Gentiana (Gentianaceae) is a species-rich plant group which rapidly radiated in the Himalaya-Hengduan Mountains in China. In this study, we tested the core plant barcode (rbcL + matK) and three promising complementary barcodes  in 30 Gentiana species across 6 sections using three methods . rbcL had the highest PCR efficiency and sequencing success (100%), while the lowest sequence recoverability was from ITS (68.35%). The presence of indels and inversions in trnH-psbA in Gentiana led to difficulties in sequence alignment. When using a single region for analysis, ITS exhibited the highest discriminatory power (60%-74.42%). Of the combinations, matK + ITS provided the highest discrimination success (71.43%-88.24%) and is recommended as the DNA barcode for the genus Gentiana. DNA barcoding proved effective in assigning most species to sections, though it performed poorly in some closely related species in sect. Cruciata because of hybridization events. Our analysis suggests that the status of G. pseudosquarrosa needs to be studied further. The utility of DNA barcoding was also verified in authenticating ‘Qin-Jiao’ Gentiana medicinal plants , which can help ensure safe and correct usage of these well-known Chinese traditional medicinal herbs.DNA barcoding of plants poses particular challenges, especially in differentiating, recently diverged taxa. The genus  For example, matK + ITS was recommended as the barcode to be used in the genus Primula [trnH-psbA + matK was demonstrated as the best barcode for discriminating Rhododendron species [DNA barcoding, a term first proposed by Hebert in 2003 , has devd plants . Howeverd plants . Limitedlex taxa –13. Sincd plants –16, the  Primula , while I species . More stCrocus and Quercus in African forests [Floristic DNA barcoding has been demonstrated to be effective for identifying species in species-rich regions that would otherwise require detailed ecological study for characterization . However forests , and low forests . This isGentiana L. (Gentianaceae) consists of 361 species with a subcosmopolitan distribution; more than half of all the species are found in southwestern China and the adjacent northeastern Himalaya-Hengduan Mountain region [Rhododendron, Primula, Pedicularis and Gentiana [Gentiana is divided into 15 sections, of which 5 are further divided into 22 series [Chondrophyllae and Cruciata [n region . This reGentiana . The gen2 series . Althoug2 series , 24. MolCruciata .Chondrophyllae is the largest and the most widely distributed section in this genus; it consists of about 163 species and is divided into 10 series [Cruciata contains 21 perennial species. Some species in this section may have diverged four million years ago, though most result from more recent speciation events [Cruciata are well known in traditional Chinese medicine and are also widely utilized for medicinal purposes in Asia (e.g. Gentiana davidii as drugs of cholesteric and hepatic diseases) [Gentiana species would therefore be invaluable.Most of the species in these sections are distributed in the mountainous regions of southwestern China and the nearby Qinghai-Tibet Plateau. Sect. 0 series . The ide0 series . Sect. Cn events . Twelve iseases) , 28. Theiseases) . Authentiseases) . FindingrbcL, matK, trnH-psbA, ITS and ITS2) were chosen for evaluation. We aimed to: i) evaluate the discriminative ability of the five barcoding regions, rbcL, matK, trnH-psbA, ITS and ITS2 and their combinations; and ii) explore the efficacy of DNA barcoding in Gentiana.In this study, five DNA barcoding candidate regions (Chondrophyllae (12 species) and sect. Cruciata (8 species), the following 4 sections were also selected for analysis: sect. Frigidae (2 species), sect. Microsperma (2 species), sect. Kudoa (4 species) and sect. Monopodiae (2 species). All specimens were collected from the wild and no specific permissions were required for the corresponding locations/activities. The field studies did not involve endangered or protected species. Vouchers specimens of the collected taxa were deposited in the South China Botanical Garden Herbarium (IBSC)  .rbcL, matK, trnH-psbA and ITS), were amplified and sequenced to test the effectiveness of their primers in Gentiana. matK required the use of two primer pairs . A 25 μl PCR reaction mixture was prepared and amplified according to the procedure described by Zhang et al. [Genomic DNA was extracted from dried leaves in silica gel using the CTAB method . Four re-Kim/-xf  and matKK-xf/-5r ). The otg et al. . PCR protrnH-psbA was aligned by section, and intraspecific inversions were found in this region. To reduce costs, we retrieved ITS2 from ITS data and re-amplified failed ITS samples.The original trace files were checked and verified by searches with NCBI’s web-based BLASTn. Sequences were assembled and inspected with Sequencher 4.1 , alignedGentiana.Genetic divergence was calculated for the five markers according to the Kimura 2-Parameter (K2P) model using MEGA 5.0. Six distance parameters were estimated, including three inter-specific distance parameters  and three intra-specific parameters  . We calcet al. [Three methods, namely a genetic distance-based method, the analysis of Best Close Match and a tree-based method, were employed to evaluate the five single markers and their combinations. The first two methods were conducted using the R package SPIDER . For theet al. . SpeciesGentiana, we retrieved all Gentianaceae sequences involving rbcL or matK and all Gentiana sequences with the internal transcribed spacer (ITS and ITS2) from GenBank. Sequences available on NCBI may not necessarily link with taxonomically validated voucher specimens, so we examined all the sequences downloaded from Genbank in an effort to ensure correct species identification. We found that almost all of the Gentiana sequences available in Genbank are associated with published phylogenetic or barcoding papers, and the sources of the sequences were identified by specialists working on this genus. Collection information for the voucher specimens was present in the relevant papers.In order to minimize the bias from incomplete sampling, we expanded our dataset using data from public databases. Since limited DNA barcode data is available for the genus trnH-psbA was not used for further analysis. We also removed sequences less than 300 bp in size and those lacking clear Gentiana species identification. We followed an established pipeline [rbcL, 274 sequences for matK, 243 sequences for ITS, and 304 sequences for ITS2. The data were analyzed with tree-based analysis, as above.Due to difficulties in sequence alignment, pipeline  to removrbcL had the highest PCR efficiency and sequencing success (100%), followed by matK (96.2%) and trnH-psbA (96.2%) . In total, 355 sequences were obtained and submitted to the GenBank database ( (96.2%) . Sequencdatabase .trnH-psbA exhibited the highest interspecific genetic distance, followed by ITS2, matK, ITS and rbcL. For the divergence among conspecifics, the rank order of theta was trnH-psbA, ITS2, ITS, matK and rbcL. An ideal barcode should possess higher interspecific variation than intraspecific variation in order to distinguish different species. ITS had one of the smallest interspecific distances and a relatively low coalescence depth.Comparative analysis of inter- versus intra-specific distances for the five regions was conducted using six parameters  Table 2Table 2. Gentiana. In the single region analysis, rbcL performed poorly, as expected (trnH-psbA (45%-71.21%), matK (52.63%-69.23%) and ITS2 (50%-67.80%). When combining two barcodes, matK + ITS gave the highest discrimination success (71.43%-88.24%). The three-region combination of rbcL + matK + ITS achieved slightly higher species identification success than matK + ITS when using the Best Close Match method (87.18%).A genetic distance-based method, the Best Close Match and a tree-based method were used to evaluate the discriminatory power of barcodes in expected . The higrbcL and matK can very reliably assign sequences to the genus Gentiana (100% success rate). Moreover, the DNA barcoding markers performed well at the section level. rbcL grouped 5/9 sections correctly, matK identified 8/10 sections, ITS identified 7/11 sections and ITS2 identified 5/11 sections  performed much better (62.50%-12.50%) than chloroplast markers (37.50%-12.50%).When the comparative analysis was restricted to each section, the mean K2P distances of all the barcodes showed significant heterogeneity . DivergeCruciata  Figs. There used . In sectrbcL and matK have been proposed as core DNA barcodes for plants [rbcL and poor primer universality of matK) have since been reported in many studies [matK, since we employed two universal pairs of primers recommended by CBOL. However, the combination of rbcL and matK had the lowest genetic divergence and could only poorly discriminate species in this genus. In sect. Cruciata, rbcL + matK was even less effective for differentiating closely related species (0–32.5%). The poor performance of rbcL + matK has been reported in many species-rich plant groups, such as Lysimachia (47.1%-60.82% discriminatory power) [Berberis (15.4%-23.1%) [Viburnum (53%) [Primula L. sect. Proliferae (50%) [rbcL + matK must be supplemented with more effective barcodes for the genus Gentiana.While r plants , their d