Looking for the clock mechanism responsible for circatidal behavior in the oyster Crassostrea gigas

Valve activity rhythm of the oyster Crassostrea gigas is mainly driven by tides in the field, but in the laboratory, only a circadian clock mechanism has been demonstrated. In an attempt to reconcile these results, the mechanisms underlying the circatidal rhythm were studied in the laboratory under different entrainment or free-running regimes and in the field at Arcachon (44°39′N/1°09′W) in February–April 2011). Results confirm the existence of a circadian clock in C. gigas. Under entrainment regimes (12-h dark/12-h light photoperiod and tidal cycles simulated by a reversing current flow), oysters exhibited both circadian and circatidal cycles. Under free-running conditions (e.g., continuous darkness), the endogenous rhythm appeared to be circadian. There was no experimental evidence for an endogenous circatidal rhythm, even in oysters just transferred from the field, where a clear tidal cycle was expressed. There are two possible mechanisms to explain tidal behavior in C. gigas: an exogenous tidal cue that drives tidal activity and masks the circadian rhythm and an endogenous circatidal clock that is sensitive to tidal zeitgebers and runs at tidal frequency.     

Photosynthesis and chlorophyll a fluorescence rhythms of marine phytoplankton

Circadian rhythms in photosynthesis were defined in field populations of phytoplankton. Measurements of carbon-dioxide fixation rates demonstrated that a diurnal periodicity of photosynthesis in samples incubated under natural light-dark (LD) cycles also were observed to continue in similar samples which had been photoadapted to constant dim light (LL) for 48 h. These changes in photosynthetic rates preceded sunset and sunrise, had daily amplitudes that ranged from 1.5 to 2.0, appeared to be independent of light-intensity, and displayed maxima about midday, while rates of dark fixation of carbon dioxide and the photosynthetic pigment content per cell were constant over the circadian cycle. Similar rhythmicity also was detected in room-temperature (22°C) chlorophyll a fluorescence yield, in both the obsence and presence of the photosynthesis inhibitor DCMU [3-(3,4-dichlorophenyl)-1, 1-dimethylurea]. However, the magnitude and timing of the fluorescence rhythm maxima seem to depend on wavelengths monitored and, in part, on the measuring technique used. Also, the circadian changes in the fluorescence intensity were abolished at low temperature (-60°C), and the shape of the emission spectra of chlorophyll fluorescence of cells in LD and LL did not change over time. The significance of the fluorescence rhythms with regard to chlorophyll a determinations and photosynthetic rates is discussed. It was concluded that there was sufficient similarity between circadian rhythms of photosynthesis in natural phytoplankton populations and in laboratory cultures of dinoflagellates to suggest that the mechanism of regulation may be the same for both of them.     

The role of the queen in circadian rhythms of honeybees (Apis mellifera L.)

Summary Colonies and smaller social groups of honeybees (Apis mellifera carnica L.) show distinct free-running circadian rhythms similar to that of individual organisms. The workers of a colony synchronize their individual rhythms to one overall group rhythm. Caste plays an important role in this synchronization process. Queens were introduced into worker groups which were entrained to a phase-shifted light/dark cycle. The introduction of the queen caused a shift in the free-running phase under constant dark conditions. Single introduced workers had no effect on the free-running rhythms. This indicates that the queen plays an important role in the synchronization of circadian rhythms of honeybee colonies.     

Circadian rhythm of swimming activity in juvenile pink salmon (Oncorhynchus gorbuscha)

The circadian rhythm of swimming activity and the role of the daily illumination cycle in the synchronization of this rhythm were studied in individual juvenile pink salmon. Sixty eight percent of all fish examined (n=38) were day-active when exposed to a 12 h L:12 h D cycle; the remaining fish were nocturnally active. One half of the fish tested under laboratory conditions of continuous, constant light intensity (LL) and constant temperature showed unambiguously endogenous activity rhythms with circadian periods for up to 10 d. The remaining fish were arrhythmic. Mean period length of the free-running activity rhythms for diurnal fish in LL shortened with constant light intensity increasing from 6 to 600 lx, as predicted by the circadian rule. In contrast, mean free-running period for nocturnal fish did not vary significantly with similarly increasing constant light intensity. Mean swimming speed (activity level) of both diurnal and nocturnal fish increased significantly with increasing light intensity. This is suggestive of a positive photokinetic response. When subjected to a phase-delayed LD cycle, the fish resynchronized their daily rhythms of activity with this new LD cycle after only one transient cycle in most instances. Hence, the timing of the daily activity rhythms appeared to occur through the direct masking action of the illumination cycle on activity, rather than through entrainment of an endogenous circadian system.     

