Pyrus bretschneideri.The circadian clock integrates endogenous and exogenous signals and regulates various physiological processes in plants. REVEILLE (RVE) proteins play critical roles in circadian clock system, especially CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) and LHY (LATE ELONGATED HYPOCOTYL), which also participate in flowering regulation. However, little is known about the evolution and function of the RVE family in Rosaceae species, especially in RVE genes in seven Rosaceae species. The RVE family members were classified into two groups based on phylogenetic analysis. Dispersed duplication events and purifying selection were the main drivers of evolution in the RVE family. Moreover, the expression patterns of ten PbRVE genes were diverse in P. bretschneideri tissues. All PbRVE genes showed diurnal rhythms under light/dark cycles in P. bretschneideri leaves. Four PbRVE genes also displayed robust rhythms under constant light conditions. PbLHY, the gene with the highest homology to AtCCA1 and AtLHY in P. bretschneideri, is localized in the nucleus. Ectopic overexpression of PbLHY in Arabidopsis delayed flowering time and repressed the expression of flowering time-related genes.In this study, we performed a genome-wide analysis and identified 51 RVE genes in P. bretschneideri.These results contribute to improving the understanding and functional research of The online version contains supplementary material available at 10.1186/s12864-023-09144-4. CCA1 and LHY, as hub genes, have attracted increasing attention.The circadian clock regulates most of the metabolic and developmental processes of plants and synchronizes plants with external environmental conditions, which greatly enhances the adaptability and competitiveness of plants , 2. The Arabidopsis, CCA1 is the first circadian clock gene to be cloned [CCA1 has a circadian rhythm, peaking at dawn [CCA1 or LHY causes arrhythmicity and late flowering, whereas both single mutants (cca1 or lhy) and double mutants (cca1 lhy) exhibit shortened rhythmic periods and early flowering [GI (GIGANTEA) and TOC1 (TIMING OF CAB EXPRESSION 1); while as transcription activators, CCA1 and LHY promote the expression of PRR7 (Pseudo-Response Regulator) and PRR9 [In e cloned . The exp at dawn . LHY is  at dawn . Constitlowering . CCA1 anand PRR9 . In addiand PRR9 . It is wand PRR9 . Moreoveand PRR9 , soybeanand PRR9 , maize [and PRR9 , and barand PRR9 .A. thaliana also belong to this group, including RVE1-RVE8 and RVE7-like, all of which share the consensus sequence SHAQK(Y/F) F [GI and TOC1 by binding with the evening element (EE), as is the case for the repressors CCA1 and LHY [GmMYB133, as an RVE homologous gene in soybean, is involved in isoflavonoid biosynthesis [PbRVE1b improves anthocyanin content in P. bretschneideri fruit skin [SgRVE6 in tobacco enhances cold tolerance [Members of the MYB transcription factor superfamily play critical roles in plant development. MYB proteins are classified into four subfamilies according to the number of adjacent MYB repeats (R) . Among tK(Y/F) F . Except K(Y/F) F . Althoug and LHY , 22. Sim and LHY . Moreove and LHY . RVE8 mo and LHY . RVE4 an and LHY . CCA1, L and LHY . To dateynthesis . Transieuit skin . Ectopicolerance . TherefoPyrus) is one of the representative fruits of Rosaceae species that is widely cultivated worldwide. Proper flowering and robust plant growth guarantee fruit quantity and quality. Although RVE proteins have been well studied in A. thaliana, the characterization of RVEs in Rosaceae species is limited. In this study, we identified 51 RVE genes from P. bretschneideri and six other Rosaceae species using published whole genome information. The phylogenetic relationship, chromosomal location, and evolutionary history were analyzed. Furthermore, we explored the protein motifs, gene structures and expression patterns of PbRVEs. Specifically, we demonstrate the function of PbLHY in repressing flowering time by ectopic transgenic analysis in A. thaliana. The results of this work contribute to further determining the functions of RVEs in Rosaceae species.Pear  and RVE protein sequences of A. thaliana were used as queries. RVE proteins have a single conserved MYB domain with a SHAQK(F/Y) F sequence. The candidate proteins were further verified by NCBI Batch CD-Search and multiple sequence alignment. In total, fifty-one RVE members were identified in this study: five in R. occidentalis, five in F. vesca, ten in M. domestica, ten in P. bretschneideri, seven in P. mume, seven in P. avium, and seven in P. persica  event (30-45 MYA) that occurred before the split of Malus and Pyrus lineages  were located on the scaffolds. We also found that five FvRVEs were located on three F. vesca chromosomes, five PmRVEs on four P. mume chromosomes, seven PaRVEs on four P. avium chromosomes, ten MdRVEs on seven M. domestica chromosomes, seven PpRVEs on four P. persica chromosomes, and five RoRVEs on three R. occidentalis chromosomes , tandem duplication (TD), proximal duplication (PD), and dispersed duplication (DSD), were detected with different contributions to the RVE family in seven Rosaceae species  and the ancient WGD (Ks ~ 1.5-1.8) of the i genome . In our 85 Table . Among tes Table .To further explore the conservation and diversity of PbRVE family members, we analyzed the features of their protein and gene sequences. The protein length of PbRVEs ranged from 226 to 777 amino acids Table . Ten putRVE genes, exon–intron compositions were investigated by the Gene Structure Display Server (GSDS) tool  were obtained and analyzed by the PlantCARE tool. The promoter region of each gene was enriched with multiple cis-elements. Next, we focused on the cis-elements related to plant hormones, low temperature and light response in the promoters of PbRVEs , and the rest of the PbRVEs contained different numbers of G-box elements.To elucidate the potential regulatory functions of PbRVE genes, we first examined their specific expression levels in five tissues using a quantitative real-time PCR (qRT–PCR) assay. PbRVE genes displayed diverse expression patterns in different tissues , then dropped steeply during the day and increased slightly before dawn. In P. bretschneideri, PbLHY, as the only homologous gene of AtCCA1 and AtLHY, shared the same expression pattern as the two genes in A. thaliana. In contrast, several PbRVE genes showed the highest expression levels before dawn (ZT 21), such as PbRVE1a and PbRVE1b. PbRVE6a showed a completely different pattern, characterized by a peak in the evening (ZT 17).Previous studies have well proven that partial RVEs  are key circadian clock components in thaliana . The expthaliana . To inveons Fig.  and consons Fig. . Under lpes Fig. . The expP. bretschneideri seedlings were treated with constant light for three days, and the expression patterns of PbRVE genes were detected. The expression of PbLHY, PbRVE1a, PbRVE7 and PbRVE8 was clock-regulated  . In our A. thaliana, CCA1 and LHY function as transcription factors and are localized in the nucleus [PbLHY fused to green fluorescent protein (GFP). When the construct was transiently transformed into leaf epidermal cells of tobacco, a fluorescent signal of PbLHY-GFP was observed in the nucleus   lines exhibited an obviously delayed flowering phenotype  Fig. d. Under ype Fig. a. The avA. thaliana, including AtGI, AtCO, and AtFT. Although the expression waveforms of AtGI and AtCO in PbLHY-OE lines were similar to those in control plants, the expression levels were changed at different time points. For example, AtGI transcripts were significantly repressed at ZT9  and M. domestica (10) had a similar number of RVE members to A. thaliana, while F. vesca (5) and R. occidentalis (5) had only half the number of A. thaliana. In addition, P. mume, P. avium, and P. persica belong to Prunoideae with the same number of RVE proteins, seven. P. bretschneideri and M. domestica belong to Maloideae with the same number of RVE proteins, ten. The phenomenon also occurred with F. vesca and R. occidentalis, both of which belong to Rosoideae [P. bretschneideri and M. domestica. To further investigate the evolutionary process of RVE genes in Rosaceae, we analyzed gene duplication events in each species. Overall, DSD was the main duplication event of RVE genes in Rosaceae. DSD is ubiquitous in plant genomes and occurs in unpredictable and random patterns, producing two gene copies that are neither adjacent nor collinear [RVE gene family in P. bretschneideri and M. domestica were only a WGD in addition to DSD, which clearly indicated that WGD was another main driving force for the evolution and expansion of these two RVE families. Meanwhile, the types of duplication events were similar between Rosaceae species that were more closely related. Since duplication events contribute greatly to the evolution and function of genes [In  domains . All RVEosoideae . It has osoideae . Therefoollinear . The eveof genes , it is wA. thaliana, RVE genes play important roles in controlling the circadian clock, photoperiod flowering, anthocyanin biosynthesis, hormone signaling, and stress responses [PbRVEs varied in different tissues, indicating that PbRVEs may have potential functions in plant development. For example, PbLHY and PbRVE8 were highly expressed in leaves, which was consistent with previous reports of homologs in poplar [P. bretschneideri. Meanwhile, we