studies , 44, 45.y power) , Berberi%-23.1%) , Viburnuum (53%) , Primulaae (50%) . TherefotrnH-psbA spacer and the availability of a universal primer have led to its successful application in many DNA barcoding studies. In this study, trnH-psbA gave the highest inter- and intra-specific divergence of all the single regions. Nevertheless, several problems limit its use in Gentiana. First, extensive variation in the size of trnH-psbA resulted in alignment ambiguities. The length of the trnH-psbA sequence varied across five of the six sections; the sequence was 199–327 bp in sect. Chondrophyllae, 410–486 bp in sect. Cruciata, 413 bp in sect. Monopodiae, 348–442 bp in sect. Microsperm, 397–470 bp in sect. Kudoa, and 360–391 bp in sect. Frigidae. We therefore had to manually adjust the algorithmically-generated alignment, which required significant effort. Second, a short 30 bp inversion was detected in trnH-psbA in Gentiana. Frequent inversions of trnH-psbA in the family Gentianaceae were reported by Whitlock et al. [G. panthaica (9 bp) and G. pseudosquarrosa (12 bp). These inversions lead to overestimation of the variation between closely related species and unite distantly related taxa, resulting in erroneous phylogenetic inferences [trnH-psbA have proved a hindrance to obtaining full length sequences in many other studies [trnH-psbA may serve as a valuable DNA barcode in plant groups where these challenges are not encountered, too much effort must be spent resolving these problems in Gentiana; trnH-psbA is therefore not recommended as a DNA barcode for Gentiana.The high variation of the  studies , althoug studies . While tGentiana at the sectional level demonstrated the phylogenetic utility of ITS sequences [Ficus [Lysimachia [Viburnum [Gentiana. This region was also capable of differentiating the closely related species Gentiana dahurica, G. decumbens and G. macrophylla. However, the sequencing success of ITS in Gentiana was low (63.7%), which may imply incomplete concerted evolution of a nuclear multiple-copy locus in this genus.ITS has been proposed as a DNA barcode because it evolves 3–4 times faster than the plastid regions, and it has been successfully used in many phylogenetic studies . A previequences . Many tas [Ficus , Lysimacsimachia  and VibuViburnum . In thiset al. [A short nuclear region (300 bp) of ITS2 was proposed as a novel DNA barcoding region for medicinal plants by Chen et al.  and has et al. , 50–52. et al. . Howeveret al.  and ‘DNAet al. .Gentiana. The use of multiple regions with more and less variation, such as combinations of plastid regions and nuclear regions, has been recommended and has been shown to improve discriminatory power in many barcoding studies [matK + ITS gave the highest species resolution (88.24%), roughly equivalent to the resolution of the three-marker combination rbcL + matK + ITS. We support the recommendation of the matK + ITS combination as a core DNA barcode for large genera of flowering plants [matK + ITS seems to be the best choice as a DNA barcode for Gentiana, with ITS2 serving as a back-up region for ITS to improve sequence recoverability.No single barcode was perfect for species identification in  studies , 16. In Gentiana barcoding study, 5S rRNA and trnL-F were successfully used as barcodes to differentiate five medicinal Gentiana species and their adulterants [trnL-F, with universal primers and high discriminatory power, also seems to be a good choice as a barcode for Gentiana species and has been suggested for other groups that have undergone a recent radiation [Gentiana.However, other highly variable regions are required to successfully identify all species. In a recent lterants . Howeveradiation , but morChondrophyllae and sect. Cruciata have both recently diverged in the Himalaya-Hengduan Mountains [Chondrophyllae (72.72%-83.33%) than for sect. Cruciata (0–62.5%)  may have caused over-estimation of the discriminatory power of DNA barcoding. In contrast, 8/12 species in sect. Cruciata were sampled and several closely related species were included, such as G. daurica, G. officinalis, G. macrophylla and G. decumbens [Although sect. ountains , 25, gre0–62.5%) . This maophyllae , 57. In ophyllae . Limitedecumbens .Cruciata. Hybridization may have occurred between G. officinalis and G. daurica [matK, which had two differences  were found in the ITS region. Additional studies sampling from more populations are needed to confirm this hypothesis.Natural hybrids and polyploidization pose a great challenge to attempts to barcode species. Hybridization events have been reported in sect.  daurica , 60, whiferences ; furtherGentiana, DNA barcoding enabled us to confidently assign most individuals at the genus and section level, even using only the rbcL/matK region or when the data set was expanded using GenBank sequences. If the section and geographical information of specimens is known, DNA barcoding will make the identification of most Gentiana species much easier for non-experts, which will greatly reduce the time and labor compared with morphological identification, especially in species-rich areas [Although limited species resolution was achieved in ch areas , 61.Lysimachia [Primula [Roscoea [Chondrophyllae were monophyletic, except G. squarrosa and G. pseudosquarrosa. All accessions of both species always clustered together with high confidence in the NJ tree and Bayesian analysis  [Aconitum sinomontanum , Salvia brzewalskii Maxim , and Veratrilla baillonii Franch. , have entered the commercial market [matK and ITS sequences of these counterfeit species from GenBank and compared them to the correct ‘Qin-Jiao’ sequences from this study using a NJ tree-based method  and detect product substitution and contamination , 30, 63.ahurica) , 65, hasl market , 29. In d method . The resrbcL + matK) is not very effective for identifying species in the genus Gentiana. Because of poor alignment and frequent inversions, the non-coding region of trnH-psbA also may not be desirable as a DNA barcode for Gentiana, even though it had the highest genetic variation and relatively high discriminatory performance. ITS was much more effective for species resolution and was capable of discriminating the closely related species in sect. Cruciata. A two-region combination of matK + ITS is recommended for use as a plant barcode in Gentiana. The ITS2 region, with its high sequence recoverability, can serve as a back-up region for ITS. Although DNA barcoding may not always be the perfect identification tool, we emphasize the practical applications of this method in biodiversity surveys, clarifying taxonomic questions and authenticating medicinal plant materials.The suggested core plant barcode  NJ trees using ITS and ITS2, respectively, with GenBank sequences for genus Gentiana.(a-b) NJ trees using (PDF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Bacillus megaterium, a Gram-positive bacterium often used as a model system in research and as a protein production strain in industrial applications. Here, we present the complete annotated genome of phage Stahl and describe its major features.Stahl is a siphophage active against  Bacillus megaterium is an important bacterium to research and industry. Due to its large size, it is extensively utilized in molecular biology studies. It is nonpathogenic and contains no endotoxins, making it ideal for large-scale protein synthesis .ynthesis . Bacterihttps://cpt.tamu.edu/galaxy-public/) . Quality-controlled trimmed reads were assembled to a single contig at 67.9-fold coverage using Velvet version 1.2.10. The contig was confirmed to be complete by PCR using primers that face the upstream and downstream ends of the phage DNA. Products from the PCR amplification of the junctions of concatemeric molecules were sequenced by Sanger sequencing . Genes were predicted using GeneMarkS (public/) , 4. MorpB. megaterium siphophages Slash (GenBank accession no. NC_022774) and Staley (GenBank accession no. NC_022767), respectively (Stahl has a double-stranded DNA (dsDNA) genome that is 80,148 bp in length, has a G+C content of 35.27%, and has a coding density of 89.9%. Based on sequence comparison using Emboss Stretcher, Stahl shares 85.6% and 84% nucleotide sequence identity to Few replication/recombination proteins were identified . Stahl encodes proteins for transcriptional regulation including two sigma factors and a transcription factor. Although the tape measure and tail fiber structural genes were identified, other structural proteins were not identified likely due to sequence divergence and lack of homology to other phages. A class II holin  was annotated along with two peptidoglycan-degrading enzymes: a secreted amidase and a soluble peptidase .Stahl encodes genes that are suggestive of a temperate lifestyle, such as a Rha superfamily protein (IPR014054), which contains a bacteriophage P1 Ant1-like C-terminal domain (IPR005039) and an integrase/recombinase . Rha, a protein encoded by a variety of temperate phages, prevents growth on bacterial strains that lack an integration host factor .Additionally, Stahl encodes a secreted LysM  and CAP  domain-containing protein. This protein has been identified in few phages, and its role in the phage infection cycle is unknown.KP696447.The genome sequence of phage Stahl was contributed to GenBank under the accession no.


