Egr-1: A Candidate Transcription Factor Involved in Molecular Processes Underlying Time-Memory

Abstract

In honey bees, continuous foraging is accompanied by a sustained up-regulation of the immediate early gene Egr-1 (early growth response protein-1) and candidate downstream genes involved in learning and memory. Here, we present a series of feeder training experiments indicating that Egr-1 expression is highly correlated with the time and duration of training even in the absence of the food reward. Foragers that were trained to visit a feeder over the whole day and then collected on a day without food presentation showed Egr-1 up-regulation over the whole day with a peak expression around 14:00. When exposed to a time-restricted feeder presentation, either 2 h in the morning or 2 h in the evening, Egr-1 expression in the brain was up-regulated only during the hours of training. Foragers that visited a feeder in the morning as well as in the evening showed two peaks of Egr-1 expression. Finally, when we prevented time-trained foragers from leaving the colony using artificial rain, Egr-1 expression in the brains was still slightly but significantly up-regulated around the time of feeder training. In situ hybridization studies showed that active foraging and time-training induced Egr-1 up-regulation occurred in the same brain areas, preferentially the small Kenyon cells of the mushroom bodies and the antennal and optic lobes. Based on these findings we propose that foraging induced Egr-1 expression can get regulated by the circadian clock after time-training over several days and Egr-1 is a candidate transcription factor involved in molecular processes underlying time-memory.

Keywords: Egr-1; anticipation; honey bee foraging; small Kenyon cells; time-memory.

FIGURE 1

Egr-1 expression in the absence of food reward. (A) Ad libitum (yellow) fed bees show elevated levels of Egr-1 all through the day, with a peak at 14:00. (B,C) Morning trained (blue) and evening trained (red) bees showed higher expression only at the trained time and significantly lower levels at all other time-points. Data shown as relative expression changes compared to 06:00 in the form of box-plots with individual data-points delineated, n = 6. KW-test with Dunn’s (“bh” method) multiple comparison was done on each experiment, p-values are shown in Supplementary Tables S1–S3, respectively.

FIGURE 2

Egr-1 expression in individuals exposed to two feeders. (A,B) Bees were trained for 2 h each in the morning and evening. (A) Those bees that visited only the morning feeder (blue) or the evening feeder (red) showed comparatively higher expression in the morning or evening, respectively, however, not significant. (B) Bees that visited both the feeders (green) showed comparatively higher expression at both the time-points. The time-point in between the 2 training times (13:00) showed a down-regulation trend, however, none of the time-points were significantly different. n = 3 per time-point since enough bees could not be caught. (C,D) Experiment was repeated with 1-h training period each to increase separation between the training times. (C) Bees that visited the morning (blue) feeder showed significantly higher expression at 09:00 compared to the “morning only” bees at 18:00 as well as “evening only” bees at 09:00. Similarly, “evening only” (red) bees showed significantly higher expression at 18:00 compared to “evening only” bees at 09:00 as well as “morning only” bees at 18:00. (D) Bees that visited both feeders (green) showed significantly higher expression at both the trained time-points compared to all other time-points. 13:00 showed significantly lower levels of Egr-1; n = 5 per time-point. Data shown as relative expression changes compared to the lowest value per group in the form of box-plots with individual data-points delineated. KW-test with Dunn’s (“bh” method) multiple comparison was done for single feeder visiting bees (“only morning” + “only evening”) and “both feeder” visiting bees, p-values are shown in Supplementary Tables S4, S5, respectively.

FIGURE 3

Cry-2 expression comparison between morning time-point (09:00) and evening time-point (18:00) in the same bees as shown in Figures 2C,D. “Morning only” (M09:00, M18:00; blue) bees showed no significant differences in Cry-2 expression whereas “evening only” (E09:00, E18:00; red) bees showed significantly higher expression in the morning compared to evening. “Both feeder” (B09:00, B18:00; green) bees showed significantly higher expression in the morning compared to evening, similar to “evening only” bees. The lower expression value in all 3 groups were not significantly different from each other. Data shown as relative expression changes compared to the lowest value in the form of box-plots with individual data-points delineated. KW-test with Dunn’s (“bh” method) multiple comparison was done on the entire data-set, p-values are shown in Supplementary Tables S4, S5, respectively.

FIGURE 4

Egr-1 expression when the bees were prevented from flying out using “artificial rain” setup. (A) Bees that were trained from 08:00 to 10:00, already showed significant up-regulation of Egr-1 by 07:00 and remained up-regulated till the end of training time with a peak at 08:30. The mRNA levels started to decline at 09:00 and was reduced significantly by 14:00 and remained low for the rest of the day. (B) Bees that were trained from 12:00 to 14:00, showed significant up-regulation by 11:00 with a peak at 12:30. The expression declined thereafter and was significantly low by 18:00. (C) Bees that were trained from 16:00 to 18:00 showed an up-regulation trend already by 14:00, although not significant. mRNA levels were significantly increased by 15:00 with a peak at 15:30 which started to decline thereafter, differing from the trends seen in morning and afternoon trained bees. Data shown as relative expression changes compared to 06:00 in the form of box-plots with individual data-points delineated, n = 5. KW-test with Dunn’s (“bh” method) multiple comparison was done on each experiment, p-values are shown in Tables 1–3, respectively.

FIGURE 5

In situ hybridization of Egr-1 on brains of foragers. (A–C) Trained bees that were collected 6 h before the trained time from the hive showed very low expression of Egr-1, with only a few cells in the antennal lobes stained. (D–F) “Active foragers”, collected from the feeder at 60 mins past the start of foraging time, showed strong Egr-1 expression in the mushroom bodies as well as other brains parts like antennal lobes and optic lobes. (G–I) Time-trained “non-active foragers,” collected from the hive with the “artificial rain”setup at 60 min past the trained time, showed specific expression only in the small Kenyon cells. MB, mushroom bodies; OL, optic lobes; AL, antennal lobes.

FIGURE 6

Focus on the mushroom bodies of the “active foragers” and the “non-active foragers”. (A) Almost all the small Kenyon cells (white stars) are stained for Egr-1 whereas only some of the large Kenyon cells (yellow stars) that are closer to the calyces show staining in the “active foragers”. (B) “Non-active foragers” showed very specific staining of the small Kenyon cells (white stars) and a few cells close to the “lip” region of the calyces only. Li, Lip; Co, Collar; BR, Basal Ring.

FIGURE 7

Proposed model for Egr-1 function in honey bee foraging: (a) Foraging/food reward leads to an up-regulation of Egr-1 in the sKCs, which in turn regulates the expression of downstream targets that are involved in learning and memory. (b) Time-Restricted foraging at one food source leads to entrainment of the molecular clock. This effect might be restricted to a specific population of clock cells. For example, different populations of clock cells might be involved in foraging entrainment and time-compensated sun compass navigation. (c) Time-training over several days leads to an anticipatory up-regulation of Egr-1 that is controlled by the circadian clock. Thus, Egr-1 expression in the Kenyon cells of the mushroom bodies might be regulated via two signaling mechanisms, one from food reward related pathways and one from the circadian clock.