Light-dark Cycle Research Articles

(057) Influence of Circadian Disruption on Erectile Function in Chronic Jet-Lagged Young Rats

Abstract Introduction Sleep is crucial during the developing stage of one’s life and has been associated with physical activity. However, as the number of 24-hour stores such as convenience stores and supermarkets have increased, along with increased internet usage, people have developed the habit of staying up late, resulting in sleep deprivation due to disruption in the light-dark cycle. Jet lag is widely used in animal models for inducing irregular circadian rhythms. Chronic jet lagged model animals are known to develop a variety of disorders. Objective To investigate the influences of chronic jet lag on erectile function in rats in order to elucidate the effects of irregular circadian rhythms on future sexual function. Methods 4-week-old rats underwent randomly assigned to remain either under the control light/dark cycle (Cont group) or under chronic light/dark shift schedule to simulate the conditions of chronic jet lag (CJL group). The shift-lag protocol consisted of 8 h light/dark phase advances repeated every 3 days for 4 weeks. Erectile function was examined at 1, 2, and 4 weeks post CJL treatment. After 4 weeks of CJL treatment, the rats were returned to control light/dark cycle for 4 and 8 weeks, as CJL(4w)+Rest(4w) group and CJL(4w)+Rest(8w) group, and erectile function was examined correspondingly. Erectile function was tested using intracavernous pharmacotherapy (ICP) measurements, and endothelial function was tested through an isometric tension study. Western blot analysis was used to assess the levels of endothelial nitric oxide (eNOS) and phosphorylated eNOS (p-eNOS). Additionally, mRNA expressions of clock genes (Bmal1 and Cry 1) and growth factors (TGF-β1 and ICAM-1) were tested using real-time PCR. Results The ICP/MAP ratio was significantly lower in the CJL group (0.35 ± 0.05) at 4 weeks than in the control group (0.66 ± 0.05) (P < 0.05). CJL group exhibited significantly lower testosterone levels, Bmal1, Cry 1, TGF-β1, and ICAM-1 expression, and p-eNOS and cGMP levels. The ICP/MAP ratio in the CJL(4w)+Rest(4w) group (0.47 ± 0.06) was lower than that in the control group (P < 0.01) whereas the ratio in the CJL(4w)+Rest(8w) group (0.81 ± 0.03) was similar to that in the control group (P > 0.05). Conclusions In this study, chronic jet lagged (4 weeks) rats exhibited a decreased ICP/MAP ratio, reduced testosterone level, and decreased p-eNOS expression, indicating the development of erectile dysfunction (ED). Upon 8 weeks of normal light/dark shift schedule, erectile function was recovered. This study suggests that irregular circadian rhythms induced by repeated jet lag, especially in childhood, may affect the production of sex hormones and erectile function in the future. Therefore, it is necessary for children to follow a regular circadian rhythm and healthy lifestyle. Disclosure No.

Occurence of leaf spot on Narrow-head ragwort (Ligularia stenocephala) caused by Alternaria cinerariae in Korea.

Narrow-head ragwort (Ligularia stenocephala (Maxim.) Matsum. & Koidz.) has been used for medicinal purposes and a leafy vegetable in South Korea (Choi et al., 2007; Debnath et al., 2017). In May 2022, brown spots and blight were observed on the leaves in a farmer's field in Namwon (35°26'58.9"N, 127°28'55.9"E), Korea. About 80% of the plants in a 3000 m2 cultivation area were infected. To isolate the causal agent, small pieces (1 mm2) surface-sterilized (1% NaOCl for 1 min) symptomatic leaf tissues were put onto a water agar (WA) plate and incubated in the dark at 25℃. After five days of incubation, two isolates (FD00021, FD00022) were obtained from diseased leaves using a single spore isolation technique. Morphological characteristics were examined after seven days of incubation at 25℃ in the dark. Colonies were 51.6 to 65.3 mm in diameter, gray-green in the center, and ivory at the edge. Conidiophores were straight or curved and 10 - 34 × 4 - 5 μm. Conidia were solitary or two to four in a chain, long ellipsoid to obclavate, one to thirteen transverse septa, 52 - 169 ×14 - 34 μm, blunt-tapered beak variable in size 4 - 56 × 3 - 10 μm (n=75). The morphological and cultural characteristics of the isolates were consistent with that of Alternaria cinerariae (Nishikawa and Nakashima, 2015; Simmons, 2007). For molecular identification, genomic DNA was extracted from 5-day-old cultures using the Maxwell® RSC PureFood GMO and Authentication Kit (Promega). Five gene regions, including rDNA ITS, GPD, Alt a, RPB2, and EF1-α were amplified and sequenced using ITS1/ITS4, gpd1/gpd2, Alt-a1-for/ Alt-a1-rev, RPB2-5F2/RPB2-7cR, and EF1-728F/EF1-986R primer sets respectively (Wang et al., 2022; Garibaldi et al., 2022). The resulting sequences were deposited in GenBank with accession no. OP785152, OP785153 and OP832000 to OP832007). The concatenated genes (rDNA ITS, GPD, Alt a, RPB2, and EF1-α) sequence identity of the FD00021 and FD00022 against the reference strain A. cinerariae CBS 116495 is 99.92% (2572/2574) and 99.84% (2570/2574), respectively. Maximum Likelihood tree was inferred based on the concatenated sequences of the five gene regions using the Kimura 2-parameter model with 1,000 bootstrap replications. The phylogenetic tree showed that the present strains and A. cinerariae CBS 116495 fell into the same clade with high bootstrap support (100%). Based on morphological characteristics and molecular analysis, the isolates were identified as A. cinerariae. To confirm their pathogenicity, drops (70 μl) of conidial suspension (1×104 spores/ml) were applied on intact healthy leaves (3 leaves/plant) of plants (3 plants/isolate) that had been cultivated for one month after transplantation as seedlings. Controls were treated with sterile distilled water. The treated plants were covered with plastic boxes to maintain humidity around 90% and were maintained in an incubator at 25℃ in a 12-hour light-dark cycle. Symptoms appeared only on inoculated leaves after four days of inoculation, while controls remained asymptomatic. The A. cinerariae isolates were re-isolated from infected tissue of the inoculated leaves, thus fulfilling Koch's postulates. Alternaria cinerariae is an important plant pathogen that can cause leaf spot and blight on a variety of host plants including Cineraria spp. and Tussilago farfara (He et al. 2020). This is the first report on leaf spot on Narrow-head ragwort caused by A. cinerariae in the world. Leaf spot disease caused by Alternaria cinerariae is a significant threat to narrow-head ragwort agriculture in South Korea. Therefore, its control strategies are necessary for increasing productivity.

