Strongylocentrotus intermedius, reached 80% in 2020. How sea urchins respond to high temperatures is of great concern to academia and industry. In this study, we examined the antioxidant enzyme activities of different color tube-footed sea urchins under heat stress and compared their transcriptome and microRNA (miRNA) profiles using RNA-Seq. The results showed that the antioxidant enzyme activities of sea urchins were altered by thermal stress, and the changes in peroxidase activities of red tube-footed sea urchins were particularly significant. Investigations revealed that 1,079 differentially expressed genes (DEGs), 11 DE miRNAs, and 104 “DE miRNA-DEG” pairs in total were detected in sea urchins under high temperature stress. Several mRNA and miRNAs were significantly changed , suggesting these genes and miRNAs exerted important functions in response to high temperature. At the transcriptional level, red tube-footed sea urchins were found to be more sensitive to high temperature and could respond to high temperature rapidly. DE miRNA-mRNA network showed that miR-92b-3p and PC-5p-7420 were the most corresponding miRNAs. Five mRNAs  may be potential markers of sea urchin response to high temperature. Possible interaction between miRNA-mRNA could be linked to protein folding in the endoplasmic reticulum, Phagosomes, and calcium transport. This study provides a theoretical basis for the molecular mechanism of sea urchin heat tolerance and information that will aid in the selection and breeding of sea urchins with high temperature tolerance.In the context of global warming and continuous high temperatures in the northern part of China during summer, the mortality rate of our main breeding species,  Global warming is profoundly affecting the nearshore and marine environments, resulting in environmental changes such as increased seawater surface temperature, acidification and hypoxia, which seriously threaten the population reproduction of marine organisms and the sustainable development of aquaculture . TemperaStrongylocentrotus intermedius is the major species of sea urchin cultured in China. However, it is a cold-water species and is sensitive to changes in temperature. The optimal water temperature for S. intermedius in its natural habitat is <20°C, but mortality can occur when the water temperature rises above 25°C , calmodulin-1 (Cam1), cyclophilins (Cyps) (Eriocheir sinensis) in terms of tolerance to nitrite stress. Meanwhile, several key genes involved in signal transduction, oxidative phosphorylation, and energy metabolism  were detected. In addition, several miRNAs mediating calmodulin-related genes  were identified, which suggested that the calcium signaling pathway is involved in the response to nitrite stress in crabs  . MicroRNs (Cyps) . Previous (Cyps) . Studyinin crabs . Qiang eS. intermedius under high temperature stress. We analyzed the physiological effects of heat shock on red and white tube-footed S. intermedius, detected and compared the dynamic changes of mRNAs and miRNAs in red and white tube-footed S. intermedius populations under high temperature stress, and provided clues to mRNA-miRNA interactions. These data help to understand the regulatory mechanism of S. intermedius on high temperature stress, provide information for screening molecular markers related to heat resistance and cultivating resistant sea urchins, and lay a foundation for healthy sea urchin farming.This study is the first report of mRNA-miRNA interactions in 1.5-year-old healthy sea urchins were purchased from Dalian Lushun Haibao Fishery Company  and immediately transferred to the Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture and Rural Affairs, P. R. China. Fifty healthy and active sea urchins with contrasting red and white tube feet of similar size  were selected for the experiment. Before conducting the experiments, the sea urchins were acclimatized in two 0.5 m³ temperature-independent recirculating pools  for 1 week. During the acclimation period, the sea urchins were fed fresh kelp daily, and the water was changed by 1/2, continuously aerated. After 48 h of fasting, the red and white tube-footed sea urchin population was randomly divided into 4 groups, namely, red tube-footed sea urchin control group (NR), white tube-footed sea urchin control group (NW), red tube-footed sea urchin high temperature group (HR), and white tube-footed sea urchin high temperature group (HW). The experimental design is shown in The control groups were the same as the acclimation groups, 15°C, and the experimental groups were 25°C (warmed at a rate of 2°C per hour), which was the lethal temperature for intermediate spherical sea urchins in our previous study. After reaching the temperature and maintaining it for 7 d, all sea urchins in each group were counted and individually weighed. Then, coelomic fluid of 15 randomly selected sea urchins from each group was extracted using 1 ml sterilized syringes. The upper fluid was obtained from the coelomic fluid by centrifugation  for determining the activities of antioxidant enzymes. The lower precipitates were used for RNA extraction. All samples were frozen in liquid nitrogen and stored at −80°C.Three sea urchin coelomic fluids were randomly selected from each group to detect antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione peroxidase (GSH-Px), according to the instructions in the kit. All antioxidant enzyme test kits were purchased from the Nanjing Jiancheng Biological Engineering Institute .Three coelomocyte samples were selected from every group of sea urchins. The extraction, quality testing, and purification of total RNA were performed according to Wang’s method . High puhttp://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression level for mRNAs by calculating FPKM  × exon_length (kB)], command line: ∼stringtie -e -B -p 4 -G merged. gtf -o samples. gtf samples. bam).To explore the changes in gene expression profiles of different colored tube-footed sea urchin populations under high-temperature stress, we obtained high-quality clean data by removing missing bases and low-quality bases  , mapped p-value using the Benjamini Hochberg’s method. However, since very few genes were screened with FDR in this study, in order to expand the number of differential genes and find more differentially expressed genes, p ≤ 0.05 and |log2FoldChange| >1 were considered as the threshold to determine DEGs. All the DEGs were annotated according to GO (GOseq 1.10.0 method) and KEGG (KOBAS 2.0.12 method)  was obtained by correcting the  method) . p < 0.0http://microrna.sanger.ac.uk) to identify miRNAs and examine their variations.The small RNA (sRNA) libraries were constructed with TruSeq small RNA sample prep kits  and sequenced using Illumina HiSeq2500 . The obtained sequences with 18–25 nt were used to perform BLAST analysis in miR Base 22.0 database (p < 0.05 was considered significant. Target genes of miRNAs were predicated by using Target Finder (http://www.targetfinder.org) and were subjected to the enrichment analysis of functions and pathways by GO and KEGG database .To identify DE miRNAs between treatment group  and control group , the expression levels of miRNAs in the whole library were normalized, and then DE miRNAs were screened using Student’s T-test. http://www.cytoscape.org/).All possible positively and negatively correlated miRNA-mRNA pairs were predicted using ACGT101-CORR1.1. Based on the comprehensive analysis of DEG and DE miRNAs, we screened the negatively associated DE miRNA-mRNA pairs and constructed interaction networks using Cytoscape software  of each primer. The reaction conditions were followed by 40 cycles of 95°C for 30 s, 95°C for 5 s, 60°C for 32 s, 95°C for 15 s, 60°C for 60 s, 95°C for 15 s and 60°C for 15 s. Compared with the control gene, fold-change of expression levels was determined using 2−ΔΔCT method (p < 0.05) for each variable were first detected using the one-way ANOVA test between different groups, followed by Tukey’s HSD test.To validate the results of RNA-seq and miRNA-seq, 10 DEGs and 10 DE miRNAs were randomly selected for real-time fuorescence quantitative PCR (qRT-PCR). Total RNA extraction and reverse transcription were performed by referring to T method , both inS. intermedius showed almost no change, whereas superoxide dismutase (SOD) and peroxidase (POD) expressions both increased significantly (p < 0.05), with greater changes in SOD in the white tube-footed S. intermedius and greater changes in POD in the red tube-footed S. intermedius. There was no significant change in glutathione peroxidase (GSH-PX) activity in the coelomic fluid of red tube-footed S. intermedius (p > 0.05), while the GSH-PX activity in the coelomic fluid of white tube-footed S. intermedius showed an increasing trend  expression in the coelomic fluid of ng trend .Twelve mRNA libraries were built and sequenced, six from 15°C control groups  and six from 25°C treatment groups . The raw reads ranged in number from 38,812,224 to 56,834,206 (Bioproject ID: PRJNA783358). After quality control, approximately 80.26–97.57% of raw reads remained as clean reads . The mapS. intermedius in the normal group at 15°C  and under high temperature stress at 25°C , gene expression comparisons between control and treatment groups were undertaken.On the basis of the sequencing data of red and white tube-footed p < 0.05). The most enriched subcategories in CC were “integral component of membrane”, “membrane” and “extracellular space” (p < 0.05). The most common subcategories in MF were “calcium ion binding”, “serine-type endopeptidase activity” and “GTP binding”. The findings of the GO enrichment analyses are displayed in In the HR vs. NR group comparison, 305 DEGs  were detected , of whicp < 0.05). Different from the HR vs. NR group, more basal metabolism-related pathways were annotated, such as “one carbon pool by folate (ko00670)”, “glyoxylate and dicarboxylate metabolism (ko00630)”, and “linoleic acid metabolism (ko00591)”  were detected, of which 296 DEGs were annotated in 1059 GO terms. The GO entries that are consistent with the HR vs. NR group were “microtubule-based process”, “protein folding” and “transport”; “extracellular space”, “extracellular exosome”, “endoplasmic reticulum”; “calcium ion binding”, “structural constituent of cytoskeleton” and so on. However, the difference was that “endoplasmic reticulum