- Research article
- Open Access
Hydra vulgaris exhibits day-night variation in behavior and gene expression levels
© The Author(s). 2019
- Received: 25 December 2018
- Accepted: 25 February 2019
- Published: 8 March 2019
Day–night behavioral variation is observed in most organisms, and is generally controlled by circadian clocks and/or synchronization to environmental cues. Hydra species, which are freshwater cnidarians, are thought to lack the core clock genes that form transcription–translation feedback loops in clock systems. In this study, we examined whether hydras exhibit diel rhythms in terms of behavior and gene expression levels without typical clock genes.
We found that the total behavior of hydras was elevated during the day and decreased at night under a 12-h light–dark cycle. Polyp contraction frequency, one component of behavior, exhibited a clear diel rhythm. However, neither total behavior nor polyp contraction frequency showed rhythmic changes under constant light and constant dark conditions. To identify the genes underlying diel behavior, we performed genome-wide transcriptome analysis of hydras under light–dark cycles. Using three different analytic algorithms, we found that 380 genes showed robust diel oscillations in expression. Some of these genes shared common features with diel cycle genes of other cnidarian species with endogenous clock systems.
Hydras show diel behavioral rhythms under light–dark cycles despite the absence of canonical core clock genes. Given the functions of the genes showing diel oscillations in hydras and the similarities of those genes with the diel cycle genes of other cnidarian species with circadian clocks, it is possible that diel cycle genes play an important role across cnidarian species regardless of the presence or absence of core clock genes under light–dark cycles.
- Day–night variation
- Transcriptome analysis
- Diel cycle genes
- Hydra vulgaris
Many organisms show changes in metabolic, physiological and behavioral states depending on the time of day. These changes are fundamentally controlled by endogenous circadian clocks. Circadian clocks are composed of transcription-translation feedback loops of clock genes [1–3]. Molecular clocks govern the expression of various genes involved in metabolic, physiological and behavioral regulation, leading organisms to alter their status at certain times of day. Circadian clocks can synchronize with various zeitgebers to adjust to the environmental time . Light, one of the strongest zeitgebers, resets the states of organisms via their circadian clocks. Clock systems are conserved from bacteria to humans, and in many organisms, the clock shows a diurnal rhythm under light–dark conditions, adjusts to new environmental conditions, and retains the circadian rhythm even under constant environmental conditions [4, 5].
Cnidarians appeared 740 million years ago and are the sister group to bilaterians [6, 7]. Cnidarians are classified into five main groups: Anthozoa (sea anemones and corals), Scyphozoa (true jellyfishes), Cubozoa (box jellyfishes), Staurozoa (stalked jellyfishes) and Hydrozoa (a diverse group including freshwater cnidarians) [8, 9]. Cnidarians’ primitive nervous system lacks a conspicuous central nervous system [10, 11]. However, even in cnidarians, day-night variation exists in behavioral and physiological states, and it has been clarified that such diurnal variation accompanies alterations in gene expression [12–17]. In both Acropora millepora and Nematostella vectensis, which are in the class Anthozoa, diel cycle genes showing rhythmic expression under light–dark cycles have been identified by transcriptome analysis [12, 16]. Although period and timeless are absent in these cnidarians, orthologs of core clock genes such as Clock, Bmal, and Cryptochrome (Type I and II) are conserved and are thought to form transcription–translation feedback loops to act as circadian clocks [18–20].
Hydra spp. are small freshwater cnidarians belonging to the class Hydrozoa that have been used as model organisms for studies on regeneration, stem cell differentiation, aging, and symbiosis . The Hydra genome was decoded in 2010, and interestingly, it was revealed that there were no core clock genes in the genome . Hydras react to light, although it has been found that hydras do not respond to red light [23–26]. For instance, light can induce polyp contraction, a typical behavior in hydras [27–29]. Discharge of cnidocytes is also affected by light . Moreover, hydras exhibit phototaxis and move toward light sources [31, 32]. However, while light is considered to be an important signal affecting the physiological states of hydras, little is known about the diurnal behavior and physiological states of these organisms. In this study, we found that hydra behavior showed day-night variation during a 12-h light–dark cycle (LD 12:12 cycle) by quantifying behavior with a video analysis system. Furthermore, we identified hundreds of genes whose expression showed diel oscillations under LD 12:12 cycles by genome-wide transcriptome analysis. Interestingly, the expression patterns of some of the genes are conserved in other cnidarian species that possess endogenous clock systems. Based on these data, we hypothesized that hydra behavior is affected not only by light–dark cycles but also by diel cycle genes.
