Amomum villosum is an important medicinal and edible plant with several pharmacologically active volatile oils. However, identifying A. villosum from A. villosum var. xanthioides and A. longiligulare which exhibit similar morphological characteristics to A. villosum, is difficult. The main goal of this study, therefore, is to mine genetic resources and improve molecular methods that could be used to distinguish these species. A total of eight complete chloroplasts (cp) genomes of these Amomum species which were collected from the main producing areas in China were determined to be 163,608–164,069 bp in size. All genomes displayed a typical quadripartite structure with a pair of inverted repeat (IR) regions  that separated a large single copy (LSC) region  from a small single copy (SSC) region . Each genome encodes 113 different genes with 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. More than 150 SSRs were identified in the entire cp genomes of these three species. The Sanger sequencing results based on 32 Amomum samples indicated that five highly divergent regions screened from cp genomes could not be used to distinguish Amomum species. Phylogenetic analysis showed that the cp genomes could not only accurately identify Amomum species, but also provide a solid foundation for the establishment of phylogenetic relationships of Amomum species. The availability of cp genome resources and the comparative analysis is beneficial for species authentication and phylogenetic analysis in Amomum. Amomum villosum Lour., which belongs to the monophyletic Zingiberaceae family, is a valuable medicinal plant in China with a history of more than 1300 years [A. villosum Lour., A. villosum Lour. var. xanthioides T. L. Wu et Senjen and A. longiligulare T. L. Wu [A. villosum than in A. villosum var. xanthioides and A. longiligulare, which is consistent with the statement that the effects of A. villosum are better than those of the other two species [A. villosum in clinical use. Given the special flower structure of A. villosum, the plant exhibits difficulty in self-pollination and must rely on a few kinds of insects or artificial pollination. This pollination characteristic greatly reduces the yield of A. villosum [A. villosum var. xanthioides and A. longiligulare, which exhibit high similarity in morphological characters and confusing Chinese names with A. villosum, are often used as adulterants or contaminants of A. villosum in the market , which comprised four , two (Sample numbers: Y17088 and Y19018), and two (Sample numbers: Y19019 and Y19020) individual plants of A. villosum, A. villosum var. xanthioides and A. longiligulare, respectively. All three species studied here included individuals from the main producing areas in China [A. villosum, A. villosum var. xanthioides and A. longiligulare were determined to be 163,608-164,069 bp in size  contained two introns  value [A. villosum were encoded by 27,730 codons, A. longiligulare was encoded by 27,720 codons, and that of A. villosum var. xanthioides was 27,726–27,732 codons , A. villosum var. xanthioides , and A. longiligulare . Conversely, cysteine, with 316–317 (1.1%) codons, was the least frequent amino acid in the cp genomes of these three species. Except for trnL-CAA encoded by UUG, amino acid codon (RSCU > 1) in the cp genomes of three species preferentially showed A- or U-endings, which corresponded to the mentioned results that were calculated based on the cp genome sequences.Codon usage patterns of coding sequences for U) value . We defi2 codons . Like moA. villosum, A. villosum var. xanthioides and A. longiligulare. A total of 157 SSRs were identified in the whole cp genome of A. villosum, including 91 mono-, 33 di-, 7 tri-, 18 tetra-, three penta-, and five hexa-nucleotide SSRs  or 33 (Y19018) di-, eight tri-, 18 tetra-, three (Y17088) or five (Y19018) penta-, and five hexa-nucleotide SSRs , also called microsatellites, are short and tandem repeat DNA sequences with sizes of 1–6 bp  that areide SSRs . Among tide SSRs . A totalide SSRs . The totide SSRs . For A. ide SSRs . Our res) or 33 Y018 di-, ide SSRs . All SSRAmomum species. A total of 74 long repeats were present in the cp genome of A. villosum, as follows: 29 forward repeats, 39 palindromic repeats, five reverse repeats, and 1 complement repeat (A. villosum var. xanthioides including 25 (Y17088) or 24 (Y19018) forward repeats, 36 (Y17088) or 32 (Y19018) palindromic repeats, two (Y17088) or 11 (Y19018) reverse repeats and zero (Y17088) or one (Y19018) compliment repeats. As for the cp genomes of A. longiligulare, there were 53 long repeats, including 16 forward repeats, 29 palindromic repeats, five reverse repeats, and three complement repeats. In all three species, the majority of these repeats showed lengths between 30 and 39 bp. Only A. villosum var. xanthioides (Y17088) exhibited eight long repeats more than 100 bp in size , palindromic repeats (P), reverse repeats (R), and complement repeats (C). These repeat structures promote the rearrangement of the cp genome and increase the population’s genetic diversity . We analt repeat . A total in size .A. villosum, two individuals of A. villosum var. xanthioides and two individuals of A. longiligulare were very small. Here, in each of the three species, one of the cp genomes was used for interspecies comparative analyses within Amomum species A. villosum (Y17085), A. villosum var. xanthioides (Y17088), and A. longiligulare (Y19019).From the above analysis, we can see that intraspecific differences between the cp genomes of four individuals of Amomum species  were conducted using mVISTA software with the annotated cp genome of A. villosum as a reference  genes were the most conserved in the two IR regions. The non-coding regions were more divergent than the coding regions. The cp genomes of A. villosum, A. villosum var. xanthioides and A. longiligulare showed little difference from one another. Comparing A. krervanh and A. compactum, the highly divergent regions among these five cp genomes mainly occurred in the intergenic regions, including atpH-atpI, trnD-trnY, accD-psaI, ycf4-cemA and trnI-ycf2, which may be potential molecular markers for species authentication.Interspecific comparisons between five eference . Among t2,766 bp . The comAmomum species. The average value of nucleotide variability (PI) of all the five Amomum species was 0.00306. Results shown in Amomum species were commonly located in the LSC and the SSC regions, which was in line with the result from mVISTA.DNA polymorphism analyses were executed to detect highly variable regions and to show divergence at the sequence level in the cp genomes of the five atpH-atpI, trnD-trnY, accD-psaI, ycf4-cemA, and trnI-ycf2), a total of 32 samples from eight Amomum species  were collected. Information about sample numbers and locations are listed in atpH-atpI, trnD-trnY, accD-psaI, ycf4-cemA, and trnI-ycf2  tree  was clustered into a monophyletic branch respectively. All five Amomum species formed a strongly supported monophyletic clade sister to Alpinia oxyphylla, which is sometimes used as an adulterant of amomi fructus. This topological structure thereby hinted at a close relationship between these taxa. In addition, among the three Amomum species studied here, branches of four A. villosum and two A. villosum var. xanthioides clustered into a clade, thereby reflecting a closer relationship of these species. Two A. longiligulare clustered into a monophyletic branch separately from A. villosum and A. villosum var. xanthioides with high support rates. This showed that common protein-coding genes based on cp genomes could be considered an efficient resource for species authentication in Amomum. Results were consistent with traditional phylogenetic theory and provide a valuable basis for the establishment of phylogenetic relationships of species in Zingiberales.To discuss the discriminatory power of cp genomes and determine the phylogenetic positions and evolutionary relationships of ML) tree  of 17 reA. villosum, A. villosum var. xanthioides and A. longiligulare were collected from their main producing areas. A total of four samples of A. villosum were collected respectively, from Yangchun City, Guangdong Province; Nanning City, Guangxi Province; Haikou City, Hainan Province; and Xishuangbanna City, Yunnan Province. Two samples of A. villosum var. xanthioides were collected from Xishuangbanna City, Yunnan Province; and two samples of A. longiligulare were collected from Haikou City, Hainan Province and Guangzhou City, Guangdong Province. A total of 32 dry leaf samples of eight Amomum species  were collected for verification. The origin information of all samples is listed in w/v) agarose gel. The pure DNA from dry leaves was used to perform polymerase chain reaction (PCR) and Sanger sequencing. The pure DNA from fresh leaves was used to construct a shotgun library with an average insert size of 500 bp and carry out next generation sequencing (NGS) by an Illumina HiSeq X 10 platform in accordance with standard protocol.Fresh leaves of 8 individuals of Raw reads obtained from NGS were first checked by FastQC and trimmed using Trimmomatic software ,46. The The complete cp genome was annotated using CPGAVAS  and DOGMThe GC content of the cp genome was analyzed using MEGA 6.0 . The disAmomum cp genomes, we compared the cp genomes of A. villosum, A. villosum var. xanthioides, A. longiligulare and two other Amomum species, namely, A. kravanh and A. compactum, which were downloaded from GenBank by mVISTA [To detect variations within the y mVISTA . All cp y mVISTA , and nucy mVISTA  softwareAmomum species. PCR amplification was performed in 25-μL reaction mixtures containing 20 ng to ~100 ng of genomic DNA template, 12.5 μL of 2 × EasyTaq PCR SuperMix, and 1 μL of each primer (2.5μM). Purified PCR products were sequenced using the specific primers in the ABI3730XL sequencer. Proofreading and contig assembly of sequencing peak diagrams were performed using CodonCode Aligner 3.7.1 and the sequences were aligned using ClustalW. To evaluate the capability of these sequences to authenticate the studied Amomum species, a phylogenetic tree was constructed using the neighbor-joining algorithm (NJ tree), and bootstrap tests were conducted using 1000 resamples to assess the confidence of the phylogenetic relationships using MEGA 6.0.In order to evaluate the species discriminatory power for the highly divergent regions, we designed the specific primers for each region  and carrAmomum species, we downloaded 35 complete cp genome sequences from NCBI’s Organelle Genome and Nucleotide Resources database  shared in these species were extracted and aligned separately using MAFFT v7 [N. tabacum and S. miltiorrhiza were set as outgroups.To discuss the discriminatory power of the cp genomes and reveal the phylogenetic position of database . The seqMAFFT v7 , and theMAFFT v7 . This adA. villosum, A. villosum var. xanthioides and A. longiligulare via high-throughput sequencing, which were collected from the different main producing areas in China. First of all, we obtained and compared eight complete cp genomes, and we found that the cp genomes of the same species from different producing areas showed little difference. Then, five divergent regions , which were detected by comparative analyses using Sanger sequencing based on 32 samples, proved to not be useful as effective molecular markers to identify species in Amomum. Further, phylogenetic analyses revealed that cp genomes could be used to identify Amomum species. The availability of cp genome sequences is expected to improve species identification and phylogenetic analyses in Amomum.This is the first study to sequence and determine the complete cp genomes of eight individual species of