Campylobacter fetus currently comprises three recognized subspecies, which display distinct host association. Campylobacter fetus subsp. fetus and C. fetus subsp. venerealis are both associated with endothermic mammals, primarily ruminants, whereas C. fetus subsp. testudinum is primarily associated with ectothermic reptiles. Both C. fetus subsp. testudinum and C. fetus subsp. fetus have been associated with severe infections, often with a systemic component, in immunocompromised humans. To study the genetic factors associated with the distinct host dichotomy in C. fetus, whole-genome sequencing and comparison of mammal- and reptile-associated C. fetus was performed. The genomes of C. fetus subsp. testudinum isolated from either reptiles or humans were compared with elucidate the genetic factors associated with pathogenicity in humans. Genomic comparisons showed conservation of gene content and organization among C. fetus subspecies, but a clear distinction between mammal- and reptile-associated C. fetus was observed. Several genomic regions appeared to be subspecies specific, including a putative tricarballylate catabolism pathway, exclusively present in C. fetus subsp. testudinum strains. Within C. fetus subsp. testudinum, sapA, sapB, and sapAB type strains were observed. The recombinant locus iamABC (mlaFED) was exclusively associated with invasive C. fetus subsp. testudinum strains isolated from humans. A phylogenetic reconstruction was consistent with divergent evolution in host-associated strains and the existence of a barrier to lateral gene transfer between mammal- and reptile-associated C. fetus. Overall, this study shows that reptile-associated C. fetus subsp. testudinum is genetically divergent from mammal-associated C. fetus subspecies. Campylobacter fetus has been recognized as a significant veterinary pathogen. Until recently, two subspecies were described: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv). Both subspecies have been isolated from multiple vertebrate hosts, mainly mammals, but the primary reservoir is considered to be ruminants. These subspecies display distinct host and niche preferences: Cff is often associated with the intestinal tract and aborted fetuses of ruminants, mainly sheep and cattle, whereas Cfv is almost exclusively associated with the genital tract of cattle  (f cattle . Next tod humans . This reum (Cft) .C. jejuni, symptoms of C. fetus-associated gastrointestinal illness are rarely reported  and amplified fragment length polymorphism (AFLP) analysis, have shown that reptile-associated Cft are genetically distinct from mammal-associated Cff and Cfv and that genetic diversity is higher in Cft . This suC. fetus . FurtherC. fetus , which sC. fetus are poorly understood. Comparing the whole-genome sequences of mammal- and reptile-associated C. fetus strains can provide valuable insights into this distinct host association, as well as further insights into speciation, taxonomy, and pathogenicity.The genetic factors underlying host differentiation of mammal- and reptile-associated C. fetus, genomes of the most closely related species Campylobacter hyointestinalis and Campylobacter iguaniorum were included in the analyses. A phylogenetic reconstruction provided insights into the distinct host association of C. fetus. Furthermore, detailed genome analyses characterized genomic regions specific to C. fetus, and to Cft in particular, and revealed multiple species- and subspecies-specific sequence variations, including a distinct putative tricarballylate catabolism locus, and a genomic region associated with human invasive strains.In this study, 20 genomes of reptile-associated Cft strains were compared with 39 genomes of mammal-associated Cff and Cfv. To determine features specific to n = 13) and human (n = 7) origin, 39 Cff and Cfv strains of bovine (n = 33), ovine (n = 1), human (n = 2), and unknown (n = 3) origin, one C. hyointestinalis strain of porcine origin, and one C. iguaniorum strain of reptilian origin. Characteristics of all strains used in this study are shown in 2, 7.1% CO2, 3.6% H2, and 6% O2) at 37 °C for 48 h.A total of 61 strains were used for this study, including: 20 Cft strains of reptilian  locus were confirmed with Sanger sequencing. Assembly was confirmed using Pacific Biosystems long reads. PacBio RS reads were assembled into contigs using Quiver .Whole-genome sequence data of Cft strains 03-427 and SP3 were obtained using a Roche 454 genome analyzer and was assembled into contigs using the Newbler assembler (v2.6). In this study, the genome of Cft strain SP3 was completed using methodology described previously for Cft strain 03-427 . In shorCampylobacter iguaniorum strain 1485E was sequenced as described previously    was usedsap locus contains a high number of repeated sequences and its assembly can prove difficult. The sap type of the draft Cft genomes was identified by mapping the sequence reads to the primer sequences of SAF01, SAR01, SBF01, and SBR01  and similar to Cff strain 82-40 (1.77 Mb) . The average G + C content of both strains is 33.1%. The Cft strain 03-427 genome is predicted to contain 1,695 putative protein-coding genes, 43 tRNA genes, and three rRNA operons. Cft strain SP3 is predicted to contain 1,767 putative protein-coding genes, 40 tRNA genes, and three rRNA operons. No obvious mobile elements or plasmids were identified in either strain. Cft strain 03-427 contained 29 variable homopolymeric GC tracts  and strain SP3 contained 24 variable homopolymeric GC tracts .The circular genome size of Cft strains 03-427 and SP3 is 1.78 and 1.82 Mb, respectively, which is within the known size range of 1.77 Mb)  . A total of 33 genes was present in all Cft strains, but absent from all Cff and Cfv strains . The most prominent difference identified between the C. fetus subspecies was a putative tcuRABC locus (CFT03427_0075-0078), present in all Cft strains, but absent from Cff and Cfv   . It has id cycle . These rsupplementary table S3, Supplementary Material online). Interestingly, an aspartate racemase-encoding gene (CFF8240_1412) was present in all of the Cff and Cfv strains, but absent from Cft strains. As aspartate racemase catalyzes the conversion of L-aspartate to D-aspartate, Cft is predicted to be unable to convert L-aspartate to D-aspartate. As this was a pseudogene due to a premature stop codon in Cff 04/554, but not in the other completed Cff and Cfv genomes, and this gene is located in the lipooligosaccharide (LOS) biosynthesis locus (CFF8240_1399-1414) bound by waa genes and containing multiple glycosyltransferases . In total, 65 genes were present in all C. fetus strains, but absent from both C. hyointestinalis and C. iguaniorum strains .Several genetic features were identified in sucDC; CFT03427_0903-0904), proline dehydrogenase  and sodium/proline symporter , hemin ABC transporter system , and serine protease (CFT03427_1733) were present in all C. fetus strains examined, but absent from its closest relatives.Genes encoding succinyl-CoA synthetase alpha and beta subunits , another catalase-like protein encoding region (CFT03427_1708) was present in all fucP and the surrounding coding region (CFT03427_1042-1047) was present in all C. fetus strains examined, but was absent from its closest relatives C. iguaniorum and C. hyointestinalis. Low homology orthologs (57–65% identity) were identified in C. coli and C. jejuni by an online BLASTP search against the nonredundant database. In some C. jejuni strains, fucP and the surrounding coding region (cj0480c–cj0490) are implicated in the uptake of the sugar L-fucose, which is released from the host’s mucin glycoproteins and has been shown to be important in colonizing hosts  of the Cff and Cfv genomes . Noteworthy, all Cfv strains were lacking the cas genes, while eight Cff strains, including strains 82-40, B0131, and JYCP01, which are most closely related to Cfv, did contain these genes. Although the cas genes were absent from Cfv strain 97/608, this strain did contain CRISPRs. The presence of CRISPRs suggests that cas genes may have been present, but have been lost in Cfv strain 97/608. Notably, the cas genes identified in C. fetus were not homologous with cas genes found in other Campylobacter species and showed highest homology with Sulfurospirillum and Sulfurovum species.CRISPRs were identified in the complete genomes of Cft strains 03-427 and SP3. Six genes coding for the CRISPR-associated proteins Cas1-6 (CFT03427_0656-0663) were conserved in all 20 Cft strains examined. However, these C. fetus strains . These genes are largely confined to C. fetus, although orthologs were identified with low homology in C. concisus and C. rectus. The exact function of these proteins is unknown. No CRISPRs were identified surrounding these genes, suggesting that this cluster of genes is unlike currently known CRISPR/Cas systems.Interestingly, four additional CRISPR/Cas