Increased juvenile hormone activity in migrants does not disrupt directed flight or time-compensated orientation
Because several aspects of migratory behavior are a consequence of continued JH deficiency, for example, reproductive quiescence and increased longevity [2
], we examined whether the oriented flight behavior characteristic of fall migrants also depends on persistent JH insufficiency. This was evaluated by increasing JH activity with the potent JH analog methoprene [12
] and then monitoring the effect on reproductive state and time-compensated flight orientation. Preliminary studies showed that the topical treatment of migrants with 200 μg of methoprene on day 1 and day 3 consistently led to summer-like reproductive development in both sexes by day 14, while vehicle (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 lighting conditions or a 6 hr-delayed LD cycle. These two lighting 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, butterflies housed in either LD or the 6-hr delayed LD cycle were tethered, and over the next 5 days individual flight direction 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 ); these directional migrants comprised the four groups that were evaluated for the time-compensated orientation analysis (Figure ).
Figure 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 simulator were analyzed using a Rayleigh test. Z, which is the critical value for the (more ...)
Figure 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 (more ...)
Regardless of treatment (methoprene or vehicle), group analyses showed that the directional fall migrants manifested time-compensated flight orientation (Figure ). Both treatment groups housed under prevailing LD conditions were oriented significantly in the south/southwesterly direction (Figure ); vehicle-treated migrants had an orientation vector (α) of 202.6° (n
= 12, r
= 0.714, p
= 0.001) (Figure , upper left, small blue circles), similar to what we have reported before for untreated migrants [4
], and methoprene-treated migrants had an α of 173° (n
= 10, r
= 0.713, p
= 0.004) (Figure , 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 α was 189.2° (n
= 22, r
= 0.69, p
< 0.00001) (Figure , lower, merge).
Both treatment groups housed under the 6 hr-delayed LD cycle were oriented significantly in the west/northwesterly direction (Figure ); vehicle-treated migrants had an α of 276.9° (n = 11, r = 0.58, p = 0.021) (Figure , small blue circles), and methoprene-treated migrants had an α of 291.6° (n = 15, r = 0.566, p = 0.006) (Figure , 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 α was 285.3° (n = 26, r = 0.567, p = 0.0001) (Figure , merge).
The direction and magnitude of the group orientation difference 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 and ). There were no significant sex differences in either the degree of individual directionality or group orientation of the migrants tested (Figure ).
Figure 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 (more ...)
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 ). Furthermore, all methoprene-treated females and 50% of the methoprene-treated males examined exhibited reproductive 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 treatments; this behavior was rarely observed in the vehicle-treated animals exposed to the same conditions (Figure ).
Figure 4 Methoprene increases reproductive organ development and mating behavior in fall migrants. (A) Reproductive development is increased in fall migrants following methoprene treatment. Numbers represent the animals examined. Postmortem reproductive development (more ...)
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 morphological 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
], until now this has not been evaluated in a flight simulator in which both individual directionality and group orientation can be assessed (see below). We tested these parameters in wild-caught summer butterflies captured in western Massachusetts (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.
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 individuals was apparent on inspection of the constructed virtual 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 ). We also compared the r-values for individual virtual flight paths among the three groups (Figure ), because r-values 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 ). 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).
Figure 5 Reproductive summer butterflies fail to consistently show directional flight. (A) Virtual flight paths of the individual summer butterflies tested (n = 18, black lines), vehicle-treated migrants (V-Migrants; n = 18, blue lines; 12 directional [from Figure (more ...)
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 directional were not significantly oriented, as previously suggested [9
Gene expression profiles in brain correlate with oriented flight behavior in fall migrants
Our behavioral data in migrants suggest that the regulation of directed flight behavior in individuals and group orientation are separable from reproductive state (Figures and ). 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 pathway. We therefore performed microarray analysis to determine 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 maintain oriented flight activity.
We tried to ensure that the animals used for the microarray analyses were handled in a way to minimize the influence of non-migratory factors on gene expression and to mimic the conditions used for our behavioral experiments (Table ). First, all animals were placed in glassine envelopes to minimize the influence of activity on the array analysis (number of days each group was in the envelopes is depicted in Table ). Second, all the animals were housed in controlled environmental conditions simulating those in the outside environment at the time of year of collection, as outlined in Table ; all butterflies were collected in the wild and the locales and times of year of collection were recorded (Table ). Third, the environmental conditions within the compartments in which the butterflies were housed (lighting cycle, temperature and humidity) were similar to those used to generate the behavioral data for the different groups shown in Figures to . Fourth, to minimize time-of-day effects on gene expression, all animals were killed within a 2-hr period encompassing 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 treatment, and 10 migrants following vehicle (acetone) treatment. We checked the reproductive status of all animals to ensure they had the expected reproductive state (Table ), which was similar to that found in our flight studies (Figure ); summer monarchs and methoprene-treated migrants had activated reproductive systems, while untreated and vehicle-treated migrants did not (Table ). 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 published 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, methoprene-treated, and vehicle-treated) for males and for females, and looked for gene regulation patterns common among the three comparisons for each sex (Table ; 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 and ).
