The Circadian Rhythms of Valve Movements in the ... - Springer Link

ISSN 10630740, Russian Journal of Marine Biology, 2010, Vol. 36, No. 6, pp. 419–428. © Pleiades Publishing, Ltd., 2010. Original Russian Text © V. F. Gnyubkin, 2010, published in Biologiya Morya.

ECOLOGY

The Circadian Rhythms of Valve Movements in the Mussel Mytilus galloprovincialis V. F. Gnyubkin Karadag Nature Reserve, Ukrainian National Academy of Sciences, Feodosia, 98188 Ukraine email: [email protected] Received January 19, 2009

Abstract—The longterm (10–30 day) continuous recording of valve movements in the mussel Mytilus gal loprovincialis was carried out in the laboratory under nearly natural conditions. Fourier analysis revealed the circadian (close to the diurnal) rhythm of the valve activity, the phase of which is readily shifted by a shift in the beginning of the daytime, since the light regime is one of the main factors determining the circadian rhythm. The circadian rhythm was manifested in the daily dynamics of mussel valve activity: in the daytime, mussels hold their valves closed more often than at night. This behavior may be a protective response, namely the “shadow reflex”: mussels close their valves upon a sudden decrease in illumination, thus protecting them selves from possible predators. Circadian activity can mask a mussel’s response to environmental pollution; therefore, regular valve closure should be taken into account in early warning systems such as “MusselMo nitor®,” with a correction for the season of the year, time of day, and other factors. Keywords: Mytilus galloprovincialis, circadian rhythm, STFT analysis, early warning system, MosselMoni tor®. DOI: 10.1134/S1063074010060039

The activities of living organisms are affected by many abiotic factors (light and darkness, warmth and cold, or high and low tides), most of which are recur rent phenomena with a specific rhythm. As a result, plants and animals are also subject to rhythmic activity that is dependent on environmental changes [4, 27].

www.mosselmonitor.n1]. The function of these sys tems is based on recording valve movements in mol lusks. Therefore, natural daily activity during the night or during daytime “sleep” may mask the reaction of mussels to contamination, which must be considered when designing an early warning system.

A rhythm of activity that is synchronous with the tides (tidal rhythm) in bivalve mollusks (in mussels among others) was first discovered when examining the rate of water propulsion in mollusks [28]. Later, both tidal and daily rhythms were recorded in mussels and clams (freshwater and marine) [6, 11, 15, 17, 21, 23, 26, 30]. In mussels, different rhythms appear dur ing recording of byssus thread production [25], varia tions of metabolism [11], valve movements [6, 23], cardiac function [3], and mantle movements [29].

The task of this work was to study the dynamic range of valve opening in the Mediterranean mussel Mytilus galloprovincialis, the repetition (rhythm) of their movements, the influence of the light mode on the parameters of the rhythm, as well as the daily dynamics of the opening/closing of mollusk valves. The materials of this study were partly presented pre viously [5].

The rhythm of valve movements in clams is the recurrent opening and closure of shell valves. In Cor bicula fluminea shell valves are synchronously closed for 2–3 hours every day [26]. Similar periods of shell closures have been recorded for other shellfish as well, for example, in Mytilus edulis [23] and Dreissena poly morpha [15]. It must be stressed that the synchronous closure of valves in 70% of the mussels used in the sys tem from 4 min to 1 h [14] is used as an alarm signal in all early warning systems for pollution of the aquatic environment, such as the biosignalizator of toxicity [9], the MusselMonitor® and DreissenaMonitor® systems [15, 22, http://www.mermayde.n1; http://

MATERIAL AND METHODS The work was performed in the Karadag Nature Reserve (Feodosia, Crimea, Ukraine) from 2007 through 2009 on the Mediterranean mussel Mytilus galloprovincialis. Mussels with a valve length of about 60 mm were collected at a depth of 0.2–0.5 m and kept for at least 1–2 days in aquaria for acclimatization to the laboratory conditions before the experiments. The water temperature was not regulated during acclimati zation and through the experiments; it was in the range of 10–24°C, corresponding to the water temperature in the coastal waters. The mussels were not addition ally fed. The buffer tank (200 l) in the laboratory was

