Comparative Analysis of The Daily Brain Transcriptomes of Asian Particolored Bat

Comparative Analysis of The Daily Brain Transcriptomes of Asian Particolored Bat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Analysis of The Daily Brain Transcriptomes of Asian Particolored Bat Guoting Zhang, Yujia Chu, Tinglei Jiang, Jingjing Li, Lei Feng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-951243/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Mar, 2022 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Circadian rhythms are found in almost all organisms, and they comprise one of the most basic characteristics of living things. Bats have attracted interest from researchers because of their unique biological rhythms. However, little is known about the molecular underpinnings of circadian signaling in bats. In this study, we used RNA-Seq to uncover the rhythms of expressed genes and biological processes in the brains of Asian particolored bats. Four collected time points corresponding to four biological states, rest, sleep, wakefulness, and active, were selected over the 24-hour day, and six comparisons of differentially expressed genes were obtained. Downstream functional enrichment analyses suggested that different physiological processes were active at various biological states, including drug metabolism, signaling pathways, and the circadian clock. Furthermore, trend analysis suggested that groups of genes are significantly clustered and are expressed at the lowest levels during the sleep state while being highly expressed during the active stat. In addition, we also identified the Per2 locus as an important circadian clock gene in the brain of Asian particolored bat. Our study provides a new overview of the molecular basis underlying the circadian clock in the brain of bats. Molecular Biology rhythms biological clock signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Essentially all organisms have evolved daily rhythms in physiology and behavior to adapt to a dynamic environment; these rhythms are driven by biological clocks 1 , 2 . The circadian pacemaker is one of the most famous clock systems that are controlled by an endogenous oscillator with a period of ~24 h, the circadian clock 3 . At the molecular level, the circadian clock not only regulates the expression of core clock genes such as Clock, Bmal1 , and Per but also drives the rhythmic expression of thousands of transcripts in specific tissues 3 , 4 . Daily cycles of gene expression patterns are responsible for the coordinated recurring changes in physiological processes and functions in multiple tissues 5 . Previous studies have demonstrated that the circadian clock is closely related to rhythmically expressed genes in the brain 1 , 6 . In mammals, the master circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus 7 . For the brain, gene expression rhythms and circadian clock genes have been reported in several mammals 8 such as the mouse, a nocturnal model organism. However, laboratory mice ( Mus musculus ) are usually used for these and related studies, while little is known about the molecular underpinnings of circadian signaling in other wild nocturnal animals. Bats belong to the order Chiroptera, the second largest group of mammals after Rodentia 9 . As totally wild nocturnal mammals, bats are more enigmatic and are known for their abilities to fly and echolocate in the dark 10 . Despite numerous studies concerning bats’ daily behavior patterns and physiological processes, little is known about the molecular circadian clock in this order. In this study, a transcriptomics approach was used to gain insights into the molecular circadian clock responsible for the regulation of daily physiological and behavioral outputs in the bat brain. We aimed to 1) identify genes expressed rhythmically over the 24-h light: dark cycle in the bat brain; 2) detect and verify the core circadian clock genes expressed in the bat brain; and 3) determine the rhythmic changes of associated important physiological processes. Therefore, this study provides evidence for the recurring variation in gene expression affecting the coordination of physiology and behavior in the brain of bat over a 24-hour period. Materials And Methods Ethics statement According to the regulations of Wildlife Conservation of the People’s Republic of China (Chairman Decree (2016) No. 47), permits are required only for species included in the list of state-protected and region-protected wildlife species. Vespertilio sinensis used in this study is widespread in China and is not an endangered or region-protected animal species, so no specific permission was required. All the studies have been approved by Laboratory Animal Welfare and Ethics Committee of Jilin Agricultural University. All efforts were made to minimize suffering of animals. We confirm that complied with the ARRIVE guidelines and all experiments were performed in accordance with relevant named guidelines and regulations. Sample Collection All animals used in this study were caught during July 2020 in Heilongjiang Province, China. Through a week of observation and recording of the daily activities of Vespertilio sinensis , four sampling time points were set, corresponding to the four typical behaviors of bats in a day. These time points were 4 a.m., 10 a.m., 4 p.m., and 10 p.m., corresponding to the rest state, sleep state, awake state, and active state of the bat, respectively. Three biological repeats were included for each time point, and 12 individuals were collected in total. To avoid any influence of sex-related differences, only females were selected for inclusion in the study. Animals were sacrificed by decapitation at the four time points of day during the course of the study to avoid unnecessary suffering. The brain tissues from each individual were collected and immediately flash-frozen in liquid nitrogen followed by placement in a −80°C freezer until processing for total RNA isolation. Rna Extraction Total RNA was isolated using the Total RNA Extractor reagent in accordance with the manufacturer’s protocol. The quantity and quality of total RNA were measured using a Qubit 2.0 Fluorometer and gel electrophoresis. RNA samples of the same volume and concentration were used during the step of converting mRNA into cDNA. Three paired-end cDNA libraries of each time point were generated using the mRNA-Seq assay. In total, 12 cDNA libraries were prepared at an equimolar ratio for transcriptome sequencing on the Illumina HiSeq 4000 platform. All raw reads were deposited in the NCBI Short Read Archive (SRA) Database under SRA accession PRJNA756631. Transcriptome Assembly And Functional Annotation The raw reads were filtered by Trimmomatic software 24 using four criteria: removing reads with adaptors; removing reads with unknown “N” bases; removing low-quality reads containing more than 50% low-quality bases (Q-value ≤ 20); and removing reads whose length is less than 35 nt. To construct a general reference transcriptome for subsequent analysis, the cDNA library sequences of 12 liver tissues sampled at the same time point and the brain tissue sequences were put together for splicing and assembly. The specific liver data analysis is published elsewhere. All high-quality raw reads from 12 individual cDNA libraries were used for de novo assembly by Trinity software 25 with the default parameters. After the transcripts were reduced for sequence redundancy, the longest transcript in each cluster was taken as a unigene for further analysis. The assembly result was evaluated by parameters such as the longest value of the sequence, the shortest value of the sequence, the N50 value, and the N90 value. Unigene annotations included protein function annotations, KOG function annotations, and Gene Ontology (GO) and pathway annotations. In detail, we used Blast+ 26 on the NCBI nonredundant protein (Nr) database, the Swiss-Prot protein database, the KOG database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The GO annotation of unigenes was based on the protein annotation results of Swissprot and TrEMBL and the annotation information of Uniprot. Differential Expression Analysis Considering the differences in library size, we first performed inter-sample normalization and used Bowtie2 software 27 to map clean reads to the assembled reference sequence. Then, we used Salmon software 28 to locate the genomic region or gene exon regions and estimated the gene expression levels by counting the reads. For the sequencing depth and gene length as well as the impact of the sample on the read count, the TPM value (Transcripts Per Million) was applied to determine the level of expression of each gene 29 . The correlation coefficient between each pair of replicates for the four time points was calculated using the R package (version 2.16.2) to evaluate the reliability of the experimental results as well as the operational stability. The differentially expressed genes (DEGs) at the four time points were evaluated by DESeq software 30 . We separately counted the expression levels at four time points in one day, and then compared the results in pairs to obtain a total of six pairwise tests. In the six comparisons, we focused on the comparison of rest vs wakefulness and sleep vs activity. To create a list of high-confidence DEGs for further analyses, the following stringent criteria were used: fold change >2 and adjusted p value < 0.05 31 . The visualization of the DEGs in the brain tissue collected at four time points was presented by creating heatmaps with the GPLOTS R package. Go Category And Kegg Pathway Enrichment Analyses Downstream functional classification was achieved through the integrated localization of GO 32 and KEGG pathway databases 33 . We conducted the GO and KEGG enrichment analysis on the DEGs in OmicShare ( http://www.omicshare.com/ ). All p values were computed using the hypergeometric test, and multiple test correction was performed using the Benjamini–Hochberg method based on an FDR (false discovery rate) cut-off of 0.05. Degs Trend Analysis First, we first screened the genes that were co-expressed at the four time points. Then, we conducted trend analysis on these genes in OmicShare ( http://www.omicsshare.com/ ). To determine the rhythmic changes of the important physiological and biochemical processes involved in the genes at four time points, we performed GO and KEGG enrichment analyses on the genes in the significant trend module. Identification Of Key Circadian-related Terms, Pathways, And Genes We focused on the circadian-related terms and pathways among those that were significantly enriched by genes from the trend analysis and six comparative comparisons. Considering the important functions in the regulation of circadian rhythm, the genes enriched in circadian-related terms and pathways were assumed to be the most important circadian rhythm genes in bat brains. To determine the circadian-related genes among the enriched genes, we searched for detailed information in published research articles based on their basic annotation information to identify their potential functions in the bat brain. Results Sequence analysis, assembly, and functional annotation The transcriptome sequencing results of brain tissue are shown in Table 1 . After filtering the raw reads, 143,936,974, 142,787,930, 139,867,950 and 138,606,658 clean reads were obtained for the four brain states, respectively, and the corresponding base numbers were 22,640,739,600 bp, 22,425,822,300 bp, 22051944000 bp and 21,898,530,000bp (Table 1 ). The proportions of clean/raw reads of the four states were between 94.49% and 95.51%, suggesting the high quality of the RNA-Seq data available for further analyses. Table 1. Sequencing and assembly statistics of brain samples for V. sinensis The clean reads of this brain area and liver were assembled together, and a total of 403,707 unigenes sequences were obtained. The unigenes sequences were used as the reference genome in the subsequent analysis of the brain. The lengths of 403,707 unigenes ranged from 201 bp to 19,402 bp, with an average length of 511 bp, an N50 value of 615 bp, and an N90 value of 244 bp. Among the 403,707 unigenes, 83,237, 60,588, 23,820 and 9,176 were respectively annotated by the NR, Swiss-Prot, KOG, and KEGG databases (Supplementary Fig. S1). For KOG annotation in particular, the term of signal transduction mechanisms was the most highly represented (Supplementary Fig. S2). Degs Analysis A correlation analysis was conducted based on the TPM values. From the correlation results (Supplementary Fig. S3), the sample had good repeatability and could be used for gene differential expression analysis. To clarify the differences in the transcription levels of genes in different activity states of V. sinensis , we compared the transcriptome data of two adjacent time points and obtained four comparisons of DEGs. In addition, we also focused on the analysis of the rest vs awake (before and after feeding) and sleep vs activity comparisons, and a total of six pairwise comparisons were obtained (Table 2 ). In the six pairwise comparisons, a total of 672 DEGs were detected. In the rest vs activity comparison, the number of DEGs was the highest, with 136 genes, and the lowest number of DEGs was detected in the awake vs active comparison, with 60 genes. The numbers of upregulated genes in the comparison groups were higher than the numbers of downregulated genes in the two comparisons. In terms of the definitions of upregulation and downregulation of specific genes in each pairwise comparison, an upregulated gene was defined as one that had a higher expression level in the former time point than in the latter, and vice versa for a down-regulated gene. In the awake vs active comparison, this may be because bats are always active after awakening to prepare for subsequent hunting behavior followed by a highly active predation state, so the numbers of DEGs in the two states were less, as similar genes were expressed. A heatmap of the hierarchical clustering of all DEGs indicated the large differences in gene expression between different time points (Fig. 1 ). Table 2. The number of DEGs in the six pairwise comparisons of brain tissue Comparable group Up Down Total Rest vs sleep 63 59 122 Sleep vs wake 65 69 134 Wake vs activity 32 28 60 Rest vs activity 63 73 136 Rest vs wake 63 57 120 Sleep vs activity 47 53 100 Go And Kegg Analysis Of Degs The six pairwise comparisons were analyzed by GO and KEGG enrichment, and the results suggested that the bats had significant differences in the metabolic and transport activities at different time points during the day (Supplementary Tables S1–S4). The rest (after feeding) vs awake (before feeding) comparison represented the two extreme states before and after predation. There were 63 genes that showed high expression levels in the after feeding state and low expression levels in the before feeding state (Table 2 ). In the cell component of GO, multiple complexes were significantly enriched by these 63 genes, including Prp19 complex, spliceosomal complex, and DNA packaging complex. In the category of molecular function, these genes were significantly enriched in terms of transferase activity, for example, histone methyltransferase activity (H4-R3 specificity), aryl sulfotransferase activity, and protein-arginine omega-N asymmetric methyltransferase activity. In the category of biological process, these genes were significantly enriched in hemoglobin synthesis-related terms such as positive regulation of hemoglobin biosynthetic process, regulation of hemoglobin biosynthetic process, and hemoglobin biosynthetic process (Fig. 2 ). Regarding the KEGG results, various metabolic pathways were significantly enriched by these 63 genes, including drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, and glutathione metabolism (Fig. 3 ). The above results indicated that hemoglobin synthesis, transferase activity, and metabolic activity of V. sinensis in the after feeding state may be higher than during the before feeding state. There were 57 genes that showed low expression levels in the after feeding state and high expression levels in the before feeding state. In the cell component category, these genes were significantly enriched in the terms of proton transport. The three top GO terms with the most significant p values were mitochondrial proton-transporting ATP synthase complex, proton-transporting ATP synthase complex, and proton-transporting two-sector ATPase complex, catalytic domain. In the molecular function category, these genes were significantly enriched in terms that were closely related to various enzyme transport pathways such as cation transporting ATPase activity, beta mannosidase activity, and ATPase coupled ion transmembrane transporter activity. For the biological process category, transport-related terms were significantly enriched by these 57 genes, including hydrogen ion transmembrane transport, hydrogen transport, and proton transport (Fig. 2 ). Regarding the KEGG results, a variety