stress”, “negative regulation of transcription” and “DNA-templated” were found among BP and “RNA binding”, “nuclide acid binding” and other structure-related terms were found in MF, while the DEGs were not annotated to the cell membrane entries in CC . KEGG eno00591)” .In the HR vs. HW group comparison, we detected 367 DEGs  of which there were 271 DEGs annotated to 851 GO terms. The most significantly enriched subcategories in BP, CC and MF were “microtubule-based process”, “integral component of membrane” and “calcium ion binding”, respectively . KEGG anp < 0.05). The most enriched subcategories in CC were “alpha DNA polymerase: primase complex”, “dense body” and “receptor complex” (p < 0.05). The most common subcategories in MF were “calcium ion binding”, “Hsp70 protein binding”, “transporter activity”, “aspartic-type endopeptidase activity” and “cell adhesion molecule binding” (p < 0.05) (p < 0.05), namely “phagosome”, “lysosome”, “other glycan degradation”, “notch signaling pathway”, “autoimmune thyroid disease”, “small cell lung cancer”, “thyroid hormone synthesis”, and “MicroRNAs in cancer”  were detected, of which 124 DEGs were annotated in 460 GO terms. “cell division”, “nucleosome assembly”, and “regulation of calcium ion-dependent exocytosis” were the most enriched subcategories under the BP ( < 0.05) . KEGG en cancer” .p < 0.05) (p < 0.05) . GO enrichment analysis revealed 76 genes annotated to 337 GO terms, among which “protein folding”, “innate immune response”, “response to endoplasmic reticulum stress” were significantly enriched in BP; “endoplasmic reticulum”, “extracellular space”, “extracellular region” were significantly enriched in CC; “metal ion binding”, “protein binding”, “unfolded protein binding” were significantly enriched in MF ( < 0.05) . KEGG an < 0.05) . The sig < 0.05) .p < 0.05) (p < 0.05) . GO enrichment analysis revealed 506 genes annotated to 1401 GO terms, among which “innate immune response”, “receptor-mediated endocytosis”, “vesicle fusion” and “multicellular organism development” were significantly enriched in BP; “membrane”, “extracellular exosome”, and “extracellular space” were significantly enriched in CC; “calcium ion binding”, “ubiquitin-protein transferase activity”, and “chromatin binding” were significantly enriched in MF ( < 0.05) . KEGG an < 0.05) . The sig < 0.05) .S. intermedius to identify the miRNAs involved in heat stress. The numbers of raw reads of these 12 transcriptomes ranged from 10 to 30 million (Bioproject ID: PRJNA783268). After filtering for reads with 3′ adapters and length filters, junk reads, Rfam, mRNA, repeats, rRNA, tRNA, snRNA and snoRNA sequences, as well as other Rfam RNA sequences (see Methods), we obtained 124,484 to 704,950 unique sequences from 12 miRNA libraries  from ibraries . Most ofibraries .Based on bioinformatic analysis, 671 miRNAs were identified; 195 known miRNAs and 476 novel miRNAs were expressed in all samples . By co-ep < 0.05). The top 10 significantly enriched GO terms  of the three categories are shown in To explore DE miRNAs in the different comparison groups and further understand the regulatory functions of the miRNAs, we screened DE miRNAs and performed GO and KEGG enrichment analysis on the predicted target genes. A total of 2,543, 1,190, 1744, 984, 3,614, and 5,345 target genes were predicted for the six, two, three, one, five, and ten DE miRNAs identified from the six comparison groups, respectively , and GO p < 0.05). Among them, more target genes were enriched in the classes of cellular processes, environmental information processing and metabolism. KEGG pathway analysis based on all of the predicted target genes revealed that DE miRNAs were involved in several pathways relevant to sea urchins exposed to high temperature, including protein processing in the endoplasmic reticulum (ko04144) and ubiquitin mediated proteolysis (ko04120), which are related to protein synthesis, and amino sugar and nucleotide sugar metabolism (ko00520) and alanine, aspartate and glutamate metabolism (ko00250), which are involved in basic metabolism. Furthermore, several immune-related pathways have also been discovered, including phagosome (ko04145), endocytosis (ko04144), autophagy (ko04140), mTOR signaling (ko04150) and FoxO signaling (ko04086)  .Combining the results for the DE miRNAs target genes and DEGs, we found that 5 DE miRNAs may have targeted regulatory relationships with 28 DEGs in the HR vs. NR group. Among these, 35 “DE miRNA-DEG” pairs exhibited a negative expression correlation, whereas 17 “DE miRNA-DEG” pairs showed a positive expression correlation. In the HW vs. NW group, we found that 1 DE miRNA may have targeted regulatory relationships with 20 DEGs. Among these, a negative expression correlation was found for 5 “DE miRNA-DEG” combinations, while a positive expression correlation was found for 15 “DE miRNA-DEG” pairs. In the HR vs. HW group, 3 DE miRNAs may have targeted regulatory connections with 27 DEGs. There was a negative expression correlation for 19 “DE miRNA-DEG” pairs, whereas there was a positive expression correlation for 8 “DE miRNA-DEG” pairs. In the NR vs. NW group, 1 DE miRNAs may have targeted regulatory connections with 10 DEGs. Among these, a negative expression correlation was found for 2 “DE miRNA-DEG” combinations, while a positive expression correlation was found for 8 “DE miRNA-DEG” pairs. In the HR vs. NW group, 5 DE miRNAs may have targeted regulatory connections with 16 DEGs. There was a negative expression correlation for 17 “DE miRNA-DEG” pairs, whereas there was a positive expression correlation for 18 “DE miRNA-DEG” pairs. In the HW vs. NR group, 10 DE miRNAs may have targeted regulatory connections with 107 DEGs. There was a negative expression correlation for 94 “DE miRNA-DEG” pairs, whereas there was a positive expression correlation for 70 “DE miRNA-DEG” pairs. This study focused on the response of sea urchins to high temperature, so we constructed mRNA-miRNA network maps for HR vs. NR, HW vs. NW, HR vs. HW groups. .CALR " and “phagosome (ko04145)" pathways, we used Cytoscape software to map the network of DE miRNAs and targeted DEGs of these two pathways and found that miR-92b-3p and PC-5p-7420 were the most connected miRNAs, and lva-miR-92b-3p_R+2 and pmi-miR-92c-3p_R+1_1ss10TC co-regulate CALR .HSP70, DnaJ11, and CALR) and 9 DE miRNAs were selected for the qRT-PCR measurements. The results of qRT-PCR analysis coincided with the results generated from RNA-Seq and showed similar expression trends to those of the RNA-Seq data  prevails in deep-water settlements (15–25 m) and the “usual” (U) form that mostly occurs in shallow-water settlements (5–10 m)  . The milp < 0.05). This finding is consistent with the study of S. intermedius with different colored tube feet, and our results confirmed the argument that the SOD activity of red tube-footed sea urchins responded to high temperature earlier than that of white tube-footed sea urchins. Meanwhile, we found no significant difference in CAT activity of the sea urchins under high temperature stress, suggesting that sea urchins use SOD more to regulate their ability to scavenge free radicals, or perhaps that enzyme activity is chronological and the magnitude of enzyme activity is related to sampling time. Therefore, we suggested that at the physiological-ecological level, red tube-footed sea urchins are more sensitive to high temperatures and have the potential to tolerate high temperatures.In response to environmental stress, organisms usually activate humoral immunity and alter their antioxidant enzyme activity to buffer the effects of temperature changes on the organism . The antS. intermedius with different tube foot colors were constructed in this study. Compared to previous studies . Heat shock proteins are chaperone proteins that promote the degradation of abnormal proteins or reactivate stress-damaged proteins by promoting the proper refolding of denatured proteins  with differential expression and that HSP70 expression increases and then decreases with stress time (GRP78 (glucose-regulated protein 78 kDa) is a member of the HSP70 family, and it is a key indicator of endoplasmic reticulum stress (CALR) and protein disulfide isomerase (PDI) were also shown to be significantly up-regulated. CALR is a Ca2+-binding protein that regulates intracellular Ca2+ dynamic homeostasis and signaling activities such as the UPR and related protein degradation pathways, as well as regulating the immunological response, shielding cells from stress and regulating the cell cycle  in this study, and the two had a negative regulatory connection. When cells are stressed by the external environment , HYOU1, as an endoplasmic reticulum molecular chaperone, protects and maintains cell viability .Since transcriptional attenuation of target genes is a conserved mechanism of miRNA regulation, we only focused on “DEG-DE miRNA” pairs with opposite relative expression trends in this study. After screening, we found that these “DEG-DE miRNA” pairs may explain the superiority of energy metabolism and immunity of red tube-footed sea urchins in response to high temperature stress. It has been shown that miR-184 is an important regulator of stem cell proliferation and growth , and thain vitro . MiR-184and VEGF . The tariability . Therefoiability . Membersiability . Additioiability . Yang etfections . Lv et aucumbers . MiR-92csplicing . Non-neusplicing . We hypom stress . Our finm stress . The resTo research the molecular processes involved in the sea urchin response to high thermal stress, we investigated gene expression differences and miRNA profiles of red and white tube-footed sea urchins under high temperature stress conditions. According to previous studies and miRNA-mRNA integration analysis, protein folding of the endoplasmic reticulum and phagosomes are the main pathways for sea urchins to respond to high temperature stress, and red tube-footed sea urchins may be more sensitive to high temperature and respond more quickly to high temperature stress. The red tube-footed sea urchin may in fact be more sensitive to high temperatures than other sea urchins and respond faster to heat stress. Red tube-footed sea urchins and “DEG-DE miRNA” pairs involved in protein processing of the endoplasmic reticulum may be considered as germplasm resources and molecular markers for future selective breeding.