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Fig. 1. Attachment of the position recording device (Hall’s sensor–magnet) to mussel valves. a, geometrical parameters of the mussel as a lever model, only the upper valve is presented (AD, the axis of rotation; C, the area of the valve most distant from the axis of rotation and maximally distant from the antipod valve at opening; R, distance from the axis of rotation to point C; BE, the line of equal distance from the axis of rotation; S, the area recommended for the installation of a Hall’s sensor and magnet); b, an example of the attachment of the Hall’s sensor and magnet to the mussel.

filled with sea water from a large external pool (about 150 m3), where water was periodically changed. Dur ing the experiment all the mussels were kept in a 25 liter aquarium with running water. The flow rate was about 2.5–3 l/h per mussel; this is close to the speed of water filtration in mussels, as reported by many authors, e.g., 1–1.5 l/h [10], 2–6 l/h [28], and 2–5 l/h [8]. The experiments were carried out in natural light (about 0 lx at night and from 2–5 to 30–40 lx in the day) in the summer of 2007 and with artificial illumi nation in 2007–2009: 12 h light (about 30 lx): 12 h dark (about 0 lx); the light was switched on at 7:00 and off at 19:00, Kiev summer time. The clock was not turned to winter time (Kiev summer time is close to the astronomical time at the location of the experi ments). To examine the potential effect of the artificial illumination mode on the activity of mussels, the illu mination period was shifted by 6 h in December 2008, so the light was on at 1:00 and off at 13:00 and then the original illumination mode was resumed. Illumination in the vicinity of mussels was measured in relative units with the use of photodiodes, viz., occasional measure ments in 2007 and continuous recording in 2008– 2009. In addition, the altitude of the sun above the horizon was calculated and recorded [1]. The altitude index of the sun (sinus of the angle between the direc tion to the sun and the horizontal plane) is shown on the graphs of the dynamics of valve movements and this makes it possible to correlate dynamics and the astronomical light regime. In order to record the opening of the valves for 4– 12 mussels simultaneously we used an electronic recorder designed by Stolbov et al. (2004) with 1 min discreteness of records (in the experiments in the autumn of 2007 and winter of 2008) or a radical mod ification designed by V.Zh. Mishurov et al. (in press)

with a record discreteness of 0.1 s and 1 min (summer 2007). A recording device designed by the author of this paper on the basis of an LA50USB analogdigital converter (Rudnev–Shilyaev) with a discreteness of 0.001–1 s was also used (winter 2008–summer 2009). All these devices are similar to other recording devices applied in early warning systems. The monitor–recording device of our own design differed from other monitors by having parts for attaching valve position sensors to animals, as well as differing in signal processing algorithms. The valve position sensor was constructed on the basis of a Hall sensor, which is sensitive to magnetic fields. A Hall effect transducer was attached to one shell valve and a permanent magnet to the other one. The Halleffect transducer and the magnet were installed outside the shell (Fig. 1a, point S) and attached on a line of equal distance (BE) from the axis of rotation (AD) of the valves; with the line of an equal distance running through the most distant point (C) on the mussel valve. This design allowed us, when necessary, to recalculate the voltage value from the Hall sensor into the absolute opening of the valves in point C. The voltage value from the Hall sensors was recorded in the monitor memory. A longterm (October 2007–June 2009) record of Hall sensor voltages proportional to the valve opening in 4 to 12 mollusks was conducted with the use of monitor recording devices, with intervals of 0.1 s ⎯1 min. The record was conducted as continuously as possible. Some interruptions in records arose due to technical reasons (nonsynchronous clock function, voltage drops, or program crashes, and the need to clean the memory device) and lasted from a few seconds to sev eral hours. Upon further mathematical processing of long data series, such interruptions in signal recording were filled with a portion of the same length taken

