María Luisa Fanjul-Moles

The crayfish Procambarus clarkii CRY shows daily and circadian variation

SUMMARY The circadian rhythms of crayfish are entrained by blue light, through putative extra retinal photoreceptors. We investigated the presence and daily variation of CRY, a protein photosensitive to blue light spectra and ubiquitous in animals and plants, in the putative pacemakers of Procambarus clarkii, namely the eyestalk and brain, at different times of the 24 h light:dark cycles. Using different experimental light protocols and by means of qualitative/quantitative immunofluorescence anatomical and biochemical methods, we identified CRY immunoreactivity in cells located in the medulla-terminalis-hemiellipsoidal complex (MT-HB) and the anterior margin of the median protocerebrum (PR). The immunoreaction varied with the time of day and the two neural structures showed a semi-mirror image. The results of the biochemical analysis matched these variations. Western blotting demonstrated statistically significant circadian rhythms in brain CRY abundance, but no daily circadian CRY abundance oscillations in the eyestalk. These immunocytochemical and biochemical results link a specific photoreceptor molecule to circadian rhythmicity. We propose that CRY may be linked to the photoreception of the clock and to the generation of circadian rhythmicity.

Antennal olfactory sensitivity in response to task‐related odours of three castes of the ant Atta mexicana (hymenoptera: formicidae)

Abstract The relationship between scent composition and antennal sensitivity in different castes of Atta mexicana is investigated under laboratory conditions. Extracts of dead ants are analysed by gas chromatography‐mass spectrometry to identify the compounds presumably responsible for the specific undertaking behaviour. Oleic acid is identified as one compound that triggers undertaking behaviour. To determine differences in odour reception between workers of different castes (i.e. foragers, undertakers and soldiers), further antennal sensitivity to task‐related odours is tested using electroantennographic techniques. Soldiers are the most specialized caste because of their low response to all odours, except odourants related to alarm pheromones. The behavioural specialization of soldiers and their reduced behavioural repertoire are discussed.

effect of variation in photoperiod and light intensity on oxygen consumption, lactate concentration and behavior in crayfish procambarus clarkii and procambarus digueti

The effects of light intensity and duration on metabolic and behavioral parameters of two species of crayfish, Procambarus clarkii and Procambarus digueti, were studied. Sixty animals of each species were submitted to high irradiance conditions of two different photoperiod lengths, one normal light/dark (LD) 12:12 and one extreme LD 20:4 for 2 weeks. Hemolymph, lactate and oxygen consumption were determined throughout the experimental period. Simultaneously in 18 additional animals of each species, motor activity was individually recorded under the same control and experimental conditions. Both species showed a decrease in oxygen uptake and an increase in hemolymph lactate concentration. The statistical significance of this finding was higher for LD 20:4. This extreme condition evoked a significant decrease of motor activity in P. clarkii and a high mortality rate in P. digueti. P. digueti did not survive after the experiment, whereas P. clarkii survived and adapted to the laboratory conditions. Changes in metabolic and behavioral parameters could indicate different adaptation abilities in these species.

Crayfish Procambarus clarkii shows Circadian Variations in Different Parameters of the GSH Cycle

The present study investigated the rhythmic changes in glutathione status in midgut gland and hemolymph as well as in glutathione reductase (GR) activity in the crayfish Procambarus clarkii. In order to determine the circadian nature of these rhythms different groups of crayfish were submitted to constant‐darkness conditions for 24 or 72 h after they had spent 15 days under light–dark 12:12 cycles. The animals of the different batches were killed at 6 h intervals during a 24 h cycle. Reduced glutathione (GSH) and oxidized glutathione (GSSG) in hemolymph and midgut as well as midgut GR activity were determined in midgut gland and hemolymph by fluorometric and spectrophotometric methods. Data analysis by chronogram and single Cosinor revealed circadian rhythmicity for GSH and GSSG concentration in both tissues as well as midgut GR activity. The rhythm parameters revealed oxidative stress induced by light. The possible correlation between the glutathione rhythm and other metabolic and behavioral rhythms of crayfish as well as the importance of the glutathione circadian temporal order in the adaptation of crayfish are discussed.

Nonparametric Effects of Monochromatic Light on the Activity Rhythm of Juvenile Crayfish

The current study was carried out to test the influence of blue and red monochromatic light upon the motor activity rhythm of juvenile crayfish as well as determine whether this effect involves extraretinal photoreception. Two groups of 46 juvenile instars were used: (1) intact control animals and (2) animals lacking retina and lamina ganglionaris. All animals were individually monitored with a motor activity recording system for 30 days. For the first 10 days the animals were maintained and kept in constant darkness (DD) and then submitted to 24-h skeleton photoperiod cycles (SP) consisting of 30 min red or blue light signals calibrated to the same irradiance (25 Wm-2) during the next 10 days. Afterwards, they were left in DD for the last 10 days of the experiment. Activity was quantitatively and qualitatively analyzed. Results show that all control intact animals synchronized to blue or red light exhibited shift advances or delays. These results indicate that both circadian responses to monochromatic light investigated in this study are mediated by extraretinal photoreceptors.

putative pacemakers of crayfish show clock proteins interlocked with circadian oscillations