Medicago sativa L.). There is growing evidence that enhancing freezing tolerance through resistance-related genes is one of the most efficient methods for solving this problem, whereas little is known about the complex regulatory mechanism of freezing stress. Herein, we performed transcriptome profiling of the leaves from two genotypes of alfalfa, freezing tolerance “Gannong NO.3” and freezing-sensitive “WL326GZ” exposure to −10°C to investigate which resistance-related genes could improve the freezing tolerance. Our results showed that a total of 121,366 genes were identified, and there were 7,245 differentially expressed genes (DEGs) between the control and treated leaves. In particular, the DEGs in “Gannong NO.3” were mainly enriched in the metabolic pathways and biosynthesis of secondary metabolites, and most of the DEGs in “WL326GZ” were enriched in the metabolic pathways, the biosynthesis of secondary metabolites, and plant-pathogen interactions. Moreover, the weighted gene co-expression network analysis (WGCNA) showed that ATP-binding cassette (ABC) C subfamily genes were strongly impacted by freezing stress, indicating that ABCC8 and ABCC3 are critical to develop the freezing tolerance. Moreover, our data revealed that numerous Ca2+ signal transduction and CBF/DREB1 pathway-related genes were severely impacted by the freezing resistance, which is believed to alleviate the damage caused by freezing stress. Altogether, these findings contribute the comprehensive information to understand the molecular mechanism of alfalfa adaptation to freezing stress and further provide functional candidate genes that can adapt to abiotic stress.Freezing stress is a major limiting environmental factor that affects the productivity and distribution of alfalfa ( Medicago sativa L.) is one of the most important cultivated perennial forage legume species in the world  initially causes ice formation in the cell wall, which directly affects cellular metabolic activities. However, as the ice crystals grow, water uptake by the call is reduced leading to cellular acute dehydration and severe damage to the cell membrane . Oxidati2+) in the cytosol  cascades and phytohormones also play a key role in the freezing signal process  is an important signal transduction pathway involved in freezing tolerance mechanism. CBFs, including CBF1, CBF2, and CBF3, are the AP2/ERF family transcription factors, which regulate COR genes to confer freezing stress. Accordingly, overexpressed CBFs increase the freezing tolerance in diverse plant species . In Arabolerance , and oveuncatula . MoreoveRecently, with the development of high-throughput RNA-sequencing (RNA-seq) technology applications, the mechanisms of freezing stress response in numerous plants have been revealed, and thousands of freezing tolerance-related genes are involved in signal transduction. These studies suggest that transcription factors regulate gene expression to defend against freezing stress . However−2 s−1, a light period of 14 h at 20°C, with 10 h dark at 18°C, and humidity ranging from 60 to 80%. The seedlings were irrigated with half-strength Hoagland solution daily after the seeds germinated. Four weeks later, the seedlings were transferred to a cold chamber at −10°C. All leaves were harvested at 0, 0.5, 1, and 2 h after freezing stress. Detailed sample names are as follows: G01-03, G11-13, G21-23, and G31-33 represent three “Gannong NO.3” samples harvested at 0, 0.5, 1, and 2 h, respectively; W01-03, W11-13, W21-23, and W31-33 represent three “WL326GZ” samples harvested at 0, 0.5, 1, and 2 h, respectively. Thus, every sample had three replicates, and the leaves were frozen in liquid nitrogen and stored at −80°C until use.Two freezing tolerance alfalfa cultivars were studied. The first was the freezing-tolerance cultivar “Gannong NO.3” , which is produced in Gansu, Ningxia, Xinjiang, and eastern margin of the Qinghai-Tibet plateau. Its seeds were provided by Gansu Agricultural University. The second was the freezing-sensitive “WL326GZ” , which was purchased from Zhengdao Ecological Technology Co. . All seeds were sown on arenaceous quartz in a growth room, and each sample with three times. The growth conditions were as follows: 200 μmol m−1with 4°C for obtaining the upper supernatant to detect the antioxidant activities. The SOD activity was measured according to method reported by 2O2 , we used 0.1 g fresh leaves which were homogenized in 1.5 ml potassium phosphate buffer  and centrifuged for 20 min at 12,000 r mind by 2O2 .Total RNA was isolated from leaf tissues using the RNAprep Pure Plant Kit  according to the manufacturer’s instructions and then treated with DNase I (NEB) to remove DNA. Quantification was performed using a Qubit RNA Assay Kit and a Qubit 2.0 Fluorimeter . To ensure the integrity of the RNA, we used an RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system  to library construction and sequencing.M. sativa L. reference genomeTwenty-four libraries were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina . Sequences were conducted by the Biomarker Technology Company  on the Illumina Hiseq™ 4,000 platform . The raw reads were first processed through in-house Perl scripts, and clean reads were obtained by moving adapter ploy-N and low-quality reads. The clean reads were mapped to the ribosome RNA (rRNA) database using the short reads alignment tool Bowtie2 (version 2.2.8), the rRNA mapped reads were removed, and the remaining clean reads were mapped to the 2 (fold change) | ≥1 as a significantly differential expression.Differentially expressed genes (DEGs) were analyzed using pairwise samples  by DEseq R package (1.18.0). The expression levels were adjusted with FDR < 0.05, and |log2 (fold change)| ≥1, p ≤ 0.05, and profile number ≤20. Then, the DEGs of each profile were annotated using the Gene Ontology (GO) and KEGG databases analysis using a Q ≤ 0.05.To under the DEG trends, the DEGs of each variety at each sampling time were clustered by similar expression patterns. Briefly, the expression of each DEG was input, normalized, and clustered using Short Time-series Expression Miner software (STEM) with |logThe co-expression network was performed using the weighted gene co-expression network analysis (WGCNA) in the R software package . A totalTo distinguish the two different freezing tolerance types of alfalfa, we analyzed the antioxidant enzymes and phenotypic changes in the leaves following exposure to freezing stress for 0, 0.5, 1, and 2 h. The alfalfas in “Gannong NO.3” were survived more than in the “WL326GZ” under the direct exposure to −10°C temperatures for 2 h . To furtIn order to detect the effects on the freezing stress, we analyze the SOD, POD, APX, and CAT activities of the alfalfas. As shown in To investigate which resistance-related genes can be beneficial to improve the freezing stress, we used RNA-seq approach to profile the leaves of “Gannong NO.3” and “WL326GZ” at 0, 0.5, 1, and 2 h exposure to −10°C. Our preliminary RNA-seq screen generated 155,133,490,050 raw data points. The raw data from each sample consisted of more than 59,000,000 data points, and all raw data were deposited in NCBI with the accession number PRJNA769225. Importantly, we cleared the raw data with GC < 43%, Q20 > 97%, and Q30 > 94%, and finally obtained 153,834,608,062 data points . To remoWe then constructed a comparative analysis between freezing stress treated and control leaves of “Gannong NO.3” or “WL326GZ.” There were 7,245 DEGs that showed significant upregulation/downregulation, among that, the G0 vs. G2 and W0 vs. W2 displayed 3,892 DEGs and 2,608 DEGs, respectively .To cluster the same trend expression genes, 20 profiles were generated using the trend analysis of “Gannong NO.3” and the top 6 profiles of the gene numbers were, 8, 11, 16, 18, 3, and 1 . We thenSimilar to “Gannong NO.3.,” there were 20 profiles were produced for “WL326GZ” by trend analyzed and the top 6 profiles of the DEGs were, 18, 11, 9, 10, 0, and 8 . The resTo construct the gene co-expression network of RNA-seq, we obtained 23,977 genes from the clean data for WGCNA. As shown in To identify the different reactions between “Gannong NO.3” and “WL326GZ” to the abrupt freezing stress, we obtained two modules with opposite expression patterns in those modules at different times . ObviousTo analyze the biological function of the genes from dark red and midnight blue modules, we utilized GO enrichment and KEGG pathways to classify the genes. The results of top 20 GO showed that the genes in the dark red  module w2 (fold change)| ≥1 and Q < 0.05 to screen the DEGs. There were 7,245 DEGs identified in ABCC8 [ATP-binding cassette (ABC) transporter C family member 8], which belongs to the ABC transporter C subfamily. MS.gene68529 , as another important candidate hub gene, also belongs to the ABC transporter C family, which indicates that the ABC transporter C subfamily may play an important role in freezing stress. However, there were only 80 DEGs identified between the genes of entire set and the midnight blue module  to confer freezing stress.Freezing stress could cause osmotic stress in plant cells and rapidly trigger Caxpressed . In thisxpressed . FurtherCBF1/DREB1B, CBF2/DREB1C, and CBF3/DREB1A , which damages the cell membrane and increases lipid peroxidation in plants. Plants activate antioxidant defense systems, including SOD, CAT, POD, and APX, to alleviate the damage caused by ROS . It is dTo explore the changes in abrupt freezing stress-related gene expression in alfalfa cultivars, we transcribed RNA from alfalfa leaves which exposure to −10°C for 0, 0.5, 1, and 2 h. In this study, we obtained 121,366 genes by RNA-seq based on the alfalfa reference genome. Our transcription assembly was more efficient than that of other alfalfa studies . FurtherCaenorhabditis elegans, which have been previously found in insects  participates in the biosynthesis of anthocyanin  act as the second messenger involved in a wide range of biological processes in eukaryotic cells and plays a key role in counteracting cold stress. Interestingly, in alfalfa, 2+ channels and elevates the concentration of Ca2+in the cytosol, which stimulates expression of cold-induced genes to resist stress. Similar Ca2+ signals were induced in this study. Ca2+ signals are relayed or respond to Ca2+ sensor proteins , CBF2 (DREB1C), and CBF3 (DREB1A) were rapidly induced under cold stress, and overexpression of CBF1, CBF2, and CBF3 increased freezing tolerance after exposure to freezing shock stress (CBF/DREB1 belongs to the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor family , which ik stress . Moreovek stress . In thisk stress . This dyk stress . The expk stress . This inABCC8 and ABCC3 are the key genes in counteracting freezing stress, and further studies should explore the exact functions of these genes. A simple working module is illustrated in 2+ signal transduction and CBF/DREB1 related genes demonstrate their critical role in counteracting freezing stress. Additionally, they may be associated with the higher antioxidant enzyme activities  in “Gannong NO.3” than in “WL326GZ”  can be found in the article/JM and SS conceived and designed the research. XW, WK, and FW conducted the experiment. XW and JM analyzed the data and wrote the manuscript. WK, FW, and SS finalized the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Fostering Foundation for the Excellent Ph.D. Dissertation of Gansu Agricultural University (YB2019001), Major Special Science and Technology Projects of Gansu Province: Germplasm Innovation and Breeding of Alfalfa and Oat (19ZD2NA002-3), and Discipline Construction Fund Project of Gansu Agricultural University (GAU-XKJS-2018-002).