Lactiplantibacillus plantarum CCFM8724 is a probiotic with the potential to prevent dental caries in vitro and in vivo. To explore the effects of this probiotic at inhibiting Streptococcus mutans-Candida albicans mixed-species biofilm and preventing dental caries, multi-omics, including metabolomics and transcriptomics, was used to investigate the regulation of small-molecule metabolism during biofilm formation and the gene expression in the mixed-species biofilm. Metabolomic analysis revealed that some carbohydrates related to biofilm formation, such as sucrose, was detected at lower levels due to the treatment with the L. plantarum supernatant. Some sugar alcohols, such as xylitol and sorbitol, were detected at higher levels, which may have inhibited the growth of S. mutans. In transcriptomic analysis, the expression of the virulence genes of C. albicans, such as those that code agglutinin-like sequence (Als) proteins, was affected. In addition, metabolomics coupled with a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and RNA-seq revealed that the L. plantarum supernatant had an active role in sugar metabolism during the formation of the S. mutans-C. albicans mixed-species biofilm, and the L. plantarum supernatant was also related to carbohydrate utilization, glucan biosynthesis, and mycelium formation. Hence, L. plantarum CCFM8724 decreased the mixed-species biofilm mass from the perspective of gene expression and metabolic reprogramming. Our results provide a rationale for evaluating L. plantarum CCFM8724 as a potential oral probiotic for inhibiting cariogenic pathogen biofilm formation and improving dental caries. Streptococcus mutans and Candida albicans have been detected in large amounts in oral plaque biofilms in children with early childhood caries (ECC), being considered to be directly related to ECC [S. mutans is a key contributor to pathogenic dental biofilms, which can convert dietary sucrose into dextrans and acid [C. albicans can robustly interact with S. mutans and has a significant impact on the virulence of dental plaque biofilms [Lactobacillus salivarius [L. paracasei NTU 101 fermented skim soy milk [Dental caries, which is one of the most prevalent oral bacterial infectious diseases, represent a significant public health problem, not only in adults, but also in children . Streptod to ECC . S. mutaand acid . C. albibiofilms . Common livarius  and L. psoy milk , can be Lactobacillus spp. L. salivarius inhibits S. mutans and C. albicans double-species caries biofilms by decreasing the amount of caries pathogenic species and reducing the biofilm mass in vitro [L. casei strain Shirota for 28 days, the abundance of Veillonella and Kingella increased significantly, while the abundance of some other bacteria related to caries was decreased in the oral cavity of young adults, as shown by the 16S rRNA sequencing results [L. plantarum CCFM8724 by reducing the dual-species biofilm mass and decreasing the caries score in dental caries rat models. However, how the L. plantarum CCFM8724 cell-free supernatant affects this cariogenic S. mutans and C. albicans mixed-species biofilm environment and how it affects the biofilm metabolome and transcriptome remains unclear.At present, there is experimental evidence of the inhibition of dental plaque biofilms by in vitro . Another results . Differe results , quorum  results , and red results . In our  results ,13, we eL. plantarum CCFM8724 inhibiting dual-species biofilms, we employed a combination strategy using untargeted metabolomics and transcriptomics, revealing the distinct metabolism and gene expression characteristics that are important for S. mutans and C. albicans mixed-species biofilm formation. This work reveals the mechanism of microbe–microbe interaction and provides promising baseline information for the potential use of this probiotic.To explore the mechanism of extracellular probiotic metabolites of Methanol (high-performance liquid chromatography (HPLC) grade) and acetonitrile (HPLC grade) used for metabolite extraction were purchased from Merck KGaA . Pyridine, N-methyl-N-(tri-methylsilyl) trifluoroacetamide with 1% trimethylchlorosilane (MSTFA + 1% TMCS) and methoxyamine hydrochloride (MeOX) used for metabolite derivatization were obtained from Sigma-Aldrich .L. plantarum CCFM8724 was cultured in MRS broth  under anaerobic conditions at 37 °C for 24 h. The supernatant was collected by centrifugation at 6000× g at 4 °C for 10 min and filtered through a 0.22 μm sterile membrane, then stored at 4 °C before use. S. mutans ATCC 25175 and C. albicans ATCC18804 were purchased from the China General Microbiological Culture Collection Center . S. mutans was inoculated in tryptic soy broth  and C. albicans was cultured by yeast extract peptone dextrose medium  under aerobic conditions at 37 °C. When generating the dual-species biofilm, 5% sucrose was added to the medium.S. mutans culture suspension and equal C. albicans culture suspension were added into each well of a 96-well microtiter plate. The number of S. mutans and C. albicans per well was 107 and 106 CFU mL−1, respectively. A total of 50 μL of Lactobacillus supernatant was added at the same time. The same volume of MRS broth instead of supernatant was used as control. Biofilms were formed in a 96-well microtiter plate for 24 h at 37 °C. Then, the contents of each well were removed and washed twice with phosphate buffer saline (PBS) to remove the planktonic cells. Next, the biofilm was scraped by using 200 μL non-enzyme pipette tips and finally pooled together to collect approximately 50 mg of biofilm mass for metabolite extraction and RNA-seq analysis [For each replicate, 75 μL of analysis . After qv/v/v). A total of 20 μL of ethyl undecanoate (C21:0) (0.3 μmol/sample) and 100 μL of magnetic beads were added into each sample before extraction. The C21:0 was used as an internal standard. The mixture was homogenized in a ball mill for 45 sec at 65 Hz and stopped for 15 s [g for 15 min at 4 °C. The supernatant of the extracts was dried in a vacuum centrifuge  and resuspended in MeOX-pyridine and MSTFA with 1% TMCS for derivatization. For each group, six parallel experiments were completed [The frozen sample was added with 0.4 mL ice-cold extraction solvent (acetonitrile:methanol:water) = 2:2:1 analysis was performed on a Thermo trace 1310 gas chromatograph coupled to an Thermo TSQ8000_evo quadrupole mass selective detector equipped with an RTX-5MS capillary column . The detection and metabolite identification using metabolomics were performed as the methods described by Lu . Brieflyhttps://www.metaboanalyst.ca/, accessed on 13 October 2021).The obtained data of GC–MS were analysed as followed. ABF converter 4.0 was used to convert “raw” format files into “abf” format. The MSDIAL3.48 equipped with the FiehnLib database was used for raw peaks exaction, retention time adjustment, peak alignment, deconvolution analysis, and peak identification as reported in a previous study . Statist® spectrophotometer , and Qubit® RNA assay kit in Qubit® 2.0 Flurometer . Library preparation for strand-specific transcriptome sequencing and RNA-seq were performed by Novogene . Briefly, the library of each sample was generated by 1 μg of RNA and then sequenced using the Illumina Hiseq platform (125 bp/150 bp paired-end reads). Raw data were processed by custom Perl scripts to obtain the clean data. The clean reads were mapped to the S. mutans reference genome (https://www.ncbi.nlm.nih.gov/genome/?term=ATCC+25175 accessed on 13 October 2021) and C. albicans reference genome  using the Hisat2 software program with default parameters. On the basis of the clean data, the edgeR package was adopted to identify differentially expressed genes (DEGs) using the following criteria: adjusted p value < 0.05 and log2FC > 1. DEGs were subjected to a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis [Total RNAs were isolated from frozen biofilms using TRIzol  and the DNA-free kit (Ambion (Thermo Fisher Scientific), Shanghai, China). Purity and concentration of RNAs were checked using the NanoPhotometeranalysis . Each gr®Green Supermix , in a CFX ConnectTM real-time system . From the real-time PCR data, Cq values were obtained to calculate each organism abundances. RT-qPCR primers are listed in DNA was extracted from the biofilm according to the protocol in FastDNA SPIN Kit for Soil 50T . Quantitative real-time PCR (qPCR) was performed according to the protocol provided by iTaq™ Universal SYBRThe OrthoMCL software program was used for the homologous gene analysis. Open reading frames in the genome were predicted to obtain the amino acid sequence of enzymes based on the Clusters of Orthologous Groups of protein (COGs) and KEGG databases. The HMMER3.1 software program was used to predict from amino acid sequences aligned to the Carbohydrate active enzymes database (CAZy).t-test. In the metabonomic analysis, a p value < 0.05 indicated statistical significance. In pathway enrichment, a false discovery rate (FDR) analysis was performed to adjust the p value to q value. A q value < 0.05 indicated statistical significance.R studio 4.0.1 with the R-packages ggplot2 and ggsci was used to visualize the results. The differences in this research between groups were determined by a L. plantarum supernatant or MRS broth and were subjected to untargeted metabolomics to determine the effect of small molecules in L. plantarum in the biofilm formation. We first used principal component analysis (PCA) to visualize the metabolomic similarities of the samples in a group and the differences in samples between groups. PCA can project complex GC–MS data into a lower dimensional space, and the biofilm samples marked with different colours were projected onto a two-dimensional space ), and clustered separately. PC1 and PC2 were responsible for 61.8% and 13% of the variation, respectively, indicating that the two groups could be distinguished based on their metabolic compounds  is a supervised extension of PCA that can clearly separate different groups of data . The scoCollectively, the data analysis of the biofilm metabolites yielded 216 metabolites. In the PLS-DA models, metabolites with a variable importance for the projection (VIP) value greater than 1 made a significant contribution to the separation between the control and treatment groups . The VIPp < 0.05, log2(fold change (FC)) > 1). As shown in p < 0.05, log2FC ≤ −1), 31 metabolites were upregulated , and the others were non-differentially expressed . To display the differences in the metabolite content and the similarities in mixed-species biofilms, only the significantly changed metabolites (top 20) were selected to construct a heatmap  and pathway topology analysis (PTA) results, were analysed  KEGG results are presented on the y-axis. The closer the circle gets to red, the more significant the change in the compound is [C. albicans, which were amino sugar and nucleotide sugar metabolism, galactose metabolism, fructose and mannose metabolism, pentose and glucuronate interconversions, and starch and sucrose metabolism, as shown in S. mutans: inositol phosphate metabolism, fructose and mannose metabolism, starch and sucrose metabolism, and amino sugar and nucleotide sugar metabolism  and polysaccharide lyases (PLs), but fewer genes encoding carbohydrate esterases (CEs), glycoside hydrolases (GHs), auxiliary activity (AA), and glycosyltransferases (GTs)  E,F. The S. mutans-C. albicans mixed-species biofilms was observed when the L. plantarum supernatant was added. In this study, we employed multi-omics to reveal the distinct metabolic reprogramming and gene expression during S. mutans-C. albicans dual-species biofilm formation with the addition of the L.plantarum supernatant.In our previous studies ,13, the L. plantarum supernatant treatment, the intracellular remaining sucrose increased. Sucrose can be utilized to generate extracellular polymeric substances (EPS) to help cells form biofilms [S. mutans in dental biofilms, especially in the presence of C. albicans in a caries rat model [C. albicans, thus promoting its growth [S. mutans has multiple pathways that use sucrose, such as cutting the α-1,2-linked bond of sucrose to generate organic acids and converting sucrose into extracellular polymer glucan via several glycosyltransferase enzymes (Gtfs) [Metabolic data revealed that most of the impacted metabolites were involved in the carbohydrate metabolism A. Sucrosbiofilms . A diet at model . Sucroses growth . Importas (Gtfs) . The bios (Gtfs) .S. mutans abundance compared to sucrose-sweetened milk. Moreover, it is clear that sorbitol can reduce the acid production of S. mutans in vitro, inhibit acid production in dental plaque in vivo, and prevent dental caries [L. plantarum may be the antimicrobial material basis, which requires further studies.In contrast, the xylitol and sorbitol levels were higher in the treatment group than in the control group C. Burt el caries . These rS. mutans can quickly lower the environmental pH, while S. mutans is also tolerant to low pH [S. mutans and identified 73 unique core genes, most of which are related to carbohydrate metabolism and acid resistance. However, some organic acids, such as 3-phenyllactic acid and p-hydroxylphenyllactic acid, were detected at higher levels in the treated group  proteins is associated with cell adhesion and biofilm formation [L. plantarum supernatant inhibited the adhesion ability of C. albicans. Furthermore, the genes associated with the cell wall components (Csp2 and Atc1) were downregulated.Contrarily, we observed that the genes related to the filamentous growth of fungi, including ivariuin . A familormation . The resC. albicans in mixed-species biofilms are listed in L. plantarum supernatant and was related to 140 DEGs. Adenosine-5-monophosphate, an organic compound consisting of adenine, ribose, and phosphoric acid, is a component of the extracellular DNA (eDNA). eDNA is an important biofilm component that was recently discovered. A study by Lucio [Pseudomonas aeruginosa, S. intermedius, and S. mutans.Apart from the genes mentioned above, other important genes related to the metabolites of by Lucio  indicateL. plantarum strains (L. plantarum CCFM8724 and CCFM361) in GH family, the highest proportion of the two strains, is a critical enzyme group accounting for the bacterial adaptation capacity to the host’s environment, through hydrolysing the dietary and host-produced carbohydrates [L. plantarum CCFM8724 has a higher CBM level, which can combine with GH family to degrade chitin or peptidoglycan to inhibit the pathogen growth, especially the CBM50  proteins, was affected. Our results strongly confirm that L. plantarum CCFM8724 can decrease the biofilm mass, regardless of the gene expression or metabolic reprogramming; therefore, it has the potential to act as a therapeutic agent for the prevention and treatment of caries.In conclusion, the multi-omics analyses provide new insights, showing the inhibition of the


Lactobacillus salivarius has drawn attention because of its promising probiotic functions. Tolerance to the gastrointestinal tract condition is crucial for orally administrated probiotics to exert their functions. However, previous studies of L. salivarius have only focused on the bile salt resistance of particular strains, without uncovering the common molecular mechanisms of this species. Therefore, in this study, we expanded our research to 90 L. salivarius strains to explore their common functional genes for bile salt resistance. First, the survival rates of the 90 L. salivarius strains in 0.3% bile salt solutions were determined. Comparative genomics analysis was then performed to screen for the potential functional genes related to bile salt tolerance. Next, real-time polymerase chain reaction and gene knockout experiments were conducted to further verify the tolerance-related functional genes. The results indicated that the strain-dependent bile salt tolerance of L. salivarius was mainly associated with four peptidoglycan synthesis-related genes, seven phosphotransferase system-related genes, and one chaperone-encoding gene involved in the stress response. Among them, the GATase1-encoding gene showed the most significant association with bile salt tolerance. In addition, four genes related to DNA damage repair and substance transport were redundant in the strains with high bile salt tolerance. Besides, cluster analysis showed that bile salt hydrolases did not contribute to the bile salt tolerance of L. salivarius. In this study, we determined the global regulatory genes, including LSL_1568, LSL_1716 and LSL_1709, for bile salt tolerance in L. salivarius and provided a potential method for the rapid screening of bile salt-tolerant L. salivarius strains, based on PCR amplification of functional genes. Lactobacillus salivarius was first isolated from children’s saliva in Maryland, USA [and, USA . Later sand, USA ,3,4.L. salivarius has drawn attention because of its promising probiotic functions, such as anti-aging, immunoregulation, and bacteriostasis [L. salivarius FDB89, isolated from the feces of centenarians, extended the mean life span of Caenorhabditis elegans by up to 11.9% compared to that of the control group [L. salivarius strain, FXJCJ7-2, was shown to have significant anti-inflammatory effects on lipopolysaccharide (LPS)-treated murine macrophages and mice treated with LPS [Staphylococcus aureus infections found that L. salivarius KCTC3156 was a potential novel anti-staphylococcal agent that could be effective against S. aureus biofilms [In recent years, iostasis . A studyol group . FurtherTolerance to the gastrointestinal tract environment is a decisive factor for probiotics to exert physiological benefits in the gut. The adverse environment in the human intestine to probiotics is mainly caused by bile salts (average concentration of 0.3%) .Bile salts can lead to high osmotic pressure and affect bacterial cells via protein denaturation, DNA damage, intracellular acidification, oxidative stress, and metabolic changes . BacteriL. plantarum WCFS1 became rough and the whole cells began to atrophy under bile salt conditions [Propionibacterium freudenreichii [L. acidophilus [L. acidophilus strains, lacking the transporter genes belonging to the efflux system, exhibited a significant increase in sensitivity to bile [L. johnsonii PF01 reported that cell wall biosynthesis was improved in a bile salt environment [Cell walls and envelopes are the first line of defense of bacteria against external agents. The cell surfaces of nditions . Similarnreichii  and L. adophilus . The spedophilus . Bacteri to bile . In addi to bile . Due to  to bile . Furtherironment .L. fermentum NCDC 400 identified 80 proteins responsive to the bile salt environment, including proteins that function in the stress response, DNA repair, peptidoglycan biosynthesis, and metabolism [Bifidobacterium longum BBMN68 [Bacillus coagulans HS243 based on comparative genomic analysis showed two groups of bile-salt-tolerance-related genes, including a single-copy chologlycine hydrolase-encoding gene and four chaperone-related genes [With the rapid development in experimental techniques, a multi-omics method has been applied to bacterial bile salt resistance research, simplifying the exploration of strain-specific biomarkers. For example, a quantitative proteomic analysis of tabolism . Anotherm BBMN68 . In addied genes .L. salivarius in this field is still limited, and most of these studies only focused on a single strain. One study of L. salivarius LI01 revealed that its bile salt tolerance was related to cell envelope modification, regulatory systems, general stress response, and the central metabolic processes [L. salivarius Ren reported similar results [However, despite this expansion in the use of multi-omics techniques, research on rocesses . Another results . NotablyL. salivarius strains in bile salts were tested. Then, the candidate functional genes for bile salt tolerance of L. salivarius were identified via comparative genomics analysis and verified via RT-PCR and gene knockout experiments. The result provided a potential method for the rapid screening of bile-salt-tolerant strains based on PCR amplification.To determine the ability of strains’ bile salt resistance more efficiently, the survival rates of 90 L. salivarius strains used in this study were isolated from 500 stool samples of Chinese populations in 17 provinces. The stool samples were incubated in the modified medium under an anaerobic condition [https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 10 November 2019)).The 90 ondition . Sanger L. salivarius were cultured in the basic MRS broth for successive three generations at 37 °C. Bacterial genomic DNA was then extracted using a Rapid Bacterial Genomic DNA Isolation Kit  following the manufacturer’s instructions. Whole-genome sequencing