The Impact of a Six-Hour Light-Dark Cycle on Wheat Ear Emergence, Grain Yield, and Flour Quality in Future Plant-Growing Systems.

Cultivating wheat (Triticum aestivum) in a closed environment offers applications in both indoor farming and in outer-space farming. Tailoring the photoperiod holds potential to shorten the growth cycle, thereby increasing the annual number of cycles. As wheat is a long-day plant, a night shorter than a critical length is required to induce flowering. In growth chambers, experiments were conducted to examine the impact of a 6 h light-dark cycle on the timing of wheat ear emergence, grain yield, and flour quality. Under equal daily light-integral conditions, the 6 h light-dark cycle promoted growth and development, resulting in accelerated ear emergence when compared to a 12 h cycle, additionally indicating that 12 h of darkness was excessive. To further stimulate heading and increase yield, the 6 h cycle was changed at the onset of stem elongation to a 14 h-10 h, mimicking spring conditions, and maintained until maturity. This successful transition was then combined with two levels of light intensity and nutrient solution, which did not significantly impact yield, while tillering and grain ripening did increase under higher light intensities. Moreover, it enabled manipulation of the baking quality, although lower-end falling numbers were observed. In conclusion, combining a 6 h light-dark cycle until stem elongation with a 14 h-10 h cycle presents a promising strategy for increasing future wheat production in closed environments. The observation of low falling numbers underscores the importance of factoring in flour quality when designing the wheat-growing systems of the future.

First Report of Colletotrichum fructicola Causing Anthracnosis on Glycine max in Brazil

Brazil is the largest producer of soybean [Glycine max (L.) Merrill], cultivated in diverse environments and systems. This scenario can contribute to emergence of new diseases or increase the severity of secondary diseases. In March 2023, elliptical to circular, brownish lesions, 5.2–6.1 cm length and 1.1–1.5 cm width, with salmon-colored masses of conidia in the center of the lesions, were observed on the stems of soybean cultivar ‘CZ 16B17 IPRO’, in the municipality of Campos Novos, Santa Catarina, Brazil (27º25'19''S and 51º14'14''05W). The presence of 210–355 μm length and 210–232 μm width acervuli was rare, with arrows larger than the mass of conidia (Figure S1). Fragments of the infected tissues were cut, disinfected and placed in Petri dishes containing Potato Dextrose Agar (PDA) or V8-agar medium and maintained at 23 ± 2ºC and a photoperiod of 12 h dark–light cycle. After 13 days, the development of grayish-white colonies was observed on both culture media, with the formation of a mass of septate hyaline, oblong, cylindrical conidia, 13.3–15.3 µm length and 2.9–3.5 µm width, with obtuse ends. One pure monosporic isolate was selected, isolate CF1. The presence of sexual structures was observed on PDA after 13 days and in V8 after 15–20 days. Perithecia were dark brown and globose, either immersed in the culture medium or on the surface between the mycelia. Inside of perithecia, unitunicate, clavate, and cymbiform asci, 39.1–61.0 μm length and 9.6–11.7 μm width were observed, containing eight spindle-shaped and slightly curved ascospores with rounded tips 13.8–18.3 μm length and 3.0–4.2 μm width (Figure S1). Pathogenicity tests were performed on young soybean plants at V1 phenological growth stage in four repetitions. PDA disks, 7mm in diameter, with growth mycelium were placed on stems while using uninfected PDA disks as a control. Plants were incubated in a chamber at 25 ± 2°C and 90% relative humidity. Anthracnose lesions were observed only on the stems of the inoculated plants. The same pattern of symptoms was observed on the stems, and the fungus were reisolated on PDA. The colony and morphological characteristics were identical to the previously isolated fungus. For molecular characterization, the growth mycelia were collected, macerated in liquid nitrogen, and DNA was extracted using the method Doyle and Doyle (1990) with CTAB. End-point PCR was performed using the GoTaq® Flexi DNA Polymerase (Promega, USA) and the primers, ITS-1F/ITS-4, T1/Bt2b, CL1C/CL2C, GDF/GDR, and SODglo2-F/SODglo2-R (Weir et al. 2012) for the amplification of internal transcribed spacer (ITS), β-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and superoxide dismutase (SOD), respectively. Amplified fragments were sequenced and compared with the available sequences in the Genbank (www.ncbi.nlm.nih.gov/genbank/). The sequences of all five-genes (Accession numbers OR883777, OR891749, OR891750, OR891751 and OR891752, respectively) of the isolate CF1 characterized in this study showed 99% nucleotide identities whith the stand isolate ICMP 18581 of Colletotrichum fructicola. A phylogenetic tree was constructed in MEGA X (Kumar et al. 2021), containing the amplified and concatenated sequences and representative species from the Colletotrichum gloeosporioides complex. The isolate grouped only with C. fructicola clade, confirming the identity of the fungus (Figure S2). To our knowledge, this is the first study reporting the infection of C. fructicola in soybeans in Brazil, which has already been reported in China (Xu et al. 2023).

