DD Conditions Research Articles

Behavioural phenotypes of Dicer knockout in the mouse SCN

AbstractThe suprachiasmatic nucleus (SCN) is the master clock that directly dictates behavioural rhythms to anticipate the earth's light/dark cycles. Although post‐transcriptional regulators called microRNAs have been implicated in physiological SCN function, how the absence of the entire mature miRNome impacts SCN output has not yet been explored. To study the behavioural consequences of miRNA depletion in the SCN, we first generated a mouse model in which Dicer is inactivated in the SCN by crossing Syt10Cre mice with Dicerflox mice to study behavioural consequences of miRNA depletion in the SCN. Loss of all mature miRNAs in the SCN shortened the circadian period length by ~37 minutes at the tissue level and by ~45 minutes at the locomotor activity level. Moreover, knockout animals exhibited a reduction in the precision of the circadian rhythm with more variable activity onsets under both LD 12:12 and DD conditions. We also observed that knockouts with higher onset variations were inclined to develop ultradian rhythms under constant light. In a second mouse model, recombination of Dicerflox via Cre delivery specifically in the SCN resulted in loss of behavioural rhythms in some animals depending on the injection efficiency. Together, our observations highlight the importance of microRNAs for a physiological SCN function and their pivotal role in robust circadian oscillations.

CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues

CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues

CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues.

The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK's functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClkΔ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth's biosphere.

Hopping from the heat: The locomotory activity patterns of the Lesser Egyptian Jerboa (Jaculus jaculus)

Arid-dwelling mammals are exposed to extreme daily temperatures, which puts these mammals at risk of overheating and dehydration, particularly while active, these risks are increased as the body size is reduced. Consequently, these small mammals often show strict nocturnality, confining their locomotor activity to the dark period of the day. One such animal is the Lesser Egyptian Jerboa (Jaculus jaculus), which occurs in the hyper-arid central regions of the Arabian Peninsula. In this study, twelve male J. jaculus were subjected to different light regimes. All the animals exhibited entrainment of their activity to the dark phase of each respective light regime. However, unlike other small arid dwelling mammals, J. jaculus confined most of its activity during the last few hours of the night rather than the initial hours of the night, a pattern which is consistent even under constant DD conditions. The last few hours of the night correlate to the coolest experienced temperatures in summer in the wild and, therefore, the ideal time for a very small mammal to be active. Larger small mammals, which may be less at risk of overheating, can afford to be active during the warmer periods of the night, namely the initial hours.

Hypnotic effects of melatonin depend on the environmental lighting conditions in the rat.

Acute effects of exogenous melatonin have been widely reported to promote sleep or induce drowsiness in human. However, testing of the hypnotic effects of melatonin in nocturnal rodents has yielded contradictory results. The latter may be associated with differences in concentration, lighting conditions, time of administration of melatonin, and possibly the type of analysis. In this study, electroencephalogram (EEG) and electromyogramwere recorded in pigmented male Brown Norway rats under both light-dark (LD) and constant dark (DD) conditions. Melatonin was administered intraperitoneally at a moderate dose of 3 mg/kg, at either 1 h after lights on under LD condition or 1 h after the activity offset under DD condition. The dosage is known to be able to entrain nocturnal rodents in DD conditions, but does not change sleep in rodents in LD. Only the rats under DD conditions showed a significant reduction in nonrapid eye movement (NREM) sleep latency, while the NREM sleep power spectrum remained unaffected. Under LD condition, melatonin did not alter NREM and rapid eye movement (REM) sleep latency, and had only minor effects on the NREM sleep EEG. Regardless of lighting conditions, melatonin administration resulted in less, but longer episodes for all vigilance states suggesting increased vigilance state consolidation. In the discussion, we compare our results with a summary of previously published data concerning the hypnotic effects of melatonin in polysomnographic/EEG-confirmedsleep in humans and nocturnal rodents. In conclusion, the hypnotic effect of exogenous melatonin in nocturnal rodents not only depends on the time of day, and concentration, but is also influenced by the lighting conditions. Regardless of inducing sleep or not, melatonin may consolidate sleep and through that enhance sleep quality.