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from the “tail” part of the signal preceding the record interruption, i.e., it was assumed that the behavior during the interruption was the same as before it. The individual reactions of the tested mussels varied sharply under the same conditions, so we averaged the values of valve openings within a mussel group of 4–12 individuals, except in the analysis of mussel reactions to changes in light conditions. The averaged value of the opening of mussel valves was subjected to mathematical processing. Spectral analysis of the activity was performed in Excel and MathCAD with the use of complex Fouriertransform [7] and STFT analysis (Short Term Fourier Transfor mation), i.e., a Fouriertransform over short time intervals (http://users.rowan.edu/~polikar). STFT analysis is a tool that uses the spectral composition of a signal over time to show the signal, not only for the entire time of the experiment, as complex Fourier analysis does, but also over any specific interval of the experimental time [19]. The data processing algorithm during STFT analy sis operated as follows: – Low and highfrequencies filters were created to purify the data from an oscillation constant of less than one third of an oscillation per day, as well as from high frequency oscillations with a frequency greater than one oscillation per hour; slow rhythms with a period greater than 3 days and ultradian rhythms detected by other authors [2, 3, 11, 29] were not considered; – We separated a portion of the signal with a certain duration with a “window” on the normal distribution density curve (Gaussian) from a signal containing a nearly circadian rhythm and other similar rhythms; its spectral composition was analyzed using a complex Fourier transform; the result was written into the matrix column; – Next, the “window” slid one time step further along the curve (without changing the Gaussian width) to separate the next portion of the signal: its spectral composition was determined; the result was written to the next column of the matrix; this proce dure was repeated until the end of the signal; and – A pseudo3D graph of the STFT (Short Term Fourier Transformation) was built in timefrequency amplitude coordinates using the results stored in a matrix; a graph presented the spectral composition of the signal at any moment of observation. In addition to STFT analysis of valve movements, we recorded the dynamics of the movements within the day and their variations. Long series of records were divided into portions of 24 hours and we super posed them to define the average opening and to cal culate the error of the average opening for intervals of 1 min and 6 h [26]. The dynamics of valve movements were defined for periods of 10 to 30 days in a light mode that was nearly natural and with a 6h shift of the light mode. To demonstrate the reaction of mussels to shadowing (“shadow reflex” after: Kennedy, 1960) RUSSIAN JOURNAL OF MARINE BIOLOGY

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[20], we recorded the reaction of the mussels to a shortterm change in illumination. RESULTS AND DISCUSSION To study the circadian rhythm and its variations, the author separated and thoroughly studied the records of 4–6 mussels, which, according to the author, most fully reflected the diversity of reactions from a practically continuous record (from October 2007 to June 2009) of the valve opening of 4–12 mus sels. A visual examination showed the following details (Fig. 2): (1) the opening of valves ranged from 0 up to 10 mm (mostly 1–4.5 mm), the valve opening in the day was smaller than at night; (2) repeated oscillations with a period of approximately 24 hours composed the circadian rhythm (Fig. 2a: the circadian rhythm has a stable frequency; Figs. 2b–2c: the rhythm is present but is less stable); and (3) very slow nonsinusoidal oscillations (Fig. 2), which apparently reflect seasonal rhythms of activity. A more detailed picture of the activity can be obtained with spectral analysis using Fouriertrans form and STFT conversion. Repeated oscillations with a frequency close to one per day, i.e., a circadian rhythm, were observed in all the experiments (Fig. 3). This rhythm was best expressed in experiment A (Fig. 3a), where it very weakly changed in frequency and amplitude. This was an exception. Usually, the frequency and amplitude of the circadian rhythm changed and, correspondingly, the rhythm underwent metamorphoses: it could be well pronounced and could die down; it could gradually evolve or divide into two oscillations, then connect again to one rhythm, accelerate, or slow down (Figs. 3b–3c). In other words, the spectral analysis shows that the frequency of valve movements is about one oscillation per day (circadian rhythm), as well as two and more oscilla tions per day. It is noteworthy that the circadian rhythm was found in all the records, but more than one oscillation per day is detected only fragmentarily, so there is no reason to claim that in our experiments the semidiurnal or tidal rhythm was regular, as was recorded for mussels elsewhere [28]. The dynamics of the circadian rhythm of valve movements, i.e., the dynamics of valves within 24 hours (with the open and closed positions of the valves corresponding to a certain time of day) becomes evident during superposition of 24hour portions of records (for 10–30 days) of an average activity of 4 to 6 mussels. Most records (represented and not repre sented in the figure) show that the opening of valves is greater at night and smaller from 9:00 to 17:00 (Figs. 4A, 4B). The same can be noted when analyzing the entries for minor intervals (every minute) and, with greater statistical reliability, for considerable intervals (6 hours), (Fig. 4). However, records No. 6

