Introduction

Social behavior of honey bee foraging has been an attractive field of research. Further exploration of the dynamics of honey bee foraging at the molecular and cellular level could help in uncovering the complex mechanisms of social behavior. Honey bee foraging comprises several behavioral components including learning, memory, social interaction and communication. In 1973 the Austrian ethologist Karl Ritter von Frisch (Austrian ethologist) was honored with Nobel Prize in Physiology or Medicine for his investigations of sensory perceptions in honey bees [1]. He translated the meaning of the bee waggle dance into a particular movement of the honey bee that looks like the form of figure eight, and revealed that foraging bees of the same colony share information with the help of this dance [2–4]. Bee foraging has gained enormous attention, since it offers the opportunity to elucidate the complexity of behavioral mechanisms involved in accomplishing the foraging task. Thanks to the efforts of many researchers, today we have a substantial amount of knowledge on this subject. However, understanding of the context of molecular and cellular mechanisms underlying foraging tasks is still poor.

During foraging, bees fly back and forth several times during the day between the hive and the food location, collecting nectar/pollen and bringing it to the hive for their colony. Foraging involves highly systematic and dynamic behavioral capacities that include long distance navigation using the sun as compass, evaluation of food quality, learning and memorizing flower cues, sense of colour, and social communication/interaction for coordination in collecting nectar, pollen, and water [2,5,6]. For decades the outdoor experiments have been routinely performed by feeding bees on pollen or sucrose solution placed in a specific place in order to mimic the natural foraging environment in close proximity. Using this experimental setup, we attempted to identify foraging regulatory genes in the European honey bee species Apis mellifera while the bees were performing their daily routine foraging. For this, the immediate early genes (IEGs) were the ultimate targets to examine, as they are well known as neural markers.

IEGs are rapidly induced by a large number of stimuli, and alterations of their expression are considered as part of the general neuronal response to natural stimuli as interpreted by normal synaptic activity [7]. The products of IEGs can activate downstream targets that typically function as part of a network of constitutively expressed proteins [7]. IEGs are also known to be the first activated genes that link cellular membrane events to the nucleus [8], and the gene expression changes are required for the late neuronal response which relates to the process of learning and memory formation [9] and which is a part of everyday brain functions [10]. In addition, depending on the type of the stimuli, the IEG-encoded proteins can be individually regulated in different parts of the brain [8,11], indicating that the same or different IEGs can, regulate different functions when expressed in different parts of the brain.

In our recent work, we have demonstrated involvement of two IEGs i.e., early growth response 1 (egr-1) and, nuclear hormone receptor 38 (hr38) and their corresponding partner genes such as ecdysone receptor (ecr), dopamine/ecdysteroid receptor (dopecr), dopamine decarboxylase (ddc) and dopamine receptor 2 (dopr2) during the daily foraging of bees [12]. Other papers have reported on the involvement of egr-1 in the transition from nursing to foraging bees [13] and the role of hr38 in caste and labor division [14], while the other genes were shown to have acted as egr-1 downstream genes involved in ecdysteroid and dopamine signaling with a role in the processing of courtship memory [15] in Drosophila and in olfactory learning and memory [16] in honey bees, respectively. The IEG c-jun (also known as jun-related antigen, jra in fruit fly) and egr-1 have been used as neuronal markers for the identification in honey bees of specific brain regions involved in the time memory as well as in innate and learned behaviours [17,18,19]. Interestingly, while most of the above mentioned genes are already well known through various research studies, a recently discovered insect IEG kakusei, a noncoding RNA, has been found to have function in the patterning of neuronal activity in the foraging bees [20,21,22]. Expression of kakusei was detected during ice-induced seizures in bees awakening from anesthesia, and its expression was prominent in the Kenyon cells of the mushroom bodies [20,21,22]. Since kakusei was found as neural marker, we were interested to extend the work on this gene in our experimental design.