of endocrine-related pathways were significantly enriched by these 57 genes, including the renin-angiotensin system, renin secretion, and cortisol synthesis and secretion (Fig. 3 ). The above results indicated that the intensity of transport and endocrine activities of bats in the before feeding state may be higher than in the after feeding state. In addition, sleep and activity were the two states that best reflected the difference between the day and night activities of V. sinensis . There were 47 genes that showed high expression levels in the sleep state and low expression levels in the active state (Table 2 ). In the cell component category, these genes were significantly enriched in organelle-related terms such as cytoplasmic part, Golgi apparatus, and organelle membrane. In the molecular function category, these genes were significantly enriched in terms related to enzyme activities, including inositol tetrakisphosphate phosphatase activity, cytochrome c oxidase activity, and heme-copper terminal oxidase activity. In the biological process category, these 47 genes were significantly enriched in nuclear-related terms including nuclear matrix organization, nuclear matrix anchoring at nuclear membrane, and cytoskeletal anchoring at nuclear membrane (Fig. 2 ). For the KEGG results, the 47 genes in the sleep state were only significantly enriched in the ribosome pathway (Fig. 3 ). In addition, there were 53 genes that were expressed at low levels in the sleep state and highly expressed in the activity state. In the cell component category, terms that were related to proton transport were significantly enriched by the 53 genes, including mitochondrial proton-transporting ATP synthase, catalytic core, and proton transporting ATP synthase. In the molecular function category, genes were significantly enriched in the terms related to DNA binding, including RNA polymerase II transcription factor activity (ligand-activated sequence-specific DNA binding), transcription factor activity (direct ligand regulated sequence-specific DNA binding), and chromatin DNA binding. In the biological process category, these genes were significantly enriched in multiple terms related to endocrine activities, and the three top GO terms with the most significant p values were response to corticotropin releasing hormone, cellular response to corticotropin releasing hormone stimulus, and regulation of type B pancreatic cell proliferation. In addition, these genes were also significantly enriched in terms related to the circadian clock, including entrainment of circadian clock, circadian regulation of gene expression, and circadian rhythm (Fig. 2 ). Regarding the KEGG results, various hormone secretion pathways and signal transduction pathways were significantly enriched by the 53 genes, including cortisol synthesis and secretion, MAPK signaling pathway, and PI3K-Akt signaling pathway. In addition, there were two circadian clock-related pathways that were significantly enriched, namely, circadian rhythm and the circadian rhythm-fly. Importantly, both circadian clock-related pathways contain the Per2 gene (Fig. 3 ). The above results showed that the two states of bats with completely opposite circadian rhythms do involve circadian clock-related genes and regulate the regular changes of some circadian physiological processes. Trend Analysis Of Degs To clarify the periodic expression and oscillation changes of genes in the brain area of V. sinensis during a 24-hour period, we examined the intersection of the six groups of DEGs, i.e., screened out genes that were expressed at all time points, to obtain a total of 557 genes. Then, we conducted trend analysis on these 557 genes, and the results are shown in Supplementary Fig. S4. Among the 20 modules obtained, the DEGs were significantly clustered in module 6 that contained 27 genes. These 27 genes had similar expression trends at the four time points during the day. The expression levels of these genes were decreased in the resting state, were the lowest in the sleep state, and gradually increased in the waking state, reaching the highest expression level in the activity state (Fig. 4 a). These genes in module 6 were used in enrichment analysis, and the results are shown in Fig. 4 and Supplementary Table S5. Numerous circadian clock-related terms and pathways were significantly enriched by these 27 genes. Through the above analysis, we believe that the change trend of the genes in module 6 was consistent with the biological habits of bats. These 27 genes showed obvious circadian rhythms and were closely associated with bat activity states. Bats in an active state require high expression levels of these genes, so the physiological and biochemical processes that these genes participate in play important roles in the activity and periodic awakening of bats. The important physiological and biochemical processes enriched by genes of module 6 are shown in Fig. 4 . The GO results for 27 genes in module 6 suggested that a total of 161 terms were significantly enriched; 238 terms were significantly enriched in the biological process category; 26 terms were significantly enriched in the molecular function category, and 18 terms were significantly enriched in the cell component category (Supplementary Table S5). In the cell component category, the top three terms with the most significant p values were transcription factor complex, varicosity, and nuclear membrane. In the molecular function category, terms related to DNA molecular binding were significantly enriched, and the top three terms with the most significant p values were RNA polymerase II transcription factor activity, transcription factor activity, and steroid hormone receptor activity. In the biological process category, terms related to endocrine activities were significantly enriched by genes of module 6, and the top three GO terms with the most significant p values were response to corticotropin releasing hormone, cellular response to corticotropin releasing hormone stimulus, and regulation of type B pancreatic cell proliferation. Additionally, terms related to circadian clocks were also significantly enriched, including entrainment of circadian clock and the entrainment of circadian clock by photoperiod (Fig. 4 b). The KEGG results of module 6 are shown in Fig. 4 . The top three pathways with the most significant p values were cortisol synthesis and secretion, aldosterone synthesis and secretion, and Cushing syndrome. The circadian rhythm and circadian rhythm-fly pathways were also significantly enriched (Fig. 4 c). The above results suggested that there were circadian clock-related genes in module 6 that have important molecular functions in the circadian rhythm. Important Circadian Clock Genes Combining the results of DEGs and trend analysis, we found that the genes in the sleep vs activity comparison and the genes in module 6 were enriched in pathways related to the circadian clock (Supplementary Table S3 and Table S5). We further analyzed the genes in the pathways related to the circadian clock and found that they all involved Per2 . In the GO results of the sleep vs activity comparison and trend module 6, Per2 was significantly enriched in terms related to the circadian clock. Therefore, we believe that Per2 may be an important circadian clock gene in V. sinensis . We marked the Per2 gene in the figure illustrating the circadian rhythm pathway to show its important role in the pathway (Supplementary Fig. S5). Discussion The circadian clock is the result of animals adapting to the environment, and the daily variation of the physiological activities of animals is closely associated with the rhythmic expression of important genes. In this study, we used transcriptome sequencing technology to analyze the changes in genes expression in the brains of V. sinensis during a day, with a view to revealing the circadian changes in gene expression in the brain of a nocturnal mammal. In addition, to clarify the differences in gene expression and physiological processes in the brain area of bats during sleep and activity before and after eating, we focused on the sleep vs activity and rest vs awake comparisons and found that bats have different degrees of changes in corresponding physiological processes in different states. This study is the first to examine the rhythmic changes in gene expression in the brains of bats. The analysis can not only reflect the daily changes in the physiological and biochemical activities of genes but also screen out genes that may be involved in regulating the circadian clock in the brain, thereby providing a reference for further understanding the mutual adaptations of animals and the environment, and at the same time enriching the research on the biological rhythms of nocturnal animals. Our results indicated that there are significant differences in the physiological processes of bats in sleep vs activity. In the active state of V. sinensis , the pathways related to protein kinase signals are likely to be important circadian clock regulation pathways in the bat brain. The MAPK signaling pathway and the PI3K-Akt signaling pathway were significantly enriched by the genes with high expression levels in the active state. Previous studies have shown that in the mammalian SCN of the hypothalamus, the MAPK pathway can be used as an input pathway to allow the endogenous clock to enter the 24-hour rhythm cycle 11 . The MAPK pathway can also interact with the components of the molecular circadian oscillator to affect the cycle of the circadian clock 11 . The PI3K-Akt signaling pathway can participate in the output pathway of animal retinal circadian rhythms 12 . The structure of the mammalian circadian clock system is composed of the input pathway of external signals, the oscillating central biological clock, and the output pathway of the rhythm 3 . Combining the results of this study, we speculate that these two signal pathways are closely related to the circadian clock regulation mechanism in the brain and may be involved in the input and output pathways of the circadian clock system, and that they play an important role in the brain when bats are active. In addition, genes with high expression levels in the active state were also involved in multiple pathways or terms related to the circadian clock such as circadian rhythm, circadian rhythm–fly, and circadian regulation of gene expression, indicating that the bat brain is in the optimal state for overall physiological process and the regulation of the circadian clock during the active state. When bats are active, the physiological processes related to various endocrine hormones in the brain are also relatively active, for example cortisol synthesis and secretion and aldosterone synthesis and secretion. Cortisol is a glucocorticoid with an endogenous circadian rhythm driven by the central circadian pacemaker (CCP) of the SCN 13 . Several previous studies have documented that the concentration of cortisol in the human body generally reaches a peak during the transition between sleep and wakefulness, and cortisol has a wakeful response. Cortisol arousal response refers to the increase in cortisol concentration in the first hour after awakening 14 . We speculate that bats also have a cortisol awakening response when they are awake and may maintain a higher concentration when they are in an active state. Aldosterone is a mineralocorticoid that plays an important role in regulating systemic blood pressure 15 . In rats, the concentration of aldosterone also showed a clear circadian rhythm, and the concentration reached its peak at night and was the lowest at noon 16 . We speculate that the rhythm in V. sinensis may also resemble that of rodents, with a higher concentration of aldosterone at night. Therefore, we suggest that the secretion of many hormones in bats occurs in the active state and prepares the bat for subsequent predation activity. The results of KEGG and GO of 47 genes with high expression levels in the sleep state indicated that the physiological processes related to protein synthesis are relatively active in the brain of V. sinensis . In the KEGG results, the ribosome pathway was significantly enriched, and in biological process category of GO, terms such as mitochondrial ribosome, mitochondrial large ribosomal subunit, and organellar large ribosomal subunit were also significantly enriched. Previous studies indicated that the circadian oscillator regulates the transcription of ribosomal protein mRNA and ribosomal RNA 17 . The circadian clock can affect the time translation of some mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors and the clock-dependent rhythm activation of signal pathways related to its regulation 18 . Therefore, we speculate that the ribosome-related pathways or terms enriched by genes with high expression levels in the sleep state are likely to be intuitively reflected by the regulation of the circadian clock in the brain, and the ribosome synthesis-related genes present a state of high expression level in the sleep state. The results of GO and KEGG analyses together indicated that the physiological processes related to drug metabolism are active after feeding for V. sinensis , and they were significantly enriched in 63 highly expressed genes. This pattern of changes in the expression of genes in the brain of V. sinensis is likely to reflect the adaptive evolution of bats over a long period of time due to their special diets. The food composition of bats is complex, and insects are the food component for most bat species 19 . According to the analyses of feces, the food composition of V. sinensis is mainly Coleoptera and Lepidoptera insects, and these insects are often concentrated in large numbers in areas of cultivated land 20 . Insects may have eaten plants containing pesticides, and owing to bioaccumulation, the pesticides can be accumulated in the insects and then in the predatory V. sinensis . Therefore, the physiological activity of drug metabolism in bats after feeding being higher than before eating is likely to be a self-protection mechanism for their bodies. In addition, our research also found that after bats feed, the pathways related to immune regulation in the brain are also enriched by genes with high expression levels and present an active state. We speculate that after bats feed, they also need to mobilize the immune regulation processes in the body to combat body damage caused by food ingredients and food contamination. In comparison of bats before and after feeding, various endocrine activity-related pathways were significantly enriched by high expression levels of genes and showed a high level of endocrine activity, for example, the renin-angiotensin system, renin secretion, and cortisol synthesis and secretion. Renin is a component of the renin-angiotensin system and can produce angiotensin II (AngII) 21 . In the central nervous system, AngII can regulate insulin-stimulating and sodium appetite responses 22 . The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure, and it has a significant circadian rhythm 16 . Before feeding, V. sinensis may actively adjust the blood pressure and blood sugar through various endocrine-related physiological processes to prepare for predation activities. This may also be the result of adaptive evolution in bats. We also found important genes that may regulate the circadian clock in the brain of V. sinensis , and these genes had obvious characteristics of daily rhythmic expression. In the trend analysis module 6, 27 genes could be significantly clustered and showed similar expression patterns. The expression levels of these genes began to decrease in the after feeding phase (4:00), were the lowest in the sleep phase (10:00), and gradually increased in the awakening phase (16:00) to the activity phase (22:00), where the expression levels were highest. Combined with previous studies, it is not difficult to imagine that the changes in daily expression levels of these genes in the brain are similar to the circadian rhythms of other nocturnal rodents, but opposite to the circadian rhythms of diurnal mammals. Therefore, our hypothesis is that the trend changes of the genes in module 6 at the four time points are consistent with the biological habits of V. sinensis that hide by day and come out at night. In addition, these genes were significantly enriched in cortisol synthesis and secretion pathways, aldosterone synthesis and secretion, and circadian rhythm pathways. Therefore, we can infer from these pathways that the physiological processes related to endocrine and circadian clock regulation in the brain of V. sinensis have clear daily periodic changes that regulate the overall behavioral rhythms of bats. In addition, we found that Per2 may be an important circadian clock regulatory gene in the brain of V. sinensis . The Per2 gene was differentially expressed in the sleep vs activity comparison, and the trend analysis results showed that the gene also has significant daily rhythmic expression characteristics. More