香京julia种子在线播放

Apis mellifera colonies were purchased from a local beekeeper and kept in an outdoor flight cage (12 m × 4 m × 4 m) on the campus of the National Centre for Biological Sciences, Bangalore, India. The colonies consisted of 4-frames within a standard commercial wooden hive box, each frame containing approximately 2500–3000 bees. The day-night length as well as the temperature conditions inside the flight cage were similar to the natural conditions in Bangalore with an approximate 12-h light-dark cycle all throughout the year. During the experimental period from November to February the time of sunrise changed from 06:15 to 06:45 and that of sunset changed from 17:50 to 18:30. The flight cage was devoid of any flora and the only food source available was the feeder provided by the experimenter.

The training regime consisted of presentation of a colored plate with sugar-water solution (1 M) without odor, unless mentioned otherwise, for 10 days. We trained the bees for 10 days (a) since we need enough bees for the experiment (practical restriction) which can be achieved by more days of training since more bees are recruited (b) to increase temporal accuracy for the trained time, since Moore and Doherty (2009) show that with increase in the number of days, temporal accuracy increases. The duration and time of presentation differed according to the experiment. After the training time, the sugar-water was washed off and the plate kept back at the feeder location, to avoid association of the feeder plate with the food reward. Foragers trained to the feeder were marked at the feeder on the 7th day and the collections, as documented for each experiment below, were done on the 11th day.

Honey bee foragers were allowed to forage ad libitum (all day) or trained to forage in the morning (08:00 to 10:00) or in the evening (16:00 to 18:00). On the 11th day, the food reward was not added to the feeder plate. Marked foragers were collected at six different time-points at 4 h intervals: 06:00, 10:00, 14:00, 18:00, 22:00, and 02:00. Foragers were collected from the hive, which involved opening the hive and temporarily removing the comb frames from the hive. Each experiment was performed on a separate colony.

The bees were immediately flash frozen in liquid nitrogen and stored at -80°C until further processing for RNA isolation.

As a pilot experiment, we trained foragers to 2 different feeders within a day that were separated in space and time in December 2017. The colonies were placed in a flight cage that was longer, but had the same height and width (24 m × 4 m × 4 m). The food sources were placed at the opposite ends, almost 24 m apart from each other. One feeder was blue colored with 1 M sucrose scented with 20 ul Phenylacetaldehyde per liter of sucrose and the other feeder was green colored with 1 M sucrose scented with 20 ul Linalool per liter of sucrose. The blue feeder was opened in the morning, from 08:00 to 10:00 whereas the green feeder was opened in the evening from 16:00 to 18:00. The feeder plates were left in their position after the training time. On the 5th day of training, foragers coming to each feeder were individually marked with numbered tags. From the 7th day onward, observations were made, and the bees were classified into groups that continuously visited either the morning or evening or both the feeders.

On the 11th day, bees that visited both the feeders were collected at 06:00, 09:00, 13:00, 17:00, 22:00 and 02:00. The time-points were chosen such that the bees were caught at 60 min after the start of the feeder training for both the feeders (09:00 and 17:00) and at an intermediate time-point (13:00). The other time-points were chosen according to the single feeder experiment, “collection without food reward” mentioned above.

Bees that visited either the morning or the evening feeder, were collected only at the 2 trained time-points (09:00 and 17:00). Since many of the marked bees were lost by the 11th day, only three bees per time-point were successfully analyzed.

This experiment was repeated in February 2018 with the training duration reduced to 1 h, i.e., 08:00 to 09:00 in the morning and 17:00 to 18:00 in the evening, in order to increase the separation between the 2 feeder times. From the 8th day onward, foragers coming to each feeder were marked with paint marks on the thorax in the morning and the abdomen in the evening. Each day was marked with a different color. On the 11th day, collections were done as per the above-mentioned time-points. Marked foragers were collected from the hive, which involved opening the hive and temporarily removing the comb frames from the hive. Feeder visits of the bees shown in Supplementary Figure S2.