Light and social effects on the free-running circadian activity rhythm in common marmosets (Callithrix jacchus; Primates): social masking,pseudo-splitting,and relative coordination

Summary The variability of period of the free-running circadian activity rhythm (CAR) and the degree to which it is affected by social factors was studied in diurnal marmosets (Callithrix jacchus: Primates, Cebidae), kept either singly or in pairs. Under continuous light intensities between 10^-1 and 10² lx, the spontaneous period was always shorter than 24h. It varied in relation to the amount of time spent under constant conditions (after-effects). There was some evidence of an effect of light intensity on free-running period, but no clear correlation between the two. Mutual acoustic social contact caused some males to exhibit pseudo-splitting, ascribable to positive social masking, and in many cases also resulted in relative coordination of the free-running CAR. True social entrainment, however, was not produced. The possibility that the latter could occur under some conditions is discussed, as well as the neural pathways that might mediate the observed acoustically induced social effects on the central nervous system pacemaker(s) of the circadian timing system.     

Circatidal rhythm of an intertidal crab, Hemigrapsus sanguineus : synchrony with unequal tide height and involvement of a light-response mechanism

The circatidal rhythm of intertidal animals may reflect the inequality of the tides. In addition, a light-sensitive mechanism may be involved in their internal timing systems. To test these hypotheses, the larval release activity of the intertidal crab Hemigrapsus sanguineus was monitored under different light conditions in the laboratory. Under a 24-h light–dark (LD) cycle with the phase similar to the field, the activity coincided with the times of high tide in the field and showed a tidal rhythm. This rhythm free-ran in constant, dim-light conditions, suggesting that the timing is controlled by an endogenous clock. When the population was exposed to a 24-h LD cycle with the phase changed from that in the field, the tidal rhythm was phase-shifted; while the light cycle advanced in phase from the field caused a phase-advance of the rhythm, that delayed in phase induced a phase-delay of the rhythm. Thus, a light-response mechanism is definitely involved in circatidal timing systems. But the population rhythm showed a large variability among individuals, associated with the phase-shift, and the magnitude of the phase-shift did not accurately correspond to that of the light cycle. These results suggest that the light-response system can control the phase of the rhythm less stronger than that in estuarine crabs. Most releases occurred at higher high tides, but the release of some females obviously occurred at lower high tides. The larval release pattern thus could not be accounted for by a simple synchrony with higher high tides. Hatching of H. sanguineus occurred after a “hatching program” of 49.5 to 52.5 h. This program is initiated by some factor (hatching-program inducing stumuli: HPIS) transmitted from the female to the embryos. We speculated that this factor is effectively transmitted to the embryos when the habitat is exposed to air, i.e., at lower low-tide periods, and that once each embryo is stimulated, hatching occurs synchronously 2 d later during high tide. The release of HPIS is probably controlled by the circatidal clock of the female, and the 24-h LD cycle may participate in shifting this timing to the opposite low tide. Received: 14 January 1997 / Accepted: 18 February 1997     

Hatching rhythms in Uca pugilator (Decapoda:Brachyura)

Gravid female Uca pugilator (Bosc) maintained in conditions of 14h L: 10h D light regime and constant temperature without simulated tides showed a significant positive correlation between time of hatch and time of the nighttime high tide. Light: Dark experiments showed no entrainment by LD cues. Larval hatching was delayed until dark when high tide occurred between 1900 and 2130 hrs. Hatch time was a positive linear function of the time of the nighttime high tide when high tide occurred after 2130 hrs. Time of hatch was delayed by the light period on days lacking a nighttime high tide. Experiments conducted under constant light (LL) and temperature, without simulated tides, indicated a persistent free-running rhythm of approximately 25 h 03 min, suggesting an endogenous timer. Rhythmic larval release on the nighttime high tide may be adaptive as a predator-avoidance mechanism for adult gravid females.Contribution No. 366 from the Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina     