Internal circadian misallignment in a mouse model of chemotherapy induced fatigue

BackgroundCancer survivors can experience long lasting fatigue resulting in a lower quality of life. How chemotherapy treatment contributes to this fatigue is poorly understood. Previously we have shown in a mouse model of cancer related fatigue that doxorubicin treatment induces fatigue-like symptoms related to disturbed circadian rhythms. However, the specific components of the circadian regulatory circuitry affected by doxorubicin treatment remained unclear. Therefore we investigated the role of the central circadian clock, the suprachiasmatic nucleus (SCN), in chemotherapy-induced fatigue. MethodsWe measured circadian controlled behavior and multiunit neuronal activity in the SCN in freely moving mice exhibiting fatigue-like behavior after doxorubicin treatment under both light–dark (LD) and constant dark (DD) conditions. Additionally, we assessed the expression of inflammation related genes in spleen and kidney as potential inducers of CRF. ResultsDoxorubicin treatment significantly reduced both the running wheel activity and time spent using the running wheel for over five weeks after treatment. In contrast to the pronounced effects on behavior and neuronal activity of doxorubicin on circadian rhythms, peripheral inflammation markers only showed minor differences, five weeks after the last treatment. Surprisingly, the circadian SCN neuronal activity under both LD and DD conditions was not affected. However, the circadian timing of neuronal activity in peri-SCN areas (the brain areas surrounding SCN) and circadian rest-activity behavior was strongly affected by doxorubicin, suggesting that the output of the SCN was altered. The reduced correlation between the SCN neuronal activity and behavioral activity after doxorubicin treatment, suggests that the information flow from the SCN to the periphery was disturbed. ConclusionOur preclinical study suggests that chemotherapy-induced fatigue disrupts the circadian rhythms in peripheral brain areas and behavior downstream from the SCN, potentially leading to fatigue like symptoms. Our data suggest that peripheral inflammation responses are less important for the maintenance of fatigue. Chronotherapy that realigns circadian rhythms could represent a non-invasive way to improve patient outcomes following chemotherapy.

The refractive behaviour of the human eye under different ambient lighting conditions

Background: Adjustment of the ocular accommodative system is an important phenomenon allowing for optimal vision at different distances.Aim: The study compared the refractive behaviour of the human eye under different ambient lighting conditions for different target brightness.Setting: Auto-refraction measurements were taken of the participants in the Department of Optometry at the University of Johannesburg.Methods: Five African participants from a single ethnic group aged between 20 years and 25 years, had 40 successive auto-refractor (Nidek AR 610) measurements taken on the right eye of each participant in a bright and dark room using the illuminated and dark targets (LL and DL); dark target in the illuminated and dark room (LD and DD).Results: A change in the refractive state of all participants was observed on LL and LD, except for one, who experienced myopic shifts. The largest significant difference in the variance-covariances and the means was approximately 2.40 dioptre (D). The other participants’ shifts in means were approximately the same and varied between 1.00 D and 1.50 D stigmatic shift. For the DL to DD conditions all participants, except for one, underwent a hyperopic shift of about 0.50 D.Conclusion: The refractive behaviour of the human eye appears to be dependent on ambient light conditions as well as target illumination or luminance.Contribution: The study is scientific and clinical, and focuses on changes in refractive behaviour under different lighting conditions, falling within the scope of the journal. Key insights are that there is a change in the refractive behaviour of the eye under different ambient conditions.