SUMMARY Although the molecular mechanisms that control circadian rhythms in many animals, particularly in the fly, are well known, molecular and biochemical studies addressing the location and function of the proteins and genes contributing to the cycling of the clock in crayfish Procambarus clarkii are scarce. In this study, we investigated whether three proteins that interact in the feedback loop of the molecular clock described for Drosophila are expressed in the putative circadian pacemakers of crayfish retina, eyestalk and brain and whether their expression cycles in a manner consistent with elements of the circadian clock. Here we identified PER, TIM and CLK immunoreactivity in the cytoplasm and nucleus of cells located in the retina as well as in clusters of cells and neuropils of the optic ganglia, lateral protocerebrum and brain. Brain clusters 6, 10, 9 and 11, in particular, showed Per, Tim and Clk-like immunoreactivity at the perikarya and nucleus, and these antigens colocalized at Zeitgeber time (ZT) 0 and/or ZT 12. A biochemical assay demonstrated circadian functionality of Per, Tim and Clk proteins. Both in the eyestalk and in the brain, these proteins demonstrated apparent daily and circadian rhythms. The presence and colocalization of these clock proteins in the cytoplasm and/or nucleus of several cells of retina, optic lobe and brain, depending on time, as well as their circadian oscillations, suggest interactions between positive and negative transcription factors and clock proteins similar to those forming the feedback loop of the canonical model proposed for different animals.

Crayfish Procambarus clarkii Retina and Nervous System Exhibit Antioxidant Circadian Rhythms Coupled with Metabolic and Luminous Daily Cycles

Based on previous work in which we proposed midgut as a putative peripheral oscillator responsible for circadian reduced glutathione (GSH) crayfish status, herein we investigated the retina and optic lobe‐brain (OL‐B) circadian GSH system and its ability to deal with reactive oxygen species (ROS) produced as a consequence of metabolic rhythms and light variations. We characterized daily and antioxidant circadian variations of the different parameters of the glutathione system, including GSH, oxidized glutathione (GSSG), glutathione reductase (GR) and glutathione peroxidase (GPx), as well as metabolic and lipoperoxidative circadian oscillations in retina and OL‐B, determining internal and external GSH‐system synchrony. The results demonstrate statistically significant bi‐ and unimodal daily and circadian rhythms in all GSH‐cycle parameters, substrates and enzymes in OL‐B and retina, as well as an apparent direct effect of light on these rhythms, especially in the retina. The luminous condition appears to stimulate the GSH system to antagonize ROS and lipid peroxidation (LPO) daily and circadian rhythms occurring in both structures, oscillating with higher LPO under dark conditions. We suggest that the difference in the effect of light on GSH rhythmic mechanisms of both structures for antagonizing ROS could be due to differences in glutathione‐system coupling strength with the circadian clock.

Changes in Hemolymph Glutathione Status After Variation in Photoperiod and Light-irradiance in Crayfish Procambarus clarkii and Procambarus digueti

This work studied the effect of light‐stressors, irradiance and photoperiod length on the status of hemolymph glutathione in two species of crayfish, Procambarus clarkii and Procambarus digueti. Adult animals of each species were submitted to two experimental approaches: (1) two batches of each species were placed under low or high light irradiant conditions of light–dark (LD) 24 h cycles of two different photoperiod lengths, one normal LD 12:12 and one extreme LD 20:4 low and high irradiance for 10 weeks. Time‐dependent light changes on hemolymph glutathione concentration were determined throughout the entire experimental period; and (2) three batches of the two species were submitted to independent treatments consisting of the same LD 12:12 cycles of high and low irradiance and 20:4 high‐irradiance LD cycles. Reduced and oxidized glutathione hemolymph concentrations were determined and total glutathione was calculated. In addition midgut glutathione reductase activity in both species was determined. The two species showed different hemolymph glutathione reactivity and glutathione status for the two light parameters. Dissimilar responses of both species, as well as the rate of mortality of P. digueti represent specific differences in the metabolic responses, as well as tolerance to photo‐oxidative stress produced by light. The role of glutathione in the tolerance of crayfish to photo‐oxidative stress is discussed.

effect of monochromatic light upon the erg circadian-rhythm during ontogeny in crayfish (procambarus-clarkii)

Abstract 1. 1. The aim of this work was to investigate if the asymmetric development of the two sets of visual photoreceptors responsible for short wave and long wave sensitivity during crayfish ontogeny influences the characteristics of the electroretinogram (ERG) amplitude rhythm. 2. 2. The parameters of the ERG rhythm of juvenile (2–12 weeks) and adult stages were studied in free-running conditions under blue, red or white light flash test. 3. 3. It was found that monochromatic light produces significant changes in the ERG circadian rhythm parameters in juvenile stages and has a synchronizing effect on the ultradian rhythm prior to the ERG circadian rhythm. 4. 4. Based on these results we propose the existence of two sets of circadian photoreceptors related to two independent systems involved in the synchronization of the ERG circadian rhythm (a short wave and a long wave detection system).

Relationship between Oxidative Stress, Circadian Rhythms, and AMD

This work reviews concepts regarding oxidative stress and the mechanisms by which endogenous and exogenous factors produce reactive oxygen species (ROS). It also surveys the relationships between oxidative stress, circadian rhythms, and retinal damage in humans, particularly those related to light and photodamage. In the first section, the production of ROS by different cell organelles and biomolecules and the antioxidant mechanisms that antagonize this damage are reviewed. The second section includes a brief review of circadian clocks and their relationship with the cellular redox state. In the third part of this work, the relationship between retinal damage and ROS is described. The last part of this work focuses on retinal degenerative pathology, age-related macular degeneration, and the relationships between this pathology, ROS, and light. Finally, the possible interactions between the retinal pigment epithelium (RPE), circadian rhythms, and this pathology are discussed.