importantly, the results of GO suggested that Per2 was significantly enriched with various terms related to circadian rhythms, including regulation of circadian rhythm, circadian regulation of gene expression, and entrainment of circadian clock by photoperiod, and the circadian rhythm and circadian rhythm-fly pathways were significantly enriched by Per2 in the KEGG results. Previous research demonstrated that Per is an important core circadian clock gene, and it was the earliest circadian clock gene to be cloned in Drosophila 23 . The mutation of Per can cause changes in the rhythm of Drosophila , so the gene was named "Period" 23 ; the gene is involved in the regulation of the periodic expression of related genes in the brain. The stable expression of the Per protein is also crucial for maintaining the length of the circadian rhythm. This study is the first to discover an important role of Per2 in regulating the biological rhythms and physiological processes in the brains of bats, revealing that bats may have the same molecular basis of circadian clock regulation as other nocturnal animals. As is well known, the molecular mechanism of the circadian clock is a complex network 1 , 19 ; in addition to Per , there are also core circadian clock genes such as Clock, Cry, Bmal1 , and Reverb that participate in circadian clock regulation of mammals 2 , 3 , 17 , 19 . Although these core circadian clock genes were not detected in this study, this does not mean that these genes are not involved in the regulation of bat circadian rhythms. This needs to be further examined through detailed experiments and analysis. Conclusion This study is the first to explore the daily rhythms of gene expression in the bat brain. Various physiological processes in the bat brain area showed different degrees of circadian variation, including various endocrine hormones, signal pathways, and physiological processes related to the circadian clock, and transport activities may be related to predation activities. This study found that Per2 may be an important gene involved in the regulation of the circadian clock in the brain of V. sinensis . In the future, a more detailed adaptive evolutionary analysis of this gene may be considered to clarify the position and role of this gene in the special circadian rhythms of bats. Although only four time points in the bat circadian rhythm were selected for analysis in this study, the results reflected the overall genetic basis of the circadian rhythms in the brain of V. sinensis . In future research, we may consider adding more time points for sampling within a day and a multi-day setting to systematically reveal the molecular basis of bat circadian rhythms. Declarations Data Availability All data generated or analysed during this study are included in this paper (and its Supplementary Information files). Acknowledgments We thank the faculty and students of the Key Laboratory of Jilin Province for the conservation and utilization of animal resources and valuable assistance in the field. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. This work was supported by the National Natural Science Foundation of China (Grant Nos. 32001089 and 32071492). Author contributions G.Z. and H.W. conceived and designed the study, analyzed the data with help from all authors, and wrote the article. Y.C. analyzed the data and provided great help to the sample collection. H.W. provides supervision during the entire project. J.L., L.F., H.W. and L.J. performed the sampling and assisted with interpretation of data. J.F. assisted with study design and provided laboratory space, reagents. All authors have read and approved the final article. Competing Interests The authors declare no competing interests. References Christie, A. E. et al. Circadian signaling in Homarus americanus: Region-specific de novo assembled transcriptomes show that both the brain and eyestalk ganglia possess the molecular components of a putative clock system. Mar. Genom , 40 , 25–44 (2018). Pilorz, V., Astiz, M., Heinen, K. O., Rawashdeh, O. & Oster, H. The Concept of Coupling in the Mammalian Circadian Clock Network. Journal of molecular biology , 432 , 3618–3638 (2020). von Schantz, M., Lucas, R. J. & Foster, R. G. Circadian oscillation of photopigment transcript levels in the mouse retina. Brain research. Molecular brain research , 72 , 108–114 (1999). Mure, L. S. et al. Diurnal transcriptome atlas of a primate across major neural and peripheral tissues., 359 , 1232–1232 (2018). Zhang, S. et al. Signalling entrains the peripheral circadian clock. Cellular Signalling 69 , (2020). Li, J. 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L., Jian, K., Shi, L. & Ko, G. Y. P. Phosphatidylinositol 3 kinase-Akt signaling serves as a circadian output in the retina. Journal of Neurochemistry , 108 , 1607–1620 (2009). Born, J. & Wagner, U. Memory consolidation during sleep: role of cortisol feedback. Annals of the New York Academy of Sciences , 1032 , 198–201 (2004). Lightman, S. L., Birnie, M. T. & Conway-Campbell, B. L. Dynamics of ACTH and Cortisol Secretion and Implications for Disease. Endocr. Rev , 41 , 470–490 (2020). Thosar, S. S. et al. Separate and interacting effects of the endogenous circadian system and behaviors on plasma aldosterone in humans. American Journal of Physiology-Regulatory Integrative and Comparative Physiology , 316 , R157–R164 (2019). Hilfenhaus, M. Circadian rhythm of the renin-angiotensin-aldosterone system in the rat. Archives of toxicology , 36 , 305–316 (1976). Jouffe, C. et al. The Circadian Clock Coordinates Ribosome Biogenesis. Plos Biology 11 , (2013). Missra, A. et al. The Circadian Clock Modulates Global Daily Cycles of mRNA Ribosome Loading. Plant Cell , 27 , 2582–2599 (2015). Cohen, Y., Bar-David, S., Nielsen, M., Bohmann, K. & Korine, C. An appetite for pests: Synanthropic insectivorous bats exploit cotton pest irruptions and consume various deleterious arthropods. Mol. Ecol , 29 , 1185–1198 (2020). Yin, Z. et al. Changes in the gut microbiota during Asian particolored bat (Vespertilio sinensis) development. Peerj 8 , (2020). Share, L. Interrelations between vasopressin and the renin-angiotensin system. Federation proceedings 38, 2267-2271(1979). Fitzsimons, J. T. The renin-angiotensin system and sodium appetite. Journal de physiologie , 79 , 461–465 (1984). Price, J. L. in Circadian Rhythms Vol. 393 Methods in Enzymology (ed M. W. Young) 35-60(2005). Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data., 30 , 2114–2120 (2014). Haas, B. J. et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nature Protocols , 8 , 1494–1512 (2013). Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research , 25 , 3389–3402 (1997). Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nature Methods , 9 , 357–354 (2012). Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nature Methods , 14 , 417–417 (2017). Alvarez, R. V., Pongor, L. S., Marino-Ramirez, L. & Landsman, D. TPMCalculator: one-step software to quantify mRNA abundance of genomic features., 35 , 1960–1962 (2019). Wang, L., Feng, Z., Wang, X., Wang, X. & Zhang, X. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data., 26 , 136–138 (2010). Korthauer, K. et al. A practical guide to methods controlling false discoveries in computational biology. Genome Biology 20 , (2019). Harris, M. A. et al. The Gene Ontology (GO) database and informatics resource. Nucleic acids research , 32 , D258–261 (2004). Ogata, H. et al. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic acids research , 27 , 29–34 (1999). Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx TableS5.xlsx SupportingInformation.pdf Cite Share Download PDF Status: Published Journal Publication published 09 Mar, 2022 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 01 Nov, 2021 Reviews received at journal 27 Oct, 2021 Reviewers agreed at journal 21 Oct, 2021 Reviewers invited by journal 21 Oct, 2021 Editor assigned by journal 21 Oct, 2021 Editor invited by journal 06 Oct, 2021 Submission checks completed at journal 06 Oct, 2021 First submitted to journal 30 Sep, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-951243","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":55656403,"identity":"491404a0-783b-463d-9879-55ad85f20092","order_by":0,"name":"Guoting Zhang","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Guoting","middleName":"","lastName":"Zhang","suffix":""},{"id":55656404,"identity":"a0dac4b6-60e9-4b67-8a60-cf640cbbf49d","order_by":1,"name":"Yujia Chu","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yujia","middleName":"","lastName":"Chu","suffix":""},{"id":55656405,"identity":"bac35120-955a-4253-bbb3-9bd6555770e8","order_by":2,"name":"Tinglei Jiang","email":"","orcid":"","institution":"Northeast Normal University","correspondingAuthor":false,"prefix":"","firstName":"Tinglei","middleName":"","lastName":"Jiang","suffix":""},{"id":55656406,"identity":"63753913-c866-46e2-9a05-42a008d74961","order_by":3,"name":"Jingjing Li","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Li","suffix":""},{"id":55656407,"identity":"c0b59ee7-c5b7-4133-a3a4-39eb5fd4d403","order_by":4,"name":"Lei Feng","email":"","orcid":"","institution":"Northeast Normal University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Feng","suffix":""},{"id":55656408,"identity":"e891d0f4-cdc1-45dd-af58-cc66f9e6d195","order_by":5,"name":"Hui Wu","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wu","suffix":""},{"id":55656409,"identity":"ef79c12b-467f-4e05-8b08-f1d67dfee684","order_by":6,"name":"Hui Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3QsQrCMBSF4Uih0xXXFvUdAoE4GPRZRIhLUNw6FhRHXRV8iI6OgYAuFdeCDoLQxQ6C4KopODildRPMvwXOB5cgZLP9aA4KGNTej0pYjsS84YdfkcpUMSzLErzbq+s45ECOk+0FEGtG0knPRhIPeXu1YUBP2wEBxEkk3RY2ESoFJdWYA00ErQNSvUiC6xnJIdNkpoAsRw9NniVIIsglJ9gTriaymHSTjDqgD/MSTv017pOVcqmR+AtB7hCwbm3ZT70s6DTnu0lqJLrPM/Kvcgr2+eRWvLHZbLa/7gUkD0aZPwdVLQAAAABJRU5ErkJggg==","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wang","suffix":""},{"id":55656410,"identity":"4d436df4-2001-424b-93cc-4abae9494a8f","order_by":7,"name":"Jiang Feng","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiang","middleName":"","lastName":"Feng","suffix":""}],"badges":[],"createdAt":"2021-09-30 08:44:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-951243/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-951243/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-022-07787-z","type":"published","date":"2022-03-09T11:58:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":14328571,"identity":"37a27f94-0b9f-46da-a027-29475fde390b","added_by":"auto","created_at":"2021-10-07 21:57:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":368684,"visible":true,"origin":"","legend":"Heatmaps based on differentially expressed genes from six pairwise comparisons of four time points. Different colors indicate relative expression levels. ","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/f339ced68e8478fb5b74d59b.jpg"},{"id":14328577,"identity":"d8c90afe-294d-45a2-9907-04e948dafe66","added_by":"auto","created_at":"2021-10-07 21:57:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":881610,"visible":true,"origin":"","legend":"The top 10 GO categories with the most significant p values (cell component, molecular function, and biological process) were significantly enriched for upregulated and downregulated genes in rest vs awake and sleep vs activity comparisons. The black and white bars represent the terms significantly enriched for upregulated and downregulated genes, respectively.","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/fdb2efe0cbf654086d25946c.jpg"},{"id":14328655,"identity":"d5989ed5-ff73-497c-8d34-8f0fdf7648ad","added_by":"auto","created_at":"2021-10-07 22:00:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":611265,"visible":true,"origin":"","legend":"KEGG pathways significantly enriched for DEGs from rest vs wake and sleep vs activity comparisons. The black and white bubbles represent pathways that are significantly enriched for upregulated and downregulated genes, respectively.","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/14394d7b311ffdb36c2f4290.jpg"},{"id":14328654,"identity":"7636e992-5548-48c7-93ba-d7f8a72e34b4","added_by":"auto","created_at":"2021-10-07 22:00:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":633518,"visible":true,"origin":"","legend":"The trend of module 6 and the associated enrichment results. (a) The trend of genes in module 6 at four time points. (b)Top 10 GO categories with the most significant p values that were significantly enriched for genes from module 6. (c) KEGG pathways significantly enriched for genes from module 6.","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/ac50a1d6362cf8254d66614a.jpg"},{"id":19023016,"identity":"59d50cae-d93b-4e70-bbc6-0e5fba29d54d","added_by":"auto","created_at":"2022-03-09 11:58:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":756010,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/7841907f-6966-4f7a-a02c-a239cb04ec9e.pdf"},{"id":14328568,"identity":"80b1e5c8-b644-4811-b5f5-1a91fd6f5af4","added_by":"auto","created_at":"2021-10-07 21:57:37","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":53071,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/745f54b7e6c6e51628348ed3.xlsx"},{"id":14328653,"identity":"9ff3b145-9368-4772-8bf1-107df659cd67","added_by":"auto","created_at":"2021-10-07 22:00:37","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":47702,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/3af1835e867c4acd893efcef.xlsx"},{"id":14328570,"identity":"b5cb3d1b-60be-4880-87c0-3fbfd70604a3","added_by":"auto","created_at":"2021-10-07 21:57:37","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":139745,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/792be20cd7e25512acc7b66a.xlsx"},{"id":14328656,"identity":"d25a39d1-bdb5-4006-a5e6-24005e5c44f9","added_by":"auto","created_at":"2021-10-07 22:00:38","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":24991,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/2e9527a43f2c03247af7931b.xlsx"},{"id":14328573,"identity":"e27a8f56-23a2-4798-8f64-a6a4412ea25c","added_by":"auto","created_at":"2021-10-07 21:57:37","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":38856,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/5e51a4654fc5ed689009f126.xlsx"},{"id":14328575,"identity":"c5d4ef5e-adcd-4fd0-bfe2-bfbadd2f898e","added_by":"auto","created_at":"2021-10-07 21:57:38","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":657193,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-951243/v1/4d5c6217acd2ed0f3580806b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eComparative Analysis of The Daily Brain Transcriptomes of Asian Particolored Bat\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEssentially all organisms have evolved daily rhythms in physiology and behavior to adapt to a dynamic environment; these rhythms are driven by biological clocks \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The circadian pacemaker is one of the most famous clock systems that are controlled by an endogenous oscillator with a period of ~24 h, the circadian clock \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. At the molecular level, the circadian clock not only regulates the expression of core clock genes such as \u003cem\u003eClock, Bmal1\u003c/em\u003e, and \u003cem\u003ePer\u003c/em\u003e but also drives the rhythmic expression of thousands of transcripts in specific tissues \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDaily cycles of gene expression patterns are responsible for the coordinated recurring changes in physiological processes and functions in multiple tissues \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Previous studies have demonstrated that the circadian clock is closely related to rhythmically expressed genes in the brain \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In mammals, the master circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. For the brain, gene expression rhythms and circadian clock genes have been reported in several mammals \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e such as the mouse, a nocturnal model organism. However, laboratory mice (\u003cem\u003eMus musculus\u003c/em\u003e) are usually used for these and related studies, while little is known about the molecular underpinnings of circadian signaling in other wild nocturnal animals.\u003c/p\u003e \u003cp\u003eBats belong to the order Chiroptera, the second largest group of mammals after Rodentia \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. As totally wild nocturnal mammals, bats are more enigmatic and are known for their abilities to fly and echolocate in the dark \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Despite numerous studies concerning bats\u0026rsquo; daily behavior patterns and physiological processes, little is known about the molecular circadian clock in this order. In this study, a transcriptomics approach was used to gain insights into the molecular circadian clock responsible for the regulation of daily physiological and behavioral outputs in the bat brain. We aimed to 1) identify genes expressed rhythmically over the 24-h light: dark cycle in the bat brain; 2) detect and verify the core circadian clock genes expressed in the bat brain; and 3) determine the rhythmic changes of associated important physiological processes. Therefore, this study provides evidence for the recurring variation in gene expression affecting the coordination of physiology and behavior in the brain of bat over a 24-hour period.