Serratia marcescens. We identified eight putative RND efflux system genes in the S. marcescens Db10 genome that included the previously characterized systems, sdeXY, sdeAB, and sdeCDE. Six out of the eight genes conferred multidrug resistance on KAM32, a drug hypersensitive strain of Escherichia coli. Five out of the eight genes conferred resistance to benzalkonium, suggesting the importance of RND efflux systems in biocide resistance in S. marcescens. The energy-dependent efflux activities of five of the pumps were examined using a rhodamine 6 G efflux assay. When expressed in the tolC-deficient strain of E. coli, KAM43, none of the genes conferred resistance on E. coli. When hasF, encoding the S. marcescens TolC ortholog, was expressed in KAM43, all of the genes conferred resistance on E. coli, suggesting that HasF is a major outer membrane protein that is used by all RND efflux systems in this organism. We constructed a sdeXY deletion mutant from a derivative strain of the clinically isolated multidrug-resistant S. marcescens strain and found that the sdeXY deletion mutant was sensitive to a broad spectrum of antimicrobial agents.We investigated the role of the resistance-nodulation-cell division superfamily (RND) efflux system on intrinsic multidrug resistance in  Serratia marcescens, a Gram-negative bacilli, is widely distributed in the environment. Although not initially regarded as a pathogen, S. marcescens is associated with occasional hospital-related outbreaks. The treatment of S. marcescens infections with antimicrobial agents is becoming more challenging because clinically isolated strains that exhibit elevated resistance against β-lactams, aminoglycosides, and fluoroquinolones have been reported2.9. RND-type efflux systems consist of three components: the inner membrane protein (IMP), periplasmic membrane fusion protein (MFP), and outer membrane protein (OMP). The electrochemical potential of H+ across the cell membrane appears to be the driving force for drug efflux associated with RND efflux systems. Three RND efflux systems in S. marcescens, SdeXY10, SdeAB11, and SdeCDE11, have been characterized to date. SdeXY was the first multidrug efflux system to be characterized from S. marcescens, and showed broad substrate specificity when expressed and characterized in Escherichia coli10. The gene expression of sdeXY was up-regulated in a tigecycline-resistant clinically isolated strain of S. marcescens12. The gene inactivation of sdeXY from the environmentally isolated S. marcescens-type strain, NCTC10211, increased susceptibilities to tigecycline, tetracycline, ciprofloxacin, and cefpirome. SdeAB also showed broad substrate specificity when expressed in E. coli11. Gene knockout analyses revealed that SdeAB conferred intrinsic multidrug resistance to fluoroquinolones, chloramphenicol, novobiocin, sodium dodecyl sulphate (SDS), and ethidium bromide in S. marcescens13. In a separate study, a S. marcescens cetylpyridinium chloride mutant showed the up-regulated expression of sdeAB and also became resistance to fluoroquinolones, tetracycline, chloramphenicol, and benzalkonium chloride14. In contrast to SdeXY and SdeAB, SdeCDE did not exhibit broad substrate specificity and only conferred novobiocin resistance to S. marcescens15.Resistance-nodulation-cell division superfamily (RND) efflux systems play a major role in multidrug resistance in Gram-negative bacteriaS. marcescens RND efflux systems that have the potential to render S. marcescens with multidrug resistance. To achieve this, we examined putative S. marcescens RND efflux system genes from S. marcescens Db10 and characterized their substrate specificities in drug-hypersensitive E. coli strain KAM3216. We identified an additional three uncharacterized RND efflux systems with broad substrate specificities. A gene deletion analysis revealed that SdeXY conferred intrinsic multidrug resistance to S. marcescens.In the present study, we aimed to identify uncharacterized S. marcescens Db11 genomic sequence database (http://www.sanger.ac.uk/resources/downloads/bacteria/serratia-marcescens.html) was the only publicly available resource for the genomic sequence of this bacterium. Using the S. marcescens Db11 genomic sequence, we searched for RND-type efflux systems in the S. marcescens Db11 genome and identified eight RND-type efflux systems 10, SdeAB (SMA1197-1196)11, and SdeCDE (SMA2945-2946-2947)11, and five putative RND-type efflux systems. We designated these putative RND efflux systems as shown in Fig. omsA, was located adjacent to sdePQ. The other RND efflux systems did not contain the adjacent OMP gene. All of the RND efflux system genes contained the periplasmic membrane fusion protein (MFP) gene, except for sdeS. SdeCDE contained the two inner membrane protein (IMP) genes, sdeD and sdeE.When we initiated this study, the S. marcescens, E. coli, Vibrio parahaemolyticus, Vibrio cholerae, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, and revealed that the IMPs from these organisms were divided into five groups  in 32 Table .Table 1M25. Group 1 also contains the RND efflux system genes that are not in an operon with an MFP gene (e.g. acrD from E. coli). This type of RND efflux system generally exhibits narrow substrate specificities and SdeS only conferred resistance to erythromycin, novobiocin, SDS, and deoxycholate. Consistent with our previous findings10, the KAM32 strain expressing sdeXY conferred resistance to a broad spectrum of antimicrobial agents   Fig. . All of sdePQ-omsA, the other eight S. marcescens RND efflux systems did not contain the adjacent OMP gene. Thus, these RND efflux systems must rely on E. coli OMP(s) when expressed in E. coli. TolC is a major OMP in E. coli and all of the E. coli RND efflux systems require TolC for their activities37. To clarify whether S. marcescens RND efflux systems utilize TolC when expressed in E. coli, we introduced S. marcescens RND efflux system genes into E.coli KAM43, a tolC-deficient strain, and tested antimicrobial susceptibilities by measuring MICs. Except for sdePQ-omsA, none of the S. marcescens RND efflux systems showed increases in MICs when expressed in KAM43, indicating that S. marcescens RND efflux systems require TolC for their activities when expressed in E. coli . When some mutations were occurred which caused the expression of SdePQ-OmsA and SdeGH systems, these pumps would contribute to the acquired resistance in S. marcescens.We identified two previously uncharacterized S. marcescens RND efflux systems utilized TolC and HasF when expressed in E. coli. However, some RND pumps when expressed with TolC of E. coli or HasF of S. marcescens showed different substrate specificities. SdeNO expressed with HasF in KAM43 showed higher MICs than that expressed in KAM32 with TolC, whereas SdeIJ and SdeS showed lower MICs. These results suggest that compatibility between outer membrane proteins and other components in RND pumps is important for its efflux activity. As our group reported previously, only VmeAB in V. parahaemolyticus showed high MICs expressed with TolC of E. coli, while other RND pumps in V. parahaemolyticus had markedly higher MICs when expressed with VpoC, an orthologue of TolC4. This result may be important for understanding the interaction between outer membrane proteins and other components.All of the S. marcescens has multiple RND efflux systems that have the potential to confer multidrug resistance. Among these systems, SdeXY plays a major role in intrinsic multidrug resistance in S. marcescens. SdePQ-OmsA and SdeGH showed broad substrate specificities similar to SdeXY; however, these systems appear to be inducible and do not play major roles in the intrinsic multidrug resistance of S. marcescens.In summary, the present study revealed that The bacterial strains and plasmids used in the present study are listed in Supplementary Table https://clustalw.ddbj.nig.ac.jp).Entire sequences of inner membrane protein were obtained from several database. The phylogenetic tree was obtained using CLUSTALW (S. marcescens RND efflux system genes using the S. marcescens Db11 genomic sequence database (http://www.sanger.ac.uk/resources/downloads/bacteria/serratia-marcescens.html). DNA fragments, which contained the open reading frames (ORFs) of S. marcescens RND efflux system genes or S. marcescens OMP, hasF (SMA3509), were amplified by PCR using the chromosomal DNA of S. marcescens Db10, the parent strain of Db1139, as a template. Primers used for cloning are listed in Supplementary Table hasF), which were located downstream of the lac promoter of the vector plasmid. Since PCR products did not include the promoter region of the genes, gene expression was controlled by the lac promoter.We identified putative sdeCDE (SMA2945-2946-2947) was performed in two steps. The 5′ half fragment was amplified with two primers (SMA2945-2947F fw EcoRI and SMA2945-2947F re BamHI), and the 3′ half was then amplified with two primers (SMA2945-2947B fw EcoRI and SMA2945-2947B re BamHI). After digestion with EcoRI and BamHI, each fragment was individually inserted into pUC18. The resultant plasmids were designated as pURS4F and pURS4B. After the digestion of pURS4B with MluI and BamHI, the fragment was inserted into pURS4F at the same sites. It was named pURS4. The plasmid pURS44 carrying sdeC and sdeD, but not sdeE was also constructed. The plasmid pURS4 carrying sdeC-sdeE was digested with NcoI and self-ligated. Similarly, the plasmid pURS45 carrying sdeC and sdeE, but not sdeD was constructed. The plasmid pURS4 was digested with Tth111I, blunted, and self-ligated.The cloning of sdePQ (SM1743-1742), two types of plasmids were constructed. The fragment including sdePQ-omsA was amplified with PCR using SMA1743-1741 fw XbaI and SMA1743-1741 re EcoRI. After digestion with XbaI and EcoRI, the fragment was inserted at the same site in pUC19, and the resultant plasmid was named pURS8. To construct pURS82 carrying incomplete omsA, pURS8 was digested with HpaI and self-ligated.To evaluate the function of OmsA, the gene of which is located downstream of S. marcescens showed higher β-lactam resistance, we were unable to utilize pURS2 carrying sdeXY. To complement sdeXY, we constructed another plasmid pSMXY. PCR was initially performed with two primers SMA0370-0369 fw EcoRI and SMA0370-0369 re XbaI using the Db10 genome as a template. PCR was then performed with two different primers, SMA0370-0369 fw EcoRI and SMA0370-0369 re BglII, to create the BglII site instead of the XbaI site. The PCR products obtained were digested with EcoRI and BglII, gel-purified, and then ligated into the EcoRI-BamHI sites of the vector pSTV28.Since 40.The MICs of various antimicrobial agents were assessed in Muller–Hinton broth (Difco) using the standard two-fold dilution method as previously describedS. marcescens KS24, a plasmid pSMC2-cured derivative of S. marcescens KS341 was used to construct a sdeXY deletion strain. The sdeXY deletion strain of S. marcescens KS24 was constructed by homologous recombination using the lambda Red recombinase system42. Two-step PCR of the gentamicin cassette flanked by long (1000 nt) homologous extensions of the target gene were essentially performed as previously described43 using pBRFRTGM and the genomic DNA of S. marcescens KS24 as the template and the primers listed in Supplementary Table sdeXY mutant strains were generated by electroporation of the purified PCR product into S. marcescens KS24/pKD46 as described previously42. The deletion of sdeXY in the mutant strain was verified by PCR.40. In the rhodamine6G efflux assay, E. coli KAM32 strains were grown in L media until O.D650 = 0.7. E. coli cells were harvested by centrifugation, washed twice using Potassium Phosphate Buffer (PPB) containing 5 mM MgSO4, and resuspended in the same buffer that contained 1 µM rhodamine 6 G and 40 µM carbonylcyanide-m-chlorophenylhydrazone (CCCP). The cell suspension was incubated at 37 °C for one hour to de-energize cells, washed twice using the same buffer that did not contain CCCP, and then resuspended in the same buffer. The resultant cell suspension was incubated on ice for two hours and used in the efflux assay.The efflux of rhodamine 6 G and ethidium was evaluated as previously describedS. marcescens KS24 and S. marcescens KS24ΔsdeXY strains were grown in L media until O.D650 = 0.7. S. marcescens cells were harvested by centrifugation, washed twice using modified Tanaka Buffer44, and resuspended in the same assay. The resultant cell suspension was incubated at 37 °C in the presence of 20 mM lactate-tetramethylammonium hydroxide (pH 7.0) for 5 min and used in the efflux assay.In the ethidium efflux assay, the Supplementary table