system-associated RAMP superfamily protein coding loci (CFT03427_1628-CFT03427_1633) were conserved in 93.2% (55/59) of the cadF, ciaB, and cytolethal distending toxins cdtABC, identified in C. fetus previously  was exclusively found in C. fetus. In Pseudomonas aeruginosa, a patatin-like protein has been linked to the development of lung injury, sepsis, and bacterial dissemination in animal models and human infections .Most known and predicted virulence determinants, such as eviously , were alfections . Type IVn 98/445 , but werC. fetus is the S-layer, which is associated with resistance to complement-mediated killing and is considered to be an important virulence factor  were sapA type, 15% (3/20) were sapB type, and 15% (3/20) were sapAB type . Considering the high variability of this region and the draft nature of the genomes, the sequence reads of these particular strains were searched for parts of these genes and were found absent in the reads as well. As these genes are essential in formation of the S-layer , whereas the conserved glycosylation region characteristic for sapB (CFF04554_0484-0487), including GDP-D-mannose dehydratase-encoding wcbK . The two different conserved glycosylation regions adjacent to the sap locus are likely involved in LPS-biosynthesis and Sap binding, as has been shown for wcbK in sapB type C. fetus  was highly divergent from all other C. fetus strains examined, and flaAB and maf showed highest homology with the related species C. hyointestinalis and C. iguaniorum , was conserved in all Cff/Cfv and most Cft strains examined, but was absent from the seven flaAB recombinant Cft strains  are found in and adjacent to this region. The strong association with flaAB in the nonrecombinant strains suggests that this region is involved in the O-linked glycosylation of flagellin. Like in the flaAB recombinant Cft strains, this region was missing in C. iguaniorum  adjacent to  strains . Within function , and genuaniorum . No otheiamABC , and showing higher than expected amino acid homology (99–100%) between all Cff and Cfv strains and human clinical Cft strains, but not reptilian Cft strains (93–97%), was observed. Recombination analysis indicated a recombination event between Cff and Cft isolated from humans. A maximum-likelihood phylogenetic dendrogram based on iamA (mlaF) illustrated this recombination event (atpA (uncA) MLST allele type 6, whereas all other Cft strains known are MLST allele type 5  detected inside recombinations was higher in Cft, whereas the number of SNPs outside recombinations was lower in Cft, compared with Cff and Cfv . Also thsap type was observed in Cff and Cfv, with sapA and sapB type strains forming separate clusters . The identification of these sap types in genetically diverse Cft strains shows that these sap types are widespread in Cft and are present in both mammal- and reptile-associated C. fetus. In contrast to previous studies  genomes. Based on the high genetic similarity between Cff and Cfv, and the lack of a clear boundary between these subspecies, the validity of these subspecies has been questioned .


Zymoseptoria tritici (synonym Mycosphaerella graminicola) is a prominent pathogen of wheat. The reference genome of the isolate IPO323 is one of the best-assembled eukaryotic genomes and encodes more than 10,000 predicted genes. However, a large proportion of the previously annotated gene models are incomplete, with either no start or no stop codons. The availability of RNA-seq data allows better predictions of gene structure. We here used two different RNA-seq datasets, de novo transcriptome assemblies, homology-based comparisons, and trained ab initio gene callers to generate a new gene annotation of Z. tritici IPO323. The annotation pipeline was also applied to re-sequenced genomes of three closely related species of Z. tritici: Z. pseudotritici, Z. ardabiliae, and Z. brevis. Comparative analyses of the predicted gene models using the four Zymoseptoria species revealed sets of species-specific orphan genes enriched with putative pathogenicity-related genes encoding small secreted proteins that may play essential roles in virulence and host specificity. De novo repeat identification allowed us to show that few families of transposable elements are shared between Zymoseptoria species while we observe many species-specific invasions and expansions. The annotation data presented here provide a high-quality resource for future studies of Z. tritici and its sister species and provide detailed insight into gene and genome evolution of fungal plant pathogens.The fungal pathogen  Zymoseptoria  comprises a group of plant pathogens that infect a variety of Poaceae species. The four pathogen species Z. tritici, Z. pseudotritici, Z. ardabiliae, and Z. brevis originated in the Middle East and have diverged recently   was sequenced and annotated by the Joint Genome Institute (JGI) and provides one of the best-assembled eukaryotic genomes with 21 complete chromosomes sequenced from telomere to telomere  (Z. pseudotritici (isolate ST04IR_2.2.1), and Z. ardabiliae (isolate ST04IR_1.1.1)  with standard settings for paired-end read assembly. This assembly is available under the NCBI BioProject (PRJNA273516) on Genbank. The assembled genomes of Z. pseudotritici, Z. ardabiliae  seedlings and one dataset obtained from axenically grown fungal cells  agar in a shaking incubator at 200 rpm at 18° using the TRIZOL reagent  following the protocol of the manufacturer. Illumina RNA-seq libraries for two axenic culture replicates per species were prepared from an input of 10 µg total purified polyA RNA . For data processing, the sequence read quality was first evaluated using FASTQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Subsequently, reads were filtered using tools from the Galaxy server, including grooming, trimming, filtering, and masking steps  transcriptome were initially filtered out using fastq_screen v0.4.1 (www.bioinformatics.babraham.ac.uk/projects/fastq_screen) as described by Two previously published RNA-seq datasets were used for Protein-coding genes were identified using the Fungal Genome Annotation pipeline described by de novo and genome-guided methods. By the de novo method, RNA-seq reads were first assembled into unique sequences of transcripts (contigs) using the Inchworm module within Trinity. Contigs were clustered by the Chrysalis module, and corresponding De Bruijn graphs that represent the possible different isoforms were constructed. In the final step, De Bruijn graphs were processed by the Butterfly module to produce full-length transcripts. In the genome-guided method, RNA-seq reads were first aligned to the genome using GMAP  (ab initio predicted gene models. Only the complete gene models predicted by GeneMark-ES with a support from the homology-based comparison (100% of coverage for each exon) and with the exact same exon–intron boundaries as in the reconstructed transcripts were selected for the training and testing of ab initio gene predictors. The 2693 selected gene models were divided into two sets: one for the training (training set) containing 1611 sequences (60%) and one for assessing prediction accuracy (test set) containing 1082 sequences (40%). The evaluation of the training process was performed using Augustus . Obtained pairwise protein alignments were processed using the software SiLiX to build families of homologous proteins  if a signal peptide was predicted by both Neural-Network and HMM methods by the software SignalP 3.0  and used for transcriptome assembly. Assemblies were based on two different but complementary approaches: (i) de novo, where the reads were assembled without a reference sequence and (ii) genome-guided, where the IPO323 reference genome was used as a template for transcript assemblies. In total, 68,653 transcripts were generated by the de novo method and 73,127 transcripts by the genome-guided method  compared to 1555 incomplete genes in the previous annotation (vs. 505 bp) and encode longer proteins (488 vs. 437 amino acids) when compared to the annotation generated by the JGI pipeline  had no homologies within the NCBI nonredundant protein database and did not include any known protein domain. These represent either species-specific genes of Z. tritici or incorrectly predicted genes. Given the fact that 55% of these have RNA-seq support, we considered that the majority of these novel genes must be correctly predicted. Characterization of the Z. tritici secretome yielded comparable results to the previously reported Z. tritici secretome  (no acids . SSPs ofar role) .Z. pseudotritici, Z. ardabiliae, and Z. brevis were filtered (Table S1) and assembled. The resulting transcriptomes include 35,132, 36,546, and 33,201 transcripts clustered into 12,027, 12,719, and 11,542 putative gene models for Z. pseudotritici, Z. ardabiliae, and Z. brevis, respectively , http://genome.jgi.doe.gov/Zymps1/Zymps1.home.html (Z. pseudotritici), http://genome.jgi.doe.gov/Zymar1/Zymar1.home.html (Z.ardabiliae), and http://www.uniprot.org/taxonomy/1047168 (Z. brevis).RNA-sequencing data obtained from axenic cultures of ectively . The traectively . The tot tritici . OverallZymoseptoria species including orthology (interspecies) and paralogy (intraspecies) were analyzed by a comparison of the predicted proteomes using the software package SiLiX  were clustered into 10,612 families. These families belong to three categories. First, there are 10,361 families of orthologous sequences present in at least two species. The different distributions of these families across the Zymoseptoria species are shown in i.e., independent duplication of a conserved gene occurred in one or more species. Third, there are 15 families of paralogous sequences, i.e., duplication of species-specific genes. The remaining 5050 sequences that could not be classified into a gene family were considered as proteins encoded by orphan genes.Homology relationships among the four Z. tritici encodes 1798 orphan genes , of which 1221 are localized on core chromosomes and 577 on accessory chromosomes. However, accessory chromosomes are significantly enriched in orphan genes because they constitute 79.3% of the genes on these chromosomes compared to 10.9% on core chromosomes . A large majority of the orphan genes (95.5%) have no in silico attributed function. In general, we could not link species-specific genes to any biological or pathogenicity-related function; however, this uniqueness supports the species-specific nature of the genes that have no homology with known proteins from organisms outside the Zymoseptoria species complex. Among the orphan sequences that could be assigned a function, we found predicted functions relating to molecule transport , primary and secondary metabolism , and organic compounds degradation . Small secreted proteins (SSPs) of plant pathogens particularly play an important role as putative effectors during the host infection . Orphan genes representing 9–11% of the predicted genes in Z. pseudotritici, Z. ardabiliae, and Z. brevis were likewise significantly enriched in SSP-encoding genes and genes that could not be assigned a function.The genome of nfection , and we Z. tritici (eight families) and Z. ardabiliae (seven families) and included 23 and 14 genes, respectively. In Z. ardabiliae, 57% of these duplicated sequences are SSP-encoding genes, whereas there were no SSP paralogs in Z. tritici. In Z. ardabiliae, only one family could be associated with a gene function (kinase), whereas in Z. tritici several families contained sequences with known protein domains of transposable elements.Families of paralogous genes were only found in Zymoseptoria species were divided into three categories. The first category comprised the core set of Zymoseptoria genes and included 7786 families containing one gene per species .Families of orthologous genes within the four  species . A largeZ. tritici, Z. pseudotritici, and Z. brevis and three copies in Z. ardabiliae.A particular category of families was also identified where orthologous sequences were conserved in two or more species with independent duplications in one or more species. This category contained 236 families with 1552 sequences. Most of the families (87%) comprised genes present in the four species. The largest family included 15 genes all encoding an alcohol dehydrogenase with four copies found in Z. tritici IPO323, 7.4 Mb (18.6%)  were found to be repetitive DNA, in agreement with the recently reported repeat content of IPO323 (>17%) in Z. tritici consists of complete chromosomes allowed a high-quality repeat annotation with only few uncategorized families. TEs of class I (retrotransposons) are the most abundant in the IPO323 genome and represent 13.1% of the total genome size (5.2 Mb), whereas TEs of class II (DNA transposons) represent 4.2% (1.6 Mb) , we identified 101 families of TEs. The majority of these families belong to the class II (62 families), mainly represented by TIRs (44 families), MITEs (11 families), and Helitron (4 families). Although present in a lower number (32 families), class I elements represent 71% of the repetitive fraction of the genome. The most abundant group of class I elements is the LTR elements from the Gypsy superfamily (14 families), followed by those of the Copia superfamily (12 families) and LINEs (7 families)  (Of the 39.7-Mb genome assembly of  (18.6%)  were fou(1.6 Mb) . The remamilies) . Interesomosomes . However 0.3692) .Z. tritici genome despite the RIP machinery.As already described by Z. pseudotritici, Z. ardabiliae, and Z. brevis, we generated improved assemblies of Illumina-sequenced genomes of the isolates: ST04IR55 of Z. pseudotritici, ST11IR611 of Z. ardabiliae, and Zb163 of Z. brevis. The assembly sizes were 38.8 Mb, 37.3 Mb, and 39.8 Mb, respectively, compared to the 39.7 Mb genome assembly of Z. tritici.To improve the repeat annotation of Z. ardabiliae genome, 15.5% of the Z. pseudotritici genome, and 23.9% of the Z. brevis genome, including a total of 117, 105, and 164 repeat families identified in the genomes, respectively  were only identified in Z. pseudotritici and were the third most represented TE family in the genome , 135 families of class II TEs (27.2%), and 242 uncategorized families (48.7%). We could improve the TE annotation using the complete Zymoseptoria TE library generated here. We found 19.9% of repeats (+1.3%) in the genome of Z. tritici, 17.6% (+2.2%) in the genome of Z. pseudotritici, 12.7% (+4.8%) in the genome of Z. ardabiliae, and 25.2% (+1.3%) in the genome of Z. brevis.In total, we found 497 repeat families in the four Zymoseptoria genomes, we found that some families identified independently in each species were derived from a common ancestral TE family. An analysis of the 497 families was performed using Blastclust (Table S2). Fourteen of these groups were composed of TIR elements, eight groups of LTR-Gypsy elements, six groups of uncategorized elements, three groups of LTR-Copia elements, two groups of Helitrons, and one group of LINE elements. Only one of these groups included TE families present in each of the four species; this suggests that this element, an LTR-Gypsy element, was present in the genome of the common ancestor of the four species (Table S2). Interestingly, this element may have expanded prior to the divergence of species because we observed a comparable size expansion in the four species comprising between 120 and 180 kb in each genome. Given the phylogenetic tree of the four Zymoseptoria species, as shown in Table S2). This analysis mainly showed that these species have undergone events of invasion and expansion of distinct TEs after their divergence.Addressing TE evolution in the Zymoseptoria species complex provides a unique model system for the application of “omics” data to study the underlying genetics of pathogenicity and host specificity of plant pathogens . Of these, 7786 represent core Zymoseptoria genes shared among the four species. Our exhaustive search for gene orthology also identified genes shared by two or three species and suggested recent gains or losses of genes among the Zymoseptoria species. Notably, we also identified 1798 orphan genes in Z. tritici enriched in putative pathogenicity-related factors. Species-specific orphan genes in Z. tritici and in the three other species may include key determinants of virulence in wheat and other grass hosts. Further functional studies will allow this hypothesis to be tested.We applied the same annotation pipeline to re-sequenced genomes of the three close relatives of Z. tritici was conducted (et al. were not categorized into known TE classes. We were able to improve the TE classification, leaving only 9% of the 111 repeat families unclassified. The TE annotation pipeline used here contains a number of supplementary steps of clustering of the REPET outputs and allowed us to identify more complete elements. These were all manually checked to avoid the generation of false or chimera elements.Recently, a new identification and classification of repeats and TEs in Zymoseptoria species genomes coupled with preliminary comparative analysis allowed us to highlight interesting features relevant to the biological life traits of these organisms. Establishment of homologous sequence families across the four species showed that a large part of genes are shared between two or more species and that the major difference of gene content relies on orphan genes. These orphan genes, in all four species, are significantly enriched in putative pathogenicity-related genes and potentially play a role in the determination of host specificity. Similar presence–absence patterns of pathogenicity-related genes were shown to be strong determinants of host range in plant pathogens, e.g., between lineages of the rice blast pathogen Magnaporthe oryzae, the wilt pathogen Verticillium dahliae, and lineages of the Leptosphaeria maculans–Leptosphaeria biglobosa species complex (Leptosphaeria maculans–Leptosphaeria biglobosa species complex (The gene and TE annotation of the four Z. tritici and its related species and provide a strong basis to understand the underlying genetics and molecular biology of host–pathogen interactions in Zymoseptoria spp.The annotation data presented here will greatly improve our understanding of gene evolution, adaptation, and the role of TEs in pathogen evolution. The predicted gene models will furthermore greatly support future studies of gene expression in