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e According to the regulations of Wildlife Conservation of the People\u0026rsquo;s Republic of China (Chairman Decree (2016) No. 47), permits are required only for species included in the list of state-protected and region-protected wildlife species. \u003cem\u003eVespertilio sinensis\u003c/em\u003e used in this study is widespread in China and is not an endangered or region-protected animal species, so no specific permission was required. All the studies have been approved by Laboratory Animal Welfare and Ethics Committee of Jilin Agricultural University. All efforts were made to minimize suffering of animals. We confirm that complied with the ARRIVE guidelines and all experiments were performed in accordance with relevant named guidelines and regulations.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eSample Collection\u003c/h2\u003e\n\u003cp\u003eAll animals used in this study were caught during July 2020 in Heilongjiang Province, China. Through a week of observation and recording of the daily activities of \u003cem\u003eVespertilio sinensis\u003c/em\u003e, four sampling time points were set, corresponding to the four typical behaviors of bats in a day. These time points were 4 a.m., 10 a.m., 4 p.m., and 10 p.m., corresponding to the rest state, sleep state, awake state, and active state of the bat, respectively. Three biological repeats were included for each time point, and 12 individuals were collected in total. To avoid any influence of sex-related differences, only females were selected for inclusion in the study. Animals were sacrificed by decapitation at the four time points of day during the course of the study to avoid unnecessary suffering. The brain tissues from each individual were collected and immediately flash-frozen in liquid nitrogen followed by placement in a \u0026minus;80\u0026deg;C freezer until processing for total RNA isolation.\u003c/p\u003e\n\u003ch2\u003eRna Extraction\u003c/h2\u003e\n\u003cp\u003eTotal RNA was isolated using the Total RNA Extractor reagent in accordance with the manufacturer\u0026rsquo;s protocol. The quantity and quality of total RNA were measured using a Qubit 2.0 Fluorometer and gel electrophoresis. RNA samples of the same volume and concentration were used during the step of converting mRNA into cDNA. Three paired-end cDNA libraries of each time point were generated using the mRNA-Seq assay. In total, 12 cDNA libraries were prepared at an equimolar ratio for transcriptome sequencing on the Illumina HiSeq 4000 platform. All raw reads were deposited in the NCBI Short Read Archive (SRA) Database under SRA accession PRJNA756631.\u003c/p\u003e\n\u003ch2\u003eTranscriptome Assembly And Functional Annotation\u003c/h2\u003e\n\u003cp\u003eThe raw reads were filtered by Trimmomatic software \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e using four criteria: removing reads with adaptors; removing reads with unknown \u0026ldquo;N\u0026rdquo; bases; removing low-quality reads containing more than 50% low-quality bases (Q-value \u0026le; 20); and removing reads whose length is less than 35 nt. To construct a general reference transcriptome for subsequent analysis, the cDNA library sequences of 12 liver tissues sampled at the same time point and the brain tissue sequences were put together for splicing and assembly. The specific liver data analysis is published elsewhere. All high-quality raw reads from 12 individual cDNA libraries were used for de novo assembly by Trinity software \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e with the default parameters. After the transcripts were reduced for sequence redundancy, the longest transcript in each cluster was taken as a unigene for further analysis. The assembly result was evaluated by parameters such as the longest value of the sequence, the shortest value of the sequence, the N50 value, and the N90 value.\u003c/p\u003e \u003cp\u003eUnigene annotations included protein function annotations, KOG function annotations, and Gene Ontology (GO) and pathway annotations. In detail, we used Blast+ \u003csup\u003e26\u003c/sup\u003e on the NCBI nonredundant protein (Nr) database, the Swiss-Prot protein database, the KOG database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The GO annotation of unigenes was based on the protein annotation results of Swissprot and TrEMBL and the annotation information of Uniprot.\u003c/p\u003e\n\u003ch2\u003eDifferential Expression Analysis\u003c/h2\u003e\n\u003cp\u003eConsidering the differences in library size, we first performed inter-sample normalization and used Bowtie2 software \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e to map clean reads to the assembled reference sequence. Then, we used Salmon software \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e to locate the genomic region or gene exon regions and estimated the gene expression levels by counting the reads. For the sequencing depth and gene length as well as the impact of the sample on the read count, the TPM value (Transcripts Per Million) was applied to determine the level of expression of each gene \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe correlation coefficient between each pair of replicates for the four time points was calculated using the R package (version 2.16.2) to evaluate the reliability of the experimental results as well as the operational stability. The differentially expressed genes (DEGs) at the four time points were evaluated by DESeq software \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. We separately counted the expression levels at four time points in one day, and then compared the results in pairs to obtain a total of six pairwise tests. In the six comparisons, we focused on the comparison of rest vs wakefulness and sleep vs activity. To create a list of high-confidence DEGs for further analyses, the following stringent criteria were used: fold change \u0026gt;2 and adjusted \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 \u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe visualization of the DEGs in the brain tissue collected at four time points was presented by creating heatmaps with the GPLOTS R package.\u003c/p\u003e\n\u003ch2\u003eGo Category And Kegg Pathway Enrichment Analyses\u003c/h2\u003e\n\u003cp\u003eDownstream functional classification was achieved through the integrated localization of GO \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and KEGG pathway databases \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. We conducted the GO and KEGG enrichment analysis on the DEGs in OmicShare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.omicshare.com/\u003c/span\u003e\u003c/span\u003e). All \u003cem\u003ep\u003c/em\u003e values were computed using the hypergeometric test, and multiple test correction was performed using the Benjamini\u0026ndash;Hochberg method based on an FDR (false discovery rate) cut-off of 0.05.\u003c/p\u003e\n\u003ch2\u003eDegs Trend Analysis\u003c/h2\u003e\n\u003cp\u003eFirst, we first screened the genes that were co-expressed at the four time points. Then, we conducted trend analysis on these genes in OmicShare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.omicsshare.com/\u003c/span\u003e\u003c/span\u003e). To determine the rhythmic changes of the important physiological and biochemical processes involved in the genes at four time points, we performed GO and KEGG enrichment analyses on the genes in the significant trend module.\u003c/p\u003e\n\u003ch2\u003eIdentification Of Key Circadian-related Terms, Pathways, And Genes\u003c/h2\u003e\n\u003cp\u003eWe focused on the circadian-related terms and pathways among those that were significantly enriched by genes from the trend analysis and six comparative comparisons. Considering the important functions in the regulation of circadian rhythm, the genes enriched in circadian-related terms and pathways were assumed to be the most important circadian rhythm genes in bat brains. To determine the circadian-related genes among the enriched genes, we searched for detailed information in published research articles based on their basic annotation information to identify their potential functions in the bat brain.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eSequence analysis, assembly, and functional annotation\u003c/h2\u003e\n \u003cp\u003eThe transcriptome sequencing results of brain tissue are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. After filtering the raw reads, 143,936,974, 142,787,930, 139,867,950 and 138,606,658 clean reads were obtained for the four brain states, respectively, and the corresponding base numbers were 22,640,739,600 bp, 22,425,822,300 bp, 22051944000 bp and 21,898,530,000bp (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The proportions of clean/raw reads of the four states were between 94.49% and 95.51%, suggesting the high quality of the RNA-Seq data available for further analyses.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1.\u0026nbsp;\u003c/strong\u003eSequencing and assembly statistics of brain samples for \u003cem\u003eV. sinensis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u003cimg 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x0aOHkldXtWL55pyQagHgVrvswQkcD4CdS0mBtSyxJPkxpXzsVeSBG5F4O/ufCsNHFcCEjgbgbppjzbyftBm4NpnVH+tC0K+Jcyhf+0SsFZ3NpgKkoAEXgnUGGFc0Skk8BwEvCA8h52d5ZMQGG3k9YDPwbqnrXra128G079/4095Dvm0z2VjJD9/CkC7mqr+1C39CVEuCEv1VabPEpDAaQTquswFYbSu6yhb9bQ1rlRiPkvgvgj8uzvfl25qIwEJHEhgtJEjgoN0NmM2bhIbfS4MW/U50Ofb/ToOciPn1AtC5G9NO/qQmyQggcsSqOs9FwRG3IobW/VZx1n3dRzjymVtqnQJbBHwgrBFyHoJPAiBejjPZaAeoOtmTX2tY4qH1NOWTT0bO/3rN4YZf1TWx+GdlF8F0rfmHBxqioxeXtv4LAEJnE7AuHI6QyVI4BEJeEF4RKupswR2SCDfJtaLQX3OlHPpyMUi5eYSkIAEOgHjSifiuwTmCHhBmONkKwlI4AoE8stAvRjkOX8alQ0/71dQyyEkIIEHJmBceWDjqfrNCHhBuBl6B5aABCoB/lyp/9kT9fyJQ34tyJ8h+adFlZzPEpDAEgHjyhIZyyWwTsALwjofayUggSsR6P9AMb8c5NJAff03D1dSy2EkIIEHJmBceWDjqfpNCXhBuCl+B5eABCQgAQlIQAISkMB9EfCCcF/2UBsJSEACEpCABCQgAQnclIAXhJvid3AJSEACEpCABCQgAQncFwEvCPdlD7WRgAQkIAEJSEACEpDATQl4QbgpfgeXgAQkIAEJSEACEpDAfRHwgnBf9lAbCUhAAhKQgAQkIAEJ3JSAF4Sb4ndwCUhAAhKQgAQkIAEJ3BcBLwj3ZQ+1kYAEJCABCUhAAhKQwE0JeEG4KX4Hl4AEJCABCUhAAhKQwH0RWLwg5P/F1PzNiwxkoA/oA/qAPqAP6AP6gD7wiD5wzNVj8YJwjDD7SEACEpCABCQgAQlIQAKPTcALwmPbT+0lIAEJSEACEpCABCRwVgJeEM6KU2ESkIAEJCABCUhAAhJ4bAJeEB7bfmovAQlIQAISkIAEJCCBsxLwgnBWnAqTgAQkIAEJSEACEpDAYxM4ywXh27dv//yXfngnffr06bHpTGr/48eP1/k/y3wnsdhMAhKQgAQkIAEJSOABCZx8Qfjy5cvr4ZhDchIHZf4zUM9yYM58mbNJAhKQgAQkIAEJSEACj0zg5BMth+J37979hwEXh2e5IPgLwn/Mb4EEJCABCUhAAhKQwIMSOMsFgUsCF4KenuWC0OftuwQkIAEJSEACEpCABB6VwMkXhA8fPvzz7w/W/swm37TThs+vX7/+wy115Fw66FN/oaj1/FuH/IlTyrtA+qauXmKq3jzXPxOq7SKv1iMvutfxo2cto239NxqM1VPVMbqSe8HqpHyXgAQkIAEJSEACErg0gZMvCChYD7X1uSqfQ3IOvTmgpw0H7vTlub7n4E3besmo/xg6fSOPvJZl/PShvh7MKa8H+1wAaBddaVPHz1ipr3rWCwXPtV/VITqmrL9nDHMJSEACEpCABCQgAQlcg8BZLggoWg/EOeTWA3MO4zkI5zBOTsohOxeIWlbljA7adexXYS8vfw77yCXlwlFlRae0QbfongtCHS+y0ybv0b3K7jplfPqGQR2PcUiRFZ0yhrkEJCABCUhAAhKQgASuQeBsF4Qom4N/DtEcjOvhOOXJcyHIey4MyMthuR6864E9B+1+GK99I7fm0XXtgpADe51P+vV8pGfXqTLIHL0gdJK+S0ACEpCABCQgAQncmsDJF4Qc8OtE6mGYg3Z9z6G+tuc5B/gcnikbHbwPvSCsfRN/6AWBeYzSSM+ZCwKyokO4hEPeR+NZJgEJSEACEpCABCQggUsROPmCwIF2dHDOoTmK5+A7ulDQJgflWh8Z1CWNLhv9ME7bUVlkJM+YuUSMvtGvZUuH9pGeffyqd70EoUv6h1F+vYie5hKQgAQkIAEJSEACErgWgbNcEDjY1sNzvuWvB+F6YM4BmJxyEm1zQM7k8750QYj8tCNPig6UZQzqqqyZC0L6ZIzIR2bmkQN+lV3nS5+lCwLcMo/INpeABCQgAQlIQAISkMCtCPw9UR+pQb59rwdiDtM5PFexvU36ps2oHln14E3bepnIBSAH+CqzXhJSn187cqhPeX+nvKZcJtI+F6KuC3JGZemXPHzy3vM6j6qHzxKQgAQkIAEJSEACErgkgX9PwZcc6QjZObT3C8IRou6yS/1VoV8QePeXhbs0m0pJQAISkIAEJCCBXRPwgnBj845+5chlgV9UTBKQgAQkIAEJSEACErgmAS8I16TdxuLPlLgM5M+eajW/muTPkGq5zxKQgAQkIAEJSEACErgkgbu9IORb9OR7/TOj/BlV5pl8dGm4pCMoWwISkIAEJCABCUhAAhC42wuC5pGABCQgAQlIQAISkIAErk/AC8L1mTuiBCQgAQlIQAISkIAE7paAF4S7NY2KSUACEpCABCQgAQlI4PoEvCBcn7kjSkACEpCABCQgAQlI4G4JeEG4W9OomAQkIAEJSEACEpCABK5PwAvC9Zk7ogQkIAEJSEACEpCABO6WgBeEuzWNiklAAhKQgAQkIAEJSOD6BLwgXJ+5I0pAAhKQgAQkIAEJSOBuCXhBuFvTqJgEJCABCUhAAhKQgASuT8ALwvWZO6IEJCABCUhAAhKQgATuloAXhLs1jYpJQAISkIAEJCABCUjg+gS8IFyfuSNKQAISkIAEJCABCUjgbgl4Qbhb06iYBCQgAQlIQAISkIAErk/AC8L1mTuiBCQgAQlIQAISkIAE7paAF4S7NY2KSUACEpCABCQgAQlI4PoEHv6C8OPHj5dPnz69fPjw4fr0BiOeW5/Pnz+/IPOZ0iEMafvly5dnwuNcJSABCUhAAhKQwEUJnHRB4HD25s2bzc8lD7hv3759Hf9eLgjn0uf3798v79+/f8rD76EMuSDCCmZ7Sj9//nzhgvju3bvFSyJzDq+sxe/fv/+DATmsD+rJed9KyP348eNrH+Sjx4gv5RmX51GiPDpip67fqM+3b99e541s5r91CSTGRN+1WLDnNTXjL5U1dhmxquzhNRu/Z3wR22NP7Mq6HfkUOsZft3yrzqc+L9l5yxer3zM2PjWzXurY9/58qJ+MWGLH2Kbn+M9aYi0TD/gsrestHalHL8ZGzpovVV2qfemHPyz5IP3W4sqsL9fxH/GZecIXXjNp5C/0gzMyqr8gey3N2GumDWMcY68aC9f2oRmfnmmzxuIWdSddEIBXFyaLCeNnQ4nhtgJGnTjOdWhizNFGd6icc7VnDqfqw8b09evXc6n0cHIOZYgf8tlTgkECatZUnx9rqwbcHsQTlONLtKcN5WuJscPz169fr5sxPlkTAS96RY++4edA1tshcykRyJGDjnx4Zo5ddvqjJ3Oifu0wtyUn8h41n/GXzA17jOIm7GEZjvgN7fKe/qM8PhB/7L7ImJTFF7DbKE7iG5GRfGb86LRk5xlfRKfoRx6mW+slYz9CnjnBNnNd0nuJJZxim56vsUIe42Nj2vFMWU9rOtKX8RNDEtNGvjSSS18S/Rmnx7X0od1SXIHbjC9H1qPmzBOusfHWPJb8hX5b8WEkG/ts2WumzTH2mt2HZnx6ps1o/rcuO+mCwKSzSJkIRuhBh8BOu5mELPofmugzExwOlXtse3Q5RR84clt95nQMQ5jBbk+JtdPXVJ0fc65rsNbxTHAlgNYE2wTdWp5nAnn3v762GbOzRm7fbNlEOZjVhD5rMSGXmdoH2cjqKfPbOkBSz7iwXBu7y3+09y1/YT45mGFjuNZEGfaviTa9Xa3P85YvUl99AT2waR8Pm/ayjLGVr9l5yxfx5+5H8fuRT27pcs/1M36yxhJ/6HEHm/X1Xxlkf+fglRS+XRb1SzoyTr+EpG23X8Yhpx8+WFPGJ69pK67M+nKV+cjP2HvrbLbmL8x9Kz50PjP2mmmTsWdiT9VhtObhUPehGZ+eaVPHvafnw0/jK9ovLbaVLn+qsmltOeGfDuWBPjMbWOly0Ud0OUUf+o6c86JK35nwYxiySZzC/c4QvKqTja9vYFQSHOP7PI8SwQwZNeFbNcjVOp7Z5Ecc6VODbO/HBlA3f+op41MTctY28to2z+GQd3LmwfxnZDEf2tG+86gyH/05nEb+krlhQ/ylr7Hw6X3DuR/KIo98yxeXZONr9VCZzTSXyLUx6/h5XrPzsb64R5+Z8ZMllthoZBfs2Nd/7EIeP6plPBMPqOtpRsf0mTl7zMa16LkUV2Z9ObrtIccXWAdraclf6LMVH0ZyZ+w10+ac9opPRt/4St6TV5+eaZN+95avW/xAbdcWKQGFjQlwOBobQA0m+XaPOj44GwnjxjlTToCqKeW1rD6jF98IIAdHRYccdNCLuugVJ0//mfGRyeaDHjgDMvgkMU/qGQN5GTv1Nc8Gic41rc1hTUd0ygfnJuWdPGXIT3kdN89r498Lw8yh+0fm8Ih5AhJz6wkfi83IWUN1444v1XWGjHDCb0YJucjqqft16pFHHeugJ8ZGNwJ5/GTUrvfr76wZZNTEemJc1i9j8B5/ru0oY12SaDdqU9s/8vOavzAvbBWO3Z7xiyV/oX4pIQu2+XRfxObUVf9EFvpit6ToHznULflp+iSn75qdj/FF9EWXWR2iy73n4bxk0y2WfX5wqnbs9bznW/leh+/EJ2vdlo61LbbdGp9xZuIacmi7FFdmfbnq9+jPWd9L89jyl/TPuu7xYSR3xl4zbc5pr74Pzfj0TJvR/O+h7GoXBAwJKAIJnyy+uhnFiSoYDtYJHgRpHIy+NVFG36XEgqcNspBB/8gkT5DkQIXj0i5pa3ycD/mRwQZFn6oP9dlgWEi1LuMkjzPnPfnaHNZ0hDVzrHNCJgx6Gc5P+1FaG/9eGKI7dobhXtLWJonPMl9sydzj18wfn6QsvhkmS+Wpz9rsF63qZ2kb/RgHv+59aMeaoB4fOtY2jF3nkTngs4xZY0oOiYzNuqtM0AOd95pij8oqc4URNsoa57nGIjjCp8fXxKTEsMjrOf2XfDF69T6jcvRjX0A39MH20bn3z/usnQ/1ReZTGWW8R8/DfeQnsywrA7h2v6n1PHd/S/1S+ZqO6ZucsWm/lmbi2kxciV59rKXy3u4R37MWR7rP+stafBjJnbHXTJsluyyVj3RJWd+Hlny3ltfnyCFfKq9tbv18lQtCvrnBQWoCNp8kgLEh1NQPFSOo9KF8LdGmf3sQvajrnwTOtfHZtHo9OjCnqg+yI496nHoprTntaA7I6Tp0Rpln1YGDVdWLuazpxTij8SObuv7JeGv6nZshOsBwLyn+EJZL84Ijdmf+WWf04b33XSqP7NTnWx5k55BYD99pT3307GuMNvgIvoUf8Nk6aEZucsbEX2vKeLWM51FMQb+kvflH5pU8XLrNqYdhLe9xgjbxIexNwlaUYbfZNPLF6NVlLJWnXeJU9El5z9Fxxs6H+CLy8Oespz7mI7+He/WHzGeWZdqTw2lrXSOXT09L5Ws6VhmMO4o7tQ3PzJX1T1tsy6fHtYzZ+9a4stRmqbzLesR3bAS7UaJuZu2lL20jb21tzdhrps2SXZbKo2fPR/vQku/W8vpcZS6V1za3fh5b/EitYizympYMkeCftgAbOWEWMgubetrVNCqr9TyP2qBXl9X78b40/tJ8u+GjN/nWRrfEamkO0XdJx9QT4OoFAPa1DL3YPNfSIzBERxjuJcUf+poazY9gy/zTlo2zvqdP/HZtQ8cX8I/YPHqsBfS0qXLxq6wx+rIGOGyuyYme5Miqfpu6jJX35DWm0Kb79N78I/NOHi7xgZTzDpuaepyijjgCbzhhJ+Rhs5ENqqz+3H2RzRWZPSF/6/KBH9JuKVE3Y+dDfZE5V19eGv8Ry2GGPbqfzLKsc8bW2Ggr8UteYkFtS1n9lS91Szqmnhx/pS/5TNqKaxmzy6px5RRf7nIf5ZZ4wLkAACAASURBVB0bLa3fmbXX59njQ6/P+5a9aLfV5hz2WtqHZnx6pk3me2/5fyP2CRoSbJaCDuV9EffFOHJCgjrBhwWKkWjDpyZk97Jaz/OoDeOzOXW9at+18Zfm23VEPmPlwMWGuzRmZ1J1Gc2B+jUd0x9+2Yhpj+4ZC11mDgCj8e+NITqi015SbNQ38qX54WP18J1DXm0fm9WyrWd8estHsh7qoaqPj6+h45Ys9KEt447WCj6Mretc6VPnRv3aZ5bpFpt7ql/yFziusaDfKMWmnfOobS+rvohPMH5njl6jw2GVha9g76W0Nq865iG+CI+1MZd0eZTyJT+ZZVnniax++az1eV46qDEmdT0t6Vjb4Ts13tS6mece12biyim+PKPTPbZJ/Oi6HeMvkVHjQ8q28m6vUfve5lR7re1DMz4902Y0j3sou8oFIYuuBwECP4flpO6EMWy9odKGT004aS+r9TyP2kQvdKhBhsDE+9b4qad9TV3HehiKs/SNMv2j0+hQNJpDdNhiVNslmOcWz5gpix6jfDR+9L0XhuiITntJM5tk5ppAlndyfK+vDd6rT9b2o2d8Y+1Smz75Jifv5Nijrw/kbR0KmQs61nWAD8dPKR/Jps/a3EZ9qr6P/nyIv/Q41ecObzbxY9bTyBeRVX2BNhzat+SjZ/WDrufofWTnUdnIF9Gn64RPHXNJGul2D2WH+MmIW53D7EEv+03d+9YuoFs6Yjt8NAkf2YoraUtO/x7XkDGab48rx/pyHf+RnlmDcJlJI369H5yReUga2av3X2pzrL3QE9uTJ9V9aManZ9pE9r3lcxaf1DqH33pYTVcWIptBggN5faddAgLGwNAJHrlY5ACCY2UM2uKQyF9KaTNySPrRv35wJtLM+MismxwbC/2Rl4MKz5kDzsV7DWxV75EzUb80hxkdIx+9mG/d/NAffcIzbXu+ND7t7oVhWMBwL4l1MLIPc2S9YBcS/oQtu19RX/0TP+S9MsJ++EYtox8+gUzqM064Rk58iXGR0f0o6wPbkMj7uu/jI2vkU3DIeMhi/lVWdKrziL7JkUG/vaYlfxnNF9vw6Qn+YVt5px1jwD22nvXF2B75+BPxETvXxLjUk5BLm65D95faP88jO8/4IuPTt3/w7T2lQ/xkxDIsslbzXvORneBLOfbHvjyjyygt6UhfLgLdRrxnz0XeaHz6rsU1+sX3E7NGcWXGl0dzetQyWMIXflup+8tsfDjWXjM23bIXOhLTajyMb4/8rMakGZ+eabPF9Rb1Z7sgjCDWCWFEFi9GoC3OkAWYdhiEeoJxNgkMRnvKaJ/NKQaq41