Effects of constant light and dark conditions on the locomotor activity, body mass, and body temperature rhythms of Eurasian Tree Sparrows (Passer montanus)

Light is an essential environmental cue influencing the endogenous circadian clocks that regulate behavioral and physiological processes in animals. Despite extensive research on the circadian rhythms of avian behavior and physiology, the mechanisms by which they adapt and adjust to abnormal photoperiod conditions, such as artificial light, have yet to be fully elucidated. Here, the circadian rhythms of the body mass, activity, and core and peripheral body temperatures of the Eurasian Tree Sparrow (Passer montanus, ETS)—a diurnal passerine—were examined under three different lighting conditions: constant light (LL), constant darkness (DD), and normal light–dark cycles (LD). Our results showed that the activity, body mass, core, tarsometatarsus, and eye temperatures of the ETSs varied significantly with treatment, time, and the interaction between treatment and time; however, there was no significant change in body mass in response to the interaction between time and treatment. Under LD conditions, the ETSs exhibited significant circadian rhythms in activity and core, tarsometatarsus, and eye temperatures. Under LL conditions, only the core temperature exhibited significant rhythmicity. Under DD conditions, there were no significant circadian rhythms in activity, body mass, and eye temperature; however, the core and tarsometatarsus temperatures remained rhythmic. Our results indicate that the core body temperature rhythm of ETS is endogenous and can be maintained under abnormal photoperiods. In contrast, the activity and body surface temperature, uncoupled from the core body temperature under abnormal photoperiod conditions, strongly depend on light. These results differ from those of other diurnal avian species, suggesting a unique coping mechanism of ETS in response to an abnormal photoperiod.

CRISPR/Cas9-mediated knockout of period reveals its function in the circadian rhythms of the diamondback moth Plutella xylostella.

Circadian clocks control the rhythmicity of many behaviors and physiological features of insects. To study the circadian clock of the moth Plutella xylostella, we employed CRISPR/Cas9-mediated genome editing to investigate the effect of loss of the clock gene period on the circadian rhythms. P. xylostella harbors a single copy of period. Phylogenetic analysis showed that P. xylostella PERIOD is more homologous to mouse PERIOD than the PERIOD proteins from bees, flies, mosquitos, and many other Lepidoptera, such as Danaus plexippus and Bombyx mori. The circadian rhythms in adult locomotor activity were altered in the period knockout strain of P. xylostella under light-dark (LD) and continuous dark (DD) conditions. Under the LD cycle, the wild-type moths displayed nocturnal activity with activity peaking very early after lights off and quickly declining after lights on. In contrast, the period knockout strain had no peak in activity when the lights were turned off and exhibited steady activity throughout the hours of darkness. Interestingly, under DD conditions, our results showed that the locomotor rhythm can be maintained without period gene, but at a lower rhythmicity ratio than wild-type. In addition, knockout of period in P. xylostella changed circadian rhythms patterns related to pupal eclosion, mating, egg-laying, and egg hatching. Mechanistically, loss of PERIOD disrupted the molecular rhythm of period and changed the clock transcription rhythm in the heads of the moths under LD and DD conditions. Together, our study indicates that the PERIOD is required for normal expression of many behavioral rhythms in P. xylostella.

Relationship between ethylene and leaf-yellowing induced by darkness in cut small-flowered chrysanthemum

Chrysanthemums (Chrysanthemum × morifolium) are the most widely produced cut flowers in Japan. During transportation, the leaves of cut small-flowered chrysanthemums grown in the summer season sometimes turn yellow. The previous study used detached chrysanthemum leaves to clarify the acceleration of leaf yellowing due to warm temperatures and dark conditions. In this study, to clarify the relationship between ethylene and leaf-yellowing senescence induced in cut small-flowered ‘Kosuzu’ chrysanthemums, ethylene production and sensitivity to ethylene were investigated. Under 24 h of continuous darkness (DD conditions), leaves turned yellow during the experimental period (days 0 to 9), with lightness (L*) and chroma (C*) values of leaves increasing from 35.8 to 53.6 and from 19.1 to 42.8, respectively, and hue values decreasing from 128.0 to 112.9. The concentrations of chlorophyll a and b decreased more under DD conditions than under storage under 12 h light/12 h dark (LD) conditions. Ethylene production by leaves decreased under both light regimes. At days 3 and 4, the leaves of LD flowers produced more ethylene than those of DD flowers. Under both light regimes, treatment with ethylene (10 µL/L for 24 h) did not accelerate leaf-yellowing compared to the control (no ethylene treatment). Hue values of leaves decreased immediately in DD flowers, regardless of ethylene treatment, but there was almost no change in LD flowers during the experimental period. The results suggest that dark conditions induce chlorophyll degradation and leaf-yellowing senescence, chlorophyll degradation under dark conditions is not associated with an increase in endogenous ethylene, and dark conditions may accelerate leaf yellowing markedly compared to exogenous ethylene application in cut small-flowered chrysanthemums.