Diurnal and circadian rhythms in the turbidity of growing Skeletonema costatum cultures

Skeletonema costatum (Grev.) Cleve grown in batch culture at low light intensity under a 14 h light: 10 h dark photocycle showed exponential cell proliferation (1.1 doublings d^-1) without significant phasing of the cell division by the light: dark cycle. The growth in carbon concentration was, however, restricted to the light period. The turbidity of the culture closely followed the carbon oattern, and was not affected by the increase in the cell number during the dark period. It was found that a trustule suspension had only approximately 1% of the turbidity of the corresponding intact algae. Culture turbidity was therefore regarded as a biomass parameter similar to the carbon concentration, without direct correlation to the timing of the cell division. The short-time variations in the turbidity of growing algal cultures were further studied in a cage culture turbidostat. The growth rate (based on turbidity) increased rapidly during the first half of the light period, decreased slightly towards the evening and was zero throughout the dark period. When transformed to continuous light, the growth of the culture continued to show damped oscillations for up to 1 wk, but with a period of 26.7 h instead of 24 h. The same circadian rhythm was observed in chlorophyll content, and is thus possibly a reflection of a freely oscillating internal biological clock. The cage culture turbidostat was found to be a suitable device for studies of the photocycle related regulation of biosynthesis in S. costatum.     

Dark survival of autotrophic,planktonic marine diatoms

A general ecological problem is considered: how long can a photoautotrophic microalga, incapable of producing a resting spore (stage), retain its viability in the dark following removal from the euphotic zone? Nine coastal diatoms, including some capable of producing resting spores, were kept in the dark for 90 days at 15°C, and their growth (viability) checked at periodic intervals upon reillumination. Seven of the 9 diatoms retained their viability for 90 days; generation time of illuminated cells then ranged from 2.5 to 10 days. Skeletonema costatum survived only 7 weeks of darkness. Based on the present and published observations, dark survival of this species is inversely related to temperature; it survives at least 24 weeks at 2°C, and from 1 to 4 weeks at 20°C. None of the species was observed to grow in the dark. The effects of temperature and light on dark survival, and of darkness on the chemical composition and photosynthesis following reillumination as reported in the scattered literature are evaluated. Together with the present observations, it is suggested that dark survival of photoautotrophic microalgae: (1) varies between species; (2) may be temperature dependent in some species, as in S. costatum; (3) may be prolonged by periodic illumination at subcompensation intensities for photosynthesis, as shown in Dunaliella tertiolecta. The potential ecological significance of these findings is also considered, should these in vitro results apply to natural populations.     

Photosynthetic activity of the non-dormant <Emphasis Type="Italic">Posidonia oceanica</Emphasis> seed

The photosynthetic adaptive features of non-dormant seeds in Posidonia oceanica were studied in order to evaluate the effects of light on germination success. Transmission electron micrographs showed the presence of chloroplasts in the epidermal cells, close to the nucleus at the periphery of the cytoplasm. The well-developed thylakoid membranes and the presence of starch granules indicated that the chloroplasts were photosynthetically active. The relationship between photosynthesis versus irradiance in P. oceanica seeds incubated at 15 and 21°C was analysed. The net photosynthesis in the non-dormant seed of P. oceanica was positive and compensated its respiration demand (90 μmol quanta m⁻² s⁻¹) at both temperatures. Net photosynthesis was negative at the other irradiance values. To test the effects of light on germination success, seeds were placed both in dark and light conditions. Germination success was significantly higher in light rather than in dark condition. The characteristics observed in the photosynthesis in P. oceanica seed could be a mechanism to guarantee seedling survival in temperate waters, demonstrating though the specialized nature of this species.     

