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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: Comp Biochem Physiol A Mol Integr Physiol. 2018 May 9;223:52–59. doi: 10.1016/j.cbpa.2018.05.002

Localization and expression of putative circadian clock transcripts in the brain of the nudibranch Melibe leonina

Victoria E Duback 1, M Sabrina Pankey 1, Rachel I Thomas 2, Taylor L Huyck 2, Izhar M Mbarani 2, Kyle R Bernier 2, Geoffrey M Cook 2, Colleen A O’Dowd 1, James M Newcomb 2, Winsor H Watson III 1
PMCID: PMC5995673  NIHMSID: NIHMS966542  PMID: 29753034

Abstract

The nudibranch, Melibe leonina, expresses a circadian rhythm of locomotion, and we recently determined the sequences of multiple circadian clock transcripts that may play a role in controlling these daily patterns of behavior. In this study, we used these genomics data to help us: 1) identify putative clock neurons using fluorescent in situ hybridization (FISH); and 2) determine if there is a daily rhythm of expression of clock transcripts in the M. leonina brain, using quantitative PCR. FISH indicated the presence of the clock-related transcripts clock, period, and photoreceptive and non-photoreceptive cryptochrome (pcry and npcry, respectively) in two bilateral neurons in each cerebropleural ganglion and a group of < 10 neurons in the anterolateral region of each pedal ganglion. Double-label experiments confirmed colocalization of all four clock transcripts with each other. Quantitative PCR demonstrated that the genes clock, period, pcry and npcry exhibited significant differences in expression levels over 24 hrs. These data suggest that the putative circadian clock network in M. leonina consists of a small number of identifiable neurons that express circadian genes with a daily rhythm.

Keywords: Gastropod, circadian rhythm, cryptochrome, biological clock, nudibranch, clock genes

1. Introduction

Many animals exhibit a daily rhythm of activity and tend to be more active either during the day (diurnal), or during the night (nocturnal). In addition, some animals have a crepuscular rhythm, expressing their strongest bouts of activity around sunrise and sunset (Dunlap, 1999; Panda et al. 2002). All of these rhythmic patterns are strongly correlated with, and typically synchronized to, environmental cues such as the 24 hr light-dark (LD) cycle (Bunney & Bunney, 2000). Moreover, in the absence of LD cues, most animals continue to show a daily (~ 24 hr) rhythm of activity, indicating the presence of an endogenous circadian clock (Aschoff, 1965). The molecular mechanisms that give rise to these ~ 24 hr clocks have been elucidated in many organisms ranging from cyanobacteria to humans (Takahashi, 1991, 1995; Dunlap, 1999; Young & Kay, 2001; Hastings et al. 2007; Allada & Chung, 2010). As a result of these studies, it is now clear that most organisms, from prokaryotes to mammals, share a transcription-translation feedback loop of clock genes and their protein products that are either identical or quite similar in principle.

While we now know a great deal about daily behavioral rhythms and the molecular basis of the clocks underlying these rhythms, less is known about the connection between the molecular clockwork pathways and the neural networks responsible for influencing the expression of specific physiological and behavioral rhythms. Gastropods, due to their large identifiable neurons and stereotyped behaviors, are often suitable model systems for these types of investigations. Therefore, one of our major long-term goals is to develop a nudibranch model system that will facilitate investigations of the interactions between clock neurons and the neural networks controlling specific behaviors, such as swimming in M. leonina (Thompson & Watson, 2005; Sakurai et al., 2014).

Daily rhythms of locomotion have been demonstrated in a number of gastropods, including Aplysia californica (Kupfermann, 1968; Jacklet, 1972), Bulla gouldiana (Block & Davenport, 1982), Bursatella leachi plei (Block & Roberts 1981), Helisoma trivolvis (Kavaliers, 1981), Limax maximus (Sokolove et al., 1977), Melanoides tuberculata (Beeston & Morgan, 1979), and M. leonina (Newcomb et al., 2004; 2014). Furthermore, the isolated eyes and retinal neurons of some of these species exhibit circadian oscillations of neural activity (Jacklet, 1969; Block & Roberts, 1981; Block & Wallace, 1982; Michel et al., 1993), suggesting that the eyes are the location of at least one clock system. Localization of putative circadian clocks has only been accomplished in two gastropods – A. californica and B. gouldiana, using immunohistochemistry with antibodies developed to a conserved region of the Drosophila melanogaster PERIOD protein (Siwicki et al., 1989). PERIOD-immunoreactive neurons were located in the eyes of each species, as well as in approximately 10 neurons in the cerebral ganglion of A. californica (Siwicki et al., 1989). Localization of period transcripts in B. gouldiana eyes has since been confirmed with in situ hybridization (Constance et al., 2002). In both of these species, period oscillates in certain tissues, based on western blots (Siwicki et al., 1989) and in situ hybridization (Constance et al., 2002). However, of all of these gastropods, the one with the best-studied central pattern generator (CPG) underlying a behavior that is expressed with a circadian rhythm is M. leonina (Thompson & Watson, 2005; Newcomb et al., 2014; Sakurai et al., 2014). Thus, it may be the most promising model system for investigating the link between clock neurons and a specific behavior.

M. leonina displays two modes of locomotion – crawling and swimming (Agersborg, 1923; Lawrence & Watson, 2002) and the CPG underlying swimming consists of four pairs of bilateral neurons (Thompson & Watson, 2005; Sakurai et al., 2014). M. leonina moves more at night (Newcomb et al., 2004) and this pattern of activity is controlled by an endogenous circadian clock (Newcomb et al., 2014). Furthermore, transcripts of clock genes in M. leonina have been recently sequenced (Cook et al., in press), making it possible to carry out experiments that were previously not possible. The goal of this project was to use these transcript sequences to accomplish two objectives: 1) determine the location of putative clock neurons that express these clock transcripts using RNA probes with fluorescent in situ hybridization (FISH); and 2) investigate the temporal expression profile of some of these clock genes.

