle), group
analyses showed that the directional fall migrants mani-
fested time-compensated flight orientation (Figure 2).
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Both treatment groups housed under prevailing LD condi-
tions were oriented significantly in the south/southwest-
erly direction (Figure 2A); vehicle-treated migrants had an
orientation vector () of 202.6° (n = 12, r = 0.714, p =
0.001) (Figure 2A, upper left, small blue circles), similar
to what we have reported before for untreated migrants
[4], and methoprene-treated migrants had an of 173° (n
= 10, r = 0.713, p = 0.004) (Figure 2A, upper right, small
red circles). The mean flight orientation did not differ
between vehicle- and methoprene-treated migrants (p =
0.18; Watson-Williams F-test) and the combined was
189.2° (n = 22, r = 0.69, p < 0.00001) (Figure 2A, lower,
merge).
Both treatment groups housed under the 6 hr-delayed LD
cycle were oriented significantly in the west/northwesterly
direction (Figure 2B); vehicle-treated migrants had an of
276.9° (n = 11, r = 0.58, p = 0.021) (Figure 2B, small blue
circles), and methoprene-treated migrants had an of
291.6° (n = 15, r = 0.566, p = 0.006) (Figure 2B, small red
circles). Again, the mean flight orientation did not differ
between vehicle- and methoprene-treated migrants (p =
0.573; Watson-Williams F-test) and the combined was
285.3° (n = 26, r = 0.567, p = 0.0001) (Figure 2B, merge).
The direction and magnitude of the group orientation dif-
ference between the merged LD and 6 hr-shifted LD
groups (a clockwise shift of 96.1°; p < 0.00001, Watson-
Williams F-test) are those expected of a time-compensated
sun compass that has been delayed by 6 hrs (compare
merged data in lower rows of Figure 2A and 2B). There
were no significant sex differences in either the degree of
individual directionality or group orientation of the
migrants tested (Figure 3).
Postmortem analysis of the oriented butterflies revealed
that the methoprene-treated male and female migrants all
had activated reproductive systems; male reproductive
organ weights were almost doubled compared with the
vehicle group (p < 0.0001), and >100 mature oocytes were
found in the methoprene-treated females (Figure 4A).
Furthermore, all methoprene-treated females and 50% of
the methoprene-treated males examined exhibited repro-
ductive behavior by forming mating pairs when exposed
to high-intensity light and increased temperature (25°C)
from day 14 to 16 after the start of methoprene treat-
ments; this behavior was rarely observed in the vehicle-
treated animals exposed to the same conditions (Figure
4B).
These data show that individual fall migratory monarchs
uniformly manifest directed flight and as a group show
robust time-compensated sun compass orientation even
when their reproductive systems are activated (at the mor-
phological and behavioral levels) by JH analog treatment.
Although JH deficiency may be involved in the induction
of directional flight and proper sun compass orientation,
it is not required for their maintenance.
Summer butterflies uniformly fail to show directed,
oriented flight behavior
Although it has been reported that 'summer' monarchs do
not exhibit oriented flight [9,10], until now this has not
been evaluated in a flight simulator in which both indi-
vidual directionality and group orientation can be
assessed (see below). We tested these parameters in wild-
caught summer butterflies captured in western Massachu-
setts (latitude 42°59'N) between 20 July and 10 August
2008 and housed indoors in a light-dark cycle that was
timed to coincide with the prevailing lighting conditions.
These butterflies were reproductive, as most were found in
mating pairs while held in screened cages outdoors prior
to being flown in a flight simulator. Moreover, fall
migrants typically are not found at this locale until after 1
September.
Relationship between virtual flight path and Z-score valueFigure 1
Relationship between virtual flight path and Z-score
value. To obtain Z-scores (shown upper right of each
graph), flight data for each butterfly tested in the flight simu-
lator were analyzed using a Rayleigh test. Z, which is the crit-
ical value for the Rayleigh test, is calculated from the
following formula: Z = nr
2
, where n is the number of observa-
tions and r is the magnitude of the mean vector. Only butter-
flies with a Z-score 500 or above have a flight path that
shows clear directionality. Virtual flight paths were con-
structed by starting in the center of the square and plotting
each direction interval consecutively as one unit length [5].
1108
571
502
380
320
224
188
32
127
N
N
N
S
SS
W
W
W
E
E
E
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In marked contrast to fall migrants, only 5 of 18 summer
butterflies (27.8%) that flew outdoors continuously for 5
to 10 min in the flight simulator flew directionally (with
Z-scores 500). This lack of directional flight among indi-
viduals was apparent on inspection of the constructed vir-
tual flight paths of the summer butterflies compared with
the individual flight paths of both the methoprene-treated
and vehicle-treated migrants housed in LD (Figure 5A).
We also compared the r-values for individual virtual flight
paths among the three groups (Figure 5B), because r-val-
ues are a measure of angular dispersion and range from 0
to 1, in which 0 represents complete dispersion of the data
and 1 represents all of the data concentrated in the same
direction. The r-values differed significantly among the
three groups (p = 0.005), and the summer butterflies had
significantly lower r-values than those in the two migrant
groups. The five summer animals with directional flight
were not oriented significantly in any one direction as a
group (p > 0.05) (Figure 5C). There were no significant sex
differences in either the directionality of flight among
individuals or group orientation in the summer butterflies
tested (sex ratio was 1:1 within the group; six males and
seven females were non-directional; three males and two
females were directional).
These data show that the majority of individual mid- to
late-summer butterflies exhibit non-directional flight
behavior. Although the numbers were small, the data also
suggest that as a group those butterflies that were direc-
tional were not significantly oriented, as previously sug-
gested [9,10].
