Listeria monocytogenes is an important pathogen that can cause listeriosis. Despite the growing recognition of Listeria spp. as a foodborne and environmental pathogen, the understanding of its prevalence and characteristics of Listeria spp. in the marine environment remains unknown. In this study, we first investigated the genetic and phenotypic characteristics of Listeria species isolated in a coastal city in China. The findings revealed that the sequence type 87 (ST87) L. monocytogenes, a prevalent clinical and seafood strain in China, dominates in recreational beach sands and possesses a notable biofilm-forming capacity in seawater. The presence of ST87 L. monocytogenes in coastal environments indicates the potential health risks for both recreational activities and seafood consumption. Moreover, the ST121 isolates from sand had a versatile plasmid encoding multifunctional genes, including uvrX for UV resistance, gbuC for salt resistance, and npx for oxidative resistance and multiple transposases, which potentially aid in survival under natural environments. Black-headed gulls potentially facilitate the spread of L. monocytogenes, with similar ST35 strains found in gulls and beach sand. As a reservoir of microbes from marine environments and human/animal excrement, coastal sand would play an important role in the spread of L. monocytogenes and is an environmental risk for human listeriosis. Listeria consists of 30 species, as listed in the List of Prokaryotic Names with Standing in Nomenclature (LPSN) [Listeria monocytogenes is the main human pathogenic species known to cause listeriosis, a serious infection that primarily affects in elderly, pregnant women, children, and immunosuppressed patients. L. monocytogenes can cause septicemia, meningitis, abortion, and other complications in humans and animals, while L. ivanovii causes similar pathological manifestations in ruminants, except for meningoencephalitis [L. monocytogenes is an environmental saprophyte that occupies a wide range of habitats, including river, soil, vegetation, and food-processing environments [L. monocytogenes has the ability to grow under harsh environmental conditions, such as high salt concentration, pH 4.5, heavy metals, and/or freezing temperature. Studies have demonstrated that this bacterium can survive on dusty sand particulates, even under conditions of 10 °C and 88% relative humidity for more than 151 days [Listeria fleishmanii was initially isolated from cheese and has been observed to persist for more than 5 years, and sporadic occurrences of this species have been found in soil and feed [Listeria aquatica was originally identified in water in the United States [L. monocytogenes has been reported to be constantly present in the lagoon and soil around pig manure treatment plants [The genus e (LPSN) . Listeriphalitis ,3. Despiphalitis . Furtherronments . L. monoand feed ,7. Listet plants .L. monocytogenes exhibits regional heterogeneity, ecological niche preference, and genetic diversity [L. monocytogenes includes four genetic lineages (I–IV) and 14 serotypes, which are further subdivided by clonal complexes (CCs) and multilocus sequence types (STs) [The species iversity ,11. L. mes (STs) . The majes (STs) . Serotypes (STs) ,13. L. monocytogenes is well documented and is known to frequently occur through the consumption of contaminated food items, such as ready-to-eat meat, soft cheese, and unpasteurized milk [L. monocytogenes infection [L. monocytogenes after swimming in a public pool [L. monocytogenes [L. monocytogenes in public access surface water along the central California coast and its occurrence in Moroccan marine environments underscores its potential as contamination sources and its implication for the human health [The infection of zed milk . Seafoodzed milk ,16. In anfection . A case lic pool . Migratoytogenes . The occn health ,21,22,23L. monocytogenes in the marine environment in China has not yet been reported. This study aimed at a preliminary exploration of the prevalence and genetic features of Listeria species in coastal environments of China.To date, an epidemiological investigation and risk assessment of TM; Minneapolis, MN, USA). Prior to bacterial isolation, the sand was subjected to socking and rubbing in roughly 100 ml of phosphate-buffered saline (PBS) . Following the natural deposition of sand, the supernatant was transferred into a 50 mL tube for centrifugation at 4000 rpm for 10 min. The pellets were subsequently inoculated into half-Fraser and Fraser broth  for the enrichment of L. monocytogenes [Listeria Agar  and incubated at 37 °C for 48 h. The potential clones were purified on a brain heart infusion (BHI) agar plate . Colonies indicative of potential Listeria presence were selected for PCR identification, employing primers targeting the Listeria genus-specific gene prs  [L. monocytogenes EGD-e was used as a positive control.A total of 769 sand samples were collected from 5 distinct bays , an island (denoted as I), and a village (denoted as V) en route to the island in a coastal city of China A. All saytogenes . The enrTTTGCGG) . The ampListeria spp. isolates obtained from sand samples were cultured in the BHI agar plate at 37 °C for 18 h. After incubation, the bacterial cells from one agar plate were collected via scraping followed by genomic DNA extraction using a Wizard® Genomic DNA Purification Kit . The extracted DNA was quantified using a Qubit 2.0 Fluorometer. The DNA library was constructed using the NEBNext® UltraTM DNA Library Prep Kit  with Illumina technology. The whole-genome shotgun sequencing of these isolates was performed on the Illumina Hiseq PE150 platform. The raw data were filtered to obtain clean data, and then high-quality paired reads were assembled using SOAP denovo (v 2.04). Genome sequencing, raw data filtration, and clean data assembly were performed using the instruments from Novogene Co., Ltd. . For species-level identification, fastANI (v 1.33) was used to calculate the average nucleotide identity (ANI) between the genomes. Prokka (v 1.14.6) was used for genome annotation tasks.The Listeria spp. The core and pan-genomes were analyzed with the Roary pipeline using a 95% identity cutoff. The phylogenetic tree based on the core genes of Listeria spp. was constructed using FastTree (v 2.1.10). The sequence types were determined with in silico multilocus sequence typing (MLST). The publicly available strains from the National Center for Biotechnology Information (NCBI) database, which shared the same sequence type as the sequenced isolates in this study, were selected for SNP analysis (https://www.chiplot.online/) tools.WGS was used for the molecular characterization of analysis . The geoanalysis . The SNPListeria virulence database in VirulenceFinder (https://bitbucket.org/genomicepidemiology/virulencefinder_db/src/master/) and previously reported resistance genes on linux server [The identification of virulence- and resistance-associated genes was accomplished through local sequence alignment with the extant x server ,27. In s7 CFUs of microbial suspensions were added to each well of the 96-well microtiter plate containing 200 μL of sterile seawater and incubated for 4 days at 25 °C. After incubation, the suspended bacteria were rinsed three times with PBS. The remaining cellular layers were stained with 1% crystal violet solution for 30 minutes and then washed with deionized water. The crystal violet bound to biofilm was dissolved in 95% ethanol, and the optical density was quantified at A595 using a microplate reader . Multiple comparisons of nonparametric tests were performed using Statistical Product and Service Solutions  .Biofilm formation assays were performed using the classical crystal violet method . The expListeria isolates were obtained, representing an observed frequency of 3.38% (26/769). The 16 L. monocytogenes strains were isolated from 2.08% (16/769) of the samples in Bays B, D, and E, as well as Village V and Island I. Seven strains of L. fleischmannii were found in Bays B, C, and E, with a total prevalence of 0.91% (7/769) and prevailing in Bay C. Three strains of L. aquatica were sporadically found in Bay E, with a total frequency of 0.39% (3/769)  B,C. L. monocytogenes isolates belonged to two lineages (I and II) and four sequence types . The prevalent ST87 strains were isolated in Bays B and D, Village V, and Island I. ST121 and ST35 strains were found in Bay E. ST85 isolates were found in Bay E and Village V.The Listeria demonstrated to have biofilm capacity in seawater  as well as their regulation genes . These genes exhibited genetic polymorphism in different sequence types of isolates. Within an identical sequence type, a consistent allelic profile was observed, and its polymorphism was found to be associated with the ST type , inlA (full length), inlB, inlC, and iap but not LIPI-3. Notably, LIPI-4 was found in the ST87 isolates. The ST121 isolates contained stress survival islet 2 (SSI-2), whereas the ST35 and ST85 isolates contained stress survival islet 1 (SSI-1). Additionally, the ST121 isolates carried the cadmium resistance gene cadA1C1, whereas the ST35 isolates had the cadA3C3 gene. No disinfectant resistance genes were detected in these isolates. According to the CARD database, all L. monocytogenes isolates inherently carried antibiotic-resistance genes norB (for fluoroquinolone resistance), fosX (for fosfomycin resistance), and mprF (for cationic peptide resistance).A total of 182 virulence and resistance genes were analyzed across the isolates. All 16 ce genes . SpecifiL. monocytogenes harbored a 57 kb plasmid. Ten plasmids sharing 100% identity and 100% coverage with this 57 kb plasmid were discovered in L. monocytogenes, L. innocua, and L. welshimeri in the NCBI database. These plasmids were distributed in the strains across various regions, including the United States, Italy, Australia, Poland, and Russia . The phylogenetic analysis showed a distinct trend toward regional clustering, with the sandy-derived isolates in this study exhibiting a closer evolutionary relationship with isolates from China than those from other countries. The isolates from clinical cases were distributed in the most evolutionary branch (n = 661) but absent in isolates from other STs (n = 1358)  .L. monocytogenes from beach sand in this study were compared with those of 1313 isolates from other regions . The sandy-derived ST121 isolates exhibited the closest genetic relationship to four isolates from three clinical cases that occurred between 2007 and 2008 in China, with 36–51 SNP differences  from the USA, China, and Australia. These isolates displayed a minor genetic variation with each other, ranging from one to nine SNPs . L. fleischmannii possessed a total of 6170 genes and 1659 core genes based on the 16 L. fleischmannii isolates, including 7 isolates from beach sand and 9 from other sources. The core genome and pan-genome of L. fleischmannii were 29.5% smaller and 40.8% larger than those of L. monocytogenes which had 2354 core genes and 4383 pan-genes according to a previous report [L. fleischmannii genome possesses a greater number of accessory genes, contributing to species diversity and conferring competitive benefits to individual organisms.L. aquatica displayed relatively small genome sizes, ranging from 2.6 to 2.7 Mbp. L. aquatica possessed a total of 3851 genes and 2063 core genes based on the five isolates of L. aquatica, including two isolates from beach sand and three from other sources. The core genome and pan-genome of