The present study represents further investigation of immediate early genes (IEG) that could play a role during the daily foraging of honey bees, and is a continuous work of our previous report by Singh et al [12]. We selected two potential IEGs kakusei and c-jun, and four other orthologous genes that have been reported to be involved in cognitive process in vertebrates: extracellular signal-related kinase (rk7), glutamate receptor (GluR), 5-hydroxy tryptamine (serotonin) receptor 2 alpha (5-HT2α) and dopamine receptor 1 (Dop1). Among the six genes tested, we observed only for the kakusei gene with a transient and prolonged upregulation during reward foraging and a short period of overexpression during unrewarded foraging. These findings demonstrate a potential role for kakusei during reward foraging and in learning of food reward foraging.

Materials and methods

Brain dissection, RNA and cDNA preparation, and qPCR

The frozen bees were lyophilized at -50°C with vacuum at 0.420 mBar for 20min, using a Freeze Zone® PlusTM 4.5 liter cascade Freeze Dry System (Labconco Corporation, Kanas City). Brain dissection was performed in a glass chamber containing 100% ethanol placed on a dry ice platform. The whole brain from each bee was dissected and placed into a micro centrifuge tube separately, and 500 μl Trizol (Trizol® Reagent, ambion RNA, life technology) was added. The brain was homogenized, RNA was extracted, and cDNA was prepared from that RNA, using the kits supplied by Invitrogen (Thermo Fisher Scientific) following the manufacturer's protocols. The cDNA from each brain was subjected to qPCR using a 7900HT Fast Real Time PCR System (Applied Biosystem, Singapore) in a 10μl reaction volume containing oligonucleotide primers (Sigma Aldrich) specific to target genes and SYBR Green (KAPA Syber® FAST PCR Master Mix (2X) ABI Prism®). The qPCR cycles followed Applied Biosystem protocol and the Rp49 gene was used as endogenous control [26] in each qPCR run and analysis. The details of the target genes and the oligonucleotide primers are provided in Table 1.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Details of primers used for quantifying the target genes.

https://doi.org/10.1371/journal.pone.0222256.t001

Results

In this study, only the immediate early gene kakusei showed a remarkable transient upregulation during the course of food reward foraging. The other five genes erk7, c-jun, glur, 5-ht2α, dop1 showed no statistically significant differences (S1 Fig).

Expression profile of kakusei during food reward foraging

The expression of kakusei was measured at eleven time points, BF (pre/before foraging), 0min, 15min, 30min, 45min, 60min, 75min, 90min, 105min, 120min and AF (post/after foraging). In order to check the consistency of the results, three experiments were performed using independent samples collected from two different bee colonies over three months. The experiment 1 and 2 were from colony 1 and experiment 3 was from colony 2. Each of the three experiments demonstrated transient increases of kakusei level during the continuous food reward foraging over the collection time of two hours. The results are summarized in Fig 1 (S1 Table).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Expression changes of the IEG kakusei during daily foraging of bees.

Data are shown as fold changes of kakusei expression level at different time points with respect to t0 (mean value was set to 1 at this time point). The blue, red and green bar graphs represent three independent experiments (experiment 1, experiment 2 and experiment 3 respectively) with each bar representing the kakusei expression level at each time point. Graphs at the left (Experiment series A) represent kakusei level from t0 (14.00 hrs) to t120 (16.00 hrs). Graphs at the right represent kakusei level at the start of foraging (SF/t0: 14.00 hrs) and before-foraging (BF: 09.00 hrs) or after foraging (AF: 18.00 hrs). Each time point has sample size of n = 5. For statistics one-way ANOVA with Turkey- Kramer post-hog multiple comparison test were performed and number of asterisk symbol represents * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