2. Materials and Methods

2.1 Collection and housing of animals used for FISH

Adult M. leonina were collected from eelgrass beds in the Puget Sound near the University of Washington’s Friday Harbor Laboratories, from kelp forests near Monterey Bay, CA (Monterey Abalone Company), and from floating kelp masses around Catalina Island, CA (Marinus Scientific). Animals were shipped overnight to New England College and maintained in aquaria containing artificial seawater (Instant Ocean, 32–35 ppt salinity). The tanks also contained eelgrass or kelp shipped from the collection sites. All aquaria were kept at 10–12°C, with a 6 am:6 pm, light/dark cycle. Animals were given at least 3 days to acclimate to the LD schedule prior to dissection.

2.2 FISH

Sequence-specific RNA probes were developed by Biosearch Technologies for clock, period, and photoreceptive and non-photoreceptive cryptochrome (pcry and npcry, respectively), based on sequences derived from RNA transcriptomes (Cook et al., in press). In brief, 48 non-overlapping, 20-nucleotide RNA probes were designed for each transcript, with each probe being conjugated to a fluorophore. Amplification of the fluorescent signal results from multiple probes hybridizing to a single transcript. If less than five probes attach to a transcript, it will tend to blend into the background. FISH experiments were done in two stages. First, RNA probes for individual clock transcripts were used in individual brains to determine if each transcript could be identified (n = 35). These were accomplished with RNA probes conjugated to CAL Fluor 610 (peak excitation = 590 nm, peak emission = 610 nm). Dissections were done initially at 6 am, 12 pm, 6 pm, and 12 am, to determine if there were any differences in labeling over time. Dissection time had no apparent effect, so subsequent dissections were typically done around noon. Second, once each transcript was successfully identified, double-label experiments with two RNA probes conjugated to different fluorophores were used in individual brains (n = 21). In these experiments, one fluorophore was CAL Fluor 610 and the other was Quasar 670 (peak excitation = 647 nm, peak emission = 670 nm). Control experiments for each set of experiments involved omitting the RNA probes (n = 3).

Brains and attached buccal ganglia were removed, pinned in a dish, and fixed overnight in a 3.7% formaldehyde fixation buffer (in phosphate buffered saline) at 4°C. RNase-sterile techniques were used throughout this entire procedure to minimize degradation of RNA and nuclease-free water was used when making all solutions. The following day, brains were rinsed and then left overnight in 70% ethanol at 4°C. The brain was then incubated in 10% deionized formamide in Wash Buffer A (Biosearch Technologies) for 3 min on a shaker at room temperature, followed by incubation in the relevant FISH RNA probe (made against clock, period, pcry, or npcry). Initially probes for clock were diluted 1:100, 1:50 and 1:25 to determine the optimal dilution. Subsequently, all probes were diluted either 1:50 or 1:25 in 10% deionized formamide, made in Hybridization Buffer (Biosearch Technologies). Incubation in the FISH probe was done overnight on a shaker at 37°C. Double-label experiments included two FISH probes, with different fluorophores, each diluted to a 1:25 concentration in the final deionized formamide/Hybridization Buffer solution. Precautions were taken for the remainder of the protocol to keep light exposure to a minimum. Following overnight incubation in the FISH probe(s), brains were washed again in a 10% deioinized formamide solution, made in Wash Buffer A, on a shaker for 30 min at 37°C, followed by a 3-min rinse in Wash Buffer B (Biosearch Technologies) on a shaker at room temperature. Brains were then dehydrated in increasing concentrations of ethanol (50%, 70%, 90%, 95%, 100%, and 100%), for 10 min each, at room temperature. Dehydrated brains were then cleared using methyl salicylate, and mounted in Cytoseal (Electron Microscopy).

2.3 Imaging of FISH preparations

Single-label FISH images were obtained on a Zeiss LSM 880 confocal microscope, using an excitation wavelength of 594 nm. Multiple images of optical sections of a brain were obtained at a high resolution and stitched together with Zen software. Double-label FISH preparations were imaged on a Zeiss Axio Scope.A1 epifluorescence microscope, using emission filter sets that prevented bleed through between CAL Fluor 610 and Quasar 670 fluorophores. Images were obtained with a Zeiss Axiocam digital camera and viewed/captured with Zen 2012. Pseudocolors of red and green were used to facilitate confirmation of double-labeled neurons. For each preparation, the location and numbers of labeled neurons were noted on a brain map.

2.4 Collection and dissections of animals for qPCR experiments

All the animals for the qPCR experiments were collected in Parks Bay off of Shaw Island, WA, (GPS: 48.565239, −122.980976), between March 2 and 11, 2017, and held in a flow-through seawater tank that was exposed to the ambient L:D cycle (which was roughly 12:12, due to the temporal proximity to the spring equinox). Animals were allowed at least 24 h after collection to adjust to their new conditions before the dissections began.