Hydra vulgaris (strain 105) without buds were used in all experiments. The hydras were maintained in a hydra culture solution (HCS; 1 mM NaCl, 1 mM CaCl2, 0.1 mM KCl, 0.1 mM MgSO4, 1 mM tris-(hydroxymethyl)-amino-methane; pH 7.4, adjusted with HCl) at 20 °C under a 12-h light–dark cycle (LD 12:12 cycle). Light intensity was maintained at approximately 450 lx. The hydras were fed with newly hatched Artemia nauplii twice per week.
Behavioral tracking and data analysis
A custom ImageJ macro  was used for frame subtraction analysis to quantify hydra behavior. The stored frames were divided into hourly datasets consisting of 720 frames each. Then, the differences in grayscale values (256 gradations) between each pair of images for all pixels were calculated, and new subtracted images were produced. To remove the noise from the newly produced images, an optimal threshold was automatically determined after median filtering. When hydras moved between the two frames, the grayscale values of some pixels exceeded the threshold, which was defined as movement. When all pixel values were lower than the threshold, hydras in these frames were defined as quiescent (Fig. 1c). No significant movement was detected when dead hydras fixed with 8% paraformaldehyde were monitored for one hour and analyzed by the macro. Thus, the custom macro was able to automatically distinguish whether hydras moved between frames. To evaluate hydra behavior, the percentage of frames in which movement was detected was calculated.
Observation of spontaneous polyp contraction
To continuously observe spontaneous polyp contraction, hydras were prepared and their movements were captured over two consecutive days as described above. Polyp contractions were manually identified using ImageJ or Fiji , and the number of contractions was quantified.
Sampling and RNA isolation
Five hydras were collected every four hours during the LD 12:12 cycles, starting at ZT1. Total RNA was isolated from each sample using TRIzol Reagent (Thermo Fisher Scientific, MA, USA) and purified using an SV Total RNA Isolation System (Promega, WI, USA) according to the manufacturer’s instructions. RNA in samples was quantified by an ND-1000 spectrophotometer (NanoDrop Technologies, DE, USA), and the quality was confirmed with an Experion System (Bio-Rad Laboratories, CA, USA). The samples were biologically replicated at each timepoint.
Transcriptome analysis by microarray
The cRNA was amplified, labeled, and hybridized to a 4 × 44 K custom-made Hydra microarray (Agilent Technologies, CA, USA) according to the manufacturer’s instructions. All hybridized microarray slides were scanned by an Agilent scanner. Relative hybridization intensities and background hybridization values were calculated using Agilent Feature Extraction Software (184.108.40.206). The raw signal intensities and flags for each probe were calculated from the hybridization intensities (gProcessedSignal) and spot information (gIsSaturated, etc.) according to the recommended procedures of the Agilent Flag criteria in GeneSpring Software. Raw signal intensities of all samples were log2-transformed and normalized by quantile algorithm with the preprocessCore library package  in Bioconductor software .
Analysis of rhythmic gene expression
Genes with rhythmic expression patterns were identified using ARSER , JTK_CYCLE  and empirical JTK_CYCLE . Log2-transformed and normalized data from two daily cycles were used as input data for all programs. In ARSER and JTK_CYCLE, P < 0.05 was used as the significance threshold, whereas a Bonferroni-adjusted P value < 0.05 was used in empirical JTK_CYCLE. The oscillating genes identified by all three algorithms were classified into four clusters by K-means clustering and analyzed for enrichment of gene ontology (GO) terms. After a search was conducted for the human homologue of each gene (e-value < 1 × 10− 3), GO enrichment analysis was performed for each cluster using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) version 6.8 . The human homologues of all the unique genes on the custom-made microarray were used as a background model.