Hyles genus exhibit a spectacular diversity of larval color patterns. These species diverged ∼7.5 million years ago, making this rather young genus an interesting system to study a wide range of questions including the process of speciation, ecological adaptation, and adaptive radiation.Adapted to different ecological niches, moth species belonging to the Hyles vespertilio, the first reference genome of a member of the Hyles genus. We generated 51× Pacific Biosciences long reads with an average read length of 8.9 kb. Pacific Biosciences reads longer than 4 kb were assembled into contigs, resulting in a 651.4-Mb assembly consisting of 530 contigs with an N50 value of 7.5 Mb. The circular mitochondrial contig has a length of 15,303 bp. The H. vespertilio genome is very repeat-rich and exhibits a higher repeat content (50.3%) than other Bombycoidea species such as Bombyx mori (45.7%) and Manduca sexta (27.5%). We developed a comprehensive gene annotation workflow to obtain consensus gene models from different evidence including gene projections, protein homology, transcriptome data, and ab initio predictions. The resulting gene annotation is highly complete with 94.5% of BUSCO genes being completely present, which is higher than the BUSCO completeness of the B. mori (92.2%) and M. sexta (90%) annotations.Here we present a high-quality genome assembly of the bat hawkmoth H. vespertilio genome provides a valuable molecular resource to study a range of questions in this genus, including phylogeny, incomplete lineage sorting, speciation, and hybridization. A genome browser displaying the genome, alignments, and annotations is available at https://genome-public.pks.mpg.de/cgi-bin/hgTracks?db=HLhylVes1.Our gene annotation strategy has general applicability to other genomes, and the  Attacus atlas with a wingspan measuring 25–30 cm, but also includes several model organisms, such as the domestic silkmoth Bombyx mori, a bombycid of great economic importance for silk production, and the tobacco hornworm Manduca sexta, which is a common pest sphingid species causing considerable damage to tobacco, tomato, pepper, eggplant, and plantations of other crops [B. mori [Bombyx huttoni and Bombyx mandarina of the Bombycidae, and M. sexta [Bombycoidea are a speciose superfamily of moths, comprising 10 families, >500 genera . This su[B. mori  togetherM. sexta  of the SHyles. This genus originated in South America and comprises 32 recognized species [Hyles an interesting genus to study a wide range of questions including the process of speciation, ecological adaptation, adaptive radiation, genetics of reproduction, and evolution in action. However, such studies are hampered by the lack of suitable molecular resources. In particular, a well-assembled reference genome for any Hyles species is currently missing.Sphingidae include the hawkmoth genus  species , with re species , speciesHyles vespertilio (NCBI:txid283848)  long sequencing reads, the annotation of this genome, and a comparative analysis to B. mori and M. sexta. All data can be visualized and downloaded from a genome browser instance at [Here we present a high-quality nuclear and mitochondrial genome assembly of the bat hawkmoth 48) Fig. , the firtance at .Schmidtea [We generated 51× PacBio long reads with an average read length of 8.9 kb and an N50 read length of 16.5 kb. PacBio reads longer than 4 kb were assembled into contigs with a customized assembler called DAmar. DAmar is a hybrid of our MARVEL approach, which was used to assemble the Axolotl  and Schmchmidtea  genomes,B. mori (460.3 Mb) and M. sexta (419.4 Mb), the H. vespertilio assembly is substantially larger by 191 and 232 Mb, respectively. As shown in Fig. M. sexta assembly  but shorter than the contigs of the B. mori assembly , which was based on a combination of different sequencing technologies including 80× PacBio long reads, 60× Illumina reads, and complete sequences of bacterial artificial chromosome and Fosmid clones. To assess and compare genome completeness, we used BUSCO [H. vespertilio assembly (95.58% complete genes) shows a level of completeness similar to that of B. mori (96.48%) and M. sexta (95.21%).Compared with the assembly of ed BUSCO  and the H. vespertilio using PacBio long reads and the DAmar assembler. The resulting circular mitochondrial contig has a length of 15,303 bases.We also assembled the mitochondrial genome of H. vespertilio assembly consists of repetitive sequences, we modelled and masked repeats using RepeatModeler and RepeatMasker. To compare repeat content with B. mori and M. sexta, we applied the same procedure to these genomes as well. We found that the H. vespertilio genome has a high repeat content of 50.3% , while the M. sexta assembly is less repeat-rich (27.5%), which may be an underestimation because similar repeat copies may not be properly assembled for this species. This analysis also suggests that expansions of transposons and other repeats contributed to the larger genome size of H. vespertilio because the H. vespertilio assembly comprises 117 Mb more in repetitive elements compared to B. mori (328 vs 210.5 Mb). Long interspersed nuclear elements (LINEs) comprise the largest repeat class in the H. vespertilio assembly, followed by short interspersed nuclear elements (SINEs) and DNA transposons  [H. vespertilio assembly and B. mori and M. sexta, and used these alignments to project annotated genes from B. mori and M. sexta to the H. vespertilio genome using CESAR [B. mori and M. sexta, respectively. Second, coding sequences (CDS) from 22 available lepidopteran species were downloaded from LepBase [H. vespertilio assembly using GenomeThreader [H. euphorbiae, a closely related species, were mapped to the H. vespertilio assembly and assembled into transcripts using StringTie [ab initio gene finders, SNAP, GlimmerHMM, Genemark-ES, and Augustus [M. sexta and B. mori CESAR projections, generating a set of 50,612 loci. Finally, models for which >50% of the CDS was contained within repeat sequence were removed. This produced a final set of 23,768 genes.To annotate genes in the er (EVM)  to produng CESAR , 17. Thi LepBase . CDS andThreader . The numtringTie . TAMA antringTie  were useAugustus , predictH. vespertilio gene annotation, we used BUSCO [B. mori (92.2% complete BUSCO genes) and M. sexta (90% complete BUSCO genes).To assess the completeness of our ed BUSCO  and the Hyles vespertilio. PacBio long reads have been instrumental to assemble long contigs, in particular because the H. vespertilio genome is longer and more repeat-rich than other Bombycoidea species. With a contig N50 value of 7.5 Mb, our assembly is the second-most contiguous Bombycoidea genome to date.Here we present a high-quality genome assembly for the bat hawkmoth M. sexta as a reference. Nevertheless, we were able to project a large number of genes  also from the more distantly related B. mori. To supplement genes predicted by homology-based approaches, we additionally aligned proteins and CDS from LepBase [H. vespertilio, we used RNA-seq data from the related H. euphorbiae species to obtain transcriptomic evidence for gene models. Finally, because homology and transcriptomic evidence may miss lineage-specific or low-expression genes, we aimed at increasing gene annotation completeness by using 4 ab initio gene prediction methods, aided by a large training set available from our high-quality gene projections. After integrating and filtering all evidence, this strategy produced a gene annotation with a higher BUSCO gene completeness than for other Bombycoidea species. Because all employed methods are usable for other species, our integrative gene annotation strategy likely has general applicability to many other genomes.To annotate coding genes, we developed a strategy that integrates multiple different pieces of gene evidence. First, we used genome alignments to project genes annotated in related species. Gene projection generally produces very accurate annotations but is by definition limited by the completeness of the gene sets of related species. Evolutionary distance is another factor influencing the completeness of results obtained from gene projections, with closely related species generally allowing for more complete projections , consist LepBase . In the H. vespertilio genome for future studies, we provide a freely available genome browser instance [B. mori and M. sexta, the gene annotation, and the underlying gene evidence is shown in Fig. To make our data accessible to the community and enable efficient use of the instance  for dataH. vespertilio genome provides a valuable molecular resource to study speciation and hybridization processes in this genus. In particular, together with newly generated molecular data, the genome will help to infer the phylogeny of the 32 recognized species, which is not yet resolved owing to a high degree of hybridization between species and incomplete lineage sorting [Hyles species exhibit a spectacular diversity of color patterns, exemplified by many different colorations of larva and adult wings  and areHyles vespertilio, collected by Alberto Zilli in Latium (Province Rieti), Mt. Terminillo, Vallonina (950 m), Italy, on 31 May 2018 (specimen accession number LG2117), in accordance with the EU's environmental and scientific legislation. A second co-captured male was deposited as a voucher in the Museum of Zoology .The DNA sample was derived from a single male individual of 2–ground abdomen in home-made lysis buffer  and standard phenol-chloroform extraction. High molecular weight genomic DNA was precipitated by centrifugation after adding ice-cold ethanol and dissolved in Tris-EDTA, pH 8.0. RNA was removed by RNase A treatment. Pulsed-field gel electrophoresis  showed that the resulting DNA molecules were ∼50–150 kb long. The genomic DNA concentration was 413 ng/µL .High molecular weight genomic DNA for the PacBio library was isolated after lysis of the liquid NTM templates" [TM device (Diagenode). PacBio SMRTbellTM libraries were size selected for fragments >20 kb with the SAGE BluePippinTM device. Single-molecule real-time (SMRT) sequencing was performed on the SEQUEL system making use of sequencing chemistry 2.1; movie time was 10 hours for all SMRT cells. A total of 6 SMRT cells were sequenced with an average unique molecular yield of 5.3 Gb.PacBio continuous long-read libraries were prepared as described in the "Guidelines for preparing size-selected 20 kb SMRTbellmplates" . In brieH. euphorbiae larval tissue samples  using the Direct-Zol RNA kit according to the manufacturer’s instructions . RNA quantity was determined using