Comparative analysis of locomotor behavior and head diurnal transcriptome regulation by PERIOD and CRY2 in the diamondback moth.

Earth's rotation shapes a 24-h cycle, governing circadian rhythms in organisms. In mammals, the core clock genes, CLOCK and BMAL1, are regulated by PERIODs (PERs) and CRYPTOCHROMEs (CRYs), but their roles remain unclear in the diamondback moth, Plutella xylostella. To explore this, we studied P.xylostella, which possesses a simplified circadian system compared to mammals. In P.xylostella, we observed rhythmic expressions of the Pxper and Pxcry2 genes in their heads, with differing phases. In vitro experiments revealed that PxCRY2 repressed monarch butterfly CLK:BMAL1 transcriptional activation, while PxPER and other CRY-like proteins did not. However, PxPER showed an inhibitory effect on PxCLK/PxCYCLE. Using CRISPR/Cas9, we individually and in combination knocked out Pxper and Pxcry2, then conducted gene function studies and circadian transcriptome sequencing. Loss of either Pxper or Pxcry2 eliminated the activity peak after lights-off in light-dark cycles, and Pxcry2 loss reduced overall activity. Pxcry2 was crucial for maintaining endogenous rhythms in constant darkness. Under light-dark conditions, 1 098 genes exhibited rhythmic expression in wild-type P.xylostella heads, with 749 relying on Pxper and Pxcry2 for their rhythms. Most core clock genes lost their rhythmicity in Pxper and Pxcry2 mutants, while Pxcry2 sustained rhythmic expression, albeit with reduced amplitude and altered phase. Additionally, rhythmic genes were linked to biological processes like the spliceosome and Toll signaling pathway, with these rhythms depending on Pxper or Pxcry2 function. In summary, our study unveils differences in circadian rhythm regulation by Pxper and Pxcry2 in P.xylostella. This provides a valuable model for understanding circadian clock regulation in nocturnal animals.

Circadian rhythm and whole gut transit in mice.

In preclinical studies whole gut transit (WGT) in mice is a gold-standard "leading-edge" approach that measures the time between orogastric gavage of carmine red and defecation of the first carmine red pellet. Transit studies in humans are performed during the active day because GI motility and transit are suppressed during the night. Since mice are nocturnal, WGT studies traditionally done during the day occur during their rest phase. How circadian rhythm affects WGT in mice is not known. We used an automated approach for high temporal resolution uninterrupted testing of mouse WGT and activity. We housed wild-type Bl6/C57 mice under the standard 12 h light-dark cycles. At 8 weeks, we performed carmine red orogastric gavage and assessed WGT during Light (rest) conditions. Then, we exposed mice to a reverse 12 h light-dark cycle for 2 weeks and tested them in the Dark (active) under red light conditions. Timelapse videos were analyzed to quantify activity and to timestamp all pellets, and multiple parameters were analyzed. When complementary light cycle reversal experiments were performed, we found a significant increase in mouse activity when mice were tested during their Dark (active) phase, compared to their Light (rest) phase. In mice tested in the Active phase compared to the Rest phase, we found a significant acceleration in WGT, increased rate and total number of pellets produced, and more pellet clustering. These data show that the mice tested in the Active phase have important differences in activity that correlate with multiple alterations in gastrointestinal transit. During the Active phase mice have faster WGT, produce more pellets, and cluster their output compared to testing in the Rest phase. Like in humans, circadian rhythm is an important consideration for transit studies in mice, and a simple reverse light cycle approach facilitates further studies on the role of circadian rhythm in GI motility.

Circadian Disruption across Lifespan Impairs Glucose Homeostasis and Insulin Sensitivity in Adult Mice.