Prior to performing the dissections, all of the lab benches, dissection tools, and microscopes were cleaned with ethanol and RNase AWAY (Thermofisher Scientific). Over the course of three days, on two occasions, five animals were dissected every 3 hrs, for a total of 80 samples (n=10 for each time point). The dissections occurred at 6 am, 9 am, 12 pm, 3 pm, 6 pm, 9 pm, 12 am, and 3 am. During the night dissections (6 pm – 3 am), red light was used, as evidence suggests that M. leonina are not sensitive to this wavelength of light. Each dissection lasted ~ 10–30 min. The esophagus, to which the brain is firmly attached, was removed from each animal and placed in a tube containing 5 mL of RNAlater. The RNAlater samples were then covered with parafilm and stored at 4°C.

All 80 samples were shipped in a cooler, on ice, to the University of New Hampshire (UNH) overnight. At UNH the brain (paired cerebropleural and pedal ganglia) was removed from the esophagus and placed in PCR-clean Eppendorf tubes. The Eppendorf tubes were immediately flash frozen in a container holding a combination of 95% ethanol and dry ice, and then stored at −80°C until RNA extraction. All samples were processed within a month of arriving at UNH.

2.5 RNA extraction and qPCR

RNA was extracted and purified from each central nervous system (CNS) using TRIzol (Ambion, Fisher Scientific) and a Purelink RNA mini kit (Invitrogen, Thermofisher Scientific). Contaminate genomic DNA was removed with the on-column PureLink DNase treatment kit (Ambion, Thermofisher Scientific). Approximately 20 ng/μl total RNA was obtained for each sample based on measurements obtained with both a Nanodrop (NanoDrop Spectrophotometer ND-1000, University of New Hampshire, Durham, NH) and Qubit 2.0 (Flourometer, Invitrogen University of New Hampshire, Durham, NH).

DNA primers were designed using Primer3 (Table 1; Koressaar and Remm, 2007; Untergasser et al., 2012) so that amplicon sizes fell within an optimal range for qPCR assays (between 75 and 150 base pairs) for the four clock transcripts of interest (clock, period, pcry, and npcry) and the reference housekeeping gene alpha tubulin (atub). After an initial denaturation step (95°C for 10 min), cDNA synthesis was performed using a qScript cDNA supermix (Quantabio) following manufacturer protocols. A total of 3 μl of diluted cDNA was used for a 10 μl PCR reaction, and samples were amplified in triplicate for each primer. Complimentary DNA was amplified using an Agilent MX3000P qPCR system (Agilent) for 40 cycles (95°C for 30 s, 55°C for 60 s, 72°C for 60 s), using an Applied Biosystems SYBR select master mix (Applied Biosystems). These data were first normalized to the housekeeping gene alpha tubulin. The fold change over time was then graphed, which represents the expression levels of the genes per time point in comparison to the average expression level for each gene.

Table 1.

Forward and reverse qPCR primer sequences, for circadian clock and housekeeping (control) transcripts.

Gene Forward primer Reverse primer
alphatubulin 5′-GCCCTACAACTCCATCCTGA-3′ 5′-GTAGGTGGGACGCTCAATGT-3′
period 5′-CTGATGACGGTGTGTTGTCC-3′ 5′- AAGACCAGAAACGGCTGCTA-3′
photoreceptive cryptochrome 5′-GGTTTGGAGACCTTTGACCA-3′ 5′-GCGAGGGCACTGTAATACCT-3′
non-photoreceptive cryptochrome 5′-ACAAACGGTTCCAGTCCATC-3′ 5′-TGACAGGTGTGACGCTCTTC-3′
clock 5′-ACCAGCACAGAGCTTGTCAC-3′ 5′-ATCCTGCAGCTTCTTGGACT-3′
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2.6 Statistical analyses

All qPCR statistical tests were performed with JMP13 (SAS, Cary, NC). Pairwise differences in expression level (ddCt values) between any two time points were determined with a students t-test.

3. Theory

Previous studies have indicated that M. leonina express a circadian rhythm of locomotion and also possess many of the core genes that control circadian rhythms in other organisms. Therefore, our working hypothesis was that we would see differences in the expression of these circadian genes in the brains of animals that were dissected in the night vs the day. Furthermore, in order to lay the foundation for future studies we sought to identify specific clock neurons using in situ hybridization. This effort was successful and makes it possible to carry out further intracellular investigations of clock neurons in M. leonina.

4. Results

4.1 Single-label FISH

Seven of 13 brains labeled with anti-clock FISH probes exhibited two clock-positive neurons in each cerebropleural ganglion (Fig. 1A). This variability was likely due to the fact that we initially used three different probe concentrations (1:100, 1:50, and 1:25); this pair of cells were clear in all four brains labeled with the 1:25 probe concentration. There were also clock-positive neurons in the anterolateral region of both pedal ganglia in 10 of 13 preparations (Fig. 1A). The number of pedal neurons in these ten preparations was quite variable: with 8.3 ± 8.2 and 8.8 ± 6.2 (mean ± standard deviation) neurons in the left and right pedal ganglia, respectively. These clock-positive pedal neurons were present in all four brains labelled with the 1:25 probe concentration, although the number of neurons was similar in preparations labelled with weaker probe concentrations. After these clock FISH trials, we concluded that a dilution of 1:100 was not strong enough to provide consistent results. Therefore, all subsequent FISH experiments were done with probe concentrations that were diluted 1:50 or 1:25, and these yielded indistinguishable results.

Figure 1.

Figure 1

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RNA FISH labeling in Melibe brains for four different circadian clock transcripts: clock (A), period (B), npcry (C), and pcry (D). All four probes consistently labeled two cerebropleural neurons in the middle of each cerebropleural ganglion (arrowheads), and a small group of neurons in the anterolateral region of each pedal ganglion (arrows). The boxed region in (B) is shown at higher magnification in Figure 2. The fluorophore was CAL Fluor 610, but images were subsequently changed to a green color to improve contrast. The scale bar represents 200 μm.