Hydras show diurnal behavioral rhythms
To investigate whether hydras show day-night variation in total behavior under a 12-h light–dark cycle (LD 12:12 cycle), we recorded hydra behavior for two consecutive days. Since hydras are not able to respond behaviorally to red light , we captured video with infrared illumination to visualize behavior even under dark conditions (Fig. 1a, b). Through frame subtraction analysis of images acquired every five seconds, we detected the frames in which hydras moved (Fig. 1c, see Materials and Methods). Based on the proportion of frames in which motion was detected among the 720 frames acquired in one hour, hydras exhibited marked day-night variation in behavior (Fig. 1d). On the first day, movement was detected in 53.5 ± 2.7% (mean ± SEM) of the total frames during the daytime (ZT0-ZT12) but only in 37.9 ± 2.4% of the total frames during the nighttime (ZT12-ZT24). On the second day, movement was detected in 46.3 ± 2.4% of the total frames from ZT0-ZT12 but only in 31.9 ± 1.8% of the total frames from ZT12-ZT24. These results suggest that hydras are more active in the daytime than in the nighttime under LD 12:12 cycles (Fig. 1e).
To know whether the behavioral variation remains under constant environmental conditions, we recorded hydra behavior under constant dark (DD) and light (LL) conditions (Fig. 1b). Unlike LD 12:12 cycles, there was no circadian variation in hydra behavior under DD condition (Fig. 1f, g.). Under LL condition, although the behavioral onset was induced by lights-on stimulus as well as LD cycles, there was no circadian variation throughout two days (Fig. 1h, i). These results suggest that the behavioral onsets and the offsets are triggered by lights-on and lights-off stimulus, respectively. In other words, the day-night variation of total behavior is induced by the light–dark cycles.
Genes showing diel expression under LD 12:12 cycles
Clustering and gene ontology (GO) enrichment analysis of the diel cycle genes
Diel cycle genes involved in neuron activity
Putative channel genes exhibiting diel expression patterns
Homologue identified by BLAST2GO
Potassium voltage-gated channel subfamily G member 2 (R. norvegicus)
Endosomal lysosomal potassium channel TMEM175 (D. rerio)
Calcium-gated potassium channel (T. volcanium)
Potassium voltage-gated channel subfamily A member (O. mykiss)
Voltage-dependent calcium channel subunit alpha-2 delta-3 (H. sapiens)
Transient receptor potential cation channel subfamily A member 1 homologue (C. elegans)
Acid-sensing ion channel 1 (R. norvegicus)
Acid-sensing ion channel 3 (H. sapiens)
Putative neurotransmitter receptor genes exhibiting diel expression patterns
Homologue identified by BLAST2GO
Neuronal acetylcholine receptor subunit alpha-6 (M. musculus)
Gamma-aminobutyric acid receptor subunit beta-3 (R. norvegicus)
Metabotropic glutamate receptor 8 (M. musculus)
Adenosine receptor A2a (C. l. familiaris)
Muscarinic acetylcholine receptor M3 (M. musculus)
Homologous genes of kinesin motor proteins (Gene IDs: Sc4wPfr_1040.g7652.t1 and Sc4wPfr_1402.g13898.t2) and cyclin-dependent kinase-like 5 (Gene ID: Sc4wPfr_126.2.g29250.t2) also showed diurnal expression rhythms. Most kinesins contribute to anterograde transport by moving along microtubule filaments to the plus end [49, 50]. In neurons, kinesin motors are critical for axonal transport of translated proteins and synaptic vesicle precursors . Cyclin-dependent kinase-like 5 not only participates in neural development but also controls axonal transport via phosphorylation of motor proteins [52–54]. The diel expression of these channels, neurotransmitter receptors, and axonal transport-associated genes may reflect day–night variation in neuron activity in hydras.