The Influences of Illumination Regime on Egg-laying Rhythms of Honey Bee Queens.

Honey bee queens show extreme fecundity, commonly laying more than a thousand eggs in a single day. It has proven challenging to study the temporal organization of egg-laying behavior because queens are typically active around the clock in the dark cavity of a densely populated nest. To contend with this challenge, we developed two novel methods allowing detailed monitoring of queen activity and egg laying. We first adapted a high-resolution, continuous, tracking system allowing to track the position of barcode-tagged queens in observation hives with colonies foraging outside. We found that the queen is active ~96% of the day with typically no diurnal rhythm. Next, we developed a new laboratory procedure to monitor egg laying at single egg resolution under different light regimes. We found that under constant darkness (DD) and temperature conditions, queens laid eggs with no circadian rhythms. Queen fecundity was severely reduced under constant light (LL). Under a 12:12 illumination regime, queen fecundity was comparable to under constant darkness, with a higher number of eggs during the light phase. These daily rhythms in egg laying continued when these queens were released to DD conditions, suggesting that egg-laying rhythms are influenced by endogenous circadian clocks. These results suggest that honey bee queens are active and lay eggs around the clock with no diurnal rhythms. Light has complex influences on these behaviors, but more studies are needed to determine whether these effects reflect the influence of light directly on the queen or indirectly by affecting workers that interact with the queen.

Developmental light deprivation transiently reduces the expression of vGluT2 and GluN2B in the rat ventral suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the most important circadian clock in mammals. The SCN synchronizes to environmental light via the retinohypothalamic tract (RHT), which is an axon cluster derived from melanopsin-expressing intrinsic photosensitive retinal ganglion cells. Investigations on the development of the nonimage-forming pathway and the RHT are scarce. Previous studies imply that light stimulation during postnatal development is not needed to make the RHT functional at adult stages. Here, we examined the effects of light deprivation (i.e., constant darkness (DD) rearing) during postnatal development on the expression in the ventral SCN of two crucial proteins for the synchronization of circadian rhythms to light: the presynaptic vesicular glutamate transporter type 2 (vGluT2) and the GluN2B subunit of the postsynaptic NMDA receptor. We found that animals submitted to DD conditions exhibited a transitory reduction in the expression of vGluT2 (at P12-19) and of GluN2B (at P7-9) that was compensated at older stages. These findings support the hypothesis that visual stimulation during early ages is not decisive for normal development of the RHT-SCN pathway.

Proasellus thrive in the dark – plasticity enabling cave colonisation?

Darkness is the hallmark of all subterranean habitats and, to some degree, of many surface habitats. Many surface animals that live in semi-dark habitats, such as freshwater benthos, are photophobic. Yet, few of them can colonize caves and form subterranean populations. We hypothesize that phenotypic plasticity induced by darkness enables surface crustaceans to colonize caves, as has been previously shown for the cavefish Astyanax mexicanus. Our target organisms were surface species that form distinct subterranean populations with different adaptations to caves and have closely related cave species. Proasellus proved to be a good model system, and we selected the surface species Proasellus coxalis and its cave relative P. anophtalmus to learn what adaptations evolve in subterranean forms. We exposed a randomly selected group of both species to complete darkness (DD) where we could establish breeding colonies. Control breeding colonies are exposed to a 12:12 h light-dark photoperiod (LD). Other conditions are kept as constant and equal as possible, including conditioned Alnus glutinosa leaves used for shelter and food. We examined the molecular phenotypes of both Proasellus species, animals originally collected from the wild and preserved after four months under the experimental conditions DD and LD. The number of observed differentially expressed genes (DEGs) was significantly higher between cohorts DD and LD of P. coxalis compared to P. anophtalmus. The direction of regulation of DEGs also showed the opposite effect, with the cohort of P. coxalis DD showing predominant upregulation and the cohort of P. anophtalmus DD showing predominant downregulation relative to their cohorts LD. A greater number of DEGs in both species were associated with various metabolic processes such as lipid regulation. In contrast, DEGs related to growth, cell division, rhythmic processes, sensory perception of taste, and sexual reproduction were observed only in P. coxalis. We examined the changes at the phenotypic level in the first generation born in the experiment. Surprisingly, P. coxalis showed better fitness under DD conditions (growth rate, fecundity, survival). Other phenotypes were altered in both adaptive and non-adaptive directions i. e. towards vs. opposite of cave adaptations. We conclude that darkness is not a limiting factor in cave colonization and suggest that it may even be a key factor in the evolution of troglomorphy via phenotypic plasticity.