Period labeling was similar to clock, with 7 of 9 preparations exhibiting two period-positive neurons in each cerebropleural ganglion (Fig. 1B). In the other two brains, one preparation had two period-positive neurons in the left cerebropleural ganglion, but only one in the right ganglion, and in the other brain, there was one period-positive neuron in the left cerebropleural ganglion and no labeling in the right ganglion. In 6 of 9 preparations, there were also period-positive neurons present in the anterolateral region of both pedal ganglia (Fig. 1B). The number of period-positive neurons was 3.9 ± 2.7 in the left pedal ganglion and 4.7 ± 2.2 in the right pedal ganglion.

In 5 of 6 brains labeled with probes for npcry, there were multiple bilaterally symmetric neurons in each cerebropleural ganglion (Fig. 1C). Three of these brains exhibited two labeled neurons in each cerebropleural ganglion, similar to clock and period, while a fourth preparation had three npcry-positive neurons in the left cerebropleural ganglion, and the fifth brain had three labeled neurons in both cerebropleural ganglia. The same five brains that exhibited npcry-positive neurons in the cerebropleural ganglia also had labeling for npcry in the anterolateral region of each pedal ganglion (Fig. 1C). There was an average of 4.8 ± 2.5 npcry-positive neurons in the left pedal ganglion, and 5.2 ± 2.3 neurons in the right pedal ganglion.

The fourth RNA probe for pcry labeled two neurons in each cerebropleural ganglion in all seven preparations (Fig. 1D). All seven brains also exhibited pcry-positive neurons in the anterolateral region of both pedal ganglia, with 9.3 ± 4.1 and 9.3 ± 4.9 neurons in the left and right pedal ganglia, respectively (Fig. 1D).

All of the FISH labeling was punctate at higher magnification (Fig. 2), illustrating labeling of individual transcripts by the accumulation of upwards of 40 custom FISH probes per transcript. This labeling was also relegated to the cytoplasm, and did not appear in the nucleus (Fig. 2).

Figure 2.

Figure 2

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Close up image of the two cerebropleural neurons (outlined in dotted lines) in the boxed region of Figure 1B. The labeling in each cell is punctate and illustrates individual RNA transcripts in the cytoplasm. The scale bar represents 5 μm.

4.2 Double-label FISH

As seen in Fig. 1, all four RNA probes labeled similar numbers of neurons in similar areas of the brain. To determine if multiple probes were labeling the same neurons, additional experiments were done with RNA probes that had different fluorophores, to enable double-labeled preparations. The following combinations of probes were used: clock and period (n = 6), period and npcry (n = 9), and npcry and pcry (n = 6). By association, these combinations enabled us to determine potential co-localization of all four RNA probes. Based on the single-label experiments above, RNA probes were used at a concentration of 1:25. In all preparations, there was 100% colocalization between probes (Fig. 3). The pattern of labeling (two bilaterally symmetric cerebropleural neurons and two small groups of bilaterally symmetric pedal neurons) and the numbers of labeled neurons in each area were similar to single-label FISH experiments (see Fig. 1). Control preparations (n = 3), that lacked RNA FISH probes, did not exhibit any labeling (not shown).

Figure 3.

Figure 3

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Double-label FISH experiments demonstrate consistent colocalization of clock and period, period and npcry, and npcry and pcry. Yellow cells in the merged images indicate overlap between the red and green fluorophores. In each instance, neurons in locations that were similar to those seen in single-label FISH experiments (see Fig. 1) were labeled – two cells in each cerebropleural ganglion and a small group of neurons in the anterolateral region of each pedal ganglion.

4.3 qPCR

The brain (cerebropleural and pedal ganglia) exhibited oscillating expression of circadian clock genes over a period of 24 hr (Fig. 4). Note that all of the expression levels are expressed in comparison to the average expression for each gene, meaning that positive expression is higher than the average and negative expression is lower. There were significant variances in expression levels overall in a 24 hr period for pcry and period (Fig. 4C, D; ANOVA; p=0.0001 and p=0.0084, respectively), and fluctuations trending towards significance for clock (Fig. 4A; ANOVA; p=0.0893). No significant differences were seen overall for npcry (Fig. 4B; ANOVA; p=0.1253). Clock tended to have high gene expression at night, between 15 h (circadian time, middle of the night) and 2 h (right after sunrise), which gradually declined until 9 h (sunset) (Fig. 4A). The circadian clock gene npcry showed weak expression between 6 h (middle of the day) and 12 h (sunset), and then it subsequently increased from 18 h (middle of night) to 21 h (end of night) (Fig. 4B). Pcry had several time points with significant differences over the 24 h period (Fig. 4C), which was similar to the pattern for period (Fig. 4D). There were two troughs, at 3 h (day) and 21 h (night), and the expression slowly increased until 18 h (midnight) for both. There also was a peak in expression at 24 h for pcry and period.

Figure 4.

Figure 4

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Average fold change (log2) values over time for M. leonina brain circadian clock gene transcripts (A) clock, (B) npcry, (C) pcry, and (D) period; n=10 for each gene and time point. The yellow and black bars represent times of day (yellow) and night (black), which was approximately 12:12 when these experiments took place. Time points represent circadian time (0 is lights on, which was 7 AM in real time; 12 is lights off, which was 7 PM). Time 0 was double plotted as time 24. Error bars represent the standard error of the mean. In addition to the average expression fold change (log2), all data points are plotted to show actual variability.