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In addition, the IEVMT thin film demonstrates good mechanical and thermal stability with a low coefficient of friction of 0.51 and a low thermal conductivity of 3.9 × 10−3 W·m−1·K−1. This reveals that reducing the thickness and utilizing the framework is effective in increasing the ionic conductivity and provides a promising stable and low-cost candidate for high-performance solid electrolytes.Ceramic electrolytes hold application prospects in all-solid-state lithium batteries (ASSLB). However, the ionic conductivity of ceramic electrolytes is limited by their large thickness and intrinsic resistance. To cope with this challenge, a two-dimensional (2D) vermiculite film has been successfully prepared by self-assembling expanded vermiculite nanosheets. The raw vermiculite mineral is first exfoliated to thin sheets of several atomic layers with about 1.2 nm interlayer channels by a thermal expansion and ionic exchanging treatment. Then, through vacuum filtration, the ion-exchanged expanded vermiculite (IEVMT) sheets can be assembled into thin films with a controllable thickness. Benefiting from the thin thickness and naturally lamellar framework, the as-prepared IEVMT thin film exhibits excellent ionic conductivity of 0.310 S·cm Therεr) of IEVMT film shows a peak at around 90 °C which refers to the ferroelectric-to-paraelectric phase transition temperature. The maximum dielectric constant is about 6.31 around 90 °C at 1 kHz and the minimum value is 2.25 at 10 MHz. The dielectric loss at a low frequency usually depends on the grain boundary polarization, and then dipolar rotation takes effect in the high-frequency range [εr at lower frequencies is mainly due to the block of electrode-electrolyte polarization at the interface, while the rotation of carriers lags behind the change in electric field, resulting in the reduction in dielectric constant at a high-frequency range. Moreover, the enhanced Li+ ion mobility through the wide gap between IEVMT sheets under thermal activation may also lower the dielectric constant [δ, −2–10−1) over a large range of temperatures, demonstrating that the increase in interlayer spacing reduces the friction between dipole molecules and therefore the polarization loss.For superior ceramic electrolytes, a lower dielectric constant and dielectric loss is also essential. The dielectric constant and loss of IEVMT film were then tested and presented in cy range . Thus, tconstant ,51. Surp2, have excellent wear resistance properties and demonstrate application prospects in electrical contact materials [2 [2/Cu-Zn composite [2·s−1 at 500 °C. According to the calculation formula for thermal conductivity k = α × Cp × ρ, where k, α, Cp, and ρ represent the thermal conductivity, thermal diffusivity, heat capacity, and density, respectively, the k of IEVMT film is 3.9 × 10−3 W·m−1·K−1. The low thermal conductivity further facilities the application of IEVMT films in middle- and high-temperature environments. Therefore, the good anti-wear and thermal resistance properties of IEVMT films provide desirable stability and durability, ensuring the safety of long-term use.The stability of ceramic electrolytes, meanwhile, will also be a prime concern. Usually, organic materials and polymers, such as rubber, have excellent wear resistance properties. Beyond that, 2D materials, such as graphene and MoSaterials . Owing taterials  and muchaterials , TiB2 [5rials [2 , Mica [5rials [2 , montmorrials [2 , MoS2/Cuomposite , and graomposite . Moreove−1 is achieved by the resulting IEVMT thin films. Simultaneously, a lower dielectric constant and dielectric loss are observed in the IEVMT films. Moreover, the IEVMT films exhibit a coefficient of friction of 0.51, which is lower than various 2D materials, and a low thermal conductivity of 3.9 × 10−3 W·m−1·K−1, demonstrating excellent stability. Combining the structure and properties analyses, the rigid nanosheets of vermiculites ensure stability, while the thin thickness and interlayer channel contribute to improved ionic conductivity. This superior comprehensive performance endows the IEVMT films with wide application prospects in medium- and high-temperature solid electrolytes.In this work, a smooth, flexible, and free-standing thin IEVMT film is successfully prepared by using natural vermiculite minerals as starting materials, which provides a promising solution for the limitation of thickness and intrinsic resistance of ceramic electrolytes. A simple procedure, including thermal expansion, ion exchange, and vacuum filtration, is designed to exfoliate the raw vermiculite. The raw vermiculite mineral is first expanded by rapid heat treatment, in which the fast evaporation of internal water reduces the strong combination force between layers and expands the interlayer space. Followed by a two-step ionic exchange process, the vermiculites further exfoliate into thin nanosheets with one or two atomic layers. Then, through vacuum filtration, the IEVMT thin film is self-assembled by the above-exfoliated vermiculite sheets to 30 μm thin ceramic films, dramatically reducing the thickness of ceramic electrolytes. The detailed structure and morphology characterization confirms that single-layer vermiculite frameworks are obtained with a 1.2 nm interlayer channel, which can provide the transport channel for lithium ions and decrease the intrinsic resistance. High ionic conductivity of 0.31 S·cm


The WT and transgenic tobacco plants were evaluated for photosynthetic performance to determine the maximum rate of carboxylation , maximum rate of electron transport (Jmax), the photosynthetic compensation point (Γ*), quantum yield of PSII (ΦPSII), and mesophyll conductance (gm). Additionally, all plants were harvested to compare differences in above-ground biomass. Overall, transformants did not perform better than the WT on photosynthesis-, biomass-, and leaf composition-related traits. This is in contrast to previous studies that have suggested significant increases in photosynthesis and yield with the overexpression of ictB, although not widely evaluated under field conditions. These findings suggest that the benefit of ictB is not universal and may only be seen under certain growth conditions. While there is certainly still potential benefit to utilizing ictB in the future, further effort must be concentrated on understanding the underlying function of the gene and in which environmental conditions it offers the greatest benefit to crop performance. As it stands at present, it is possible that ictB overexpression may be largely favorable in controlled environments, such as greenhouses.In this study, four tobacco transformants overexpressing the inorganic carbon transporter B gene ( By the year 2050 it is projected that global food supply will need to increase by 50–85% to keep pace with a growing human population and shifting dietary preferences with greater emphasis on the consumption of animal products  is a highly conserved gene among cyanobacteria that was proposed to be involved in inorganic carbon accumulation in Synechococcus PCC 7942  than the wild type (WT) , the maximum rate of electron transport (Jmax), leaf CO2 uptake rate (A), and stomatal conductance (gsw) (ictB expression may help boost photosynthetic performance in field conditions. Paddy-grown rice expressing ictB had significantly (10.5%) higher mesophyll conductance (gm) and 13.5% higher A compared with the WT  assess the potential of ictB to improve water use efficiency in rain-fed field conditions. We subsequently discuss why benefits might be seen in greenhouses and controlled environments for ictB transformants but not in field trials.However, these gains may not always translate when grown in field conditions where improvements to crops would be most relevant towards improving food production. Indeed, previous studies have shown that ictB single construct was placed in the tobacco (Nicotiana tabacum) cv. Samsun background (–2 s–1) were measured throughout the field season  were produced at the University of Essex where the ckground . Temperad season . Once ind season . Measure2 uptake and modulated chlorophyll fluorescence were measured on the youngest fully expanded leaves using portable open gas exchange systems incorporating CO2 and water vapor infra-red gas analyzers . Light was provided through an integrated LED light source and modulated fluorometer, incorporated into the head of the temperature- and humidity-controlled leaf measurement chamber .Leaf CO2 uptake  to intercellular CO2 concentration  and to photosynthetic photon flux density (PPFD) was measured twice during the experiment. Response curves were performed 47 d after sowing (from 24 to 27 August 2020) and once again later in development at 61 d after sowing (from 7 to 10 September 2020). Response curves were measured for each genotype once per each block (n=12).The response of COA to Ci (A/Ci curves), photosynthesis was measured at saturating light (2000 µmol m–2 s–1) and CO2 concentrations in the following order: 400, 250, 150, 100, 50, 400, 550, 700, 900, 1100, 1300, and 1500 µmol mol–1. Additionally, the block temperature was set at 28 °C, the average relative humidity was between 66% and 77%, and the vapor pressure deficit (VPD) at leaf temperature was between 0.79 kPa and 1.74 kPa. The gas exchange systems were matched before each curve, and steady-state fluorescence (Fs) and maximal light-adapted fluorescence (Fmʹ) were recorded at each measured Ci.To examine the response of Vc, max (µmol m–2 s–1) and apparent Jmax (µmol m–2 s–1) were calculated utilizing the equations from Vc, max and apparent Jmax at 28 °C. The ‘apparent’ term is used because the parameters are based on Ci instead of CO2 concentration inside the chloroplast (Cc). The photorespiratory CO2 compensation point , carboxylation efficiency , and the maximum rate of CO2 uptake in saturating light and CO2  were calculated from the A/Ci curves that were fitted at 28 °C. CE was the initial slope of curves with Ci ≤250 µmol mol–1.The apparent J method to calculate gm (mol m–2 s–1) (Cc (µmol mol–1), Vc, max, and Jmax. For this analysis, the scaling constant (c) and the enthalpies of activation (ΔHa) to calculate the Michaelis constant of Rubisco for CO2 , the inhibition constant , and Γ* at 25 °C were taken from Vc, max, Jmax, and gm were adjusted to 28 °C using the equations in A non-linear analysis with the Marquardt method  for A/Q curves) were measured at ambient [CO2] (400 µmol mol–1) and the following PPFDs: 2000, 1700, 1400, 1100, 800, 600, 425, 250, 150, 100, 50, and 0 µmol m–2 s–1. The gas exchange systems were matched before each curve, and Fs and Fmʹ were recorded at each PPFD. The A/Q curves were fitted for quantum