Expression patterns of clock genes in the kidney of two Lasiopodomys species

Abstract Previous studies showed that the kidney has its own molecular circadian clock expression regulation that maintains the homeostasis of physiological processes. However, limited information is available on the molecular mechanisms of the kidney circadian rhythm in subterranean rodents. Here, we report circadian gene expression in the kidney of subterranean Mandarin voles and the related aboveground Brandt’s voles, reared under 12L:12D (LD) or dark (DD) conditions, respectively. The results showed that the rhythmic genes were represented in Brandt’s voles in higher numbers under LD than DD conditions, but the number of rhythmic genes in Mandarin voles was similar between the two treatment conditions. The gene expression levels at different timepoints all showed reduced results under DD conditions compared with those in the LD cycle in Brandt’s voles, whereas the expression levels of the tested genes at certain Zeitgeber timepoints showed higher results than in the LD cycle in Mandarin voles. The gene expression peak showed chaotic resetting under DD conditions in both voles. We thus suggest that Mandarin and Brandt’s voles have different molecular circadian clock expression adjustment patterns in the kidney as an adaptation to different living environments. Mandarin voles seem to be more adapted to the dark environment, while Brandt’s voles are more dependent on external light conditions.

Hundreds of LncRNAs Display Circadian Rhythmicity in Zebrafish Larvae.

Long noncoding RNAs (lncRNAs) have been shown to play crucial roles in various life processes, including circadian rhythms. Although next generation sequencing technologies have facilitated faster profiling of lncRNAs, the resulting datasets require sophisticated computational analyses. In particular, the regulatory roles of lncRNAs in circadian clocks are far from being completely understood. In this study, we conducted RNA-seq-based transcriptome analysis of zebrafish larvae under both constant darkness (DD) and constant light (LL) conditions in a circadian manner, employing state-of-the-art computational approaches to identify approximately 3220 lncRNAs from zebrafish larvae, and then uncovered 269 and 309 lncRNAs displaying circadian rhythmicity under DD and LL conditions, respectively, with 30 of them are coexpressed under both DD and LL conditions. Subsequently, GO, COG, and KEGG pathway enrichment analyses of all these circadianly expressed lncRNAs suggested their potential involvement in numerous biological processes. Comparison of these circadianly expressed zebrafish larval lncRNAs, with rhythmically expressed lncRNAs in the zebrafish pineal gland and zebrafish testis, revealed that nine (DD) and twelve (LL) larval lncRNAs are coexpressed in the zebrafish pineal gland and testis, respectively. Intriguingly, among peptides encoded by these coexpressing circadianly expressed lncRNAs, three peptides (DD) and one peptide (LL) were found to have the known domains from the Protein Data Bank. Further, the conservation analysis of these circadianly expressed zebrafish larval lncRNAs with human and mouse genomes uncovered one lncRNA and four lncRNAs shared by all three species under DD and LL conditions, respectively. We also investigated the conserved lncRNA-encoded peptides and found one peptide under DD condition conserved in these three species and computationally predicted its 3D structure and functions. Our study reveals that hundreds of lncRNAs from zebrafish larvae exhibit circadian rhythmicity and should help set the stage for their further functional studies.