https://doi.org/10.1371/journal.pone.0222256.g001

We observed an increase of kakusei expression from 0min (start of foraging, 14.00 hrs) to 15min (P = <0.0001) and from 15min to 30min (P = <0.0001) (Fig 1 Exp 1A). While the increase held at about 30min (P_30m-45m = not significant) and a decrease was apparent from 45min, there was a sustained increased in expression of kakusei further down along the time series until 120min during the reward foraging as compared to the level at 0min (Fig 1 Exp 1A and S1 Table). A similar pattern was also observed in the subsequent two experiments with independent samples (Fig 1 Exp 2A/3A and S1 Table). The kakusei level was also significantly higher (P = <0.0001) at the start of foraging (0min) when compared to the level at pre foraging (09.00 hrs) or post foraging (18.00 hrs), while there was no significant difference between the before and after foraging samples (Fig 1 Exp 1B). This observation was further validated by two further independent experiments (Fig 1 Exp 2B & 3B). We then examined whether kakusei overexpression pattern was statistically different among the three independent results. We found significantly different overexpression of kakusei among the three independent experiments (P = 0.0001). The difference in kakusei levels between Exp-1/Exp-3 vs Exp-2 (P = 0.0001) was much higher than Exp-1 vs Exp-3 (P = 0.044). This difference might be due to age differences among the group of bees in Exp-1A/1B, Exp-2A/2B and Exp-3A/3B as we did not maintain the age of foraging bees during sample collection. The graphical results of the analysis are presented in Fig 2A. Similarly, in the case of the start/pre/post foraging groups, among the three independent experiments, we observed difference between Exp-2 vs Exp-1/-3 (P = 0.0001) and no difference between Exp-1 and Exp-3 (Fig 2B).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2.

Comparison of three independent experiments during two hours of foraging (A) and before/after foraging (B), and expression changes of IEG kakusei with unrewarded (C) and different foraging time (D). A&B: The three independent experiments shown in Fig 1 were examined for interaction using two-way ANOVA. The bars represent mean kakusei expression in each experiment and the dots represent the mean level of kakusei at various time points. The blue, red and green color bars/dots represent experiment 1, experiment 2 and experiment 3 respectively. C&D: The kakusei expression profile is presented as fold changes with respect to t0 (mean value was set as 1 at this time point) and each green bar graphs represents kakusei levels at each time point. Fig 2C and 2D represent data for unrewarded and time-shifted foraging respectively. Each time point has sample size of n = 5. For statistics one-way ANOVA with Turkey- Kramer post-hog multiple comparison test were performed. The number of asterisk symbol represents * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

https://doi.org/10.1371/journal.pone.0222256.g002

Discussion

The importance of using immediate early genes as tool for finding molecular and cellular mechanisms underlying neuronal network and behaviors in honey bees has been well emphasized in the recent studies [11,12,28,29]. Keeping this idea as central, this study extends our recent report [12] on the search for immediate early genes (IEGs) that could be used as research tool for finding the molecular and cellular mechanisms underlying social behavior using foraging of honey bees as model system. As the foraging of bees consists of systematically well organized social behaviors that include learning, memory, social interaction and communication etc; honey bee foraging has been extensively studied in various research objectives in connection to social behavior [2,5,6]. Noting that honey bees learn food sources, identify the food location, memorize the place, interact and communicate among the foragers, as can be clearly observed during the time of their daily foraging; it is a promising approach to find the genes whose expression is increased upon foraging and to examine their roles in these behavioral routines. In the present study the possible function of the newly discovered insect IEG kakusei is presented, suggesting its role during the foraging of bees. A possible function of the gene in learning of food reward during foraging had also been observed.