Primula L. is a typical plant group with which to understand genetic variance between species in different levels of relationships. Chloroplast genome sequences are used to be the information resource for quantifying this difference and reconstructing evolutionary history. In this study, we reported the complete chloroplast genome sequence of Primula sinensis and compared it with other related species. This genome of chloroplast showed a typical circular quadripartite structure with 150,859 bp in sequence length consisting of 37.2% GC base. Two inverted repeated regions  were separated by a large single-copy region  and a small single-copy region . The genome consists of 112 genes, including 78 protein-coding genes, 30 tRNA genes and four rRNA genes. Among them, seven coding genes, seven tRNA genes and four rRNA genes have two copies due to their locations in the IR regions. The accD and infA genes lacking intact open reading frames (ORF) were identified as pseudogenes. SSR and sequence variation analyses were also performed on the plastome of Primula sinensis, comparing with another available plastome of P. poissonii. The four most variable regions, rpl36–rps8, rps16–trnQ, trnH–psbA and ndhC–trnV, were identified. Phylogenetic relationship estimates using three sub-datasets extracted from a matrix of 57 protein-coding gene sequences showed the identical result that was consistent with previous studies. A transcript found from P. sinensis transcriptome showed a high similarity to plastid accD functional region and was identified as a putative plastid transit peptide at the N-terminal region. The result strongly suggested that plastid accD has been functionally transferred to the nucleus in P. sinensis.Species-rich genus  Gaertnera  has been widely used in phylogenetic studies, because of its recombination-free and maternal inheritance . More imaertnera . Recentlaertnera , which oaertnera .infA, accD, rpl22 and rpl32  from P. sinensis plastid and search clue in its transcriptome to get lines of evidences for functional transfer to nucleus.In this study, we released a complete chloroplast genome of an endemic P. sinensis were collected from the South China Botanical Garden, Chinese Academy of Sciences (CAS). Modified CTAB method was used to isolate whole-genome DNA . PCR products mixture was fragmented into ∼300 bp size by the Nextera XT transposome. Library Sequencing acquired 2× 250 bp paired reads using Illumina Miseq Desktop Sequencer at Kunming Institute of Botany, CAS. All reads data were deposited to NCBI SRA database with an accession number P. poissonii (NC_024543). Then, relative position and direction of each hitting contig were determined. Subsequently, hitting contigs were assembled manually to acquire complete chloroplast sequence in Geneious R7 . The resulting plastome sequence was used as the reference, which was subsequently verified by remapping initial reads. Regions of four SC-IR junctions were identified by Sanger sequencing using four pair primers. Primer sequences used in this study can be found  after removing adaptors and trimmed low quality reads. Assembly was conducted twice separately with two different k-mer value 60 and 64. Contigs generated by assembling were subjected to BLAST searches against the complete chloroplast sequence of be found .http://dogma.ccbb.utexas.edu/) . Finally, a circular genome map was illustrated using OGDRAW (http://ogdraw.mpimp-golm.mpg.de/) (Plastome annotation was performed using DOGMA (as.edu/)  and compto pafII . The annmpg.de/) .Primula, sequences of P. sinensis and P. poissonii were compared according to variability (Pi). Alignment was performed with MAFFT version 7 , GMATo v1.2  was usedSRX1665905. The cleaned reads were assembled de novo using Trinity with the default parameters to obtain 48,887 unisequences. Eight different software, namely TargetP  was delivered to Majorbio , where cDNA sequencing was performed with Illumina Hiseq4000 platform. Raw data were filtered and deposited in the Sequence Reads Archive (SRA) database under accession number  TargetP , Protein TargetP , BacelLo TargetP , CELLO2G TargetP , Euk-mPL TargetP , EuLoc  using GTR + Γ nucleotide substitution model  region, 17,725 bp of small single-copy (SSC) region, and two inverted repeat (IR) regions with 25,535 bp of one copy in length. The nucleotide composition of this genome has a GC content of 37.2%. Comparative analysis revealed that the genome structure of P. sinensis shared a high similarity structure to other Primulaceae species  (The chloroplast genome of C465962)   regions. Highly divergence regions are potential molecular genetic markers for population genetics studies. We therefore compared the regional divergence of chloroplast genome sequences of these two Primula species. The Pi value generated by DnaSP version 5 was used to indicate the level of divergence between P. sinensis and P. poissonii  for further phylogenetic use. We therefore recommend rpl32–trnL, trnS–trnG, pafII–cemA, trnC–petN, trnT–trnL, trnK–rps16 as efficient chloroplast markers, considering their balanced PICs and length size, although additional researches of their utilities are necessary in future.The result shows the genetic diversity (Pi) varied from 0–0.47 . Most ofeviously . Remarkaeir PICs . CompariAccD gene encodes β subunit of the Acetyl-CoA Carboxylase, which is unique related to fatty acid synthesis within the chloroplast. Gene knockout experiments in tobacco indicated that accD gene function is indispensable, suggesting it should be an essential gene (accD (pt-accD) gene or pseudogenization of pt-accD are widely present in multiple distant lineages, including Acoraceae, Campanulaceae, Ericaceae, Fabaceae, Geraniaceae and Poaceae  originated from plastid have been found in different taxa, such as Sciadopitys verticillata (Sciadopityaceae) (Trifolium repens (Fabaceae) (Trachelium caeruleum (Campanulaceae)  , Trifoliabaceae) , Trachelulaceae) . These nrd cells , which paccD locus in Primula sinensis shows the truncated gene with incomplete ORF. We found a portion of pt-accD gene near the start position was absent with about 400 bp in length by comparing with other plastomes from Primulaceae. This deletion has been verified by PCR amplification using a pair of primers located on both flanks. Reads mapping also showed high coverage levels on flanking regions of the deletion. Due to the presence of the deletion, pt-accD coding sequence was terminated prematurely with the introduction of the stop codon. The remaining ORF with residual sequences does not include the conserved functional region. Actually, pt-accD has become a pseudogene, with a deletion involved  for the transferability of accD gene with lines of solid evidences.Owing to its important function in plastid development, activity . We theric sites . In contic sites . In ordeic sites . The chlaccD transcript in P. sinensis with pt-accD from other 30 species, which belongs to six families in Ericales, was performed by RAxML. This dataset contained multiple alignment of 31 sequences of the C-terminal functional regions, since the regions are relatively conserved as discussed above. The maximum likelihood tree showed n-accD from P. sinensis was located within the clade of the Primulaceae with P. poissonii as the sister group , Vaccinium macrocarpon (JQ757046), and Chamaedaphne calyculata (KJ463365) in Ericaceae. It is likely that the missing or pseudogenization of pt-accD genes occurred accidently and independently in Ericales.Phylogenetic analysis of the n-er group . ConsideAgrostemma githago as outgroup. Phylogenetic relationships were inferred using 57 plastid protein-coding genes. As we know, the third base position of codon evolves faster than the rest of two positons with higher substitution rate. ML trees were produced by using three different datasets, all coding sequence, codon 1 + 2 and codon3, respectively. The result showed that topologies of three datasets were highly congruent with one another and all nodes were well supported (The family interrelationships within the large order Ericales, constrained by the data available, have remained unclear . Recentlupported . Primuloupported .10.7717/peerj.2101/supp-1Supplemental Information 1Click here for additional data file.