examined the expression of PbRVE genes under light/dark cycles and constant light conditions to explore their expression characteristics in leaves. PbLHY, PbRVE1a, PbRVE7, and PbRVE8 exhibited both typical diurnal and circadian rhythms, peaking around dawn. These genes not only have highly conserved structures and motifs but also have the same expression patterns as their homologs in A. thaliana [P. bretschneideri. Additionally, transcripts of many RVE genes showed relatively high levels in roots, such as PbRVE3b and PbREVE6b. The function of RVE genes in roots has rarely been reported before. Recently, transcriptome analysis of Juglans regia and Medicago truncatula found that RVE genes may be involved in the nitrogen metabolic pathway [RVE genes had diverse expression patterns in different developmental stages of jujube fruit [PbRVE1b promoted anthocyanin accumulation in P. bretschneideri peel [PbRVE3b and PbRVE6c were also highly expressed in fruit, implying that they may be involved in P. bretschneideri fruit development.In esponses , 24–26. n poplar , rice [4n poplar , and munn poplar . Leaves n poplar , and thethaliana , 19, sug pathway , 51. Morbe fruit , and PbReri peel . In our P. bretschneideri, was filtered in the P. bretschneideri genome due to the lack of conserved residues. However, the full-length PbLHY gene containing the complete conserved domain was cloned. Our results complemented the conservation of circadian clock genes in plants [A. thaliana [A. thaliana, the dynamic interaction of photoperiodic flowering is largely controlled by the level of GI-CO-FT module activity [PbLHY could delay flowering in transformed A. thaliana. The expression of GI, CO, and FT was repressed at different time points in PbLHY-OE lines. Thus, we proposed that PbLHY was the candidate gene as a circadian clock component and regulated flowering in P. bretschneideri. Further studies will focus on confirming this hypothesis and exploring more functions of the PbRVE genes.PbLHY, which was the only homolog of CCA1 and LHY in n plants . Furtherthaliana . In longactivity . In shoractivity . In bothactivity , 47. OveRVE genes in seven Rosaceae species and investigated their evolutionary characteristics, such as phylogenetic relationship, chromosomal locations, and duplication events. Then, a systematical analysis was carried out to study RVE members in P. bretschneideri. Based on the analysis of gene structures, conserved motifs, and phylogenetic tree, PbRVE genes were clustered into two subfamilies. The expression levels of ten PbRVE genes varied among P. bretschneideri tissues. Under 12-h light/12-h dark conditions, ten PbRVE genes exhibited diurnal rhythms; among them, four PbRVE genes also exhibited rhythms under constant light conditions. In addition, overexpression of PbLHY could delay flowering time in transgenic A. thaliana plants. These results provide evidence for the functional verification of the P. bretschneideri clock genes and represent a foundation for further functional characterization of RVE genes in Rosaceae.In this study, we identified 51 Pyrus bretschneideri) was downloaded from the Pear Genome Project (http://gigadb.org/dataset/100083) [M. domestica [P. persica [https://phytozome-next.jgi.doe.gov). The genome sequences of R. occidentalis [F. vesca [P. avium [P. mume [https://www.rosaceae.org/). The genome sequence of A. thaliana was downloaded from the Arabidopsis Information Resource  [P value <1e− 10). Protein sequences of candidate RVEs in the seven Rosaceae species were analyzed by Pfam and SMART to verify the MYB domain. The sequences without the MYB domain were deleted.The genome sequence of Chinese white pear (/100083) . The genomestica  and P. p persica  were dowdentalis , F. vescF. vesca , P. aviuP. avium , and P. [P. mume  were dowis.org/) . ProteinA. thaliana were aligned by MAFFT v7.0  [http://www.jalview.org/). The maximum likelihood phylogenetic tree of the RVE family was created by iqtree (http://www.iqtree.org/) (bootstrap =1000) and visualized by iTOL (https://itol.embl.de/).The RVE protein sequences of the seven Rosaceae species and ftware/) . The reshttp://chibba.agtec.uga.edu/duplication/) [RVE gene pairs were identified using DIAMOND software with the parameters --bin 2 --max-target-seqs 5 --e-value 1e− 10. Then, the RVE gene pairs and their chromosome location information were fed into MCScanX software to identify the collinear chains with default parameters. Genes located on the scaffolds were removed. The result was visualized by TBtools [RVE gene family were identified using the DupGen_finder pipeline [RVE gene pairs in each Rosaceae species were calculated by KaKs_Calculator 2.0 using the Nei-Gojobori method [Collinearity analysis among the seven Rosaceae genomes was conducted locally using a method similar to that developed for the Plant Genome Duplication Database (cation/) . Candida TBtools . Duplicapipeline . The noni method .http://meme-suite.org/tools/meme) [RVEs in the seven Rosaceae species were plotted by the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/) [PbRVE genes were downloaded from the Pear Genome Project [Cis-acting regulatory elements of PbRVE genes were obtained using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [Conserved motifs of RVE proteins in the seven Rosaceae species were analyzed by MEME tool v5.0.5 (ls/meme)  with theedu.cn/) . These r Project . Cis-acte/html/) .Pyrus bretschneideri Rehd. cultivar ‘Dangshansuli’ trees grown in the Nanjing Agricultural University Experimental Field, China. The harvested seeds from ‘Dangshansuli’ trees were vernalized at 4 °C in sand for 2 months. Then, the rooted seedlings were selected and transferred to a room with settings of 22 °C, 100 μmol m− 2 s− 1 light, and 65% relative humidity. All seedlings had grown under 12-h light/12-h dark conditions for 30 days. The time of the light beginning to turn on was defined as zeitgeber time zero (ZT 0). For diurnal rhythm analysis, leaves of seedlings were collected every 4 h for one day. For circadian rhythm analysis, seedlings were released to constant light, and the leaves were collected every 4 h for 3 days. The leaves of three seedlings with uniform growth were randomly selected as a mixed sample at the designated time point. Three independent biological repeats were collected for the experiments. All samples were frozen immediately in liquid nitrogen and stored at − 80 °C.For tissue-specific expression analysis, all materials were harvested from ten-year-old A. thaliana plants were grown under long-day conditions (16-h light/8-h dark) in the controlled room with settings of 22 °C, 100 μmol m− 2 s− 1 light, and 65% relative humidity. Seeds of transgenic A. thaliana plants were sterilized and screened on Murashige and Skoog (MS) medium with 20 mg L− 1 hygromycin. Flowering phenotypes were observed in the T3 generation. For gene expression analysis, seeds were grown in MS medium for 12 days, and then the seedlings were collected every 4 h for one day.PbRVEs were designed by Primer Premier 5.0 software, and the specificity was verified by the Primer search program against the P. bretschneideri genome (Table -ΔCT method. PbUBQ (POLYUBIQUITIN) of P. bretschneideri or AtACT (ACTIN) of A. thaliana was used as the reference control to normalize the expression of the target genes. Three biological replicates and three technical replicates were used for each experiment. All qRT–PCR experiments followed MIQE guidelines [Total RNA was extracted from frozen tissues using a Plant Total RNA Isolation Kit  specifically designed for plants rich in polysaccharides and polyphenols. One microgram of total RNA was reverse transcribed in a 20 μl reaction volume using One-Step gDNA Removal and cDNA Synthesis SuperMix . Gene-specific primers of me Table . To testidelines .PbLHY was amplified by PCR. The product was inserted into the pCAMBIA1300-35S: CDS-GFP vector [Nicotiana benthamiana) leaves was performed according to a published protocol [A. thaliana transformation assay, the above Agrobacterium strains were transformed into A. thaliana (Columbia-0) plants using the floral dip method [PbLHY transgenic lines were confirmed by PCR with gene-specific and vector primers  Coding sequence of PbLHY. (b) Alignment of PbLHY and AtCCA1/AtLHY protein sequences. The red box indicates the MYB domain. The blue backgrounds correspond to the percent identity of the multiple alignment. Fig. S2. Alignment of conserved domains from PbRVE proteins. (a) Alignment of MYB domains from ten PbRVEs and eleven AtRVEs. Red box indicates MYB domain, and blue box indicates SHAQK(Y/F) F sequence. (b) Alignment of LCL domains from subfamily II members. The red box indicates the MYB domain. The green box indicates the LCL domain. The blue backgrounds correspond to the percent identity of the multiple alignment. Fig. S3. The motif details of ten conserved domains identified in RVE proteins and the logos of these domains created using the MEME. Amino acids are expressed in the standard single letter code. The size of the letters at each position represents their frequency.Additional file 2: Table S1. Characteristics of RVE family members in seven Rosaceae species. Table S2. Numbers of RVE gene pairs from different duplication events in seven Rosaceae species. Table S3. Ka and Ks calculation of duplicated RVE gene pairs in seven Rosaceae species. Table S4. The primers used in the assays.