bjRiZ52iC7f8OAXjXY8Jyx6bs1fubFGMhHL2QwV5yOhHMk6KTNa8XC/zAm7Wtam8OWjpEDu77RoS86baW18e+FIYEcv9pLCvPk1b/xLWxJHfbD37DDKNVAh4zq37SnP7Lic+TIhWXWWZeLjDp+Xzdpn/XBGJGZg2Xa1PETqDPnnvc5Zl3RbjS3jJGcdplnyvaSd1bVX0ZzpL63QUb8KfGr98XWtCEnHeKL+FP8ZuSz6JN5LPlU9ZeuW95Hdt7yxRxIM37NqdtLqvPiuftAnydtltYMXIi7o7RkJ/pQx2dJ7pqOxKVen/caW/r4jEW7tbiWedCW/rSHT4+ZtNvy5ch65JwzV9gmX7JZ5km72mY2Phxjr0NsumYv7IvexCbmfOg+NOPTM23C8F7ys10Q7mVCe9ADB8VRTYcRgFndIA7r/dytOTzVoH5tGrce/9rz3cN4bHi3SvrLrcgfNu6t7XTr8Q+jZetb24vzA+cv0/8IeEG4U0/g2zU+pjkCfIuVbzTnetiqEoDf0jfGtd2lnm89/qXmtVe5bKK3vIzrL4/hWbe2063Hfwwr3Y+Wt7bXLb/0uB8r/NXEC8JfFnf1xE2an0K9JGybhW++YQUz02EEuBQQlEc/oR8m6bjWtx7/OK2fuxf+cqvLgf7yGL53azvdevzHsNL9aHlre/GFB3HNM8S/PuEF4V8ed/fG4defvJbNAhtu/S7sZUbWSEACEpCABCQggUMIeEE4hJZtJSABCUhAAhKQgAQksHMCXhB2bmCnJwEJSEACEpCABCQggUMIeEE4hJZtJSABCUhAAhKQgAQksHMCXhB2bmCnJwEJSEACEpCABCQggUMIeEE4hJZtJSABCUhAAhKQgAQksHMCXhB2bmCnJwEJSEACEpCABCQggUMIeEE4hJZtJSABCUhAAhKQgAQksHMCXhB2bmCnJwEJSEACEpCABCQggUMIeEE4hJZtJSABCUhAAhKQgAQksHMCXhB2bmCnJwEJSEACEpCABCQggUMIeEE4hJZtJSABCUhAAhKQgAQksHMCXhB2bmCnJwEJSEACEpCABCQggUMIeEE4hJZtJSABCUhAAhKQgAQksHMCXhB2bmCnJwEJSEACEpCABCQggUMIeEE4hJZtJSABCUhAAhKQgAQksHMCD39B+PHjx8unT59ePnz4sHNT3X56P3/+fGX9+/fvl69fv768e/fuBf6npI8fP74gzyQBCUhAAhKQgAQkcB8ETrogcDh88+bN5ufUQ+Qaqrdv376O7wVhjdLpdd++fXt5//79C5eEL1++vIT7qbZFLrKQu/f0+fPnP9xg+f379/9MGR5cvFhX5LA+JP369ev1Ehf7LPVlbMboicsaF+6MP9Kx94msUSxgPkmxNe1Yr6OLIX4Am4zPRfSQtDZ/ZDNuxh/53DHzP0S/Q9rO+MJMm60xY7/ebsZf6TPaB0a2rfKrnfFVfK73mRl/pk0dN88zvjjjCzNtMual8hkGx/rJzJphXsSpuv5ZwzXBCR34QuiQvbrqjcyt/QYWW/KX/L3qm+eZ+c+0uQc/yZxm/OUc9lrav2ZsOhMfMp+eJ8bHH5lvTYyf/ZG2zLWnGZv2PrzjW9m/RzGNNjOyZ9qMxj932X9PCAeMAOgKIRtFFjHgCQi0m009sMz0y4Y/09Y2hxPAWXH6upCyIcTWh0v92wP/wO5V/t/afTzBK+uAQyzrAr+t/AgutIMDnzAmn0nIRyZrssrtfZGdANnrCJhZ09iddmuy6E/7BOOex6bMLYd95OFPfSOHC+OFE+14T7+ua39fm3/mHFm0RXb0i6xj5p++58xnfGGmzZZO4YLdaprx17Tv9se311LsHD9DB9Y/70kz48+0ibyaz/gi7Wd8YaZNHfvczzMMjvWT+MbWmmFOORRl/adP5ss7Nqa+r/u06Tl6s0aJQyRk0D/vvT3xYkt+5kS7rZS2mcsoZsy0YZxb+0nmOuMvtD3WXnANr+wfiefInbHpTHzIfHpOX3Son+ovM2t/1qZ97OxX5CTiWff1GdkzbfrYl3rfXiUrI+NsGCQpCzSAKMc4tJtJMe5M29oGZ+iGqPU+n0YAtnWRIw2bwr3a+pRRWEyzfnLKOLfqm6CZ8RME6mGqt6Et7Nkkt1I2LwLgVmLMbNa1LTKwKbolYRM2/7WEjjUO0BZZdW4j/+nzwgfqIRE5jE+7qtNIl635I5c514Tedbxj519lnut5xhdm2mzps+QLXfbIX5GN3bf8o+uA7O5n+C1lSTPjz7SJvJrP+OKML8y0qeNe4nmGQW+DHjNxZWbNIAsOdR2tzRMbM/ZMwq+6reg76o9/sr7pM6rPeEv+nvqaz8x/ps09+Enm1X1haV2n/SH2Ctv0Je+/6MzYFB234kMdoz5jj+4ztb7XZX+pbWZsWtvnmbnVXyvCto45I3umTca8dP43Ip9hpNEFYVZsFnjdJGb7HuLEszJt9z8C2LQf5KhhYcH9XBeEjIMfPEta2uzq/MO5lvVnLuHYogf/3o53DmIEoJFcAnzfXCO7fgtT5XJAHNkMWWuXFXSowRSZ+Bl61ZTx12Slzdr8R7JpX337mPlXXS/9PLJZH3OmTfqs+ULa1Hzkr9gRhrCbjQU5MNWNE1sgYy2Nxu/tZ9r0PiNfnPGFmTZ9rGu8zzCY8ZOZNcN8OBjxgWP/oqDPd3avzpruPoWfIKPHHGIJ/rQ290P9fWb+M23u1U9imzVms/ZCFvanffUB5p64PGvTY+MD4zI+F0X8u/tI5lvz0dqfsWmVwfPS3Jg/n6QZ2TNtIu/S+dUuCBiLRczkY8S66ecbTer44LQkwPNcy6sD0qa2XwKGwzA249Cf9yR0w1GiG+Mxbk3oGh3JeY/hq37pU/VNGXmVUx0BHVhIBFrahBVteK+pyqB93Whph+7oFh2YWxZL5kod5cjqQbiORZvYopZng0lQRl50T7vU0bbOp7JPW3Jk9LnU+r0940cJnktzg1v8bKkN9fGl+HC1efphe8Ykj/1SR479GK8n7LKlZ+2DfPQYJdYeuvHpiXGWxl/yGWRszT8bR19H+D1jZq2fa/59Xud6n/GFmTbos+ULI527v8bOMMxnZNclWYltifHIW0t9/FHbmTbpt+aLM74w0yZjXTOfYbDlJ7NrJmso9idfi+HUj/aTzidyl9Zs3bN4ToxE9kj+of4+M/+ZNszrXv0kzNf8ZdZeyIIHaxp5rGn8oMaDQ2yKjEPjQ/a0+CL9E9sz1+ToOtqHZm0aOcmZK+P2GIZO6EGakT3TJmNeI7/aBYFFi0EAyIdngNYAQBvKamJxZfFjbOqr09GWslFQiBz6ISdjI68eOHhPwMFAOCftkzA+Rk6bOHodkz5Vd8airLZhrgRm6kgcuuiD/DgS7+iDHrSjf9UFGVUX2tInC4F+6c8Y6Er7MGNM6km0RXbm9VrY/of6yirVlDFubIosdKWM5zDinfLox/wpY849wSt69rq9vcMIu8QXlua3ZR/6dcbxq9g5snmPHWK/1EXOyNbIH5XXvvWZ8Ud2rD4xmhdlfGqCE+MjcyltzT/jdj/v5UvzXCpf0udS5SNmfayZNvTZ8oUud81f8Sn8A3/eslXkIi8xk/iwtQ7Wxq8yZ9YU7WN79B0xW7J5La/P0YF8qby2udTzDCfGHs256hQ+W2smfWjHmmfufBJnUp+cOuy9lZgHbXscyUEs8rNHxn+QPZJ/qL/PzH+mDfNkHqP4uVS+xeac9Vv+Mmuv6AQT1iCfbrtZmyKLtofEh4yPH3BGwgfQHT+Pb6RN7JZ63pNSV8uoWypPP+yLvJ5q+ZKMWl6fq6yl8trmEs//ndEJoyxNAoMBD6PXhPH4JMWoeSfH0ephchQAkD0KCpGDXoyT8cmzYKMbMvqHfjhX1wG5XY+R7r1NHL6PE93jTIyblLK8M4/Kg0CJfgmYOYD3MXgnIY9gmUWDrDpexklOv7BKWeRQV/sikzJ0IFE36s8cMucqs/OqdXt7xgb43lriUByWS+3CuNqBttms4/OMVe3Y/Yo+I1utlS/phJ/HH0dt8Dl8lk/0o110ysZCXfx5Sd7M/Jfa9PJzzX8051PLZnxhpg16zPhC13fGX7EXNq0xvcvJO7ECOyMX7rF56ns+M/5Mmy53yRdnfGGmTR/v0u8zDGb8pK+N6L1Unvrsp0v2hNko9qd/zWlH++x3xADK8LEk4gM6JY32kGP8fWmetbw+Z3zyXn6PfhJ9t/zlEHshExth+5x1YruMN2NT2h4aHyK/5tk7ug5pM1r73XZpu1Se+uxdeU9ey5dk1PL6HBnkS+W1zSWer3JBqJDqJGLAlMV58p4cZ8GYcTra1bTlxNm4aMeCAHYSunV5qSNfMgx9aj+ekV9Tb0N9Hbu25TmcapuUpS0yCHhLiTHps5QSwAmyLOR6QBv1YbyRvOhVdaV/nXPY9f7d7hm39k3ZHnN8mU16LSXQrrWhLoy7HWJnylk/sK0p9qtl+ES3FfX4wJa+kYM/zRwQo3eXy/jowZj4J581eZGzNn9YIq+3SV/qSeeYfzicM5/xhZk26DTrC1X/GX9Ne9rCei2hA7E89sAH6ENcGKWZ8WfajGRTFj+ovjjjCzNtlsa8RPkMg1k/od3MmhnNgzXb403aIXOpLm2S4yfIok9Y4zeUkbBb95m+hxzj78iemf9MG2RF91ely/8wr+pzpeoqjzP+coi94EGshnm1XT2v1PJwqTZl4rQ5JD6swUKf0Z6WPn3tz9o0/ZMnhuU9efYz3mdkz7SJ7Gvk65H8QA0Cm7wmIOFoGL6mlKeMxU27mnBijEwgAF4PALSdcWIOLgQWnJL2CTIxYNctOizNqesx0r23YVzGW0rhUfmlLH2q7imreR+z1uUZ+VyUkMUHxktpSefoVXVFRl3sYdfnzPvo0Dej+5Kej1IOk/jeks74IiyWfLL2w69HNgx71kxsFXv3nLFI+ESeM0aVk7K1nLH6pr3UHj3WNkjmz3pd88+Z+TN+NqOqC7pSnnSO+UfWufIZX5hpE31mfSHtZ/w1bclnLoijzTRl9K9pZvyZNlXm6Ln74owvzLQZjXWJshkGh/gJOs6smdFcWK9LMQ7OPcaMZIzKmCP94yPI4X3pg68f6u913Jn5z7S5Jz/J/Gb8hbaH2Gs0T8pGe33Vo9qU8sSCtKllsX2tW3vGD9f2D/oyft2HZmzax1w62OOjMEiakT3TJvIunf97Gj9xtCxg8powUDcC9RiPA2VSFnzeA73eQGnDp6YtJ0afOAlBknHpQ4pu6MF4SQQW3jOn9E9914NDUWQutWGhhAN6kKIPzwlmlV/KIhPniYyUsWhyKMvcyOtiipMiL+XkzLvzjFzyXM5qGc/Rq+qKPHQLq7CjbU31ElHL0QO995rwp9gpc4RVeFEWf4h/UDbql/7kIxsicy0wx35VDn3wrzo27dbk1P480zb+1evqO2PgK0ttqZ/1h5n541fdz7v8c8y/zvHUZxigd7VH94WZNlt6jHyBPn0symDEZykRq/t6721H4zEP/IExk2bGn2kTeUt5xq6+OOMLM22Wxjxn+QyDY/xkZs2M5kGMq/tCbYON+zqs9UvPzJHYsuZ79EX2lvyR/43GnZn/TJt78ZPMccZf0vYQe43YEw/YU0ZpyaYj+2SN0ueQhE70XUqRW9f+jE1H8vDPGvuQzdyrz87InmkzGv8SZWe9IOTmVw/0UZpNHFgJHOT1nXZxDMAmyOCgud0hFyNg9IwRAyN/KfWxkFedlr6MUz+Mk9R1x5koq4EouuPA6MQYyKgLDJ3rGHmOAzFnyjI3xk8ZMklhnL7kjBOu6Mbcaj3POB0JPdE9C4KLQy4Prw3a/4yclSbojFz0Qzc+yM04tEEn2tTxwj7j1+FoGxa1fA/P2HRkF+YcFvgOrCjrn3CJD9VAFM6wJcXfqx91hvHXXo5Px4YjOaPxIyP65z055cyHMeMrjIPv9AQL5opPR4/ahj5wrHObmT/j0i8cl/xwa/5Vl0s+h2X3A94zh5k28GTeNVZ1vUe+AN8tf4V7fA6Z9GEcWCeN/CX2wr7xB56rjjPjz7aJ76HTIb444wszbcLiEvkMgxk/GdlpZs3gi/Ql0R5/GK3b1GMLYlxPo/Fpg+74Z127vW99xx7Vj2pdnkf+PoorM/OfacO4t/aTzH3GX9KWuR1ir3BNTCD2YGvKa9qy6Ux8GNmLcZBNYmz8MLGSMuoSC5gbH+zS96EZm47GR2/8lHGQwfjd12dkz7SpPC/5fLYLAuD7pyoeYNl0AAfQmgBLPYeDGBoDIjeH4Bgmhq9jLgUGxmFhYLDIinzGRzcOyZHFc6/vevBex0NG2pDTn5wxqyz0Zi7RI4sniys6oHPk1TL0ZQFWGQnQYcl48KUfPNEB/UhZRJFNnrr0rzl6IGeUGDfjoE/mkrbpC8/oS/vKI20pY5w1XdL20XI4xYY9hwcpB7len/dwwfaUwbMmxghj8u4TtS3P8bdezjhZCyM5S+Mjh7WZzaHKRWb8Dd15HumXNoyP74wSdfg0eU0z86/rgrFGfrg1/zrmpZ5nfGGmDfplXWHLJabdF2AZv+t5/BXZtEs8R35f/7RZ8hf6Jnb0GDUz/kyb0fizvkjfGV+YaYOsS6QZBrN+smSnrTUT38FPsCc6jVJtF5+q/jgan3bxDeYxk1jXfNZSdKltluLK1vyRMdPmln6Sec74S9qGUWxFvmUv+tKPWED7UUyYtSm6LsUHxhnZK/sHY1CPXWo6ZO1v2XQ0PmPV8109d1U9tmTTdqZNlXmp5/HJ71Kj7UjuTCDay3SZay5kh8yJgMJiJWhsJRZTv8lv9Xnm+luzevbxH8332HDrBn9t/fWXaxM/brxnt9Ot53+c1W7X69a8nn38S1veC8KRhJ/pgsBtlm8DyA9JsxeEyOeGb9omAKeZS9e2pONa3Hp8/Grpm8rjZrT/XrfcSG/tL7ce/1G869acbj2+ceUwT312ez2Dv3hBOGxN/GnNgbn+3P6nYqcP/ILAz72HXBLyk+ba4YQ2x1w+dop5c1rwGv0Zz2bHMzW49fjMHR1McwTYxGDGZn6LdGt/ufX4t2B+zJi35nTr8Y0rh3nNs9vrWfzFC8Jh6+K1NX82Uz9HiHjILlwO1g77dVIcTCqjpW+8+SVm9u9Lq3yfJSABCUhAAhKQgAQuQ8ALwmW4KlUCEpCABCQgAQlIQAIPScALwkOaTaUlIAEJSEACEpCABCRwGQJeEC7DVakSkIAEJCABCUhAAhJ4SAJeEB7SbCotAQlIQAISkIAEJCCByxDwgnAZrkqVgAQkIAEJSEACEpDAQxLwgvCQZlNpCUhAAhKQgAQkIAEJXIaAF4TLcFWqBCQgAQlIQAISkIAEHpKAF4SHNJtKS0ACEpCABCQgAQlI4DIEvCBchqtSJSABCUhAAhKQgAQk8JAEvCA8pNlUWgISkIAEJCABCUhAApch4AXhMlyVKgEJSEACEpCABCQggYck4AXhIc2m0hKQgAQkIAEJSEACErgMAS8Il+GqVAlIQAISkIAEJCABCTwkAS8ID2k2lZaABCQgAQlIQAISkMBlCFz9gvDjx4+XT58+vXz48OEyM7pDqb9+/Xr5/Pnzy9u3b+9QO1WSgAQkIAEJSEACEpDAXwJnuyBwCObgzyH4zZs3L+/evXv58uXLy+/fv1/LM2Tqn+mC8PHjx1cmcDFJYJbAz58/Xy+WrCUu1lvp/fv3r2uut/v27dufdcm6Y01uJdrEb1mzXHBH/SjHr/nwvJaYQ2Rurf+Z8evY0SE58WiUlhiN2j5a2aH+Ar9uh8odltgLuVtp1ha0yx6ALb5///6PaN5jw57jx2upykbvkQ9UPXkeJeYLF8Ynn5n/SM69ls34yQwn7EFsghO2nIlRh/rXUsyo+nU/Gdk9tqjjr8U12sVPI3/kq5k/Zx/67DEx75ztluZX7bG0rigP09HaX5KdcvyNftiDs2VNvCObT6+r7Xhe8qnerr+vjY/M+Eny6g/V76ifjatdh2u/n+XECrg+aYBQHoeoE6Nt35hq/R6fs4D2ODfndBkCBMOsn63Nl6DIuurBkeD+9evXVwWRwYY2s/YYm02BxIbLO0GtJsaKXokBffy0zwZD/cyBa2b8bM7Mu37oO0qMPWI0avuIZcx71l+yoXVfwE6xKXlk1s1uxGbGFjkgRH58ph7oGL/asj6v6YBsdKUNH+SgU+0z46+0h2HWTPawKmc0/0cqi01hG1tU/Wc4EVfglLUML+Tlvcqrz4zNJzaPnWqbPFPHGKOYMeNvkVNzxkYuaSmuURffjP+hR01woyz8kNnXUm3/qM/Mj3mFw2geM/4ys/ZHsilj7WE3bI5d4jtpz/jxqbSlbJTWfGrUnrLIXBqfNsgNI/K+V6JfdEx75N17OvmCgAONgGTiBAzqa+J9j4upzrE/47CdQ2/juwQ6gfhNNqJezztrjOCDf/XASECtifq+2dV6nunTg1fWefQgSOc5/VnTPTBSR/BEv63DQ+TMjI8s5PaDG2U53EUe+Rqj2u7Rn2f8pW54NQ5jz24jyvCrEdOwmrUFfsdBoSb8ovos+vQDAP4w8qvIoT061n7MkfEie9Zf46uRTY5OlO8pLfnJLKcclioTOFV/qnU84yfYqX4Tj50ow89qih26P0YO9bNrP3Jn4kraMr/qTylPTn315fgbY+wxYVfs1NOsv8ys/S6bd7gSI1j/3d7UM373qcSsbr81nxqNPTN+dOj7ZZV3iN/Xfvfw/F+LH6hVHKcbo4rpwRWDrgWS2ncvzwnIe5mP87gOgfhN30Dr6KylBKEciGp9fWYt1o2t1uWZYDxan6Mgnz7kBMm6+VM2+81ilTMz/ujggAx0HG0khzCqujza84y/YH8OMjAZ2bnPmXi95leztsA/+kaKvdKfPWRkO/yh+1XVMT5Wy3imH4eLpTTyV/Tpc0U+5XtKM36S+XZOiTU9JsUOIxsiK4e5eh5AVmcbOfGL6JF8qRw5S2PTdyau0I61kTPK6MC/NH/k89ljIk7AZCZ1f6EPZXxqwl5Ltkw7eNJvya7xlbRPjmzqktJua7y0T741Pu3wZ8ajbV8T1M/6fca8p3zO4gsaZ+JrQXjUNYuv1rEBIIc6YNcgjXNgBOr44DDVENns6IMjUE+7KqOOxTO6s1HGSelT53GKPsjHETMfHCcBOXrUOTE3xqtzSjvz5yYQv1nyjfg8lNZ8Hn/Hz/hsJTaDuhbSfulAiW7UjTZT1hd1Wb99bUd2zQ8dP31ZQ6y1nmYZ9X6P+L7lL9gqjJbsWedNnMKvDt1YR7agDFmMn/g38pk+Pj6zljJnZNaU8lrG85K/Zj9Dz5pofwyDKuPensOGuS2lJU7hscRpTSZ7buIU9sIHu28dEzNG/tbnNRtXaIeO+RALq2/hs9TVMsaC6Zavdp0e5T1M1vRd8hf6HLP28Qs4s3dkfM5p1e+oW9qrEucY/xifmhkfH0B2fCX6dk4zft/73MP7SReEBAqMd0gCYu2DwQGYBZfbXjYPnADHoJ4Pz3xI6BAD4SgJTjEIQX+UcJ4YlfEZK4v7FH0YC8dCVm6w6IRujJdEXRwYHZlPdE8bcwmsbeT4WXwIUvgX7XvCr+LrM36Ww3xfO/St4zFO9EM+jncwjgAAIABJREFUa7r2ybisRcpZu5GdtdF15T1tqizKR+PX/uiWmJHyWUZp/+h57DGKJfDHRuQknmscHs0dnlttRv1GtqBdYjvxsdtqJIf2+MNaQg7+1+XRLzE9/cNnzV87u/hxL4/MR8zDYWlOqR9xYl1S3u0SO7Dm1hK+QX/Wc28b1ofGjCV/q3ocEleYI/NBR3RFflLY5D35UnnqHzknBsBhKWXutKFtj930O3TtRyZ2IGYhM+eo+A1jjeJTLT/Wp2bGDw/0oT3xBgaj/W3N7yPn3vJli09oGvAjA611jxOlTYxOef1ELmBrMIrh0j96UJ6UMvKlhHzGy4aZdqfqg9yqL3ITnDIGujKvjM0iWNM1/cyfi0B8feQb+Fn8Byr4cl0DnRQ+RgDjMwrgaZ+1wzpAPh/6In8U+KiPnvRJSlnek7Pp8llKh46PHHRgXj0dyqj3f7T3MB/5C4euWg4bPksJpthzzVdGfZdsQVu+fCEWxg+z0Y/kUMb4W20yHjLTlnHwsXqwyxi0D6fqr/G7yog+S+WR94h55t/nWueyxIk2+A3xgLhAgjtlozVYZfLM2NkP4c84SdEr78nXYkbsn7ZLeeyYMem3FteQQ5vMNetgScel8iV9Hqk8DNZ0hlUY1HWVPoeufcbkUxM2wO9yvhq1oX0tj05VDs9rPtVlpG8fP+XJqWcNjPY39Fjy+/S/t/ykCwJBAWN1I25NsvfhfS1QRR4OllsYfZKy8DFAUsrW5KJ3lZO+p+pD/6oLcruTMhfa4Uw4TYJPdDCXQPWb7sf4Ez5U08jvaj3PWRejg35ti2yCHDJZJ/HfNT9NmxzS8l7l8pxf93p5fT90fDb6bBqRcyyj9H/EPMy7v/AO95rqJlrL8wzP2DJlM/nIFvSjPHsFfsQhgvi35FOUjzbakQ45oOKv9MnBj3wphVXmSE7/ETvK025J3iOVZ+59rqM5pG2dP4fBHHawIW2wZ1+DXR71tCUxNn3rYTJj9X5rMWPJ37oM3g+NK/TBD6tf5JvwLh/dmc8e09JZaTTX2LD6y6FrH7lL8Ql/SRzhPJjnqgtl+XIg+tR6ntd8anb8LpP3xJ5at+X3te09Pf89ZR+pVQ4RBIzZxGKrRuUdIy4lHI1xgMwC7wYn0HQZKVsLgOhAv566rF6/pc+of9cZmehWLzxrm1nXwffnIBC/6X6Mj619evtKi35bF4TanmfWytbmz5jIzsaQQNkPgMzp0I10a3zqiQ01rfGhbo1RlfNIz0v+kli3xIR+NfF+bDwa2QLZOUhmHPaMxPWU1Rwd+qWm1q890xfZa6n7K227jpQh61B/XRv3HuqY0+waGHHqc0ibvtZrO+JCHzNl8bVjYsaSv9Wxl57puxXX6IsvZW7RuccPZOVQujTeo5YzN2w3k+IL2Qfo09fV1tqnD3YZreHKeemyhq7Z447xqdnxRzzwk6r3yF9SFr8fybmHsjmLr2jKpowx1hYZQT6LC1G0x8hJwIxBc9Egj0ycq24UCW7pH4ekPCllfRGnnnzJ6U/VB33r/Bir68x7mJDXW3HV0efnJhC/WfPjEGIN1TWQ8pqzrmgX36t1S8+sPfwza3OpXb6dS33G6jqxrrO203Yt3xqfudSAvCZrhtFa/3uvO8RfiFE9TjE/Nq2+cWGvGZ9Zs8WIPbZdOlRh05kxu03wQ2JwPaD0Nrx3f6WMeXYmvB/ir6Ox7q3sED8ZcarzgTO26j5T2/C8tCcTW3KYOzRmrPlbH7+/b8WVtEen7hPMt8Y12uBzWwwi89Fy5s/6nUkjfzl07TMOcujX977qa9i/t4mfJXYc6lOZ48z4aVtz+lXfiD59HtXva/97ep6z+IbGBE+MRKCvQZln6gCWFGMBJymGQEb9ZLFFNu2RGWfFAXhPfxZ8Usrq2KlLjg7I7il9qy48z+qT4EvOfNExY+HcJOooixPDbmmj7Pr5/jwE8Gl8b82PQ4N2NTDhdynDD/mwduo6oS9+iF/GFymjLWPSnnrea2JDrxsiYyGj64k+tEtwTL861jHjd136nGp9fQ6PWran50P8BdvyqQl7wah/ErdoyxjYtNuaOuy7ZAvGqr6AT9T3qkdiZi3L88hfqEMe+w0y6V9T/C4xfMlf8XP6p136VX+tch/1eclPMt/Mf4kT86Yu6zvtK49up7ClPDwzXo0vkbkWMzIO/Zf8rY9PH8ZZi2voxfjRhznit+Q1xXcppy1+x3h7TcyNmBAumWfsF/vDY7QPzKz9kb3oRzl2WeKMvWhDPe147j6x5VP0Y90zXk1b4+MHMEiKb1VOPCM786BtuNV2kXFP+X9Px0dqBygOuNlYAMKi6Qsr9eTVGDgYjkU5OQZN4plyZAIWI/COEzAuz/nQNu1rWWQlZ+zU180v9cfqk/7ogL6MgZ68wwd9SbzDJnqQ37uzZG7m1yEQ/0xe18tIA9rhV0n4U/yLOp5Hhzr8tK45ZNCegJbAH5nJsxHQjv74dl/raYu8rAV06O2OGT+yydGzy6z19bkzqnWP/szc6mfLX6ivbYhTtX99rhsuto7NO7M1W+CPOcAjm7Yjf0Qm49WNt47T/SV7QHx4FEcP8Vfaohs6jvy16vKIz9WumWPmMcuJftgBe3K4GqVuJ9ogv54TluLGVszIeGv+1sefiWvMBT+q8xv5E+PXMwIcltpF10fMs7aqz8AxadZfZtZ+txdjpF/GX+JMvKA/n6pf9CRf8ynmwRjYPme0mfGJX4yZvktjz/p91fcens92QbiHyaiDBCRwHAEC8VJwO07iYb1uPf5h2toaAvXScG0i+su1iR833q3tdOvxj6P2vL1ubS8O/PWC8LyW+N/MvSA8uwc4fwn8/0+eS98EXgMQ3xbfcvxrzHFPY7CJLn37f4156i/XoHz6GLe2063HP53gc0m4tb1u+aXHPVraC8I9WkWdJHAlAhzKCcr8BHqLdOvxbzHnRx8Tf7nV5UB/eQzvubWdbj3+Y1jpfrS8tb34woO4xi8Ypr8EvCD8ZeGTBCQgAQlIQAISkIAEnp6AF4SndwEBSEACEpCABCQgAQlI4C8BLwh/WfgkAQlIQAISkIAEJCCBpyfgBeHpXUAAEpCABCQgAQlIQAIS+EvAC8JfFj5JQAISkIAEJCABCUjg6Ql4QXh6FxCABCQgAQlIQAISkIAE/hLwgvCXhU8SkIAEJCABCUhAAhJ4egJeEJ7eBQQgAQlIQAISkIAEJCCBvwS8IPxl4ZMEJCABCUhAAhKQgASenoAXhKd3AQFIQAISkIAEJCABCUjgLwEvCH9Z+CQBCUhAAhKQgAQkIIGnJ+AF4eldQAASkIAEJCABCUhAAhL4S8ALwl8WPklAAhKQgAQkIAEJSODpCXhBeHoXEIAEJCABCUhAAhKQgAT+EvCC8JeFTxKQgAQkIAEJSEACEnh6Al4Qnt4FBCABCUhAAhKQgAQkIIG/BB7mgvD+/fuXz58//9X8Ck+/f/9++fTp08ubN29e3r59e9T43759e/nw4cPLly9frqDxeAi4/fr1a1xpqQQkIAEJSEACEpCABAqBky4IHHw5PPcPh+mPHz++/Pz5swx12uMtLgjM7+vXr6+Kc8hmXocctH/8+PHaBz63vCBgC5MElgiwTllf+Om7d+/++Hxvjz/3tc4lei1xQWbd0I/1tNaeOtYJOtCe8Ujol1hDvrQGmcM5Y87avJ65DjsRU7BRvjgZ2fUYf4ErPlD9DLvWxFj4FTrgDz1Rny928KX4UW9HTE9873W+n07gknGFGIDt8I2lvZXxE0vIe2zg3bhyup3PJWHWXzLe9+/fX+NE3tdy4kV8AZ9ZigmRQWzoseUZ/eWkCwIwgUYwzyIlOLNws3ksbeYxxCjHOFsGHPU7Z1nmdapM5lH5nCrv0P7Mw03wUGrP0571ySGPAErCX3kf+UwOXTm8bV08CeCRg1wCdA+6IY2fMi719KsHTsoyFs8jGR72QvLyORssvkDCf3iPferoh/pL+mYjj5/Fh1LPO2NSP/KFXDLxIZ7xq+pPyMHHRjpnDPPTCFwyrqAZvhU/ydmjasz4xATszjN+0v2AsvgAzyNfMq5Uqpd7PsRf0AK7Yk9iwFZirdOWPYZE/KBf3nv/nNm6Pzyjv2zT7fQG78Dui5SFNSofdP9PEQsfI90yMZ8Z59vSMc7W+Wz1O1c9dmDxmSQwIsBGm8Ne6vHVvpniQ6zLQ1IuHekTuXlPnsvB0hqpcYQ2fV2yxnowj2zz8xLIN3FVamJcjdnH+Asykd/9sY5Vn/GDkd0pS/lINw4X+HK/NFTZPp9G4JJxJZrFtqO40WNP2tZDoXElJG+fz/pLNOVily8JUraUs9a7P9QYUfsRE5BLn8SQ1D+jv1zsgpCNfLR4A3yU4ygYom42o3aXLov+p46TwHQoh1PHTf98Q5J3cwlUAlwEum+yibIG+eYliXVJW/zp2LWJDC6sNeWwtuana4E5/T3sVaqXe8ZOfeNkNHyj2vZYf2Fj5kP/rS828IuRLnXzT/ytPkt9fb8creeVfI24Etv2+DWizgGRg19NxpVK47bPs/6CluxLxIeZM1r2sr7e8ytC3zeIYfhKjSEh84z+crELAgYEaL+5YZDUUc9mEONhHMrqB+NQT5++GbCBsGGlPc/1G4IYtuc4B+Nm/B5gIq/m0bHLynxwcNqjQ3U6+lHex+C96lAPY8yBuWZ8nrNZIjv60wdmjM2nykBP5NCW1Pshkz6Riz6ZA3n6UR49yEmjMsqjM/WmxyCATevBLlpTHjviO/GN+ALrcTbhY7Qf9cHPMlbWA35U1zHvrCsSz3ySeF5am2ljfj4C8O4HLaRXuxzrL4mV8THyvn/UmVBffSF1+C2+hB4847s8k3iPX6e9+fkJYJtLx5X4y5o94wP4SXwgs6XMuBIat81n/AUNsSHxJ3al31qKj/SzUcrJk3he8gfaPKO/rNMNuY0cI2WRsrFzEKBsdCCgLMEbI/PMJwk59K2Gy+Gkbgb0rQdZDiE4DmU59EZmzZFPu7TJAaUHs+hR+46ekcWc0Ie5o3ucjPZxxPChjLGy8dEvvKITPCIjMsMSOeFBG/ogAzaVY8aJzNqP8ZGLHHJ0QW+eScjlPX0zh+hMG+po08vqPF+F+T93TaCvv2rbXBIzAfyj+lGvT7uax3fwFcaq65p28dsEcOrxS9ri1yTGpR0yyOOnjN/XbR3b5/MT6LEqI2CvxKyUHeMv9MUHMg42j70jN3n8Ie/Ja0xFr/gWcojXpssTgDufmrJn9Lhxip/gA2t7DvV8iCmjcY0r1UK3e571F2JM4gF2x7ZrKT6X81Pa9jNPzlDZc/ALPjUx7rP5yzrdSmflOYswOUE4Qbl3w8DVWN3Iee8HCWRXg3EwWApAVX4dH+Mjpx5qqc9mlAMxZdGj9u/PyOk6IKtulMyDMZFHig5hVfO0IZhVHbuzRrfKKGVVx6oH5WnTbUPgrPOIzlU+Nq38M49ahk0qw6qLz/dJID6RNYP9sCN+mUDcNadNDvG9bukdf6YPn+ojdW2kL20pr2sgdcnRrR72aIsP089LQyidP09sgD0xgE/s1Q9gGf0Yf6EvcQp7xjcjLzl1Nf6kfJSjJzrH9+I/kUG96XwErhFX4ouMtZZoh+2xdd/7er/4RcqNKyFx2XzGX7BdtXX6bGlGjMD22U+wMWXsRUnsGfW8Q/1MbNm7v5ztghDDZdFuLUTqOcBiOD5JMXo1FnW0qQZbMiCBoLaLXPLo1mWjC/JrefSo/fszbRhvLWXMzmetD3XZeBPY6pyi25q+OG7fsEf9qh70YTNm4XQeOQRkg0V25KWsX0iqbJ/vlwB2jM2xP596YRxpHn8Y1S2VZS1Uv8TPGL8mfH9UnjbUsx7id+S0R26esxmkj/n5CBAvcxnDDj0OjEY6xl+Qgy/W2FdlY/OlutoucuqehP7EK3yJGMs4pvMSuHRcSTzp8WM0C+xMjFvbo4wrI3LXK1vzl9imapO4U8tGz/RlfRMv8AH61TWPH/UvlYgrW7ElOu15H/p7Mh+RnSzrm3kMzYGzJ8oIzhiMgN2NnPd6+EVG3www3uhwvmbYBJS6USA75XXM6NH1r+9pEwepdXmObNqS8l7HStvkbKYwwmnh1eeUcauMlEUGfbteaVP70T6OzjiMDR9413a0oSyHO+xHGQsueqYuOpg/HoHYdOuAjW/ho4em6kP0TSzocnq7Wo/vVf3wO9qjO6mvl9rX5/MTgDc2WUvH+gt2XpKNzRl7K3UZxFT6Zh9IXNySY/3xBC4RV9ifsCP2m0n4ytoFwbgyQ/E6bbq/ZI1i79FnJg5E8/hNzkf0HclM2ZJ/PYO/XOSCgCGAzgE+m3aMkwNl3mP4/l4Pp9RhrOoEGIeyfgnJwTryao5D0KcHCTYQyquuXa8qJ885mDDPOBt19XAeZ4yTRQc4ZIOiD+2Q1zcv6ph3nXt0q4xSFt36HClPm9qPctrWy1Z0XmqH3qnDDjBH98ogepg/DgH8Hz/DplsJH4hPb7VNPfJZZ9VPWCushbr2skZqu8hg3O7bWYeR0ddL+pqfnwD2G8X5PtIx/oIM5CfWdJn4Uo2LvZ53fKjr12Ns4uKov2WnE7hUXMk+NRuH8IP6xUKdmXGl0rjt86y/HLNuWfucV5b8IDPf2kOexV9OviAk2BLIa8LIbPwYA5hJ9YBOXwyRQwPvWfT0yaETWbSph1gCP/Ipy0GC9v2wkXGToyeyaEvKBtKDTC4gkZ3+NUcv5oe8fHivmxbzoK7yiQ7pk5yxMv/oR//IDMf0zzs6pQyd4Jj+Vd+06Rsu+jIGfdGhtqtj5CJVD2jRt9qmjunz/RPA5tgWHxhdDrBx9Sd8Ap/BX5J4Zu3hO6TEBdYVdXzok/rej3Fpgy74Um9H+9TRribKWUPomHGrvrWtz6cTgH98AFt1e8z4C1rgDzVu4IOJN8jEhiN/pC/12Lz2H82MenyiJ3ydOIacPPc2vp9G4BJxpWqEr+ADo1hBLMK+6ECizZKvGFcq1ds9b/lL12x0QWA9130ofYgBtKdu63JAH2ITn1F6Jn856YIAwBxuk7M5JPGccnJSjIqh2AD6IsfALORqyCqjGg2jEwRSXwNCdBjl6MCmQD/yfpiIvJqP5FCGs1QdctChrs+fcZMIWMyRMZhvNkbqwxXdkJG2OHb4RTfq0z5lyEOvmmq/yE09Y0cXxoIr78jFHkk8M0ZfYCOG6WN+3wTiO/gwvjRK1T+wdfXjtO+BmffIxmd4rj6efuT4W9ridyP5tKMNbUcJ2ejGWPiw6TIEEkeIMT0OZMQZf6Et9kROUmRjwx4T04a8tqMtn5Hv0q7H9sipPoceNc6ljfnxBLKezx1XolHkx/7kNREDUocv4QtLCVnGlSU61ymPPdf8pWuSOFDL+z5EHX7AvsLZrJ+Lat/6jD58RumZ/OXfVTWiYZkEJCABCUhAAhKQgAQk8DQEvCA8jamdqAQkIAEJSEACEpCABLYJeEHYZmQLCUhAAhKQgAQkIAEJPA0BLwhPY2onKgEJSEACEpCABCQggW0CXhC2GdlCAhKQgAQkIAEJSEACT0PAC8LTmNqJSkACEpCABCQgAQlIYJuAF4RtRraQgAQkIAEJSEACEpDA0xDwgvA0pnaiEpCABCQgAQlIQAIS2CbgBWGbkS0kIAEJSEACEpCABCTwNAS8IDyNqZ2oBCQgAQlIQAISkIAEtgl4QdhmZAsJSEACEpCABCQgAQk8DQEvCE9jaicqAQlIQAISkIAEJCCBbQJeELYZ2UICEpCABCQgAQlIQAJPQ8ALwtOY2olKQAISkIAEJCABCUhgm8DiBeHNmzcvfmSgD+gD+oA+oA/oA/qAPqAPPK4PbF8H/tvCC4IXIS+C+oA+oA/oA/qAPqAP6AM79YH/Hv+3SxYvCNtdbSEBCUhAAhKQgAQkIAEJ7I2AF4S9WdT5SEACEpCABCQgAQlI4AQCXhBOgGdXCUhAAhKQgAQkIAEJ7I2AF4S9WdT5SEACEpCABCQgAQlI4AQCXhBOgGdXCUhAAhKQgAQkIAEJ7I2AF4S9WdT5SEACEpCABCQgAQlI4AQCZ78g/Pr16/U/k/Xjx48T1Lp+13fv3v35z3t9+vRpUQHq8t8C/vDhw2I7KyQgAQlIQAISkIAEJPCIBM5+Qfjy5cvrAXrtkH2voHLw39I9lwkvCPdqSfWSgAQkIAEJSEACEjiWwNkvCDlkkz9aiu5eEB7NcuorAQlIQAISkIAEJHAuAmc9xX/79u3Pn99w2Ob9kZIXhEeylrpKQAISkIAEJCABCVyCwFkvCPzJDf/2IAftpT/B6RcJ2ifV/pHDv2uoKeXk9RLCeKnjVwA+9R0ZtU3tS11tW9v1f0/R/8Qo/ZJHbt6XONQ5+SwBCUhAAhKQgAQkIIF7IPD3ZH6iNhzicxBeO1wzDAfn+mc8vCdx+I4cynjPBSH/AJoyUv69Qz3A51BOzkG9Xzh4T786LvJqX9rRP2XRITpRHj2jF2V1XrRNG55NEpCABCQgAQlIQAISuHcCf0/mJ2rKoTvfnNeDdT8w18N0Pdhn+BzIIyvl5PlFIDJz+K+H8PTvbSiPzHpBqAf/3pcxU0afpP4LAuV1zpkXZVV++ptLQAISkIAEJCABCUjgXgmc7YLAQbqmHKx7OW3qAT2/BqRvPWj3vjmYV9k8VxmpO/cFIfLQM3rUi8movPbJ/MwlIAEJSEACEpCABCRwzwT+PdUfqWk/1OeQnpz6nuovCbTr37SnL3m+kc/BfO3gnX5pk18ZKI8e9YJSx+190XlUFj36BaFy4Dl697n7LgEJSEACEpCABCQggXslcJYLAgflfhiuB/N+kK4wOMhzCK9/wpP6HORz2EcObesvBmmbvB/oqx6nXBDq/JYuCOiQ8dd0jK7mEpCABCQgAQlIQAISuDcCJ18QOLwvXQByoO8XAN6TcoDP4Z2Ddb7Vz68MuTzUb+hThpx6GM8BPZeKyKc8Y+TiQVnGQk76Zj4ZL+/Ree2CENlVv/Qzl4AEJCABCUhAAhKQwL0T+HtSP0LTHJRzsM4BHFEpq3kO2vnVIHX1ME1dlZuDftTLoT19yZPqhYTyPs5SWfpHVpUTndOmy+z60S5y0sdcAhKQgAQkIAEJSEACj0Lg7+n6UTR+AD1Hl4YHUFsVJSABCUhAAhKQgAQk8OIF4QxOkD8r4s+Z8jmDWEVIQAISkIAEJCABCUjg6gS8IJwBef7siD9/8teDMwBVhAQkIAEJSEACEpDAzQh4QTgD+voPoXk2SUACEpCABCQgAQlI4FEJeEF4VMuptwQkIAEJSEACEpCABC5AwAvCBaAqUgISkIAEJCABCUhAAo9KwAvCo1pOvSUgAQlIQAISkIAEJHABAl4QLgBVkRKQgAQkIAEJSEACEnhUAl4QHtVy6i0BCUhAAhKQgAQkIIELEPCCcAGoipSABCQgAQlIQAISkMCjEvCC8KiWU28JSEACEpCABCQgAQlcgIAXhAtAVaQEJCABCUhAAhKQgAQelYAXhEe1nHpLQAISkIAEJCABCUjgAgS8IFwAqiIlIAEJSEACEpCABCTwqAS8IDyq5dRbAhKQgAQkIAEJSEACFyDgBeECUBUpAQlIQAISkIAEJCCBRyXgBeFRLafeEpCABCQgAQlIQAISuAABLwgXgKpICUhAAhKQgAQkIAEJPCqBu74gvH///uXz58+Pyla9JSABCUhAAhKQgAQk8HAETrogvHnz5qV/KoEvX778U//hw4davfnsBWETkQ0kIIEHIPDr16+Xt2/fvvz48eM/2n7//v3l3bt3r7Hy06dPL79///5PG74oSayd/dKE+Jk+NWesmojT6MaH557q2FUOz8zLJAEJSEAC+yNw0gUBHGxubBRfv34d0mGzY6P6+fPnsN5CCUhAAnsnwJcjxMl+QeC9Xhy4IPQvUji0p9+3b99e5YwO8pUhB/d+mM97vWAgh/hM+8TqLjuXl/RPTj+TBCQgAQnsk8DJFwQ2LjaMbGAjTH3DG7WxTAISkMAeCfDlSb7N73GSw3c9sHNI58LARYDEwb33IZ5+/PhxFRVjjr60Ybx8WZNLBF/yJCWeU0ei7ehXDcpG8iPHXAISkIAEHpuAF4THtp/aS0ACd0yAgzYH+vzSWg/7HL5HX65w+F+7AHDIr4f60fRzCah1lNVv/TngM35PXFBy+B/JoT1tRn8K1WX5LgEJSEACj0ngv7vDgfPIN0514+si6i8IbCpsPtnk+PaMzYZP3fSQN/q5nTb0ZWNjsxttYJSxwdKGT/0GjG/m0Ief0aMHbfrP6uhJP/Sinj51LNpThw4cAnr/zsB3CUjg+QgkboziZP5cqB+0E1s6LWQgL78u9Pqtd2JtDv60Jb7VC0P6M8baBYUYvFYfOeYSkIAEJPC4BK5+Qcjmx6GbTYbDNRskmxIH/6R6ME9ZNtlskGx2yMkBnnbIi1ze6YMsNsM8056NkXcSGydl9E1CRuopp33046LAM3rzoa0XhJAzl4AEIEB8yoGcWEKMSUyhnphBWU+j8pTRnlhZY1Xvv/ROHKz9kMOnp6XytCPeJQanzFwCEpCABPZF4L+704HzG218XUTfhLLZbW2W2QwjL/3yTs7BnfKkHPbp2z+0ib61T8qiD9+Q9b55pw0fLgjZbMmrvOhiLgEJPCcBYgIH6aQeYygfxbO1cr6MSJ/RN/8Za5SPvvVfuggslSMXHbhomCQgAQlIYN8EHv6C0Dcz3tcO69moa5uUkZOoQ85SYvPPLxz1l4al9pZLQALPRYC4kC8QmHmPMZTlF9BOhvizdginni8s6p88dhn9nV9Q+7f+6DiKc5TVy02VhQxkmSQgAQlIYN8ETr4g8I0Sm1U9cHdk+dOclGeDy4Gc8pSlDTly6waWg3nGyqabd/rQvvap8nge9UlZ9EEeGzRzW0rowkaZi4Kb5hIpyyXwXAQST/Kr4yiHCAei/cMxAAAgAElEQVR86hJ3QmntgE6byJ+9IORb/x7Pli4o6JQ/jYpOydGNXyNMEpCABCSwbwInXxDAwwVg6VDOZta/jcploG6MKau42ai6XDYu5FHH4bxeDujLQZ068voNXnTI5lr7pSz68C0ZMvgZv27C9OGddvk2jk03Y1bdfZaABCQQAj3GpJzYWWNRDvOJL2lXcw7o/UuXWt+fl771Jz4S5xL36Bc9a+yMPMoOGTf9zCUgAQlI4PEInOWCkL/Z5+//s7GQs/GxodRDNojy7wTqN1Epy7dc5DmkBytytv72lnHzrT7988k3/FXXyE1Z1Ydx0jd5Nkc2UcbIxsqlhXeTBCQggRGBHLwTM9ImsYTYli8baoxLbMmFgXbEoRqrkEUfyhN/I5+cL0d6+9QTo+nL2PTlmVg8SuiyVDdqb5kEJCABCTwugbNcEJg+GxDf9ucwzYGZjalfDtiQ0oacDbL2S1ltk18Ret+06ZeQXCSoRw8uB2yA2aTTD3ldJu8k2ueXCtrXuSCH+eaXgz7+47qDmktAApcgkNhD3hOHf2IIcSaxKm1yIUgsq3EobciJc8hI/EodcWzrywsO/bTh0/tHDjmXhx7Pa73PEpCABCSwHwJnuyBcAwmbE5tYDvg1Z+M0SUACEnhWAlwG1g74z8rFeUtAAhKQwOEEHuqCwDdto2+w8udMh0/fHhKQgAT2QYA/ARr9idE+ZucsJCABCUjgmgQe5oLAxYBfDLgk1E2QZzfGa7qMY0lAAvdEIDFw9OXJPempLhKQgAQk8DgEHuaCAFIuAvwdbP60iL+57X+z+zjo1VQCEpCABCQgAQlIQAL3R+ChLgj3h0+NJCABCUhAAhKQgAQksC8CXhD2ZU9nIwEJSEACEpCABCQggZMIeEE4CZ+dJSABCUhAAhKQgAQksC8CXhD2ZU9nIwEJSEACEpCABCQggZMIeEE4CZ+dJSABCUhAAhKQgAQksC8CXhD2ZU9nIwEJSEACEpCABCQggZMIeEE4CZ+dJSABCUhAAhKQgAQksC8CXhD2ZU9nIwEJSEACEpCABCQggZMIeEE4CZ+dJSABCUhAAhKQgAQksC8CXhD2ZU9nIwEJSEACEpCABCQggZMIeEE4CZ+dJSABCUhAAhKQgAQksC8CXhD2ZU9nIwEJSEACEpCABCQggZMIeEE4CZ+dJSABCUhAAhKQgAQksC8CXhD2ZU9nIwEJSEACEpCABCQggZMIeEE4CZ+dJSABCUhAAhKQgAQksC8CXhD2ZU9nIwEJSEACEpCABCQggZMIHH1B+PHjx8ubN29eP+/fv3/5/fv3fxT58uXLnza0/fDhwz9tej1yakLmp0+fXmW8e/fu5fv377XaZwlIQAJPRYC4+/Hjx9eY2OPpr1+/Xr5+/fpCHCW2LiX6JXaTf/78eamp5RKQgAQk8KQEjr4gwKse4PtmFZ60YcP6+fNniv7kHPrrRsXmVhMyuSAgg/5v3759YYM0SUACEng2AsRCYiCH/1E8pT4xdemCwCWixlyeR7Keja3zlYAEJCCBfwmcdEGIqGw4bFCjNLo8fPv27fXwP2pPGfXI5XKQxKbHBmiSgAQk8EwEiK1LX7RUDvlld+mCgBxiq0kCEpCABCSwRuAsFwQuAPnZe7QxjS4IHPT5sGHxrVZPyOv9+KbLb7w6Kd8lIIE9E+CX1dm4t3ZByK8H+ROk+uXLnvk5NwlIQAISOJzA2S4I+VMiNrL+DVU/6GcTo20+vQ+Xh9HfxtK+/ynS4dO2hwQkIIHHIMCfFRFD+TKF+Jc/Mxppn9g6+qKGssTbyPHPi0YULZOABCQggbNdEEDJJYHNi82nbjz9ghDsbGbZ9Hof3keb3FJ5ZJpLQAIS2AuBHPj5soRfAIixiZmjL0rSfhQ7YUJ//mMPxGRiKV/EUGaSgAQkIAEJVAJnvSAgmIsBlwQ+uSQsXRCiCBsWmxUbX9LSRWCpPP3MJSABCeyFQL717/PJn2j28q0LQm3PpYN42n+9rW18loAEJCCB5yRw9gsCGHPg529d+XZq64JAHy4Htd3Sz+hsaKNvzp7TfM5aAhLYM4GlC0IO933uh1wQ6MtFY+nXhi7bdwlIQAISeB4CF7kggI9vpTjMc0ngs5VoX39BGP0j5Wx++WViS6b1EpCABB6ZQOJo/w85cKjnS5SeEiNnD/3EXH9B6BR9l4AEJCCBi10QQMvmwyWBz1biGzE2tyQ2LTbA+vexbHp842WSgAQk8AwEiH/Ez37gJ7bWL1TC4tALAr/a1hgbOeYSkIAEJPDcBLZP7ht8+GZr7R+65T9/WsVw+M//KzKbE38yNNrs2LxSzsbHhSH9qjyfJSABCeyVQH4tyBcoxEtiYf9Vgfnnzzv5wqUn5OTXV/oSW/31oFPyXQISkIAEIHDSBYEDfH4hGH3LxQBcAPqfGLFRpR91S4d++uaCwSVkqZ2mlIAEJLBnArkkEDeJuzno1zn3eEzbmmo9cXUko7b3WQISkIAEnpfAvzvIhTj4E/aFwCpWAhKQgAQkIAEJSEACZyZwlQvCmXVWnAQkIAEJSEACEpCABCRwIQJeEC4EVrESkIAEJCABCUhAAhJ4RAJeEB7RauosAQlIQAISkIAEJCCBCxHwgnAhsIqVgAQkIAEJSEACEpDAIxLwgvCIVlNnCUhAAhKQgAQkIAEJXIiAF4QLgVWsBCQgAQlIQAISkIAEHpGAF4RHtJo6S0ACEpCABCQgAQlI4EIEvCBcCKxiJSABCUhAAhKQgAQk8IgEvCA8otXUWQISkIAEJCABCUhAAhci4AXhQmAVKwEJSEACEpCABCQggUck4AXhEa2mzhKQgAQkIAEJSEACErgQAS8IFwKrWAlIQAISkIAEJCABCTwiAS8Ij2g1dZaABCQgAQlIQAISkMCFCHhBuBBYxUpAAhKQgAQkIAEJSOARCXhBeESrqbMEJCABCUhAAhKQgAQuRMALwoXAKlYCEpCABCQgAQlIQAKPSMALwiNaTZ0lIAEJSEACEpCABCRwIQJeEC4EVrESkIAEJCABCUhAAhJ4RAJHXxB+/Pjx8ubNm9fP+/fvX37//v2f+X/58uVPG9p++PDhT5ufP3++0I/yd+/evXz9+vVPXR6Q+enTpz9tvn//nipzCUhAAk9BoMbKt2/fvsbEUbytMD5//vxPvK11eZ5pk7bmEpCABCTwXASOviCAqR7g6+G/IqQNFwE2uaRfv369sNF9+/bttYjLBu/9koBMLgjIoD9taGuSgAQk8AwEiJXEQHISMZM4uBRvaZMvb05t8wx8naMEJCABCYwJnHRBiMj8ksBGNkp9o6Jdb8uvDWx8+WaMjRC5eUcubfi1wSQBCUjgGQgQB2sMZM75ZbZ+6RIWtOULGeJkj7uHtElbcwlIQAISeE4CZ7kgsBF9/Pjx9UDP5tVT36i4CPR2bHZcCPJnRMjr/dJmtDH2MX2XgAQksEcC+YVg9GsqfzbEpYLY2eNnWMy0SVtzCUhAAhJ4TgJnuyDkmysO+WxQNfWNijZsUj1RnosD34Attel/itTl+C4BCUhgrwT4EoUvWXriwsAXK6SlC8JMmy7XdwlIQAISeD4CZ7sggI5LAhsXB/36LX+/IHD4738qxN/Y0i+H/3pZqGZZKq9tfJaABCSwVwL8eWa+SMkcib3EWXLS6IIw0ybyzCUgAQlI4LkJnPWCAEouBlwS+OSS0C8IbG4c9PPvELgc8GsBZemzdBFYKn9uMzp7CUjgGQgQH/k3Bj0RP+ufHI0uCDNtulzfJSABCUjgOQmc/YIARn4C5yDPRpZvrTpeLgn5tSH/aLn+qkBd/5YMGcjNrwxdpu8SkIAE9kqAWMqfEOVXgsyTi0H/c8x+QZhpE3nmEpCABCQggYtcEMCa/woRl4TRN14VPRseF4L6bxdG/0iZTY4LQn5lqDJ8loAEJLBnAsTEUezjMkBcXPrwRctMmz2zc24SkIAEJHAYgYtdEFCDXwayaS2plV8Y8udGacdlgUtD/baMja7+ypC25hKQgAT2TIBfCOrlgLiYf5A8mnf/BeHYNqN+lklAAhKQwP4JnHxB4N8PcGivB/mKjU2MS0JP9OMSQN9+OUhbNrnU8esBF4b8Z1DTxlwCEpDAXgnkIpAvWmqe2DiauxeEERXLJCABCUhglsB/T+6zPf//v5RRN6zRvxlgg+t/YpSfu7k8cPBfSnVz5CLh5WCJlOUSkMAeCRA7a4ytz2vx0AvCHr3BOUlAAhK4HoGTLgizai79ujDb33YSkIAEJCABCUhAAhKQwHUIXOWCcJ2pOIoEJCABCUhAAhKQgAQkcCoBLwinErS/BCQgAQlIQAISkIAEdkTAC8KOjOlUJCABCUhAAhKQgAQkcCoBLwinErS/BCQgAQlIQAISkIAEdkTAC8KOjOlUJCABCUhAAhKQgAQkcCoBLwinErS/BCQgAQlIQAISkIAEdkTAC8KOjOlUJCABCUhAAhKQgAQkcCoBLwinErS/BCQgAQlIQAISkIAEdkTAC8KOjOlUJCABCUhAAhKQgAQkcCoBLwinErS/BCQgAQlIQAISkIAEdkTAC8KOjOlUJCABCUhAAhKQgAQkcCoBLwinErS/BCQgAQlIQAISkIAEdkTAC8KOjOlUJCABCUhAAhKQgAQkcCoBLwinErS/BCQgAQlIQAISkIAEdkTAC8KOjOlUJCABCUhAAhKQgAQkcCoBLwinErS/BCQgAQlIQAISkIAEdkTAC8KOjOlUJCABCUhAAhKQgAQkcCqBp7kg/Pr16+Xz588vb9++PZXZWfqjz6dPn+5Gn7NMSiESkIAEJCABCUhAAg9P4KQLwocPH17evHnz+pk5eL9///5Pe/peM338+PHP2Nccd2mse9NnSU/LJSABCUhAAhKQgASei8BJFwRQ/fz588/B+9u3b4v0fvz48acdfW6RcqG5xdiMyQWpplvrU3XxWQISOD+B79+/b/5SmF82+bKFLw74dfGQRHu+oCHG9vTly5fXOup57omY/e7du9fYTD5q0/v4LgEJSEAC+ydw8gUBRBx82YDYYJYSG9+tD8S3HJ9NnANATbfUp+rhswQkcH4CHNizxvvaz2hcDoifv3//fv3wZ4fEUd5nU8boFwQO+8gm9iCP53oB4PLCe8bmGT1rm1kdbCcBCUhAAvsi8O+J9ci5sUFlcxn9isAGlW+nljbKI4c+qFs20oM6naFxNuc+91vpc4YpKUICEpgksLTO86UBeRKxYunb/rSp+devX18P/sSWekGIbC4BSdTTLuPRtyd0ZXyTBCQgAQk8N4GzXRCysfEtVU98K8bFIZeIWk8/6tm4+HCRyEaXjTV19Ktlh37Tlb51fP7cqf57AHRBJxJ68E4/npkbuvBeU50DmyvfCtaUfplH+kcf+ueZttnAqwyfJSCBxySQtd2154BOTOiJeDSKo70dcQLZXAKQk7hJuyXZxKfRxSCyRzE6deYSkIAEJPA8BP67Ox0xdzYpEgfjvlFx+M03UqPNhwN4flKnLc98krLRsWmS2AzrJSLtZvK+UbPBIjcHcjZYdEUnEs/Mh/GyqWYzrr+UIJcNHf2RVS8E2bT72MhPGVzSt44/MyfbSEAC900g67xrmXjI2q8p5bVs9IxcvuAgxvS4SwwbXTLok1g6kkkMX6sf9bFMAhKQgAT2R+CsFwQOx2xUbEJJbHZ8SKONj80oB/KlNhzGkUtbZPcNNWNt5fRFTlIuNJT1T9pQXudDOWWZ0+idSwQH/apnH5t+S2V9vOhiLgEJPB6B0TpnFolr9csGyomHxI+1xBcW+dJidEFgzFEcWSrPWMd++ZL+5hKQgAQksA8Cf0/LJ8ynbkRsbhyg8805G04OyqMLQoblUM0FIAf1lCeP3GyKKT8kR0/kJ/FeD/oprznt6/yoo6z26+9LG3YdGzldn5T18ao+PktAAo9FYLTOmQFxkYsAn/yX3fIL6dq3+PnlMxSW4s0ojlA2KkcWsbX/eWTGMJeABCQggeci8Pe0fMK864aTXxHY4PhmbOvXATZGLhG0Y3NcukQgLxeIXD4OVRk96yF9bbOMbNrX+VHeLwRsqsyBubPpo6e/IISguQSem0CPO5UG8S/1xJClXxVqH+ILsSZpdEGgTY9btKeMup7Qo8bqXu+7BCQgAQk8F4GzXxDAl2/7c2gO0tHhn4N0/dZq1AZ5bJwcvvm72vqNW2TP5NmI0zZ6ktcNt26gMxeEXAqYb9rnG8GM1cemfKmMcpMEJLAPAqN1vjQz4h9x5P/auwMjqXEtCqCkQAykQA6EQAykQAZkQAZEQAQkQAJkQA7z61J1qbda98x0T++3McdVs3bLsiQfd0l6tpu9tDQYSD9z6S/H5mlA9q9L0tansOm/0sasLQQIECBAIAL/HkGudMlEuD/Q7aEdxDLxnkvf+Z+T5wxYnZAnvYNpJuz5nDJmAJFBLAFCBtGW07SZb9bb7f54uANh6khZ60Dbdidf9s0f+zVt1pX25+nHY0sDnxzfY9f25PikzfoeK9M+AgSOL9A+7amWpg+55eZH+9usu6RvS98105pv3gxJf5T+Lusu6VfbRzXNmgABAgT+LoEXBQgd+DrBziS4S/Z1cOrA1HxZZ3+WTpwzMObOVgbJ7M8A1X353LLWOpOewS3HPzaorce1nRkMO1FPGXOwXNu7nkfPYeab27M9qSflN7DZas88NtsWAgT+fIH2L3MSPs8q/Ur6nfQPvenR/b2J0b6m6XPdfinruaT/TN2pN+Vke+2T2ra171l/OD3LtU2AAAEC5xcwC73DNc7gvg6w/bwO+HeoThEECPwBAp24ty/Iet5E6f7cNEj6VgCR/iPHJc8aAJSg5WztT0CQwCN/s+4GHrNtc3urLa3PmgABAgTOLyBAeOE1zkA6B95ZXNLn4/y5zzYBAgSeK5Anq1sBwHOPl48AAQIECFwjIEC4Rmsjb35/kMf/6+CdAd1j+g0wSQQIXC0wXw26+mAHECBAgACBKwUECFeCrdnzCkDfH+4j+gQMCRAsBAgQeIlAbjzkt1le+XmJomMJECBA4FoBAcK1YvITIECAAAECBAgQOLGAAOHEF9epESBAgAABAgQIELhWQIBwrZj8BAgQIECAAAECBE4sIEA48cV1agQIECBAgAABAgSuFRAgXCsmPwECBAgQIECAAIETCwgQTnxxnRoBAgQIECBAgACBawUECNeKyU+AAAECBAgQIEDgxAIChBNfXKdGgAABAgQIECBA4FoBAcK1YvITIECAAAECBAgQOLGAAOHEF9epESBAgAABAgQIELhWQIBwrZj8BAgQIECAAAECBE4sIEA48cV1agQIECBAgAABAgSuFRAgXCsmPwECBAgQIECAAIETCwgQTnxxnRoBAgQIECBAgACBawUECNeKyU+AAAECBAgQIEDgxAKHCBDevn378PHjx7sz//jx41e5r1+/vnvZtxSY9nz48OHhKO255RwcQ4AAAQIECBAgcG6BFwUI7969e3j16tWvv+dMehMINH+O7fJfBQjv37//XV/r2nN9tPbsaaFuAgSeJ/D9+/eH9p3pZ3OT4efPn/86OPnaJ2edz+uSmxSfP3/+Vd6nT5/W3T4TIECAAIFfAi8KEFJCBqFO+r98+XKR9du3b7/zbQ1cFw984Y4OmC8s5ubDM7DPZe/2zLbYJkDg2AJ96ph1lvSxCRLSj8wlAUPSM/mf+dZAIsHFmzdvfvXFAoQpaJsAAQIEpsCLA4QUlklwBqcMPJeW3D3fY3K8R501yKCe4Gkue7ZntsM2AQLHF0hAsE7yM7FPvzJvtGTiv3UzIunr0ps1AoRVxmcCBAgQqMA/Z69NvXKdSW8Hra2nCJkoJ3honiuLf1H2vSbkGdT7WsA8gb3aM9tgmwCBP1egE/ysu+QGzTrhz9OEpK9Lj1/zr/l8JkCAAIG/V+BuAUIfca93sUKbu1gJHLYChAxW2Z+Jc5aUk4EtAcXXr19//8g4A10+X7tsTchz523+HiD19y7dbE+2O8lv+1p/8ue43MlL29YfWfe4vn7V49ueHN/t5O0rBC3fmgABAlsC6QfnxL9PKtf+sYHAfNKQ8pouQNjSlUaAAAECEbhbgJDCMknOhDgDUJdMhDuYbQUI2ZdjOoFOnqZlEp/Br5Ppx15han3rupPwpqe8lpu0tDX1ZbKfpXWnrr7Pm4E3bZxPR1JuJvZpW8qcAUHPf6075Tct59ljZ/2/GuE/BAgQuCCQvmpO7jvhb7/Tw55Kn2X0GGsCBAgQIBCBuwYIvZOVSXCXDEIdiLLORHtdZoCQfc03B7ymrcc+9bkT8uZrEJM617/mWduT9KT1PLY+965eJv1d1rqTfiltmvV4awIECEyBPA3IzYi5PBUIzH40xzX/7M9mebYJECBAgMC/Z+s3mMzJbV+76aCUO/GdNF+a5K8T8uZrGWlS065t3johz+enBsa1PanzqQChg+5s81p3yrmUlnQLAQIELgmkH83Tz/anzZegIf3T7Huyr32SV4wqZU2AAAECzxW4e4DQpwgZyPJKTl/dSYMuTfLXCXnzzQGvac89seZbJ+T5/NRkfG1PyloDhDyJSPCT8+3AnVeF5uC91p1yLqU91aaejzUBAn+nQPrUdbJfifQ9642PfE76ujRwWPOv+XwmQIAAgb9X4O4BQij7FKET6PJemuSvE/Lm+y8ChLYt60zuu2Tw7bK2J+lrgNCgIOfY/OvgfSkYSP65JJ8AYYrYJkBgCuSGxOxf2v80T/qztQ/J56SviwBhFfGZAAECBFaBf85U173P+JxBqz/WbfYOQOvg1Pf/14EuE+b5Xm3zzX+Vo2m9Q5917o4l/bGlPx7ucQkKclzqnH9ta/Kt7WnarCsBxWzfVhsa6OT4Hru2J8clbZ7/VlnSCBD4+wTSd6SvmX1Vt9tnRSX50q/1H1LoP3E6b4JUr//oQvukplsTIECAAIEKvChAyB2qDlZZz0fW2dcnAA0YZt7e7VrTOqlueo5d60laB8THBrn1uJ50g5rUkUE1A23Ky9J6s+45rGlrvrl/tif1pPw8Zcj2Vnvmsdm2ECBAoAK9obD2E/m83qCY/Vr6mnxel7UP0uesQj4TIECAQATMSG/8HiSo2Bq0k7Y1MN9YjcMIECBAgAABAgQI/F8FBAg3cOdpw3xaMotI+tZj/ZnHNgECBAgQIECAAIGjCggQbrgyeSe4rx/Nw/PIv+8Az3TbBAgQIECAAAECBP4UAQHCDVcqrxDlFaP5Y+cEDOs7wTcU7RACBAgQIECAAAECuwoIEHblVzkBAgQIECBAgACBYwkIEI51PbSGAAECBAgQIECAwK4CAoRd+VVOgAABAgQIECBA4FgCAoRjXQ+tIUCAAAECBAgQILCrgABhV36VEyBAgAABAgQIEDiWgADhWNdDawgQIECAAAECBAjsKiBA2JVf5QQIECBAgAABAgSOJSBAONb10BoCBAgQIECAAAECuwoIEHblVzkBAgQIECBAgACBYwkIEI51PbSGAAECBAgQIECAwK4CAoRd+VVOgAABAgQIECBA4FgCAoRjXQ+tIUCAAAECBAgQILCrgABhV36VEyBAgAABAgQIEDiWgADhWNdDawgQIECAAAECBAjsKnDaAOHbt28PHz58eHj37t2uwK38y5cvD2/fvn349OlTk6wJECBAgAABAgQIHE7g7gFCJuavXr16+P79+64n+/r161/tOEKAEJO2R4Cw69dC5QQIECBAgAABAk8I3D1AyF37BAgfP358our/fnfasVeA8PXr1388LWjgJED476+7GgicTeDnz5+/bzKkX8tf+pi5/Pjx4+Hz58+PPqnMMemjc8PCQoAAAQIELgncNUDIAPXmzZtff0cYgPYMEDIIz2BAgHDpKyidAIGnBPKKYgODrLf61/Q56X+zf/Y9LTt9UG6YtJymWxMgQIAAgVXgrgFCBqXcwcpfBqEMansuacMeTxA6mM9BWoCw5zdB3QT+bIFM/HMD5qnlOf1Mg4SnyrKfAAECBP5egbsGCBnE8ig8f5mcv3///rdsB67evZoT96bNCXW2ezcs6/Vx+u+CH9nYChBSTn4snH25C9c60+YENq0rr0hlf/7WuvP7ig6yyT8DoeTt+XSdc+/5p77kT7nZnzotBAgQuCTQGw7pc2Zfs5V/9jNb+5PWvuvSfukECBAgQOBuAUIGrjzi7pLgIBPgedcrk/AMTpmgzyUT7jlRzuS8A2GOSblrWfP4S9s5JvV1yeQ9ZafMLPNJRybunbSn7Wl325sgoEvSkq/nmgG5x6W+LFuDdNNy7tnOkrbccl6/DvYfAgT+CoFO6NNX5C99SPuwFaD9TG98rPvzueVt7ZNGgAABAgQicLcAIZPoGQz0Tvo6UCU9E+o5wM3gIOkdCNf1WtZTlzDHzwChTw7Wcpsn5WdfJ/Apv2mtqwPwzJOJfstIvuaZ7X0sbZbVeqwJECBQgfStuWmSfjZ91Hw62zxZb/Uzc3+2BQiriM8ECBAgsArcJUDooLROvPN53n1v5UmbQcHcblnN+5J16p8T93x+bDLeYGDmaVrb0fY9J48AoWrWBAjcQyA3UDrBnzdkWnb7p9n3dF/XPb6frQkQIECAwCpwlwAhd7PmhLmVdHKdpwZzyR33Bg65Kzb/nwkd4LbKm2U8Z3srQHhs4Gx7Z91Nm/XlSUTOOYN1BumcywxEeg6zrsfSZn2zHtsECBBYBdLnXLrZsdXPrMcLEFYRnwkQIEBgFXhxgNAJ8lpwPncg6/v6zdP09XcL85i8hjQDiwx880lDy3psvQYImcgnLeX0Faes274GA3PC3rRZT3+k3N8e5PiWl3xbg/RjabO+WY9tAgQIbAmkL0s/ui5b/cyaR4CwivhMgAABAqvAiwKEDFC5m54nApeWvve/Tu4zSGWQW9NTTn+8m8n8/OuAmEl7Juf9vFV3Juw5NvV36e8iZvNNTtIAAAO2SURBVJnZ7g+iW+8MTJrWACDrtLufW/a6Trk5NsFEym/dSevStFlf91kTIEBgSyB9z3xiOfMIEKaGbQIECBC4VeBFAcKcaG8NWHN/tmeeTJqTdmminYl079Bnkj8n0c8JEGbda719kpB1ysrSJwU9LgNtjuvnrJPWAXimZzttbaCR8vJUIek5j/WY1LXW13bceiEdR4DA+QRyEyR9Q/vJPr2cr2XOs+5Nh3kjYu7Pdm/atMx1v88ECBAgQOBFAcLfyJdBtQPsVpDwN5o4ZwIE/huBBAi9oZGbEOvrjLPW9YZG+qe5rDcqst+NiSlkmwABAgQq8M8RpKnWFwX6ytCaIYHDY3ft1vw+EyBAgAABAgQIEDiigADhyquSu3gJBOYj/gQHeb0od+gsBAgQIECAAAECBP5kAQHClVcv7/jmnzjt60UJGPJ5BgxXFik7AQIECBAgQIAAgcMICBAOcyk0hAABAgQIECBAgMD+AgKE/a+BFhAgQIAAAQIECBA4jIAA4TCXQkMIECBAgAABAgQI7C8gQNj/GmgBAQIECBAgQIAAgcMICBAOcyk0hAABAgQIECBAgMD+AgKE/a+BFhAgQIAAAQIECBA4jIAA4TCXQkMIECBAgAABAgQI7C8gQNj/GmgBAQIECBAgQIAAgcMICBAOcyk0hAABAgQIECBAgMD+AgKE/a+BFhAgQIAAAQIECBA4jIAA4TCXQkMIECBAgAABAgQI7C9wMUB49erVgz8GvgO+A74DvgO+A74DvgO+A74Df+534JZwQ4AgEBII+g74DvgO+A74DvgO+A74Dpz0O3DXAOGWwhxDgAABAgQIECBAgMCfLXDxCcKffVpaT4AAAQIECBAgQIDALQIChFvUHEOAAAECBAgQIEDgpAIChJNeWKdFgAABAgQIECBA4BYBAcItao4hQIAAAQIECBAgcFIBAcJJL6zTIkCAAAECBAgQIHCLgADhFjXHECBAgAABAgQIEDipgADhpBfWaREgQIAAAQIECBC4RUCAcIuaYwgQIECAAAECBAicVECAcNIL67QIECBAgAABAgQI3CIgQLhFzTEECBAgQIAAAQIETiogQDjphXVaBAgQIECAAAECBG4RECDcouYYAgQIECBAgAABAicVECCc9MI6LQIECBAgQIAAAQK3CAgQblFzDAECBAgQIECAAIGTCggQTnphnRYBAgQIECBAgACBWwQECLeoOYYAAQIECBAgQIDASQUECCe9sE6LAAECBAgQIECAwC0C/wP1mTsnY6IYkwAAAABJRU5ErkJggg==\"\u003e\u003c/em\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eThe clean reads of this brain area and liver were assembled together, and a total of 403,707 unigenes sequences were obtained. The unigenes sequences were used as the reference genome in the subsequent analysis of the brain. The lengths of 403,707 unigenes ranged from 201 bp to 19,402 bp, with an average length of 511 bp, an N50 value of 615 bp, and an N90 value of 244 bp.\u003c/p\u003e\n \u003cp\u003eAmong the 403,707 unigenes, 83,237, 60,588, 23,820 and 9,176 were respectively annotated by the NR, Swiss-Prot, KOG, and KEGG databases (Supplementary Fig. S1). For KOG annotation in particular, the term of signal transduction mechanisms was the most highly represented (Supplementary Fig. S2).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eDegs Analysis\u003c/h2\u003e\n\u003cp\u003eA correlation analysis was conducted based on the TPM values. From the correlation results (Supplementary Fig. S3), the sample had good repeatability and could be used for gene differential expression analysis.