was performed using an Illumina HiSeq platform  to generate 350-bp paired-end reads. High-quality sequences were assembled using SOAPdenovo2 (https://github.com/aquaskyline/SOAPdenovo2 (accessed on 28 December 2019)) [https://github.com/BGI-Qingdao/TGS-GapCloser (accessed on 28 December 2019)) [http://exon.gatech.edu/GeneMark/license_download.cgi (accessed on 28 December 2019)) [L. salivarius strains have been deposited in the NCBI database. The accession numbers of each strain are listed in Strains classified into r 2019)) . The innr 2019)) . Genes wr 2019)) . The genL. salivarius strains used in this study is listed in This study was approved by the Ethics Committee in Jiangnan University, China (SYXK 2012-0002). All donors or their legal guardians provided written informed consent before sample collection. The collection of fecal samples caused no harm or discomfort to the participants. Detailed information about the 90 Bile salt solutions were prepared according to a published method, modified by changing the final concentration of bile salts to 0.3% .v/v) inoculum of each strain was inoculated into 10 mL of fresh MRS broth and was grown for 12 h. The OD600 was measured, and the culture of each strain was diluted with MRS to the same absorbance. Cells in the diluted culture of each strain (1 mL) were collected via centrifugation  and washed twice with sterilized normal saline.Strains were grown overnight in basic MRS broth at 37 °C for 12 h. After subculturing twice, a 2%  and mixed with a 0.3% bile salt solution (0.5 mL). The mixture was then incubated at 37 °C for 4 h. The total number of viable bacteria was determined after the tolerance assay. All experiments were conducted in triplicate. The survival rates of L. salivarius strains in bile salt solutions were calculated according to a previous method [An aliquot (0.5 mL) of each washed cell suspension was used for the original viable count test. Cells in another 0.5 mL aliquot were collected via centrifugation ) [https://www.megasoftware.net/dload_win_gui (accessed on 28 February 2020)) [L. johnsonii PF01 were used as an outgroup to construct the phylogenetic trees (www.evolgenius.info/evolview (accessed on 15 March 2020)).Three subtypes of BSHs , as described in a previous study , were idy 2020)) . The phyy 2020))  through ic trees . The phyL. salivarius strains with high survival rates in the bile salt solutions (the tolerant group) and the bottom six strains with low survival rates (the non-tolerant group) were selected for comparative genomic analysis.The top eight https://orthomcl.org/orthomcl/ (accessed on 10 February 2020)) [https://github.com/ncbi/pgap (accessed on 15 February 2020)) [The core gene database of the tolerant group was built using OrthoMCL (y 2020))  with they 2020)) , pickinghttp://exon.gatech.edu/GeneMark/license_download.cgi (accessed on 9 February 2020)) [https://ftp.ncbi.nih.gov/blast/db/ (accessed on 25 February 2020)), and COG (http://archive-dtd.ncbi.nlm.nih.gov/COG/ (accessed on 25 February 2020)) databases using BLAST with the default parameters. KEGG pathway annotations were conducted using DAVID 6.7 online (https://david.ncifcrf.gov/ (accessed on 25 February 2020)).The protein sequences of all the genes picked above were predicted using Genemark (y 2020)) . Next, tThe strains with the highest and lowest survival rates were incubated in the bile salt solutions for 4 h after three subcultures, and a parallel culture grown in fresh MRS broth for the same duration was used as a control.g, 10 min) in an enzyme-free tube and then mixed with 200 μL of lysozyme solution. The mixtures were incubated at 37 °C for 30 min to break the cell walls. Cells without cell walls were collected via centrifugation . Total RNA from each strain was extracted using the TRIzol Plus RNA Purification Kit according to the manufacturer’s protocol . The RNA concentrations were measured using an RNA assay with a NanoDrop spectrophotometer . cDNA was synthesized in a 20 μL reaction using a HiFiScript gDNA Removal cDNA Synthesis Kit . qRT-PCR was performed in a 96-well plate following the TransStart Tip Green qPCR SuperMix kit  instruction. The thermocycling program was set as previously described [http://www.primer3plus.com/ (accessed on 7 May. 2020)) and were synthesized by the Sangon Biotechnology Laboratory  (Cells in the culture of each strain (1 mL) were collected via centrifugation  . The 16SEscherichia coli Top10 cells.A knockout experiment based on the Cre-lox system was performed according to a previously reported method, with minor modifications . BrieflyE. coli Top10 cells harboring the constructed plasmid were grown aerobically in Luria–Bertani (LB) broth at 37 °C. The plasmids were extracted using the GeneJET Plasmid Miniprep Kit , following the manufacturer’s instructions. The plasmid concentrations were measured using a dsDNA assay with a NanoDrop spectrophotometer .L. salivarius FWXBH36M1 cells were prepared according to a previously published method [2, 0.1 mM CaCl2, and 75.2 mM sorbitol) was added, and the cells were incubated for 4 h at 37 °C. Bacteria were grown on MRS plates containing 20 μg/mL chloramphenicol as a selective marker. The transformants were confirmed via PCR using vector-specific primers . The survival rates of six strains were ≤0.7%, and L. salivarius FYNDL5M1 exhibited the lowest survival rate (0.02%).To compare the tolerance of different al rates . Among tp < 0.10). Strains from the Guangdong, Gansu, Anhui, and Jiangsu provinces showed higher survival rates. In contrast, strains from the Fujian, Chongqing, Yunnan, and Zhejiang provinces showed lower survival rates. This tendency suggests that the tolerance of strains to bile salt solutions varied with their isolated regions. However, the area-dependent influence disappeared when the strains were classified according to the isolated parts of China (p > 0.10).Notably, this phenotypic variation was significant when the analysis was performed according to the isolated region of each strain . Among them, FWXBH9M2 and FSDHZD3L5 had double BSHB-encoding genes. The other four strains only had one type of BSH-encoding gene (three with BSHA only and one with BSHB only). However, the numbers and types of BSHs in each strain did not show region-dependent diversity  and six non-tolerant  strains was performed.To determine the potential mechanisms of how functional genes were involved in the bile salt tolerance of LSL_1709) was highly homologous with the Ydr533c protein-encoding genes in Saccharomyces cerevisiae, which are upregulated in response to various stress conditions [The results showed that there were 15 tolerant core genes deleting in at least two non-tolerant strains a. Among nditions .LSL_1716 and LSL_1715) encoding the PTS system transporter subunits IIB and IIC, respectively, were involved in mannose transmembrane transport. Two genes (LSL_1714 and LSL_1713), which encode for the PTS system transporter subunits IIABC and IIBC, respectively, are involved in N-acetylglucosamine transmembrane transportation  b. In theortation .Next, the whole genomes of the tolerant strains were compared with the core genes of the non-tolerant group using PGAP to determine the redundant genes under bile salt solutions. The results showed seven different genes between the two groups, including one TetR/AcrR family transcriptional regulator-encoding gene, one site-specific integrase-encoding gene, one aspartate carbamoyltransferase-encoding gene, and four genes that encode hypothetical proteins . The copLSL_0261, LSL_0166, and LSL_0167 were related to substance metabolism and transportation   and non-tolerant strains (FYNDL5M1 and FCQHC3ML6) to determine the transcriptional changes of the functional and redundant genes identified above upon culture in bile salt solutions. According to their genome coordinates and functions, the tolerance-related genes could be classified into five groups , and onend xerC) a.LSL_1568, LSL_1716, LSL_1709, and LSL_0951 were increased in both strains when strains were grown in bile salt solutions (rfaG showed a slight decrease in FWXBH36M1 (0.18 on average); in FJLHD9M1, it showed a maximum increase (41.12 on average).For the tolerance-related genes, the results showed that the relative expression level of olutions a,b. HoweLSL_1568, LSL_1716, and LSL_1709 showed remarkable changes compared with that of LSL_0951. There was an opposite trend in the relative expression level of these functional genes in FJLHD9M1, where LSL_0951 demonstrated a higher expression level than others. The differences in the fold-changes of the functional genes upon growth in a bile salt system may explain the intra-strain diversity in the bile salt tolerance of L. salivarius.The four genes with increased relative expression levels were changed to different extents in the two tested strains. In FWXBH36M1, the relative expression of In addition, an inconsistency in the relative expression of the redundant genes was found in strains FYNDL5M1 and FCQHC3ML6, despite the noticeable growth of all four genes c,d. ThisL. salivarius to bile salt solutions, in-frame deletions of the top three most highly relatively expressed genes in FWXBH36M1 were performed. Confirmation of positive transformants via DNA electrophoresis is shown in To prove that the functional genes obtained above influenced the tolerance of p > 0.05), indicating that gene knockout did not affect the growth of the strains under normal conditions.Compared with that of the wild-type strain, the growth profile of the three mutant strains in MRS did not show any significant differences (p < 0.05). Additionally, the survival rates of the three mutant strains showed a consistent trend with the relative expression of the corresponding genes. Specifically, the knockout of the most highly expressed gene in FWXBH36M1 also led to the most significant drop-in survival rate.However, the survival rates of the three mutant strains were significantly lower than that of the wild-type strain under bile salt conditions (L. salivarius strains in bile salt solutions.Therefore, the functional genes obtained through the comparative genomic analysis were related to the survival rates of L. salivarius via comparative genomic analysis and verified the results via qRT-PCR and gene knockout experiments.In this study, we identified the potential functional genes that confer bile salt tolerance in L. salivarius strains showed intra-strain differences in