The mechanism of calcification and its relation to photosynthesis and respiration in the scleractinian coral <Emphasis Type="Italic"> Galaxea fascicularis</Emphasis>

The mechanism of calcification and its relation to photosynthesis and respiration were studied with Ca²⁺, pH and O₂ microsensors using the scleractinian coral Galaxea fascicularis. Gross photosynthesis (Pg), net photosynthesis (Pn) and dark respiration (DR) were measured on the surface of the coral. Light respiration (LR) was calculated from the difference between Pg and Pn. Pg was about seven times higher than Pn; thus, respiration consumes most of the O₂ produced by the algal symbiont's photosynthesis. The respiration rate in light was ca. 12 times higher than in the dark. The coupled Pg and LR caused an intense internal carbon and O₂ cycling. The resultant product of this cycle is metabolic energy (ATP). The measured ATP content was about 35% higher in light-incubated colonies than in dark-incubated ones. Direct measurements of Ca²⁺ and pH were made on the outer surface of the polyp, inside its coelenteron and under the calicoblastic layer. The effects on Ca²⁺ and pH dynamics of switching on and off the light were followed in these three compartments. Ca²⁺ concentrations decreased in light on the surface of the polyp and in the coelenteron. They increased when the light was switched off. The opposite effect was observed under the calicoblastic layer. In light, the level of Ca²⁺ was lower on the polyp surface than in the surrounding seawater, and even lower inside the coelenteron. The concentration of calcium under the calicoblastic layer was about 0.6 mM higher than in the surrounding seawater. Thus Ca²⁺ can diffuse from seawater to the coelenteron, but metabolic energy is needed for its transport across the calicoblastic layer to the skeleton. The pH under the calicoblastic layer was more alkaline compared with the polyp surface and inside the coelenteron. This rise in pH increased the supersaturation of aragonite from 3.2 in the dark to 25 in the light, and brought about more rapid precipitation of CaCO₃. When ruthenium red was added, Ca²⁺ and pH dynamics were inhibited under the calicoblastic layer. Ruthenium red is a specific inhibitor of Ca-ATPase. The results indicated that Ca-ATPase transports Ca²⁺ across the calicoblastic layer to the skeleton in exchange for H⁺. Addition of dichlorophenyldimethylurea completely inhibited photosynthesis. The calcium dynamics under the calicoblastic layer continued; however, the process was less regular. Initial rates were maintained. We conclude that light and not energy generation triggers calcium uptake; however, energy is also needed.     

Vertical migratory rhythms of benthic diatoms in a tropical intertidal sand flat: influence of irradiance and tides

Vertical migratory behavior of benthic diatoms is one of the adaptive strategies employed for a life in intertidal habitats. Irradiance and tides are considered to be the key factors governing vertical migration. Experiments were carried out to determine the influence of these factors in a tropical intertidal sand flat. Rising to the sediment surface for fulfilment of their light requirements for photosynthesis was the first priority. If not fulfilled during the low-tide exposure, diatoms could withstand the tidal effects and stay up at the surface even during the high-tide coverage. In the laboratory experiments, where the effects of tides were removed, the endogenous clock continued to operate in a similar fashion to that in the field when under 12-h light:12-h dark conditions, whereas continuous darkness induced a tidal rhythm. In continuous light, diatoms preferred to stay up longer than was observed in field. The above-mentioned observations reveal that irradiance has a stronger effect than tides in controlling/regulating microscale migrations in benthic diatoms. In addition, temporal differences in the irradiance and the resulting changes in diatom migration can have implications for littoral primary productivity.Communicated by O. Kinne, Oldendorf/Luhe     

Self-organization of circadian rhythms in groups of honeybees (Apis mellifera L.)

Workers in social groups of honeybees (Apis mellifera L.) synchronize their individual free-running circadian rhythms to an overall group rhythm. By monitoring the activity of bees by recording the oxygen consumption and intragroup temperature, it is shown that the rhythm coordination is in part achieved by temperature fluctuations as an intragroup Zeitgeber. Trophallaxis was shown to have only a minor (if any) effect on circadian rhythm synchronization. A model incorporating a feed back loop between temperature and activity can plausibly explain the observed synchronization of individual rhythms in social groups as a self-organization phenomenon. Correspondence to: R.F.A. Moritz     

Relative importance and interactive effects of photosynthesis and food in two solar-powered sea slugs