L. aquatica were 12.4% and 12.1% smaller than those of L. monocytogenes according to a previous report [L. aquatica, over half of the genes in the pan-genome are classified as core genes, highlighting the relatively high conservation of the L. aquatica genome.s report . In L. aL. fleischmannii and L. aquatica was obviously less than that in L. monocytogenes (L. fleischmannii harbored some virulence and resistance genes associated with adherence (lap and fbpA), intracellular survival , invasion (recA), peptidoglycan modification (oatA), regulation , cold resistance (cspB and cspD), low pH resistance (lmo0796), lysozyme resistance (spoVGII), nisin resistance (liar), and salt resistance (gbuA). L. aquatica contained the genes associated with adherence (lap), immune modulation (tcsA), intracellular survival , regulation (fur and sigB), cold resistance (lmo0866 and yycG), and nisin resistance (liar). L. fleischmannii harbored the fluoroquinolone resistance gene norB, while L. aquatica did not carry antibiotic-resistant genes. Furthermore, the plasmid was not found in L. fleischmannii and L. aquatica isolates in this study.The number of virulence genes in ytogenes . L. fleiEscherichia coli O157:H7, a pathogenic bacterium responsible for hemorrhagic colitis and hemolytic uremic syndrome, has been detected in marine environments and is known to persist for up to a year in various water environments [L. monocytogenes is an important foodborne pathogen that is capable of infecting both human beings and animals. In addition to food contamination, multiple transmission modes have been reported, such as contact with animals, exposure to contaminated water and environment, etc. [L. monocytogenes, leading to multiple internal lesions and a fatal infection [L. monocytogenes poses a potential threat to human and animal health. Therefore, investigating the contamination of Listeria in the marine environment, and elucidating the prevalence and molecular characteristics of L. monocytogenes in beach sand, are important for the evaluation of infection risk for humans, and would be helpful to prevent listeriosis in people contacting this environment.Water-related illnesses impose a significant financial burden in California, with an annual cost of USD 3.3 million, attributed to coastal water pollution at just two beach locations . Choleraronments . L. monont, etc. ,33,34. Rnt, etc. ,36. A lonfection . ExposurListeria from beach sand within a popular tourist destination, Listeria spp. were identified in 26 out of the 769 samples collected from the coastal environment, including four sequence-type isolates  of L. monocytogenes and two other species (L. fleischmannii and L. aquatica). These findings suggest that marine environments could be a potential reservoir for Listeria species. In this pioneering investigation of L. monocytogenes in raw seafood and fresh aquatic products in China [L. monocytogenes in beach sand and their enhanced biofilm formation indicate a potential risk for the persistent contamination of aquatic products and food security. Notably, the regional heterogenicity of the ST87 isolates was observed, and the sandy isolates showed a closer association with other isolates previously found in China (150 isolates from humans and 84 isolates from food and environments). A majority of virulence genes, including LIPI-4, a cluster of six genes associated with transplacental and central nervous system infection, were found in 661 ST87 isolates from this study and NCBI database, including 276 isolates from the United States . This finding is consistent with a previous study on the ST87 strain in China [L. monocytogenes in coastal ecosystems signifies potential health risks for people through recreational activities and contaminated seafood consumption.ST87 is a common sequence type of in China ,39. It win China ,40,41. IL. monocytogenes are predominant in food and have the demonstrated ability to persist within food-associated environments [inlA gene encoding a full-length InlA with identical amino acid sequence, while 97.1% (68/70) of ST121 strains possessed a truncated inlA gene [ST121 strains of ronments . Interesronments . AlthougnlA gene .L. monocytogenes could augment the survival and virulence of LM populations [L. monocytogenes against sunlight exposure and sea salt in beach environments.The plasmids within ulations ,45. A coulations . In thisulations ,47. TherL. monocytogenes through migration [lmo0651 , suggesting the possibility that these isolates originated from the same source and therefore highlighting the potential for long-distance spread meditated by black-headed gull migration. Fecal contamination with L. monocytogenes dropped in the sand might result in the persistence of the pathogen in recreational marine environments, thus leading to an infection risk for people around beaches.Seabirds can carry pathogenic microbes and serve as a long-distance disseminator of infectious agents such as igration . Accordiigration . Our stuListeria detection. No Listeria spp. was detected in samples of Bay A, which was located upstream and had the cleanest coastal water and the least population density among these bays. Bay B, approximately 10 kilometers from the downtown area, featured pristine white sand beaches and had the isolation of L. monocytogenes and L. fleischmannii. Bay C, a niche tourism hotspot enriched with coral reefs, had the highest isolation of L. fleischmannii and was located approximately 4 kilometers away from the downtown area. Bay D, located in the downtown area with a public bathing beach of visa-free access, had a relatively high isolation rate of L. monocytogenes. Bay E, located downstream of the other bays, had a relatively high species diversity of Listeria. Additionally, Island I and Village V, both popular leisure resorts located between Bay A and Bay B, were contaminated with L. monocytogenes and offered various maritime entertainments and sports for tourists. Our finding suggests that the prevalence of L. monocytogenes increased with the degree of human activity, in agreement with studies conducted in Danish aquatic and fish-processing environments [The bacterial community in the seawater and river environments is subjected to geographical location, with greater species abundance observed downstream, potentially attributable to diverse factors such as human activity and algal blooms ,50. In tronments .Listeria isolates could generate biofilm in seawater, with the L. monocytogenes ST87 and L. fleishmanii isolates showing particularly robust biofilm formation. L. monocytogenes isolates harbored many biofilm formation genes with genetic polymorphism, while L. fleischmannii isolates may have yet unidentified biofilm formation genes, thus warranting further study. Additionally, the presence of genes resistant to environmental adaptation, especially those related to salt resistance (gbuABC and opuCABC) and UV resistance (uvrX), likely enhances the survival and proliferation of L. monocytogenes within marine environments. The continuous emergence of seafood contamination by L. monocytogenes has been well documented, with 0.5–1.0 % of listeriosis cases associated with the consumption of ready-to-eat seafood [L. monocytogenes infection through accidental ingestion.Biofilms of marine bacteria are ubiquitous in marine ecosystems, facilitating rapid adaptation to environmental challenges and participating in a set of functions . In this seafood . FurtherL. monocytogenes on the central California coast showed a prevalence of 41.9% to 62% in public access surface water, with serotype 4b strains accounting for most of the isolates [L. monocytogenes isolates from the central California coast contain the inlA gene encoding for intact InlA, which differs significantly from the isolates found in foods and food-processing environments [L. monocytogenes isolates in Morocco showed a prevalence of 2.5% in the marine environment, with serotype 1/2b strains in the majority [L. monocytogenes in a variety of marine habitats presents a potential contamination source and a threat to human health.Environmental surveys of isolates ,21,22. Fronments . Anothermajority . The preL. monocytogenes contamination in foods in 1998 and 2014–2015 in this coastal region [Listeria can exist and produce biofilm in marine environments, and the potential persistent colonization would cause seafood contamination and, consequently, human infection. The continuous monitoring of pathogenic bacteria, including L. monocytogenes, in these environments is crucial for public health in coastal cities. Susceptible individuals should avoid engaging in beach recreational activities or exposing their wounds to marine environments.As an accumulation substrate and source of bacterial pollution from diverse origins, beach sand could serve as a significant reservoir for bacterial pathogens, with 10–100-fold greater bacterial abundance than that of adjacent recreational waters . Studiesl region ,57. TherListeria from the coastal environment in China, highlighting the potential risk of L. monocytogenes infection in the marine environment. Notably, ST87 L. monocytogenes isolates emerged as the predominant sequence-type strains in recreational sand beaches, exhibiting robust capacity for biofilm formation in seawater. This finding highlights marine environments as significant reservoirs for the virulent strains of L. monocytogenes. However, this study was a preliminary exploration and had limited sample coverage of the entire coastal environment; thus, the sampling range and sample size should be expanded for a more comprehensive risk assessment of L. monocytogenes in a future study. The routine surveillance of pathogens in highly populated coastal regions and marine fishing areas is important for public health. Effective epidemic monitoring by public health authorities is essential to evaluate the potential risk of L. monocytogenes infection in marine environments and to develop strategies for listeriosis prevention for visitors and residents in coastal regions.Our study first reveals the genetic and phenotypic profiles of diverse species isolates of