Aedes aegypti. Here, the effects of developmental siRNA-mediated dsx silencing were assessed in adult females. Targeting of dsx during A. aegypti development resulted in decreased female wing size, a correlate for body size, which is typically larger in females. siRNA-mediated targeting of dsx also resulted in decreased length of the adult female proboscis. Although dsx silencing did not impact female membrane blood feeding or mating behavior in the laboratory, decreased fecundity and fertility correlated with decreased ovary length, ovariole length, and ovariole number in dsx knockdown females. Dsx silencing also resulted in disruption of olfactory system development, as evidenced by reduced length of the female antenna and maxillary palp and the sensilla present on these structures, as well as disrupted odorant receptor expression. Female lifespan, a critical component of the ability of A. aegypti to transmit pathogens, was also significantly reduced in adult females following developmental targeting of dsx. The results of this investigation demonstrate that silencing of dsx during A. aegypti development disrupts multiple sex-specific morphological, physiological, and behavioral traits of adult females, a number of which are directly or indirectly linked to mosquito reproduction and pathogen transmission. Moreover, the olfactory phenotypes observed connect Dsx to development of the olfactory system, suggesting that A. aegypti will be an excellent system in which to further assess the developmental genetics of sex-specific chemosensation.The development of sex-specific traits, including the female-specific ability to bite humans and vector disease, is critical for vector mosquito reproduction and pathogen transmission. Doublesex (Dsx), a terminal transcription factor in the sex determination pathway, is known to regulate sex-specific gene expression during development of the dengue fever vector mosquito  doublesex (dsx) during development of the human disease vector mosquito Aedes aegypti. Targeting of dsx resulted in decreased length of the female wing and proboscis, ovary and reproductive defects, and disruption of olfactory system development. Female lifespan, a critical aspect of mosquito pathogen transmission, was also significantly reduced in adult females following developmental targeting of dsx. The results of this investigation demonstrate that silencing of dsx during A. aegypti development disrupts multiple sex-specific morphological, physiological, and behavioral traits of adult females, a number of which are directly or indirectly linked to mosquito reproduction and pathogen transmission. The results obtained also connect Dsx to development of the mosquito olfactory system, suggesting that A. aegypti will be an excellent system in which to further assess the developmental genetics of sex-specific chemosensation.Only adult female mosquitoes, which require blood meals for reproduction, bite humans and spread diseases. The genes that regulate development of sex-specific traits may therefore represent novel targets for mosquito control. Here, we examine the effects of silencing the sex-determination gene  Most animal species display sexually dimorphic behaviors, the majority of which are linked to sexual reproduction . DiseaseDrosophila melanogaster identified a mutation in the doublesex (dsx) gene that transformed males and females into intersexes [dsx gene encodes a key terminal transcription factor in the sex-determination pathway that controls Drosophila male and female sexual differentiation [Drosophila dsx pre-mRNAs are spliced in a sex-specific manner [M) and female (DsxF) proteins with a common N-terminus and DNA-binding domain, but distinct male and female C-termini that differentially regulate sex-specific gene expression . Stiewed by , all relntiation –25. The ntiation , 21, 26.s taurus  and the chotomus . Dsx funhatching . More re viruses .(dsxM) and female (dsxF) splice variants of dsx in A. aegypti. Recently, Hall et al. [Nix, a male-determining factor (M factor) in A. aegypti that is required and sufficient to initiate male development, and which encodes a potential splicing factor. The absence of Nix shifts the alternative splicing of dsx toward the female-specific dsxF splice form, suggesting that Nix normally promotes splicing of dsxM. Although the sex-specific dsx splice forms likely direct sexually dimorphic mosquito development, functional analysis of dsx in A. aegypti is lacking. In a recent study from our laboratory [dsx expression in the pupal brain suggesting that sexually dimorphic neural development in A. aegypti may require dsx function. In support of this, a search of the A. aegypti genome sequence uncovered 732 Dsx consensus binding sites, 48 of which flank dimorphically expressed genes identified in male vs. female pupal head transcriptome microarray experiments [A. aegypti genes flanked by Dsx consensus binding sites group under a number of significant gene ontology terms, many of which are linked to neurological processes or neural development, suggesting that Dsx may regulate sex-specific gene expression in the developing mosquito brain. To examine this, we [dsx during A. aegypti pupal development. In our study, siRNAs corresponding to different target sequences in exon 2, which is common to male and female splice variants, were injected into A. aegypti pupae. These targeting experiments demonstrated that Dsx is required for the regulation of sex-specific gene expression during A. aegypti neural development. The results of our initial investigation [A. aegypti. Here we test this hypothesis by examining the impact of larval and pupal dsx silencing on the development of sex-specific traits in adult female mosquitoes.Salvemini et al.  detectedl et al.  describeboratory , we deteeriments . A. aegythis, we  used smatigation , in conjtigation ), supporA. aegypti dsx (dsxF) during development. Their dsx targeting protocol differed from that which we used [dsx exons  that was delivered by soaking mosquito larvae in dsRNA or by feeding the larvae E. coli expressing dsx-targeting dsRNA (rather than through microinjection). Although the Whyard et al. [dsx targeting strategies resulted in highly male-biased populations of mosquitoes, the number of dsx dsRNA-treated larvae that developed into adults was halved relative to the negative controls, and with no significant increase in the number of males observed, suggesting that the majority of females simply failed to survive to adulthood, which did not permit analysis of sex-specific characters in these animals. Our ability to analyze late pupae microinjected with siRNAs targeting a separate region of the dsx gene (exon two) suggested that this targeting strategy [A. aegypti larvae, silenced dsx while permitting female survival. Use of these targeting strategies allowed us to examine adult female morphological, physiological, and behavioral phenotypes that result from developmental silencing of dsx.Whyard et al.  recently we used  in that d et al.  dsx targstrategy , which dstrategy  procedurstrategy , as wellstrategy –34 to deA. aegypti Liverpool-IB12 (LVP-IB12) strain , from which the genome sequence [dsx-silenced adults were further confirmed in a subset of animals through dissection to assess the presence of testes or ovaries.The sequence  was genesequence , except sequence . Adults sequence . Sexes oIn situ hybridization was performed as previously described [OR 2 (AAEL005999), OR 9 (AAEL006005), OR 62 (AAEL011796), and OR 123 (AAEL017537) and dsx . At least 20 tissue specimens were processed for each in situ hybridization experiment, and at least two replicate experiments were performed. A sense riboprobe was used as a control in all hybridization experiments. Immunohistochemical staining was performed as described previously [OR transcript analyses, mean gray values  were calculated for digoxigenin-labeled OR transcript signal in 25 control or experimental antennae combined from two replicate experiments. Data were statistically analyzed using one-way ANOVA followed by the Bonferroni post hoc test.escribed . Riboproescribed  protocoleviously , 40 usindsx (AAEL009114) was performed as described previously [dsx-KD A and dsx-KD B, that correspond to different target