Honey bee foragers were trained to forage either in the morning (08:00 to 10:00), afternoon (12:00 to 14:00), or evening (16:00 to 18:00) during the months of December 2016, February 2017 and November 2017, respectively. On the 11th day, the “artificial rain” setup was started at 06:00. The “artificial rain” setup consisted of a box made of plexiglass which would hold water, called the water basin. The water flowed continuously through pores on the underside of the water basin. The entire setup was positioned such that the hive entrance was completely blocked by the falling water and hence prevented the bees from flying out. Any marked bee that crawled out of the hive and escaped, was caught and chilled on ice until the completion of the collections. Collections were made from 5 equidistant holes on the inner lid of the hive that was covered with a black chart paper. The holes had a flap cover which was opened at the time of collection and a 50 ml tube was placed over the hole. Bees that crawled up the tubes, being attracted to light, were chilled on ice and then the marked foragers were separated out and flash frozen. This collection method was adopted to prevent bees from flying out during collections. Since we were interested in the expression pattern in and around the trained time, we collected bees at half hour intervals starting from an hour before the trained time up until an hour after the trained time. The rest of the time-points corresponded to previous collection time-points.

Collection time-points for morning trained bees: 06:00, 07:00, 07:30, 08:00, 08:30, 09:00, 10:00, 14:00, 18:00, 22:00. Collection time-points for noon trained bees: 06:00, 10:00, 11:00, 11:30, 12:00, 12:30, 13:00, 14:00, 18:00, 22:00. Collection time-points for evening trained bees: 06:00, 10:00, 14:00, 15:00, 15:30, 16:00, 16:30, 17:00, 18:00, 22:00.

Frozen brains were dissected on a dry ice platform in a glass cavity block in 100% ethanol. Brains were homogenized in TRIzol (Invitrogen, Life Technologies, Rockville, MD, United States) using a motorized homogenizer and RNA was extracted using the standard Trizol-chloroform method. Glycogen (20 mg/ml, Thermo Scientific, Life Technologies, Rockville, MD, United States) was added for increased recovery of RNA. cDNA was prepared using the SuperScript^TM III First-Strand Synthesis System (Invitrogen, Life Technologies, Rockville, MD, United States).

Primers for Egr-1 and RP49 qPCR were the same that were used in Singh et al. (2017). Egr-1 primers recognise a region in exon 3, hence amplify all 3 isoforms of Egr reported by Sommerlandt et al. (2016). Primers for Cry-2 were Forward: 5′-AGGTCTCACATACTCTTTACA-3′; Reverse: 5′-ACTGTTGGTACTGGTGGT-3′. The qPCR was performed following the same protocol as in Singh et al. using Kapa SybrGreen (KapaBiosystems, Wilmington, Massachusetts, United States). The standard curve method was followed and RP49 was used as the internal control.

To generate riboprobes for Egr-1, primers (Forward: 5′-AAAGGGAGAGAGAGGATGAAG-3′; Reverse: 5′-TAATGCGGTGGTGTGAGTTC-3′) were generated to amplify a 1096 bp fragment of the gene in exon 3. RNA was isolated and converted to cDNA following the procedure as described above and the cDNA was used as a template to amplify the fragment. The fragment was then purified using PCR Purification kit (Qiagen, Hilden, Germany) followed by cloning of the fragment into the pCR^TMII-TOPO^® vector using the TOPO TA Cloning Kit (Invitrogen, Life Technologies, Rockville, MD, United States) following the manufacturer’s protocol. The cloning mix was transformed into E. coli (DH5-alpha) and screened using the blue-white screening regime. The plasmids were then isolated and sequenced for confirming the presence and orientation of insert.

Time-trained “active foragers” were collected from the feeder at 60 min after the onset of foraging. Time-trained “non-active foragers” were caught at 60 min after onset of the training time from the hive using the “artificial rain” setup. Time-trained control bees were caught at 6 h before the trained time from the hive. The bee brains were freshly dissected on DEPC water and immediately embedded into the Jung Tissue Freezing Medium (Leica Microsystems, Nussloch, Germany). The embedded brains were then sectioned using a HYRAX C-25 cryostat into 12 μm thin sections and collected on Superfrost Plus Microscope slides (Fisherbrand, Hampton, NH, United States). The slides were allowed to dry at room temperature for about 10 min and kept on dry ice until further processing.

RNA probes were synthesized using SP6 Polymerase or T7 Polymerase using DIG RNA labeling mix (Roche, Indianapolis, IN, United States) incubated for 2 h at 37°C. The probes were then purified using the Qiagen Micro kit (Qiagen, Hilden, Germany) and stored at -80°C.