In general, when comparing all circadian clock genes, there was a tendency for peak expression around 18 h (night). However, the expression patterns for clock and npcry indicate relatively moderate changes throughout the night whereas pcry and period show greater fluctuations in expression. For all of the genes, expression was fairly low during the day.

When comparing individual time points to one another, for each circadian gene of interest, some significant differences were apparent between specific pairs of time points (Fig. 5). For example, gene expression for clock quickly up-regulated from hour 12 (sunset) to hour 18 (midnight) (Fig. 4A, Fig. 5A) and clock gene expression at hour 18 (midnight) and 21(early morning) was very different than the expression at hour 6 (midday) and 9 (afternoon). These trends signify differences in expression from the day hours to the night hours for clock. Non-photoreceptive cryptochrome also showed gene up regulation from right after midnight to early morning with limited to no expression seen throughout the day (Fig. 5B). Trends toward expression differences were seen between hour 21(early morning) and the daytime hours (Fig. 5B) as the expression was slowly increasing over time until its peak at hour 21. The expression of Pcry at hour 18 (midnight) and hour 24 (sunrise) was significantly higher than every other hour, including all of the day hours (Figs. 4B, 5C). However, there was no difference between the expression at midnight and sunrise, suggesting that the expression began to down regulate from the early morning until sunrise (Fig. 5C). Like pcry, the significant differences in expression levels over time for period were between high levels at hour 18 (midnight) and 24 (sunrise) in comparison to all the other hours (Fig. 5D). Both pcry and period had troughs in expression at hour 3 (10 am; real time), and then leveled off until after sunset, before rapidly up regulating during the night. Every gene of interest showed significant differences between the expression during at least one daytime hour and one nighttime hour, suggesting differential expression over a 24-hour period and potentially daily rhythmic expression over time.

Figure 5.

Figure 5

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Heat maps showing time points were gene expression was significantly different for each clock gene of interest, clock (A), npcry (B), pcry (C), and period (D) (student’s t-test). The scale is from dark blue to white. The middle of the color scale signifies 0.05 (light blue), anything less than that value (darker blue) is significantly different, whereas anything lighter (white) is not different.

5. Discussion

5.1 Clock transcript localization

FISH labeling revealed a small group of neurons in the brain of M. leonina that consistently exhibited the presence of four putative clock gene transcripts (Figs. 1–3). These included two bilaterally symmetric neurons near the center of each cerebropleural ganglion, and a group of < 10 neurons in the anterolateral region of each pedal ganglion. Thus, the putative circadian clock in M. leonina likely consists of a network of ~20 neurons in two bilaterally symmetric regions of the brain.

A putative circadian circuit of ~20 neurons would rank M. leonina’s clock as one of the smaller clocks in bilaterians, with the exception of several Lepidopteran insects that appear to have a central clock of only ~8 neurons (Sauman & Reppert, 1996; Wise et al., 2002; Sehadová et al., 2004; Sauman et al., 2005; Zhu et al., 2008). The central circadian clock is more than 2–3 times larger in crickets (~50 neurons; Shao et al., 2006, 2008), cockroaches (~50 neurons; Wen & Lee, 2008), and the housefly, Musca domestica (~50 neurons; Codd et al., 2007), more than seven times larger in D. melanogaster (~150 neurons; Allada & Chung, 2010) and three orders of magnitude larger in the suprachiasmatic nucleus of mammals (~20,000 neurons; Stephan & Zucker, 1972; Inouye & Kawamura, 1979; Ralph et al., 1990). Immunohistochemistry data from several decades ago suggest that ~10 neurons in the cerebral ganglion of A. californica contain PERIOD (Siwicki et al., 1989). However, western blots indicated that the antibodies bound to proteins that were 48 and 66 kDa in size, which is significantly smaller than most PERIOD proteins. There are two predicted PERIOD-like proteins for A. californica now available on NCBI (Accession numbers XP_005093378.1 and XP_012944985.1), and the predicted size of these proteins is 127 and 176 kDa, respectively (as determined by the ExPASy Compute pl/Mw tool [https://web.expasy.org/compute_pi/]). Furthermore, the only other known PERIOD protein in gastropods is for B. gouldiana (101 kDa; Constance et al., 2002). Therefore, it is possible that the antibody was not labeling PERIOD in the study by Siwicki et al., (1989), and that the number of neurons in a putative central clock in the brain of A. californica still remains to be determined.

Most prior electrophysiological and histological evidence point to the eyes as the location of the circadian clock in gastropods (Jacklet, 1969; Block & Roberts, 1981; Block & Wallace, 1982; Siwicki et al., 1989; Michel et al., 1993; Constance et al., 2002). In this study, our use of wholemount preparations meant that the FISH probes probably did not have access to retinal neurons inside the eye due to lack of permeabilization of the tough connective tissue surrounding the eye. Furthermore, M. leonina eyes exhibit strong autofluorescence over a wide range of excitation/emission wavelengths, including those of our fluorophores. Therefore, an additional clock in the eyes of M. leonina is still a distinct possibility, although M. leonina without eyes are still capable of maintaining a circadian rhythm of locomotion (Newcomb et al., 2014). There is also prior evidence to suggest that gastropods may have multiple circadian clocks. For example, in A. californica, some animals can maintain circadian rhythms of locomotion in constant darkness after eye removal, suggesting that there must be additional clocks outside of the eyes (Lickey et al., 1977). There is some evidence to suggest that the abdominal ganglia may contain a circadian clock in A. californica (Audesirk & Strumwasser, 1975; Beiswanger & Jacklet, 1975), but the ability of such a clock to maintain circadian activity in constant darkness has not been definitely demonstrated. Furthermore, lesion studies suggest that the circadian system in A. californica lies within the cerebral and buccal ganglia, and sensory structures associated with these regions of the brain (Roberts & Block, 1982).