Sacoglossans use chloroplasts taken from algal food for photosynthesis (kleptoplasty), but the adaptive significance of this phenomenon remains unclear. Two con-generic sacoglossans (Elysia trisinuata and E. atroviridis) were collected in 2009–2011 from Shirahama (33.69°N, 135.34°E) and Mukaishima (34.37°N, 133.22°E), Japan, respectively. They were individually maintained for 16 days under four experimental conditions (combination of light/dark and with/without food), and their survival rate and relative (=final/initial) weights were measured. Both light and food had positive effects on the survival in E. trisinuata, whereas no positive effects of light or food on survival were detected in E. atroviridis. Both light and food had positive effects on relative weights in both species, but light had smaller effects than food. A significant interaction term between light and food was detected in E. trisinuata (but not in E. atroviridis) in that only the presence of both resulted in weight gains. This result suggests that E. trisinuata can obtain sufficient additional energy from photosynthesis for sustaining growth when fresh chloroplasts are continuously supplied from algal food. In addition, fluorescence yield measurements showed that unfed individuals of both E. trisinuata and E. atroviridis lost photosynthetic activity soon (<4 and 4–8 days, respectively). In conclusion, photosynthesis may function to obtain supplementary nutrition for sustaining growth when food is available in sacoglossans with short-term functional kleptoplasty.     

Effects of endogenous and some exogenous factors on the activity of the juvenile banana prawn Penaeus merguiensis

In conditions of alternating light and dark, juvenile Penaeus merguiensis de Man are more active during the dark phase. The rhythm persists in continuous dim red light, but not in continuous bright white light. The period of circadian rhythm shown in continuous dim red light varies between individuals from 22.75 to 26.0 h, with a mean of 23.8 h. The response of prawns to an artificially produced tidal situation is mediated by the presence or absence of water flow. No endogenous component of this tidal rhythm was demonstrated. There is an irregular short-term rhythm (period 2 to 3 h). It is suggested that this allows starved prawns to conserve energy.     

Light-dependency of nitrate uptake by phytoplankton over the spring bloom in Auke Bay,Alaska

The effect of light intensity on nitrate uptake by natural populations of phytoplankton was examined by ¹⁵N traceruptake experiments during the spring (March–May 1987) in Auke Bay, Alaska. The data were fit to a rectangular hyperbolic model which included a term for dark uptake. Three types of curves described nitrate uptake as a function of light intensity. The first (Type I) had a low half-saturation light intensity (K _I), low chlorophyll-specific uptakes rates, no dark uptake and occasional photoinhibition. These were observed during a period of biomass decrease, accompanied by low daily light and strong wind, prior to the major bloom. The second type (Type II) had relatively high K _I, high chlorophyll-specific uptake rates, and no dark uptake. Type II curves were observed during most of the period prior to nitrate depletion in the surface waters. Types I and II both appeared prior to nitrate depletion in the water and reflected variations in the light history of the phytoplankton population. The third type (Type III) occurred in nitrate-deplete conditions, when nitrate uptake was less dependent on light intensity (i.e., high rates of dark uptake and lower K _I). Decreased light-dependency during this period was coupled with physiological nitrogen deficiency in the population. Comparing these parameters to those of photosynthetic carbon fixation, K _Ivalues of nitrate uptake were generally higher than those of photosynthesis prior to nitrate depletion, and lower during nutrient-deplete conditions.     

Effects of non-circadian light/dark cycles on the growth and moulting of Palaemon elegans reared in the laboratory

The growth and moulting of Palaemon elegans Rathke has been compared under a circadian (12 h light: 12 h dark) and two non-circadian (8 h light:8 h dark and random light:dark) light/dark cycles. In prawns reared individually from hatching to the late juvenile phase, growth, measured as increase in total length, was significantly retarded in the non-circadian regimes, during zoeal, post-larval and early juvenile development. This effect was primarily the result of reduced increments at moulting in the non-circadian regimes. Growth of prawns reared from hatching to the post-larval phase, measured as wet and dry weights, was similarly reduced in a non-circadian regime. These effects support the hypothesis that the integrity of an animal's physiology is partially dependent on maintaining, through the action of daily environmental cycles, correct timing relationships between its oscillating sub-systems.     