Cerasus (Rosaceae) contain numerous fruit trees and ornamentals with high economic values. The origin and genetic divergence among various types of fruiting cherries always remain a perplexing issue. We employed three plastom fragments and ITS sequence matrices derived from 912 cherry accessions to elucidate the phylogeographic structure and genetic relationship among fruiting cherries, as well as the origin and domestication of cultivated Chinese cherry. The integration of haplotype genealogies, Approximate Bayesian computation (ABC) approach and estimation of genetic differentiation within and between different groups and lineages has facilitated the resolution of several previously unresolved questions. Firstly, distant phylogenetic relationships between Cerasus and Microcerasus accessions, as indicated by both nuclear and chloroplast data, suggested independent origins and evolution for these two taxa. Moreover, two distinct geographic origin centers (Europe and China) have been confirmed, with significant phylogeographic signals and high genetic differentiation observed between cherries from these regions. This may be attributed to long-term geographic isolation caused by Himalaya-Hengduan Mountains. Our phylogeographic analyses and ABC analysis suggested that cherries inhabiting in China may have undergone multiple hybridization events during the glacial refugia of the eastern edge and southern Himalaya-Hengduan Mountains, followed by rapid radiation throughout their current habitats during interglacial period. The discrepancy between nuclear and chloroplast data may be attributed to hybridization events and incomplete lineage sorting. Furthermore, we speculated that the domesticated Chinese cherries were derived from wild accessions in Longmenshan Fault Zones approximately 2600 years ago. We have also traced the domestication processes and dispersal routes of cultivated Chinese cherries.The subgenus  Cerasus of the family Rosaceae [Cherry is an economically important group, belonging to the subgenus Rosaceae . Cherry Rosaceae ,2. MajorRosaceae ,2. TheseRosaceae . As pereP. pseudocerasus, P. avium, P. cerasus, P. tomentosa, other species in subgenus Microcerasus etc., fruiting cherry) and ornamentals , both of which have high economic and aesthetic values [P. avium), sour cherry (P. cerasus), Chinese cherry (P. pseudocerasus) and dwarf cherry (P. tomentosa) [Cherry species comprise a diverse range of fruit trees  . Among tmentosa) ,2,5.Cerasus, there remains limited understanding regarding the origin and genetic relationship among various cultivated species, particularly with respect to Chinese cherry and sweet cherry. Whether all these distinct cultivated types have originated from a single or multiple location(s) is still unclear. In one scenario, it is plausible that the most recent wild common ancestor in China gave rise to geographically isolated lineages. China was once considered as a primary center of agriculture origins [Malus domestica) has also been traced back to Northwest China, specifically near the Tianshan mountain range, along the Silk Route that spans from Asia to Western regions [Despite the widespread distribution and diverse species of subgenus  origins ,7,8. The origins ,10. The  regions ,12. Furt regions . The alt regions ,15, with regions  and apri regions . HoweverMany molecular investigations have been conducted to explore the genetic diversity, population structure and phylogenetic relationships of cherry species. These studies have revealed that the evolutionary history of cherry species is highly complex and involves multiple hybridization events ,21,22,23matk and ndhF gene sequences, intergenic spacer regions of trnQ-5′rps16, trnL-trnF and atpB-rbcL as well as the introns of and rpl16 and rps16 [Recently, the utilization of nuclear and chloroplast DNA sequences has played a crucial roles in analyzing the phylogeography and population genetics of plant crops . Previound rps16 ,26,27,28rpl16, trnL-trnF and trnQ-rps16) to conduct comprehensive phylogeographic analyses on 912 cherry accessions from 96 populations in 88 towns across 12 provinces of China. The accessions included 535 cultivated and wild Chinese cherries, 120 European cherry accessions, 174 accessions from other subgenus Cerasus taxa, and 79 accessions from the subgenus Microcerasus  with high variation were selected for further analyses. The aligned DNA regions of rpl16, trnL-trnF and trnQ-rps16 were 1255 bp, 1048 bp and 1098 bp in length, respectively. Since no significant heterogeneity was detected by the partition homogeneity test, the sequences from the three regions were merged into a single cpDNA dataset consisting 3401 bp in length. After gap removal, the final concatenated cpDNA dataset consisted of 2313 bp with a GC content of 30.7%. By treating the indels as substitutions, we identified a total of 145 polymorphic sites and 107 parsimony-informative sites. A total of 113 haplotypes were identified, with corresponding haplotype and nucleotide diversity indexes of h = 0.7702 and π = 0.0824, respectively.Following preliminary testing, three chloroplast DNA sequences  and Bayesian inference (BI) methods. The ITS datasets were fitted with the best-fit GTR model, while the chloroplast haplotypes were modeled using the best-fit GTR + G model. The BI and ML trees generated with the same dataset exhibited the highly congruent topologies. Therefore, we have only presented the BI tree for each dataset due to its more robust statistical supports. K = 5 for 69 haplotypes  were present in lineage II, with the exception of P. mahaleb accessions which formed a distinct lineage IV. The basal lineage V comprises all MC accessions, including P. tianshanica, P. tomentosa, P. humilis and P. glandulosa. In general, Microcerasus accessions are significantly genetically distinct from those of Cerasus. European cherries exhibit relatively distant genetic relationships with their Chinese counterpart. The ITS haplotype phylogenetic tree was classified into five well-supported lineages (designated as I–V) a. The saplotypes b. These plotypes c. Among Cerasus accessions. The EC accessions were classified into three distinct lineages  that exhibited relatively genetic divergence from other Chinese native cherries. Lineage III primarily comprised the hybrids of European cherries, while lineage IV corresponds to the majority of European sweet cherry cultivars. P. mahaleb accessions were also clustered into a distinct lineage (VI) separate from other European cherries. Moreover, the majority of CC, WC and RC accessions from NEC, QLM, LFZ and YGP were classified into lineage I. A few CC and WC accessions from LFZ and QLM were combined with RC accessions from LFZ, QLM and YGP in lineage II. The remaining accessions of LFZ, QLM and YGP were grouped into lineage V.Chloroplast phylogenetic analysis yielded seven distinct lineages (I–VII) with robust support values a, which Molecular diversity indices were calculated for various groups  as well as lineages defined from haplotype analyses  based onD  value were exclusively detected in group EC, and significantly negative Fu’s Fs were observed in lineages I (CC + WC + RC)  and II (EC)   and II (EC), while multimodal patterns were observed in the remaining groups and lineages  and lineage II (CC + WC + RC)  exhibited significantly negative Tajima’s D and Fu’s Fs values as well as unimodal mismatch distribution curves (p = 0.0000) exhibited significantly negative Fu’s Fs value but multimodal patterns  were performed on both ITS and chloroplast sequences . For ITS 0.0030) . Unimodalineages . For chln curves . The linpatterns . These rFCT values were observed among groups (ITS/cp: 0.7196/0.5792) and lineages (ITS/cp: 0.8402/0.7506)  were also observed among almost all lineages  between groups CC and WC for the ITS data and was 0.1681 (p < 0.01) between groups WC and RC for the chloroplast data  was found among different groups and lineages, with a smaller proportion detected among populations within groups and within lineages (10.75–18.63%), while an even smaller percentage was observed within populations (5.24–26.04%) . Meanwhi/0.7506) . The reslineages , betweenlineages , and betlineages . The lowast data .The ABC analysis was performed on all datasets to infer the divergency history of cultivated cherries. Six possible scenarios were designed for three groups a, and moIn order to elucidate the origin of cultivated Chinese cherry, a phylogeographic analysis was further conducted on Chinese cherry and its relatives . Forty ITS haplotypes were identified in the three groups, which could be divided into three sub-lineages  based on haplotype network using BAPS a. The suA total of 80 chloroplast haplotypes were identified in the CC, WC and RC groups, which were also classified into three sub-lineages  based on haplotype network and BAPS analysis a. Sub-liD values in ITS sequences. In chloroplast data, NEC-C and LFZ-C exhibited significantly negative Tajima’s D values, while YGP-R had significantly negative Fu’s Fs value. Considering the sub-lineages, low genetic diversity, significantly negative Tajima’s D and Fu’s Fs values as well as unimodal MDA curves were detected in sub-lineage i for both ITS and chloroplast data  . In the ast data .st values (Fst < 0.2500) in both ITS and chloroplast data. However, moderate Fst values were observed between NEC-C and LFZ-C  as well as between QLM-C and LFZ-C  in the ITS data (st = 0.0025–0.2103) was observed among all geographic subgroups of CC and WC in ITS data. However, low levels of genetic differentiation were only observed between LFZ-W and four subgroups of CC (Fst = 0.0894–0.2016), while the four cultivated subgroups exhibited significant genetic differentiation from YGP-W (Fst = 0.3691–0.8558) and QLM-W (Fst = 0.3117–0.6204). Furthermore, the majority of the geographic subgroups within WC and RC exhibit low or moderate levels of genetic differentiation (ITS/cp: Fst = 0.0203–0.3065/0.1737–0.4754) with one another  were formulated to infer the domestication history of Chinese cherry . The resDue to the parallel evolution of morphological traits, frequent intra- and inter-specific hybridization, and complex biogeographic histories ,21,23,27Cerasus and Microcerasus accessions  (Both nuclear and chloroplast data indicate distant phylogenetic relationships between subgenus cessions . Previouocerasus . Meanwhie of MC)  indicateCerasus. In both nuclear and chloroplast sequences, all European P. mahaleb accessions formed a basal clade in Cerasus  divergced from a mixed ancestral group (CC + WC + RC + EC) approximately 3.52 Ma (95% CI: 0.89–6.50 Ma), which lends further support to this hypothesis  in chloroplast data  . The cenP. tomentosa from the subgenus Microcerasus exhibits high genetic differentiation from other fruiting cherries and evolved independently. The QTP and HHM may intensify genetic differentiation, resulting in the divergence of domestication between cultivated fruiting cherries in Europe  and China . In terms of chloroplast haplotype network, a noticeable divergence was observed between P. avium and most hybrid types among European fruiting cherries, while no significant difference was detected in nuclear haplotype network. Considering the maternal inheritance of chloroplast sequences in higher plants [Malus [Crataegus [Overall, our data suggests that the economically cultivated fruiting cherry species have independent origins. r plants , it is ps [Malus  and Cratrataegus .As mentioned above, multiple instances of hybridization events might occur among cherry species in China. Under this scenario, chloroplast sequences are powerful tools for exploring the origin and domestication of plant species, owing to their maternal inheritance. In chloroplast data, the genetic differentiation between cultivated Chinese cherry  and LFZ-W was much lower compared to other wild subgroups (QLM-W and YGP-W). This provides molecular evidence supporting our recent hypothesis ,51 that C. pseudocerasus) The domestication and migration of woody fruit trees have been demonstrated to be closely associated with anthropogenic civilization, especially the revolutionary improvements and dissemination of agricultural techniques  [Fst values (Fst = 0.4165–0.5494) were detected between the CC and RC, indicating few gene introgression between cultivated Chinese cherries and their close relatives from other Cerasus taxa. This is different from those of other Rosaceae fruit trees, such as apple [Based on nuclear and chloroplast data, we traced the domestication processes and potential dispersal routes of cultivated Chinese cherry (rafting) ,55,56,57rafting)   ,53,58,59as apple ,56,57 anFst = 0.45–0.5494 Microcerasus (MC). In subgenus Cerasus, the sample set consists of 535 accessions of Chinese cherry, 120 European cherry accessions and 174 accessions of other wild relatives. All 535 Chinese cherry specimens, comprising 331 cultivated (CC) and 204 wild