Regardless of whether or not there is an additional clock in the eyes of M. leonina, the evidence presented here, as well as in a previous study (Newcomb et al., 2014) suggests that there are putative clock neurons in both the cerebropleural and pedal ganglia that may be important in regulating circadian rhythms of locomotion, such as swimming. The neural circuit underlying swimming in this animal consists of four bilateral pairs of neurons, one of which is in the cerebropleural ganglia and the other three are located in the pedal ganglia (Thompson & Watson, 2005; Sakurai et al., 2014). The two pairs of cerebropleural neurons identified in this study, which contain clock transcripts, are in the same region of the cerebropleural ganglia as Swim Interneuron 1 (Thompson & Watson, 2005). In contrast, the pedal clock neurons identified here are anterior and lateral to the pedal swim interneurons, although these pedal swim interneurons are known to have extensive arborizations in the pedal ganglia and neuropil (Thompson & Watson, 2005; Sakurai et al., 2014). Future studies are planned to investigate whether or not the newly identified clock neurons connect to, or modulate, the swim interneurons in M. leonina.

5.2 Clock transcript expression

In general, the qPCR data obtained is consistent with the hypothesis that there are clock neurons in the M. leonina brain that utilize much of the same molecular machinery found in other animals. Npcry was up regulated at night compared to the day, and both period and pcry had peaks of expression around the same time, and slowly ramped up to this peak. Thus, in general, we see expression levels of clock genes that tend to increase in the late evening, and then decline late in the day and into the early evening.

The expression of M. leonina bmal was not measured in this study, but we hypothesize that bmal oscillates independently of period and npcry and, in turn, works with clock to shut off the expression of period; similar to what happens in the mammalian clock (Shearman et al., 2000). Although all of the circadian clock genes show low levels of expression during sunset, period and pcry have slight peaks shortly thereafter, and timeless (data not shown) shows trends mimicking this pattern. This could be when the genes are translated into proteins in the cytoplasm before beginning the negative feedback, shutting off their own transcription and promoting the upregulation of the other genes (Dunlap, 1999; Shearman et al., 2000; Allada & Chung, 2010).

The molecular machinery driving circadian rhythms has now been elucidated in a number of organisms. Based on these studies, there are several different ways that gene expression can lead to circadian rhythms of activity (Bunney & Bunney, 2000; Young & Kay, 2001). The two most well-studied and common patterns are found in the fruit fly and mouse. Both the molecules and oscillation patterns we have documented in brain of M. leonina are more similar to the mouse model than the D. melanogaster model. For example, the D. melanogaster model contains cycle, a circadian gene that binds with clock at the E-box promotor and helps regulate the transcription of period and timeless. In the Mus genus bmal, a homolog of cycle, binds to clock and regulates the transcription of period and npcry (Shearman et al., 2000). Although not included in this study, Cook et al., (in press) have demonstrated the presence of bmal in the M. leonina transcriptome. Therefore, the molecular mechanisms involved in the M. leonina circadian clock(s) contains the same genes present in the mammalian system and the connection/pathway of these genes is also similar. The times of up and down regulation for each gene vary from a mouse to M. leonina, but this is to be expected, because they live in different environments and express a different range and pattern of behaviors.

6. Conclusions

  1. In the gastropod, M. leonina, clock gene transcripts are expressed in a relatively small number of neurons in the brain. There are a pair of putative clock neurons in the cerebropleural ganglia as well as a cluster of 5–10 neurons in each pedal ganglia.

  2. M. leonina transcripts from the circadian genes cryptochrome (both photoreceptive and nonphotoreceptive), clock and period are all expressed differentially over time.

  3. These data indicate that M. leonina might be a good model system for investigating the influence of identifiable clock neurons on the neural networks responsible for generating stereotyped behaviors at specific times of the day or night.

Acknowledgments

We thank Mark Townley (UNH) for his assistance with the confocal microscope, Robert Calin-Jageman for his suggestions of where to get custom in situ hybridization probes, Lou Tisa for providing the qPCR machine, and the staff at the Friday Harbor Laboratories for providing the facilities required to collect and maintain the animals used for the qPCR experiments. We are also grateful to Paul Katz and his colleagues at Georgia State University for access to their transcriptome for M. leonina.

Funding

This work was supported by a grant to JMN and WHW from the New Hampshire-INBRE through an Institutional Development Award (IDeA), P20GM103506, from the National Institute of General Medical Sciences of the NIH.