\u003c/p\u003e\n\u003cp\u003eTo clarify the differences in the transcription levels of genes in different activity states of \u003cem\u003eV. sinensis\u003c/em\u003e, we compared the transcriptome data of two adjacent time points and obtained four comparisons of DEGs. In addition, we also focused on the analysis of the rest vs awake (before and after feeding) and sleep vs activity comparisons, and a total of six pairwise comparisons were obtained (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In the six pairwise comparisons, a total of 672 DEGs were detected. In the rest vs activity comparison, the number of DEGs was the highest, with 136 genes, and the lowest number of DEGs was detected in the awake vs active comparison, with 60 genes. The numbers of upregulated genes in the comparison groups were higher than the numbers of downregulated genes in the two comparisons. In terms of the definitions of upregulation and downregulation of specific genes in each pairwise comparison, an upregulated gene was defined as one that had a higher expression level in the former time point than in the latter, and vice versa for a down-regulated gene. In the awake vs active comparison, this may be because bats are always active after awakening to prepare for subsequent hunting behavior followed by a highly active predation state, so the numbers of DEGs in the two states were less, as similar genes were expressed. A heatmap of the hierarchical clustering of all DEGs indicated the large differences in gene expression between different time points (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eThe number of\u0026nbsp;DEGs\u0026nbsp;in the six pairwise comparisons of brain tissue\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.05084745762712%\"\u003e\n \u003cp\u003e\u003cstrong\u003eComparable group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.220338983050848%\"\u003e\n \u003cp\u003e\u003cstrong\u003eUp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.316384180790962%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDown\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.412429378531073%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.05084745762712%\"\u003e\n \u003cp\u003eRest vs sleep\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.220338983050848%\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.316384180790962%\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.412429378531073%\"\u003e\n \u003cp\u003e122\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.05084745762712%\"\u003e\n \u003cp\u003eSleep vs wake\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.220338983050848%\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.316384180790962%\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.412429378531073%\"\u003e\n \u003cp\u003e134\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.05084745762712%\"\u003e\n \u003cp\u003eWake vs activity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.220338983050848%\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.316384180790962%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.412429378531073%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.05084745762712%\"\u003e\n \u003cp\u003eRest vs activity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.220338983050848%\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.316384180790962%\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.412429378531073%\"\u003e\n \u003cp\u003e136\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.05084745762712%\"\u003e\n \u003cp\u003eRest vs wake\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.220338983050848%\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.316384180790962%\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.412429378531073%\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.05084745762712%\"\u003e\n \u003cp\u003eSleep vs activity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.220338983050848%\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.316384180790962%\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.412429378531073%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003eGo And Kegg Analysis Of Degs\u003c/h2\u003e\n\u003cp\u003eThe six pairwise comparisons were analyzed by GO and KEGG enrichment, and the results suggested that the bats had significant differences in the metabolic and transport activities at different time points during the day (Supplementary Tables S1\u0026ndash;S4). The rest (after feeding) vs awake (before feeding) comparison represented the two extreme states before and after predation. There were 63 genes that showed high expression levels in the after feeding state and low expression levels in the before feeding state (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In the cell component of GO, multiple complexes were significantly enriched by these 63 genes, including Prp19 complex, spliceosomal complex, and DNA packaging complex. In the category of molecular function, these genes were significantly enriched in terms of transferase activity, for example, histone methyltransferase activity (H4-R3 specificity), aryl sulfotransferase activity, and protein-arginine omega-N asymmetric methyltransferase activity. In the category of biological process, these genes were significantly enriched in hemoglobin synthesis-related terms such as positive regulation of hemoglobin biosynthetic process, regulation of hemoglobin biosynthetic process, and hemoglobin biosynthetic process (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Regarding the KEGG results, various metabolic pathways were significantly enriched by these 63 genes, including drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, and glutathione metabolism (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The above results indicated that hemoglobin synthesis, transferase activity, and metabolic activity of \u003cem\u003eV. sinensis\u003c/em\u003e in the after feeding state may be higher than during the before feeding state.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThere were 57 genes that showed low expression levels in the after feeding state and high expression levels in the before feeding state. In the cell component category, these genes were significantly enriched in the terms of proton transport. The three top GO terms with the most significant \u003cem\u003ep\u003c/em\u003e values were mitochondrial proton-transporting ATP synthase complex, proton-transporting ATP synthase complex, and proton-transporting two-sector ATPase complex, catalytic domain. In the molecular function category, these genes were significantly enriched in terms that were closely related to various enzyme transport pathways such as cation transporting ATPase activity, beta mannosidase activity, and ATPase coupled ion transmembrane transporter activity. For the biological process category, transport-related terms were significantly enriched by these 57 genes, including hydrogen ion transmembrane transport, hydrogen transport, and proton transport (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Regarding the KEGG results, a variety of endocrine-related pathways were significantly enriched by these 57 genes, including the renin-angiotensin system, renin secretion, and cortisol synthesis and secretion (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The above results indicated that the intensity of transport and endocrine activities of bats in the before feeding state may be higher than in the after feeding state.\u003c/p\u003e\n\u003cp\u003eIn addition, sleep and activity were the two states that best reflected the difference between the day and night activities of \u003cem\u003eV. sinensis\u003c/em\u003e. There were 47 genes that showed high expression levels in the sleep state and low expression levels in the active state (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In the cell component category, these genes were significantly enriched in organelle-related terms such as cytoplasmic part, Golgi apparatus, and organelle membrane. In the molecular function category, these genes were significantly enriched in terms related to enzyme activities, including inositol tetrakisphosphate phosphatase activity, cytochrome c oxidase activity, and heme-copper terminal oxidase activity. In the biological process category, these 47 genes were significantly enriched in nuclear-related terms including nuclear matrix organization, nuclear matrix anchoring at nuclear membrane, and cytoskeletal anchoring at nuclear membrane (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). For the KEGG results, the 47 genes in the sleep state were only significantly enriched in the ribosome pathway (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn addition, there were 53 genes that were expressed at low levels in the sleep state and highly expressed in the activity state. In the cell component category, terms that were related to proton transport were significantly enriched by the 53 genes, including mitochondrial proton-transporting ATP synthase, catalytic core, and proton transporting ATP synthase. In the molecular function category, genes were significantly enriched in the terms related to DNA binding, including RNA polymerase II transcription factor activity (ligand-activated sequence-specific DNA binding), transcription factor activity (direct ligand regulated sequence-specific DNA binding), and chromatin DNA binding. In the biological process category, these genes were significantly enriched in multiple terms related to endocrine activities, and the three top GO terms with the most significant \u003cem\u003ep\u003c/em\u003e values were response to corticotropin releasing hormone, cellular response to corticotropin releasing hormone stimulus, and regulation of type B pancreatic cell proliferation. In addition, these genes were also significantly enriched in terms related to the circadian clock, including entrainment of circadian clock, circadian regulation of gene expression, and circadian rhythm (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Regarding the KEGG results, various hormone secretion pathways and signal transduction pathways were significantly enriched by the 53 genes, including cortisol synthesis and secretion, MAPK signaling pathway, and PI3K-Akt signaling pathway. In addition, there were two circadian clock-related pathways that were significantly enriched, namely, circadian rhythm and the circadian rhythm-fly. Importantly, both circadian clock-related pathways contain the \u003cem\u003ePer2\u003c/em\u003e gene (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The above results showed that the two states of bats with completely opposite circadian rhythms do involve circadian clock-related genes and regulate the regular changes of some circadian physiological processes.\u003c/p\u003e\n\u003ch2\u003eTrend Analysis Of Degs\u003c/h2\u003e\n\u003cp\u003eTo clarify the periodic expression and oscillation changes of genes in the brain area of \u003cem\u003eV. sinensis\u003c/em\u003e during a 24-hour period, we examined the intersection of the six groups of DEGs, i.e., screened out genes that were expressed at all time points, to obtain a total of 557 genes. Then, we conducted trend analysis on these 557 genes, and the results are shown in Supplementary Fig. S4. Among the 20 modules obtained, the DEGs were significantly clustered in module 6 that contained 27 genes. These 27 genes had similar expression trends at the four time points during the day. The expression levels of these genes were decreased in the resting state, were the lowest in the sleep state, and gradually increased in the waking state, reaching the highest expression level in the activity state (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). These genes in module 6 were used in enrichment analysis, and the results are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Table S5. Numerous circadian clock-related terms and pathways were significantly enriched by these 27 genes. Through the above analysis, we believe that the change trend of the genes in module 6 was consistent with the biological habits of bats. These 27 genes showed obvious circadian rhythms and were closely associated with bat activity states. Bats in an active state require high expression levels of these genes, so the physiological and biochemical processes that these genes participate in play important roles in the activity and periodic awakening of bats.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe important physiological and biochemical processes enriched by genes of module 6 are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The GO results for 27 genes in module 6 suggested that a total of 161 terms were significantly enriched; 238 terms were significantly enriched in the biological process category; 26 terms were significantly enriched in the molecular function category, and 18 terms were significantly enriched in the cell component category (Supplementary Table S5). In the cell component category, the top three terms with the most significant \u003cem\u003ep\u003c/em\u003e values were transcription factor complex, varicosity, and nuclear membrane. In the molecular function category, terms related to DNA molecular binding were significantly enriched, and the top three terms with the most significant \u003cem\u003ep\u003c/em\u003e values were RNA polymerase II transcription factor activity, transcription factor activity, and steroid hormone receptor activity. In the biological process category, terms related to endocrine activities were significantly enriched by genes of module 6, and the top three GO terms with the most significant \u003cem\u003ep\u003c/em\u003e values were response to corticotropin releasing hormone, cellular response to corticotropin releasing hormone stimulus, and regulation of type B pancreatic cell proliferation. Additionally, terms related to circadian clocks were also significantly enriched, including entrainment of circadian clock and the entrainment of circadian clock by photoperiod (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). The KEGG results of module 6 are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The top three pathways with the most significant \u003cem\u003ep\u003c/em\u003e values were cortisol synthesis and secretion, aldosterone synthesis and secretion, and Cushing syndrome. The circadian rhythm and circadian rhythm-fly pathways were also significantly enriched (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). The above results suggested that there were circadian clock-related genes in module 6 that have important molecular functions in the circadian rhythm.\u003c/p\u003e\n\u003ch2\u003eImportant Circadian Clock Genes\u003c/h2\u003e\n\u003cp\u003eCombining the results of DEGs and trend analysis, we found that the genes in the sleep vs activity comparison and the genes in module 6 were enriched in pathways related to the circadian clock (Supplementary Table S3 and Table S5). We further analyzed the genes in the pathways related to the circadian clock and found that they all involved \u003cem\u003ePer2\u003c/em\u003e. In the GO results of the sleep vs activity comparison and trend module 6, \u003cem\u003ePer2\u003c/em\u003e was significantly enriched in terms related to the circadian clock. Therefore, we believe that \u003cem\u003ePer2\u003c/em\u003e may be an important circadian clock gene in \u003cem\u003eV. sinensis\u003c/em\u003e. We marked the \u003cem\u003ePer2\u003c/em\u003e gene in the figure illustrating the circadian rhythm pathway to show its important role in the pathway (Supplementary Fig. S5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe circadian clock is the result of animals adapting to the environment, and the daily variation of the physiological activities of animals is closely associated with the rhythmic expression of important genes. In this study, we used transcriptome sequencing technology to analyze the changes in genes expression in the brains of \u003cem\u003eV. sinensis\u003c/em\u003e during a day, with a view to revealing the circadian changes in gene expression in the brain of a nocturnal mammal. In addition, to clarify the differences in gene expression and physiological processes in the brain area of bats during sleep and activity before and after eating, we focused on the sleep vs activity and rest vs awake comparisons and found that bats have different degrees of changes in corresponding physiological processes in different states. This study is the first to examine the rhythmic changes in gene expression in the brains of bats. The analysis can not only reflect the daily changes in the physiological and biochemical activities of genes but also screen out genes that may be involved in regulating the circadian clock in the brain, thereby providing a reference for further understanding the mutual adaptations of animals and the environment, and at the same time enriching the research on the biological rhythms of nocturnal animals.