individuals (WC), were systematically collected from a total of 70 natural populations (534 accessions) and Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences (ZFRI) (1 accession). The European cherry (EC), including cultivars of P. avium, P. cerasus, P. mahaleb and their inter- and intra-specific hybrids, were collected from ZFRI in a professional manner. All the wild relatives (RC) were collected from 16 natural populations (155 accession) and Kunming Institute of Botany, Chinese Academy of Science  (19 accession) , comprising 48 individuals of P. tomentosa from 8 natural populations, 26 samples of P. tianshanica from 2 natural populations, and 3 and 2 specimens of P. japonica var. salicifolia and P. japonica var. glandulosa respectively from ZFRI  were collected and classified into five groups: cultivated Chinese cherry (CC), wild Chinese cherry (WC), wild relatives (RC), European cherries (EC) and cession) . In subgrom ZFRI . Four plrom ZFRI . Additiomatk and ndhF gene; intergenic spacer of trnQ-5′rps16, trnL-trnF and atpB-rbcL; introns of rpl16 and rps16) were selected to examine their variation by using six cultivated and four wild P. pseudocerasus accessions. Subsequently, three regions of chloroplast genome  exhibiting high variation were remained and utilized for genotyping all 912 accessions . The sequences from the three chloroplast regions and nuclear ITS fragment were aligned separately with CLUSTAL_X 1.81  and manu6 generations with one cold and three heated chains, starting with different random trees and sampling trees every 100 generations. The first 8000 generations were discarded as burn-in. The ML analysis was performed using IQ-TREE V.1.5.5 [https://github.com/rambaut/figtree, accessed on 26 November 2018).The identification of Nuclear DNA (nrDNA) and Chloroplast DNA (cpDNA) haplotypes was performed separately for each datasets in DNAspV5 , with de V.1.5.5  with 100K). Ten algorithm repetitions were performed for each value of K (1–20). The K value with highest marginal log  indicates the optimal number of groups that best fits the data. Meantime, the genetic structures within lineages were also detected using Structure 2.3 [K (1–10) using admixture model and correlated allele frequencies. The MCMC algorithm was run for 100,000 burn-in itertions followed by 100,000 additional generations. The mean membership coefficients (Q) matrix was calculated for each K by using the estimated Q-matrix from 20 runs of each K in CLUMPP software [K) was determined using the method proposed by Evanno et al. [http://taylor0.biology.ucla.edu/structureHarvester/, accessed on 1 July 2014) [Bayesian Analysis of Population Structure version 5.4 (BAPS 5.4) was employed to deduce the genetic structure of each designated lineage based on haplotype data from both nrDNA and cpDNA. The BAPS method uses MCMC simulation to partition different populations or individuals into variable groups  .Cerasus (RC) and Microcerasus (MC) , wild Chinese cherry (WC), European cherries (EC), other members of the subgenus sus (MC) . Distincdistance  to accouD [Fs statistics [D and Fu’s Fs, indicating strong evidence for demographic growth. To further investigate the historical dynamics of both geographic and genetic groups, a mismatch distributions analysis (MDA) was performed using a sudden demographic expansion model with 5000 permutations in ARLEQUIN 3.5. Statistical significance was tested by PRag and PSSD, respectively. This analysis compared the observed frequencies of pairwise differences in haplotypes with those expected under a single sudden expansion model [Neutrality tests were utilized to identify potential historical population events. Tajima’s D  and Fu’satistics  were calon model . It is kon model .Microcerasus and other species, as well as its base location in the phylogenetic tree, we first tested six potential scenarios of divergence to determine whether cultivated cherries originated from Microcerasus. Subsequently, we classified the mix group into CC, WC and RC categories and established three possible scenarios to estimate the probable domestication history of cultivated Chinese cherry  algorithm using DIYABC v.2.1.0 software to establish the most probable scenarios for their domestication history. Based on the taxonomic classification, PCA and phylogenetic tree analysis, all the accessions were categorized into three groups: mix group (CC + WC + RC), group EC, and group MC. According to the significant divergence between e cherry . The Gendy trees . A conseCerasus and Microcerasus. The distinct genetic divergence and separate domestication processes observed between cultivated cherry species from Europe and those from China can be attributed to the geographic isolation caused by the Himalaya-Hengduan Mountains. Two distinct geographic origin centers (Europe and China) were established. Gene tree conflict between nuclear and chloroplast data may result from frequent hybridization among cherry species, as well as the incomplete lineage sorting. The high-altitude regions located at the eastern edge and southern parts of the Himalaya-Hengduan Mountains are likely to have served as glacial refuges for cherries inhabiting in China. Meanwhile, we propose that cultivated Chinese cherries most likely originated in Longmenshan Fault Zone, and subsequently spread throughout four geographic regions, undergoing distinct domestication and divergent selection. This study provides valuable information for the effectively conserving and utilizing cherry resources worldwide.Phylogeographic analyses of 912 cherry accessions indicated distinct genetic differentiation and evolutionary process among cherry species, especially between those belong to the subgenus


Lactobacillus amylovorus has been reported to reduce weight and fat content in humans. To identify and understand the mechanism underlying this process, the complete genome of CICC 6090, which was isolated from pig intestines, was sequenced using PromethION and next-generation sequencing. Lactobacillus amylovorus is a probiotic candidate organism. Nakamura et al.  and 1,110,786,305 bp of raw data were generated. Guppy (version 5.0.16) was used for ONT base calling, and a mean_qscore_template of ≥7 and sequence length of ≥1,000 bp were used for quality control. In all, 981,310,309 bp of data passed quality control. Flye version 2.9  was used for genome assembly  was used for correction with next-generation sequencing data  was used to move the original protein DnaA sequence to the start site of the genome replication to obtain the final genome sequence  broth under anaerobic conditions at 37°C. The steps in the Oxford Nanopore Technologies (ONT) sequencing workflow were as follows. DNA was extracted with a QIAgene bacterial genome extraction kit; DNA quality was assessed by electrophoresis, Nanodrop spectrophotometers, and a Qubit fluorometer. The 20-kb DNA fragments were selected using Blue Pippin (Sage Science). Adapter ligation was performed using NBD103 and NBD114 kits (ONT). The DNA library was quantified using Qubit. Real-time single-molecule sequencing was performed on a Nanopore PromethION sequencer (R9.4.1). In summary, 131,900 reads   were predicted using Prodigal  with intact CDS (https://card.mcmaster.ca/) (http://www.mgc.ac.cn/VFs/)  and no plasmids. The average nucleotide identity (ANI) based on BLAST+ was analyzed online using the system default parameters by comparison with the other five ciesws/) . The ANICP110363 (https://www.ncbi.nlm.nih.gov/nuccore/CP110363). The raw sequences obtained from the Nanopore PromethION sequencer were deposited in the Sequence Read Archive under the BioProject no. PRJNA895949 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA895949). The raw sequences obtained from the DNBSEQ-T7RS platform were deposited in the Sequence Read Archive under the BioProject no. PRJNA898672 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA898672).The complete genome sequences were deposited in the National Center for Biotechnology Information (NCBI) under the accession number


Coptodon camerunensis, was determined by next-generation sequencing. The mitogenome was 16,557 bp long and encoded with 37 genes . The C. camerunensis mitogenome is AT-biased (52.63%), as exhibited in its congener, Coptodon zillii (52.76% and 53.04%). The majority of PCGs start with an ATG initiation codon, except COI, which starts with a GTG codon and five PCGs and ends with the TAA termination codon and except seven PCGs with an incomplete termination codon. In C. camerunensis mitogenome, most tRNAs showed classical cloverleaf secondary structures, except tRNA-serine with a lack of DHU stem. Comparative analyses of the conserved blocks of two Coptodonini species control regions revealed that the CSB-II block was longer than other blocks and contained highly variable sites. Using 13 concatenated PCGs, the mitogenome-based Bayesian phylogeny easily distinguished all the examined old-world cichlids. Except for Oreochromini and Coptodinini tribe members, the majority of the taxa exhibited monophyletic clustering within their respective lineages. C. camerunensis clustered closely with Heterotilapia buttikoferi (tribe Heterotilapiini) and had paraphyletic clustering with its congener, C. zillii. The Oreochromini species also displayed paraphyletic grouping, and the genus Oreochromis showed a close relationship with Coptodinini and Heterotilapiini species. In addition, illustrating the known distribution patterns of old-world cichlids, the present study is congruent with the previous hypothesis and proclaims that prehistoric geological evolution plays a key role in the hydroclimate of the African continent during Mesozoic, which simultaneously disperses and/or colonizes cichlids in different ichthyological provinces and Rift Lake systems in Africa. The present study suggests that further mitogenomes of cichlid species are required, especially from western Africa, to understand their unique evolution and adaptation.The mitogenomic evolution of old-world cichlids is still largely incomplete in Western Africa. In this present study, the complete mitogenome of the Cameroon endemic cichlid,  The family Cichlidae (order Cichliformes) is one of the most species-rich groups, with 1750 recognized species classified under 252 genera . CichlidIn the Neotropical region, multi-locus phylogeny and exon-based phylogenomic approaches have been employed to resolve the taxonomic conundrum, systematics position of competing clades, and evolutionary trend ,16,17,18Molecular data were to determine the population structure of cichlid fish from different river basins and East African lakes ,37,38,39Coptodon includes 32 valid species that are endemic to Africa and the Middle East [Coptodon was carried out based on morphological characteristics, including dentition, body shape, coloration, and meristic and morphometric traits [Coptodon [Coptodon species classification and placed them in the newly defined tribe ‘Coptodonini’ [Coptodon species were recognized in Africa by the rapid success of molecular methods [Coptodon species is sparse in the worldwide GenBank database, with two mitogenomic sequences of a single species, Coptodon zillii (KM658974 and MW194077), which were produced to understand mitogenomic characteristics and phylogeny [Coptodon camerunensis from Cameroon in order to address the gap in mitogenomic-based phylogenetic evaluations. This species is endemic to Cameroon and is classified as ‘Vulnerable’ by the International Union for Conservation of Nature’s (IUCN) Red