Gene expression profiles in brain correlate with oriented
flight behavior in fall migrants
Our behavioral data in migrants suggest that the regula-
tion of directed flight behavior in individuals and group
orientation are separable from reproductive state (Figures
2 and 4). It consequently seemed possible that there
might be a set of genes that regulates oriented flight
behavior in migrants that is independent of the JH path-
way. We therefore performed microarray analysis to deter-
Figure 2
0
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o
Natural light:dark cycle
(lights on 0600-1800 hrs)
0
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6-hour phase delay
(lights on 1200-2400 hrs)
A
B
Vehicle
Merge
MethopreneVehicle
Merge
Methoprene
Reproductive migrants show time-compensated sun compass orientationFigure 2
Reproductive migrants show time-compensated sun
compass orientation. (A) Vehicle- and methoprene-
treated migrants housed under normal fall conditions show a
flight orientation in the south/southwesterly direction.
Migrants were housed in a light-dark cycle with lights on
from 0600 to 1800 hours EST with a temperature cycle of
23°C during light-12°C during dark before being tested out-
doors in a flight simulator. The migrants were flown between
1230 and 1530 hours from 19 September to 15 October
2007. The large circles represent the 360° of possible direc-
tion (0° = north). The small solid circles on the perimeter
represent the mean orientation of individual butterflies. Blue,
vehicle-treated migrants; red, methoprene-treated migrants;
merge, combined data. The arrow indicates the mean vector,
and the length of the arrow represents the strength (r value).
(B) Vehicle- and methoprene-treated migrants housed under
a 6-hr phase delayed lighting cycle show a flight orientation in
the west/northwesterly direction. Migrants were housed in
light-dark cycle with lights on from 1200 to 2400 hours EST
with a temperature cycle of 23°C during light-12°C during
dark before being tested outdoors in a flight simulator. The
migrants were flown between 1230 and 1530 hours from 19
September to 15 October 2007. The large circles represent
the 360 of possible direction (0° = north). The small solid cir-
cles on the perimeter represent the mean orientation of indi-
vidual butterflies. Blue, vehicle-treated migrants; red,
methoprene-treated migrants; merge, combined data. The
arrow indicates the mean vector, and the length of the arrow
represents the strength (r value).
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mine whether there are differentially expressed genes
between summer butterflies and fall migrants, irrespective
of their reproductive status. These genes might provide
insights into brain changes necessary to initiate and main-
tain oriented flight activity.
We tried to ensure that the animals used for the microar-
ray analyses were handled in a way to minimize the influ-
ence of non-migratory factors on gene expression and to
mimic the conditions used for our behavioral experiments
(Table 1). First, all animals were placed in glassine enve-
lopes to minimize the influence of activity on the array
analysis (number of days each group was in the envelopes
is depicted in Table 1). Second, all the animals were
housed in controlled environmental conditions simulat-
ing those in the outside environment at the time of year of
collection, as outlined in Table 1; all butterflies were col-
lected in the wild and the locales and times of year of col-
lection were recorded (Table 1). Third, the environmental
conditions within the compartments in which the butter-
flies were housed (lighting cycle, temperature and humid-
ity) were similar to those used to generate the behavioral
data for the different groups shown in Figures 1 to 5.
Fourth, to minimize time-of-day effects on gene expres-
sion, all animals were killed within a 2-hr period encom-
passing the mid-light time of the light-dark cycle in which
the various butterfly groups were housed.
We collected total brain RNA from 10 summer butterflies,
10 fall migrants, 10 migrants following methoprene treat-
ment, and 10 migrants following vehicle (acetone) treat-
ment. We checked the reproductive status of all animals to
ensure they had the expected reproductive state (Table 1),
which was similar to that found in our flight studies (Fig-
ure 4A); summer monarchs and methoprene-treated
migrants had activated reproductive systems, while
untreated and vehicle-treated migrants did not (Table 1).
The brain RNAs were amplified and then used to probe a
custom Nimblegen array that was designed to analyze the
9417 unique cDNA sequences established in our pub-
lished brain EST library [11].
To discover genes that might be involved in oriented
flight, but not reproduction, we compared the summer
group with each of the three fall groups (untreated, meth-
oprene-treated, and vehicle-treated) for males and for
females, and looked for gene regulation patterns common
among the three comparisons for each sex (Table 2A; see
Additional file 1 for specific ESTs and annotation). The
rationale for this approach was that methoprene would
only regulate JH-dependent genes and should not affect
genes that regulate directional flight and orientation, as
our flight experiments showed that oriented flight was not
altered by increasing JH activity (Figures 2 and 4).
As the comparisons were done separately for males and
females, and our behavioral data did not show significant
sex differences in flight directionality and orientation
(Figure 3), we focused on the common differentially reg-
ulated genes that were shared between males and females
(Table 2A, right column). Accordingly, we identified 40
cDNAs that were differentially regulated between summer
butterflies and fall migrants, irrespective of sex. Further-
more, hierarchical clustering analysis using the individual
animal expression data for only these 40 cDNAs showed
that all 10 summer animals were clustered together cor-
rectly, whereas the three fall groups formed their own sep-
arate cluster; bootstrap values supported this trend (Figure
6). The expression of 14 genes was increased in migrants,
while the expression of 26 genes was increased in summer
butterflies (Figure 6, listed from top to bottom of heat
map, respectively). The magnitude of the differences was
Flight direction and orientation do not differ between male and female migrantsFigure 3
Flight direction and orientation do not differ
between male and female migrants. A. R-values of flight
direction were similar between treatment groups (vehicle =
blue; methoprene-treated = red) and between sexes. B.
Mean orientation of all directional butterflies did not differ
between males and females (p > 0.05). The data were stand-
ardized to orientation relative to LD for both the LD and the
6-hr phase delayed LD groups to directly compare orienta-
tion of all the animals of each sex used for the studies in Fig-
ure 2.