The slides were fixed in 4% PFA overnight at 4°C. The slides were washed for 20 min in 0.1 M phosphate buffer (PB), followed by treatment with 10 mg/ml Proteinase K solution for 15 min at room temperature (RT), re-fixation in 4% PFA for 15 min at 4°C, followed by treatment with 0.2 M HCl for 10 min and 0.25% acetic anhydride in TEA for 10 min. Each step was followed by a 5 min wash with 0.1 M PB. The slides were dehydrated through an ethanol gradient of 70% → 95% → 100% and air-dried for 1 h. The slides were pre-hybridized in the hybridization buffer (50% formamide, 10 mM Tris–HCl pH 7.6, 200 ug/ml tRNA, 1X Denhardt solution, 10% Dextran sulfate, 600 mM NaCl, 0.25% SDS, 1 mM EDTA) without riboprobes for 1 h at 60°C. The riboprobes were added to the hybridization buffer followed by denaturation at 85°C for 5 min. The denatured probes were added to the slides and allowed to hybridize overnight at 60°C in a mineral oil bath.

The slides were then washed through a series of SSC buffer, starting with 5X SSC (rinse), 1:1 solution of formamide and 2X SSC for 30 min at 60°C, followed by 2X SSC and 0.2X SSC for 20 min each at 60°C and finally, 3 washes with TNT (0.1 M Tris, 0.15 M NaCl, 0.05% Tween) at RT.

For detection, the slides were first blocked with 5% BSA for 30 min at RT, followed by incubation in Anti-DIG POD (Roche, Indianapolis, IN, United States) overnight at 4°C. After incubation, slides were washed in TNT and incubated in Tyramide-Cy5 (Perkin Elmer, MA, United States) for 15 min followed by washing and mounting with Vectashield with DAPI (Vector Laboratories, CA, United States). The fluorescent images were captured using Olympus FV1000 at a magnification of 10 × with 1 um thick optical sections. Post hoc adjustments of brightness and intensity were made using ImageJ analysis software (NIH, United States).

All statistical analyses were performed using R [R 3.4.1 GUI 1.70 El Capitan build (7375)] (R Core Team, 2017). Since the data-points were not normally distributed, Kruskal Wallis (KW) tests were done. When the KW-test was significant, post hoc analyses for comparison amongst the groups was done using the dunn.test package in R (Dinno, 2017) with p-values adjusted for multiple comparisons using the Benjamini-Hochberg (“bh”) method (Benjamini and Hochberg, 1995). The alpha was set at 0.05. All data are represented as box-plots with individual data-points indicated.

Honey bee foragers were allowed to forage ad libitum or were trained either to a morning feeder (08:00 to 10:00) or to an evening feeder (16:00 to 18:00) for 10 days. On the 11th day, the marked foragers were collected from the hive in the absence of food reward. Honey bees that foraged at the ad libitum feeder showed elevated expression of Egr-1 throughout the day with highest expression at 14:00 (Figure 1A, p-values in Supplementary Table S1). Foragers that were trained to a morning or an evening feeder showed significant up-regulation in the mRNA levels of Egr-1 at the time of feeder training, i.e., 10:00 in the morning trained and 18:00 in the evening trained foragers compared to most of the other time-points (Figures 1B,C, p-values in Supplementary Tables S2, S3). The time point directly following the training time in the morning experiment and the one preceding the training time in the evening experiment showed p-values that were slightly above the cut off (morning 14:00: p = 0.06; evening 14:00 p = 0.07). Restricted foraging for a short time of the day led to a restricted Egr-1 expression occurring only around the time of training.

Next, we tested the dynamics of Egr-1 expression in individual bees trained to 2 different feeders separated in space and time. Both feeders differed in color and odor and one was opened in the morning while the other was opened in the evening. Presenting a colony with 2 feeders at different times of the day resulted in 3 foraging groups: (a) bees that visited only the morning feeder (“only morning”), (b) bees that visited only the evening feeder (“only evening”), and (c) bees that visited both the feeders (“both feeder”).

Egr-1 brain expression levels of the bees that visited only one feeder showed a peak at the time they had been trained to visit the feeder similar to our previous experiments (Figure 2A, KW test: ns; Figure 2C, p-values in Supplementary Table S4). In contrast, Egr-1 expression levels of the bees that visited both feeders showed two peaks, one at each training time (Figure 2B, KW test: ns; Figure 2D, p-values in Supplementary Table S5). When the bees were trained to visit the feeders for only 1 h starting at 08:00 and 17:00, expression of Egr-1 was significantly down-regulated at the intermediary time-point of 13:00 (Figure 2D). In the 2 h training experiment, the Egr-1 expression was not down regulated at 13:00 (Figure 2B). A greater temporal separation of the training period led to a more distinct regulation of Egr-1 expression.