RNA processing-associated diel cycle genes
RNA processing-associated genes exhibiting a diel expression pattern
Homologue identified by BLAST2GO
Tuftelin-interacting protein 11 (X. laevis)
Serine/arginine-rich splicing factor 4 (M. musculus)
Heterogeneous nuclear ribonucleoprotein 1 (A. thaliana)
Comparative analysis of diel cycle genes in hydras and other cnidarians
Homologous pairs of diel cycle genes in three cnidarian species (Hydra vulgaris, Nematostella vectensis and Acropora millepora)
Homologue identified by BLAST2GO
Heat shock 70 kDa protein 12A (H. sapiens)
Heme-binding protein 2 (M. musculus)
Protein disulfide isomerase A5 (M. musculus)
Transcription factor HES-1-B (X. laevis)
Homologous diel cycle genes present in both Hydra vulgaris and Nematostella vectensis but not in Acropora millepora
Homologue identified by BLAST2GO
Dual serine/threonine and tyrosine protein kinase (R. norvegicus)
Elongation of very long chain fatty acids protein (H. sapiens)
Serine/threonine-protein phosphatase 6 catalytic subunit (R. norvegicus)
Hydra behavior can entrain to light–dark cycles
In this study, we clarified that day-night variation exists in hydra behavior. Based on our behavioral recordings, hydras show high behavioral activity in the daytime and less activity in the nighttime (Fig. 1d, e). Although hydras exhibit stable behavior repertories , we found that the behavior of hydras entrained to the light–dark cycles. However, the activity pattern was not similar to typical circadian rhythms shown under LD 12:12 cycles because there was a lack of anticipatory behavior prior to the environmental cues (Fig. 1d). If organisms possess endogenous circadian clocks, they are able to show anticipatory behavior, one of the important features of circadian rhythms . As shown in Fig. 1d, hydra activity increased rapidly at lights-on (ZT0) and then slightly decreased until approximately midday (ZT6). From midday to lights-off (ZT12), the activity level was almost constant, but at lights-off, the level dramatically decreased and then remained constant until the end of the night (ZT24). Given that the various light-stimulated behaviors of hydras reported so far started at ZT0 and ceased at lights-off, the day-night variation might be produced by the light-on/off stimulus itself. In fact, hydra activity did not increase at CT0 under DD condition and kept constant throughout two days without circadian oscillations (Fig. 1 f, g). Moreover, since CT0-CT12 under LL is (the) same condition as ZT0-ZT12 under LD cycles, it is unsurprising that these activity patterns were similar to each other; however, there were neither a rapid decrease at CT12, nor circadian rhythms in hydra activity under LL (Fig. 1 h, i). These behavior patterns we observed thus do not contradict previous reports that there are no canonical clock genes in the Hydra genome . However, upon separate analysis of polyp contraction behavior, a typical behavior of hydras, it seems that the frequency of contraction may be regulated by endogenous oscillation systems (Fig. 2b). Contraction frequency initially increased at lights-on but then gradually decreased without showing any response to the lights-off stimulus, as shown in Fig. 2b. Then, the frequency mildly increased slightly before the next lights-on stimulus. This pattern closely resembles anticipatory activity, which can be shown in organisms with endogenous circadian clocks . Nevertheless, the frequency of contraction did not show circadian rhythms under DD and LL (Fig. 2d, f). These findings suggest that hydras may possess a circadian system which is only driven by light–dark cycles to regulate polyp contraction behavior even though there are no canonical clock genes in the hydra genome. Although we have clearly shown in this study that hydra behavior at least entrains to LD 12:12 cycles, further studies are necessary to address whether hydras possess endogenous circadian systems for specific behaviors.
Day–night variation in gene expression levels
Through genome-wide transcriptome analysis, we comprehensively investigated gene expression in hydras under LD 12:12 cycles. This analysis allowed us to identify genes showing diel expression patterns in hydras. HyDCGs corresponding to 1.6% of the total number of genes exhibited striking diel expression patterns. Because we used three analytic algorithms to detect genes with diel oscillation and extracted only the common genes, it is probable that there are more diel cycle genes in hydras than we identified. By K-means clustering, the HyDCGs were classified into four groups with distinct peak phases: dawn, midday, dusk and midnight. This finding suggests that the diel expression patterns are not controlled solely by lights-on or lights-off stimuli. However, considering that the number of genes in each cluster was not evenly distributed (P < 0.01, chi-square test), hydras might have a specific time of day where rhythmic transcriptions are actively performed to induce physiological day-night variations.