Circadian rhythm disruption is associated with impaired glucose homeostasis and type 2 diabetes. For example, night shift work is associated with an increased risk of gestational diabetes. However, the effects of chronic circadian disruption since early life on adult metabolic health trajectory remain unknown. Here, using the "Short Day" (SD) mouse model, in which an 8 h/8 h light/dark (LD) cycle was used to disrupt mouse circadian rhythms across the lifespan, we investigated glucose homeostasis in adult mice. Adult SD mice were fully entrained into the 8 h/8 h LD cycle, and control mice were entrained into the 12 h/12 h LD cycle. Under a normal chow diet, female and male SD mice displayed a normal body weight trajectory. However, female but not male SD mice under a normal chow diet displayed glucose intolerance and insulin resistance, which are associated with impaired insulin signaling/AKT in the skeletal muscle and liver. Under high-fat diet (HFD) challenges, male but not female SD mice demonstrated increased body weight gain compared to controls. Both male and female SD mice developed glucose intolerance under HFD. Taken together, these results demonstrate that environmental disruption of circadian rhythms contributes to obesity in a sexually dimorphic manner but increases the risk of glucose intolerance and insulin resistance in both males and females.

Transcriptome analysis of the harmful alga Heterosigma akashiwo under a 24-hour light-dark cycle

The photoperiod, which is defined as the period of time within a 24-hour time frame that light is available, is an important environmental regulator of several physiological processes in phytoplankton, including harmful bloom-forming phytoplankton. The ichthyotoxic raphidophyte Heterosigma akashiwo is a globally distributed bloom-forming phytoplankton. Despite extensive studies on the ecological impact of H. akashiwo, the influence of the photoperiod on crucial biological processes of this species remains unclear. In this study, gene expression in H. akashiwo was analyzed over a 24-hour light-dark (14:10) treatment period. Approximately 36 % of unigenes in H. akashiwo were differentially expressed during this 24-hour treatment period, which is indicative of their involvement in the response to light-dark variation. Notably, the number of differentially expressed genes exhibited an initial increase followed by a subsequent decrease as the sampling time progressed (T0 vs. other time points). Unigenes associated with photosynthesis and photoprotection reached their peak expression levels after 2–4 h of illumination (T12-T14). In contrast, the expression of unigenes associated with DNA replication peaked at the starting point of the dark period (T0). Furthermore, although several unigenes annotated to photoreceptors displayed potential diel periodicity, genes from various photoreceptor families (such as phytochrome and cryptochrome) showed unique expression patterns. Collectively, our findings offer novel perspectives on the response of H. akashiwo to the light-dark cycle, serving as a valuable resource for investigating the physiology and ecology of this species.

Effects of circadian clock disruption on gene expression and biological processes in Aedes aegypti

BackgroundThis study explores the impact of disrupting the circadian clock through a Cycle gene knockout (KO) on the transcriptome of Aedes aegypti mosquitoes. The investigation aims to uncover the resulting alterations in gene expression patterns and physiological processes.ResultsTranscriptome analysis was conducted on Cyc knockout (AeCyc-/-) and wild-type mosquitoes at four time points in a light-dark cycle. The study identified system-driven genes that exhibit rhythmic expression independently of the core clock machinery. Cyc disruption led to altered expression of essential clock genes, affecting metabolic processes, signaling pathways, stimulus responses and immune responses. Notably, gene ontology enrichment of odorant binding proteins, indicating the clock's role in sensory perception. The absence of Cyc also impacted various regulation of metabolic and cell cycle processes was observed in all time points.ConclusionsThe intricate circadian regulation in Ae. aegypti encompasses both core clock-driven and system-driven genes. The KO of Cyc gene instigated extensive gene expression changes, impacting various processes, thereby potentially affecting cellular and metabolic functions, immune responses, and sensory perception. The circadian clock's multifaceted involvement in diverse biological processes, along with its role in the mosquito's daily rhythms, forms a nexus that influences the vector's capacity to transmit diseases. These insights shed light on the circadian clock's role in shaping mosquito biology and behavior, opening new avenues for innovative disease control strategies.

Preparation of solid-solid phase change composites and their solar-interfacial water evaporation performance

Solar powered interfacial evaporation materials are widely used in seawater desalination. But solar energy is intermittent. To solve this problem, researchers combined phase change materials (PCMs) with interfacial evaporation materials. Many PCMs are loaded into the interface evaporation matrix material through physical blending, which may cause leakage problems during long-term use, leading to secondary water pollution. We have developed a novel interfacial evaporation material that continuously and efficiently vaporizes clean water under intermittent sunlight to address these issues. The solid-solid PCMs (BC/PVA/GO-g-LA-SSPCMs) were obtained by chemical grafting of PCMs lauric acid (LA) and graphene oxide (GO), and the substrate material bacterial fibrin (BC) was modified by polyvinyl alcohol (PVA). The material achieved light absorption of 88.8% in the full spectrum range. Under 1 kW m−2 light, the evaporation rate reaches 3.4 kg m−2h−1, and the solar-thermal conversion efficiency is 94.2%. In the dark, the evaporation rate is 1.05 kg m−2h−1, and the solar-thermal conversion efficiency is 49.5%. 15 light-dark cycle experiments and harsh environments (high concentration brine, acidic solution, alkaline solution, dye wastewater) showed high efficiency and stable evaporation performance. The material cost of the evaporator is about 0.98 USD·m−2, and the solar-thermal quality factor is 4.64, which is higher than that of most reported materials. In short, BC/PVA/GO-g-LA-SSPCMs provides a new idea for the future design and development of solar-thermal conversion materials to achieve efficient and continuous saltwater desalination and wastewater purification under intermittent solar-irradiation.

Elovl4b knockout zebrafish as a model for ocular very-long-chain PUFA deficiency

Very-long-chain PUFAs (VLC-PUFAs) are a group of lipids with chain lengths >24 carbons, and the ELOVL4 (elongation of very-long-chain FA-4) enzyme is responsible for vertebrate VLC-PUFA biosynthesis. Studies on the role of VLC-PUFAs in vision have been hindered because of the need for adequate animal models to capture the global loss of VLC-PUFAs. Since homozygous Elovl4 ablation is lethal in neonatal mice because of catastrophic drying from the loss of their protective skin barrier, we established a zebrafish (Danio rerio) model of Elovl4 ablation. We generated Elovl4b KO zebrafish by creating a 56-bp deletion mutation in exon 2 of the Elovl4b gene using CRISPR-Cas9. We used GC-MS and LC-MS/MS to analyze the VLC-PUFA and lipid profiles from wild-type and Elovl4b KO fish eyes. We also performed histology and visual-behavioral tests. We found that heterozygous and homozygous Elovl4b KO zebrafish eyes had altered lipid profiles and a significantly lower C30 to C36 VLC-PUFA abundance than wild-type fish. Moreover, Elovl4b+/− and Elovl4b−/− KO larvae had significantly lower motor activity in response to light-dark cycles than their age-matched controls. Elovl4b−/− adult fish showed no obvious differences in gross retinal morphology and lamination compared with wild type, except for the presence of lipid droplets within the retinal pigment epithelial cell layer of Elovl4b−/− fish. Our data indicate that the loss of Elovl4b in zebrafish changes ocular lipid profiles and leads to visual abnormalities and subtle retinal changes. These findings highlight the use of zebrafish as a model for VLC-PUFA depletion and ELOVL4-related dysfunction.

Mathematical Analysis of Light-sensitivity Related Challenges in Assessment of the Intrinsic Period of the Human Circadian Pacemaker.

Accurate assessment of the intrinsic period of the human circadian pacemaker is essential for a quantitative understanding of how our circadian rhythms are synchronized to exposure to natural and man-made light-dark (LD) cycles. The gold standard method for assessing intrinsic period in humans is forced desynchrony (FD) which assumes that the confounding effect of lights-on assessment of intrinsic period is removed by scheduling sleep-wake and associated dim LD cycles to periods outside the range of entrainment of the circadian pacemaker. However, the observation that the mean period of free-running blind people is longer than the mean period of sighted people assessed by FD (24.50 0.17 h vs 24.15 0.20 h, p 0.001) appears inconsistent with this assertion. Here, we present a mathematical analysis using a simple parametric model of the circadian pacemaker with a sinusoidal velocity response curve (VRC) describing the effect of light on the speed of the oscillator. The analysis shows that the shorter period in FD may be explained by exquisite sensitivity of the human circadian pacemaker to low light intensities and a VRC with a larger advance region than delay region. The main implication of this analysis, which generates new and testable predictions, is that current quantitative models for predicting how light exposure affects entrainment of the human circadian system may not accurately capture the effect of dim light. The mathematical analysis generates new predictions which can be tested in laboratory experiments. These findings have implications for managing healthy entrainment of human circadian clocks in societies with abundant access to light sources with powerful biological effects.

Artificial light at night alters progression of cold neuropathy in a sex-dependent manner in a mouse model of type II diabetes mellitus

Artificial light at night (ALAN) is a pervasive circadian rhythm disruptor. Exposure to ALAN is associated with detrimental effects on physiology and behavior, including disrupted metabolism, immune function, endocrine function, and pain behavior. Given the detrimental effects of ALAN and other circadian rhythm disruptors on pain, we sought to understand how ALAN may alter the progression and severity of diabetic neuropathy. To do this, we used a previously reported high-fat diet and streptozotocin injection protocol to induce a type II diabetic phenotype in ∼8 week old female and male mice and then exposed the mice to either control or ALAN lighting conditions in 14:10 h light-dark cycles for 4 weeks. Male mice housed in control conditions exhibited reduced responsiveness to cold pain; in contrast, ALAN blunted this effect in male mice. ALAN exposure also elevated blood glucose and altered body mass loss in male mice. These effects were not present in female mice. The results of this study highlight the need to consider and study ALAN exposure and sex as a biological variable as risk factors in the treatment and mitigation of pain.

Effects of cage type and the light-dark cycle on the behavior of hens subjected to forced molting.