Naeger et al. (2011) showed that morning and evening trained foragers differ in the expression pattern of Cry2 and Per indicating that the foragers likely developed different circadian rhythms according to their foraging activity. Therefore, we got interested in the question how foragers visiting one or two feeders over the day differ in Cry2 expression. In the “morning only” bees, Cry-2 expression levels at 09:00 and 18:00 were very similar, whereas in the “evening only” bees, Cry-2 expression levels were significantly higher at 09:00 compared to 18:00 (Figure 3, p-values in Supplementary Table S6). These results are consistent with those of Naeger et al. (2011) who showed a similar expression pattern for Cry-2, when they trained bees from 09:00 to 10:15 or from 17:00 to 18:15 and looked at transcripts levels at the 2 trained time-points for both the groups. The bees that visited “both feeders” in our experiments showed a Cry2 expression pattern similar to the “evening only” foragers, with significantly higher expression at 09:00 compared to 18:00.

Different to the earlier experiments, in which the training time was forced onto the bees, bees in our experiments could choose when to forage and this decision then influenced their circadian clock.

To test whether Egr-1 expression in time-trained foragers is regulated by the circadian clock, we tested Egr-1 expression in time-trained foragers that were prevented from flying out. If Egr-1 is under the influence of the circadian clock, it would be up-regulated at the trained time even in the absence of flight activity and other environmental cues. To reduce any stress responses that might occur in bees mechanically restricted from leaving the colony, we used an “artificial rain” setup (Supplementary Figure S1A) (Riessberger and Crailsheim, 1997). As in the case of natural rain, the foragers would not fly out. The feeder-trained foragers were collected from the hive.

Morning trained foragers prevented from flying out showed a slight but significant up-regulation of Egr-1 about an hour before the trained time, i.e., at 07:00 and the up-regulation was maintained till the end of training time with a peak at 08:30. The expression levels dropped after the trained time, and at 18:00 the expression was significantly lower compared to the highest level of Egr-1 expression i.e., 08:30 and hence had dropped to levels equivalent to 06:00 (Figure 4A, p-values in Table 1).

In the afternoon trained foragers, Egr-1 showed an expression pattern similar to morning trained foragers with significant elevation at 11:00 compared to the 10:00 and 06:00. The elevated expression was maintained till the end of training time with a peak at 12:30 and then dropped significantly by 18:00 (Figure 4B, p-values in Table 2).

In the evening trained foragers, the Egr-1 expression was very low in the morning, with no difference in levels at 06:00, 10:00 and 22:00. An up-regulation trend was observed at 14:00, however, it was not statistically significant. A statistically significant up-regulation was observed at 15:00_, and the up-regulation was maintained till the end of training time with a peak at 15:30. The expression levels dropped down to minimum values at 22:00 (Figure 4C, p-values in Table 3; Supplementary Figure S1B, p-values in Supplementary Table S7).

Together, our artificial rain experiments clearly showed that Egr-1 expression is up-regulated in time-trained foragers without any foraging or flight activity. This molecular response resembles anticipatory behavior of time-trained honey bee foragers (Moore et al., 1989).

To identify the brain regions that could be involved in foraging and time-training related Egr-1 up-regulation, we performed brain in situ hybridization for Egr-1. Specifically, we compared the Egr-1 expression pattern of brains from actively foraging honey bees caught 60 min after the onset of foraging (“active foragers”) (see Singh et al., 2017) and time-trained but not flying foragers caught 60 min after onset of the training time (“non-active foragers”). As a control we used foragers caught from the hive, 6 h before the training time.

The control bees showed very low staining with only a few cells in the antennal lobes stained (Figures 5A–C). In “active foragers”, predominant expression of Egr-1 was seen in the small Kenyon cells (sKCs) compared to large Kenyon cells (lKCs), where only few cells showed staining (Figures 5D–F, 6A). “Non-active foragers” also showed Egr-1 expression in the sKCs. The expression was lower compared to “active foragers” and more specifically expressed in the sKCs. Very few lKCs were stained in the “non-active foragers” suggesting anticipatory up-regulation of Egr-1 specifically in the sKCs (Figures 5G–I, 6B). We limited our analysis to the mushroom bodies, because they allow a clear identification and comparison of neuron populations between different individuals. Our stainings suggest that there might be additional neuron populations in other brain areas involved in these processes.