Footnotes

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Declarations of interest

None

References

  1. Agersborg HPK. The morphology of the nudibranchiate mollusc Melibe (syn. Chioraera leonina) (Gould) Quarterly Journal of Microscopical Science. 1923;67:507–592. [Google Scholar]
  2. Allada R, Chung BY. Circadian organization of behavior and physiology in Drosophila. Annual Review of Physiology. 2010;72:605–624. doi: 10.1146/annurev-physiol-021909-135815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aschoff J. Circadian rhythms in man. Science. 1965;148:1425–1432. doi: 10.1126/science.148.3676.1427. [DOI] [PubMed] [Google Scholar]
  4. Audesirk G, Strumwasser F. Circadian rhythm of neuron R15 of Aplysia californica: photoentrainment. Proceedings of the National Academy of Science, USA. 1975;72:2408–2412. doi: 10.1073/pnas.72.6.2408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beeston DC, Morgan E. A crepuscular rhythm of locomotor activity in the freshwater prosobranch, Melanoides tuberculate (Müller) Animal Behavior. 1979;27:284–291. [Google Scholar]
  6. Beiswanger CM, Jacklet JW. In vitro tests for a circadian rhythm in the electrical activity of a single neuron in Aplysia californica. Journal of Comparative Physiology. 1975;103:19–37. [Google Scholar]
  7. Block GD, Davenport PA. Circadian rhythmicity in Bulla gouldiana: role of the eyes in controlling locomotor behavior. Journal of Experimental Zoology. 1982;224:57–63. [Google Scholar]
  8. Block GD, Roberts MH. Circadian pacemaker in the Bursatella eye: properties of the rhythm and its effect on locomotor behavior. Journal of Comparative Physiology. 1981;142:403–410. [Google Scholar]
  9. Block GD, Wallace SF. Localization of a circadian pacemaker in the eye of a mollusc, Bulla. Science. 1982;217:155–157. doi: 10.1126/science.217.4555.155. [DOI] [PubMed] [Google Scholar]
  10. Bunney WE, Bunney BG. Molecular clock genes in man and lower animals: possible implications for circadian abnormalities in depression. Neuropsychopharmacology. 2000;22:335–345. doi: 10.1016/S0893-133X(99)00145-1. [DOI] [PubMed] [Google Scholar]
  11. Codd V, Dolezel D, Stehlik J, Piccin A, Garner KJ, Racey SN, Straatman KR, Louis EJ, Costa R, Sauman I, Kyriacou CP, Rosato E. Circadian rhythm gene regulation in the housefly Musca domestica. Genetics. 2007;177:1539–1551. doi: 10.1534/genetics.107.079160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Constance CM, Green CB, Tei H, Block GD. Bulla gouldiana period exhibits unique regulation at the mRNA and protein levels. Journal of Biological Rhythms. 2002;17:413–427. doi: 10.1177/074873002237136. [DOI] [PubMed] [Google Scholar]
  13. Cook GM, Gruen AE, Morris J, Pankey MS, Senatore A, Katz PS, Watson WH, III, Newcomb JM. Sequences of circadian clock proteins in the nudibranch molluscs Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea. Biological Bulletin. doi: 10.1086/698467. (in press) [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dunlap JC. Molecular bases for circadian clocks. Cell. 1999;96:271–290. doi: 10.1016/s0092-8674(00)80566-8. [DOI] [PubMed] [Google Scholar]
  15. Hastings M, O’Neill JS, Maywood ES. Circadian clocks: regulators of endocrine and metabolic rhythms. Journal of Endocrinology. 2007;195:187–198. doi: 10.1677/JOE-07-0378. [DOI] [PubMed] [Google Scholar]
  16. Inouye ST, Kawamura H. Persistence of circadian rhythmicity in a mammalian hypothalamic “island” containing the suprachiasmatic nucleus. Proceedings of the National Academy of Sciences USA. 1979;76:5962–5966. doi: 10.1073/pnas.76.11.5962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jacklet JW. Circadian rhythm of optic nerve impulses recorded in darkness from isolated eye of Aplysia. Science. 1969;164:562–563. doi: 10.1126/science.164.3879.562. [DOI] [PubMed] [Google Scholar]
  18. Jacklet JW. Circadian locomotor activity in Aplysia. Journal of Comparative Physiology. 1972;79:325–341. [Google Scholar]
  19. Kavaliers M. Circadian and ultradian activity rhythms of a freshwater gastropod, Helisoma trivolvis: the effects of social factors and eye removal. Behavioral and Neural Biology. 1981;32:350–363. doi: 10.1016/s0163-1047(81)92411-0. [DOI] [PubMed] [Google Scholar]
  20. Koressaar T, Remm M. Enhancements and modifications of primer design program Primer3. Bioinfomatics. 2007;23(10):1289–91. doi: 10.1093/bioinformatics/btm091. [DOI] [PubMed] [Google Scholar]
  21. Kupfermann I. A circadian locomotor rhythm in Aplysia californica. Physiology and Behavior. 1968;3:179–181. [Google Scholar]
  22. Lawrence KA, Watson WH., III Swimming behavior of the nudibranch Melibe leonina. Biological Bulletin. 2002;203:144–151. doi: 10.2307/1543383. [DOI] [PubMed] [Google Scholar]
  23. Lickey ME, Wozniak JA, Block GD, Hudson DJ, Augter GK. The consequences of eye removal for the circadian rhythm of behavioral activity in Aplysia. Journal of Comparative Physiology A. 1977;118:121–143. [Google Scholar]
  24. Michel S, Geusz ME, Zaritsky JJ, Block GD. Circadian rhythm in membrane conductance expressed in isolated neurons. Science. 1993;259:239–241. doi: 10.1126/science.8421785. [DOI] [PubMed] [Google Scholar]
  25. Newcomb JM, Lawrence KA, Watson WH., III The influence of light on locomotion in the gastropod Melibe leonina. Marine and Freshwater Behaviour and Physiology. 2004;37:253–269. [Google Scholar]