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(Fig. 4C) where valve openings are nearly the same in the daytime and at night also occurred (20–25% of all records). A diurnal activity pattern in clams was also reported by other researchers. Thus, in experiments with Mediterranean mussels, Slatina [11] noted the most active breathing during the afternoon and evening (but not at night), as well as variations of activ ity during the year. In Corbicula, the greatest opening of valves was recorded in the afternoon, while at night the mollusks were completely closed for 2–3 h [26]. In contrast, the blacklip pearl oyster Pinctada marga ritifera, like the mussels in our experiments, were open at night more widely than in the daytime [17]. An increased production of byssus thread as a sign of activity occurred in mussels at night [25]. The fresh water mollusks Anodonta anatina and Unio tumidus were also more open at night than in the morning and in the daytime [18]. The observed behavior of mussels is probably rele vant to their response to changing light conditions, although they have no anatomically pronounced eyes [8]. As an experiment showed (Fig. 5), dimming of illumination and light cutoff (even for a fraction of a second) caused the rapid (with a latency period of about 1 s) closing of valves for 1–5 min (Fig. 5b), while

an increase in illumination did not induce a visible reaction (Fig. 5a). Thus, a distinct response to changes in light conditions, especially to shadowing, the so called “shadow reflex” [16, 20], was recorded in Med iterranean mussels, as in other clams (Mytilus edulis, Dreissena polimorpha, Unio pictorum, representative of the genus Spisula). Evidently, under natural condi tions and in vitro, mussels associate reduced lighting (due to local shadowing from waves or clouds or move ments of neighbors and enemies) with the approach of a predator and close their valves for 1–5 min. There fore, closing of the valves in the daytime and opening of the valves at night can be considered as an adaptive response of the mollusks, i.e., protection from visu allyoriented predators [13, 17]. Illumination and changes in its intensity cause not only an immediate reaction to light, including “the shadow reflex,” but also serve as a factor that defines the overall mode of activity and synchronizes the activity of the animals with daily and seasonal envi ronmental changes [4, 27]. In order to test the impact of the light regime on the pattern of the circadian rhythm, we studied the dynamics of the valve move ments in manydaylong experiments with changing phases of the light regime during shifts of the daylight hours (Fig. 6): 30 days, when the light was on and off,


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respectively, at 7:00 and 19:00 (Fig. 6A); 24 days, the same at 1:00 and 13:00 (Fig. 6B), and 20 days with the light mode returned to the original one. Shifting the beginning of the daylight hours led to a shift in mussel activity, i.e., the phase of the diurnal RUSSIAN JOURNAL OF MARINE BIOLOGY

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rhythm changed. So, for daylight beginning at 7:00 and during the artificial light mode “12 h light : 12 h dark,” the dynamics of the activity were the same as that under natural light conditions: during astronomi cal daytime, the valve gap was smaller than at night (Figs. 4A–4B, Fig. 6A). If the rhythms were deter No. 6

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Fig. 4. Daily dynamics of valve openings in the experiments of 2007–2008. A, July 13–28, 2007, 4 mussels; B, July 31–August 10, 2007, five mussels; C, September 12–22, 2008, six mussels. The beginning and termination of recording: 0:00 Kiev summer time. Ordi nate axis: a and b, valve opening, mm; c, the altitude of the sun above the horizon presented as sinus of the angle (on the vertical plane) between the direction to the Sun and the horizontal plane); d, illumination (relative units) measured with a photocell. The average gap (correspondingly, solid lines and heights of columns) and the error of average gap (correspondingly, dash lines and figures at the columns) were calculated at the 0.95 level. RUSSIAN JOURNAL OF MARINE BIOLOGY

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Fig. 6. The dynamics of daily openingclosure of valves in 6 mussels (a) at shifts of the beginning of the day time. A, November 1–December 1, 2008, light on at 7:00, light off at 19:00; B, December 2–December 25, 2008, light on at 1:00, light off at 13:00; C, December 26, 2008–January 15, 2009, light on at 7:00, light off at 19:00; b, the altitude of the Sun above the horizon presented as sinus of the angle (on the vertical plane) between the direction to the Sun and the horizontal plane (dash line); c, illumination, rel. units: about 0.3, “daytime,” about 0, “night” (solid line). Dark time of artificial days is marked by a dark back ground. Kiev summer time. No. 6