efficiency (ΦPSII), leaf CO2 uptake in saturated light , and light compensation point utilizing the {photosynthesis} R-package (Light response curves (n=12/genotype/time point). Within the cuvette, the flow rate was 500 µmol s–1, [CO2] was maintained at 400 µmol mol–1, relative humidity was maintained at 70%, and actinic PPFD was 10% blue light. The PPFD and block temperature were changed at each time point to reflect ambient conditions throughout the day. The gas exchange systems were matched before each time point measurement, and Fs and Fmʹ were logged. The parameters of A, stomatal conductance , Ci, and intrinsic water use efficiency  throughout the course of a day were obtained from these data.Diurnal measurements were made every 2 h on 3 September 2020, from 08.00 h to 18.00 h. On this day, sunrise was at ~06.23 h, while sunset was at ~19.20 h. One plant of each genotype was measured in each of the 12 blocks per time point  from one plant per tobacco genotype per block (n=12 per genotype). After the samples were ground, total RNA and protein were extracted from the same leaf discs using the NucleoSpin RNA/Protein Kit . Once the protocol was completed, the RNA concentration was diluted to 200 ng μl–1.Leaf discs were collected into liquid NictB was normalized with the values obtained for the housekeeping gene for protein phosphatase 2A  primer (Invitrogene) according to the protocol in the RevertAid Reverse Transcriptase kit . The cDNA was diluted 10 times. For semi-quantitative reverse transcription–PCR (RT–PCR), 10 μl of cDNA in a total volume of 25 μl was used with HS VeriFi Mix  according to the manufacturer’s recommendations. The PCR products were fractionated on 2.0% agarose gels. qPCRs were prepared with the 2× qPCRBIO SyGreen Mix Lo-ROX  with 1 µl of cDNA and 0.5 µM of each primer in a total volume of 10 µl. The amplification reaction included 40 cycles of 5 s at 95 °C, 10 s at 60 °C, and 15 s at 72 °C. The expression level of 2A PP2A; . Primers13C, ‰) using an elemental analyzer  in conjunction with an isotope ratio mass spectrometer  on continuous flow. The carbon ratios were then measured relative to laboratory standards and calibrated relative to the international Vienna Pee Dee Belemnite (VPDB) standard.Leaf discs were collected on 4 September 2020. Samples were freeze-dried and ground. Then, ~2 mg of each leaf sample was used to determine the carbon content  and the carbon isotopic composition (δ2), and stem height (cm) per plant. Total leaf area was measured using a leaf area meter . Biomass samples were dried to a constant weight at 50 °C to determine leaf dry and stem dry weight (g per plant). The above-ground biomass was the combined sum of leaf and stem dry weight. Leaf area ratio  was determined by dividing the total leaf area by the total above-ground biomass.All tobacco plants (~48 plants per genotype) were harvested on 16 September 2020 to obtain the total number of leaves, number of leaves on the main stem, total leaf area . The rest of the analyses were done in SAS , by using the PROC UNIVARIATE procedure to assess for normality and for the discovery of outliers, and by using the PRO MIXED procedure for the ANOVA. Pair-wise comparisons were done by the least square means test (t-test) with significance determined as a P-value ≤0.05.After testing for normal distribution, homogeneity of variances by the Shapiro–Wilk test and Levine test, variables were analyzed with a mixed model ANOVA with or without repeated measurements. ‘Day’ was the repeated measurement factor when a variable was collected multiple times throughout the season. The fixed effects were the genotype (tobacco lines), day, and their interactions, while the block was the random effect. The Kenward–Roger method was used to calculate the degrees of freedom. Mean discrimination analysis was performed utilizing Tukey’s honest significant difference (HSD) with significance determined as ictB-expressing plant lines ictB1, ictB3, ictB4, and ictB6. No transcript was detected in WT control plants, and different levels of transgene expression were observed among transgenic lines, with ictB6 showing the highest transgene expression . No significant differences were found between genotypes for Asat, ΦPSII, and light compensation point for any of the A/Q curve measurements throughout the season . The apparent Vc, max, apparent Jmax, CE, Amax, and Γ* in the transgenic lines were not significantly higher than in the WT. The overall values of these parameters increased throughout the duration of the season, but without significant differences between lines  against the δ13C in the WT , showing a significantly more negative δ13C in the WT .Leaf carbon content (leaf C) and δenotypes . None ofenotypes . The WT genotype . The δ13Significant differences were found among the genotypes for most measured biomass-related traits, including above-ground biomass, leaf and stem dry weights, total number of leaves, number of leaves on the main stem, total leaf area, and stem height . DespiteictB gene indicated higher photosynthesis and biomass compared with the WT plants from which they were derived  had a lower apparent Vc, max, CE, and gm than the WT in at least one of the sets of measurements  and the apparent Jmax (between 130 µmol m–2 s–1 and 170 µmol m–2 s–1) obtained in the greenhouse study of ictB soybean study  than the WT because A and gm tend to be higher while gsw does not change  had lower biomass  and fructose-1,6-bisphosphate aldolase (FBPase)  regeneration (3 plants, which can lead to yield penalties of up to 36% in important food crops (ictB in plants resulted in a decrease of Γ* (ictB transformants under conditions that are known to affect the CO2 compensation point could help us to better understand the underlying function and see if improved performance is significantly associated with specific environmental factors. For example, if ictB transformants can maintain a lower Γ* under drought or heat stress conditions, then it could be a promising application for the future, especially as temperature and drought stress are projected to increase with global climate change. However, as mentioned, further effort would need to be put into understanding under which specific conditions ictB might be most beneficial.Drought and heat conditions adversely affect photosynthetic performance in crops, including increasing the photorespiratory COJXB online.The following supplementary data are available at Fig. S1. Layout of the field experiment.Fig. S2. Weather data for the growing season.A/Ci curves.Fig. S3. Raw data for Fig. S4. Semi-quantitative RT–PCR and qPCR results of transgenic lines.Vc, max, Jmax, gm, and Γ*_adjusted between transgenic lines and the wild type at 28 °C.Fig. S5. Comparison of Fig. S6. Stem height and number of leaves on the main stem.Table S1. Summary of traits measured, their abbreviations, and units.Vc, max and Jmax at 25 °C .Table S2. Apparent erac193_suppl_Supplementary_MaterialClick here for additional data file.


Prunus genus. Plum × apricot  are the most widespread interspecific hybrids, which include plumcots, pluots, and apriums. In this work, 115 accessions of interspecific hybrids from different origins and 27 reference genotypes of apricot and other diploid plum species were analyzed using eight simple sequence repeat (SSR) markers to assess the population structure and current genetic diversity. A total of 149 alleles were obtained, with an average of 19 alleles per locus. The overall polymorphic information content (PIC) mean value of SSR markers was 0.81, indicating a high degree of polymorphism of the SSR. The genetic analysis revealed 141 unique genotypes and two synonyms. The unweighted pair group method with arithmetic averages (UPGMA) dendrogram and the population structure with five groups inferred through the discriminant analysis of principal components (DAPC) revealed a clear genetic differentiation between apricot genotypes and the rest of the accessions since the interspecific hybrids clustered with the Japanese plum genotypes. Repeated backcrosses between interspecific hybrids with plum genotypes could be the cause of the higher genetic proximity of the hybrids with respect to plum than with apricot genotypes. This corresponds to the fruit morphology and agronomic behavior observed in most interspecific hybrids, which also resemble plums more than apricots.The main objective of many fruit-breeding programs around the world is the release of new cultivars from interspecific hybridizations between species of the  Prunus comprises 400–430 species and is one of the most diverse genera within the Rosaceae family, with species of great commercial interest such as Prunus armeniaca L. (apricot), Prunus avium L. (sweet cherry), Prunus domestica L. (European plum), Prunus dulcis Mill. , Prunus persica (L.) Batsch (peach), and Prunus salicina Lindl. (Japanese plum) [Prunus that have been cultivated for generations in several regions of the world, such as the hybrids P. armeniaca × Prunus cerasifera Ehrh., traditionally grown in southwest Asia [Prunus dasycarpa Ehrh. [Prunus species allows the development of interspecific hybridizations for breeding purposes [The genus se plum) . The cloest Asia . These hP. salicina and at least 15 different diploid plum species between the late 19th and early 20th centuries [Luther Burbank obtained more than 100 cultivars from interspecific crosses between enturies , introduenturies  and ‘Rutenturies .Prunus species for objectives such as self-compatibility and resistance to Sharka disease  [P. salicina and P. cerasifera are usually used as female parents to ensure fruit set and obtain a greater number of offspring [P. armeniaca, P. persica, and P. avium as male parents [Currently, many fruit-breeding programs around the world use interspecific hybridizations between us, PPV)  as well us, PPV) . In thesffspring ,11, and  parents . Plumcot parents . These h parents . However parents ,12. In t parents . In 1991 parents . However parents ,15.Plumcot fruits have a higher content of sugars, such as glucose and fructose, than plums or peaches, although they do not exceed the sugar content of apricots . Pluot fPrunus [Prunus [Although most of the currently grown Japanese plum-type cultivars are actually hybrids , modern Prunus . The relPrunus ,21,22,23 [Prunus ,21,24,25Prunus species, mainly apricot. For this purpose, 115 accessions of interspecific hybrids, including commercial cultivars and advanced selections, and 27 reference genotypes have been analyzed using eight simple sequence repeat (SSR) markers.The objective of this work is to assess the genetic diversity and population structure of interspecific hybrids of plum with other AN), with an average value of 19. These values were similar to those reported in previous works in apricot , which can be related to the genetic bottleneck as a consequence of the use of common parents in breeding programs of apricot [Prunus species of the eight SSR markers used in this study and previously observed in other works [All the SSR markers used in this study showed correct amplification and turned out to be polymorphic in the analysis of 115 accessions of interspecific plum × apricot hybrids and 27 reference