In this study, the effects of cage type and the light-dark cycle on the behavior of hens before, during, and after forced molting were determined. For this purpose, 73-week-old hens were placed in the two different cage types used in the experiment. The barley method was used to induce molting at 75 weeks of age. The molting period lasted 35 days in total. The frequencies of walking, feeding, comforting, and preening behaviors were affected by the forced molting period. The addition of enrichment materials to the cage did not cause any changes in hen behavior. The light-dark cycle was important for the frequency of all examined behaviors except fighting behavior. No interaction was found among forced molting period, cage type, and light-dark cycle regarding behaviors. It is thought that there is a decrease in welfare-related behaviors at the end of the laying period and this decrease does not increase with forced molting using barley; on the contrary, appropriate forced molting applied to hens during this period will increase welfare. It was observed that cage type did not cause a significant difference in hen behavior during the forced molting period.

Light-dark cycles may influence insitu soil bacterial networks and diurnally-sensitive taxa.

Soil bacterial taxa have important functional roles in ecosystems (e.g. nutrient cycling, soil formation, plant health). Many factors influence their assembly and regulation, with land cover types (e.g. open woodlands, grasslands), land use types (e.g. nature reserves, urban green space) and plant-soil feedbacks being well-studied factors. However, changes in soil bacterial communities insitu over light-dark cycles have received little attention, despite many plants and some bacteria having endogenous circadian rhythms that could influence soil bacterial communities. We sampled surface soils insitu across 24-h light-dark cycles (at 00:00, 06:00, 12:00, 18:00) at two land cover types (remnant vegetation vs. cleared, grassy areas) and applied 16S rRNA amplicon sequencing to investigate changes in bacterial communities. We show that land cover type strongly affected soil bacterial diversity, with soils under native vegetation expressing 15.4%-16.4% lower alpha diversity but 4.9%-10.6% greater heterogeneity than soils under cleared vegetation. In addition, we report time-dependent and site-specific changes in bacterial network complexity and between 598-922 ASVs showing significant changes in relative abundance across times. Native site node degree (bacterial interactions) at the phylum level was 16.0% higher in the early morning than in the afternoon/evening. Our results demonstrate for the first time that light-dark cycles have subtle yet important effects on soil bacterial communities insitu and that land cover influences these dynamics. We provide a new view of soil microbial ecology and suggest that future studies should consider the time of day when sampling soil bacteria.

Humanized dopamine D4.7 receptor male mice display risk-taking behavior and deficits of social recognition and working memory in light/dark-dependent manner.

The dopamine D4 receptor 7-repeat allele (D4.7 R) has been linked with psychiatric disorders such as attention-deficit-hyperactivity disorder, autism, and schizophrenia. However, the highly diverse study populations and often contradictory findings make it difficult to draw reliable conclusions. The D4.7 R has the potential to explain individual differences in behavior. However, there is still a great deal of ambiguity surrounding whether it is causally connected to the etiology of psychiatric disorders. Therefore, humanized D4.7 R mice, with the long third intracellular domain of the human D4.7 R, may provide a valuable tool to examine the relationship between the D4.7 R variant and specific behavioral phenotypes. We report that D4.7 R male mice carrying the humanized D4.7 R variant exhibit distinct behavioral features that are dependent on the light-dark cycle. The behavioral phenotype was characterized by a working memory deficit, delayed decision execution in the light phase, decreased stress and anxiety, and increased risk behavior in the dark phase. Further, D4.7 R mice displayed impaired social recognition memory in both the light and dark phases. These findings provide insight into the potential causal relationship between the human D4.7 R variant and specific behaviors and encourage further consideration of dopamine D4 receptor (DRD4) ligands as novel treatments for psychiatric disorders in which D4.7 R has been implicated.

First report of sweet corn root rot caused by Pythium graminicola in southeast China.