an Implen Nanophotometer and integrity of RNA samples was verified using an Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit . Transcriptome sequencing was carried out by GATC Biotech on an Illumina HiSeq2500 Genome Analyzer using paired-end (2 x 125 bp) read technology. Sequencing libraries were generated from mRNA fragmented to an average of 180 bp using the TruSeq RNA library preparation kit  yielding ~12-20 million read pairs for each sample.Total RNA was isolated from De novo genome assembly was performed with DAmar [th DAmar . This asth DAmar , 13 and th DAmar , 31.To assemble the genome, we performed the following 4 steps: set-up, read patching, assembly, and error polishing. In the set-up phase, PacBio reads were filtered by choosing only the longest read of each zero-mode waveguide and requiring subsequently a minimum read length of 4 kb. The resulting 2.2 million reads (45× coverage) were stored in a Dazzler database .k-mer seeding in repetitive regions when computing all local alignments between all reads. Because masking repeats can lead to missing low-quality or noisy regions within PacBio reads, we used LAseparate to find proper alignment chains that prematurely end in repeat regions. For those alignment chains, we recomputed local alignments with the repcomp tool without using the repeat mask. Then we applied LAfix to detect and correct read artefacts.The patch phase detects and corrects read artefacts including missed adapters, polymerase strand jumps, chimeric reads, and long low-quality read segments that are the primary impediments to long contiguous assemblies. To this end, we first computed local alignments of all raw reads. Because local alignment computation is by far the most time- and storage-consuming part of the pipeline, we reduced runtime and storage by masking repeats in the reads as follows. First, low-complexity intervals, such as microsatellites or homopolymers, were masked with DBdust . Second,Manual inspection of the overlap graph  revealedIn the assembly phase, we first calculated all overlaps between patched reads using the same alignment strategy of the patch phase. The subsequent steps of (i) computing a quality track for all reads, (ii) computing a detailed repeat mask, (iii) filtering overlap piles, (iv) computing the overlap graph, and (v) touring the overlap graph to obtain primary contigs follow the steps of the original MARVEL assembly pipeline , 13.In the error-polishing phase, we polished all contigs using the raw PacBio reads and 2 rounds of Arrow . All comTo assess completeness of the genome assembly, we used BUSCO v3  and the H. vespertilio compared to other Bombycoidea.To investigate whether some contigs may represent alternative haplotypes, we determined and plotted the per base read coverage. As shown in Ampelophaga rubiginosa . The resulting overlaps were filtered for proper circular alignment chains  with the tool LAfilterMito. The filtered reads were then processed according to the general assembly pipeline . After read patching, the reads were split into shorter reads with a 1,500-bp overlap to ensure that the assembly creates a circular contig that consist of >1 read. Error polishing was done by running Arrow [To assemble the mitochondrial genome, the corresponding PacBio reads were extracted by mapping them with daligner to the mitochondrial reference sequence of the related ng Arrow  with filng Arrow  to circu-engine ncbi”) to identify repeat families in genomes of H. vespertilio, B. mori, and M. sexta. We used RepeatMasker with default parameters to soft-mask the 3 genomes with their respective repeat library. Tandem Repeat Finder [We first used RepeatModeler   and B. mori . Pa LepBase  with par LepBase , chainCl LepBase , and Rep LepBase   and query genome (here H. vespertilio), coding transcript annotations for the reference species and a file linking gene and transcripts isoforms. For each gene, TOGA identifies the chain(s) that aligns the putative ortholog in the query using synteny and the amount of aligning exonic and intronic sequence. To obtain the locations of coding exons of this gene, TOGA then extracts the genomic region corresponding to the gene on this chain from the query assembly and uses CESAR 2.0 [B. mori gene models from SilkBase [M. sexta models from LepBase [H. vespertilio. Using a 10% overlap, 15,169 (77%) of the 19,760 M. sexta projections overlap a B. mori projection and 9,996 (85%) of the 11,721 B. mori projections overlap an M. sexta projection. A total of 3,336 of the M. sexta and B. mori projections are identical. Projected genes were assigned a weight of 8 and classed as “Other prediction” within EVM.As the first piece of evidence, we used TOGA (last commit: 2 May 2019) to project annotations of coding genes from multiple reference genomes to a query genome. Briefly, TOGA takes as input pairwise genome alignment chains between a designated reference  with parameters “–dta –no-unal –mp 4,1 –score-min L,0,-0.125,” which resulted in mapping 65.82% of the reads. Transcripts were assembled with StringTie [As a third piece of evidence, we used short-read RNA-seq data that were generated from larval tissue of the closely related tringTie  with deftringTie  with partringTie  was alsotringTie  against ab initio gene prediction tools were used to predict genes in the H. vespertilio genome. As training data, we used a set of non-overlapping, full-length and intact genes that were projected from B. mori and M. sexta and resulted in an identical gene model in H. vespertilio . This set was randomly divided into 80% training data and 20% test data. SNAP [H. vespertilio using optimize_augustus.pl (–cpus = 12 –kfold = 12) and the training gene set, and Augustus was further trained using these parameters with the etraining tool. Genes were then predicted with Augustus, providing the intron positions as extrinsic hints. Finally, intron hints were provided to Genemark-ES [ab initio prediction.”As a fourth piece of evidence, 4 ta. SNAP  and Glimta. SNAP  were trata. SNAP , we firsemark-ES  for selfemark-ES . SNAP anM. sexta and B. mori with no CDS overlap to any consensus model were included into the consensus set. Consensus gene models with >50% CDS overlap within a single repeat region, as annotated by RepeatMasker, were removed. To assess completeness of the gene annotation, we applied BUSCO v3 [H. vespertilio protein set and the annotated B. mori and M. sexta proteins, using the Endopterygota dataset comprising 2,442 genes.EVM  was thenBUSCO v3  in proteH. vespertilio are available at the NCBI under the Bioproject ID PRJNA574010. H. euphorbiae RNA-seq data have been submitted to the EBI SRA (accession numbers: ERS4198286–ERS4198293). The genome, our gene annotations including the gene evidences, and genome alignments to B. mori and M. sexta are available for download [GigaScience repository, GigaDB [All raw sequencing data and the genome assembly of download  and for download . Other d, GigaDB .Supplementary Table 1: BUSCO statistics for 3 Lepidoptera genomesSupplementary Table 2: Repeat contentSupplementary Table 3: Number of alignments produced using GenomeThreader from 22 Lepbase speciesSupplementary Table 4: Contamination analysisHyles assemblySupplementary Table 5: purge_dups classification of contigs in the Supplementary Figure 1: Final overlap graph without the additional chimeric read removal stepSupplementary Figure 2: Final overlap graph after applying the additional chimeric read removal stepSupplementary Figure 3: Per base read coverage histogramSupplementary Text 1: DALIGNER - Fast and sensitive detection of all pairwise local alignmentsSupplementary Text 2: Detecting and masking repeatsHyles assemblySupplementary Data File 1: Instructions to Supplementary Data File 2: DAmar coverage estimation configuration fileSupplementary Data File 3: DAmar assembly configuration filegiaa001_GIGA-D-19-00361_Original_SubmissionClick here for additional data file.giaa001_GIGA-D-19-00361_Revision_1Click here for additional data file.giaa001_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giaa001_Reviewer_1_Report_Original_SubmissionReuben William Nowell, Ph.D. -- 11/8/2019 ReviewedClick here for additional data file.giaa001_Reviewer_2_Report_Original_SubmissionSimon Baxter -- 11/23/2019 ReviewedClick here for additional data file.giaa001_Supplemental_FilesClick here for additional data file.BLAST: Basic Local Alignment Search Tool; bp: base pairs; BUSCO: Benchmarking Universal Single-Copy Orthologs; CESAR: Coding Exon-Structure Aware Realigner; CDS: coding sequence; EDTA: ethylenediaminetetraacetic acid; EVM: Evidence Modeler; Gb: gibabase pairs; HMM: hidden Markov model; kb: kilobase pairs; LINE: long interspersed nuclear element; Mb: megabase pairs; NCBI: National Center for Biotechnology Information; NMD: nonsense-mediated decay; ORF: open reading frame; PacBio: Pacific Biosciences; RNA-seq: RNA sequencing; SINE: short interspersed nuclear element; SMRT: single-molecule real-time; SNAP: Semi-HMM-based Nucleic Acid Parser; SRA: Sequence Read Archive; TAMA: Transcriptome Annotation by Modular Algorithms; TOGA: Tool to infer Orthologs from Genome Alignments.The authors declare that they have no competing interests.This work was funded by the Max Planck Gesellschaft , the Federal Ministry of Education and Research (grant 01IS18026C), and the German Research Foundation . It benefitted from the sharing of expertise within the DFG priority program SPP 1991 Taxon-Omics.Author contributions:GM, AKH and MH conceived the study. MP assembled the genome and mitogenome. DJ aligned genomes, analysed transposons and annotated genes. FP contributed to material sampling and data analysis. SW extracted gDNA and sequenced the genome. HV sequenced and provided RNA-seq data. GM designed the sequencing and assembly strategy. MH contributed to data analysis and set up the genome browser. MP, DJ, AKH and MH made figures. GM and AKH provided funding for DNA sequencing. AKH and MH wrote the manuscript with input from all authors.