List of Threatened Species [C. camerunensis mitochondrial genes, as well as their comparison with the C. zillii mitogenome, can contribute to our understanding of genetic evolution. The large-sized DNA sequences of the targeted species will aid in estimating the genetic diversity and population structure of this unusual cichlid fish species in the near future. Furthermore, continental drift and subsequent geological processes, such as drainage evolution, disseminate and colonize fish variety, including cichlids, are also explored throughout African continents [The genus dle East . This grc traits . Later, Coptodon . The intodonini’ ,64. The  methods ,66,67,68hylogeny ,70. Thus Species . We anti Species . The strntinents ,74,75. TCoptodon species specimen was procured from the Nyong River in Cameroon (3.765 N 12.245 E)  [C. camerunensis and C. zillii were acquired from the IUCN (https://www.iucnredlist.org/ (accessed on 15 July 2023) and mapped using ArcGIS 10.6 software .The 2.245 E) . The Inseristics . The synJ938224) . The ran® Genomic DNA extraction kit  and a conventional procedure. The quality and quantity of gDNA were examined through a NanoDrop spectrophotometer . The TruSeq Nano DNA High Throughput Library Prep Kit (Illumina) manufacturer’s instructions were followed in the preparation of the sequencing libraries. Briefly, 100ng of genomic DNA was trimmed using adaptive focused acoustic technology (Covaris), and the unplugged DNA was end-repaired to make 5′-phosphorylated, blunt-ended dsDNA molecules. The TruSeq DNA UD Indexing adapters were ligated to these DNA fragments after a single ‘A’ nucleotide was added. To construct the final DNA library, the products were cleaned and enriched by PCR. The libraries were quantified using qPCR and certified using the Agilent Technologies 4200 TapeStation D1000 screentape (Agilent Technologies). Paired-end (2 × 150 bp) sequencing was executed using the NovaSeq (Illumina) available at Macrogen (https://dna.macrogen.com/), Daejeon, Republic of Korea. High-quality paired-end reads were assembled by the Geneious Prime version 2023.0.1. Further, to obtain the full length of the control region, species-specific primers (5′-GCAACGAGGATTGACGTTCC-3′ and 5′-GGCTAAGCAAGGTGTTATGG-3′) were designed in the present study. The PCR was carried out by a TaKaRa verity thermal cycler containing a 1X PCR buffer, 1 U Taq polymerase, 10 pmol primers, 2.5 mM dNTPs, and 1 µL template DNA. The purification of the PCR products was performed by an AccuPrep® PCR/Gel purification kit . The amplicons were amplified with the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and sequenced bi-directionally using the ABI PRISM 3730XL DNA analyzer housed at Macrogen (https://dna.macrogen.com/), Republic of Korea. The control region was assembled with the complete mitogenome by assuring the overlying regions’ alignment through MEGA X after eliminating the noisy parts through SeqScanner version 1.0 . The direction and boundary of each gene were confirmed through MITOS v806  and MitoAnnotator  web servers [https://www.ncbi.nlm.nih.gov/WebSub/, accessed on 15 July 2023) along with the standard five-column feature table. The genomic DNA were extracted using the AccuPrep servers ,80. The C. camerunensis mitogenome was designed through a MitoAnnotator . The mitogenomic structure and variations were compared with the two available mitogenomes of a single congener, C. zillii . We calculated the overlapping regions and the intergenic spacers between the neighboring genes manually. The nucleotide compositions of PCGs, ribosomal RNA (rRNA), transfer RNA (tRNA), and the control region (CR) were estimated using MEGA X [https://tandem.bu.edu/trf/trf.html, accessed on 15 July 2023) [The spherical view of the g MEGA X . The basg MEGA X . The stag MEGA X ,84. The g MEGA X , and tanly 2023) . Mugil cephalus (AP002930) (family Mugilidae) was incorporated into the dataset as an outgroup. All 13 PCGs were concatenated using the iTaxoTools 0.1 tool to build the dataset for phylogenetic analysis [https://itol.embl.de/login.cgi, accessed on 15 July 2023) [To understand the matrilineal phylogenetic relationships, all accessible African Cichlids mitogenomes were obtained from the GenBank database (Accessed on 19 April 2023) . The mitanalysis . The besanalysis . The conly 2023) .C. camerunensis mitogenome  was determined (GenBank accession number OQ696044) for the first time. A total of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a non-coding AT-rich regulatory region (CR) were found in the circular mitogenome of C. camerunensis. The heavy strand had 28 genes , whereas the light strand contained NAD6 and eight tRNAs   found between the tRNA-Pro (P) and control regions. C. zillii (KM658974) also showed six overlapping regions (24), while the other mitogenome (MW194077) showed seven overlapping regions (25), with the longest overlapping region (10) detected between ATP synthase 8 (atp8) and ATP synthase 6 (atp6) genes. Furthermore, C. camerunensis included 10 intergenic spacer regions totaling 58 bp in length, with the longest stretch (35 bp) located between trnN and trnC. C. zillii mitogenome (KM658974) had nine intergenic spacer regions with a total length of 55 bp, whereas the other mitogenome (MW194077) was marked with the highest (11with 59 bp) among the three Coptodon species  . The C.  species .C. camerunensis mitogenome was AT-biased (52.63%), with 27.12% A, 16.38% G, 30.99% C, and 25.51% T. A Similar AT richness was detected in the nucleotide composition of the other two Coptodon mitogenomes ranging from 52.76%  to 53.04%   . The mitto 0.306 . A similher fish . The worC. camerunensis, 13 PCGs accounted for 69.29% of the mitogenome, measuring 11,472 bp. The shortest PCG was ATP8, with a length of 168 bp, while the largest was NAD5, with a length of 1839 bp. Furthermore, C. zillii (KM658974) and C. zillii (MW194077) both marked the same size as the C. camerunensis with 11,472 bp for a total of 13 PCGs. The total PCGs of C. camerunensis were AT-biased (51.59%), whereas the AT skew and GC skew were −0.067 and −0.332, respectively. The majority of the C. camerunensis PCGs began with an ATG (Methionine) initiation codon, whereas COI began with a GTG  codon , but the incomplete stop codons (TA-) and (T--) were found among NAD2 and ATP6, and among COII, COIII, NAD3, NAD4, and CYTB, respectively. Moreover, a unique feature was observed in C. camerunensis ND6 PCG, which was terminated by the TAG stop codon, while in other mitogenomes of C. zillii, this gene was terminated by the TAA stop codon. The termination codon for most of the PCGs was the same in Coptodon mitogenomes; however, the COI of C. zillii (KM658974) was terminated by the TAG stop codon, and the C. zillii mitogenome (MW194077 and KM658974) was detected with the TAG codon as a termination codon for NAD1 gene  codon . A similAD1 gene . By addiillii MW1077 both C. camerunensis were 2636 bp long , comprised a short ribosomal RNA (12S rRNA) and a big ribosomal RNA (16S rRNA), with lengths of 945 bp and 1691 bp, respectively. The length of the rRNAs of C. camerunensis was the lowest when compared to the other two mitogenomes: 2637 bp in C. zillii (KM658974) and 2638 bp in C. zillii (MW194077)  and 53.14% . The AT skew in the ribosomal RNA varied from 0.210  to 0.212 (C. camerunensis), while the GC skew ranged from 0.126  to 0.123 (C. camerunensis) (C. camerunensis mitogenome consisted of 22 tRNA genes whose length ranged from 66 bp (trnC) to 74 bp (trnK), with a total length of 1554 bp , which was similar to that recorded for C. zillii (MW194077). Moreover, the lowest complete length (1553 bp) for transfer RNA Genes was recognized in C. zillii (KM658974). The 22 tRNA genes of all three Coptodon mitogenomes were AT-biased, ranging from 54.7% (C. camerunensis) to 54.93% . The range of AT skew was 0.020  to 0.031 (C. camerunensis), and GC skew was 0.037  to 0.049   . AT richunensis) . The strM658974) . Except M658974) .C. camerunensis CR was 929 bp, with a 62.86% AT and 37.14% GC content. The full-length sequence of the CR was recovered from two separate mitogenomes of the C. zillii species at two different locations, spanning from 852 bp (MW194077) to 924 bp (KM658974). The AT skew ranged from −0.020  to 0.027 (C. camerunensis), and GC skew was −0.212 (Coptodon camerunensis) to −0.142   were determined to be comparable in both C. camerunensis and C. zillii, as documented in other teleost fishes [Coptodon species using these variable nucleotides. As demonstrated in other species, similar studies on conserved domains may be required to understand the replication and transcription of the mitochondrial genome [The overall length of W194077) . Neithert fishes ,91,97. Tl genome ,91.C. camerunensis depicted close clustering with H. buttikoferi (tribe Heterotilapiini) and showed paraphyletic cladding with its congener, C. zillii  to be endemic to Lake Tanganyka, placed in the basal node of the current topology  showed a close association with the species distributed in different river basins in the present BA topology , Lake Ejagham (4 species), Sanaga River, Wouri River, Nyong River, and Lake Barombi Koto. Based on the observed endemicity, it is reasonable to believe that the West African rift system has had an important influence on the development of Coptodon species in Western Africa. From the known distribution of the studied species, C. camerunensis in the Wouri River originates at the meeting of the Nkam and Makombe River systems northeast of Yabassi City and empties into the Gulf of Guinea. A current expedition in the Nyong River, however, explains the C. camerunensis’ expanded range distribution in south-eastern Cameroon. The current study proposes that correct geographical information, prehistoric drainage evolution, coupled with genomic-based cladistics pattern, would clarify a concrete scenario of old-world cichlids development, including Coptodon lineages in Africa. This is because of the current distribution and diversification.The west-central nation of Cameroon significantly contributes to the continent of Africa’s distinctive faunal makeup. Due to Cameroon’s location at the confluence of the Congo Basin and the West African rainforest, several aquatic habitats have converged there. Due to the range of habitats and ecological niches created by such geographic diversity, numerous organisms, including fish populations, have evolved and adapted ,132. A vCoptodon camerunensis, which are endemic to Cameroon. The structure and variation in different genes could help us to gain knowledge on the mitogenomic evolution of studied species compared with its congener, Coptodon zillii. The functions of the mitochondrial genome and its related genes can be inferred from such empirical molecular data. The mitogenome-based Bayesian phylogeny readily delineated all of the investigated African cichlids, including Coptodon camerunensis, and revealed paraphyletic clustering with Coptodon zillii, in contrast to the earlier hypothesis. However, in order to identify the evolutionary path of Coptodon genus members and confirm their systematic classification, additional mitogenomes from West Africa must be generated. Nuclear genome or whole genome data may help with future research on the phylogeny and population genetics of cichlid fishes. The current study also indicates that geological opportunity has a considerable impact on the African continent’s hydroclimate, which has resulted in the diversification and/or colonization of cichlid species in various ichthyological provinces and rift lakes.The present study decodes the complete mitogenome of