Effects of insemination and blood-feeding on locomotor activity of wild-derived females of the malaria mosquito Anopheles coluzzii

BackgroundBehavioural shifts in the canonical location and timing of biting have been reported in natural populations of anopheline malaria vectors following the implementation of insecticide-based indoor vector control interventions. These modifications increase the likelihood of human-vector contact and allow mosquitoes to avoid insecticides, both conditions being favourable to residual transmission of the malarial parasites. The biting behaviour of mosquitoes follows rhythms that are under the control of biological clocks and environmental conditions, modulated by physiological states. In this work we explore modifications of spontaneous locomotor activity expressed by mosquitoes in different physiological states to highlight phenotypic variability associated to circadian control that may contribute to explain residual transmission in the field.MethodsThe F10 generation progeny of field-collected Anopheles coluzzii from southwestern Burkina Faso was tested using an automated recording apparatus (Locomotor Activity Monitor, TriKinetics Inc.) under LD 12:12 or DD light regimens in laboratory-controlled conditions. Activity recordings of each test were carried out for a week with 6-day-old females belonging to four experimental treatments, representing factorial combinations of two physiological variables: insemination status (virgin vs inseminated) and gonotrophic status (glucose fed vs blood fed). Chronobiological features of rhythmicity in locomotor activity were explored using periodograms, diversity indices, and generalized linear mixed modelling.ResultsThe average strength of activity, onset of activity, and acrophase were modulated by both nutritional and insemination status as well as by the light regimen. Inseminated females showed a significant excess of arrhythmic activity under DD. When rhythmicity was observed in DD, females displayed sustained activity also during the subjective day.ConclusionsInsemination and gonotrophic status influence the underlying light and circadian control of chronobiological features of locomotor activity. Overrepresentation of arrhythmic chronotypes as well as the sustained activity of inseminated females during the subjective day under DD conditions suggests potential activity of natural populations of A. coluzzii during daytime under dim conditions, with implications for residual transmission of malarial parasites.Graphical abstract

Diel and Circadian Patterns of Locomotor Activity in the Adults of Diamondback Moth (Plutella xylostella).

The Diamondback Moth (Plutella xylostella) is a highly destructive lepidopteran pest of cruciferous crops. However, there still is relatively little known about the locomotor activities of diel and the circadian patterns in P. xylostella. Here, we present an analysis of the diel locomotion of P. xylostella under several laboratory settings. We established a system for measuring the individual locomotor activities of P. xylostella and found that both males and females showed a nocturnal pattern of activity under 26 or 20 °C LD conditions, with activity peaking immediately after lights off and quickly declining after lights on. In addition, we showed that it is difficult to assess the free-running circadian rhythms of P. xylostella under 26 °C DD conditions. However, we found that males showed a higher power, rhythm index (RI) and rhythmic ratio than females under 20 °C DD conditions, which indicated that males in low-temperature conditions are much more suitable to study the free-running circadian rhythms of P. xylostella. The findings of this study will help us to have a better understanding of the diel activity of P. xylostella and may provide a foundation for the development of an effective pest management strategy.

VRILLE shows high divergence among Higher Diptera flies but may retain role as transcriptional repressor of Clock

In the circadian system, the clock gene vrille (vri) is an essential component of the second feedback loop, being responsible in Drosophila for the rhythmicity of the Clock (Clk) gene transcription by its repression. Here we studied vri in a fruit fly pest, the Tephritidae Anastrepha fraterculus, aimingtoinvestigate its molecular evolution and expression patterns from whole-head extracts. We used a combination of transcriptomic, genomic and gene walking strategies to sequence and characterize Afravri in male and female head transcriptomes of A. fraterculus and detected two putative isoforms that may correspond to A and D vri isoforms of Drosophila. Both isoforms produced a full-length sequence that translates to 842 amino acids. While the protein sequence showed significant divergence to orthologous sequences from other organisms, the bZIP domain was highly conserved. Molecular evolutionary analyses showed that vri in higher Diptera flies has been evolving under positive selection. A more detailed analysis showed positive selection also in Tephritidae with 29 sites evolving under positive selection in comparison with Drosophilidae. Real time expression analysis in LD and DD conditions showed cyclic expression of Afravri mRNA with oscillation opposite to AfraClk, suggesting that VRI may also behave in Anastrepha as a transcriptional repressor of Clk, providing another indication that higher Diptera might share common interlocked transcript-translation feedback loops (TTFLs) mechanisms that differ from other insects in target genes.