Sweet corn (Zea mays L.) is widely consumed as fresh or frozen vegetable worldwide, and Zhengtian68 is a popular commercial variety cultivated extensively in southeast China. In May 2021, 40% of the inbred line YK063 (the female parent of Zhengtian68) showed early yellowing of the leaves at flowering time in a commercial seed production field with a total area of 0.5 ha in Guangzhou, Guangdong Province after a heavy rain. Black and rotten roots were observed in the diseased plants after digging the whole plant out of the soil. Grain filling was also severely affected, adversely impacting seed production. Diseased plants were more easily found in the lower section of the field, where water accumulated after rainfall. Three plants with rotten roots were collected randomly from the field to identify the causal pathogen. The diseased roots were cut into 2-3 mm sections, washed in 75% ethanol for 2 minutes and rinsed three times in sterile distilled water. Four to five sections per plant were placed on potato dextrose agar (PDA) and incubated at 28℃ in the dark for three days. Three isolates GF1, GF2, and GF3 from different plants were purified by hyphal tip isolation and transferred to new PDA and 10% V8 juice agar (16 g agar, 3 g CaCO3, 100 ml V8 juice, and 900 ml distilled water) and incubated at 28℃ for 10 days in darkness for further investigation. Translucent, glassy mycelial growth was observed on the PDA media. Morphological characteristics of the 3 isolates were observed under a microscope from the 10%V8 media. The hyphae were aseptate and 2.7 to 4.5 μm wide (mean±SD,3.3±0.44μm, n=44). Sporangia were inflated, or lobulate, terminal, or intercalary. Oogonia were globose, smooth-walled, terminal, or occasionally intercalary, with a diameter of 17.2-24.1 μm (mean±SD, 21.3±2.14μm, n=29). Oospores were globose, plerotic, smooth, and 14.5-21.2 μm (mean±SD, 18.7±2.07μm, n=35) in diameter. The antheridia were diclinous or monoclinous, not intercalary, and one to six antheridia were attached to each oogonium. Based on these morphological characteristics, 3 isolates were identified as Pythium spp. including Pythium graminicola (Van der Plaats-Niterink 1981). Genomic DNA was extracted from the mycelia grown on PDA using a Fungal Genomic DNA kit (Scintol, Beijing, China) according to the manufacturer's instructions. The cytochrome oxidase II (Cox II) gene and internal transcribed spacer (ITS) region of the rDNA were amplified using the primers FM58/FM66 (Martin 2000) and ITS4/ITS5 (White et al. 1990) respectively. Amplification was performed in a 50μl reaction volume using 25 μl PCR Mix (Trans Gene, Beijing, China), 3 μl genomic DNA (50 ng/μl), 1 μl each forward and reverse primer (10 μM), and 20 μl ddH2O. The PCR program was as follows: initial denaturation at 95°C for 30 s, 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 60 s, extension at 72°C for 60 s, and a final extension at 72°C for 10 min. PCR products were sequenced and submitted to GenBank (accession no. OQ504322, OQ933130, and OQ933212 for ITS; OQ512002, OQ942203, and OQ942204 for Cox II). BLASTn analysis revealed that the ITS and Cox II sequences showed more than 98.62% similarity (721/724bp, 722/724bp,723/724bp for ITS; 514/514bp, 506/507bp, 500/507bp for Cox II) to P. graminicola ATCC96234 (accession no. AB095045 for ITS, and AB160849 for Cox II), respectively, supporting the morphological analysis. A neighbor-joining phylogenetic analysis of the ITS and Cox II concatenated sequence further confirmed that the isolates were P. graminicola. To test the pathogenicity of GF1, GF2, and GF3 a wheat seed inoculum was prepared as previously described (Qu et al. 2016). Sweet corn YK063 plants were planted in sterilized nutrient soil in plastic pots (one plant per pot) and grown in a greenhouse at 28℃ with 60% humidity and a 12-h/12-h light-dark cycle. For each isolate,10 plants were inoculated with 20 infected wheat seeds around the roots at the V5 stage, while 10 other YK063 plants were inoculated with the non-infected wheat seeds as a control. The experiment was repeated once. Three weeks later, the non-inoculated plants were asymptomatic. In contrast, inoculated plants showed stunning, yellowing of the leaves, root rot, and decreased production of lateral roots, exhibiting symptoms similar to those originally described for the disease. P. graminicola was successfully reisolated from the diseased roots and identified by morphological characteristics and sequencing of the ITS and Cox II as the causal agent for this root rot disease, fulfilling Koch's postulate for defining a causal agent. P. graminicola was reported as a causal agent of damping-off on dent corn in Georgia (Li et al. 2018). To our knowledge, this is the first report of P. graminicola causing root rot in sweet corn in southeast China. Identification of this pathogen will facilitate further research on this disease and the development of effective strategies to control the disease.

First Report of Leaf Spot Caused by Paramyrothecium foliicola on Peanut in China.