Stenotrophomonas maltophilia hospital-acquired pneumonia (HAP) in critically ill patients. The optimal modalities of antimicrobial therapy remain to be determined. Our objective was to describe the epidemiology and prognostic factors associated with S. maltophilia pneumonia, focusing on antimicrobial therapy.There is little descriptive data on S. maltophilia HAP during intensive care unit stay. Primary endpoint was time to in-hospital death. Secondary endpoints included microbiologic effectiveness and antimicrobial therapeutic modalities such as delay to appropriate antimicrobial treatment, mono versus combination therapy, and duration of antimicrobial therapy.This nationwide retrospective study included all patients admitted to 25 French mixed intensive care units between 2012 and 2017 with hospital-acquired S. maltophilia HAP diagnosis for duration of 11 [5–18] days. The Simplified Acute Physiology Score II was 47 [36–63], and the in-hospital mortality was 49.7%. Underlying chronic pulmonary comorbidities were present in 14.1% of cases. Empirical antimicrobial therapy was considered effective on S. maltophilia according to susceptibility patterns in only 30% of cases. Delay to appropriate antimicrobial treatment had, however, no significant impact on the primary endpoint. Survival analysis did not show any benefit from combination antimicrobial therapy  or prolonged antimicrobial therapy for more than 7 days . No differences were noted in in-hospital death irrespective of an appropriate and timely empiric antimicrobial therapy between mono- versus polymicrobial S. maltophilia HAP (p = 0.273). The duration of ventilation prior to S. maltophilia HAP diagnosis and ICU length of stay were shorter in patients with monomicrobial S. maltophilia HAP (p = 0.031 and p = 0.034 respectively).Of the 282 patients included, 84% were intubated at S. maltophilia HAP occurred in severe, long-stay intensive care patients who mainly required prolonged invasive ventilation. Empirical antimicrobial therapy was barely effective while antimicrobial treatment modalities had no significant impact on hospital survival.clinicaltrials.gov, NCT03506191 Stenotrophomonas maltophilia is one of the 10 most frequently isolated pathogens responsible for hospital-acquired pneumonias (HAPs) in intensive care unit (ICU) patients in western countries  for continuous variables.S. maltophilia to death. The follow-up was censored at discharge from the ICU and/or the hospital. Baseline prognostic factors were age, SAPS II, mechanical ventilation at diagnosis, VAP, duration of mechanical ventilation before the diagnosis, SOFA score at diagnosis, bacteremia, mono/polymicrobial pneumonia, use of empirical antimicrobial therapy, and use of empirical antimicrobial therapy effective against S. maltophilia. Other antimicrobial therapy-related variables were not defined as baseline and were thus entered in the model as time-dependent variables, including time elapsed between sample and effective antimicrobial therapy, use of effective combination antimicrobial therapy, and duration of effective antimicrobial therapy against S. maltophilia (monotherapy or combination therapy).Prognostic factors associated with time to in-hospital death were studied using the Cox proportional hazard model. Time to in-hospital death was calculated from the diagnostic date of p < 0.05) with outcome in the univariate analysis and that were present at the diagnosis were considered for the multivariate model, and the final model was selected using backward stepwise regression (p < 0.05). Hazard ratios (HR) were calculated accordingly with their 95% confidence intervals (CI).Baseline and time-dependent variables associated .Significance was defined as S. maltophilia pneumonia were included  with a S. maltophilia pneumonia. Forty patients (14.2%) presented with a chronic underlying pulmonary disease. Other characteristics of patients are described in Table Patients had been hospitalized in the ICU for 11 [5–19] days at the time of onset of ients 14.% presentS. maltophilia HAP are described in Table S. maltophilia pneumonias were monomicrobial and 80.8% were VAP. Blood culture was concomitantly positive in only 7.1% of cases.Characteristics of S. maltophilia are presented in Additional file S. maltophilia VAP had a duration of mechanical ventilation before the onset of pneumonia of 11 [5–18] days. S. maltophilia pneumonia-related septic shock was present in 123 patients (43.6%) within 48 h of S. maltophilia HAP . Among these patients who developed septic shock, 38 (30.8%) did not receive initial empirical antimicrobial therapy. Forty-nine percent of patients fulfilled moderate or severe ARDS criteria.Microbiological diagnosis methods for isolation of S. maltophilia pneumonia, patients received 3 [2–4] prior antimicrobial therapies for at least 5 consecutive days in ICU. Empirical antimicrobial therapy was a posteriori effective against S. maltophilia in 30.1% of cases. The duration of effective antimicrobial therapy on S. maltophilia was 11 [7–15] days. A combination of antimicrobials effective against S. maltophilia was used in 59.4% of patients for 7 [5–12] days.The description of antimicrobial therapy modalities is reported in Table S. maltophilia strains is presented in Fig. S. maltophilia strains. The main antimicrobial therapies prescribed to treat S. maltophilia HAP after identification were TMP-SMX (29%), ciprofloxacin (25%), and ticarcillin–clavulanate (24%) . Co-infection with another pathogen, mostly Pseudomonas aeruginosa (n = 30), was diagnosed in 70 patients (24.8%) during recurrence . Recurrence of p = 0.0001), SAPS II , and SOFA score at pneumonia diagnosis  (Table p < 0.0001).In univariate analysis, variables associated with the primary endpoint  were age , prior antimicrobial therapy) were not statistically associated with time to in-hospital death.Neither the duration of treatment nor the use of combination therapy directed against nt Table . Other cp = 0.001) and SOFA score at S. maltophilia pneumonia diagnosis  were associated with in-hospital death  .In multivariate analysis, only age  . Comparisons of characteristics and outcomes between mono- and polymicrobial S. maltophilia HAP are provided in Table S. maltophilia HAP. A similar number of VAP occurred in both groups, 91 (77.8%) versus 136 (82.9%) (p = 0.280) for mono- and polymicrobial HAP respectively. The duration of ventilation prior to S. maltophilia HAP diagnosis and ICU length of stay were shorter in patients with monomicrobial S. maltophilia HAP.The aforementioned results remained unchanged when considering only monomicrobial S. maltophilia HAP. Regarding the large screening, the prevalence of S. maltophilia HAP remained very low. The majority of S. maltophilia HAP was VAP and occurred in patients ventilated for more than 10 days and previously exposed to several antimicrobial therapies. The mortality rate of these patients remained high, but surprisingly, the treatment delay in adequate antimicrobial therapy targeting S. maltophilia was not found to be associated with mortality. This observation may be the result of (i) a low virulence of the pathogen, (ii) the underlying condition of the critically ill patient being more contributive to the outcome than S. maltophilia HAP itself, or (iii) a 24- to 48-h delay in the treatment of S. maltophilia HAP had no real impact. Finally, if SAPSII and SOFA score were independently associated with mortality, no specific pneumonia or antimicrobial therapy-related factors impacted the outcome.Herein, we report the largest cohort study of critically ill patients developing S. maltophilia pneumonia develops in high-risk phenotypes patients, i.e., long ICU/hospital length of stay and duration of mechanical ventilation. Despite a mortality rate of approximately 50%, it is difficult to delineate direct attributable mortality of S. maltophilia HAP from mortality linked to underlying diseases [S. maltophilia isolation and the number of prior antimicrobial therapies suggest noticeable patient frailty and complicated medical history. These factors have been associated to high mortality in patients with resistant bacteria in ICU [S. maltophilia HAP could also be perceived as a final septic insult in long-stayer patients, promoting care withdrawal from the medical team.Our study population shares common features with previously published reports , 19–23. diseases . Indeed,a in ICU . S. maltS. maltophilia HAP. Conversely, our large series of mixed ICU patients did not confirm these elements. This implies that the involvement of S. maltophilia in late onset HAP should be considered and be kept in mind by all critical care physicians. However, in our series, initial antimicrobial therapy inactive against S. maltophilia was not a risk factor for in-hospital mortality, arguing against a systematic coverage of S. maltophilia by empirical antimicrobial therapy in this setting.Previous studies that included a small number of patients , 26–31 sS. maltophilia HAP diagnosis without apparent consequences on S. maltophilia emergence and susceptibility profiles. Soubirou et al. found that the increase in use of antimicrobial class was an independent predictor of S. maltophilia emergence in VAP [S. maltophilia pneumonia [Although the prolonged duration of antimicrobial treatment is a well-known risk factor for emergence of multidrug resistant (MDR) bacteria , it did e in VAP . It is hneumonia , 4.Despite clinical signs of HAP, only 59% of patients readily received empirical antimicrobial therapy  versus prolonged (8 days and greater) antibiotic course could not properly be investigated in our study because of its design. Chastre et al. demonstrated that an 8-day course of antibiotic therapy for VAP was not inferior to a longer duration, but only 0.8% of patients had an S. maltophilia VAP [S. maltophilia strains may partially explain these findings. In a recent retrospective study focused on the interest of combination therapy, Shah et al. reported that combination of antibiotic therapies yielded similar clinical efficacy and resistance development compared to monotherapy [The duration of ilia VAP . Howeverotherapy .S. maltophilia HAP may raise some concerns. The S. maltophilia strains in our study had a preserved susceptibility to ticarcillin–clavulanate and TMP-SMX, 73 and 88% respectively as expected [S. maltophilia HAP were treated with TMP-SMX. These discrepancies may be related to ICU physicians’ habits, the fear of TMP-SMX-related side effects, and the type of antibiotics administered prior to occurrence of S. maltophilia HAP. The use of fluoroquinolones could have been considered easier. When prescribed, TMP-SMX was combined with another antibiotic effective on S. maltophilia in 80% of cases. In addition, antimicrobial agent shortages change antimicrobial therapy armamentarium, with a cessation of manufacture of ticarcillin–clavulanate in 2015 [S. maltophilia [Optimal antimicrobial against expected . However in 2015 . Althougtophilia , it may tophilia . The clitophilia .Augmented renal clearance can alter the pharmacokinetics and pharmacodynamics (PK/PD) of several antimicrobial agents, mainly ß-lactams . The detS. maltophilia infection were compared. We did not consider S. maltophilia colonizations but only HAP, unlike previous studies [S. maltophilia. This was a pragmatic study that describes different ICU practices, and to date, there is no formal evidence of improved outcomes depending on the diagnosis method used [Our study differs from previous studies, where patients with and without  studies . In the hod used . DespiteS. maltophilia HAP had a very low incidence in critically ill patients but was associated with high mortality rate in this large multicenter study. Its onset is hard to predict because of lack of specific risk factors but occurs mainly in long-stay ICU patients. The present study did not provide evidence of a significant effect of delay, duration, or combination of antimicrobial therapy on mortality. Efforts in developing novel and effective approaches for prevention are warranted.Additional file 1.List of participating centers, collaborators andcase-mixof ICU patients.Additional file 2: Table S1.Invasive devices inserted at the diagnosis ofStenotrophomonas maltophiliahospital-acquiredpneumonia. Description of invasive devices inserted at the diagnosis of Stenotrophomonas maltophilia hospital-acquired pneumonia.Additional file 3: Table S2.Diagnosis methods for isolation ofStenotrophomonas maltophilia. Description of diagnosis methods for isolation of Stenotrophomonas maltophilia.Additional file 4: Table S3.Treatment failure ofStenotrophomonas maltophiliahospital-acquiredpneumonia. Description of treatment failures of Stenotrophomonas maltophilia hospital-acquired pneumonia.Additional file 5: Table S4.Variables associated with the time toin-hospitaldeath in patients withS. maltophiliaventilator-associatedpneumonia. Variables associated with the time to in-hospital death in patients with S. maltophilia ventilator-associated pneumonia.Additional file 6: Table S5.Propensity Score Matching. Time to in-hospital death was compared between matched groups using a Cox proportional hazard model.Additional file 7: Table S6.Variable associated withtime-to-deathin the propensity matched population. Variable associated with time-to-death in the propensity matched population.