survival rates in bile salt solutions, indicating that intrinsic bile salt tolerance is strain-specific. Similar results have been reported in previous studies on Bifidobacterium [L. acidophilus strains [L. salivarius [Lactobacillus [L. plantarum [Ninety acterium  and L. a strains . Considelivarius , phenotybacillus . Notablylantarum , thus suL. salivarius strains, thus indicating that intra-strain phenotypic differences in bile salt tolerance were induced by mechanisms other than the presence of the BSHs. Similar results have been reported in previous studies on L. johnsonii [L. salivarius [Further analysis of BSH variations did not reveal any associations between the BSHs and the bile salt resistance of ohnsonii  and L. slivarius , indicatlivarius .L. salivarius strains was associated with the peptidoglycan synthesis process. Peptidoglycan is the main component of the cell wall [L. fermentum [L. johnsonii [L. plantarum [Comparative genomic analysis based on the whole bacterial genome showed that the bile salt resistance of ell wall . The celell wall . Therefoermentum , L. johnohnsonii , and L. lantarum  found siL. salivarius strains. PTS is a complex kinase system that catalyzes sugar transport and phosphorylation [Comparative genomic analysis and KEGG enrichment analysis indicated that bacterial PTS also played an important role in the bile salt resistance of rylation . It is crylation . In a strylation . TherefoLSL_1709 in the strain with maximum stress tolerance increased dramatically in the bile salt condition, and the survival rate of LSL_1709-mutant strain significantly lowered in the bile salt solutions. All of these indicated this gene has a strong association with the strains’ bile salt-tolerant ability. Considering the chaperone activity of LSL_1709, its encoding product may involve the strains’ bile salt tolerance by inhibiting the misfolding and denaturation of protein caused by the stress condition [Additionally, the relative expression of ondition .Analysis of the strains’ redundant genes related to bile salt tolerance showed that four potential redundant genes were related to the repair of DNA damage, substance transportation and transcription regulation. All of these can involve repairing bacterial damage caused by bile salts ,50. ThisS. cerevisiae [Bacillus mycoides [Interestingly, the relative expressions of the possible functional and redundant genes varied from strain to strain, indicating that even the same gene has different responses to bile salt conditions in different strain’s genomic backgrounds. This phenomenon can also be observed in revisiae  and Bacimycoides , which mHowever, it should be noticed that this research was based on in vitro experiments only, lacking the corresponding in vivo confirmation. Previous studies have shown that the maximum concentrations of bile salts tolerated by probiotics differ in vivo and in vitro. Besides, the types of bile salts used and the bile conditions in the in vitro experiments were different from those in the human intestine . TherefoL. salivarius. The results indicated that the bile salt resistance of L. salivarius strains was mainly related to the chaperones, PTS and peptidoglycan synthesis. Our work also provided a potential method for rapidly screening the bile-salt-tolerant strains of L. salivarius based on PCR of the functional genes.Overall, in this study, we applied comparative genomic analyses to determine the mechanism of how the gene variations involved in the bile salt tolerance of


Environ. Res. Lett. 10, 114016 (2015)]. The Indus River in South Asia alone supplies water for over 300 million people . When light-absorbing particles (LAP) darken the snow/ice surfaces, melt is accelerated, affecting the timing of runoff. In the Indus, dust and black carbon degrade the snow/ice albedos . During the COVID-19 lockdowns of 2020, air quality visibly improved across cities worldwide, for example, Delhi, India, potentially reducing deposition of dark aerosols on snow and ice. Mean values from two remotely sensed approaches show 2020 as having one of the cleanest snow/ice surfaces on record in the past two decades. A 30% LAP reduction in the spring and summer of 2020 affected the timing of 6.6 km3 of melt water. It remains to be seen whether there will be significant reductions in pollution post−COVID-19, but these results offer a glimpse of the link between pollution and the timing of water supply for billions of people. By causing more solar radiation to be reflected, cleaner snow/ice could mitigate climate change effects by delaying melt onset and extending snow cover duration.Melting snow and ice supply water for nearly 2 billion people [J. S. Mankin, D. Viviroli, D. Singh, A. Y. Hoekstra, N. S. Diffenbaugh,  Snow and ice melt supply water to nearly 2 billion people . The IndThe COVID-19 epidemic has resulted in decreased emissions, especially in the spring of 2020 when many countries were under strict stay at home orders. Air quality in cities worldwide, for example, Delhi, India , improveUsing two remote sensing approaches –8, snow-There were differences between the approaches for the magnitude of LAP concentration, with approach 1 showing overall higher concentrations and a minimum around 33 ppmw, while approach 2 showed lower concentrations and negligible minimum concentration. The snow-covered area and the snow grain size were not significantly different in 2020 than the mean  for both approaches D and 3D 3.An energy balance model, forced with snow cover from approaches 1 and 2, was used to model melt using the observed cleaner snow compared to simulated dirtier snow with mean 20-y LAP concentration. Compared to the dirtier snow, the average volume of melt retained in 2020 is 6.55 kmTwo independent approaches show significant decreases in LAP over the Indus Basin in 2020. This decrease was presumably caused by the COVID-19 lockdowns and associated decreased economic activity, but a more thorough analysis, for example, with in situ measurements of pollutant composition, would be needed to establish causality. Assuming the lockdowns were the cause, this study demonstrates how changes in human behavior can affect the water supply for billions of people.Approach 1 is the MODIS Snow-Covered Area and Grain Size  plus DusThe ParBal model is detailed in previous publications , 13. To ftp://snowserver.colorado.edu/pub/fromRittger/products/Indus and ftp://ftp.snow.ucsb.edu/pub/org/snow/products/SPIRES/Indus/. The source codes for approach 1 are not publicly available. The source codes for approach 2 and the ParBal model are at https://github.com/edwardbair/SPIRES/releases/tag/v1.0 and https://github.com/edwardbair/ParBal/releases/tag/v1.0.The snow cover time space data cubes are available at


The results of this study show the effects of socio-economic determinants, such as higher income levels, high school education, private insurance, and married marital status, have favorable survival in patients with young-onset colorectal cancer (YoCRC). Moreover, most of the positive social factors are often interrelated. The inclusion of these factors could further prognosticate and help with healthcare resource allocation for successful interventions through public health measures. Colorectal cancer awareness, knowledge, and even utilization of medical services would differ with the education and health literacy.Introduction: We aimed to assess the impact of socio-economic determinants of health (SEDH) on survival disparities within and between the ethnic groups of young-onset (<50 years age) colorectal adenocarcinoma patients. Patients and Methods: Surveillance, epidemiology, and end results (SEER) registry was used to identify colorectal adenocarcinoma patients aged between 25–49 years from 2012 and 2016. Survival analysis was performed using the Kaplan–Meir method. Cox proportional hazards model was used to determine the hazard effect of SEDH. American community survey (ACS) data 2012–2016 were used to analyze the impact of high school education, immigration status, poverty, household income, employment, marital status, and insurance type. Results: A total of 17,145 young-onset colorectal adenocarcinoma patients were studied. Hispanic (H) = 2874, Non-Hispanic American Indian/Alaskan Native (NHAIAN) = 164, Non-Hispanic Asian Pacific Islander (NHAPI) = 1676, Non-Hispanic black (NHB) = 2305, Non-Hispanic white (NHW) = 10,126. Overall cancer-specific survival was, at 5 years, 69 m. NHB (65.58 m) and NHAIAN (65.67 m) experienced worse survival compared with NHW (70.11 m), NHAPI (68.7), and H (68.31). High school education conferred improved cancer-specific survival significantly with NHAPI, NHB, and NHW but not with H and NHAIAN. Poverty lowered and high school education improved cancer-specific survival (CSS) in NHB, NHW, and NHAPI. Unemployment was associated with lowered CSS in H and NAPI. Lower income below the median negatively impacted survival among H, NHAPI NHB, and NHW. Recent immigration within the last 12 months lowered CSS survival in NHW. Commercial health insurance compared with government insurance conferred improved CSS in all groups. Conclusions: Survival disparities were found among all races with young-onset colorectal adenocarcinoma. The pattern of SEDH influencing survival was unique to each race. Overall higher income levels, high school education, private insurance, and marital status appeared to be independent factors conferring favorable survival found on multivariate analysis. Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer deaths in both men and women in the United States, with an estimated 147,950 people diagnosed and 53,200 deaths in 2020 ,7. AmongThe influence of these factors on mortality within each racial/ethnic group has not been studied in YoCRC. Resource allocation for successful interventions through public health measures requires a better understanding of the impact of SEDH on YoCRC survival within each vulnerable racial/ethnic group of a diverse population. Outcome disparities exist for CRC between African Americans and Caucasians and can be attributed to the socioeconomic status ,15. AnotThis is a retrospective cohort study of Surveillance Epidemiology and End Results Program-American Census Survey (SEER-ACS) data. Since it is a de-identified dataset, the study was exempted for a review/approval by the institutional review board of the Wright Center for Graduate Medical Education (IRB reference—1698777-1).