Levilactobacillus brevis) versus lack of an operon structure (Lactiplantibacillus plantarum). Within species molecular population genetic analyses of GDP genes in L. brevis and L. plantarum sampled from diverse fermented food and other environments showed abundant synonymous and non-synonymous nucleotide diversity, mostly driven by low frequency changes, distributed throughout the coding regions for all genes in both species. GAD genes showed higher level of replacement polymorphism compared to transporter genes (gadC and YjeM) for both species, and GAD genes that are outside of an operon structure showed even higher level of replacement polymorphism. Population genetic tests suggest negative selection against replacement changes in all genes. Molecular structure and amino acid characteristics analyses showed that in none of the GDP genes replacement changes alter 3D structure or charge distribution supporting negative selection against non-conservative amino acid changes. Phylogenetic and between species divergence analyses suggested adaptive protein evolution on GDP genes comparing phylogenetically distant species, but conservative evolution comparing closely related species. GDP genes within an operon structure showed slower molecular evolution and higher conservation. All GAD and transporter genes showed high codon usage bias in examined LAB species suggesting high expression and utilization of acid resistance genes. Substantial discordances between species, GAD, and transporter gene tree topologies were observed suggesting molecular evolution of GDP genes do not follow speciation events. Distribution of operon structure on the species tree suggested multiple independent gain or loss of operon structure in LABs. In conclusion, GDP genes in LABs exhibit a dynamic molecular evolutionary history shaped by gene loss, gene transfer, negative and positive selection to maintain its active role in acid resistance mechanism, and enable organisms to thrive in acidic environments.Glutamate decarboxylase (GAD) pathway (GDP) is a major acid resistance mechanism enabling microorganisms’ survival in low pH environments. We aimed to study the molecular evolution and population genetics of GDP in Lactic Acid Bacteria (LAB) to understand evolutionary processes shaping adaptation to acidic environments comparing species where the GDP genes are organized in an operon structure ( Organisms are exposed to a variety of stress factors in their surroundings. Acid stress is a main stress factor in several bacterial species, and only a limited number of them evolved mechanisms to cope with high acidic environments. The bacteria that produce lactic acid as part of their natural metabolic processes are called Lactic Acid Bacteria (LAB) . LABs arLABs can be isolated from natural environments such as water and soil, however, their abundance in these environments has not been found at high levels . LABs arIn LABs, lactic acid is generated through several pathways of carbohydrate metabolism, mainly by the glycolysis pathway . Lactic gad) and antiporter (gadC) gene, while others may not have the antiporter gene together with the enzyme gene  gene . During this process the protons present in the intracellular environment participate in the reaction. Thus, the bacteria can regulate the decreasing pH level . Since tyme gene . The GADbacillus , Lactocotococcus , and Strtococcus  LAB genedC) gene . Among t. brevis , L. planlantarum , L. buchbuchneri , and L. aracasei  reportedL. brevis is the best-examined species in the genus and has a high GABA-producing capability. Only L. brevis possesses two GAD genes that produce isozyme GADs among the LABs examined so far  gene, positioned downstream of the gadA gene, is also considered a part of the GAD cluster structure  or any GAD operon structure in L. plantarum genome  versus a species that lacks an operon structure (L. plantarum). First, we performed molecular population genetics and molecular evolution analyses with L. brevis and L. plantarum strains with whole genome data isolated from diverse food and non-food environments to compare the natural selective forces shaping the GDP genes in LABs with and without an operon structure. Second, the GAD system gene organization in diverse LAB species is compared via phylogenetic analyses. Based on phylogenetic relationships, divergence patterns of L. brevis and L. plantarum GAD system genes are compared with respect to other LAB species. Finally, the functional consequences of amino acid changes observed in L. brevis and L. plantarum genes is investigated with molecular structural analyses.We aimed to study the molecular evolution and population genetics of GDP in LABs to understand evolutionary processes shaping adaptation to acidic environments comparing species where the GDP genes are organized in an operon structure (http://www.ncbi.nlm.nih.gov/Entrez/) web site (last accessed on 15/08/2022). Only strains having the whole genome or contig data containing all of the GAD pathway genes, and with clearly indicated isolation sources were selected from the results of the NCBI search. GeneMark.hmm prokaryotic web tool (http://exon.gatech.edu/GeneMark/gmhmmp.cgi) was used to identify and distinguish the GAD operon genes. Incomplete and pseudogene sequences were excluded from the analyses. Based on reported isolation sources, 30 strains from five different isolation groups (populations) were formed for L. brevis and 88 strains from nine isolation groups were formed for L. plantarum. One of the groups created for both bacterial populations is the feces group, which is a non-food source. All other groups contain strains isolated from various food environments. L. brevis and L. plantarum strains used for analyses. Due to discrepancy for the nomenclature of L. brevis GAD genes  against all available L. plantarum genomes. The glutamate/gamma-aminobutyrate transporter family protein coding gene yjeM was identified as the homolog of gadC gene. Similarly, gadC and yjeM homolog transporter genes of other LABs were identified by amino acid and nucleic acid BLAST searches against their respective genomes. Since there is no report of a specific L. brevis and L. plantarum strains, and 32 bacterial species were performed by the MUSCLE alignment algorithm  were predicted with MEGA-X/Models by using sequence alignments. The model with the lowest Bayesian Information Criterion (BIC) score was chosen, and the Maximum Likelihood (ML) method was applied. In addition, the 1000 bootstrap method was employed to evaluate the phylogeny, and the complete deletion option in MEGA-X was used to treat the sequence alignments. 16S rRNA sequences were used for reconstructing the phylogenetic relationships of bacterial species. Phylogenetic relationships of bacteria based on each individual GAD pathway gene was also evaluated.L. brevis and L. plantarum populations for nucleotide diversity included segregating sites (S), total number of mutations (Eta), pairwise comparison of nucleotide diversity  (θ)  , and ave, π) (θ) . Allele ) (θ)  , Neutralhttp://genomes.urv.es/CAIcal/), a web-based tool, was used to calculate the CAI values. The codon usage tables required for CAI estimates were retrieved from http://www.kazusa.or.jp/codon/.Codon usage bias analyses included codon bias index (CBI)  and codowww.ncbi.nlm.nih.gov/Class/Structure/) and literature. To investigate the impact of replacement changes on the protein isoelectric points (pI) Prot Pi (www.protpi.ch), a web-based tool, was used. The pKa values used in the pI calculation were estimated by ExPASy (https://www.expasy.org/). The effect of amino acid change on the protein function  was estimated by PROVEAN (http://provean.jcvi.org/)  . Selectivi.org/)  and its vi.org/) .Several protein structure prediction servers were employed to investigate the structure of GDP proteins as there was very limited structure information on GDP proteins in literature. I-TASSER  was usedhttps://www.r-project.org/).Distribution of synonymous and non-synonymous changes among different protein domains