\u003c/p\u003e \u003cp\u003eOur results indicated that there are significant differences in the physiological processes of bats in sleep vs activity. In the active state of \u003cem\u003eV. sinensis\u003c/em\u003e, the pathways related to protein kinase signals are likely to be important circadian clock regulation pathways in the bat brain. The MAPK signaling pathway and the PI3K-Akt signaling pathway were significantly enriched by the genes with high expression levels in the active state. Previous studies have shown that in the mammalian SCN of the hypothalamus, the MAPK pathway can be used as an input pathway to allow the endogenous clock to enter the 24-hour rhythm cycle \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The MAPK pathway can also interact with the components of the molecular circadian oscillator to affect the cycle of the circadian clock \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The PI3K-Akt signaling pathway can participate in the output pathway of animal retinal circadian rhythms \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The structure of the mammalian circadian clock system is composed of the input pathway of external signals, the oscillating central biological clock, and the output pathway of the rhythm \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Combining the results of this study, we speculate that these two signal pathways are closely related to the circadian clock regulation mechanism in the brain and may be involved in the input and output pathways of the circadian clock system, and that they play an important role in the brain when bats are active. In addition, genes with high expression levels in the active state were also involved in multiple pathways or terms related to the circadian clock such as circadian rhythm, circadian rhythm\u0026ndash;fly, and circadian regulation of gene expression, indicating that the bat brain is in the optimal state for overall physiological process and the regulation of the circadian clock during the active state.\u003c/p\u003e \u003cp\u003eWhen bats are active, the physiological processes related to various endocrine hormones in the brain are also relatively active, for example cortisol synthesis and secretion and aldosterone synthesis and secretion. Cortisol is a glucocorticoid with an endogenous circadian rhythm driven by the central circadian pacemaker (CCP) of the SCN \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Several previous studies have documented that the concentration of cortisol in the human body generally reaches a peak during the transition between sleep and wakefulness, and cortisol has a wakeful response. Cortisol arousal response refers to the increase in cortisol concentration in the first hour after awakening \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. We speculate that bats also have a cortisol awakening response when they are awake and may maintain a higher concentration when they are in an active state. Aldosterone is a mineralocorticoid that plays an important role in regulating systemic blood pressure \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In rats, the concentration of aldosterone also showed a clear circadian rhythm, and the concentration reached its peak at night and was the lowest at noon \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. We speculate that the rhythm in \u003cem\u003eV. sinensis\u003c/em\u003e may also resemble that of rodents, with a higher concentration of aldosterone at night. Therefore, we suggest that the secretion of many hormones in bats occurs in the active state and prepares the bat for subsequent predation activity.\u003c/p\u003e \u003cp\u003eThe results of KEGG and GO of 47 genes with high expression levels in the sleep state indicated that the physiological processes related to protein synthesis are relatively active in the brain of \u003cem\u003eV. sinensis\u003c/em\u003e. In the KEGG results, the ribosome pathway was significantly enriched, and in biological process category of GO, terms such as mitochondrial ribosome, mitochondrial large ribosomal subunit, and organellar large ribosomal subunit were also significantly enriched. Previous studies indicated that the circadian oscillator regulates the transcription of ribosomal protein mRNA and ribosomal RNA \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The circadian clock can affect the time translation of some mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors and the clock-dependent rhythm activation of signal pathways related to its regulation \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore, we speculate that the ribosome-related pathways or terms enriched by genes with high expression levels in the sleep state are likely to be intuitively reflected by the regulation of the circadian clock in the brain, and the ribosome synthesis-related genes present a state of high expression level in the sleep state.\u003c/p\u003e \u003cp\u003eThe results of GO and KEGG analyses together indicated that the physiological processes related to drug metabolism are active after feeding for \u003cem\u003eV. sinensis\u003c/em\u003e, and they were significantly enriched in 63 highly expressed genes. This pattern of changes in the expression of genes in the brain of \u003cem\u003eV. sinensis\u003c/em\u003e is likely to reflect the adaptive evolution of bats over a long period of time due to their special diets. The food composition of bats is complex, and insects are the food component for most bat species \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. According to the analyses of feces, the food composition of \u003cem\u003eV. sinensis\u003c/em\u003e is mainly Coleoptera and Lepidoptera insects, and these insects are often concentrated in large numbers in areas of cultivated land \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Insects may have eaten plants containing pesticides, and owing to bioaccumulation, the pesticides can be accumulated in the insects and then in the predatory \u003cem\u003eV. sinensis\u003c/em\u003e. Therefore, the physiological activity of drug metabolism in bats after feeding being higher than before eating is likely to be a self-protection mechanism for their bodies. In addition, our research also found that after bats feed, the pathways related to immune regulation in the brain are also enriched by genes with high expression levels and present an active state. We speculate that after bats feed, they also need to mobilize the immune regulation processes in the body to combat body damage caused by food ingredients and food contamination.\u003c/p\u003e \u003cp\u003eIn comparison of bats before and after feeding, various endocrine activity-related pathways were significantly enriched by high expression levels of genes and showed a high level of endocrine activity, for example, the renin-angiotensin system, renin secretion, and cortisol synthesis and secretion. Renin is a component of the renin-angiotensin system and can produce angiotensin II (AngII) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In the central nervous system, AngII can regulate insulin-stimulating and sodium appetite responses \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure, and it has a significant circadian rhythm \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Before feeding, \u003cem\u003eV. sinensis\u003c/em\u003e may actively adjust the blood pressure and blood sugar through various endocrine-related physiological processes to prepare for predation activities. This may also be the result of adaptive evolution in bats.\u003c/p\u003e \u003cp\u003eWe also found important genes that may regulate the circadian clock in the brain of \u003cem\u003eV. sinensis\u003c/em\u003e, and these genes had obvious characteristics of daily rhythmic expression. In the trend analysis module 6, 27 genes could be significantly clustered and showed similar expression patterns. The expression levels of these genes began to decrease in the after feeding phase (4:00), were the lowest in the sleep phase (10:00), and gradually increased in the awakening phase (16:00) to the activity phase (22:00), where the expression levels were highest. Combined with previous studies, it is not difficult to imagine that the changes in daily expression levels of these genes in the brain are similar to the circadian rhythms of other nocturnal rodents, but opposite to the circadian rhythms of diurnal mammals. Therefore, our hypothesis is that the trend changes of the genes in module 6 at the four time points are consistent with the biological habits of \u003cem\u003eV. sinensis\u003c/em\u003e that hide by day and come out at night. In addition, these genes were significantly enriched in cortisol synthesis and secretion pathways, aldosterone synthesis and secretion, and circadian rhythm pathways. Therefore, we can infer from these pathways that the physiological processes related to endocrine and circadian clock regulation in the brain of \u003cem\u003eV. sinensis\u003c/em\u003e have clear daily periodic changes that regulate the overall behavioral rhythms of bats.\u003c/p\u003e \u003cp\u003eIn addition, we found that \u003cem\u003ePer2\u003c/em\u003e may be an important circadian clock regulatory gene in the brain of \u003cem\u003eV. sinensis\u003c/em\u003e. The \u003cem\u003ePer2\u003c/em\u003e gene was differentially expressed in the sleep vs activity comparison, and the trend analysis results showed that the gene also has significant daily rhythmic expression characteristics. More importantly, the results of GO suggested that \u003cem\u003ePer2\u003c/em\u003e was significantly enriched with various terms related to circadian rhythms, including regulation of circadian rhythm, circadian regulation of gene expression, and entrainment of circadian clock by photoperiod, and the circadian rhythm and circadian rhythm-fly pathways were significantly enriched by \u003cem\u003ePer2\u003c/em\u003e in the KEGG results. Previous research demonstrated that \u003cem\u003ePer\u003c/em\u003e is an important core circadian clock gene, and it was the earliest circadian clock gene to be cloned in \u003cem\u003eDrosophila\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The mutation of \u003cem\u003ePer\u003c/em\u003e can cause changes in the rhythm of \u003cem\u003eDrosophila\u003c/em\u003e, so the gene was named \"Period\" \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e; the gene is involved in the regulation of the periodic expression of related genes in the brain. The stable expression of the \u003cem\u003ePer\u003c/em\u003e protein is also crucial for maintaining the length of the circadian rhythm. This study is the first to discover an important role of \u003cem\u003ePer2\u003c/em\u003e in regulating the biological rhythms and physiological processes in the brains of bats, revealing that bats may have the same molecular basis of circadian clock regulation as other nocturnal animals. As is well known, the molecular mechanism of the circadian clock is a complex network \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e; in addition to \u003cem\u003ePer\u003c/em\u003e, there are also core circadian clock genes such as \u003cem\u003eClock, Cry, Bmal1\u003c/em\u003e, and \u003cem\u003eReverb\u003c/em\u003e that participate in circadian clock regulation of mammals \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Although these core circadian clock genes were not detected in this study, this does not mean that these genes are not involved in the regulation of bat circadian rhythms. This needs to be further examined through detailed experiments and analysis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study is the first to explore the daily rhythms of gene expression in the bat brain. Various physiological processes in the bat brain area showed different degrees of circadian variation, including various endocrine hormones, signal pathways, and physiological processes related to the circadian clock, and transport activities may be related to predation activities. This study found that \u003cem\u003ePer2\u003c/em\u003e may be an important gene involved in the regulation of the circadian clock in the brain of \u003cem\u003eV. sinensis\u003c/em\u003e. In the future, a more detailed adaptive evolutionary analysis of this gene may be considered to clarify the position and role of this gene in the special circadian rhythms of bats. Although only four time points in the bat circadian rhythm were selected for analysis in this study, the results reflected the overall genetic basis of the circadian rhythms in the brain of \u003cem\u003eV. sinensis\u003c/em\u003e. In future research, we may consider adding more time points for sampling within a day and a multi-day setting to systematically reveal the molecular basis of bat circadian rhythms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this paper (and its Supplementary Information files).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the faculty and students of the Key Laboratory of Jilin Province for the conservation and utilization of animal resources and valuable assistance in the field. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. This work was supported by the National Natural Science Foundation of China (Grant Nos. 32001089 and 32071492).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.Z. and H.W. conceived and designed the study, analyzed the data with help from all authors, and wrote the article. Y.C. analyzed the data and provided great help to the sample collection. H.W. provides supervision during the entire project. J.L., L.F., H.W. and L.J. performed the sampling and assisted with interpretation of data. J.F. assisted with study design and provided laboratory space, reagents. All authors have read and approved the final article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChristie, A. 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A. \u003cem\u003eet al.\u003c/em\u003e The Gene Ontology (GO) database and informatics resource. \u003cem\u003eNucleic acids research\u003c/em\u003e, \u003cb\u003e32\u003c/b\u003e, D258\u0026ndash;261 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOgata, H. \u003cem\u003eet al.\u003c/em\u003e KEGG: Kyoto Encyclopedia of Genes and Genomes. \u003cem\u003eNucleic acids research\u003c/em\u003e, \u003cb\u003e27\u003c/b\u003e, 29\u0026ndash;34 (1999).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":" rhythms, biological, clock, signaling","lastPublishedDoi":"10.21203/rs.3.rs-951243/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-951243/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCircadian rhythms are found in almost all organisms, and they comprise one of the most basic characteristics of living things. Bats have attracted interest from researchers because of their unique biological rhythms. However, little is known about the molecular underpinnings of circadian signaling in bats. In this study, we used RNA-Seq to uncover the rhythms of expressed genes and biological processes in the brains of Asian particolored bats. Four collected time points corresponding to four biological states, rest, sleep, wakefulness, and active, were selected over the 24-hour day, and six comparisons of differentially expressed genes were obtained. Downstream functional enrichment analyses suggested that different physiological processes were active at various biological states, including drug metabolism, signaling pathways, and the circadian clock. Furthermore, trend analysis suggested that groups of genes are significantly clustered and are expressed at the lowest levels during the sleep state while being highly expressed during the active stat. In addition, we also identified the Per2 locus as an important circadian clock gene in the brain of Asian particolored bat. Our study provides a new overview of the molecular basis underlying the circadian clock in the brain of bats.\u003c/p\u003e","manuscriptTitle":"Comparative Analysis of The Daily Brain Transcriptomes of Asian Particolored Bat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-10-07 21:57:35","doi":"10.21203/rs.3.rs-951243/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-11-01T05:46:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-10-28T02:11:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"fb5bcf8e-09ce-4a2c-9d94-8bc9a6a302f5","date":"2021-10-21T05:24:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-10-21T04:18:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-10-21T04:16:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-10-06T04:49:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-10-06T04:34:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-09-30T08:29:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f13592b0-7be5-4689-9f30-20e7eb860a08","owner":[],"postedDate":"October 7th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":7693386,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2022-03-09T11:58:26+00:00","versionOfRecord":{"articleIdentity":"rs-951243","link":"https://doi.org/10.1038/s41598-022-07787-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2022-03-09 11:58:26","publishedOnDateReadable":"March 9th, 2022"},"versionCreatedAt":"2021-10-07 21:57:35","video":"","vorDoi":"10.1038/s41598-022-07787-z","vorDoiUrl":"https://doi.org/10.1038/s41598-022-07787-z","workflowStages":[]},"version":"v1","identity":"rs-951243","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-951243","identity":"rs-951243","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}