The HyDCGs included several channels, neurotransmitter receptors, and axonal transport-associated genes. Thus, it is possible that neuron activity changes during the daytime and nighttime in hydras. Neuropeptides are important for the regulation of neuron activity in hydras , and recent genome sequencing has revealed that hydras have various neurotransmitter receptors [22, 60]. Previous pharmacological and electrophysiological studies have suggested that several neurotransmitters regulate pacemaker activity involved in spontaneous contraction and elongation in hydras [46, 61]. The diel expression patterns of neuron-associated genes observed in this study may contribute to transitions in behavior.
The diel cycle genes of hydras share common properties with the diel cycle genes of other cnidarians. Through comparative analysis of the genes of hydras and other cnidarians, we found that four genes commonly showed diel oscillations in expression among three cnidarian species (Hydra vulgaris, Nematostella vectensis and Acropora millepora) (Table 4). In addition, several genes showing diel expression patterns were shared between Hydra vulgaris and Nematostella vectensis (Table 5) or between Hydra vulgaris and Acropora millepora. Despite their lack of major clock genes, hydras may have systems to produce diel oscillations in gene expression to enable proper biological functioning at certain times of the day that are similar to the systems of other cnidarians with circadian clocks.
Absence of canonical clock systems in hydras
Several cnidarians in the class Anthozoa possess typical core clock genes and exhibit endogenous circadian rhythms in both behavior and gene expression [12, 16]. Moreover, core clock genes are also encoded in the genome of Clytia hemisphaerica, a cnidarian of the class Hydrozoa . It is thus probable that the loss of clock genes in hydras was secondary. In addition to lacking a central clock system, hydras also rely on photoreception to trigger behavior. However, we cannot exclude the possibility that hydras have novel endogenous systems supporting day-night variation. For example, a recent multiomics study revealed that hundreds of genes, proteins and metabolites show robust circadian oscillations in cultured Drosophila S2 cells even though it is known that S2 cells do not express core clock genes . Such findings clearly suggest that there is another oscillator independent of canonical clock systems.
In some cnidarians possessing orthologs of the core clock genes, it has been reported that behavioral circadian rhythms and circadian gene expression tend to dampen and then resolve within a few days under constant darkness   . A recent study also revealed that differential gene expression between midday and midnight disappears under constant darkness conditions in Nematostella vectensis . In other words, clock systems might be not as robust in cnidarians as in other taxonomic groups, indicating that cnidarians, especially hydras, are intriguing subjects for understanding the evolutionary aspects of circadian clocks.
In this study, we demonstrated that hydras exhibit diel rhythms in terms of behavior and gene expression despite a lack of typical clock genes under light–dark cycles. Through genome-wide transcriptome analysis, we identified 380 genes with clear diel oscillations in expression, and some of these genes shared common features with the diel cycle genes of other cnidarian species with endogenous clock systems. These results suggest that the identified genes might play an important role in inducing diel rhythms and that these types of genes are common throughout cnidarians regardless of the presence or absence of core clock genes. This is the first report of a comprehensive analysis regarding diel rhythms in hydras.
We thank Dr. Kaori Yasuda and Dr. Atsushi Doi (Cell Innovator Co., Ltd.) for their assistance with microarray analysis. We also thank Kyuya Matsumoto and Dr. Ichiro Kodama for their helpful advice on setting up the behavioral experiments and Sakura Nishimura, Misako Hori and Kazumasa Sumida for their assistance with behavioral analysis.
This work was supported by the Yamakawa Prize and Grant (Kyushu University Fund) to HJK, the KIKAN Education Award and Scholarship (Kyushu University) to HJK, and in part by the Grant Basic Science Research Project from the Sumitomo Foundation (No.180788) to TQI.
Availability of data and materials
The datasets used/produced during the current study are available from the corresponding author on reasonable request.
HJK and TQI designed the study and performed the experiments and data analysis. HJK, YK and TQI wrote the paper and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare they have no competing interests.
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