Peanut (Arachis hypogaea) is an important economic and oil crop in China. In September 2022, leaf spots wereobserved on peanut in Luoyang city, Henan province, China (34°49'N, 112°37'E). The disease occurred on about 30% ofthe peanutleaves in only one 0.5-acre field. Symptoms appeared primarily as brown spots, that varied in shape, and appeared round, oval or irregular. In addition, some disease patches exhibited a concentric ring pattern. Small pieces (5×5 mm) of five diseased leaves were surface disinfected in 3% NaClO for 2 minutes, rinsed three times in sterile distilled water, dried on sterilized filter paper, and cultured on potato dextrose agar (PDA) at 25°C for 3 days. Five isolates with uniform characteristics were obtained and subcultured by transferring hyphal tips to fresh PDA. The colonies of the isolates were circular and the margins were clean. The colonies showed white coloration, and after 5-7 days of incubation on PDA plates, concentric rings with dark green sporodochia appeared on the surface of the colonies. The conidiophores branched repeatedly. The conidiophore stipes unbranched, hyaline, 10.0 to 23.2×1.5 to 3.3 μm (n=50). The conidia were rod-shaped or long oval and single-celled, measuring 4.6 to 8.6×1.4 to 3.1 μm (n=100). Based on these characteristics, the five isolates were identified as Paramyrothecium foliicola (Lombard et al 2016). Genomic DNA was extracted from the representative isolates LH-1-1 and LH-1-2. The internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), calmodulin (CmdA), and translation elongation factor 1-alpha (tef1) loci were amplified and sequenced using the following primer pairs: ITS1/ITS4 (White et al. 1990), RPB2-5F2/RPB2-7cR (O'Donnell et al. 2007), CAL-228F/CAL-2Rd (Carbone & Kohn 1999), and EF1-728F/EF2 (O'Donnell et al. 1998), respectively. BLASTn analysis revealed that the sequences of ITS (OR352397.1 and OR417392.1), RPB2 (OR413573.1 and OR420678.1), CmdA (OR413572.1 and OR420677.1), and tef1 (OR413574.1 and OR420679.1) had 99 to 100% (553/558 bp, 721/721 bp, 597/598 bp, and 384/389 bp) similarity to P. foliicola (MN593634.1, MN398038.1, OM801785.1, MK335967.1). A phylogenetic tree based on the Maximum Likelihood method also confirmed that the two isolates converge on the same branch as P. foliicola. Pathogenicity tests were performed using leaves of 60-day-old peanut plants (cv. Zhonghua 8). Briefly, uninfected healthy leaves (non-wounded) were inoculated with 30-µl drops containing a spore suspension (5×105 conidia/ml) of LH-1-2, and peanut leaves inoculated with sterile distilled water served as controls. All treatments were incubated in an incubator at 25℃ and high relative humidity with a 12:12 hour light-dark cycle. After 5-7 days, inoculated leaves showed symptoms similar to those observed in the field, while no symptoms were observed on control leaves. The pathogenicity tests were repeated three times. The fungus was reisolated from the infected leaves and identified as P. foliicola based on morphological and molecular characteristics, thus fulfilling Koch's postulates. P. foliicola has previously been reported to cause leaf spot of tomato and mung bean, stem canker of cucumber (Huo et al. 2022; Sun et al.2020; Huo et al. 2021). To our knowledge, this is the first report of P. foliicola causing leaf spot on peanut in the world. Identification of this pathogen will be helpful in monitoring peanut diseases and developing disease control strategies.

Addressing Circadian Disruptions in Visually Impaired Paralympic Athletes.

Transmeridian travel is common for elite athletes participating in competitions and training. However, this travel can lead to circadian misalignment wherein the internal biological clock becomes desynchronized with the light-dark cycle of the new environment, resulting in performance decrement and potential negative health consequences. Existing literature extensively discusses recommendations for managing jet lag, predominantly emphasizing light-based interventions to synchronize the internal clock with the anticipated time at the destination. Nevertheless, visually impaired (VI) athletes may lack photoreceptiveness, diminishing or nullifying the effectiveness of this therapy. Consequently, this invited commentary explores alternative strategies for addressing jet lag in VI athletes. VI athletes with light perceptionbut reduced visual acuity or visual fields may still benefit from light interventions in managing jet lag. However, VI athletes lacking a conscious perception of light should rely on gradual shifts in behavioral factors, such as meal timing and exercise, to facilitate the entrainment of circadian rhythms to the destination time. Furthermore, interventions like melatonin supplementation may prove useful during and after travel. In addition, it is recommended that athlete guides adopt phase-forward or phase-back approaches to synchronize with the athlete, aiding in jet-lag management and optimizing performance.

MR Spectroscopy Assessment of Daily Variations of GABA Levels within the Parietal Lobe and Anterior Cingulate Gyrus Regions of Healthy Young Adults.

The changes that occur in the gamma-aminobutyric acid (GABA) levels within specific brain regions throughout the day are less clear. To evaluate the daily fluctuations of GABA levels within the parietal lobe (PL) and anterior cingulate gyrus (ACC) regions and explore their association with melatonin (MT) levels, heart rate (HR), and blood pressure. Prospective. 26 healthy young adults (15 males and 11 females aged 22-27 years). 3.0T, T1-weighted imaging, Mescher-Garwood point resolved spectroscopy (MEGA-PRESS) sequence. The acquired GABA signal contained the overlapping signals of macromolecules and homocarnosine, hence expressed as GABA+. The creatine (Cr) signal was applied as an endogenous reference. The GABA+, GABA+/Cr were measured at six different time points (1:00, 5:00, 9:00, 13:00, 17:00, and 21:00 hours) using MEGA-PRESS. The blood pressure, HR and sputum MT levels, were also acquired. The one-way repeated-measures analysis of variance (ANOVA) was used to evaluate the GABA, blood pressure, HR, and MT levels throughout the day. A general linear model was used to find the correlation between GABA and blood pressure, HR, and MT. P < 0.05 was statistically significant. Significant variations in GABA+/Cr and GABA+ levels were observed throughout the day within the PL region. The lowest levels were recorded at 9:00 hour (GABA+/Cr: 0.100 ± 0.003，GABA+:1.877 ± 0.051 i.u) and the highest levels were recorded at 21:00 hour (GABA+/Cr: 0.115 ± 0.003, GABA+:2.122 ± 0.052 i.u). The MT levels were positively correlated with GABA+/Cr (r = 0.301) and GABA+ (r = 0.312) within the ACC region. GABA+/Cr and GABA+ in ACC are positively correlated with MT. GABA levels in the PL have diurnal differences. These findings may indicate that the body's GABA level change in response to the light-dark cycle. 1 TECHNICAL EFFICACY: Stage 2.