Vibrio harveyi.Horizontal gene transfer (HGT), which is affected by environmental pollution and climate change, promotes genetic communication, changing bacterial pathogenicity and drug resistance. However, few studies have been conducted on the effect of HGT on the high pathogenicity and drug resistance of the opportunistic pathogen V. harveyi 345 that was multidrug resistant and infected Epinephelus oanceolutus was isolated from a diseased organism in Shenzhen, Southern China, an important and contaminated aquaculture area. Analysis of the entire genome sequence predicted 5678 genes including 487 virulence genes contributing to bacterial pathogenesis and 25 antibiotic-resistance genes (ARGs) contributing to antimicrobial resistance. Five ARGs  and one virulence gene (CU052_28670) on the pAQU-type plasmid p345–185, provided direct evidence for HGT. Comparative genome analysis of 31 V. harveyi strains indicated that 217 genes and 7 gene families, including a class C beta-lactamase gene, a virulence-associated protein D gene, and an OmpA family protein gene were specific to strain V. harveyi 345. These genes could contribute to HGT or be horizontally transferred from other bacteria to enhance the virulence or antibiotic resistance of 345. Mobile genetic elements in 71 genomic islands encoding virulence factors for three type III secretion proteins and 13 type VI secretion system proteins, and two incomplete prophage sequences were detected that could be HGT transfer tools. Evaluation of the complete genome of V. harveyi 345 and comparative genomics indicated genomic exchange, especially exchange of pathogenic genes and drug-resistance genes by HGT contributing to pathogenicity and drug resistance. Climate change and continued environmental deterioration are expected to accelerate the HGT of V. harveyi, increasing its pathogenicity and drug resistance.V. harveyi pathogenesis and antimicrobial resistance and developing pollution control measurements for coastal areas.This study provides timely information for further analysis of  Vibrio harveyi is a halophilic, luminescent Gram-negative γ-proteobacteria, as an important pathogen of vibriosis [V. harveyi has been extensively studied for more than a decade and is reported to be a serious pathogen for a range of marine vertebrates and invertebrates [V. harveyi causing serious infections was to common snook  in Florida, USA [V. harveyi was shown to cause disease in Penaeus monodon and Penaeus japonicas in Thailand [Dentex dentex) in Spain [Holothuria scabra  in Toliara, Madagascar [Lates calcarifer) in Malaysia [Epinephelus spp. in China [With the rapid expansion of aquaculture, vibriosis has become one of the most serious diseases to endanger sustainable aquaculture development , 2. Vibribriosis –5. V. hatebrates –7. The eida, USA . SubsequThailand , brown-sThailand , common in Spain , Holothudagascar , Asian sMalaysia , and Epiin China –15.Vibrio spp., including V. harveyi. Several studies show that drug resistance and multidrug resistance (MDR) are occurring in aquaculture worldwide, including in shrimp ponds in Thailand, Malaysia, India, and China [V. harveyi infection [V. harveyi antibiotic resistance, elevating the toxicity of V. harveyi. The pathogenic and drug-resistant genes of V. harveyi are a key to the fundamental cause of pathogenicity and drug resistance. Therefore, studying the pathogenic and drug-resistance genes of V. harveyi will provide an important foundation for determining pathogenic and drug-resistance mechanisms.The wide use of antibiotics, increased environmental pollution and global climate change are leading to the enhancement of drug resistance of nd China –19; shelnd China –24; and nd China –27. Enhanfection , 16. Fornfection  found thV. harveyi a recognized pathogen of marine animals, different strains vary in their ability to cause disease [V. harveyi are extracellular proteases, outer membrane proteins, hemolysins, esterases, phospholipases, exotoxin and secretion systems [Staphylococcus aureus infection. Le et al. [Rickettsiales genomes are associated with strong bootstrap support for HGT with function as ATPases, aldolases, transporter activities, cystathionine beta-lyases, sugar phosphate permeases, growth inhibitors and antitoxin activities. For horizontal transfer, exogenous DNA must evade the bacterial immune system such as restriction modification systems (RM systems) and CRISPR-Cas systems [Although  disease . The mai systems , 30. Pro systems . Inactiv systems . Virulen systems . Park et systems  found the et al.  reported systems , 37.V. harveyi strains to determine pathogenic and antimicrobial-resistance mechanisms. Evidence is growing that HGT is an important driving force for prokaryotic evolution affecting pathogenicity and drug resistance [V. harveyi by affecting HGT [V. harveyi is the dominant species that causes serious infection and mortality of farmed fish in Guangdong, Southern China [V. harveyi pathogenesis and drug resistance.Serious infection and multidrug resistance lead to the emergency of the availability of whole genome sequences for different sistance , 38. Coating HGT , 40. V. rn China , 15. HowV. harveyi 345 is multidrug resistant to ampicillin, rifampicin, tetracycline, pediatric compound sulfamethoxazole tablets, vancomycin, doxycycline, trimethoprim, streptomycin, kanamycin, sulfamethoxazole, furazolidone, cefixime, and chloramphenicol. This strain was isolated from a diseased E. oanceolutus in Shenzhen, Southern China. V. harveyi 345 is suspected to lead to kidney enlargement and softening, spleen enlargement, and anal bleeding of E. oanceolutus and has a median lethal dose (LD50) of 9.83 × 105 CFU·g− 1 [V. harveyi pathogenesis and drug resistance, controlling V. harveyi disease and reducing economic losses. In this study, we present the entire genome sequence of V. harveyi 345 with comparative genomics analysis of its pathogenesis, antimicrobial resistance and genome expansion caused by HGT.The predominant strain CFU·g− 1 . TherefoV. harveyi 345 contained two circular chromosomes, named ChI  and ChII , and two circular megaplasmids named p345–185  and p345–67    were further classified into Clusters of Orthologous Groups (COG) , two S subunits (hsdS) and five M subunits (hsdM). The other two belonged to type III RM systems, encoding a methyltransferase (mod) and a restriction enzyme (res).Twelve RM system genes were annotated in 45 Table . Ten belVibrio such as Escherichia, Pseudomonas, Salmonella, Aeromonas, and Mycobacterium. T3SS1, which is widely reported to be involved in Vibrio pathogenesis, was found on ChI. T6SSs are implicated in cell targeting and virulence, and are believed to mediate antibacteria [tlh gene encoding a thermolabile hemolysin (TLH) was on ChII. In addition, four virulence genes  were on plasmid with CU052_28670 on p345–185, and CU052_00025, CU052_00190, and CU052_00400 on p345–67.A total of 487 putative virulence factors were predicted , penicillin , tetracycline , chloramphenicol  and trimethoprim resistance (dfra26). In addition, five genes  were on the plasmid p345–185. These results were consistent with antibiotic-susceptibility assay data for strain 345 showing resistance to streptomycin, ampicillin, tetracycline, chloramphenicol, trimethoprim, and sulfamethoxazole.We identified 25 genes predicted to have > 40% identity to well-characterized ARGs  and were involved in recombination, peptide synthesis, conjugation and integration  suggesting recent acquisition of this gene from a Pseudovibrio strain . The top 15 similar strains were selected for muti-alignment and phylogenetic analysis  and Aeromonas sobria (75.53% identity), suggesting HGT of this gene from P. aeruginosa or A. sobria . The top 15 similar strains were selected for muti-alignment and phylogenetic analysis , and an OmpA family protein in V. crassostreae (97.04% identity), suggesting recent acquisition of this gene from V. splendidus or V. crassostreae  was not found on 345 chromosomes and showed 100% amino acid identity to sul2 in Acinetobacter baumannii, Escherichia coli, Actinobacillus pleuropneumoniae, Histophilus somnim, and Salmonella enteric  of nucleotide (Blastn) results showed high homolog (query cover > 40% and identity > 40%) of p345–185 with plasmids pVPS62, V36, pVPS114, pVPH2, and pVPS91 from V. harveyi 345 was deposited in GenBank with accession numbers CP025537 (ChI), CP025538 (ChII), CP025539 (plasmid p345–185) and CP025540 (plasmid p345–67).The complete genome sequence of V. harveyi 345 was isolated and studied because of its multidrug resistance and serious virulence to E. oanceolutus in Shenzhen, Southern China. The complete genome sequence of V. harveyi 345 was determined and compared with 30 V. harveyi strains. V. harveyi 345 was assembled into two circular chromosomes and two plasmids. It had a larger genome than 27 strains other than KC13.17.5, E385, and 74F and had a consistent G + C content with the 30 others strains. Compared to the 30 V. harveyi strains, V. harveyi 345 had more predicated genes than the others strains except for ZJ0603. In total, 487 virulence genes encoding proteins in flagella, iron uptake, pili, LPS, CPS, chemotaxis and type II/III/IV/VI secretion involved in fish disease in clinics, were identified in V. harveyi 345. In addition, 25 ARGs including genes for resistance to aminoglycoside , penicillin , tetracycline , chloramphenicol  and trimethoprim (dfra26) were found in V. harveyi 345, consistent with its resistance to streptomycin, ampicillin, tetracycline, chloramphenicol, trimethoprim, and sulfamethoxazole.V. harveyi strains was conducted to analyze HGT events by identifying strain-specific genes and strain-gene families. Considering the average gene number of 5087 for the 31 V. harveyi strains, the 3338 core genes represented approximately 66% of the total genome, meaning that approximately two-thirds of the genome was conserved among all strains. However, flower plots and dispensable heatmaps showed that each stain contained strain-specific genes, probably obtained by HGT. A total of 217 genes were specific to V. harveyi 345, which was more than other strains except for ZJ0603. This result suggested that a large number of HGT events happened in 345. In addition, seven gene families were specific to V. harveyi 345. Most  of the strain-specific genes had > 40% identify to other Vibrio species. The remaining came from other genera such as Shewanella, Photobacterium, Pseudovibrio, and Escherichia. We focused on the characterization of three strain-specific genes, CU052_00920, CU052_13320, and CU052_14535. CU052_00920 encoded a class C beta-lactamase and was transferred from Pseudovibrio spp. Beta-lactamase enzymes are reported to inactivate beta-lactam antibiotics by hydrolyzing the peptide bond of the characteristic four-membered beta-lactam ring, rendering the antibiotic ineffective [V. harveyi 345. CU052_13320 encoded a virulence-associated protein D and closes to a virulence factor in Pseudomonas aeruginosa (73.40% identity) and Aeromonas sobria (75.53% identity). CU052_13320 probably contributed to the virulence of 345. CU052_14535 encoded an OmpA family protein and was acquired from V. splendidus or V. crassostreae. Among pathogenic bacteria, OmpA proteins are important for pathogenesis including bacterial adhesion, invasion, or intracellular survival and evasion of host defenses or stimulation of proinflammatory cytokine production [V. harveyi 345. DNA can also be horizontally transmitted between bacteria through plasmids, phages or uptake of naked DNA from environment [V. parahaemolyticus. PVPS62 is reported to be a pAQU-type plasmid and emerge MDR conjugative plasmid among important pathogens [tetm, tetb, qnrs, dfra17, and sul2) involved in resistance to tetracycline, fluoroquinolone, trimethoprim, and sulfonamide, and one virulence gene (CU052_28670) involved in immune evasion were located on plasmid p345–185. These genes could be horizontally transferred, promoting drug resistance and virulence. Especially, CU052_29140 (sul2) showing 100% amino acid identity to sul2 in Acinetobacter baumannii, Escherichia coli, Actinobacillus pleuropneumoniae, Histophilus somnim, and Salmonella enteric, should contribute to the resistance of pediatric compound sulfamethoxazole tablets and sulfamethoxazole. Similarly, Klein et al. [Virulence genes and antimicrobial-resistance genes are acquired by bacterial replication and by HGT. Horizontal transfer is the exchange of genetic material within species without any sexual mechanism . This phffective . This reironment . Homologathogens . Five ann et al.  found thV. harveyi 345. These genetic units probably acted as HGT delivery tools, contributing to pathogenesis, drug resistance and environmental adaptation of V. harveyi 345. Several type III/VI secretion proteins along with IS family transposases were predicted on the same GI that could be easily transmitted. Dissemination of these genes could further compromise Vibrio infections, limiting treatment options. These results further indicated that HGT contributed to virulence and antibiotic resistance of V. harveyi 345.Except for plasmids, HGT encompasses a variety of genetic units, collectively known as MGEs  and inclE. oanceolutus in Shenzhen, Southern China. Recent increasing aquaculture density has led to frequent breeding diseases and increased use of antibiotics in Southern China, resulting in serious antibiotic residues and pollution [-related enzyme activity, and damaging immune systems. These factors regulate bacterial resistance and pathogenicity [V. cholera encoding genes that confer resistance to chloramphenicol, sulphamethoxazole, trimethoprim and streptomycin. The mechanism is increasing expression of SXT activators, enhancing drug resistance. Similarly, high environmental temperatures in combination with UV irradiation accelerate the spread of stx (Shiga toxin) genes by enhancing Stx prophage induction and Stx phage-mediated gene transfer [V. harveyi and affect the ecological safety of aquaculture ecosystems.The 345 strain was isolated from a diseased ollution . Intensiollution . Many stgenicity –54. For genicity  found thtransfer . TherefoV. harveyi 345 which has a multidrug-resistant phenotype. We did comparative genomics between this strain and 30 other V. harveyi strains. The genome was determined to study V. harveyi 345 virulence and antimicrobial resistance. Multiple virulence factors and resistance genes were predicted in this strain, consistent with results of Jiang et al. [Vibrio pathogenesis and antibiotic resistance. These studies could improve seafood quality and reduce economic loss. We recommend that more research be done on the response mechanism of HGT relative to environmental change and antibiotic use. This research will be an important scientific basis for predicting outbreaks and controlling V. harveyi disease. As environmental pollution including use of antibiotics and climate change enhance the virulence and drug resistance of pathogens, research should explore management approaches, for example by bacteriophages, to manage the occurrence of V. haveyi in the environment [We present the first complete genome of the serious disease-causing g et al. . We searironment .V. harveyi 345 was grown overnight in 2216E medium  at 28 °C with vigorous shaking. Overnight cultures were inoculated with a 1:1000 dilution into 20 ml fresh 2216E medium and grown until OD600 = 0.5. Cells were collected by centrifugation and genomic DNA extracted using the CTAB method [B method . The quaA PacBio RSII 20-kb template library was constructed from at least 10 μg genomic DNA at the Beijing Genomics Institute (BGI). The library was subjected to quality control (QC) with Qubit Fluorometer  and Nanodrop 2000  to check the concentration, purity and integrity of library templates. Libraries were sequenced on Illumina Hiseq 4000 and Pacbio RSII sequencing platforms. Genomes were primarily assembled with a variety of software for short sequence assembly based on clean data from the Illumina Hiseq 4000 platform. RS_HGAP Assembly3 of SMRT Analysis v.2.3.0 was used to assemble data from the Pacbio RSII platform. Contigs were error corrected successively by soapSNP and soapIndel software and GATK analysis. Gaps between contigs were filled by PCR.− 5 . COG, KEGG, GO and NR databases were used to search domain architecture. The tRNA genes were identified by tRNAscan-SE v.1.23 [ORFs were predicted by Glimmer v.3.02 , and genE v.1.23  and rRNAE v.1.23 . The sRNE v.1.23 . Tandem E v.1.23 .V. harveyi 345 were predicted by blasting against the Virulence Factor Database (VFDB) (http://www.mgc.ac.cn/VFs/main.htm) [http://ardb.cbcb.umd.edu/) [Putative virulence factors of ain.htm)  and ARGsmd.edu/) .GIs, prophages, and CRISPR-Cas systems were respectively identified using online tools IslandViewer 4, PHAge Search Tool (PHAST), and CRISPR Finder software –66.V. harveyi strains with V. harveyi VH2 as the reference strain and an average gene number of 5087  matrix of the 31 strains with the maximum-likelihood method, bootstrapped 1000 times via Treebest-1.9.2 software [Comparative genome analysis was conducted among 31 software .V. harveyi VH2 as a gene pool. Genes predicted in one strain among other 30 strains (Query samples) were BLAST with the gene pool, and results filtered by length and identity (> 40%). BLAST coverage ratios  of genes from the gene pool and Query samples were calculated separately. If BCR values from the reference and Query sample were smaller than the setting value (40%), the reference gene did not have homology with the Query gene. The nonhomologous gene from the Query genome was added to the gene pool. Query samples were processed and the final gene pool used as the pan gene pool. The non-homolog genes from the reference genome were the strain specific genes against the Query sample. Nonhomologous genes were BLAST with the gene pool of another Query sample, repeated for all samples, and the final nonhomologous gene pool was used as the strain-specific gene pool.Core-pan genes were analyzed using a BLAST method : Genes wAfter aligning to genes from samples, BCR values of genes from the pan gene pool were calculated for each sample. The coverage array was generated for this pool. If the BCR value of a gene in each sample was larger than the setting value, the gene was a core gene. If the gene was predicted from an assembled result, BLAST results were filtered and the sequence was removed if the number N (N means uncertain nucleotides in gene) was larger than the setting (30%) for the gene. Dispensable genes were included in the pan-gene pool but not the core-gene pool.Gene families were constructed using genes from the reference strain and the target bacterium. Protein sequences were aligned with basic local alignment search tool of protein (Blastp) and redundancy was eliminated. Gene family TreeFam clustering was carried out with alignment results and Hcluster_sg software. Alignment results for proteins were converted into multiple sequence amino acids in the CDS area after multiple sequence alignments with the clustered gene family using Muscle software . Gene faV. harveyi 345. The genes were compared to the NR protein database of NCBI using the Blastp tool. The results were filtered manually to find significant hits to proteins belonging to species other than V. harveyi. Multiple sequence alignment was conducted using ClustalW [Of the strain-specific genes, we extracted CU052_00920, encoding a beta-lactamase class C; CU052_13320, encoding a virulence-associated protein D; and CU052_14535 encoding an OmpA family protein for further analysis on contribution to the antibiotic resistance or virulence of ClustalW . An aligClustalW  with theClustalW .sul2) on p345–185 as for CU052_00920, CU052_13320, and CU052_14535.In addition, the entire sequence of p345–185 was compared to the NR nucleotide database of NCBI using a Blastn tool. A phylogenetic tree was constructed with the neighbor-joining method using max seq difference = 0.75 and BLAST pairwise alignments. The characterization method was the same for CU052_29140  concentrations of water samples.Additional file 2: Table S2. Gene information and gene statistics for 31 V. harveyi strains.Additional file 3: Figure S1. KEGG pathway classifications of V. harveyi 345 coding sequences.Additional file 4: Figure S2. GO functional classifications of V. harveyi 345 coding sequences.Additional file 5: Table S3. Details of virulence factors predicted with the VFDB database.Additional file 6: Table S4. Genomic islands in a single chromosome (CP025537) of V. harveyi 345.Additional file 7: Table S5. Genomic islands in a single chromosome (CP025538) of V. harveyi 345.Additional file 8: Table S6. Prophage sequences present in V. harveyi 345 chromosomes.Additional file 9: Table S7. Strain-specific genes of V. harveyi 345.


For this, posterior odds were used to test whether species from Reunion did form a monophyletic group and we estimated divergence times between species. Three out of the four previously described Reunion black fly species could be sampled, namely Simulium ruficorne, Simulium borbonense and Simulium triplex. The phylogenies based on nuclear and mitochondrial markers showed that S. ruficorne and S. borbonense are the most closely related species. Interestingly, we report a probable mitochondrial introgression between these two species although they diverged almost six million years ago. Finally, we showed that the three Reunion species did not form a monophyletic group, and, combined with the molecular datation, the results indicated that Reunion black fly diversity resulted from multiple colonization events. Thus, multiple colonizations, rather than in-situ diversification, are likely responsible for an important part of black fly diversity found on this young Darwinian island.True oceanic islands typically host reduced species diversity together with high levels of endemism, which make these environmental set-ups ideal for the exploration of species diversification drivers. In the present study, we used black fly species (Diptera: Simuliidae) from Reunion Island as a model to highlight the main drivers of insect species diversification in this young and remote volcanic island located in the Southwestern Indian Ocean. Using local and regional (Comoros and Seychelles archipelagos) samples as well as specimens from continental Africa, we tested the likelihood of two distinct scenarios,  The rfication –7. Islanfication –10. Due fication ,8,11,12.fication , anoles fication  and Darwfication ). In-sitene flow ,17.de novo on volcanic hotspots. Hence, these islands have never been connected to continental masses and constitute suitable systems for investigating evolutionary processes involved in the assembly of species communities, as illustrated by several studies using plants [Located in the Southwestern Indian Ocean (SWIO) region, Reunion Island is a young island, 2.1 million years (Myr), belonging to the Mascarene archipelago, which also includes Mauritius and Rodrigues islands . The SWIg plants ,22, reptg plants ,24, nemag plants , birds [g plants  and aviag plants ,28.Onchocerca volvulus (responsible for river blindness in human populations) or avian blood parasites of the genus Leucocytozoon [ruficorne species-group (subgenus Nevermannia), have been previously described based on morphological characters by Giudicelli [Simulium ruficorne  was originally described from Reunion Island while this species is currently known to have a large spatial distribution extending from the SWIO  to the Middle East and the Mediterranean sea [Simulium borbonense , Simulium indoceanicum  and Simulium triplex . These three species were considered as endemic to Reunion Island although S. triplex was later reported on Mauritius Island [S. ruficorne population. According to this hypothesis, each colonization has conducted to reproductive isolation before the arrival of another group of immigrants from African continental S. ruficorne population (multiple colonizations). This process would have taken place three times on Reunion Island.Black flies (Simuliidae) are insects of both medical and veterinary importance ,31. Somecytozoon –34. The cytozoon ,36. Furtcytozoon ,38. Intecytozoon . Black fcytozoon –46. The cytozoon . On Reunudicelli . Simuliunean sea ,48. Thres Island . Based os Island  suggesteS. ruficorne. We then investigated the processes of diversification possibly involved by confronting alternative scenarios including (i) in-situ diversification within Reunion Island following a single colonization event of a S. ruficorne population and (ii) multiple independent colonizations.In this study, we sequenced mitochondrial and nuclear genes from Reunion black fly species in order to investigate their phylogenetic relationships and evaluate divergence times between each of these species and African continental No endangered or protected species was included in the present study. Most specimens were sampled on Reunion Island and did not require specific permits as the study did not include endangered species and did not involve sampling in protected areas. On Comoros archipelago, the samples were provided by an entomological survey carried out in the context of disease investigation for which the research protocol was approved by the Vice-Presidency of Agriculture, Fisheries and Environment of the Union of Comoros (see ). Sample2. These traps were placed on the sampling sites  between 5.00 and 6.00 PM and picked up the next day between 7.00 and 8.00 AM. As this method lures the flies for a blood meal, mostly unfed females were captured. In addition, adults were captured using a butterfly net held out of a car driving at 30 Km.h-1 along perennial rivers. This method allowed trapping males and (fed/unfed) females. The sampling was opportunistically complemented with larvae specimens from other areas in the region (Comoros and Seychelles archipelagos) and from Africa  (The main sampling was conducted on Reunion Island from January to May 2011 . Larvae Morocco) . All samDNA extraction, amplification and sequencing section). Differences in amplicon size were visualized by electrophoresis on 2% agarose gels stained with 1X GelRedTM (Biotium Inc.) and further confirmed by Sanger sequencing.All Reunion black fly larvae could be identified morphologically using previously published keys . For larc oxidase subunit I encoding gene (COI) and a nuclear fragment encompassing 18S rDNA (3’ end), Internal Transcribed Spacer 1 (ITS1), 5.8S rDNA, Internal Transcribed Spacer 2 (ITS2) and 28S rDNA (5’ end) locus (thereafter referred as 18S/28S) was amplified and sequenced for phylogenetic reconstructions. COI was amplified with LCOI490/HCO2198 primers [A sample of 14 adults and 62 larvae (comprising at least one larva per species and sampling station in Reunion) was used for molecular analyses. DNA