genotypes. The genetic profiles of each accession obtained with the eight SSR markers are included in NA = 4.1 ; 890 acc = 24.36 ) and Jap[NA = 13 ). Howeve apricot . The smaer works  and the P. salicina , P. domestica , and P. insititia  [Ho and He) of the analyzed accessions was lower than that observed in apricot cultivars, a crop in which a decrease in genetic diversity has been observed [FIS values ranged from −0.13 (CPPCT033) to 0.37 (CPSCT026), with a mean of 0.08, indicating that there is no inbreeding in the whole population since values were observed close to zero. The FST ranged from 0.06 (BPPCT007) to 0.29 (BPPCT039), with a mean value of 0.13, showing low genetic differentiation due to the gene flow between accessions [The polymorphic information content (PIC) ranged from 0.61 (CPPCT033) to 0.90 (CPSCT005), with a mean value of 0.81; therefore, the alleles were considered to be highly polymorphic, showing their usefulness for the study of genetic diversity . The obs = 0.70) , indicatobserved . The FISThe analysis for the detection of homonyms and synonyms allowed the identification of 141 unique genotypes and 2 synonyms: ‘IBG047’ and ‘IBG057’, both accessions from the Ibergen breeding program, which were grouped in the subgroup B6. The genetic relationships between the interspecific hybrids and the reference genotypes were assessed using the unweighted pair group method based on arithmetic averages (UPGMA) to generate a dendrogram based on the Nei and Li similarity index. The dendrogram showed the first node with a high bootstrap value (100), separating the accessions into two groups: group A, formed by 12 accessions (8.5%); and group B, formed by 130 accessions (91.5%) a. These Group A was divided into two subgroups. The subgroup A1 was formed by the ten reference genotypes of apricot. In subgroup A2, two advanced selections from different breeding programs were grouped, ‘IBG024’ and ‘Z029’, which could be aprium hybrids since they showed a greater genetic relationship with the apricot reference genotypes than with those of plum . P. cerasifera, P. salicina, and P. simonii). Plumcots, pluots, and the reference genotypes were allocated into different groups, mixing and grouping mainly according to their genealogical origin. This grouping trend has been found between pluots, plumcots, plums, and apricots in a previous study, suggesting that the grouping is mainly due to the relationship between the accessions and their parents [Group B, the larger of the two groups, was made up of the rest of the interspecific hybrids and the reference genotypes of diploid plums ( parents . P. persica) with yellow flesh [P. persica. Subgroup B2 was a small but diverse group, grouping the reference genotypes ‘Kelsey’ , ‘Mitard’ (P. cerasifera), and ‘Songold’ (‘Golden King’ (unknown origin) × ‘Wickson’ ). ‘Songold’ could be a hybrid between P. salicina and P. cerasifera since it is grouped close to ‘Mitard’ (P. cerasifera). ‘Songold’ was developed by ARC-Infruitec, a public breeding program of South Africa that frequently uses ‘Methley’  as a parent [P. salicina), and the pluots of Zaiger Genetics: ‘Flavor Fusion’, ‘Flavor Finale’, and ‘Flavor Grenade’. These accessions were located near to ‘Red Beaut’ (‘Burmosa’ × ‘Eldorado’) [P. simonii). Subgroup B5 contained some commercial cultivars from Zaiger Genetics. In subgroup B6, 16 accessions were allocated, including the reference genotype ‘Sweet Treat’ (pluerry). Subgroup B1 was separated a greater distance from the rest, grouping five Zaiger Genetics accessions. Two of them were reference genotypes: ‘Honey Top’, a nectarine  , which wP. salicina) [n = 35), including four reference genotypes  and eight commercial cultivars. The genealogy of ‘Glory Red’ includes ‘Queen Ann’ [‘Red Beaut’ is one of the cultivars most used by Zaiger Genetics as a parent , which calicina) ,34,35. ‘een Ann’ , which ween Ann’ . The threen Ann’ . FinallyThe advanced selections were allocated in the subgroups A2 and B1–B9, showing the same diversity observed among the commercial cultivars. K = 5) according to the lowest BIC value (166.17) (K = 5) would be obtained with 25 principal components (PCs)  was performed. Despite the high degree of introgression in this type of interspecific hybrids ,12,34,36(166.17) A. The crts (PCs) b, so 25 In the scatterplot of the DAPC analysis , groups K = 5) , the reference genotypes ‘Dapple Jack’ (pluot) and ‘Mariposa’  were grouped with ‘Queen Rosa’ and’ Santa Rosa’, which are considered simple hybrids of P. salicina [n = 32, 23%) included reference genotypes of different species, such as ‘Abundance’ and ‘Kelsey’ , ‘Honey Sun’ and ‘Honey Top’ (P. persica), and ‘Mitard’ (P. ceracifera), and some hybrids of P. salicina, such as ‘Angeleno’, ‘Methley’ , and ‘Red Beaut’. Group G4 included the largest number of accessions , including four reference genotypes: ‘Queen Ann’, ‘Simon’ (P. simonii), ‘Songold’, and ‘Sweet Treat’ (pluerry). Finally, group G5 was formed exclusively by the 10 reference genotypes of P. armeniaca (apricot). The accessions were allocated into five groups (K = 5) . Group G n = 38, 7%, the rThe composition of groups G1, G2, G3, and G4 was homogeneous, probably due to their accessions being obtained from the selection of crosses and backcrosses in which common parents such as ‘Mariposa’, ‘Queen Ann’, ‘Queen Rosa’, ‘Friar’, and ‘Red Beaut’ were used ,31,37.K = 5) was attributed to the variability within accessions with significant differences (p < 0.01)  on genetic differentiation among the five groups based on DAPC and within accessions revealed that 80% of the total variation in the genetic structure ( < 0.01) , a perceK = 5) (NA PER LOCUS) ranged from 6 (G5) to 12 (G3). The total number of alleles for each group ranged from 49 (G5) to 95 (G3). The same trend was observed in allelic richness (AR), with values between 6.25 (G5) and 8.14 (G3). Alleles observed in only one group were considered private alleles (PA), with the smallest value (4) in group G2 and the largest value (24) in group G5. These results showed moderate and relatively homogeneous levels of genetic diversity, except in group G3, which showed a greater number of alleles (NA PER LOCUS and NA TOTAL), greater allelic richness (AR), and a value of the coefficient of inbreeding notably higher than the rest (IFS = 0.21), revealing a heterozygosity deficit, as observed in a diverse group of apricots from different geographical origins [Ho) ranged from 0.61 (G2) to 0.75 (G1 and G5), and the expected heterozygosity (He), from 0.67 (G2) to 0.86 (G3). Ho was slightly lower than He in groups G1, G2, G3, and G4, which could be attributed to the exhaustive breeding activity developed in these hybrids. The heterozygosity (Ho and He) of G5, which includes the apricot cultivars, was similar to that observed in a previous work including traditional and new apricot cultivars [The parameters of genetic diversity were calculated for each of the five groups (K = 5) . The numultivars .STF) were determined , ‘Simon’ (P. simonii), and ‘Robinson’ (P. munsoniana)), and the first simple hybrids  [Although many species are involved in the genealogy of the interspecific hybrids, our results suggest that the observed diversity is lower than expected, probably due to the use of the same parents, such as ‘Friar’, ‘Mariposa’, ‘Queen Ann’, ‘Queen Rosa’, and ‘Red Beaut’, in different breeding programs . In addiickson’) . In previckson’) ,11. ThisA group of 142 accessions was analyzed, including 27 reference genotypes , 18 commFor each accession, DNA was extracted from young leaves collected in spring and preserved in silica gel . Once drPrunus species in previous reports [For SSR analysis, eight SSR markers were selected from the previously reported studies in Japanese plum  and peac reports ,20,24,25The amplification was performed using five sets of multiplex PCR reactions (M01 to M05). Each multiplex reaction was designed according to the protocol described by Guerrero et al. . All reaThe data of alleles generated by the SSR markers were converted into an object of the class ‘genind’ using the ‘df2genind’ function of the ‘adegenet’ package v. 2.1.2.  and anal(NA), polymorphism information content (PIC), private alleles (AP), allelic richness (AR), observed heterozygosity (Ho), expected heterozygosity (He), inbreeding coefficient (FIS), and Wright’s fixation index (FST)) were determined on the whole population using the packages ‘adegenet’ v. 2.1.2 [The genetic diversity parameters  cluster analysis was used to determine the genetic relationships among accessions according to Nei and Li ; a UPGMAK) in the whole population was inferred using the ‘find.clusters’ function and according to the lowest Bayesian information criterion (BIC) value. The correct numbers of principal components (PCs) to be retained were determined using the cross-validation function ‘xvalDapc’. The membership probabilities were obtained from the DAPC objects in the slot posterior (‘class(dapc1$posterior)’). The slot ‘assign.per.pop’ was used to indicate the proportions of successful reassignment (based on the discriminant functions) of accessions to their original groups [The population structure was analyzed by a discriminant analysis of principal components (DAPC) using the ‘adegenet’ package v. 2.1.2. ,57. The ST values using the ‘pp.fst’ function. The genetic diversity analysis at the group level was performed using the same parameters determined in the whole population (3.4.1.). The package ‘hierfstat’ v. 0.5-7  was usedFinally, the variance components among the inferred groups and the accessions were calculated with an analysis of molecular variance (AMOVA) using the ‘poppr’ package v. 2.8.5 .Prunus due to their commercial potential [The molecular characterization allowed structuring the interspecific plum × apricot hybrids into five groups that clearly corresponded with the genealogical background. The growing interest in obtaining new interspecific hybrids of otential  led to totential ,12, whicotential  and Japaotential . These r