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mined only by endogenous factors and illumination had no effect the behavior then the same pattern of the mussel activity would have been observed during an artificial shift of the daylight start time, i.e., when the light was switched on at 1:00 and off at 13:00. How ever, the shift of the beginning of the day lighting was followed by a shift of the relative opening of the valves, and the shell valves were more closed than in the dark, regardless of the astronomical time (Fig. 6B). A return to the initial lighting regime (light on at 7:00, light off at 19:00) led to almost a return of the dynamic activity of mussels, which is observed at natural lighting (Fig. 6C). In other words, the curves of the daily open ing of valves almost repeat (in antiphase) the curve of the sun’s height above the horizon (Figs. 4A–4B; Figs. 6A, 6C), if the daylight hours coincide with the astronomical day. However, if the daylight hours are shifted, the dynamics of mussel activity followed this shift (Fig. 6B) and markedly differed from that observed for lighting close to the natural astronomical day. It is noteworthy that sharply turning off the lighting “in the evening” induced (Fig. 4C, Fig. 6) two reac tions: a quick “shadow reflex” and a smooth increase in valve opening. Shortterm responses to changes in lighting (a quick response to shading) and longterm responses (the activity follows the general illumination in antiphase with a period of approximately 24 hours, i.e., a circadian rhythm) are probably based on differ ent neurological mechanisms. This assumption can be confirmed by the very rapid (milliseconds) closure of the valves that is caused by switching off the light (with a latency of about 1 s), with subsequent opening in 1– 5 min (Figs. 5, 6). However, if illumination was kept constant after switching off the light (or switching it on), we also observed other reactions: the valves gently closed with increased light and smoothly opened upon a light decrease, regardless of how fast the lighting changed, for fractions of a second for the artificial lim its of daylight hours or for a few hours during natural lighting. The founders of “rhythmology” [4, 27] believe that circadian rhythms are inherent to all living organisms and have a distinct endogenous component with a period close to the duration of a day. To define the endogenous component of rhythmic activity in pure conditions experiments are conducted under constant ambient conditions (light, water flow, and food avail ability), these requirements are rarely actually met. The rhythms were examined in several mollusks under constant ambient conditions. Thus, K.P. Rao, who was the first to observe tidal rhythmicity in the rate of water propulsion in Mytilus and who did not find a diurnal rhythm, stated: “No indications of a diurnal rhythm in the rate of water propulsion were observed” [28, page 358]. In contrast, other authors [13] have noted a weak diurnal rhythm in the absence of tidal rhythm when recording the valve movements of mus sels under constant conditions. In experiments under

constant experimental conditions, Langton and Gab bott found neither daily nor tidal rhythms in the oyster Ostrea edulis; the state of the crystalline cone depended only on the availability of food [24]. We note that not all the potential synchronizing factors were eliminated in our experiments on the role of lighting. For example, the water temperature changed with the water temperature in the sea, the concentration of food depended on the flow of water from the sea, etc. However, regardless of these external factors we observed lightdependent behavior of the mussels: a greater valve gap in the dark, with phase shifts of the circadian rhythm following shifts of the early daylight hours (Fig. 6). Therefore, we can assume that the behavior of the Mediterranean mussel is mostly determined by the lighting regime and not just by socalled endogenous mechanisms, whose contri bution remains unknown. At the same time, we cannot exclude the influence of some destabilizing and syn chronizing factors such as diet, the presence of neigh bors, etc. [13, 24, 25]. The susceptibility of the circa dian rhythm of the Mediterranean mussels to external influences may result in a lesspronounced rhythm, in comparison, for example, with Corbicula fluminea [26]. The rhythm was distinctly pronounced in approximately 75–80% of all records and the differ ence between night and daytime valve gaps was some times insignificant (Fig. 4C). Regardless of the nature of the circadian rhythm that also occurs under natural conditions, it brings about the regular closure of valves to less than 30% of the normal opening (Fig. 2). Such a gap is comparable to the opening that is accepted as a threshold to trigger the alarm signal in the early warning systems of an unfavorable state of the environment, in a similar manner to the MosselMonitor® system. This regular closure of valves caused by rhythms should be consid ered when adjusting a warning system; this can be done using appropriate software or with the use of coefficients that are dependent on the time of the day, season, and other factors of the environment. ACKNOWLEDGMENTS The author extends his thanks to A.L. Morozova, Director of the Karadag Natural Reserve of the Ukrai nian National Academy of Sciences, for the opportu nity to work under contract; to M.A. Polyakov, Head of the Laboratory of Bioacoustics of Karadag Natural Reserve for the opportunity to work with MathCAD software, as well as to my colleagues for their coopera tive efforts during the preparations for recording valve movements of the mussels: V.Zh. Mishurov and K.N. Kuzmin, researchers of the Hydrological Physi cal Institute of the Ukrainian National Academy of Sciences for the modification of one recorder; V.N. Nikolsky, A.Ya. Stolbov, and V.V. Trusevich, researchers of the Institute of Biology of the Southern Seas of the Ukrainian National Academy of Sciences


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