extraction was realized with the Qiagen EZ1 DNA Tissue kit  according to the manufacturer's protocol. The mitochondrial cytochrome  primers  and 18S/ primers . AmplifiSimulium aureohirtum  (ruficorne species-group) from Asia . For both the mitochondrial and nuclear analyses, two independent MCMC chains were run for a total of 30 million generations each, of which 10% were discarded as burn-in. Samples were kept every 1,000th generation. Convergence was verified by effective sample size (ESS) values exceeding 200 for all parameters, using Tracer v1.6 for continuous parameters. For the tree topology parameter, we calculated the pseudo- and approximate ESSs, as implemented in the functions topological.pseudo.ess and topological.approx.ess of the R package rwty [Dated phylogenetic trees were estimated with BEAST v2.3.2  for nucl-1 Myr-1 . For theage rwty , based oSequences were available for both nuclear and mitochondrial markers only for a small number of individuals. Consequently, we chose to keep both nuclear and mitochondrial analyses separate.in-situ diversification, we assessed the likelihood of all Reunion species forming a monophyletic clade. As the Comorian specimens were identified as S. triplex (see the Results section), these specimens were synonymized with Reunion S. triplex in this monophyly analysis, effectively testing a hypothesis of in-situ diversification in the SWIO region (Hypothesis H1) rather than on Reunion Island alone.In order to assess the likelihood of a scenario with a single colonization of Reunion by black flies, with a subsequent S. ruficorne specimens (Hypothesis H2), as the mitochondrial phylogeny suggested the paraphyly of S. ruficorne (see the Results section).We further evaluated the monophyly of Reunion H1 and H2) was assessed using its posterior odd, calculated as: P(H|D) / (1-P(H|D)), where P(H|D) is the posterior probability of hypothesis H [H|D) was estimated as the frequency of trees in the posterior distribution that included the clade considered, when this frequency was more than 0. When the clade of interest is not comprised in any tree of the posterior sample (zero frequency), we estimated that P(H|D) was less than 1/ESStopology.Based on Bergsten et al. , we did thesis H . The posSimulium ruficorne, Simulium borbonense and Simulium triplex. No Simulium indoceanicum specimen was captured. For each sampled species, the amplification of the ITS1 locus produced amplicons with different sizes: 82, 110 and 116 bp for S. triplex, S. ruficorne and S. borbonense, respectively  identify black flies collected on Comoros as S. triplex. Thus the collected Comorian specimens will be referred to as S. triplex. Larvae from the Seychelles archipelago showed specific morphological characters and the ITS1 nucleotides sequence was clearly different from that obtained with specimens from Reunion, Africa or the Comoros, and therefore represent a species otherwise absent from our sample.Sampling on Reunion Island allowed the collection of three out of the four previously described species, namely ectively . This siectively . The amppecimens . Similarame size . MoreoveS. triplex have entirely black antennae except for a white first segment. In contrast, S. borbonense and S. ruficorne females have white antennae with a black first segment. These two latter species can be distinguished according to their legs: S. borbonense has a large black patch on each femur while S. ruficorne exhibits tiny spots. Simulium borbonense and S. ruficorne males could not be morphologically distinguished.Based on the detected length polymorphism of ITS1, we also proposed additional discriminating morphological characters allowing identification of adults from Reunion. Both females and males of n = 20) or within Comorian (n = 4) S. triplex, nor within Seychelles specimens (n = 4) (S. borbonense (n = 19) or from Reunion (n = 16) and African S. ruficorne (n = 12) showed nucleotide polymorphism comprising respectively 12, ten and six distinct haplotypes. Reunion and African S. ruficorne did not share any mitochondrial haplotype. Nuclear amplification (18S/28S) produced 29 sequences ranging from 576 to 650 bp for S. borbonense (n = 4), Reunion (n = 3) and Comorian (n = 1) S. triplex, Reunion (n = 8) and African (n = 12) S. ruficorne and only one sequence was obtained from a Seychelles specimen  . In contspecimen . InteresS. triplex, which clustered with Reunion S. triplex (Clade A). In contrast, the specimens from Seychelles formed a clade of their own (Clade B), related to but quite divergent from S. triplex. All Reunion and African S. ruficorne grouped together. The S. aureohirtum specimens fell within the ruficorne species-group. Interestingly, a number of sequences obtained from Reunion S. ruficorne specimens , as did sequences from African S. ruficorne (Clade E).The mitochondrial phylogeny  confirmegeny see . The remS. triplex. The topology further showed that the S. ruficorne specimens harbouring S. borbonense haplotypes (designated by stars in S. ruficorne nuclear DNA. It is noteworthy that an African S. ruficorne specimen (BB013) is embedded within S. aureohirtum clade. This topology likely results from a poor resolution of the nuclear phylogeny. Indeed, a visual inspection of the nuclear sequences further suggests that S. ruficorne BB013 belongs to the S. ruficorne clade with the presence of a single deletion detected only in S. ruficorne sequences (data not shown).The nuclear phylogeny  was lessH1, posterior odd ≈ 0, The mitochondrial and nuclear phylogenies indicated that the three Reunion black fly species do not cluster together, suggesting that they originated through multiple colonizations Figs  and 3. TS. ruficorne is paraphyletic as some sequences obtained from Reunion S. ruficorne belong to the S. borbonense clade . Altogether, these results indicate that Reunion black flies identified as S. ruficorne can also host S. borbonense haplotypes.The mitochondrial phylogeny suggested that se clade . This paS. triplex and the two other species from Reunion Island is estimated at 12.87 Myr ago (95% CI: 11.11–15.75), while S. borbonense and S. ruficorne diverged 6.46 Myr ago (95% CI: 4.71–8.30) (S. triplex and specimens collected in Comoros diverged more recently: 0.67 Myr ago (95% CI: 0.31–1.11) (S. ruficorne diverged 2.24 Myr ago (95% CI: 1.46–3.04), which is close to the estimated age of Reunion Island emergence . Specifically, the divergence of 71–8.30) . In cont31–1.11) . Interesmergence .Simulium ruficorne, Simulium borbonense and Simulium triplex. The fourth species, Simulium indoceanicum, was not observed in our samples. The sampling was carried out during the austral summer and not through the whole year, thus we cannot rule out that the absence of S. indoceanicum resulted from either a restricted sampling period or geographic distribution of this species. Environmental perturbations are known to have considerable impact on black fly communities. These perturbations include hydrological modifications, deforestation, tourism development, habitat destruction and pollution [S. indoceanicum, originally described from samples collected in 1983, can be hypothesized even though it remains difficult to address.The investigation of Reunion black fly fauna allowed sampling three out of the four species described on the island: ollution ,62. Reunollution ) that inollution  and dataollution . Thus, tS. triplex in the black fly fauna of Comoros archipelago. Consequently, S. triplex may not be considered as endemic to Mascarene archipelago anymore. On Seychelles archipelago, only one endemic Simulium species has been reported so far: Simulium speculiventre  belonging to the ruficorne species-group (Nevermannia) [S. speculiventre.In addition to the investigation of Reunion black flies, our study brings some original information on regional black fly diversity. We describe rmannia) . Thus, tThe identification of black fly species from Reunion Island was thus far mainly based on morphological characters of larvae, pupae and the genitalia of male adult . AlthougS. ruficorne and S. borbonense are the most closely related species. Based on mitochondrial data, S. ruficorne appeared paraphyletic. Such paraphyly has been reported for other black fly species, complex species or species-group: Simulium fenestratum  [Simulium articum  [Simulium tuberosum  [S. ruficorne is only supported by the mitochondrial phylogeny  [Simulium articum complex. To explain this situation, the authors proposed possible introgressive hybridization with the sharing of alleles between members of sympatric species. In our case, the hypothesis of mitochondrial introgression requires interspecific hybridization between S. borbonense and Reunion S. ruficorne, two sympatric species belonging to the same species-group (ruficorne). Interestingly, this interspecific hybridization appears to be asymmetrical, and would result from interspecific crosses between Reunion S. ruficorne males and S. borbonense females. Indeed, no introgressed specimens descending from the reciprocal hybridization  were observed. Such asymmetrical introgression has been previously reported for other organisms: birds, Flycather [Culex pipiens [Wolbachia-mediated selective sweep. To test this last hypothesis, the presence of Wolbachia wsp gene was investigated by using previously published PCR amplification test [Wolbachia in any of the tested flies (data not shown). The presence of paraphyletic species feeds the controversy on the use of mitochondrial markers alone to describe the taxonomy and phylogeny of invertebrates [Phylogenetic reconstructions allowed the description of Reunion black fly species relationships. Among the Reunion sampled species, s, 1934) , Simuliuh, 1914)  and Simum, 1911) . Intereshylogeny . Indeed,d, 1903)  or otherd, 1903) . Kruegerd, 1903)  proposedd, 1903)  also replycather , spiderslycather , Honey Blycather  and mosq pipiens  and has ion test  and we ctebrates ,77.S. ruficorne and S. borbonense, while the times of coalescence of all mitochondrial haplotypes for each of these two species are much more recent (< 1.00 Myr). Incomplete lineage sorting would be associated with more ancient mitochondrial lineages for each species although we cannot rule out possible colonization-dependent bottlenecks that would lead to the loss of a significant part of ancestral polymorphism. Interestingly, one nuclear sequence (18S/28S) was identically recovered from both African and Reunion S. ruficorne populations. This pattern is suggestive of a divergence time that is not long enough for lineage sorting to complete or gene flow between Reunion and African S. ruficorne populations.In this study, we estimated a divergence time of 6.46 Myr between i.e. African S. ruficorne) [Simulium triplex is present on different islands across the SWIO , suggesting that this species has a large spatial distribution and has potentially colonized Reunion Island from another island of the region. Altogether, we propose that the black fly diversity of Reunion Island has been built by a recurring influx of new colonists from the regional species pool, all belonging to ruficorne species-group.Speciation by geographical isolation following colonization is commonly accepted as a general source of endemism in oceanic island ecosystems ,78. The ficorne) . Our phySimulium oviceps  and Simulium dussertorum , two species belonging to the oviceps species-group (subgenus Inseliellum). Interestingly, Giudicelli [S. triplex is a stenothermic species, restricted to headwaters whereas S. borbonense and S. ruficorne are eurythermic species evolving within the lowland stream [S. borbonense and S. triplex result from speciation from a hypothetical S. ruficorne ancestor. Such speciation process could have taken place on nearby areas such as Mauritius Island, Rodrigues, Madagascar or Africa. In Madagascar, several black fly species are present and notably species belonging to the ruficorne species-group [ruficorne species-group are known to be ornithophilic [In Pacific Ocean archipelagos, it has been shown that dispersal and speciation processes may explain the current diversity and distribution of black fly species. Within these islands, adaptive radiation represents the major mode of speciation with specialization of species to specific habitats ,43,46,79udicelli  has showd stream . The impes-group . Such roes-group ,41,42,80hophilic ,81.in-situ diversification, which improves our understanding of processes driving the biodiversity composition on young oceanic islands. Interestingly, we highlighted that two Reunion species, separated by approximately six million years of independent evolution, displayed an intriguing case of asymmetrical mitochondrial introgression. It is interesting to note that the evolutionary histories of some of these black flies’ hosts (i.e. Zosterops birds) and the blood parasites (i.e. Leucocytozoon) transmitted by black flies to these birds, have been investigated in the SWIO region [In this study we showed that the Reunion black fly diversity resulted from multiple colonizations of distinct fly species rather than O region –28. The S1 FigTM (Biotium Inc.) The visualization was realized under UV.The colors red, blue, green and orange represent species from Morocco, Reunion Island, Comoros and Seychelles archipelagos, respectively. The electrophoresis was performed during 2 hours on a 2% agarose gel stained with 1X GelRed(TIF)Click here for additional data file.