Psychosis is associated with increased subjective and altered endocrine and autonomic nervous system stress-reactivity. Psychosis patients often experience auditory verbal hallucinations, with negative voice content being particularly associated with distress. The present study developed a voice-simulation paradigm and investigated the effect of simulated voices with neutral and negative content on psychophysiological stress-reactivity, and the effect of mindful voice-appraisals on stress-reactivity.Eighty-four healthy participants completed the Montreal Imaging Stress Task with simultaneous presentation of one of three randomly allocated auditory stimuli conditions: negative voices, neutral voices or non-voice ambient sounds. Subjective stress-levels and mindful voice-appraisals were assessed using questionnaire measures, and cortisol and α-amylase levels were measured using saliva samples.ANOVA revealed a significant effect of condition on subjective stress-levels (p = .002), but not cortisol (p = .63) or α-amylase (p = .73). Post-hoc analyses showed that negative voices increased subjective stress-levels relative to neutral voices (p = .002) and ambient sounds (p = .01), which did not differ from each other (p = .41). Mindful voice-appraisals were associated with less distress across conditions (p = .003), although negative voices were also associated with less mindful appraisals (p < .001).Negative voice content, rather than voices or auditory stimuli per se, is linked to greater subjective but not physiological stress-reactivity. Mindful appraisals may partially moderate this effect. These findings highlight the importance of voice content for the impact of voice-hearing, and highlight the potential value of mindfulness training to treat voice distress in psychosis. People The negative content of psychotic experiences may be particularly important in elevating the stress response. AVHs occur in healthy members of the general public as well as people with psychotic disorders  and do not necessarily cause distress or imply need for care in the healthy voice hearers (HVHs) . A consiCognitive models of psychosis have posited that the appraisal of anomalous experiences, and not simply the content, shapes their impact . MindfulThe evidence discussed above suggests that both the content of voices and the way an individual appraises experiences may play a role in the impact of voice hearing on the stress response. In view of this, we aimed to test the effect of simulated voices and their content on stress-reactivity in general population volunteers. To this end, we developed an AVH simulation paradigm that exposes participants to either negative or neutral voice content, a potentially valuable tool currently lacking in AVH research (freely available from the authors upon request). We hypothesised that simulated AVHs with negative voice content would increase the subjective, cortisol and α-amylase reaction to a psychosocial stress paradigm over and above the levels produced by neutral voices or a non-voice ambient control. The latter two were hypothesised not to differ. It was further hypothesised that a more mindful appraisal of voices during the paradigm would be associated with a lower subjective, cortisol and α-amylase stress response.22.1The sample consisted of 84 participants from the general population. Participants were primarily recruited through opportunity sampling. Participants were excluded if they were: under 18, not fluent in English, hearing-impaired, had previously experienced AVHS, had received secondary care for any mental health issue, or scored 10 or above on the depression subscale of the Hospital Anxiety and Depression Scale . The lat2.22.2.1The Montreal Imaging Stress Task (MIST)  is a wel2.2.2Three 10 min audio materials were developed for the present study: a) negative simulated voices , b) neutral simulated voices , and c) non-voice neutral sounds . To ensure validity of the voice simulation, an initial longlist of negative and neutral voice comments was drawn from a) online first-person reports of voice content in service user forums, b) service user literature and c) reports from clinicians with expertise in working with psychosis patients (sources listed in Supplement A). The final shortlist was established excluding comments that were performance- and task-specific, or too derogatory and/or potentially risky . Voice-tracks were identical in the number of statements, the number of first-person statements, as well as the frequency of words within the English language (assessed using Subtlex-UK ). The tr2.2.3The Southampton Mindfulness Questionnaire (SMQ) is a 16-item self-report measure that assesses mindful awareness of distressing voices . For the2.2.42 (28) = 259.8, p < .001). Only one component had an eigenvalue over Kaiser's criterion of 1, explaining 50.49% of the variance. Since no item had a coefficient below 0.5  was created to assess subjective stress-reactivity before and after the task, ranging from 0 cm to 16.5 cm, with total scores ranging from 0 to 132 and higher scores indicating greater subjective stress levels. Participants were asked how stressed, anxious, angry, relaxed (reverse coded), threatened, embarrassed, socially judged and expecting of positive vs negative consequences they were, to reflect an array of possible stress responses. A Principal Component Analysis was carried out to determine whether the scale represented a latent factor indicative of a general subjective stress reaction, using VAS scores at post-MIST. The Kaiser-Meyer-Olkin measure was used to verify sampling adequacy (KMO = 0.84) and Bartlett's test of sphericity indicated sufficiently large correlations . All Salivettes were frozen at −20 °C in a secure laboratory freezer within 2 h of collection. Participants were asked to gently chew the Salivettes for each collection, and were instructed not to touch samples with their hands. All samples were analysed at the StressLab  using ELISA-assays for cortisol and α-amylase, with an inter-assay variability coefficients of 5.2% and 3.0%, respectively.2.3www.sealedenvelope.com; blocks of 12) so that there were 28 participants in each condition, and organised by a collaborator  so that the experimenter was blinded to individual conditions. Participants received no instructions on a particular response style  to the auditory stimuli and were asked not to comment on the content of the auditory stimuli during their performance to avoid unblinding the experimenter. Procedures are detailed in Participants were told that the aim of the study was to test the effects of auditory stimuli on cognitive abilities. Eligible participants completed an initial VAS and saliva sample, and were then invited to practice on the MIST  for 10 min. All participants were instructed to use the keyboard with their non-dominant hand to increase difficulty. During the actual trial, participants wore a headset with the headphone covering their left ear (with the experimenter sitting to their right) to present auditory stimuli, with equal volume settings for all participants. Auditory stimuli were block-randomized  were undertaken for all analyses. For all cortisol and α-amylase analyses, associations of dependent variables with all potential covariates were assessed and included as covariates if significant. For cortisol, bivariate correlations revealed gender and menstrual cycle as covariates. For α-amylase, no covariates were identified. A repeated-measures mixed 6 (sampling timepoints) × 3  ANCOVA was carried out on the total VAS scores, cortisol and a-amylase. Lower-order repeated-measures ANOVAs using total VAS scores were used for post-hoc testing of condition-specific effects . For tests for associations, the area under the curve with respect to the ground (AUCg), i.e., the total amount of biomarker secretion across all timepoints and statistical power of 0.8. Effect size was based on meta-analytic findings of differences in cortisol peak response to acute stress between schizophrenia patients and healthy controls . This waimepoints was calc33.1One-way ANOVAs showed effective randomization, with no significant differences in age  = 0.03, p = .97), gender  = 1.16, p = .32), BMI  = 1.4, p = .25) or number of native English speakers  = 0.96, p = .39) between conditions. One-way ANOVA also confirmed that VAS scores did not differ between conditions at pre-MIST  = 0.64, p = .85), and neither did cortisol  = 0.51, p = .51) or α-amylase  = 0.77, p = .47). Demographics and questionnaire scores by condition are presented in 3.2Repeated-measures ANOVA confirmed a significant effect of timepoint on VAS scores  = 234.1, p < .001), and there was a significant interaction with condition  = 4.8, p < .001). Tests of between-conditions effects showed no significant effect of condition  = 0.04, p = .961). Lower-order ANOVAs were carried out including pre- to post-MIST VAS scores, to capture the dynamic of the stress response, using FDR-adjusted p-values to adjust for multiple comparisons. This showed significant condition differences in VAS change  = 9.7, p = .002), with significantly greater change in NEG compared to NEU (p = .002) and NEG compared to AMB (p = .01), but no differences between NEU and AMB (p = .41). VAS scores throughout the paradigm are shown in 3.3Repeated-measures ANCOVA controlling for gender and menstrual phase confirmed a significant effect of timepoint on cortisol levels  = 5.7, p = .005), with post-hoc analyses confirming a significant increase in cortisol after the MIST paradigm for all conditions. However, there was no significant interaction with condition  = 1.0, p = .41). No significant effect of condition was found  = 0.46, p = .63). Repeated measures ANOVA also confirmed a significant effect of timepoint on α-amylase levels  = 23.0, p < .001), but there was no significant interaction with condition  = 0.1, p = .71). No significant between-conditions effect was found  = 0.32, p = .73). Cortisol and α-amylase scores throughout the paradigm are shown in 3.4t-test revealed a significant effect of voice condition on SMQ scores (t(54) = 3.9, p < .001), with significantly lower SMQ scores, indicating lower mindfulness, in the negative voice condition compared with the neutral voice condition. Further bivariate correlations showed no significant association between SMQ scores and either logn AUCg cortisol  or logn AUCg α-amylase .Bivariate correlation showed that SMQ-voices scores were negatively associated with total VAS delta scores  in actual voice-hearers as well. Alternatively, HPA- and ANS-parameters may not be sufficiently sensitive to reveal subtle differences in stress levels, which are better captured by subjective reports. A further limitation is the possibility that verbal feedback from the experimenter may have interfered with the simulated voices. Lastly, the lack of a no-stress control group means the present analysis did not investigate the effects of voices alone on stress response. However, it seems unlikely that simulated voices alone would initiate a stress response, beyond some mild negative affect in the negative voice condition.4.3In the context of the existing literature on voice content in psychosis, the present findings suggest that negative voice content may be an important factor in the subjective stress-reactivity and distress of clinical voice-hearers. Future research should investigate the predictive value of negative voice content in transition rates of at-risk populations, rather than merely the presence of voices, potentially aiding early identification of at-risk individuals and allowing for early intervention. The present subjective stress findings may have further implications for understanding the development and maintenance of psychotic disorders. According to a recent stress feedback model proposed by Howes and colleagues , stress 5Participants exposed to voices with negative content showed an increased subjective stress reaction to a psychosocial stressor compared with those who were exposed to voices with neutral content or an ambient sounds condition. No significant effect of voice condition on either HPA- or ANS-function were observed. The present study underscores the importance of addressing the content of voices in psychosis research and psychological therapies for voices and adds support to the emergence of mindfulness-based therapies for negative, distressing voices.