is compared by Chi-square tests. For continuous variables, deviations from normal distribution is tested by Shapiro test. Population genetic parameter estimates per gene were compared among populations by non-parametric Kruskal-Wallis one-way ANOVA followed by non-parametric Wilcoxon pairwise tests. For two group comparisons non-parametric Wilcoxon test is conducted. ggplot2 package  was usedL. brevis GAD pathway consists of transcription regulator (gadR), glutamate/GABA transporter (gadC), and glutamate decarboxylase enzyme (gad1 and gad2) genes. The operon is formed by gadR, gadC, and gad1. In total, sequence data of 30 strains from five isolation environments (populations) were analyzed. The transporter gene, gadC, showed the highest number of overall (synonymous and replacement/non-synonymous sites combined) segregating sites and mutations based on all (n = 30) L. brevis samples. gadC also had the highest overall nucleotide diversity based on segregating sites (θ), and average pairwise nucleotide difference (π) estimates  estimate. However, the glutamate decarboxylase enzyme coding gene, gad2, outside the operon showed significantly higher number of replacement/non-synonymous changes and non-synonymous site diversity compared to other genes and operon  gad2 again showed the highest number of replacement changes and non-synonymous site diversity  and glutamate decarboxylase enzyme (gadB) genes form the GAD pathway. In total, sequence data of 88 strains from nine isolation environments (populations) were analyzed. gadB had higher overall, synonymous, and replacement polymorphism nucleotide diversity compared to yjeM (gadB had higher number of replacement polymorphisms and non-synonymous nucleotide diversity (non-parametric Wilcoxon p = 0.0002), and yjeM was the most conserved gene , Fu-Li’s D*, and Fu-Li’s F* test results  and its functional domain information is available  of vailable . The GADvailable . L. brevee genes . Then, oequences . Replaces domain . SimilarL. brevis strains a total of seven aa changes were observed in gadR. Eighty six percent (6/7) of the changes were singletons (observed only once), and 57% (4/7) of changes were observed in sourdough strains. Seventy one percent (5/7) of the aa changes were conservative , and only one of the singleton aa changes that leads to charge, class (basic to sulfuric aa), polarity, and interaction mode change was identified as deleterious with PROVEAN analysis  of the changes were singletons. Fifty percent (8/16) of the changes were observed in the feces strains. Only four of the aa changes  that results in class, polarity, and interaction mode changes were identified as deleterious changes  isolation groups. Over 85% (14/16) of the aa changes were biochemically conserved changes and only one change is identified as deleterious  position change in different populations is summed to determine the total number of aa changing mutations on the genes. Among all analysis . For the changes . Sixteeneterious . With 41er genes .gadB among all L. plantarum strains was higher than L. brevis GAD genes combined. Most replacement changes were non-singletons observed in more than one population. The number and percentage of biochemically unconserved and deleterious changes was also higher in gadB than L. brevis GAD genes combined. The unconserved and deleterious aa changes were observed in all L. plantarum populations  and charge distribution of GAD pathway proteins is investigated. For each gene/protein a sequence without any aa change is selected as a wild type, and its pI and charge at pH values ranging from 4.00 to 7.00 is estimated. Then, pI and charge at various pH value estimates for each gene’s protein sequences with different aa changes are calculated. Despite abundance of aa changes with different biochemical properties, the pI and charge estimates of all proteins with aa changes were nearly identical to each other and to their respective wild types  indicatiL. brevis Gad2, there was no secondary or three-dimensional (3D) structure information for any of the GAD pathway proteins examined. I-TASSER was used for secondary structure predictions, Phyre2 was used for transmembrane helix structure predictions, and AlphaFold was used for 3D structure predictions. First, GAD enzyme protein structures were analyzed. There was 84% aa sequence similarity between Gad2 and GadB, and 52% similarity between Gad2 and Gad1 (L. brevis or L. plantarum GAD genes (detailed above in π non-synonymous) and non-synonymous site Tajima’s D values. Whereas higher non-synonymous site diversity driven by rare/singleton replacement changes  on the second half of Gad1 was observed, higher non-synonymous site diversity driven by both rare and common  replacement changes on the first half of Gad2 was observed (Except for and Gad1 . Despiteand Gad1  except tand Gad1  importanand Gad1 . None ofobserved . For Gadobserved .E. coli glutamate/GABA antiporter (PDB code 4DJI) (Structure prediction programs identified the de 4DJI)  as the bde 4DJI) , the secde 4DJI) . Slidingde 4DJI) .There is very limited structure information on GAD system regulatory proteins, and potential regulatory proteins have not been annotated in most LABs. Structure analyses identified a simple 3D structure consisting of alpha-helices with a possible single TM-helix . The smaWe also examined the distributions of singleton vs. non-singleton, and deleterious vs. non-deleterious replacement changes on the alpha-helix, beta-sheet, and coil regions for all GDP genes. None of the results was significant suggesting negative selection against replacement changes is evident for all secondary structures.L. brevis and L. plantarum were similar, and greater than 0.77 (range 0.77–0.83) indicating high expression of GAD pathway genes (Most amino acids are coded by more than one codon (codon redundancy), and codon preference can be under selection, leading to non-random codon usage bias. Selection on codon usage is generally observed in rapidly growing bacterial populations, and genes with high expression levels . One of ay genes  in both ay genes .L. brevis and L. plantarum populations, molecular evolution and divergence patterns of L. brevis (with operon structure) and L. plantarum (without operon structure) GAD system genes are compared with respect to other LAB species. These interspecies analyses are conducted using 32 bacterial species (31 LABs and the outgroup E. coli) that are commonly found in acidic fermented food environments. These species are chosen so that each one has at least one high coverage whole genome sequence with annotated GAD pathway genes, and they represent species with and without GAD operon structure.Following molecular population genetic analyses within L. brevis and L. plantarum samples to the ratio of non-synonymous to synonymous divergence between L. brevis and L. plantarum, and other LAB species. For most comparisons, between species divergence (non-synonymous/synonymous fixed difference ratio) was greater than within species non-synonymous/synonymous polymorphism ratio, indicated by statistically significant MK test results, suggesting positive selection (adaptive protein evolution) on the genes  tests, comparing the ratio of non-synonymous to synonymous variation within he genes . We alsohe genes  further L. brevis gad1 and gadC with respect to gad and gadC in L. spicheri, L. zymae, L. sakei, L. angrenensis, and L. cerevisiae. L. brevis gad2 with respect to L. plantarum gadB, and with respect to gad genes in L. paraplantarum, L. herbarum, L. argentoratensis also did not show positive selection. Similar neutral evolution results were observed for L. plantarum gadB with respect to gad genes in L. argentoratensis, L. herbarum, and S. paracollinoides. Moreover, negative selection (high conservation) was observed when L. plantarum gadB is compared with L. paraplantarum gad. To get a broader perspective on the between species sequence comparisons, a phylogenetic tree based on 16S rRNA sequences is constructed to examine the phylogenetic relatedness