0
o
90
o
180
o
270
o
0
o
90
o
180
o
270
o
n=28
r=0.743
p<0.00001
n=20
r=0.504
p=0.005
0
0.2
0.4
0.6
0.8
1.0
11
9
12
16
A
B
Males Females
r-value
Males
Females
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uniformly modest, with a mean expression difference
among the 40 genes of 1.70-fold (range = 1.28-fold to
3.21-fold). Nonetheless, the expression profile of these 40
genes appears to predict a critical behavioral characteristic
of fall migrants in individual butterflies – oriented flight
behavior, independent of reproductive state. It is impor-
tant to note, however, that some of the differences in gene
expression could reflect non-migratory aspects of mon-
arch biology that were hard to control in our studies,
including the age of the butterflies and the mixed genetic
background inherent in collecting wild animals.
Of the 40 cDNAs, only 25 had matches with other data-
bases, with 24 being annotated with function (Figure 7).
The annotated brain cDNAs that were upregulated in
migrants included those involved in cytoskeletal organi-
zation (Actin related protein 5), ATP dependent proteolysis
(CG5045), immune responses (Eukaryotic translation initi-
ation factor 3 subunit and CG6359) and the initiation of
translation (Eukaryotic translation initiation factor 3 subu-
nit). The annotated cDNAs that were upregulated in sum-
mer butterflies included those that may be involved in
neural and behavioral plasticity, including those involved
in neuronal development (abrupt), synaptic transmission
(Synapse-associated protein 47 kD), and cell proliferation
(will die slowly).
The two differentially regulated cDNAs that appeared to
be most directly related to time-compensated orientation
were tyramine beta hydroxylase, whose protein regulates
octopamine biosynthesis, which is involved in motor
behavior, and vrille, which encodes a circadian clock com-
ponent (based on studies in Drosophila); both cDNAs were
upregulated in summer butterflies. The protein VRILLE is
an important negative regulator of Clock transcription,
and CLOCK is a critical transcriptional regulator of the cir-
cadian clock mechanism of insects and mammals [13].
There were also several differentially regulated cDNAs,
upregulated in summer butterflies, that were involved in
more general cellular processes, which included steroid/
cholesterol metabolism (Cytochrome P450 and HMG
Coenzyme A synthase), lipid metabolism (CDP-diglyceride
synthetase and CG31140-PB), electron transport (Cyto-
chrome P450 and CG8032-PA), and intracellular signaling
pathways (growl, capa receptor, and CG42450-PA).
The one annotated cDNA without assigned function can
now be classified as being involved functionally in ori-
ented flight behavior, along with the other 39 cDNAs.
Interestingly, 15 cDNAs had no annotation with other
databases. Their lack of identity based on available
genomic and EST resources could mean that the non-
annotated cDNAs contain incomplete sequence informa-
tion for orthologous matches with other databases. A
more exciting possibility is that the non-annotated cDNAs
represent unknown genes whose functions are unique to
the migratory state in monarch butterflies.
JH-regulated gene expression patterns in brain correlate
with reproductive state
In addition to 'orientation' genes, we were also interested
in evaluating the JH-response genes. These genes are likely
involved in reproductive status and longevity. Since these
genes are expected to be regulated by JH, the methoprene-
treated fall butterflies should have expression profiles
similar to those in the summer animals. Again, we per-
formed sex-specific statistical analyses, and compared the
summer and the fall groups, and the methoprene-treated
and vehicle-treated migrants (Table 2B). We then screened
for shared genes between the two groups for each sex.
Of the sex-specific groups of differentially regulated genes
(Table 2B, # shared in each sex; for complete list of JH-reg-
ulated, sex-specific ESTs and annotation see Additional
File 2), we focused on three genes involved in increased
JH activity for which we previously showed significant
increased expression in summer butterflies (compared
with fall migrants) by real-time polymerase chain reaction
(qPCR): juvenile hormone acid methyltransferase (jhamt),
which encodes the enzyme that mediates the last step in
JH biosynthesis [14];allatotropin, which encodes a neu-
ropeptide that can increase JH synthesis [15]; and takeout,
which encodes a potential JH-binding protein [16]. The
mRNA levels for both jhamt and allatotropin were upregu-
lated significantly in summer males compared with
untreated male migrants by microarray; mRNA levels were
also higher in female summer butterflies but the levels did
not reach significance (Figure 8). Combining the sexes
and reanalyzing the microarray differences between sum-
mer butterflies and untreated migrants showed significant
up-regulation of both jhamt and allatotropin expression in
summer butterflies (p < 0.01), which was consistent with
our previous qPCR results, as animals of mixed sex were
used in that analysis [11]; as both jhamt and allatotropin
are associated with the corpora allata, it is likely that the
expression profiles of each represent expression mainly in
corporal allata tissue, which was likely included in our
brain dissections. The mRNA levels of takeout were mar-
ginally upregulated in summer butterflies by microarray
analysis (Figure 8), but were not upregulated significantly,
as those reported by qPCR in our previous work [11]. It is
noteworthy that the qPCR results were performed on
mRNA from whole heads, while the microarray analysis
was performed using mRNA from dissected brains; this
disparity in tissue composition likely contributed to any
expression discrepancies between the previous qPCR
study and our current microarray results.
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We next examined cDNAs that were differently regulated
in both males and females (Table 2B, # shared by both
sexes), to determine whether we could identify JH-regu-
lated genes involved in more global processes that would
not be expected to be sex-specific, such as longevity and
fatty acid metabolism. We identified 23 putative JH-
response genes that were common between the males and
females. Hierarchical clustering using the 23 JH-regulated
genes showed that summer and methoprene-treated
migrants clustered together, as expected (Figure 9A). The
other two groups, the untreated and vehicle-treated
migrants, formed their own cluster (Figure 9A). The
expression of 11 genes was increased in the summer and
methoprene-treated migrants, while the expression of 13
genes was increased in the untreated and vehicle-treated
migrants (Figure 9A, listed from top to bottom of heat
map, respectively). Similar to the orientation genes, the
magnitude of the differences was uniformly modest for
the JH-regulated genes, with a mean expression difference
among the 23 genes of 1.67-fold (range = 1.24-fold to
3.28-fold). Thus, the hierarchical clustering predicted cor-
rectly the reproductive status of all individual animals
tested, using the brain expression pattern of these 23
genes that are common between the sexes.