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 ), we focused on the common differentially regulated genes that were shared between males and females (Table , right column). Accordingly, we identified 40 cDNAs that were differentially regulated between summer butterflies and fall migrants, irrespective of sex. Furthermore, hierarchical clustering analysis using the individual animal expression data for only these 40 cDNAs showed that all 10 summer animals were clustered together correctly, whereas the three fall groups formed their own separate cluster; bootstrap values supported this trend (Figure ). The expression of 14 genes was increased in migrants, while the expression of 26 genes was increased in summer butterflies (Figure , listed from top to bottom of heat map, respectively). The magnitude of the differences was 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 important to note, however, that some of the differences in gene expression could reflect non-migratory aspects of monarch 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.
Figure 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). Animal ID code for clustering is as depicted in Table 1. Bootstrap analysis (more ...)
Of the 40 cDNAs, only 25 had matches with other databases, with 24 being annotated with function (Figure ). The annotated brain cDNAs that were upregulated in migrants included those involved in cytoskeletal organization (Actin related protein 5), ATP dependent proteolysis (CG5045), immune responses (Eukaryotic translation initiation factor 3 subunit and CG6359) and the initiation of translation (Eukaryotic translation initiation factor 3 subunit). The annotated cDNAs that were upregulated in summer 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).
Figure 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 (more ...)
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 component (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 circadian 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 (Cytochrome 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 oriented 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 information 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 performed sex-specific statistical analyses, and compared the summer and the fall groups, and the methoprene-treated and vehicle-treated migrants (Table ). We then screened for shared genes between the two groups for each sex.
Of the sex-specific groups of differentially regulated genes (Table , # shared in each sex; for complete list of JH-regulated, 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
, which encodes a neuropeptide that can increase JH synthesis [15
]; and takeout
, which encodes a potential JH-binding protein [16
]. The mRNA levels for both jhamt
were upregulated 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 ). Combining the sexes and reanalyzing the microarray differences between summer butterflies and untreated migrants showed significant up-regulation of both jhamt
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
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 marginally upregulated in summer butterflies by microarray analysis (Figure ), 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.
Figure 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 (more ...)
We next examined cDNAs that were differently regulated in both males and females (Table , # shared by both sexes), to determine whether we could identify JH-regulated 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 ). The other two groups, the untreated and vehicle-treated migrants, formed their own cluster (Figure ). 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 , 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 correctly the reproductive status of all individual animals tested, using the brain expression pattern of these 23 genes that are common between the sexes.
Figure 9 Cluster and annotation of JH-response genes. (A) Hierarchical clustering of individual butterflies (top) based on expression profiles of the 23 regulated genes expressed as heat maps (below). Animal ID code for clustering is as depicted in Table 1. Bootstrap (more ...)
Of the 23 JH-related cDNAs, 21 had matches with other databases, with 15 annotated with biological function (Figure ). Predictably, they included genes involved in longevity (rosy), fatty acid metabolism (CG6543-PB), and, interestingly, immune responses (hemolin, TGF beta-inducible 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).
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 annotation based on available genomic and EST databases, which could mean that they contain incomplete sequence information for orthologous matches with other databases or that the non-annotated cDNAs represent unknown genes whose functions are unique to the migratory 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 generations of reproductively active spring and summer butterflies, 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 disappearance bearing of released monarchs showed that summer 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 consistent with our longer flight recordings in a flight simulator. 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 behavioral difference with fall migrants, as fall migrants consistently exhibit directional flight, which is why we used them in our gene expression studies.
Another distinguishing feature between summer butterflies and fall migrants is reproductive state. Summer butterflies are reproductively competent, while fall migrants are JH deficient, which leads to reproductive diapause, with decreased weight of reproductive organs and quiescent reproductive activity [2
]. 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
]. It is unclear whether the entire repertoire of migratory behaviors (including reproductive diapause and directional flight behavior) are initiated by the same environmental 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 activity 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 ). It will be interesting to determine in future experiments whether JH antagonist treatment can convert summer 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 prolonged exposure to high environmental temperatures during 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 September and early October supports this idea, because adult monarchs are not seen in the southern United States throughout most of the summer [20
An exciting aspect of our work was the discovery of a suite of 40 genes whose differential expression in brain distinguishes 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 especially 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.