kakusei is an insect immediate early gene recently identified by Kubo and his group in the seizure induced bees by cold or CO₂ treatment, and is transcribed into a non-coding neuronal RNA [20]. They found that the transcript was prominently increased in the small-type Kenyon cells of the dancers and foraging bee brains, suggesting involvement of the gene in foraging behavior. However, its involvement during the course of foraging had never been studied before. This is the first report on transient overexpression of kakusei during foraging. We have further examined the possible role of the gene before and after foraging. After food reward kakusei was immediately overexpressed over a short period of time from 15 to ca. 45 to 60 min, and then started decreasing. Interestingly, without food reward, there was a short time overexpression at 15min and no further increase of the gene level. This indicates that food reward is essential for the increase and sustaining of higher kakusei levels during foraging, and suggests a possible role of kakusei in learning and memorizing of food reward during foraging. The possibility of kakusei involvement in learning and memory was previously indicated in the earlier report by Kiya et al., 2007 [20] as they reported overrepresentation of kakusei among the re-orienting Kenyon cells of bees assumed to be foragers. This is in line with the notion that re-orientation flight was the mechanism of flight behavior that was practiced by those bees for memorizing the hive location, thus indicating a role of kakusei in the information integration during foraging flight, which is an essential part for dance communication [20]. We suspect that the kakusei gene might also be involved in social interaction and communication among the foraging bees, linking through different molecular pathways as these behaviors were clearly observed among the foragers over the two hours of foraging. Future research on this direction will be able to give a valid comment with evidence on it. Moreover, the suggestive evidence of the present study on the role of kakusei in learning needs to be rigorously tested further, using other experiments such as proboscis extension reflex (PER), in order to draw a conclusive answer which will be carried out in our further research. Our recent study by Singh et al., 2018 [12] had demonstrated involvement of two IEGs, egr-1and hr38, and downstream genes such as ecr, dopecr, ddc, and dopr2 during daily foraging. Moreover, the role of egr-1 and hr38 in learning of food reward during foraging and memory processing was also suggested. Therefore, coordination of kakusei and the two IEGs egr-1 and hr38 during foraging behavior and learning and memory of food reward during foraging is highly assumable that would be tested in our future works. Since kakusei is a non coding gene, this study also clearly supports the dynamic role of non-coding genes in daily routine behavior.

On the other hand, earlier studies on songbird (Zebra finch) showed profound involvement of egr-1 gene in learning and memory [30]. In the egr-1 transgenic cricket (Gryllus bimaculatus), behaviorally relevant neural circuits was also visualized at cellular resolution [31]. In rodent system, several studies had revealed Egr-1 roles in synaptic plasticity and memory formation in a variety of memory systems including amygdala-dependent memory consolidation processes [32]. Moreover, the functions of hr38 in neural activity in Bombyx mori (silkmoth) and Drosophila melanogaster (fruit fly) and in honey bees were also revealed [14,33]. It is highly promising to do further research with egr-1, hr38 and kakusei using honeybees as model system for further understanding on learning and memory and the finding on honeybees will be useful across animal kingdom.

Conclusion

Previous reports have already indicated involvement of immediate early genes could be used as neural marker and their association in social behaviors such as learning and memory or memory processing. Subsequently, the importance of using immediate early genes as tool for finding molecular and cellular basis of these behaviors in honey bees had been thoroughly discussed [11,12,28,29]. Our recent report by Singh et al., has provided further evidence that the two IEGs egr-1 and hr38 are immediately and transiently expressed during honey bee foraging and further that they are involved in learning and memory processing [12]. The present finding on the recently discovered IEG kakusei is an addition to the circle, thus increasing the number of IEGs in elucidating molecular and cellular signaling underlying social behaviors, using honey bee foraging as model system. In addition to the available reports from different studies, further studies on how the three IEGs egr-1, hr38 and kakusei coordinate in completing the foraging task via their specific role in the learning, memory and their roles in communication and interaction could contribute in mapping molecular and cellular pathways to these behavior. Thereby the mechanisms of social behavior in honey bees could be opened up in more details and mapped which could be further translated to cognitively complex animals and even to human [34].

Supporting information

Acknowledgments

Dr. Ned Mantei, Department of Biology, Molecular Health Sciences, ETH Zurich, carefully and thoroughly read the manuscript. He provided valuable suggestions and advices and immensely helped in editing the manuscript. It is great honor to thank Dr. Ned Mantei for his kind and wholehearted support in making the manuscript to this form.

Dr. Axel Brockmann is gratefully acknowledged for providing the facilities in his lab to work and complete this project. Dr. Brockmann also read the manuscript and provided valuable comments. Ms. Neha Tanwar and Dr. Sophia Liyang kindly supported during experimental sample preparation.