of 32 bacterial species  genes in bacteria with a true operon structure (clade I) are more closely related to each other and descend from a common ancestor, and all transporter genes in bacteria with a smaller operon like structure (clade III) are more closely related to each other and descent from a different common ancestor. Within group pairwise genetic distance was lowest for clade I group, followed by clade III and clade I groups (gadC and yjeM gene sequences from distant genus and species show less divergence (appear to have not changed much) compared to transporter sequences from more closely related species, as these gadC and yjeM sequences are phylogenetically more closely related to each other than indicated by the 16S rRNA gene phylogenetic tree of the species that they belong to.However, closer examination of interspecies tests showed neutral evolution (no sign of positive selection) in certain comparisons such as  species . This spe clades . Clade II groups . The CAII groups . BecauseI groups  one cannL. antri, L. reuteri GADs) clustered together with GAD sequences that are not in an operon structure  forming a separate cluster within clade II cluster. Within group pairwise genetic distance was lowest for clade I, followed by clades II and III , whereas L. brevis gad2, which is present outside the operon structure, clustered with GADs from species without an operon structure (purple colored clade II lineages). Moreover, there is over 80% amino acid sequence similarity between L. brevis gad2 and other gad sequences in the purple group. Taken together with the fact that gad1 and gad2 sequences within L. brevis populations cluster into two well supported distinct clades strongly suggest that the origin of gad2 in L. brevis is not a gene duplication event, but a horizontal gene transfer of a gad gene from a LAB without an operon structure. CAI estimates for all GAD genes were high in all species suggesting high expression of decarboxylase enzyme genes in all species  versus lack of an operon structure (L. plantarum). Within species analyses focused on gene sequences belonging to specific strains with whole genome sequence sampled from diverse food and non-food environments. Between species analyses examined the distribution of operon structure on the LAB species tree, and compared the divergence and molecular evolution of GAD pathway genes between closely and distantly related LAB species.We aimed to study the molecular evolution and population genetics of GAD pathway genes in LABs to understand selective processes shaping adaptation to low pH environments comparing gene sequences from species where the GAD pathway genes are organized in an operon structure . The C-terminal regions of L. brevis Gad2 and L. plantarum GadB form a C-plug structure that curls toward the active site (L. brevis Gad2 and L. plantarum GadB were nearly identical, both having a C-terminal region curving towards the active site. Moreover, the C-terminal regions of Gad2 and GadB were highly conserved. A similar C-terminal structure that curls toward the active site is not observed in Gad1, and the C-terminal region of Gad1 was less conserved compared to the C-terminal region of Gad2. The quaternary structures of Gad2 and GadB enzymes in the active state are reported to be monomer (L. brevis Gad1 is reported to be active in a tetramer structure (Several of our findings are in agreement with functional studies. e operon . We obsevis Gad1 . We alsoive site . The C-tive site , indicat monomer  and dime monomer , respecttructure . N-termitructure . The impE. coli GadC transporter 3D structure in the databases. E. coli GadC has a C-plug structure that goes through a conformational change according to intracellur pH and controls transport of ions (L. brevis GadC is similar to E. coli GadC, and a possible C-plug structure is observed. The possible C-plug residues in L. brevis GadC was also highly conserved. A possible C-plug structure was not observed in the YjeM transporter, and most amino acid changes were observed on the C-terminal residues of YjeM proteins.We could only find the  of ions . Our 3D E. coli (Shigella sp. (Listeria sp. (Brucella sp. (There are several limitations of our study. First, we only focused on LABs primarily found in fermented food environments. Because LABs not only survive but thrive and out compete other bacteria in low pH environments, we hypothesized that LABs would be one of the best bacteria to study adaptation to acidic environments. However, GAD pathway is also a major acid resistance pathway utilized by pathogenic bacteria such as E. coli , Shigellella sp. , Listerieria sp. , and Bruella sp. . Moreoveella sp.  so similella sp. . The genWe conclude that the evolution of acid resistance in LABs is shaped by adaptive protein evolution and acquisition of new genes during adaptation to low pH environments, followed by conservation of protein function and activity. Our results present another step in understanding the evolutionary mechanisms driving the molecular mechanisms behind acid resistance, and have potential broader implications by stimulating similar studies in other microorganisms.


Glandora prostrata (Loisel.) D.C.Thomas  D.C.Thomas  is 150,041 bp in length with 37.5% GC content, displaying a quadripartite structure that contains a pair of inverted repeat regions , separated by a large  and small  single-copy regions. It has 131 annotated genes including 86 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The phylogenetic analysis performed confirms that G. prostrata subsp. lusitanica is placed under the Boraginaceae family, which belongs to the Boraginales order. This study will contribute to conservation, phylogenetic, and evolutionary studies that comprise this traditional species relevant to the landscape of aromatic, medicinal, and condiment plants from Portugal. Glandora D.C.Thomas, Weigend & Hilger  D.C.Thomas  D.C.Thomas  under the voucher LISE: 96377 (identified by: Jorge Capelo).The plant material of Young leaves were selected from the collected specimen, frozen in liquid nitrogen immediately after collection, and stored at −80 °C until further processing. Total genomic DNA was extracted using an adaptation of the Doyle and Doyle  methodolERR10047929) were used to assemble the complete chloroplast genome (sequence coverage: 993×) using the GetOrganelle pipeline (v1.7.3.1)  while setting the flags ‘-max-reads’ and ‘-reduce-reads-for-coverage’ to 25 million and one thousand, respectively . The plastome annotation was performed using the GeSeq tool  while comparing with the results obtained from a BLAT  .The about 24 million high-quality paired-end reads obtained (SRA: ol) Kent  search aG. prostrata subsp. lusitanica from this study. Concatenated nucleotide sequences coding for the shared proteome (75 coding sequences) extracted from the dataset were used in the phylogenetic analysis . Four additional sequences were used as outgroups in the phylogenetic analysis: Ehretia dicksonii Hance  A.T.Richardson  also belonging to the lamiids clade; and Tilia platyphyllos Scop.  from the malvids clade.The dataset used for the phylogenetic analysis was obtained from GenBank (accession date: 1 June 2022) and composed of all 12 verified and complete chloroplast genomes that belong to the Boraginaceae family together with the plastome of The IQ-TREE 2 software package . When performing the phylogenetic analysis using the concatenated amino acid sequences of the shared proteomes , the same tree results are supported.The maximum-likelihood tree obtained from the phylogenetic analysis performed  showed tG. prostrata subsp. lusitanica isolate BPTPS049, the first described plastome belonging to the Glandora genus. This complete genome will contribute to conservation, phylogenetic, and evolutionary studies that comprise this traditional species relevant to the landscape of aromatic, medicinal, and condiment plants from Portugal.This study describes the chloroplast genome of Click here for additional data file.Click here for additional data file.