® NCI) run on the combined SEER 18 incidence registries to obtain demographic, tumor characteristics, and survival data on histologically proven (adenocarcinoma) YoCRC patients . The SEDH data were assessed whether above or below the 50th centile [An area-based measure of seven SEDH parameters available in SEER was used in concurrence with the survival data. [ centile . The par centile .t-test for continuous variables. Median survival (months) was calculated using the Kaplan–Meier analysis. The 5-year survival was analyzed using Log-rank tests and Cox proportional hazards models. Survival time was determined from the date of diagnosis to the last date of follow-up or until the date of death that was cancer-specific as made available through SEER. Hazard ratios (H.R.s) and 95% C.I.s were estimated for univariate and multivariate analysis, using the Cox proportional hazard model. Cancer-specific survival was evaluated for all patients stratified by race/ethnicity and combined as univariate analysis for each SEDH. Cox proportional hazards assumption in the multivariate models were also tested by adding an interaction term with race and follow-up time to the final models. This interaction term was not significant for the overall model or for the models stratified multiple SEDH. All data were analyzed using SPSS v27 for Macintosh (® IBM). As per the data user agreement with SEER, any cell value <11 was censored to prevent re-identification of the rare events.The demographic and tumor characteristics stratified by race/ethnicity were compared by chi-square test for categorical variables and A total of 17,145 young-onset colorectal adenocarcinoma patients were studied . A summaOverall and race-specific survival Overall cancer-specific survival was, at 5 years, 69 m. NHB (65.58 m) and NHAIAN (65.67 m) experienced worse survival when compared with NHW (70.11 m), NHAPI (68.7 m), and H (68.31 m). This is illustrated in p = 0.010), NHB (p = 0.024), and NHW (p < 0.0001). The effect size of improved HR was 0.795 (0.738–0.857). However, no difference was noted with H (p = 0.315) and NHAIAN (p = 0.237)  .p = 0.009), NHW (p = 0.315) and NHAPI (p = 0.002). The effect size of improved HR was 0.811 (0.753–0.875). However, no difference was noted with H (p = 0.075) and NHAIAN (p = 0.304)  .p = 0.001) and NHAPI (p < 0.0001), but not with NHAIN (p = 0.903), NHB (p = 0.337) or NHW (p = 0.835)  .p < 0.0001), NHB (p = 0.012), NHW (p < 0.0001), but not NHAIN (p = 0.106)) ) .p < 0.0001), NHB (p = < 0.0001) and NHW (p = 0.0001) but not in NHAIN (p = 0.335) or NHAPI (p = 0.069)  .p = < 0.0001), NHB (p < 0.0001), NHW (p < 0.0001), NHAIN (p = 0.021), and NHAPI (p = 0.05)  .p = 0.028), but not in other ethnic groups  when compared with NHB . Household income in larger families may contribute to reduced affordability to seek access to health care and accompanying costs. As per the 2018 U.S. Census Bureau, an estimated 21% of NHB and 8% of NHW live under the poverty line . It was Married patients may have potentially greater financial resources and social support, especially if the spouse is employed, and are believed to have better CRC survival when compared with single or divorced patients. In our study, being married conferred a survival advantage in H, NHB, and NHW but not in NHAIN or NHAPI. In a comparative study from the SEER database, Wang et al. demonstrated marriage to be associated with lower mortality for married patients in the patients diagnosed with CRC after adjusting for age, race, cancer stage, and surgery . In thatOur analysis revealed recent immigrant status to confer a survival disadvantage only amongst NHW, but not in other ethnic groups. As per the National Health Interview Survey from 2018, CRC screening was low amongst individuals with lesser than high school education (at 52%), uninsured (at 30%), and recent immigrants  . For YoCDespite a declining trend of overall CRC incidence and mortality in the U.S., NHB patients continue to experience a higher CRC mortality burden than NHW patients ,39. MentIn contrast to CRC over 50 years, YoCRC has not been traditionally detected through screening modalities but through symptomatic presentation to primary care, emergency rooms, or specialist services. Wu et al. demonstrated worse overall survival for YoCRC patients for NHB patients compared with NHW patients . In our Our study has a few limitations. SEER cancer registry data do not include detailed information about chemotherapy regimens, the number of cycles, or radiotherapy treatments, making it impossible to determine which subjects received adjuvants chemotherapy or radiation therapy. Incomplete and underreported data in SEER is also a limitation; however, we excluded entries with incomplete data. Moreover, the SEER insurance variable does not subdivide those with private insurance , and the differences between these subsets could not be analyzed. SEER registries also lack data on age or ethnicity matched controls, co-morbidities, environmental history, family history, tumor genotype (microsatellite instability), which are linked with treatment and outcomes for YoCRC. Despite these limitations, our study has important implications for resource allocation for eliminating barriers determining unfavorable outcomes for YoCRC. We also analyzed the socioeconomic status for different age groups and there was no statistically significant difference for all age ranges between those patients either above or below the 50th centile for poverty, clarifying the lack of relationship between age and socioeconomic status. A robust program of equal access to high-quality, well-coordinated cancer care, greater social support, and comprehensive interventions is required but may not be sufficient unless specific socioeconomic barriers that are unique to each group are effectively overcome to improve cancer-specific survival disparities between different racial groups and within each racial group based on socioeconomic determinant risk stratification.An important and proven cost-effective interventional strategy would be to deploy a patient navigation program which refers to support and guidance offered to patients who come into contact with the health system with a presumptive diagnosis of cancer, either through screening or symptoms to ensure timely coordination and access to complex cancer therapies and to identify and remove socioeconomic barriers to care . PatientWe anticipate our study will generate widespread interest among multidisciplinary teams to risk-stratify patients and facilitate efforts to address barriers in SEDH to achieve favorable cancer-specific survival outcomes.This study showed the effects of SEDH, such as higher income levels, high school education, private insurance, and married marital status, have favorable survival in YoCRC patients. Some of these SEDH are also directly and indirectly related to the patients’ ethnicity. Most of the positive social factors are often interrelated. The presence of these factors should ultimately result in timely diagnosis and better CRC survival prognosis. CRC awareness, knowledge, and even utilization of medical services would differ with the education and health literacy. Further studies are recommended to assess the impact on cancer survival in YoCRC with specific targeted interventions to remove socioeconomic barriers among YoCRC patients.


Cerasus humilis is an important fruit species widely planted for soil and water conservation in northern China due to its strong tolerance to drought and salinity stresses. In this study, a total of 170 LecRLK family genes  were identified in the newly released whole-genome sequences of C. humilis. Furthermore, nine representative LecRLK genes in young plants of C. humilis under varying drought and salinity stresses were selected for qRT-PCR analysis. Our systematic comparative analyses revealed the active participation of these nine LecRLK genes in the salt and drought stress responses of C. humilis. The results from our study have provided a solid foundation for future functional verification of these LecRLK family genes and will likely help facilitate the more rapid and effective development of new stress resistant Cerasus humilis cultivars.Lectin receptor-like protein kinases (LecRLKs) have been shown to be involved in plants’ responses to various biotic and abiotic stresse factors.  Arabidopsis thaliana [Oryza sativa [Pisum sativum [Solanum lycopersicum [Pyrus bretschneideri [Solanum tuberosum [During the whole life cycle of plants, both biotic and abiotic factors have significant influence on the growth, development and even survival of higher plants during their life cycle. Membrane localized receptor-like kinases (RLKs), together with other signal receptors like phytochromes, act as the first site of signal perception, which allows the plants to communicate between cells and to respond to changes in their environment ,2. Lectithaliana , and wera sativa , Pisum s sativum , Solanumpersicum , Pyrus buberosum .GsSRK (encoding a G-type LecRLK) in Arabidopsis boosted seed germination rate, primary root and rosette leaf growth under salt stress conditions [Arabidopsis thaliana LecRK-V.5 (an L-type LecRLK encoding gene) led to early stomatal re-opening, enabling Arabidopsis to have stronger susceptibility to pathogen infection [Based on the lectin domains’ structures, the LecRLK family has been divided into three categories: G-types, L-types, and C-types . G-type nditions . L-type nditions . It was nfection . As the nfection . Contrarnfection ,21.Cerasus humilis is a small woody shrub that is native to northern China [C. humilis [C. humilis have not identified and reported yet.rn China . It has rn China . During rn China ,25. Even humilis ,26–28, tC. humilis have been revealed and published [C.humilis genome and tissue-specific transcriptomes was carried out. Phylogenetic relationship, chromosomal distribution, gene structure, and gene duplication events were further analyzed with a series of bioinformatic tools. Moreover, nine representative genes encoding 5 G-type, 3 L-type and 1 C-type LecRLK genes were selected for qRT-PCR analysis in plants of C. humilis under salinity and drought stresses. The results of this study not only will likely help establish a soild foundation for future functional analysis of the LecRLK gene family in C. humilis, and will also help to speed up the targeted breeding efforts for stress-resistant C. humilis cultivars.Using the