Of the 23 JH-related cDNAs, 21 had matches with other
databases, with 15 annotated with biological function
(Figure 9B). Predictably, they included genes involved in
longevity (rosy), fatty acid metabolism (CG6543-PB), and,
interestingly, immune responses (hemolin, TGF beta-induc-
ible nuclear protein 1, Gp150, and inhibitor of kappa light
polypeptide gene enhancer), which were all upregulated in
untreated and vehicle-treated migrants. There were also
two genes involved in translation (ribosomal protein L35A
and polyA-binding proteins) and one involved in calcium-
dependent phospholipid binding (Annexin IX) that were
upregulated in JH-deficient migrants. Genes that were
upregulated in reproductive butterflies included those
involved in steroid biosynthesis (Cytochrome P450-18a1),
notch signaling (Enhancer of spilt mgamma), and calcium
homeostasis (CG2165-PC).
Figure 4
Mass of reproductive
glands (mg)
Mean number mature oocytes
12
16
0
20
40
60
80
100
Vehicle
Methoprene
Percent in mating pair
A
B
88
8
10
0
50
100
150
200
Vehicle
Methoprene
16
0
10
20
30
40
50
60
Vehicle
Methoprene
11
9
Methoprene increases reproductive organ development and mating behavior in fall migrantsFigure 4
Methoprene increases reproductive organ develop-
ment and mating behavior in fall migrants. (A) Repro-
ductive development is increased in fall migrants following
methoprene treatment. Numbers represent the animals
examined. Postmortem reproductive development was
assessed for all migrants depicted in Figure 2; vehicle-treated
migrants (blue bars) and methoprene-treated migrants (red
bars). Reproductive development of male migrants (upper
panel) was quantified by extracting and weighing reproduc-
tive glands (tubular gland and ejaculatory duct), while repro-
ductive development of female migrants (lower panel) was
quantified by counting all mature oocytes (that is, those with
well-defined ridged chorion). (B) Mating behavior was
increased in vehicle-treated migrants (blue bars) compared
to methoprene-treated migrants (red bars). Numbers repre-
sent the animals tested. To assess mating behavior, male and
female butterflies from each group were placed together in
flight cages under high intensity light of 2.6 × 10
5
photons/
cm
2
/s during the light period of LD at 25°C, and the number
of animals in a mating pair was recorded over a 2-day period.
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The six cDNAs that were annotated but without assigned
function can now be classified functionally as being
involved in JH-related activities, along with the other 17
cDNAs. Two differentially regulated cDNAs lacked anno-
tation based on available genomic and EST databases,
which could mean that they contain incomplete sequence
information for orthologous matches with other data-
bases or that the non-annotated cDNAs represent
unknown genes whose functions are unique to the migra-
tory state in monarch butterflies.
Defining the migratory state
The thrust of this work was to more precisely define the
behavioral and molecular differences between summer
butterflies and fall migrants. As there are several genera-
tions of reproductively active spring and summer butter-
flies, we chose to focus on the mid- to late-summer
butterflies whose offspring likely give rise to fall migrants.
We contrast this generation with those in the spring and
early summer, which are moving north/northwesterly to
repopulate the upper ranges of their habitat in Eastern
North America. Observations of those monarchs suggest
that they may have oriented flight behavior [7], and these
earlier generations need to be more rigorously evaluated
in a flight simulator, as we have done in our studies with
mid- to late-summer butterflies.
A significant aspect of our behavioral work with summer
butterflies shows that their individual flight patterns are
uniformly non-directional. A prior study using the disap-
Reproductive summer butterflies fail to consistently show directional flightFigure 5
Reproductive summer butterflies fail to consistently
show directional flight. (A) Virtual flight paths of the indi-
vidual summer butterflies tested (n = 18, black lines), vehicle-
treated migrants (V-Migrants; n = 18, blue lines; 12 direc-
tional [from Figure 2A]/6 non-directional) and methoprene-
treated migrants (M-Migrants; n = 12, red lines; 10 direc-
tional [from Figure 2A]/2 non-directional) flown in flight sim-
ulator. The summer butterflies were housed under simulated
summer conditions in a light-dark cycle with lights on from
0430 to 1900 hours EST at 20°C for at least three days
before being tested outdoors in a flight simulator (flown
between 1230 and 1530 hours). Virtual flight paths were con-
structed by starting in the center of the square and plotting
each direction interval consecutively as one unit length [5].
(B) R-values for individual virtual flight paths shown in (A). R-
values differed among summer, vehicle-treated migrants and
methoprene-treated migrants (p = 0.005). Tukey's pairwise
comparisons revealed that summer butterflies had lower r-
values than vehicle- and methoprene-treated migrants. (C)
Flight orientation of summer butterflies. Of the 18 summer
butterflies flown in the flight simulator, only five showed sig-
nificant directional flight. The large circles represent the 360°
of possible direction (0° is north), with the small solid circles
on the perimeter representing mean orientation for an indi-
vidual flight.
A
B
Summer (18) V-Migrants (18)
M-Migrants (12)
0
o
90
o
180
o
270
o
Summer
C
5/18
Summer
0.0
0.2
0.4
0.6
0.8
1.0
r-value
18
18
12
V-Migrants
M-Migrants
S
WEW
W
EE
S
S
N
N
N
Clustering of orientation genesFigure 6
Clustering of orientation genes. Hierarchical clustering
of individual butterflies (top) based on expression profiles of
the 40 regulated genes expressed as heat maps (below). Ani-
mal ID code for clustering is as depicted in Table 1. Boot-
strap analysis provides confidence values at the nodes. The
heat maps show estimated gene expression levels (red upreg-
ulated; green downregulated). The genes have been ordered
according to k-means clustering.