Staphylococcus aureus (MRSA) in the food sector is relatively low. However, their presence in seafood is a significant public health concern. In India, fish and fishery products are maximally manually handled compared to other food products. In this study, 498 fish samples were collected under various conditions  and representatives from their surroundings. These samples were screened for the prevalence of Staphylococcus aureus, determining its antimicrobial resistance, MRSA and genetic profile. It is observed that 15.0% and 3.0% of the total samples were screened positive for S. aureus and MRSA, respectively. The S. aureus strain MRSARF-10 showed higher resistance to linezolid, co-trimoxazole, cefoxitin, ofloxacin, gentamicin, rifampicin, ampicillin/sulbactam and Piperacillin–tazobactam. This MRSA, spa type t021 and SCCmec type V strain isolated from dried ribbon fish (Family Trachipteridae) carried virulence factors for exoenzymes such as aureolysin, serine, toxin genes and a novel MLST ST 243, as revealed from its draft-genome sequence. This highly pathogenic, multidrug-resistant and virulent S. aureus novel strain is circulating in the environment with chances of spreading among the seafood workers and the environment. It is further suggested that Good Hygienic Practices recommended by World Health Organization need to be followed during the different stages of seafood processing to provide pathogen-free fish and fishery products to the consumers.Compared to the clinical sector, the prevalence of methicillin-resistant The online version contains supplementary material available at 10.1186/s12866-022-02640-9. Staphylococcus aureus (S. aureus) is commonly found asymptomatic in the human skin and noses of 25% of healthy people and animals. Methicillin-resistant Staphylococcus aureus is common in a hospital environment; it has been reported in fish and fishery products [S. aureus is regarded as one of the potential foodborne pathogens. [S. aureus strains, are heat-stable, and do not degrade even when cooked [products , 2. The products . The exiproducts , 5. Becathogens. , 7. Stapn cooked . The SEsn cooked , 10. Stan cooked . Antibion cooked . MRSA isn cooked . MRSA wan cooked . MRSA isn cooked , 50.18% n cooked  and 70% n cooked . Furthern cooked .S. aureus, the presence of S. aureus is due to a post-harvest contamination [MRSA-contaminated food can be a severe health risk to consumers, and this resistance can be transmitted to the consumers , 22. Sinmination . Recentlmination . The scrA total of 498 seafood samples, including ice and water from varying sources, were collected from the fish market and processed seafood from the fish processing Industries consisting of 108 fresh (raw), 79 chilled, 64 frozen and 124 processed fish samples and 76 water and 47 ice. The study was carried out from 2012 to 2017 in the Veraval region, Gujarat state, India, to monitor the existence of MRSA in different seafood and its associated environmental samples.S. aureus on Baird Parker Agar  [S. aureus colonies with distinct characteristics were selected for coagulase tests. The MRSA isolates were confirmed using MRSA plates from HiCrome MeReSa  and BBL CHROM agar MRSA II .Fish samples were processed according to ISO 6888–1 and ISO 6888–2:2003  to isolate co, USA) . S. aureS. aureus and MRSA [S. aureus thermostable nuclease, is required for S. aureus confirmation. The mecA gene (278 bp), which is used to identify MRSA, is a gold standard for confirmation. For the identification of Staphylococcus genes, a 16SrRNA primer unique to Staphylococcus genes has also been included (750 bp). The GenElute Bacterial Genomic DNA Kit was used to isolate DNA . Initially, the monoplex PCR with each primer separately was carried out for initial standardization then gene amplification for DNA sequencing purposes. The reference strain’s DNA of S. aureus ATCC 43,300 (MRSA) and ATCC 25,923 (MSSA) were tested for positive and negative control. Multiplex PCR was carried out as per Al-Talib et al., Protocol [Multiplex PCR was used to detect and MRSA . Since tProtocol . The reaS. aureus ATCC 25,923 and ATCC 43,300 were employed.The Minimum Inhibitory Concentration (MIC) was performed according to CLSI recommendations  using BDStaphylococcus aureus subsp. aureus NCTC 8325) strain was obtained from a public database (NCBI). The variants were annotated using the pipeline comprising bwa version 0.7.17-r1188 for indexing and mapping to reference genome, samtools 1.10 and bcftools 1.10.2 for variant calling [http://www.genomicepidemiology.org/) was performed. [http://tree.bio.ed.ac.uk/software/figtree/) was employed to visualize the phylogenetic tree [https://crisprcas.i2bc.paris-saclay.fr/; [The bacterial genomic DNA was obtained using an isolation kit , and the DNA quality was verified using the NanoDrop spectrophotometer and Qubit Fluorometer . The paired-end Whole Genome Sequencing (WGS) was performed in Illumina HiSeq 2500 (paired end). The number of paied- end reads was approximately 7 billion short- read sequences in pairs of ~ 300 bp, the number of bases (Mb) was 1447.5, and there was 35.11% G + C content. The FastQC v0.11.9 quality check was performed, and low-quality reads (Phred score < 20) were trimmed using cutadapt 2.8. , 29. SPA calling , 34. Therformed. . The strrformed. . The Beatic tree , 38. CRIlay.fr/;  and the S. aureus and MRSA  and MRSA (3.0%). While comparing the worldwide incidences of MRSA in seafood products, variation in MRSA percentage was observed from very low to high. Similarly, a higher incidence of 30.0% and 22.2% MRSA were found in raw fish and prepared fishery foods in public hospitals in Salvador, Bahia, and Brazil, respectively [., found a higher level of MRSA (50%) in the 173 market fish samples in Assam, India. Almost 60% of the shrimp aquaculture settings samples were positive for the MRSA in Kerala, India [., did not find any MRSA in fish and fishery products [S. aureus and MRSA transmission from food products to humans [Total of 498 samples collected were screened for the presence of SA Table . Out of ectively . At the a, India , 41. Howproducts . The varproducts –45. The o humans .Table 1IM50 automated bacterila identification and AST system . The resistant pattern was varied between the type of samples collected; processed fish (6), chilled (4), fresh (3), and frozen samples (2) and there were no MRSA isolates in the water and ice samples which revealed the chance from the workers  were carried out for these 15 number of MRSA isolates using BD Phoenix samples  and therThis might be due to the frequent manual handling of the fish.Manal et al. recommended Cefoxitin as a marker for detecting methicillin resistance and they found 16- 60% of isolates were showed varied level of resistance to cefoxitin from the Riyadh hospital armed forces hospital clinical samples.Further they suggested that Cefoxitin could be considered a surrogate marker for the detection of MRSA .The Paired-end sequencing of the MRSA isolate performed was later subjected to a quality check by Fastqc and trimmed to generate high-quality raw reads with phread score ≥ 30 excluding adaptor sequences  and superantigenic toxins are linked to their virulence  Table . This MR seafood . Hemolys seafood , 49. BetS. aureus) degrades numerous proteins, including insulin B, and inactivates PSMs, resulting in osteomyelitis [Virulence factors for secreted exoenzyme genes, including aureolysin (aur) and serine protease (spIE) destroy host compounds or disrupt host metabolic and signalling pathways. The protease aureolysin  allow researchers to study clonal diversity and MRSA transmission in hospitals and community settings. T021 and its repetitions were the most prevalent spa Type in the strain 15–12-16–02-16–02-25–17-24, contig position  and plus orientation. Sivaraman et al., reported on the prevalence of CA- MRSA in seafood [The standardized nomenclature and availability of spa types on the central spa server  & serine and toxin genes such as hlgA, hlgB, hlgC, lukF-PV, lukS-PV, seg, sei, sem, sen, seo and seu.. So, the presence of highly pathogenic, MDR and virulent MRSA strains in the fish and fishery products could pose a severe threat to the consumers, which will guide us to design a better surveillance protocol and control measures. It further suggested that Good Hygienic Practices, as recommended by WHO, need to be followed strictly during various stages of handling and processing of fish and fishery products to provide wholesome fish to the consumers [The present study revealed that 13.65% of samples were contaminated with onsumers , 54.Additional file 1:


HTRA1) elicits cerebral autosomal recessive arteriopathy with subcortical infarcts and white matter lesions (CARASIL). The relationship between some heterozygous mutations, most of which are missense ones, and the occurrence of cerebral small vessel diseases (CSVD) has been reported. Recently, heterozygous HTRA1 nonsense mutations have been recognized to be pathogenic.Homozygous or compound heterozygous mutations in the high-temperature requirement A serine protease 1 gene  showed diffuse white matter lesions (WMLs) and microbleeds in both of them. Genetic sequencing revealed two novel heterozygous nonsense mutations: c.1096G>T (p.E366X) and c.151G>T (p.E51X).We described two Chinese patients diagnosed with HTRA1-CSVD. Attention should be paid to young patients with ischemic stroke as the first clinical manifestation. Genetic screening for such sporadic CSVD is recommended, even if the symptoms are atypical.This case report expands the clinical, radiographic, and genetic spectrum of  HTRA1) which is located on chromosome 10 (10q26) and encodes the high-temperature requirement serine protease.Cerebral small vessel diseases (CSVD) are a series of clinical, imaging, and pathological syndromes that affect small arteries, arterioles, venules, and capillaries within the brain. It increases the risk of acute cerebrovascular events and mainly manifests in cognitive dysfunction . CerebraHTRA1 mutations include heterozygous, homozygous, and compound heterozygous ones. There are two possible phenotypes related to HTRA1 mutations: classic CARASIL (OMIM 600142) and CADASIL2 which is also known as HTRA1-CSVD (OMIM 616779)  and microbleeds. Genetic sequencing revealed two novel heterozygous nonsense mutations of c.1096G>T (p.E366X) and c.151G>T (p.E51X), supporting HTRA1-CSVD. Our findings provide further insights into the relationship between genotypes and phenotypes.Recent studies have found that nonsense mutations are also pathogenic. Thus, there are 11 heterozygous nonsense mutations according to the ClinVar database, but clinical features and the possible correlation between genotypes and phenotypes of A 44-year-old man presented to our department due to weakness of the left upper limb over the past 4 days. He had no prevailing risk factors for cerebral infarction such as hypertension, diabetes mellitus, atrial fibrillation, or smoking. He denied any memory loss, dysarthria, or loss of consciousness. He had no history of alopecia or lumbago. None of his family members had relevant health problems or hereditary diseases.Neuropsychological tests showed no signs of cognitive impairment. His blood indicators, cervical vascular ultrasound, and dynamic electrocardiogram showed no significant abnormalities. Brain MRI showed an acute infarct in the right basal ganglia on DWI images , diffuseDuring the follow-up, this patient was admitted to the hospital because of recurrent ischemic stroke despite antiplatelet therapy with no complaint about cognitive impairment. Brain MRI showed acute infarct in the region next to the left lateral ventricle on DWI , diffuseHTRA1 nonsense mutation   . The varA 53-year-old man presented to our department due to dysarthria and weakness of the left limb for 17 hours. He had no past medical history and vascular risk factors. The patient could not perform some simple tasks in daily life and lost his previous interests at the age of 49. He did not have migraine, alopecia, or lumbago, which are characteristic features of monogenic CSVD. His family history was unremarkable and his family members were all healthy.On neurological examination, the patient showed a pronounced slow response to cognitive tests. The Mini-Mental State Examination (MMSE) score was 24 and the Montreal Cognitive Assessment (MoCA) score was 17. His cerebral vessels showed no significant abnormalities on cerebrovascular examination. Brain MRI showed acute infarct of the left corona radiata on DWI images, diffuse WMLs and brain atrophy, Fazekas scale score 3 on T2W/FLAIR images, chronic microbleeds in the lobar regions, and deep white matter on SWI images.Given the presentation of early-onset progressive dementia, ischemic stroke at a young age, and brain MRI demonstrating extensive WMLs and brain atrophy –E, wholeHTRA1-CSVD patients with ischemic stroke at a young age, with two unique nonsense mutations: c.1096G >T (p.E366X) and c.151G>T (p.E51X). To date, only 11 heterozygous nonsense mutation sites have been reported. Coste et al. (HTRA1 stop codon variants are not restricted to a specific domain but are present throughout the gene. The present p.E366X and p.E51X mutations may lead to nonsense-mediated mRNA decay (NMD) or truncation of the protein in front of the functional protease domain. These conditions may result in the loss of protein functions. Coste et al.  for the publication of any potentially identifiable images or data included in this article.Preparing the original draft: WC. Reviewing, editing, supervision, and conceptualization: DW. Genetic data analysis: LS and XY. Clinical data acquisition: YW and SH. All authors have read and agreed to publish this report.


Dicentrarchus labrax) hatcheries with noticeable high mortality. This study was conducted on tank-cultured D. labrax frys at a private marine hatchery near Mariout Lake . Frys showed a high mortality rate (70%), lethargy, darkening, asphyxia, ascites, and velvety skin appearance. Both infectious agents were presumptively identified in all investigated frys. The identities of the two recovered agents were confirmed by molecular assay and phylogenetic analysis. On the tissue level, histopathological examination of skin, splenic, and renal tissue indicated severe alterations due to the direct impacts of both infections. On the cellular level, scanning electron micrographs showed both protozoal and bacterial pathogens on/in gill epithelial cells in solitary and colonial forms. Vibrio alginolyticus showed variable results for tested antibiotics, with a higher sensitivity to florfenicol. A successful control strategy was strictly adopted to overcome infections and stop mortalities. Copper sulphate and hydrogen peroxide were efficiently applied to tank water to overcome A. ocellatum infections. Further, florfenicol was effectively used to overcome systemic V. alginolyticus infections. The efficacy of treatments was confirmed by the absence of infectious agents in randomly collected fish samples. To the best of the authors’ knowledge, this study is one of the earliest Egyptian studies that dealt with the dilemma of mass kills associated with external parasitic/systemic bacterial infections among hatchery-reared European seabass.Amyloodiniosis and vibriosis are serious diseases in European seabass ( The majDicentrarchus labrax L.) is one of the most important commercial fish species in the Mediterranean basin  was applied to control infections affecting European seabass frys. According to Wang et al. [2O2 was added to tank water at 25 mg/L for 30 min/day for 3 successive days. According to Francis-Floyd and Floyd [An improved therapeutic regimen using a combination of treatments [florfenicol, hydrogen peroxide .The results of gene expression were analysed using one-way-ANOVA test. The data were considered statistically significant when 2.13.This study was approved by the Institutional Animal Care and Use Committee of Cairo University .3.3.1.D. labrax frys demonstrated lethargy, dark discoloration, and sluggish swimming behaviour. Velvety skin appearance was commonly noticed. Fish showed signs of respiratory distress, including piping behaviour and rapid opercular movement. Abdominal swelling and ascites were also noticed. Some fish died without showing any apparent gross lesions.Eighty-five of 100 frys were clinically diseased. Clinically diseased 3.2.3.2.1.A. ocellatum were spherical, oval, or elliptical to pear in shape and attached firmly to the gills or the skin surface. Its length ranged from 50 to 135 (90.69 ± 2.58). The cytoplasm was opaque brown and appeared as a sac filled with granules. The protozoan presents with rhizoids or stalks through which it attaches firmly to the host tissue . The phylogenetic tree of the ITS regions of this A. ocellatum revealed two main clusters . Depending on the analysis of the present sequence, this parasite is firmly embedded within the family Oodiniaceae and identified as clusters . The fir3.3.D. labrax specimens (85%) were infected with A. ocellatum. The trophont of A. ocellatum was detected in the gills and skin of infected D. labrax. The different parasitic intensities were recorded in infected fish, ranging from mild (<5), moderate [Most of the examined moderate , and sevmoderate  trophont3.4.3.4.1.V. alginolyticus was detected in all tested specimens (pooled samples), except for three groups that were negative (15 fish). Fifteen V. alginolyticus isolates were obtained from the investigated specimens. All fish infected with V. alginolyticus were coinfected with A. ocellatum (75%), whereas only 10% was infected only with A. ocellatum. Isolates were Gram-negative, bacilli, oxidase-positive, and sensitive to O/129 vibriostatic disc. The isolates produced yellow colonies on TCBS agar.3.5.V. alginolyticus strains was submitted to GenBank (accession nos. OK340656 and OL604431). Depending on sequence analysis, the current sequences were confirmed to be V. alginolyticus. V. alginolyticus (OK340656 and OL604431) from diseased fish samples exhibited 100% to 99.93% identity with V. alginolyticus (MN874161.1-MN841280.1-MK102583.1-MN166179.1-MN945277.1). The neighbour-joining phylogenetic tree constructed based on the sequenced 16S rRNA genes of V. alginolyticus was grouped with their relevant V. alginolyticus sequences which were genetically apart from other related species (V. vulnificus (99%) and V. parahaemolyticus (97%) depending on their degree of similarities , except for the three negative groups. Similarly, Moustafa et al. [V. alginolyticus prevalence in rigorous mortality outbreaks affecting maricultured D. labrax in earthen ponds and floating net cages in Northern Egypt. This bacterial agent is a critical pathogen for numerous fish species, including seabass and seabream in the Mediterranean either in natural fisheries or captivity, causing colossal mortality [a et al.  recordedortality ,12.A. ocellatum, can produce wounds and