high-throughput sequencing technology, the whole genome and several transcriptomes of ublished ,29. In tC. humilis (variety ‘Jinou 1’) was uploaded to the Figshare (accessed with https://doi.org/10.6084/m9.figshare.11669673) from the previous study of our lab  [Arabidopsis thaliana (Araport11) and Prunus persica (Genome v2.0.a1) were downloaded from TAIR database (https://www.arabidopsis.org/) and GDR database (https://www.rosaceae.org/).The assembled genome of ., 2020) . The proLecRLK gene family members: Firstly, LecRLK genes in Prunus persica and Arabidopsis thaliana were used for local BLASTP in Genome of C. humilis. Direct search for LecRLK genes in C. humilis Genome was then conducted by using the Hidden Markov Model (HMM) method [http://pfam.xfam.org/). By running an online hmmsearch program (https://www.ebi.ac.uk/Tools/hmmer/search/hmmsearch) and the local hmmsearch software (HMMER 2.3.2), the sequences in that match PF01453, PF00139 and PF00059 were selected. The NCBI CDD online tool was used for the domain verification (https://www.ncbi.nlm.nih.gov/cdd) and final screening of LecRLK family members was conducted by following the above steps.Two methods were used to generate the maximum number of ) method ,14. The C. humilis LecRLKs obtained in the previous step were used to construct the phylogenetic tree. Using the MEGA software (version 7.1) and the parameters were set as follows: NJ method, P-distance model, Pairwise deletion and 1000 bootstrap replicates [Arabidopsis thaliana LecRLK gene family, C. humilis LecRLKs (chLecRLKs) were divided into different classes. The molecular weight (MV) and isoelectric point (pI) of chLecRLKs were predicted using online tool ProtParam (https://web.expasy.org/protparam/). The subcellular localization of chLecRLKs was predicted using the Protein Subcellular Localization Prediction website  and double checked with CELLO v2.5: subcellular LOcalization predictor (http://cello.life.nctu.edu.tw/).Protein sequences of All plicates ,30. WithLecRLK genes were searched in C. humilis genome, and a schematic diagram of gene structures was drawn using Tbtools [http://meme-suite.org/tools/meme) was used to conduct protein motif analysis, and default parameters were used except the maximum number of motifs for the conserved domain, which was set to 10.The distribution of introns and exons of the  Tbtools . Online LecRLKs were obtained from C. humilis Genome by using NCBI local BLASTN, and the chromosomal distribution were visualized by using software Tbtools [LecRLKs with default parameters and visualized by Tbtools.The chromosomal positions of  Tbtools . The mulC. humilis were downloaded from NCBI database (https://www.ncbi.nlm.nih.gov/) under accession number PRJNA417674 (fruits), PRJNA420878 (seed), PRJNA684437 (leaf) and PRJNA683804 (root). Profiles of expressed LecRLKs in different tissues were then analyzed with Venn’s diagrams tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html) and the heatmaps were drawn based on the FPKM values of all the differentially expressed LecRLKs.Transcriptome sequences of C. humilis  with were grown in pots (15 cm in diameter) filled with peat moss and perlite (1:1) in a greenhouse. With salt stress treatments, 30 plants (10 for each treatment) were subjected to three different NaCl concentrations ; With drought stress treatments, 30 plants (10 for each treatment) were put under three different watering regimes . The substrate’s water holding capacity (WHC) were determined as follows: Firstly, the total weight of pot and substrate (Peat: vermiculite: perlite: river sand = 1: 1: 1: 1) were measured; Secondly, water the substrate throughly and measure the total weight again; Finally, the weight gap was defined as the WHC.One year old plants of After ten days of being under salt or water stress conditions, leaf MDA , SOD (Superoxide dismutase), POD (Peroxidase) and CAT  activity measurements were conducted according to Li et al . ProlineLecRLKs were designed using Premier 5.0 software   according to the manufacturer’s protocols. RNA extractions were carried out in three biological replicates. The cDNAs were then synthesized using a high-capacity RNA-to-cDNA Kit . Primers sequences of nine selected al, USA) . Quantitt method . Real-tiLecRLK genes were identified from the C. humilis genome database, and the phylogenetic tree was constructed using all the LecRLK protein sequences of C. humilis and A. thaliana , the molecular weight (MW), the isoelectric point (PI), and the subcellular localization. Among the 170 LecRLK proteins, the lengths of the amino acid were between 351 and 1627, the MWs were between 38.35 and 180.30 kDa, and the PIs were between 5.01 and 9.56. Although subcellular localization predictions of some chLecRLKs (61 members) were not consistent between WoLF PSORT and CELLO, 63.5% of the chLecRLKs (108 out of 170) were shown to be located on the Plasma Membrane .chLecRLK genes ranged from 1 to 14 , and the chLecRLK-L34 gene has the most exons (11).As shown in the schematic diagram of gene structures, exon number of the 170  1 to 14 . Among 1Meanwhile, a total of 14 conserved motifs were obtained from all chLecRLK proteins . ComparechLecRLK genes were shown to be unevenly distributed on eight chromosomes (chLecRLK genes (56), while only three chLecRLK genes were located on chromosome 8 (Ch8). Additionally, gene tandem duplication events (marked in red lines) of chLecRLKs were discovered from Chr1 to Chr5, and the tandem repeats contained 83 members consisting of 67 G-types and 16 L-types. Moreover, WGD (whole genome duplication)/segmental duplicated gene pairs were also found containing 6 G-type and 4 L-type chLecRLKs  were not detected in any of the samples examined  and the genes with the highest FPKM values in different tissues were chLecRLK-G36 (seed), chLecRLK-G51 (root), ChLecRLK-G77 (leaf), and chLecRLK-G82 (fruit)  .C. humilis plants, the content of leaf Proline and MDA, and enzyme activities of leaf POD, SOD and CAT were measured. The Proline content increased significantly with the elevation of NaCl concentration and drought while the leaf MDA contents showed different changing patterns  and their expression patterns in leaf and root of NaCl and WHC treated C. humilis plants were studied.There were 33 chLecRLK genes under mild stresses were shown to be significantly different from that under severe stress levels  were significantly elevated in the leaves of plants under severe salt stress (treated with 180 mmol/L NaCl), while only five chLecRLK genes (4 G-types and 1 L-types) showed significant increases in expression under light salt stress (treated with 120 mmol/L NaCl)  . Express/L NaCl) . Howevert stress .chLecRLK genes  were significantly higher than those of the control in roots of plants treated with 120 mmol/L NaCl solutions while the relative expression of all nine genes in roots under 180 mmol/L NaCl treatment were significantly higher than those of the control  had significantly higher expression levels in roots of plants under light drought stress compared to those of the control (The relative expression levels of six  control . The exp control .Populus trichocarpa had the largest LecRLK family of 325 members while Physcomitrella patens only had 21 family members [C. humilis (170) is similar to that of Pyrus. Bretschneideri (172) and Oryza sativa (173) [LecRLK proteins are widely distributed in the plant kingdom, and the size of this protein family varies in different plant species. Previous reports showed that  members . In our humilis 10 is simiC. humilis, G-type chLecRLKs are the major chLecRLKs. Similar results were reported in a few other plant species [G-type lecRLK was reported to be the largest subgroup in most plant species ,11. One LecRK-I.9 (an L-type lecRLK) in Arabidopsis leads to enhanced resistance to Phytophthora. brassicae [AtLPK1) is related to elevated resistance to Botrytis cinerea [L-type LecRLKs are structurally similar to those of the legume-lectin proteins which exhibit glucose-Mannose specificity and they are mostly involved in plants’ responses to biotic stresses ,37,38. F cinerea ,39. Seve cinerea ,41.C.humilis genome while only one C-type was identified in the rice and Arabidopsis genomes [To date, only one to three C-type LecRLKs were found in different plant species . Only tw genomes . Unlike  genomes .LecRLKs [C. humilis, the amplification of the chLecRLK gene family is mainly caused by gene tandem duplications and therefore promotes their evolution. It is worth noting that G-type LecRLKs have been more evolutionary expanded compared to L-type chLecRLKs, which is in agreements with the findings on LecRLK gene family evolution of potato, Taxodium, tomato, pear and Populus [Duplication of genes contributes to the functional variations in plants . PreviouLecRLKs ,43. In C Populus ,11,14.chLecRLK in different organs of C. humilis have special expression patterns, transcriptomic analyses of different organs including seed, root, leaf and fruit of C. humilis were conducted to determine the organ-specific expressions of all members of chLecRLK family. The highest expressed and commonly expressed LecRLK genes in four different organs were all G-types suggesting the close involvement of G-type genes in various physiological processes. It is also interesting to note that numbers of expressed chLecRLKs in leaf and fruit were much higher than those of in roots and seeds. One possible explanation is that the leaf and fruit are constantly exposed to drastic changes in temperature, light, humidity, and other environmental conditions, more chLecRLK proteins may be needed to “process” external stress signals and their corresponding internal responses, while the roots and seeds are protected by soil and -seed coats respectively, therefore there may not be as big of a need to synthesize a large amount of chLecRLK proteins under relatively more stable environmental conditions. Similar results were also found in two woody plants including Populus and Taxodium [To determine whether the family members of Taxodium ,44. AlthTaxodium ,45.C. humilis plants treated with different NaCl concentrations and watering regimes were measured. The proline content was shown to be the best indicator for evaluating the severity of salt and drought stresses in C. humilis plants.Several biochemical indicators including the contents of MDA and proline, activities of SOD, POD and CAT are widely used for evaluating the effects of