S-M11
S-F21
S-F20
S-F19
S-M03
S-M10
S-M07
S-M04
S-F23
S-F22
M-M08
M-M13
M-M04
M-M03
F-M07
F-M29
F-F18
F-F13
F-M10
M-M15
F-F03
F-F21
M-F24
F-F14
M-F23
V-F29
V-F27
M-F30
M-F20
M-F27
V-M19
F-M32
F-M23
V-M10
V-M13
V-M14
V-M18
V-F04
V-F24
V-F22
-3.0 3.00
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pearance bearing of released monarchs showed that sum-
mer butterflies collected at a similar time of year as ours
(early August at latitude 38'9°N) were not significantly
oriented [10]. Only short flight paths can be assessed in
the disappearance bearing studies, but the results are con-
sistent with our longer flight recordings in a flight simula-
Table 1: Animal Characteristics
Reproductive Status
Category ID* Sex Male
(glands weight, g)
Female
(# of mature oocytes)
Source/Date Collected Housing Conditions
S-M03 0.0217
S-M04 0.0223
S-M07 Male 0.0218 Near 2 days LD cycle
S-M10 0.0313 Greenfield, Lights on: 4:38 AM
Summer (S) S-M11 0.0259 Massachusetts Lights off: 7:09 PM
(Lat 42°59'N) Temperature: 20°C
S-F19 70 (Long 72°60'W) Humidity: 70%
S-F20 88 7/30/07
S-F21 Female > 100
S-F22 > 100
S-F23 > 100
F-M07 0.0084 Near 2 days LD cycle
F-M10 0.0056 Port Lavaca, Lights on: 6:09 AM
F-M23 Male 0.0087 Texas Lights off: 4:54 PM
F-M29 0.0070 (Lat 28°36'N) Temperature: 20°C
Fall (F) F-M32 0.0065 (Long 96°37'W) Humidity: 70%
10/30/07
F-F03 0
F-F13 0
F-F14 Female 0
F-F18 0
F-F21 0
M-M03 0.0357 14 days LD cycle
M-M04 0.0557 Lights on: 6:00 AM
M-M08 Male 0.0341 Lights off: 5:00 PM
Fall methoprene (M) M-M13 0.0559 Temperature (light):
M-M15 0.0578 23°C
Temperature (dark):
M-F20 > 100 12°C
M-F27 > 100 Humidity: 70%
M-F30 Female > 100
M-F23 > 100
M-F24 > 100
V-M10 0.0179
V-M13 0.0198
V-M14 Male 0.0117
Fall vehicle (V) V-M18 0.0121
V-M19 0.0106
V-F04 0
V-F22 0
V-F24 Female 0
V-F27 0
V-F29 0
*Animal ID code: Group(S, F, M or V)-sex (M or F) animal number
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tor. This non-directionality is an important behavioral
trait that characterizes mid- to late-summer butterflies
from the other generations that occur over the course of
the year. These animals also provide the clearest behavio-
ral difference with fall migrants, as fall migrants consist-
ently exhibit directional flight, which is why we used them
in our gene expression studies.
Another distinguishing feature between summer butter-
flies and fall migrants is reproductive state. Summer but-
terflies are reproductively competent, while fall migrants
are JH deficient, which leads to reproductive diapause,
with decreased weight of reproductive organs and quies-
cent reproductive activity [2,3]. Reproductive diapause
usually persists over the course of the migratory journey
and for months at the overwintering sites [8]. However,
reproductive diapause can be readily manipulated in fall
migrants; diapause can be broken by exposing migrants to
elevated temperatures and increasing day length [10,17].
It is unclear whether the entire repertoire of migratory
behaviors (including reproductive diapause and direc-
tional flight behavior) are initiated by the same environ-
mental cues, which may include decreasing day length,
sun angle and temperature, and the age of the larval food
source [18].
Our studies show clearly that directional flight activity
and time-compensated sun compass orientation persist
independent of reproductive state; oriented flight activity
has also been shown in disappearance bearing studies of
reproductively active migrants [10], but persistent time-
compensation had not been shown previously. It is still
possible that JH deficiency is involved in the induction of
directed flight for sun compass orientation, but it is clear
from our results that persistent JH deficiency is not
required for maintenance. Moreover, increasing JH activ-
ity in migrants is unlikely to explain the reversed flight
direction of migrants (in the northerly direction) as they
leave the overwintering grounds in the spring, because the
methoprene-induced increase in reproductive activity in
fall migrants did not alter the normal south/southwesterly
flight direction manifested when studied in LD (Figure
2A). It will be interesting to determine in future experi-
ments whether JH antagonist treatment can convert sum-
mer butterflies to migrants.
Consistent with our findings of persistent migratory flight
in reproductive migrants, ecological observations have
suggested that a small number of migrating monarchs,
who have broken reproductive diapause because of pro-
longed exposure to high environmental temperatures dur-
ing their migration south, may give rise to a subsequent
'backup' generation of migrants, originating from the
southern range late in the fall (See [19]). Indeed, a peak in
monarch egg and larva abundance in Texas during Sep-
tember and early October supports this idea, because
adult monarchs are not seen in the southern United States
throughout most of the summer [20].
Table 2: Statistical Comparisons
A. Orientation Genes
Sex Comparison Test* # of significant # shared in each sex+ # shared by both sexes
Summer vs. Fall 1009
Male Summer vs. Fall methoprene 985 410
Summer vs. Fall vehicle 3832 40
Summer vs. Fall 995
Female Summer vs. Fall methoprene 960 212
Summer vs. Fall vehicle 1465
B. JH-response Genes
Sex Comparison Test* # of significant # shared in each sex† # shared by both sexes
Male Summer vs. Fall 1009 270
Fall methoprene vs. Fall vehicle 3122 23
Female Summer vs. Fall 995 115
Fall methoprene vs. Fall vehicle 1195
*All statistical tests were done using student's t test with the numbers of significant genes (p < 0.05) shown.