Phytophthora infestans is the most devastating pathogen of potato and a model organism for the oomycetes. It exhibits high evolutionary potential and rapidly adapts to host plants. The P. infestans genome experienced a repeat-driven expansion relative to the genomes of Phytophthora sojae and Phytophthora ramorum and shows a discontinuous distribution of gene density. Effector genes, such as members of the RXLR and Crinkler (CRN) families, localize to expanded, repeat-rich and gene-sparse regions of the genome. This distinct genomic environment is thought to contribute to genome plasticity and host adaptation.in silico approaches to predict and describe the repertoire of P. infestans secreted proteins (the secretome). We defined the "plastic secretome" as a subset of the genome that (i) encodes predicted secreted proteins, (ii) is excluded from genome segments orthologous to the P. sojae and P. ramorum genomes and (iii) is encoded by genes residing in gene sparse regions of P. infestans genome. Although including only ~3% of P. infestans genes, the plastic secretome contains ~62% of known effector genes and shows >2 fold enrichment in genes induced in planta. We highlight 19 plastic secretome genes induced in planta but distinct from previously described effectors. This list includes a trypsin-like serine protease, secreted oxidoreductases, small cysteine-rich proteins and repeat containing proteins that we propose to be novel candidate virulence factors.We used This work revealed a remarkably diverse plastic secretome. It illustrates the value of combining genome architecture with comparative genomics to identify novel candidate virulence factors from pathogen genomes. Phytophthora infestans, the causal agent of the potato and tomato late blight disease, is a successful cosmopolitan plant pathogen. Ever since the Irish potato famine in the middle of the nineteenth century, P. infestans has been recognized as one of the most problematic plant pathogens with a global impact on both commercial and subsistence agriculture [GS]GG repeats. It has one close paralog in P. infestans, PITG_05807 , genome organization appears to be a good indicator of virulence genes in P. infestans. Can this strategy be extended to explore and identify novel effectors from other pathogens? Effector genes often occur in plastic genomic regions. A remarkable example is the plant pathogenic fungus Leptosphaeria maculans in which the AvrLm1, AvrLm6 and AvrLm4-7 effector genes reside in 100 kb or larger AT-rich gene-poor isochores [Alternaria alternata [Mycosphaerella graminicola [Fusarium graminearum [Plasmodium are often found near telomeric regions of chromosomes [We exploited genome organization to augment other criteria for selection of candidate virulence genes in the oomycete plant pathogen sochores -60. In olternata , Mycosphminicola , and Fusminearum , some efomosomes . These somosomes . In summP. infestans stands out by its dramatic uneven distribution in FIR lengths that results in a clear demarcation of GDRs vs GSRs  can be considered a signature of variable genome regions. TEs have long been considered "selfish genes" for causing chromosomal breaks, deletions, or translocations . But sevs Figure . This exn Figure .P. infestans is perhaps surprising. What role may such catalytic enzymes play in the interaction between P. infestans and host plants? How do polymorphisms in these enzymes affect host interactions? BBEs are flavoenzymes that catalyze carbohydrate oxidation in plants, either for the biosynthesis of berberine type alkaloids, or for the generation of hydrogen peroxide (H2O2). Plant BBEs are highly induced during various defense responses, when they may contribute to the oxidative burst leading to cell death, through H2O2 synthesis. CAs typically function in acid-base balance control by rapidly converting carbon dioxide to bicarbonate. CA activity is also required for the onset of disease resistance in tobacco. Silencing of a CA gene in the plant Nicotiana benthamiana results in enhanced susceptibility to P. infestans [Agrobacterium tumefaciens [P. infestans growth. Alternatively, H2O2 production may contribute to plant cell wall degradation by P. infestans. The ability to degrade alkaloids may also contribute to virulence of various plant pathogens [P. infestans secreted BBE and α-CA enzymes. Plant secondary metabolites are structurally highly diverse, and their corresponding biosynthetic genes are frequently associated with divergent genome regions [Among the novel candidate virulence genes we identified, there were two types of oxidoreductases . The presence of enzymes catalyzing conversion of rather simple molecules within the plastic secretome of nfestans  and a saefaciens . Therefoathogens , for insathogens ,73. In a regions ,75. PlanP. infestans genes from the plastic secretome, PITG_02524 and PITG_08563, are predicted pectin lyases, which are known in other pathogens as virulence factors that degrade the pectic components of plant cell walls [P. infestans secreted CWDEs to the complexity of the plant cell wall. But how to explain the high divergence observed in the CWDEs in plastic regions? First, plant cell walls are highly variable from one plant species to another and between different stages of plant development [P. infestans secreted CWDEs may have been driven by arms race coevolution with host inhibitors. Third, cell wall degradation products can act as damage-induced molecular patterns (DAMPs) and trigger plant immune responses [P. infestans CWDEs may therefore evolve to minimize DAMP induction. In summary, localization of particular carbohydrate binding protein genes in plastic genomic regions may have contributed to the pathogenic success of P. infestans.Cell wall degrading enzymes (CWDEs) are a hallmark of filamentous pathogen secretomes ,76,77. All walls . Anotherll walls  or functll walls . In planll walls . It is telopment . Therefoelopment . Rapid eesponses . P. infeP. infestans but lacking in P. sojae and P. ramorum. Similar properties are typical of the virulence genes of a variety of fungal and oomycete pathogens [AvrLm1 that is responsible for gain of virulence on Rlm1 plants in L. maculans [Avr9 and avr4E of Clasdosporium fulvum [SIX1 of Fusarium oxysporum [Avr1-CO39 and Avr-Pita of Magnaporthe grisea [Plasmodium spp. [It is well accepted that due to metabolic costs and spatial constraints, genome expansion is globally selected against unless it provides an important functional advantage . Althougathogens ,86. Thismaculans . Similarm fulvum , SIX1 ofxysporum  and Avr1e grisea ,90. Addiium spp. . Such geium spp. . Anotherium spp. ,92, possium spp. . Future P. infestans using in silico approaches. We quantitatively described P. infestans genome organization by delimiting gene dense and gene sparse regions. We used genome organization as a novel approach that augments previously established criteria to mine for candidate virulence factors. Occurrence of secreted protein genes in GSRs, in combination with comparative genomics and transcriptomics, implicated 19 previously overlooked genes in virulence. These include cell wall degrading enzymes, trypsin-like serine protease, carbonic anhydrase, berberine bridge enzyme, several repeat containing proteins, and small cysteine-rich proteins.In this study, we predicted and annotated the secretome of the Irish potato famine pathogen et al. (2003) [et al. [et al. (2009) [P. infestans T30-4 genome assembly were submitted to SignalP v2.0 [P. infestans secretome.Signal peptide predictions were performed following the methods of Torto . (2003)  and Win  [et al. . The 18,. (2009)  from thealP v2.0 . A SignaalP v2.0 , RPSP [9alP v2.0 , TargetPalP v2.0 , WolfPSoalP v2.0  and TMHMalP v2.0  , or tomato at 2 or 5 dpi, compared to in vitro grown mycelia. In Figures 2 (linear expression in sample/average linear expression in control mycelia).Whole genome expression data used in this work were previously described by Haas et al.  and are P. infestans BBEs were modeled based on homology with the template protein structures of Acremonium strictum 1ZR6A [Eschscholzia californica 3D2H [3D structure of PITG_02935, PITG_02930 and PITG_06585 um 1ZR6A  and Eschica 3D2H . The aliica 3D2H  were useica 3D2H  and 1JD0ica 3D2H . Renderiica 3D2H . To compBBE: Berberine Bridge Enzyme; CA: Carbonic Anhydrase; CNV: Copy Number Variation; CRN: Crinkler effector; CWDE: Cell Wall Degrading Enzyme; DAMP: Damage induced Molecular Pattern; dpi: days post inoculation; FIR: Flanking Intergenic Region; GDR: Gene Dense Region; GIP: Glucanase Inhibitor Protein; GOOX: Glucooligosaccharide oxidase; GSR: Gene Sparse Region; OS: Orthologous Segments; RCP: Repeat-Containing Protein; SCR: Small Cysteine-Rich protein; TE: Transposable Element.SR carried out most analyses, contributed to the design of the study and drafted the manuscript. JW performed the secretome predictions, provided guidance with some of the analyses and contributed to the writing of the manuscript. LC helped with the gene expression analyses and contributed to the writing of the manuscript. SK conceived and designed the study, edited the manuscript and supervised the work. All authors read and approved the final manuscript.P. infestans predicted secretomeComplete list of . Microsoft Excel spreadsheet containing complete list of P. infestans secretome genes, including annotation steps, biological processes and biological function categories, genomic and expression data.Click here for fileP. infestans complete proteome and secretome according to protein length, cysteine and glycine contentThe distribution of . Graphs showing the distribution of protein length, cysteine and glycine content among P. infestans genes.Click here for fileP. infestans plastic secretomeGene conservation in . A) The different levels of orthology and conservation for P. infestans genes. 5994 P. infestans genes are absent from genome segments orthologous with P. sojae and P. ramorum genomes, 2396 of them do not have orthologs  in P. sojae or P. ramorum genomes. B) Frequency of P. infestans genes with orthologs in P. sojae and P. ramorum. The level of orthology is given by % of sequence identity between P. infestans protein and its best BlastP hit in P. sojae or P. ramorum proteome. Frequencies among non-secreted proteins (grey), plastic secretome (green) and other secretome proteins (yellow) are shown.Click here for fileFrequency of secretome genes, genes from non-OS, GDR and GSRs. table showing the number and % of genes from various groups matching specific conditions related to genome environment and secretome featuresClick here for filein plantaNumber and frequency of genes induced . table comparing the distribution of genes induced in planta at different time point from the whole genome and the plastic secretomeClick here for filein planta described in details in this manuscriptGenomic context of the 19 genes from the plastic secretome induced . Figure showing genes, orthologous gene pairs, and repeated sequences in the genomic environment of 19 genes from the plastic secretome.Click here for fileP. infestans berberine bridge enzymes and alpha carbonic anhydraseAlignments used for 3D modeling of .Click here for fileP. infestans Small Cysteine-rich proteinsGlobal analysis of . A) Distribution of P. infestans proteins according to their length and cysteine content. B) Position of PITG_04202 and other P. infestans secreted SCRs on the FIR heat map. C) Frequency of SCR sequences as a % of P. infestans whole proteome, secretome and non-secreted proteins; frequency of SCR genes induced as a % of SCR genes in P. infestans whole genome, secretome and non-secretome genes.Click here for file


Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguinis are members of the Mitis group of streptococci and agents of oral biofilm, dental plaque and infective endocarditis, disease processes that involve bacteria-bacteria and bacteria-host interactions. Their close relative, the human pathogen S. pneumoniae uses pilus-islet 2 (PI-2)-encoded pili to facilitate adhesion to eukaryotic cells.PI-2 pilus-encoding genetic islets were identified in S. oralis, S. mitis, and S. sanguinis, but were absent from other isolates of these species. The PI-2 islets resembled the genetic organization of the PI-2 islet of S. pneumoniae, but differed in the genes encoding the structural pilus proteins PitA and PitB. Two and three variants of pitA (a pseudogene in S. pneumoniae) and pitB, respectively, were identified that showed ≈20% difference in nucleotide as well as corresponding protein sequence. Species-independent combinations of pitA and pitB variants indicated prior intra- and interspecies horizontal gene transfer events. Polyclonal antisera developed against PitA and PitB of S. oralis type strain ATCC35037 revealed that PI-2 pili in oral streptococci were composed of PitA and PitB. Electronmicrographs showed pilus structures radiating >700 nm from the bacterial surface in the wild type strain, but not in an isogenic PI-2 deletion mutant. Anti-PitB-antiserum only reacted with pili containing the same PitB variant, whereas anti-PitA antiserum was cross-reactive with the other PitA variant. Electronic multilocus sequence analysis revealed that all PI-2-encoding oral streptococci were closely-related and cluster with non-PI-2-encoding S. oralis strains.This is the first identification of PI-2 pili in Mitis group oral streptococci. The findings provide a striking example of intra- and interspecies horizontal gene transfer. The PI-2 pilus diversity provides a possible key to link strain-specific bacterial interactions and/or tissue tropisms with pathogenic traits in the Mitis group streptococci. Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis are close relatives of the important human pathogen Streptococcus pneumoniae. They are members of the normal human oral flora, but in contrast to S. pneumoniae only occasionally cause acute or chronic disease as opportunistic human pathogens. But as early colonizers in the development of oral biofilms  S. sanguinis resembles the PI-1 pilus islet in S. pneumoniaeS. pneumoniae encode two antigenically different pili pitA, encodes signature motifs of a Gram-positive surface protein, i.e. an N-terminal signal sequence and a C-terminal CWSS, but it is a pseudogene due to a stop-codon causing its premature termination. The PitB-based pilus has been shown to be present as a single structure on the bacterial surface, in contrast to the typical multitude of pilus structures per bacterial cells observed for other Gram-positive bacteria srtG1 and srtG2, the latter is a pseudogene in most of the analyzed PI-2 gene clusters pepT and hemH genes In Mitis group streptococci, pili have been identified in S. oralis and S. mitis, with several strains expressing pili that, in addition to the pilus backbone protein, have a putative adhesin protein attached to the pilus backbone structure. Genetic variability between genes encoding the PI-2 pilus proteins showed that the PI-2 islet in oral streptococci was subject to horizontal gene transfer between these species. Further, by immunogold electron microscopy (EM), pili were found to share the morphology of the pneumococcal PI-2 pili. The sequence variation in PitB was shown to result in antigenic variation. The variability of the pilus proteins within and beyond the species suggests a role for PI-2 pili in the strain-specific tissue tropism of the Mitis group of streptococci.We report the first identification of PI-2 pili in S. pneumoniae, the publicly available genomic sequences of oral Mitis group streptococci and selected additional strains , and S. oralis Uo5 S. oralis strain ATCC10557 and strain 34 , but closer to S. pneumoniae strains (39%), suggesting that the PI-2 islet may have evolved in a different species, with slightly lower GC-content.In a search for pilus-encoding islets that resemble PI-2 of  strains , isolateectively . The ovepitA gene, which is a pseudogene in the S. pneumoniae PI-2 pilus islet, was the first gene of the oral streptococci PI-2 islet  encoded a protein with a non-canonical LPXTG-like motif (VTPTG) in the CWSS. The same motif is also present in PitB of S. pneumoniaeS. oralis 34, the start codon was missing and alternative start codons were not apparent, suggesting that pitB was not transcribed in this strain  throughout the entire 6.8 kb region and pitB . Two varsequence . SurprisATCC6249 . A sequeATCC6249 . The passe genes . The DNAequences . The strS. mitis ATCC6249 , were prepared and analyzed for the presence of pilus protein-containing polymers by Western blots. Gram positive pilus polymers produce a characteristic high molecular weight (HMW) banding pattern in Western blots developed with an antibody against a pilus protein ATCC6249 , the str strains . In addiS. oralis ATCC35037, ATCC10557, and S. sanguinis ATCC49296  were detected, demonstrating that PitA was expressed, but not polymerized , the lack of detection of HMW bands or PitA monomer in the latter strain suggests that PitA may not be expressed in ATCC6249. Taken together, these findings demonstrated the formation of pilus polymers in S. oralis ATCC35037, S. oralis ATCC10557, S. mitis 6249 and S. sanguinis ATCC49296. In addition, the results confirmed that PitA, at least in ATCC35037, ATCC10557 and ATCC49296, was part of PI-2 pilus polymers. The PitB variants were antigenically different from each other, whereas the PitA variants showed some cross-reactivity with the used polyclonal antiserum.To determine whether PitA was present in pilus structures, the cell wall extracts of the five strains and the two PI-2 deletion mutants were examined for HMW bands with the polyclonal anti-PitA antiserum . HMW banTCC49296 . The HMWTCC49296 . The twoTCC49296 . Detectiymerized . Since pS. oralis strain ATCC35037 and S. mitis ATCC6249 . Anti-PitA antiserum revealed no pilus structures on the surface of any of the tested strains (data not shown), suggesting that PitA was not present along the pilus backbone. Pilus structures were not visible by negative staining alone and other defined surface structures were not discernible (not shown).To confirm and evaluate the morphology of pilus structures on the surface of the streptococcal isolates that encode PI-2 islets, bacteria were analyzed by EM. Immunogold-labeled anti-PitB-antiserum and anti-PitA-antiserum were used to study the wild type strains and deletion mutants. Anti-PitB-antiserum reacted with pili on the surface of ATCC6249 . The labS. oralis strain ATCC35037 and S. mitis ATCC6249. Strong evidence for the presence of PI-2 pili was obtained for S. oralis ATCC10557 and S. sanguinis strain ATCC49296 in Western blots using anti-PitA antiserum. The anti-PitB antisera lacked cross-reactivity with pilus protein variants different from ATCC35037.Taken together, pilus structures were observed on the surface of S. pneumoniae, S. oralis, S. mitis and S. sanguinis, and also in the unassigned streptococcal strain C300, and the islet was always inserted between the pepT-hemH genes , the close relative of S. parasanguinis, Streptococcus australisStreptococcus cristatus (formerly S. crista) and an isolate of Streptococcus infantis  a 4.1 kb region, which is unrelated to PI-2, was inserted between pepT and hemH . PitA is a putative adhesin, which is suggested by the presence of a vWA domain. The vWA domain is present in both PitA variants, such as in ATCC35037 and ATCC6249 despite their 20% sequence variation. Further, the second sortase gene encoded in the PI-2 pilus islet, srtG2, also a pseudogene in most pneumococci PI-2 pili have formerly been identified in pitA and pitB variants, respectively, is consistent with the variation in previously identified antibiotic resistance-conferring mosaic genes in penicillin binding proteins (PBPs) of S. pneumoniae that originated from PBPs of S. mitis or S. oralisAnalysis of the PI-2 islets provided evidence for horizontal gene transfer (HGT) events, which were not limited to the acquisition of the entire PI-2 islet but also involved recombination events within the islet, as evident by the presence of combinations of particular PitA or PitB variants found within and beyond species . The obsS. pneumoniae mediates adhesion to eukaryotic cells, although with far less efficiency than observed for other Gram-positive pili et al. have shown that the vWA domain in the pilus protein Gbs1474 (PilC) of S. agalactiae is critical for adherence to epithelial cells The modular structure of the pilus proteins, with highly conserved signal sequences and motifs in the cell wall sorting signal, in combination with conserved biosynthetic genes, provides a framework to incorporate variable domains that may result in pili with different functions. The PitA variants as well as the PitB variants may facilitate adhesion. The PitB-based PI-2 pilus in S. pneumoniae PI-2 islet is the inactivation of pitA and srtG2. In general, gene inactivation was not observed in the streptococcal PI-2 gene clusters. But the pitB gene of S. oralis strain 34 lacked a start codon, and pilus structures were not seen. However, PitA monomer was identified in cell wall extracts of this strain and may exert an adhesive function on the bacterial surface, even though not as part of a pilus structure. Also, the absence of PitA in strain ATCC6249 requires further evaluation. Further studies will have to determine whether selective gene inactivation but retention of parts of the biosynthetic machinery of the PI-2 pili may provide an additional molecular mechanism to enhance variation in structure and function of PI-2 pili, and whether this may modulate the tissue tropism of certain strains.A striking feature in the S. oralis, S. mitis and the more distantly related S. sanguinis suggested a distribution of PI-2 throughout the entire streptococcus Mitis group. However, eMLSA revealed that all PI-2-containing strains were closely related to each other and to PI-2 lacking strains of S. oralis. This included S. sanguinis ATCC49296, which was in fact identified as S. oralis based on biochemical characterization in an earlier study S. sanguinis. Although HGT and the ability to develop competence for genetic transformation S. pneumoniae, S. mitis and S. oralisS. pneumoniae, S. mitis, and S. oralis.Identification of the PI-2 islet in species of S. oralis, S. mitis and S. sanguinis (most of them then classified as S. sanguis) by electron microscopy S. oralis strains ATCC35037, ATCC10557, and 34 were described to express fibrils, but no fimbriae S. gordoniiS. oralis and S. sanguinis strains S. oralis and S. mitis, but due to major sequence variation in the binding domain, their function remains to be determined Filamentous surface structures have long been observed on the surface of oral streptococci, including strains of S. oralis and S. mitis. The observed intra- and interspecies variation in the genes encoding these pili provide evidence for horizontal gene transfer events between and within PI-2 islets of these