injuries on the external surfaces of fish, which are believed to be effective portals of entry for Vibrio spp [V. alginolyticus were coinfected with A. ocellatum, suggesting a role for such protozoal infections in the emergence of vibriosis affecting D. labrax. Previous studies showed a direct link between bacterial and parasitic infections [A. ocellatum can adversely affect the health status and feeding behaviour of fish, with consequent death due to acute anoxia and asphyxia [A. ocellatum can potentiate numerous opportunistic bacterial infections like V. alginolyticus. Parasites often gain entry to aquaculture facilities due to insufficient quarantine and biosecurity measures. Infected broodstocks, contaminated water, and food are potential sources of infections [A. ocellatum can be transmitted through direct contact with live dinospores and via aerosolized droplets [External protozoal infections, such as brio spp . In thisfections ,48. Heavasphyxia . Attachmfections ,51. A. odroplets .D. labrax specimens (70%) was infected with A. ocellatum. The protozoan was identified in fish skin and gills, and the parasite intensity ranged between 5 and 20/microscopic fields). Frys kept at high densities in tanks are commonly confined to a small area providing favourable circumstances for parasitic infections [Vibrio infection starts with bacterial adhesion to fish mucus, proliferation, and colonization of epithelial tissues. Vibrios secrete numerous toxic hydrolytic enzymes, potentiating further pathogenic invasions into tissues and the initiation of septicaemic infections . The grefections .V. alginolyticus was genetically identified by sequencing the 16S rRNA gene. The 16S rRNA gene sequence of V. alginolyticus was virtually (>99%) identical to its correlated bacteria. The genetic characterization of the current protozoan took place by sequencing the ribosomal internal transcribed spacer regions (ITS rDNA) for A. ocellatum. The ITS rDNA regions flanking the small subunit (18S rDNA) and large subunit rDNA genes (28S rDNA) are significant genetic markers for recognizing fish protozoans. Several studies successfully used these genes to genetically identify A. ocellatum [In this study, the identity of cellatum .A. ocellatum due to the immune reaction of the fish body against infection with either protozoal parasites alone or coinfection with V. alginolyticus. Fish acquire immunity by identifying different foreign bodies, such as bacteria, viruses, and parasites, with the aid of different immune cells, such as macrophages, eosinophils, and lymphocytes (T or B cells) [A. ocellatum for presenting the antigen to T cells to complete the immunological function and produce different cytokines. The results were consistent with Nozzi et al. [A. ocellatum-infected gills.To express the immune genes, the three different genes, IL-1β, IL-6, and TNF-α, were up-regulated during infection with B cells) . The thri et al. , who disV. alginolyticus isolates; therefore, it was used as the core systemic therapy with H2O2 and copper sulphate to control infections in the studied hatchery. Previous research showed that florfenicol is effective against vibriosis in vivo [A. ocellatum in aquaculture [2O2 was used with copper sulphate to competently control A. ocellatum infestations in this study. According to Montgomery-Brock [2O2 is highly efficient against A. ocellatum. The therapeutic regime  controlled infections and reduced fish mortality from 70% to 10% after receiving the prescribed chemotherapeutics.Frequent and irresponsible applications of antibiotics in fish farming to control infections resulted in the emergence of uncountable resistant bacterial strains. The retrieved isolates exhibited different patterns of sensitivity against tested antibiotics. According to antibiotic sensitivity testing, florfenicol 30 µg was the most effective antibiotic against  in vivo ,53. Coppaculture . Howeveraculture . Therefory-Brock , H2O2 isA. ocellatum attached to secondary gill lamellae [Vibrio infection. Histopathological lesions exhibited through internal organs are frequent in bacterial septicaemic infections. Congestion, haemorrhage, degeneration, and necrosis were observed in tissue, correlating with previous findings in Asian seabass, Lates calcarifer [In this study, histopathological alterations suggest mixed bacterial and parasitic infections. Sections prepared from gills revealed many different developmental stages of lamellae ,44. Gramlcarifer . Tissue lcarifer ,56,57.A. ocellatum is a very common protozoal parasite that extensively hammers marine fish frys collected from the wild or reared on natural seawater. It is known to swiftly damage the skin’s immune barriers, with consequent pathogenic bacterial invasions, V. alginolyticus ubiquitously exists in the shallow seawater supply of the Egyptian coastal marine basin, complicating the above parasitic invasion with the ultimate result of heavy mass kills among vulnerable hatchery-reared frys. Ultimately, wise and/or swift therapeutic intervention is vital in restoring hatchery resources and securing a successful rearing cycle.Economic losses associated with marine fry mortalities are very costly when fry rearing costs  are considered. Thus, investigating the triggers and real aetiologies behind such mass mortalities is highly mandated to decisively propose the best corrective action. There are too many marine hatcheries relying on open seawater in Egyptian coastal provinces are found, and these hatcheries are very vulnerable to pathogenic biological invasions originating from natural open water. 5.A. ocellatum is a serious parasitic invader that drastically suppresses the primary physical barriers, which triggers vigorous attacks of the ubiquitous pathogenic V. alginolyticus. Erratic managerial practices and unfavourable water quality parameters drive fish frys to be highly susceptible to various pathogenic invasions, including the nasty V. alginolyticus. Erratic/irresponsible applications of antibiotics in aquaculture together with random dumping off municipal sewage into water streams will possibly result in the emergence of resistant bacterial strains among fishes reared in facilities irrigated by such streams. The adjusted application of water dependent disinfectants as well as an oral therapeutic regimen utilizing FDA-approved oral antibiotics such as florfenicol was very effective in controlling the disease outbreak. Ultimately, adopting strict biosecurity measures pertaining to biological filtering and UV treatment of seawater before passage into hatchery rearing tanks will sharply minimize these pathogenic invasions.


Elatostema stewardii is an important medicinal plant endemic to China. In this study, the complete chloroplast genome of E. stewardii was sequenced and assembled using next-generation sequencing technology. The complete chloroplast genome length of E. stewardii was 150,263 bp, including two inverted repeats (IRs) of 24,681 bp, which are separated by LSC and SSC of 83,791 bp and 17,110 bp, respectively. A total of 129 genes were included in the genome, consisting 85 protein-coding genes, eight rRNA genes, and 36 tRNA genes, the overall GC content of this genome was 36.3%. There are few studies on the genus Elatostema of Urticaceae, this chloroplast genome sequence will provide useful data for further research on solving the generic and familial relationships in Urticaceae. Elatostema stewardii Merrill 1925 (Urticaceae) is a perennial plant native to China. It is commonly found in shady, damp areas near woodlands and streams. As a traditional Chinese medicine, E. stewardii promotes blood circulation, disperses silt, and has detumescence and detoxification effects. Its roots can be used to repair fractures, and its stems and leaves can be used to treat coughs . We used DNA Plantzol Reagent  to extract DNA from silica dried leaves. The leaf samples and extracted DNA were stored at Zhejiang Province Key Laboratory of Plant Secondary Metabolism and Regulation, Zhejiang Sci-Tech University (http://sky.zstu.edu.cn) under the voucher number ZSTU01186 (collected by Zhe-Chen Qi and zqi@zstu.edu.cn). The sequencing libraries were prepared using Illumina's TruSeq Nano DNA Library preparation kit (350 bp median insertion) according to the manufacturer's protocol. Plastid sequences were generated using Illumina Hiseq 2500 platform . Overall, approximately 14.8 million high-quality clean reads (150 bp PE read length) were processed by Trimmomatic  has a total length of 150,263 bp, and consists of a large single-copy region , a small single-copy region , and two reverse repeat regions ). The total GC content of E. stewardii chloroplast genome was 36.3%. The genome contains 129 genes . There were 17 genes with two copies, including six PCG genes , seven tRNA genes , and all four rRNA species . In the genome, 11 protein-coding genes  contain one intron, and three protein-coding genes  contain two introns.The complete Elatostema and 11 other Urticaceae species from NCBI GenBank to study the phylogenetic position of E. stewardii in Urticaceae. Urtica lobatifolia was used as outgroup for constructing the phylogenetic tree. The sequence alignment was conducted by MAFFT v7.450 (Katoh and Standley E. stewardii is sister to a clade formed by E. qinzhouense (E. stewardii will provide necessary genetic resource and background data for further phylogenetic study of the Urticaceae.We obtained chloroplast genome data of all three published zhouense . The com