Locomotor activity rhythm in catfish Heteropneustes fossilis as a function of shoal size under different light regimens

ABSTRACT The information on the circadian characteristics of catfish in shoals is scanty. We examined the circadian locomotor activity rhythm of catfish Heteropneustes fossilis either singly housed (SS1) or in shoals of four (SS4) and six (SS6) under different light regimens. We carried out the study sequentially under LD1 (12:12), constant darkness (DD), LD2, continuous light (LL), LD3, and DL (12:12). Each condition was for at least 10–12 consecutive days. We analyzed the time-series data by employing Cosinor rhythmometry to detect circadian rhythm characteristics in locomotor activity at a fixed time window with a τ = 24 h. Results indicated that singly housed or shoals exhibited statistically significant (p < .001) circadian rhythm in locomotor activity under LD conditions with a higher activity level during the dark phase. Further, we observed free-running rhythms in locomotor activity under DD and LL, irrespective of the shoal sizes. In phase inverse DL, both singly housed and shoals demonstrated higher activity in the dark phase. The two-way ANOVA results revealed a significant effect of the factor ‘light regimen’ on amplitude and acrophase; the factor ‘shoal size’ produced a statistically significant impact on the mesor only. Both shoals showed significantly higher mesor than singly housed fish. The circadian amplitude declined under constant conditions of DD and LL. The locomotor activity rhythm exhibited a free-running pattern with a τFR greater than 24 h under both DD and LL conditions. We conclude that light is a more prominent factor for the entrainment of circadian activity in catfish H. fossilis. However, the extent of social aggregation (shoal size) has little or no effects on the characteristics of circadian locomotor activity rhythm in H. fossilis.

Light stimulation during postnatal development is not determinant for glutamatergic neurotransmission from the retinohypothalamic tract to the suprachiasmatic nucleus in rats.

The hypothalamic suprachiasmatic nucleus (SCN) is the leading circadian pacemaker in mammals, which synchronizes with environmental light through the retinohypothalamic tract (RHT). Although the SCN regulates circadian rhythms before birth, postnatal synaptic changes are needed for the RHT-SCN pathway to achieve total functional development. However, it is unknown whether visual experience affects developmental maturation. Here, we studied the effects of constant darkness (DD) rearing on the physiology (at pre- and postsynaptic levels) of glutamatergic neurotransmission between RHT and SCN during postnatal development in rats. Upon recording spontaneous and evoked excitatory postsynaptic currents (EPSCs) by electrical stimulation of RHT fibers, we found that DD animals at early postnatal ages (P3-19) exhibited different frequencies of spontaneous EPSCs and lower synaptic performance (short-term depression, release sites, and recruitment of RHT fibers) when compared with their normal light/dark (LD) counterparts. At the oldest age evaluated (P30-35), there was a synaptic response strengthening (probability of release, vesicular re-filling rate, and reduced synaptic depression) in DD rats, which functionally equaled (or surmounted) that of LD animals. Control experiments evaluating EPSCs in ventral SCN neurons of LD rats during day and night revealed no significant differences in spontaneous or evoked EPSCs by high-frequency trains in the RHT at any postnatal age. Our results suggest that DD conditions induce a compensatory mechanism in the glutamatergic signaling of the circadian system to increase the chances of synchronization to light at adult ages, and that the synaptic properties of RHT terminals during postnatal development are not critically influenced by environmental light.