species. Further work will have to determine whether the pilus protein variants confer different adhesive properties to the respective strain. The discovery of PI-2 pili in oral Mitis group streptococci may provide a key to dissect the heterogeneity of interspecies and host interactions associated with these bacteria.Taken together, genetically variable PI-2 pili were identified and characterized in multiple strains of Escherichia coli and 800 µg/ml for streptococcal strains.All streptococcal strains used in this study are listed in sipA,pepT and hemH. All primers were designed based on the sequence of S. oralis strain ATCC35037.To identify PI-2 islets primers sipA_so_up and sipA_so_dn , designewww.viridans.emlsa.net). Multilocus sequence information of strains from genomic sequencing projects was retrieved from the according loci of the genomic sequences, in case the data was not already included in the eMLSA database, and from strains with not yet available sequence information by amplification and sequencing of the corresponding loci. Primers and conditions were used according to the online instructions. Alignment of concatenated sequences was performed with MEGA version 5.0 The phylogenetic relationship between strains used in this study was determined by multilocus sequence analysis S. oralis strain ATCC35037 were amplified using primer pairs pepT_so_F/PitA_so_3 and SrtG2_2/hemH_so_R, respectively (MfeI restriction site between the two fused fragments. The PCR product was cloned into the pCR-8/GW-TOPO vector (Invitrogen), resulting in pCR8-ΔPI-2. The kanamycin-cassette of pSF151 EcoRI digestion, and ligated into MfeI-digested pCR8-ΔPI-2. The resulting plasmid pCR-ΔPI-2::km was used to transform strains ATCC35037 and ATCC10557 following established protocols 3′- and 5′-flanking regions of PI-2 of ectively . Both PCpitA and pitB, corresponding to the mature proteins  were amplified from chromosomal DNA of S. oralis ATCC35037 using primer pairs PitA_so_SmaI_F/PitA_so_NotI_R, and PitB_so_BamHI_F/PitB_so_Xho_R, respectively (E. coli expression vector pET-32a+ (Stratagene). The thioredoxin/6xHis-fusion proteins were purified using the B-PER 6xHis Fusion Purification Kit (Pierce). The N-terminal thioredoxin/6xhis-tag was removed by cleavage with recombinant enterokinase (Novagen) according to the manufacturer's recommendations. Purified PitA and PitB proteins were used to produce polyclonal antisera in rabbits (Covance Research Products Inc.).The regions of ectively  and clon2. Bacteria were collected from plates by adding 750 µl 10 mM Tris, pH 8 buffer to the plate surface, followed by resuspension and recovery of the bacteria. Samples were centrifuged for 5 min at 13000 x g and bacterial pellets were used for further processing. Bacterial pellets (approximately 40 mg per strain) were resuspended in 250 µl 10 mM Tris; pH 8.0 supplemented with 400 U/ml mutanolysin  and 30% raffinose. After 2 h of incubation at 37°C, cell wall extracts were collected by centrifugation at 12,000 × g for 15 min. 25 µl cell wall extract were mixed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiled for 10 min immediately before separation on a 3%–8% Tris-Acetate polyacrylamide gel . For immunostaining, proteins were transferred to a nitrocellulose membrane and detected with polyclonal anti-PitA  or anti-PitB antiserum .Cell wall extracts of streptococcal strains were prepared from bacteria grown on trypticase soy agar (TSA) plates containing 5% sheep blood (BD) overnight at 37°C and 5% CO2. Bacteria were resuspended in PBS before being applied to carbon-coated copper grids , and stained with 1% methylamine tungstate for 30 seconds. Alternatively, strains were applied to carbon-coated nickel grids  and immunostained according to established protocols Strains were grown on TSA-blood agar plates  overnight at 37°C and 5% COS .oralis ATCC10557: JF496566 and S. oralis 34: JF496567.The following GenBank accession numbers were assigned to PI-2 of Figure S1Alignment of PitB proteins of oral Mitis group streptococci and S. pneumoniae. PitBs of S. oralis Uo5 and S. sp. C300 are not shown . Only residues different from the consensus sequence are indicated. Sequence positions based on the S. oralis ATCC35037 sequence are indicated above the alignment. The Signal peptides (SP) and cell wall sorting signals (CWSS) are underlined and the LPXTG-like motif (VTPTG) is shown in bold. Signal peptides were predicted by the SignalP program. Conserved amino acids involved in intramolecular isopeptide bonds in S. pneumoniae PitB are indicated by an asterisk followed by the number for bond 1 and 2, respectively. GenBank accession numbers are as in (PPT)Click here for additional data file.Figure S2Evidence for past recombination events in PI-2 pilus islets of Mitis group streptococci. A. Schematic overview of PI-2 pilus islets. Sequences of S. oralis Uo5 and S. sp. C300 are not shown . Gene regions encoding conserved motifs are indicated by a black bar below the gene. SP: Signal peptide, vWA: von Willebrand A domain, CWSS: cell wall sorting signal. Red squares indicate the regions of sudden, major changes in sequence polymorphisms aligned in panels B.-E. In panels B.-E. Regions of major sequence polymorphisms are indicated in grey. GenBank accession numbers: S. oralis strains ATCC35037: AEDW01000020; ATCC10557: JF496566; and 34: JF496567; S. mitis ATCC6249: AEEN01000012; S. sanguinis ATCC49296: AEPO01000013; and S. pneumoniae GA47884: GU256423. Alignment was performed with ClustalW software.(PPT)Click here for additional data file.Table S1Primers used in this study.(DOC)Click here for additional data file.


Anopheles salivary proteins can indicate individual exposure to bites of malaria vectors. The extent to which these salivary proteins are species-specific is not entirely resolved. Thus, a better knowledge of the diversity among salivary protein repertoires from various malaria vector species is necessary to select relevant genus-, subgenus- and/or species-specific salivary antigens. Such antigens could be used for quantitative (mosquito density) and qualitative (mosquito species) immunological evaluation of malaria vectors/host contact. In this study, salivary gland protein repertoires  from several Anopheles species were compared using in silico analysis and proteomics. The antigenic diversity of salivary gland proteins among different Anopheles species was also examined.Antibody responses against In silico analysis of secreted salivary gland protein sequences retrieved from an NCBInr database of six Anopheles species belonging to the Cellia subgenus  and Nyssorhynchus subgenus  displayed a higher degree of similarity compared to salivary proteins from closely related Anopheles species. Additionally, computational hierarchical clustering allowed identification of genus-, subgenus- and species-specific salivary proteins. Proteomic and immunoblot analyses performed on salivary gland extracts from four Anopheles species  indicated that heterogeneity of the salivary proteome and antigenic proteins was lower among closely related anopheline species and increased with phylogenetic distance.Anopheles genus at the protein level. This work demonstrates that a molecular diversity is exhibited among salivary proteins from closely related species despite their common pharmacological activities. The involvement of these proteins as antigenic candidates for genus-, subgenus- or species-specific immunological evaluation of individual exposure to Anopheles bites is discussed.This is the first report on the diversity of the salivary protein repertoire among species from the  Anopheles genus are vectors of Plasmodium parasites, the causal agents of malaria. This major vector-borne disease affects around 216 million individuals annually and leads to more than 600,000 deaths, mainly in tropical and sub-tropical countries , An. arabiensis [7173], An. stephensi [30069], An. funestus [62324], An. darlingi [43151] and An. albimanus [7167], using the search term “salivary” in any fields of their description text. Signal peptides were predicted by submission of the protein sequences to the SignalP server 3.0 , An. arabiensis [7173], An. stephensi [30069] and An. albimanus [7167] together . The following search parameters were used: precursor-ion mass tolerance of 0.8 Da, fragment ion tolerance of 0.8 Da with methionine oxidation and cysteine carboxyamidomethylation specified as differential modifications, and a maximum of one missed cleavage site allowed. Scaffold  was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm [All MS/MS spectra from one species were gathered and sequence database searched using the Bioworks 3.1 (ThermoFinnigan) or Mascot Daemon v.2.2.2 . The MS/MS spectra were searched against the non-redundant protein database  of lgorithm . Proteinlgorithm .The authors declared no conflict of interest concerning the work in this paper.Conceived and designed the experiments: AL, FA, RC. Performed the experiments: FA, AL, BE. Analyzed the data: FA, AL, BuS. Contributed reagents/materials/analysis tools: BrS, PM, VC, GS, FT. Wrote the paper: FA, AL. All authors read and approved the final manuscript.Average percentage identity between local alignments of secreted salivary proteins from six Anophelesspecies. All secreted salivary protein sequences from each Anopheles species  matching at least one other salivary protein in another species at 40% identity threshold  protein sequence of each cluster. The percentage identity between the representative protein sequence (*) and other protein sequences is given for each cluster. Protein in bold are new clusterised proteins at each identity threshold. Results from this table are graphically represented on Figure 2.Click here for fileProteins identified by MS in salivary gland extracts of four Anophelesspecies. All MS/MS spectra resulting to every protein bands from each species  were gathered and searched on sequence databases of the four Anopheles species together. A list of all unique proteins identified in salivary gland extracts in both replicates is presented for each Anopheles species. Salivary gland proteins were sorted according to their signal peptide prediction  [Network) ,65 to diClick here for fileHierarchical clustering of putative secreted proteins identified in Anophelessalivary gland extracts. Proteins from An. gambiae, An. arabiensis, An. stephensi and An. albimanus SGEs were identified by mass spectrometry after in-gel trypsin digestion. Protein sequences were submitted to SignalP 3.0 server [i.e., longest) protein sequence of each cluster. Anopheles species in which secreted salivary proteins were identified are indicated. The last common taxon encompassing homologous proteins at the genus level is indicated according to in silico results . Lines in bold indicate proteins identified in antigenic bands ; AAR, An. arabiensis; AST, An. stephensi; AAL, An. albimanus; MW: Molecular weight.0 server  to selec0 server . * indics Figure . AGA, AnClick here for fileProteins identified by MS in antigenic protein bands from salivary gland extracts from Anophelesspecies. Salivary gland proteins identified in each antigenic protein band from An. gambiae, An. arabiensis, An. stephensi and An. albimanus SGEs are indicated. Band numbers correspond to those indicated on Figure Click here for fileAlignment of members of the GE-rich/30 kDa/anti-platelet protein family from An. gambiae, An. stephensi and An. albimanus. The numbers in the sequence titles indicate the NCBI accession number.Click here for file