Light regulation of nitrate reductase in Ulya fenestrata (Chlorophyceae)

Observations were made on the behavior of nitrate reductase activity in the green alga Ulva fenestrata under controlled light:dark regimes. The activity of nitrate reductase (NR) was examined in response to normal seasonal photoperiods as well as in response to shortened or extended periods of darkness. NR activity exhibits a light-dependent diurnal rhythm under both normal summer and winter photoperiods, with a maximum in the early morning (2 to 2.5 h after the start of illumination). This peak of activity is followed by a lower steady-state level of activity which is sustained throughout the light period. There is a sustained minimal level of activity in darkness. The morning peak in activity is always observed as long as tissue is illuminated, irrespective of the previous light or dark treatments. As such, it appears that nitrate reductase activity in U. fenestrata is under circadian control. There is no major difference in the NR activity pattern between summer and winter plants, except that the peak activity values in winter plants are consistently much higher (5 times) than in summer plants. The study also suggests that illumination prior to the normal start of photoperiod triggers a different set of regulatory mechanisms, indicating that the physiological state of plants is important in dictating the NR activity response to illumination.Please address all correpondence and requests for reprints to G. J. Smith at his present address (Hopkins Marine Station)     

Biochemical fractionation of primary production by phytoplankton in Belgian coastal waters during short- and long-term incubations with 14C-bicarbonate

Short- and long-term time course studies of radiocarbon accumulation in the intracellular end-products of photosynthesis (proteins, polysaccharides, lipids, small metabolites) and extracellular monomers and polymers were conducted at natural light intensity during a 24-h period in Belgian coastal waters dominated by large diatoms species in September, 1983. It is shown that carbon losses observed during the long-term incubation are due to the catabolism of reserve products (polysaccharides and lipids), which occurs both during the light and dark periods and provides carbon and energy for pursuing protein synthesis during the dark. Catabolism rates, as calculated by means of a simple mathematical model, indicate reduced rates of lipid catabolism (1–2% h^-1, respectively for the light and dark periods), although polysaccharide catabolism proceeds at much higher rates, namely 20% h^-1 during the light and 8% h^-1 during the dark period. Assuming that protein synthesis proceeds at a constant rate during the 24-h period and that 1–3 glucan constitutes the main storage product of this diatom population, it is shown that at least 65% of the gross primary production is catabolized by the cells. From this, only 16% are mobilized for dark protein synthesis. The remaining is respired, especially during the light period.     

Modeling seasonal course of carbon fluxes and evapotranspiration in response to low temperature and moisture in a boreal Scots pine ecosystem

Environmental conditions act above and below ground, and regulate carbon fluxes and evapotranspiration. The productivity of boreal forest ecosystems is strongly governed by low temperature and moisture conditions, but the understanding of various feedbacks between vegetation and environmental conditions is still unclear. In order to quantify the seasonal responses of vegetation to environmental factors, the seasonality of carbon and heat fluxes and the corresponding responses for temperature and moisture in air and soil were simulated by merging a process-based model (CoupModel) with detailed measurements representing various components of a forest ecosystem in Hyytiälä, southern Finland. The uncertainties in parameters, model assumptions, and measurements were identified by generalized likelihood uncertainty estimation (GLUE). Seasonal and diurnal courses of sensible and latent heat fluxes and net ecosystem exchange (NEE) of CO₂ were successfully simulated for two contrasting years. Moreover, systematic increases in efficiency of photosynthesis, water uptake, and decomposition occurred from spring to summer, demonstrating the strong coupling between processes. Evapotranspiration and NEE flux both showed a strong response to soil temperature conditions via different direct and indirect ecosystem mechanisms. The rate of photosynthesis was strongly correlated with the corresponding water uptake response and the light use efficiency. With the present data and model assumptions, it was not possible to precisely distinguish the various regulating ecosystem mechanisms. Our approach proved robust for modeling the seasonal course of carbon fluxes and evapotranspiration by combining different independent measurements. It will be highly interesting to continue using long-term series data and to make additional tests of optional stomatal conductance models in order to improve our understanding of the boreal forest ecosystem in response to climate variability and environmental conditions.