biotic and abiotic stresses . In our chLecRLKs under salt and drought stresses, qRT-PCR experiments of nine selected chLecRLKs were conducted in leaves and roots of the treated C. humilis plants. The relative expression levels of most chLecRLK genes were elevated in the leaves and roots of plants under severe salt and drought stresses thus indicating their participation in responses to severe stresses.To further investigate the potential roles of chLecRLKs genes were identified from the the newly published genome of woody shrub C. humilis, and they are comprised of 125 G-, 43 L-, and 2 C- types. Gene tandem duplication events were found to be the main cause of expansion of chLecRLK genes, mostly G-types and L-types. Numbers of expressed chLecRLKs in leaves and fruits were much higher than those of in roots and seeds of C. humilis based on the tissue-specific transcriptomic analyses. The qRT-PCR analysis of nine selected LecRLK genes revealed their active participation in salt and drought stress responses. The results from our study will provide a strong foundation for future functional analysis of chLecRLK family genes and will also help facilitate more effective development of new stress-resistant C. humilis cultivars.One hundred seventy S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 – 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae , but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease.IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy. Porcine pleuropneumonia is an important disease with high economic impact for the swine industry , 2. EconActinobacillus pleuropneumoniae, a Gram-negative bacterial pathogen of the pig respiratory tract. This species currently consists of 19 serovars  can be highly specific, allowing discrimination of different species when the targeted DNA sequences are sufficiently unique. Amplification of A. pleuropneumoniae-specific DNA in pig-derived samples  is therefore exploited for disease diagnosis  family protein, are A. pleuropneumoniae-specific . Depending on the primers used, such insertions can affect the results of apxIVA-based PCR assays, possibly leading to misinterpretation  database and the WGS database of Pasteurellaceae, which include 34 A. pleuropneumoniae genomes covering 13 serovars, 30 genomes of 13 other Actinobacillus and [Actinobacillus] species, 116 genomes of G. parasuis, and 291 genomes of P. multocida , PCR amplicons of expected sizes were detected in all A. pleuropneumoniae strains and isolates but were absent in other species  identified in this study are not identical to those previously described. As there have been reports of atypical A. pleuropneumoniae isolates failing to amplify the predicted target with existing apxIVA-specific primers  identified herein are in the 3′ part of apxIVA and are in close proximity to the A. pleuropneumoniae-specific region previously identified in hybridization experiments and some previously published primer pairs  are within the central part of apxIVA . In. InapxIVn silico , Table 3 present . CoampliA. lignieresii genome contains sequences (comprising multiple open reading frames) sharing 73% identity over 71% of the A. pleuropneumoniaeapxIVA sequence (71% query cover), as determined by BLASTn. Five A. pleuropneumoniae-specific apxIVA marker candidates identified here do not share significant similarity with the apxIVA-like sequences in A. lignieresii, as determined by default parameters of MegaBLAST search against databases which include three complete and incomplete A. lignieresii genomes , the NCTC 10568  genomes . In shornd sheep .apxIVA, seven novel marker candidates that map to various genes were identified. Six newly identified A. pleuropneumoniae-specific markers, namely, eamA, nusG, sppA, ybbN, ycfL, and ychJ, share 100% identity among all 11 complete A. pleuropneumoniae genome assemblies and 99.29–100% identity among all 23 incomplete A. pleuropneumoniae genome assemblies, confirming their highly conserved nature among A. pleuropneumoniae genomes. These six sequences are also A. pleuropneumoniae-specific compared in silico with available databases , suggesting that the ATCC 33377 genome may not contain xerD or the contigs that contain whole xerD marker sequence are absent in the genome assemblies. The xerD sequence identified is only 149 nucleotides in length. Assembling contigs to contain this short sequence should not be difficult unless the genome contains multiple sequences homologous to xerD. As seen in the case of apxIVA-2, when performing MegaBLAST searches against the Pasteurellaceae WGS database, only 19 out of 23 matches with A. pleuropneumoniae incomplete genomes (38–100% query cover and 96.8–100% identity) were observed  reaction with S. aureus, and catalase and oxidase tests. Species validation and molecular serotyping were performed using multiplex PCR targeting apxIVA and cps genes  at 37°C with 5% CO2. Bibersteinia trehalosi, Mannheimia haemolytica, Salmonella enterica serovar Choleraesuis, and Streptococcus suis were grown on brain heart infusion (BHI) plates at 37°C with 5% CO2.Bacterial strains used to test the presence of DNA markers in this study are either in the bacteria . Bacteribacteria . Some [Aew genus . These sA. pleuropneumoniae-conserved sequences.” In the second step, these A. pleuropneumoniae-conserved sequences were used as queries to search for highly similar sequences using MegaBLAST  available for in silico comparison is shown in In the initial step, 11 complete genome assemblies covering serovars 1–5 and 7–8  were selegaBLAST –28. SearA. pleuropneumoniae species that share more than 70% identity with A. pleuropneumoniae-specific marker candidates from the nucleotide collection (nr/nt) database. Multiple alignment of A. pleuropneumoniae-specific marker candidates and similar sequences from other species was performed using Clustal Omega  according to the manufacturer’s protocol. Briefly, PCRs were prepared to contain final concentrations of 200 μM dNTPs, 0.2 μM each primer . PCR products were visualized by agarose gel electrophoresis followed by ethidium bromide staining. Alternatively, Luna qPCR Master Mix  was used according to the manufacturer’s protocol. Briefly, qPCRs were prepared to contain final concentrations of 0.25 μM each primer and 1 ng/μL of bacterial genomic DNA. Forty-five cycles of 95°C for 15 s and 60°C for 30 s were performed using a Bio-Rad CFX96 real-time PCR machine. Fluorescence signals indicative of the presence of PCR products were measured.Genomic DNA of various bacterial species was extracted using a standard DNA purification protocol . PCR wash primer , 0.025 U


Brucella canis. In the present study, we isolated a Brucella strain (CD3) from a subclinically infected pet dog in Sichuan Province, Southwestern China. Classical biotyping methods and molecular biological tests (BCSP31 and BcSS PCR) proved that the strain belonged to B. canis. Furthermore, B. canis CD3 and another two B. canis strains (WJ5 and YA4), which were all isolated from pet dogs in Sichuan, were genotyped using multilocus sequence typing (MLST). Our results showed that the three B. canis strains were identified as the same sequence type (ST21). The present study is the first to report B. canis strain from a subclinically infected pet dog in China, indicating a potential threat to public health posed by subclinical infections in pet dogs. We suggest that screening for B. canis should be incorporated into routine medical examination of pet dogs and other companion animals in areas with a history of animal or human brucellosis.Canine brucellosis, a worldwide zoonotic disease, is mainly caused by  Brucella, is regarded as one of the most common zoonotic diseases worldwide [Brucella canis is generally recognized as the pathogen of canine brucellosis, which primarily causes reproductive failure in dogs, including infertility and abortion [B. canis infections in dogs have been increasing in China and other countries in recent years [B. canis can also infect humans and cause severe diseases [B. canis infection in pet dogs in China was reported by our lab in 2015, in which the infected dog had clinical symptoms such as fever, enlarged lymph nodes and enlarged testicle [Brucellosis, caused by Gram-negative, aerobic and facultative intracellular bacteria of the genus orldwide ,2. Bruceabortion . B. canint years ,4,5. B. diseases ,10,11,12diseases . As the diseases . China idiseases . The occBrucella in 2007 and the result showed high differentiation among different species of Brucella strains [Brucella in China mainly focused on B. abortus, B. melitensis, and B. suis. The information of MLST of B. canis in China is relatively few. In 2019, Liu et al. had genotyped seven B. canis strains in Guangxi Province as ST21, enlarging our understanding of the genetic structure and the genetic diversity of B. canis population [B. canis in Sichuan Province.Multilocus sequence typing (MLST) is a DNA sequence-based typing method that has been widely applied to microbial typing and epidemiological studies ,16,17. I strains . Recent pulation . HoweverB. canis isolation (strain CD3) from a subclinically infected pet dog, and MLST method was then used to genotype the strain CD3 and another two B. canis strains (WJ5 and YA4), which were also isolated from pet dogs in Sichuan Province, China. For the first time, we isolated a B. canis strain from a subclinically infected pet dog in China, revealing a potential threat of subclinical infections in pet dogs to public health.In this study, bacteriological and molecular biological tests (BCSP31 and BcSS PCR) were used to identify a Brucella infection in Veterinary Medical Teaching Hospital of Sichuan Agricultural University at the request of the pregnant owner. Physical examination of the pet dog showed no clinical symptoms . Blood samples were aseptically collected from a cephalic vein of the pet dog for serological tests and strain isolation.In August 2019, one male pet dog (golden retriever), aged three years and seven months, was tested for B. canis strains  were successfully isolated from the two cases and were stored in our lab [In December 2015, one male dog (golden retriever), aged two years and five months, presented with low fever, enlarged lymph nodes, and unilateral testis. In October 2016, a male poodle, aged three years, presented with obviously enlarged testicle and undulating fever. Both pet dogs were diagnosed with canine