+ See Additional file 1 for specific ESTs and annotation.
† See Additional file 2 for specific ESTs and annotation.
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An exciting aspect of our work was the discovery of a suite
of 40 genes whose differential expression in brain distin-
guishes individual summer butterflies from fall migrants,
independent of reproductive status. Any single gene or
combination of genes within the 40 could be essential for
the initiation and/or maintenance of directional flight in
migrants. The genes that were not annotated may be espe-
cially interesting targets for further studies, as we have
shown their importance in predicting oriented flight.
Although the fold changes in gene expression between
non-oriented and oriented butterflies were small, they do
not rule out larger differences in expression of individual
genes in specific neural subpopulations that have been
diluted by whole brain analysis [21] – a possibility that
needs to be evaluated for each gene. We expect that with
further study the number of differentially regulated genes
will grow, as the sequencing and annotation of the entire
monarch genome progresses.
Conclusion
Our data are the first to provide a link between alterations
in gene expression profiles in brain and migratory state in
any animal which undergoes long-distance migration.
Moreover, our results also provide the first insights into
gene expression patterns in brain that may underlie time-
compensated sun compass orientation, a complex process
involving brain integration of information about time
and space.
Our gene expression profiles resemble those reported to
be involved in behavioral plasticity in honey bees in
which a small collection of genes, most of which did not
show a greater than 2-fold change by microarray analysis,
reliably predict behavioral state (nurses from foragers) in
individual bees [21]. Further evaluation of the 40 genes
we have identified in monarchs will likely provide novel
insights into their individual and/or collective importance
for migration and the brain changes necessary to initiate
and maintain oriented flight behavior.
Methods
Animal housing
Monarch butterflies were housed in the laboratory in glas-
sine envelopes in Percival incubators with controlled tem-
Annotation of orientation genesFigure 7
Annotation of orientation genes. The sequence ID, annotation, and Biological Function based on gene ontology (GO) of
the orientation genes are shown. They are listed in the same order they are represented on the vertical axis of Figure 6. NA
means the gene is either not annotated or annotated but without GO function assigned.
SEQ ID Annotation/homologue to Biological Function
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Page 12
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Page 12 of 14
(page number not for citation purposes)
perature, humidity (70%), and lighting. The butterflies
were fed 25% honey every other or every third day.
Methoprene/vehicle treatments
Animals were treated topically on their abdomens with
either 200 g of methoprene in 5 l acetone or acetone
alone on day 1 and day 3.
Evaluation of reproductive status
For males, the ejaculatory duct and tubular gland were
weighed. For females, the presence of mature oocytes was
recorded.
Flight analysis
Butterflies were tethered as previously described [5], and
flight behavior was monitored using a modified Mourit-
sen and Frost flight simulator as described [22]. Butterflies
were flown outdoors under sunny skies when the sun
could be seen from their position in the flight barrel. Data
were analyzed to determine the significance of orientation
and the mean direction using circular statistics [23].
Microarray sample preparation
Over a 2-hour period bracketing (± 1 hr) the middle of the
light period, individual monarchs were taken from their
envelopes, and the heads were removed with scissors. The
severed heads were immediately placed in 0.5× RNAlater
(Ambion), and each brain was dissected, with the pho-
toreceptor layer removed, and placed on dry ice; brains
were stored at -80°C. RNA from individual brains was iso-
lated using the Qiagen RNeasy Mini Kit with the optional
on-column DNase treatment according to the manufac-
turer's instruction. Approximately 100 ng RNA from each
brain sample was used to synthesize amplified cDNA
using the Ovation Amplification System V2 (Nugen).
Amplified cDNAs were purified using Qiagen PCR purifi-
cation kit. All 40 samples were successfully amplified with
yields between 10 and 15 g of cDNA. As required by
Nimblegen microarray service, 6 g of amplified cDNA
from each sample were converted into doublestrand
cDNA using Klenow (NEB) reaction with random hex-
amer priming (Promega). Doublestrand cDNAs were
purified again using Qiagen PCR purification kit before
submitting to Nimblegen for labeling and hybridization.
Microarray design and analysis
A 4-plex Nimblegen custom monarch expression array
was designed based on our previously published EST data
to include all unique ESTs and contigs. In addition, 18
previously cloned monarch genes were included on the
array. A total of 9417 unique sequences were incorporated
into the array design. Each sequence was represented by
seven non-overlapping oligos on the array. Nimblegen's
service department carried out array design, synthesis,
probe labeling and hybridization. They also performed
data pre-processing including array scan, data extraction
and normalization [24]. Normalized values for each gene,
which provide a measure of expression levels, were used
in all data analyses. All normalized values and processed
data are available at GEOdatabase http://
www.ncbi.nlm.nih.gov/geo/query/
acc.cgi?acc=GSE14041.
Data analysis
To determine if gene expression levels differed between
butterflies in different behavioral states, we used a Stu-
dent's t-test to compare normalized values for each gene
between treatment groups (Table 2). We compared differ-
Comparison of sex-specific microarray expression profiles of juvenile hormone acid methyltransferase (jhamt), allatotropin, and takeoutFigure 8
Comparison of sex-specific microarray expression
profiles of juvenile hormone acid methyltransferase
(jhamt), allatotropin, and takeout. The profiles were
compared across the four experimental groups. For each
sex, each of the three fall groups was compared with sum-
mer, using unpaired 2-tail t test (with unequal variance). ns,
not significant, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.