This study aims to examine the relationship between physical activity, eating attitude, and bigorexia nervosa among university students.  A questionnaire form consisting of sociodemographic characteristics, eating habits, the Eating Attitude Scale (EAT-40), the International Physical Activity Questionnaire (IPAQ), and the Muscle Appearance Satisfaction Scale (MASS) was conducted on undergraduate students at Üsküdar University.  There were 1006 students who participated in this study with a mean age of 22.58 ± 2.87 years. The median “muscle appearance satisfaction” score of the obese students was statistically higher than the normal weight and underweight students. The median score for “Obesity Anxiety” was statistically higher in underweight students than in obese ones. A one-unit increase in IPAQ scores was related to an elevation of 24.9% in the MASS and a decrease of 17.9% in the EAT-40 scores, while a one-unit increase in MASS scores was related to a reduction of 12.5% in the EAT-40 scores.  Eating attitude is associated with bigorexia nervosa, and as MASS scores increase, EAT-40 scores decrease and as IPAQ scores increase, and EAT-40 scores decrease. In other words, as physical activity increases, muscle strength satisfaction elevates, and in parallel with this increase, a positive eating attitude improves. The World Health Organization (WHO) defines health as “a state of physical, social and mental well-being and not merely the absence of disease or infirmity.”. The “phBigorexia nervosa is a body dysmorphic disorder characterized by individuals' desire to have less fat mass and obsession with increasing muscle mass in this context. It includes environmental factors such as media influence, insecurity, peer influence, and dietary attitudes addition to personality traits such as perfectionism , 4. EvidEating attitude is a complex phenomenon affected by environmental factors as well as motor, cognitive, social, and emotional development and is described as beliefs, thoughts, feelings, and behaviors towards foods , 11. A rThe population of this cross-sectional and descriptive study consists of undergraduate university students enrolled in Üsküdar University in the 2021-2022 academic year. The sample of the study was obtained by choosing a simple random sample, while the sample size was calculated using the sample formula with a certain universe, and it was calculated that 400 university students would be sufficient for the study to be carried out based on a sample size calculation made with sampling error of 0.05 and 95% confidence. The study was begun after the necessary permission was received from the Üsküdar University Non-Interventional Research Ethics Committee (numbered 61351342/January 2022-10). A questionnaire form consisting of demographic characteristics, questions about disease status and eating habits , the Eating Attitudes Test-40 (EAT-40), the Muscle Appearance Satisfaction Scale (MASS), and the International Physical Activity Questionnaire (IPAQ) was conducted face-to-face to 1006 voluntary participants after consent forms were signed.2). According to the WHO, BMI value below 18.5 kg/m2 signifies that the individual is underweight, while BMI value between 18.5 and 24.99 kg/m2 is defined as normal weight, between 25.0 and 29.9 kg/m2 as overweight, and over 30 kg/m2 defined as obesity  were higher compared to students in health science departments [47 (19–90)] . Students consuming more than 2.5 L/d of water [57 (27–88)] and 2–2.5 L/d of water [54 (22–89)] had higher MASS median score than students consuming less than 1.5 L/d of water [34 (19–85)] . Finally, the MASS median score of students who skipped meals [51 (19–89)] was higher compared to those who did not skip meals [49 (21–90)] . Students who skipped night meals had a statistically higher MASS median score [57.5 (28–74)] than the students who skipped lunch [41 (19–85)] .The median MASS score of male students [55 (21–88)] was higher than that of females [46.5 (19–90)] , and students who skipped meals [165 (41–240)] were found to be statistically higher EAT-40 score than students who did not skip meals [159 (71–240)]  (The median EAT-40 score of students consuming less than 1.5 L/d of water [168 (41–240)] was higher compared to the students consuming more than 2.5 L/d of water [150.5 (58–240)] (< 0.001) .U = 78742; p < 0.001); “Muscle Checking” median score for males [11 (4–19)] compared to females [8 (4–20)] ; “Substance Use” median score of male students [11 (4–20)] compared to female students [8 (4–20)] ; “Injury” median score of male students [8 (3–15)] compared to female students [7 (3–15)] ; and “Muscle Satisfaction” median score of males [10 (3–15)] compared to female students [9 (3–15)] .When the median scores for subscales of MASS were evaluated, it was found to be statistically higher “Bodybuilding Dependence” median score for male students [14 (5–24)] compared to female students [11 (5–25)]  ] was found to be statistically higher than for female [16 (4–24)] ( < 0.01) .H = 20.996; p < 0.001), and the median score for “Obesity Anxiety” subscale of underweight students [17 (4–24)] was found to be statistically higher than obese students [15 (10–22)]  (It was found that the median score of the obese students [12 (8–15)] in the “Muscle Satisfaction” subscale was statistically higher compared to normal weight [10 (3–15)] and underweight students [10 (3–15)] ( < 0.05) .s = 0.249; p < 0.001 ). On the other hand, a statistically significant, but very weak correlation was found between IPAQ and EAT-40 scores . Likewise, there was a statistically significant but very weak correlation between MASS and EAT-40 scores . Finally, when the IPAQ score increased, it was found to be a 24.9% increase in the MASS score and a 17.9% decrease in the EAT-40 score. When the MASS score increased, EAT-40 score decreased by 12.5%.The correlation between the IPAQ, MASS, and EAT-40 scores of the university students participating in the study is given in β = 0.001; t = 2.965; p < 0.01) explained 0.9% of the variance of MASS scores . In addition, MASS score was found to be significantly related to EAT-40 score , and it was found that EAT-40 scores explained 0.9% of the variance of MASS scores .The relationship between IPAQ, EAT-40, and MASS score is given in It is essential to evaluate and improve eating attitudes and physical activity in the young population in order to achieve the “healthy society” goal. In this regard, our study aims to examine the eating attitudes of university students and to evaluate their relationship with bigorexia nervosa.2, and the prevalence of obesity was higher in male students compared to female students. Another recent study on medical students stated higher overweight and obesity rates in males than females [Most of the participants in this study had normal BMI in terms of WHO classification, and the percentage of female students classified as normal weight were found more in number than male students. In a comprehensive research conducted by Peltzer et al. , the bod females . This caIn our study, the majority of students consumed 2–2.5 L/d of water at recommended levels. A recent study with university students in Czechia and Slovakia reported that water consumption in this population was over 1.5 L/d ; a diffeIn this study, it was found that almost half of the students skipped their meals and the most skipped meal was found to be breakfast. According to a meta-analysis of 35 studies, it was observed that the frequency of meal skipping among young adults ranged between 5 and 83%, and the most frequently skipped meal was breakfast . AnotherStudies investigating the motivation of university students to workout have revealed that appearance anxiety and the desire to be healthy were the most important reasons pushing this population to be physically active , 36. LikIndividuals with signs of muscle dysmorphia may have a normal-looking or rather muscular body but are thought to have a very small, fat, or muscleless body structure. These individuals, in response to appearance concerns, repetitive behaviors, and mental actions, as well as muscularity and weakness become obsessed . In thisIn our study, no significant difference was found in bodybuilding addiction between normal weight and obese students. While there was no significant difference in bodybuilding addiction and muscle control scores of obese and normal-weight students, muscle dissatisfaction was found to be higher in obese students than in normal weight and underweight students. In a study, a strong positive relationship was reported between body dissatisfaction and obesity in adolescent girls . In anotIn this study, it was observed that students' eating attitudes did not differ according to gender. Similarly, in a cross-sectional study conducted, although male students were more prone to eating behavior disorders, no significant difference was found between the EAT-40 scores of female students . SimilarIn our study, no significant difference was found between the EAT-40 scores of normal weight and obese students. Similar to our results, Sanlier et al.  found thIn this study, there was no significant difference between the EAT-40 scores of health science and other science students. It has been observed that the muscle dysmorphia indicators of the students studying in health-related fields are lower than those in nonhealth fields. In a study, the risk of eating attitude disorder was found to be higher in students studying in the field of sports than in students receiving nutrition education . In anotIn our study, it was found that the median EAT-40 score and muscle dysmorphia indicators of the students who skipped meals were statistically higher than the students who did not skip meals. While muscle dysmorphia indicators were observed to be higher in those who skipped meals at night; there was no difference between the skipped meal and the EAT-40 scores. In the studies conducted with university students, it was observed that students who skip meals tend to have a higher risk of eating disorders , 62. TheThis study suggested that as students' physical activity levels increased, so did their satisfaction with their muscle strength and their eating attitudes. On the other hand, as eating attitudes improve, muscle satisfaction may increase as a measure of body image. These findings are beneficial in creating health and education programs that can support university students develop a positive body image and encouraging healthy eating habits. First, although the EAT-40 scale was developed based on attitudes, feelings, and behaviors related to eating, it is mostly used to define the risk of eating disorders, and it may not be sufficient for the accurate evaluation of general eating attitudes or specifically bigorexia nervosa. Second, the subjective nature of the EAT-40, the MASS, and the IPAQ inventories used in this study may have created a bias in the data shaped by participant statements. In this regard, comprehensive studies supported by objective measurements on the effects of physical activity, eating attitudes, and other factors on body image in university students are needed.


Due to a production error, the word “measured” was removed from a sentence.Data and Methods, subsection Drought Definition, Paragraph 2, first sentence:A correction has been made to the section “In this study, we measured drought using the Hutchinson Drought Severity Index (HDSI) (27) and the Standardized Precipitation Evapotranspiration Index (SPEI) (28).”The publisher apologizes for this mistake. The original version of this article has been updated.