brucellosis by Veterinary Medical Teaching Hospital of Sichuan Agricultural University. Two Brucella in blood samples, as described previously [The Rose Bengal plate test (RBPT) and rapid slide agglutination test (RSAT) were performed with rough antigen to detect antibodies against eviously .2 requirement, H2S production, hydrolysis of urea, agglutination in sera, growth on dyes and phage lysis tests) [B. canis RM6/66 was used as a reference strain. All studies involving living bacteria isolation were performed under biosafety level 3 (BSL-3) laboratory (DYY-2019303075).Blood samples were plated and streaked on tryptic soy agar  for strain isolation. Plates were inoculated at 37 °C over 5 days [s tests) ,21 were Brucella DNA was extracted using TIANamp Bacteria DNA extraction kit  according to the manufacturer’s instructions [Before DNA extraction, specimens were boiled for 15 min. Brucella genus-specific (BCSP31) and B. canis species-specific (BcSS) primers, respectively. PCR amplification was conducted as described previously [B. abortus 544A, B. melitensis 16M, B. suis S2, and B. canis RM6/66 were used as reference strains. The PCR products (6 µL from each reaction mixture) were loaded for electrophoresis using a 1.5% agarose gel, after which the gel was stained with ethidium bromide and photographed [The isolated strain (CD3) was identified by two different PCR protocols using eviously ,22 and pB. canis strain (CD3). Two B. canis strains (WJ5 and YA4) from clinical cases of canine brucellosis in Sichuan were also analyzed [gap, aroA, glk, dnaK, gyrB, trpE, and cobQ), one outer membrane protein (omp25), and one intergenic fragment (int_hyp) [Brucella Base, https://pubmlst.org/Brucella, accessed on 9 August 2021) [MLST genotyping was performed on the isolated trains WJ and YA4 int_hyp) . PCR ampint_hyp) . Primersint_hyp) . Distincst 2021) , and eacB. canis strains  from pet dogs and 66 Brucella isolates in MLST database , including four most common species  and two emerging species from marine mammals , were chosen for phylogenetic analysis using MEGA X. Neighbor joining trees were constructed using the Jukes–Cantor model and the percentage bootstrap confidence levels of internal branches were calculated from 1000 resamplings of the original data [Sequences of the nine loci were concatenated to produce a 4396 bp sequence for each genotype (ST). Sequence data of 3 nal data .2 requirement, H2S production and phage lysis tests were negative, while hydrolysis of urea, growth on thionin and agglutination in sera R were positive. The biotyping characteristics of the strain (CD3) were in accord with that of B. canis RM6/66 (RBPT and RSAT assays showed positive results for the tested blood samples. A suspected Brucella strain (CD3) was successfully isolated from the blood sample by tryptic soy agar. Further bacteriological tests showed that the results of COs RM6/66 .Brucella genus-specific PCR (BCSP31-PCR), using all five strains , successfully produced 224-bp PCR amplicon, indicating that CD3 strain belongs to the genus Brucella. Further B. canis species-specific PCR (BcSS-PCR) amplified a specific 300-bp amplicon  and two B. canis strains stored in our lab (WJ5 and YA4) have the same allelic profile, and all three B. canis strains from pet dogs in Sichuan belong to genotype ST21.Details of allelic profile of each B. canis strains  isolated from pet dogs, sequences of the nine loci were concatenated to conduct phylogenetic analysis with 66 Brucella isolates in the MLST database. As the dendrogram shows  were clustered together with B. canis and B. suis strains. B. ceti and B. pinnipedialis were all grouped into cluster B together. B. abortus were grouped into cluster C, which split into two subclusters (cluster C1 and C2). B. melitensis were clustered into cluster D.In order to better understand the genetic relationship of the three am shows , four maB. canis infection in Sichuan Province, China, and a B. canis strain (CD3) was successfully isolated. Canine brucellosis is a zoonotic disease that has been underestimated worldwide, posing a public health risk to dogs and humans [B. canis strains have been identified from dogs after the first isolation in 1984 [B. canis. The dog showed healthy on physical examination, while B. canis infection was revealed by serological screening and confirmed by subsequent bacteriological and molecular biological tests. This hidden case of B. canis infection in pet dog was reported in China for the first time, which will raise public health concerns about contracting B. canis. According to other reports, B. canis can be transferred from pet dogs to humans through direct contact with infected dogs or their blood or reproductive samples , and even the spayed or neutered dogs can still shed bacteria in their secretions and urine [B. canis from asymptomatically infected pet dog to human has been reported previously [B. canis infection because of the genetic similarity between B. canis isolates from the child and the pet dog [B. canis infection have been reported in many countries, including China, the United States, Argentina, and Japan [B. canis from pet dogs to humans, we suggest incorporating B. canis screening into the routine medical examination of pet dogs. Reproductive failure (including infertility and abortion) is the main problem in dogs infected with B. canis [In this study, we diagnosed a pet dog without clinical symptoms as strain CD was succd humans ,23,24. Id humans . In 2012d humans . These d in 1984 ,26,27. Ind Japan ,11,12. CB. canis strains  isolated from pet dogs in Sichuan Province. Our results showed that the allelic profile of the three B. canis strains were identical to each other and all three B. canis strains were identified as existing sequence type ST21, which has been identified in B. canis strains in Guangxi, China [B. canis isolates  were clustered together with other B. canis strains and B. suis strains. The result indicates the close relationship between B. canis and B. suis, which is consistent with findings of Whatmore et al. [B. abortus and B. melitensis were grouped into individual clusters, respectively, and strains from marine mammals  were grouped into a cluster together. These findings show high resolution of MLST method for phylogenetic genotyping of Brucella. In addition, the three B. canis strains in our study were isolated in 2015, 2016 and 2019, respectively, and they were from three different cities in Sichuan Province . Therefore, we speculated that ST21 may be the dominant genotype of B. canis infecting pet dogs in Sichuan Province in recent years. However, due to the small number of cases, surveys of B. canis infection on a larger scale in epidemic areas are needed to better understand the genotype of B. canis.MLST and phylogenetic analysis was further performed to understand the molecular characteristics and genetic relationship of the three i, China , and thei, China ,28. All e et al. . MeanwhiB. canis strain from a subclinically infected pet dog in China, indicating a potential threat to public health posed by subclinical infections in pet dogs. We suggest that screening for B. canis should be incorporated into routine medical examination of pet dogs and other companion animals in areas with a history of animal or human brucellosis.We first isolated a


Anopheles stephensi mosquitoes share urban breeding sites with Aedes aegypti and Culex quinquefasciatus mosquitoes in the Republic of Djibouti. We present evidence that A. stephensi mosquitoes might be responsible for an increase in malaria incidence in this country. We also document resistance of Plasmodium falciparum to dihydroartemisinin/piperaquine. Plasmodium falciparum (>80%) or P. vivax. Before 2013, researchers considered the Anopheles arabiensis mosquito to be the primary vector  and >100,000 suspected cases  have served in Djibouti for decades. Service members and their families  live in the capital. Despite malaria prevention and treatment measures described elsewhere  (http://vectormap.si.edu/downloads/VHazardReports/VHR_Anopheles_stephensi_2018.pdf). We extracted DNA from the legs of 103 An. stephensi mosquitoes and sequenced the cytochrome oxidase C subunit I gene to confirm morphologic identification. In addition, we conducted a phylogenetic analysis  . We condanalysis  Figure 2P. falciparum and P. vivax cocirculated . Among the country’s population, incidence increased from 25.5 cases/1,000 persons in 2018 to 49.8 cases/1,000 persons in 2019 , confirming good regimen adherence and absorption (https://apps.who.int/iris/handle/10665/162441). We sequenced molecular markers of resistance to antimalarial drugs for 9 P. falciparum isolates  were near livestock areas, mainly in the Ambouli Gardens district. We confirmed morphologic identification of adult An. stephensi mosquitoes by cytochrome oxidase C subunit I sequencing, which identified 8 haplotypes. Phylogenetic trees did not clearly indicate the origin of An. stephensi mosquitoes in Djibouti . We collected 1,835 adult mosquitoes and larvae: 1,500 tephensi . We caug or both  Table 2.er tanks . In miliDjibouti  Figure 2pfhrp2 and pfhrp3 gene deletion among P.falciparum parasites in the city of Djibouti. In the Republic of Djibouti, malaria transmission has increased since 2013. Even populations with strong malaria control programs, such as the FAF, are now affected. In 2018, the country notified the World Health Organization of ≈100,000 suspected cases, mainly among febrile patients with negative results on a rapid diagnostic test  Figure 1P. falciparum resistance to artemisinin derivatives in Djibouti.We documented an early treatment failure of dihydroartemisinin/piperaquine in an isolate lacking a K13 mutation. This finding could signal the emergence of An. stephensi mosquitoes are well-established in Djibouti and have been observed in Sudan and Ethiopia (Ae. aegypti and Cx. quinquefasciatus mosquitoes, highlighting its adaptation to urban areas. Models predict broad expansion of An. stephensi mosquito distribution into major cities in Africa, where large malaria outbreaks could occur among growing resident populations susceptible to the disease (An. stephensi mosquitoes. To control malaria and limit the spread of this anopheline species, communities and governments should prioritize larval control and access to the water distribution network.Anopheles stephensi in malaria outbreak, Djibouti, 2019.Additional information on role of