0
500
1000
1500
2000
0
20000
40000
60000
Male Female
0
10000
20000
30000
Summer
Fall
Fall methoprene
Fall vehicle
jhamt
allatotropin
takeout
**
*
***
***
***
ns
*
**
**
ns
ns
ns
ns
ns
ns
ns
ns
ns
signal intensities
Page 13
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Page 13 of 14
(page number not for citation purposes)
ences in gene expression levels at the conventional value
of p < 0.05. Although the use of an unadjusted significance
level may increase false positives due to multiple testing,
the number should be minimal in our case, because
results were drawn from comparisons of shared gene
across multiple groups and not a single comparison (see
Table 2). For those genes that showed a significant change
in expression level, we labeled them as either 'upregu-
lated' or 'downregulated' depending on the means for
each group. For example, if the mean normalized value
was higher in the summer group than the migrant group,
then the gene was considered upregulated in summer but-
terflies. Statistical comparisons and gene cluster analyses
were done using ArrayStar software. Animal cluster analy-
Cluster and annotation of JH-response genesFigure 9
Cluster and annotation of JH-response genes. (A) Hierarchical clustering of individual butterflies (top) based on expres-
sion profiles of the 23 regulated genes expressed as heat maps (below). Animal ID code for clustering is as depicted in Table 1.
Bootstrap analysis provides confidence values at the nodes. The heat maps show estimated gene expression levels (red upreg-
ulated; green downregulated). The genes have been ordered according to k-means clustering. (B) The sequence ID, annotation,
and Biological Function based on gene ontology (GO) are shown below. They are listed in the same order they are repre-
sented on the vertical axis of panel A. NA means the gene is either not annotated or annotated but without GO function
assigned.
SEQ ID Annotation/homologue to Biological function
BF01029A2C12.f1 Cytochrome P450-18a1 steroid biosynthetic process, monooxygenase activity
BF01051B1F12.f1 NA
BF01060A2C03.f1 Enhancer of split mgamma Notch signaling pathway, compound eye development, nervous system development
BF01034B1E11.f1 NA
BF01011B2D07.f1 similar to Tribolium CG11212-PA NA
BF01044B2A07.f1 defective proboscis extension response8 (dpr8) NA
BF01044A1F09.f1 CG5656-PA alkaline phosphatase activity, metabolic process
BF01021A2H08.f1 CG2165-PC (CG42314) calcium ion transport, cellular calcium ion homeostasis
BF01027B2C03.f1 similar to unknown Tribolium gene NA
BF14.478.C1.Contig543 CG4050-PB UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase activity
BF14.55.C1.Contig64 Similar to Bombyx hemolin Antibacterial immune response
BF01049A1G05.f1 rosy xanthine dehydrogenase activity, determination of adult life span
BF01027A2F05.f1 rudimentary
process
BF01031B1B02.f1 inhibitor of kappa light polypeptide gene enhancer Defense response, signal transduction
BF01028A1A09.f1 Gp150 compound eye development, transmembrane receptor protein tyrosine phosphatase signaling pathway
BF01050B1G12.f1 CG17508-PA NA
BF14.1224.C1.Contig1313 CG8086-PD NA
BF14.1952.C1.Contig2018 Bombyx TGF beta-inducible nuclear protein 1 Phagocytosis, engulfment
BF14.2386.C1.Contig2427 lethal (2) k09913 NA
BF01061A1G03.f1 Ribosomal protein L35A translation
BF14.158.C1.Contig173 polyA-binding protein positive regulation of translation, synaptic transmission
BF14.51.C2.Contig58 Annexin IX calcium-dependent phospholipid binding, actin binding
BF14.990.C1.Contig1076 CG6543-PB enoyl-CoA hydratase activity, fatty acid beta-oxidation
S-F21
S-F20
S-F19
M-M13
M-M04
M-M08
M-M03
S-M10
S-M04
S-M07
S-M03
S-F23
S-F22
M-M15
S-M11
M-F30
M-F27
M-F20
M-F24
M-F23
V-M10
F-M07
F-M29
F-M10
F-F13
F-F21
V-M19
V-F29
V-F27
V-M14
F-F14
F-M23
F-F18
V-F04
F-M32
V-F24
V-M18
F-F03
V-M13
V-F22
-3.0 3.00
A
B
Page 14
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ses were done using MultiExperiment Viewer http://
www.tm4.org/mev.html. Gene annotation was done
using the previously published ESTIMA monarch EST
database http://titan.biotec.uiuc.edu/cgi-bin/ESTWeb
site/estima_start?seqSet=butterfly.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
All authors contributed to experimental design, execution,
data analysis and writing the paper. All authors have read
and approved the final manuscript.
Additional material
Acknowledgements
We thank Lauren Foley and Danielle Metterville for technical assistance;
Carol Cullar and Fred Gagnon for supplying butterflies; and Adriana Briscoe
and Christine Merlin for comments and discussions. Supported in part by
NIH grant R01GM086794 and NSF grant IOB-0646060.
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ysis using oligonucleotide arrays produced by maskless
photolithography. Genome Res 2002, 12:1749-1755.