  26. Newcomb JM, Kirouac LE, Naimie AA, Bixby KA, Lee C, Malanga S, Raubach M, Watson WH., III Circadian rhythms of crawling and swimming in the nudibranch mollusc Melibe leonina. Biological Bulletin. 2014;227:263–273. doi: 10.1086/BBLv227n3p263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Panda S, Hogenesch J, Kay S. Circadian rhythms from flies to humans. Nature. 2002;417:329–335. doi: 10.1038/417329a. [DOI] [PubMed] [Google Scholar]
  28. Ralph MR, Foster RG, Davis FC, Menaker M. Transplanted suprachiasmatic nucleus determines circadian period. Science. 1990;247:975–978. doi: 10.1126/science.2305266. [DOI] [PubMed] [Google Scholar]
  29. Roberts MH, Block GD. Dissection of circadian organization of Aplysia through connective lesions and electrophysiological recording. Journal of Experimental Zoology. 1982;219:39–50. [Google Scholar]
  30. Sakurai A, Gunaratne CA, Katz PS. Two interconnected kernels of reciprocally inhibitory interneurons underlie alternating left-right swim motor pattern generation in the mollusc Melibe leonina. Journal of Neurophysiology. 2014;112:1317–1328. doi: 10.1152/jn.00261.2014. [DOI] [PubMed] [Google Scholar]
  31. Sauman I, Reppert SM. Circadian clock neurons in the silkmoth Antheraea pernyi: novel mechanisms of period protein regulation. Neuron. 1996;17:979–990. doi: 10.1016/s0896-6273(00)80220-2. [DOI] [PubMed] [Google Scholar]
  32. Sauman I, Briscoe AD, Zhu H, Shi D, Froy O, Stalleicken J, Yuan Q, Casselman A, Reppert SM. Connecting the navigational clock to sun compass input in monarch butterfly brain. Neuron. 2005;46:457–467. doi: 10.1016/j.neuron.2005.03.014. [DOI] [PubMed] [Google Scholar]
  33. Sehadová H, Markova EP, Sehnal F, Takeda M. Distribution of circadian clock-related proteins in the cephalic nervous system of the silkworm, Bombyx mori. Journal of Biological Rhythms. 2004;19:466–482. doi: 10.1177/0748730404269153. [DOI] [PubMed] [Google Scholar]
  34. Shao Q-M, Sehadová H, Ichihara N, Sehnal F, Takeda M. Immunoreactivities to three circadian clock proteins in two ground crickets suggest interspecific diversity of the circadian clock structure. Journal of Biological Rhythms. 2006;21:118–131. doi: 10.1177/0748730405283660. [DOI] [PubMed] [Google Scholar]
  35. Shao Q-M, Hiragaki S, Takeda M. Co-localization and unique distributions of two clock proteins CYCLE and CLOCK in the cephalic ganglia of the ground cricket, Allonemobius allardi. Cell and Tissue Research. 2008;331:435–446. doi: 10.1007/s00441-007-0534-z. [DOI] [PubMed] [Google Scholar]
  36. Shearman L, Sriram S, Weaver D, Maywood ES, Inés C, Zheng B, Kume K, Lee CC, Van der Horst GTJ, Hastings MH, Reppert SM. Interacting molecular loops in the mammalian circadian clock. Science. 2000;288(5468):1013–1019. doi: 10.1126/science.288.5468.1013. [DOI] [PubMed] [Google Scholar]
  37. Siwicki KK, Strack S, Rosbash M, Hall JC, Jacklet JW. An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla. Neuron. 1989;3:51–58. doi: 10.1016/0896-6273(89)90114-1. [DOI] [PubMed] [Google Scholar]
  38. Sokolove PG, Beiswanger CM, Prior DJ, Gelperin A. A circadian rhythm in the locomotor behavior of the giant garden slug Limax maximus. Journal of Experimental Biology. 1977;66:47–64. doi: 10.1242/jeb.66.1.47. [DOI] [PubMed] [Google Scholar]
  39. Stephan FK, Zucker I. Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proceedings of the National Academy of Sciences USA. 1972;69:1583–1586. doi: 10.1073/pnas.69.6.1583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takahashi JS. Circadian rhythms: from gene expression to behavior. Current Opinion in Neurobiology. 1991;1:556–561. doi: 10.1016/s0959-4388(05)80028-5. [DOI] [PubMed] [Google Scholar]
  41. Takahashi JS. Molecular neurobiology and genetics of circadian rhythms in mammals. Annual Review of Neuroscience. 1995;18:531–553. doi: 10.1146/annurev.ne.18.030195.002531. [DOI] [PubMed] [Google Scholar]
  42. Thompson S, Watson WH., III Central pattern generator for swimming in Melibe. Journal of Experimental Biology. 2005;208:1347–1361. doi: 10.1242/jeb.01500. [DOI] [PubMed] [Google Scholar]
  43. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG. Primer3-new capabilities and interfaces. Nucleic Acids Research. 2012;40(15):e115. doi: 10.1093/nar/gks596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wen C-J, Lee H-J. Mapping the cellular network of the circadian clock in two cockroach species. Archives of Insect Biochemistry and Physiology. 2008;68:215–231. doi: 10.1002/arch.20236. [DOI] [PubMed] [Google Scholar]
  45. Wise S, Davis NT, Tyndale E, Noveral J, Folwell MG, Bedian V, Emery IF, Siwicki KK. Neuroanatomical studies of period gene expression in the hawkmoth, Manduca sexta. Journal of Comparative Neurology. 2002;447:366–380. doi: 10.1002/cne.10242. [DOI] [PubMed] [Google Scholar]
  46. Young MW, Kay S. Time zones: a comparative genetics of circadian clocks. Nature Reviews Genetics. 2001;2:702–715. doi: 10.1038/35088576. [DOI] [PubMed] [Google Scholar]
  47. Zhu H, Sauman I, Yuan Q, Casselman A, Emery-Le M, Emery P, Reppert SM. Cryptochromes define a novel circadian clock mechanism in monarch butterflies that may underlie sun compass navigation. PLoS Biology. 2008;6(1):e4. doi: 10.1371/journal.pbio.0060004. [DOI] [PMC free article] [PubMed] [Google Scholar]

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