Additional file 1
Table S1
Sex-specific orientation genes.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1741-
7007-7-14-S1.xls]
Additional file 2
Table S2
Sex-specific juvenile hormone-regulated genes.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1741-
7007-7-14-S2.xls]
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    • "The most extensive study comparing differential expression patterns between phenotypes used microarray analyses of monarch butterflies Danaus plexippus to compare summer and migratory phenotypes. This study identified 40 differentially expressed genes between seasons using brain tissue [15]. Two genes of particular interest were the clock gene vrille and the locomotionrelevant TβH (tyramine beta hydroxylase) gene as their presumed functions directly link to migratory components (timing and movement respectively). "
    [Show abstract] [Hide abstract] ABSTRACT: Background: We still have limited knowledge about the underlying genetic mechanisms that enable migrating species of birds to navigate the globe. Here we make an attempt to get insight into the genetic architecture controlling this complex innate behaviour. We contrast the gene expression profiles of two closely related songbird subspecies with divergent migratory phenotypes. In addition to comparing differences in migratory strategy we include a temporal component and contrast patterns between breeding adults and autumn migrating juvenile birds of both subspecies. The two willow warbler subspecies, Phylloscopus trochilus trochilus and P. t. acredula, are remarkably similar both in phenotype and genotype and have a narrow contact zone in central Scandinavia. Here we used a microarray gene chip representing 23,136 expressed sequence tags (ESTs) from the zebra finch Taeniopygia guttata to identify mRNA level differences in willow warbler brain tissue in relation to subspecies and season. Results: Out of the 22,109 EST probe sets that remained after filtering poorly binding probes, we found 11,898 (51.8 %) probe sets that could be reliably and uniquely matched to a total of 6,758 orthologous zebra finch genes. The two subspecies showed very similar levels of gene expression with less than 0.1 % of the probe sets being significantly differentially expressed. In contrast, 3,045 (13.8 %) probe sets were found to be differently regulated between samples collected from breeding adults and autumn migrating juvenile birds. The genes found to be differentially expressed between seasons appeared to be enriched for functional roles in neuronal firing and neuronal synapse formation. Conclusions: Our results show that only few genes are differentially expressed between the subspecies. This suggests that the different migration strategies of the subspecies might be governed by few genes, or that the expression patterns of those genes are time-structured or tissue-specific in ways, which our approach fails to uncover. Our findings will be useful in the planning of new experiments designed to unravel the genes involved in the migratory program of birds.
    Preview · Article · Feb 2016
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    • "The ability to assess and respond to changes in one's environment allows many organisms to exist successfully in a range of social scenarios and physical environments. Research suggests ture [26], and many insects exhibit seasonal migratory behavior that is adaptive and hypothesized to be induced by a seasonal cue [30, 31] . However, the effect that abiotic developmental cues associated with season and climate change, such as temperature, have on adult behavioral plasticity remains ambiguous. "
    [Show abstract] [Hide abstract] ABSTRACT: Rearing environment can have an impact on adult behavior, but it is less clear how rearing environment influences adult behavior plasticity. Here we explore the effect of rearing temperature on adult mating behavior plasticity in the butterfly Bicyclus anynana, a species that has evolved two seasonal forms in response to seasonal changes in temperature. These seasonal forms differ in both morphology and behavior. Females are the choosy sex in cohorts reared at warm temperatures (WS butterflies), and males are the choosy sex in cohorts reared at cooler temperatures (DS butterflies). Rearing temperature also influences mating benefits and costs. In DS butterflies, mated females live longer than virgin females, and mated males live shorter than virgin males. No such benefits or costs to mating are present in WS butterflies. Given that choosiness and mating costs are rearing temperature dependent in B. anynana, we hypothesized that temperature may also impact male and female incentives to remate in the event that benefits and costs of second matings are similar to those of first matings. We first examined whether lifespan was affected by number of matings. We found that two matings did not significantly increase lifespan for either WS or DS butterflies relative to single matings. However, both sexes of WS but not DS butterflies experienced decreased longevity when mated to a non-virgin relative to a virgin. We next observed pairs of WS and DS butterflies and documented changes in mating behavior in response to changes in the mating status of their partner. WS but not DS butterflies changed their mating behavior in response to the mating status of their partner. These results suggest that rearing temperature influences adult mating behavior plasticity in B. anynana. This developmentally controlled behavioral plasticity may be adaptive, as lifespan depends on the partner's mating status in one seasonal form, but not in the other.
    Full-text · Article · Feb 2016 · PLoS ONE
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    • "Comparisons with other insects that display adult reproductive diapause such as D. montana and the mosquito Culex pipiens, where limited transcriptome analyses have been performed [149, 152], revealed some overlap, though restricted by the small number of transcripts assessed in these studies. However, general comparisons of transcript changes in components of signal pathways by KEGG or GO analyses suggest similarities between reproductive diapause in Drosophila and dormancy in other insects and C. elegans, including IIS and TOR signaling, stress responses and metabolism, as well as energy storage [62,153154155156. This comparison can be extended to mammals, where the hibernation phenotype involves altered metabolism and TOR signaling [157, 158]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: In models extensively used in studies of aging and extended lifespan, such as C. elegans and Drosophila, adult senescence is regulated by gene networks that are likely to be similar to ones that underlie lifespan extension during dormancy. These include the evolutionarily conserved insulin/IGF, TOR and germ line-signaling pathways. Dormancy, also known as dauer stage in the larval worm or adult diapause in the fly, is triggered by adverse environmental conditions, and results in drastically extended lifespan with negligible senescence. It is furthermore characterized by increased stress resistance and somatic maintenance, developmental arrest and reallocated energy resources. In the fly Drosophila melanogaster adult reproductive diapause is additionally manifested in arrested ovary development, improved immune defense and altered metabolism. However, the molecular mechanisms behind this adaptive lifespan extension are not well understood. Results: A genome wide analysis of transcript changes in diapausing D. melanogaster revealed a differential regulation of more than 4600 genes. Gene ontology (GO) and KEGG pathway analysis reveal that many of these genes are part of signaling pathways that regulate metabolism, stress responses, detoxification, immunity, protein synthesis and processes during aging. More specifically, gene readouts and detailed mapping of the pathways indicate downregulation of insulin-IGF (IIS), target of rapamycin (TOR) and MAP kinase signaling, whereas Toll-dependent immune signaling, Jun-N-terminal kinase (JNK) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways are upregulated during diapause. Furthermore, we detected transcriptional regulation of a large number of genes specifically associated with aging and longevity. Conclusions: We find that many affected genes and signal pathways are shared between dormancy, aging and lifespan extension, including IIS, TOR, JAK/STAT and JNK. A substantial fraction of the genes affected by diapause have also been found to alter their expression in response to starvation and cold exposure in D. melanogaster, and the pathways overlap those reported in GO analysis of other invertebrates in dormancy or even hibernating mammals. Our study, thus, shows that D. melanogaster is a genetically tractable model for dormancy in other organisms and effects of dormancy on aging and lifespan.
    Full-text · Article · Jan 2016 · BMC Genomics
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