sequences in exon 2, which is common to both the male and female dsx splice variants, were used in dsx silencing experiments. The sequences of these siRNA duplexes, which were purchased from Integrated DNA Technology  and confirmed through BLAST searches to have no significant homology to A. aegypti genes other than dsx, are as follows:Targeting of eviously . Two siRDsx-KD A: 5’ rCrArGrGrArArCrArGrArCrGrArCrGrArArCrUrUrGrUrCAA3’ / 5’rUrUrGrArCrArArGrUrUrCrGrUrCrGrUrCrUrGrUrUrCrCrUrGrArG3’, and Dsx-KD B: 5’rCrArArGrArUrCrGrCrUrGrGrArUrGrGrUrArArArGrArUGT3’ / 5’rArCrArUrCrUrUrUrArCrCrArUrCrCrArGrCrGrArUrCrUrUrGrCrG3’. All phenotypes were confirmed following knockdown (KD) with both dsx-KD A and dsx-KD B, suggesting that none of the phenotypes reported herein were the result of off-site targeting by either siRNA. A scrambled version of dsx KD B, an siRNA duplex lacking significant sequence homology to any genes in the A. aegypti genome, was used for control experiments:5’rGrArArGrArGrCrArCrUrGrArUrArGrArUrGrUrUrArGrCGT3’ / 5’rArCrGrCrUrArArCrArUrCrUrArUrCrArGrUrGrCrUrCrUrUrCrCrG3’. None of the phenotypes reported were observed in control-injected animals, which were not significantly different than wild type animals for any of the phenotypes assessed.dsx was performed using the procedure described by Mysore et al. [dsx was confirmed through in situ hybridization as discussed in the recent siRNA-mediated dsx gene targeting study [dsx transcript signal in brains and antennae from minimally 20 control or experimental specimens combined from two separate replicate experiments. These data were statistically analyzed using one-way ANOVA followed by the Bonferroni post hoc test.siRNA was microinjected into pupae  as descre et al. , which we et al.  and is de et al. . Silencing study . To quanng study  for digodsx as described above. For these experiments, structures were dissected from sugar-fed ~10 day old adult female mosquitoes, mounted and analyzed with a Zeiss Axioimager equipped with a Spot Flex camera. Areas and lengths were measured using Fiji Image J software. Wing lengths were measured from the apical notch to the axillary margin, excluding the wing fringe as described in [dsx-KD olfactory structures were visualized under SEM and assessed for numerical and structural anomalies. Data from replicate experiments were combined and statistically analyzed with one-way ANOVA followed by the Bonferroni post hoc test.Wing length and area, proboscis, antenna, and maxillary palp lengths were assessed in females following chitosan/siRNA nanoparticle mediated targeting of ribed in . To miniribed in . BrieflyBlood feeding behavior was visually assessed through analysis of engorged female abdomens following plasma membrane blood feedings which were one hour in duration. The number of eggs produced per female (fecundity) and eggs produced per female that generated first instar larvae (fertility) were assessed as described by Hill et al. . The numA. aegypti OR genes were exported from VectorBase [Introns as well as 5’ flanking sequences 0–5 kb upstream of the open reading frames of ctorBase . These sctorBase  Dsx consdsx (AAEL009114), OR 2 (AAEL005999), OR 9 (AAEL006005), OR 62 (AAEL011796), and OR 123 (AAEL017537).The following genes were studied in this investigation: dsx function in the developing A. aegypti brain [dsx during A. aegypti larval and/or pupal development. For larval silencing experiments, dsx-KD A, dsx-KD B, or control chitosan/siRNA nanoparticles were fed to larvae [dsx in chitosan/siRNA nanoparticle-fed or siRNA-injected animals was confirmed through in situ hybridization experiments. Quantification of dsx signal through mean gray value analyses in control vs. dsx-silenced animal tissues indicated that significant knockdown levels were achieved , and the mosquitoes could still be identified as females on the basis of the external morphology. However, as detailed herein, fertility and fecundity defects that correlated with ovary defects were observed in these animals. Olfactory phenotypes were also detected, and female lifespan decreased.Chitosan/siRNA larval le pupae . As withA. aegypti body size, which is larger in females, can be assessed through analysis of wing size, a proxy for adult body size [dsx-KD A or dsx-KD B siRNA nanoparticles as larvae. On average, the areas of dsx-KD A and dsx-KD B wings are 19% and 21% smaller than control-fed animal wing areas, respectively . The antennae of animals fed with dsx-KD A nanoparticles are 23% shorter than those of control-fed animals, while those fed dsx-KD B nanoparticles are reduced by 14%  is significantly reduced (P<0.0001) in adult females that were injected as pupae with dsx-KD A or dsx-KD B siRNA as compared to control-injected females  were observed in the percentages of females that took blood meals .To explore the cause of this reduced fertility and fecundity, mating, blood feeding behavior, and ovary histology were assessed in od meals  or matedod meals  followinood meal . These eas pupae . The len ovaries . Five daod meals  and ovarod meals  are alsodsx (Trichoid sensilla (sTrichodea) were quantified through analysis of SEM images of the adult female antenna  were assessed through analysis of SEM images of the adult female maxillary palp  genes was assessed in A. aegypti adults. Bohbot et al. [OR 62 and OR 123 that was detected through qRT-PCR studies. WhoIe-mount in situ hybridization detected expression of OR 62  or dsx-KD B (P<0.001) siRNA  siRNA . These dA. aegypti dsx, a terminal transcription factor in the insect sex determination pathway, during development of female mosquitoes. The adult phenotypes examined fell into four general categories that are discussed in more detail below: growth , a positive regulator of cellular growth in D. melanogaster [A. aegypti female pupal brain. Dsx consensus binding sites flank the A. aegypti cdk 4/6 gene, and sexually dimorphic expression of this gene in the pupal brain is disrupted by dsx knockdown [D. melanogaster [Drosophila [dsx in both sexes of A. aegypti. Additionally, Gotoh et al. [dsx and the juvenile hormone (JH) signaling pathway, demonstrating that female-specific splice variants of C. metallifer dsx contribute to the insensitivity of female mandibles to JH. It would therefore be interesting to determine if sex-specific Dsx splice forms regulate JH-responsiveness in A. aegypti.Our recent study demonstrated that genes linked to the cell cycle are upregulated in  females . These gnogaster , 52, is nockdown . Interesnogaster , was recosophila . It is th et al.  identifiD. melanogaster [Bombyx mori [dsx during pupal development in T. casteneum was found to result in decreased fertility and fecundity [dsx was silenced during female A. aegypti pupal development  and yp2 genes are targets of Dsx [T. casteneum YPP vitellogenin (vg) and vitellogenin receptor (vgr) genes were identified as targets of Tc Dsx [YPP genes is conserved in insects. However, the A. aegypti YPP genes lack any obvious Dsx binding sites, and if Dsx regulates expression of these genes in A. aegypti, it is likely to do so indirectly. Interestingly, juvenile hormone (JH) is known to regulate expression of A. aegypti Vg-A [A. aegypti, Gotoh et al. [C. metallifer dsx contribute to the insensitivity of female mandibles to JH. It would therefore be interesting to determine if links between Dsx and JH responsiveness exist in A. aegypti.Vitellogenesis, the synthesis and secretion of yolk protein precursors (YPPs), is a critical event in the mosquito reproductive cycle that is activated in response to the blood meal , 56. In s of Dsx , 58. Simf Tc Dsx . Thus, opti Vg-A . Although et al.  recentlyA. aegypti female reproduction in many additional ways. Although we did not detect any significant impacts of Dsx silencing on membrane blood feeding behavior or the ability to mate in the laboratory setting  and its receptor (Dh44R1), is suppressed, the brain expedites sperm ejection from the uterus, resulting in decreased fecundity. Thus, Dsx signaling could have multiple impacts on female reproduction in A. aegypti. Gaining a fuller understanding of these impacts will likely require much more detailed knowledge of A. aegypti reproductive behaviors.Dsx likely regulates  setting , it is nOR genes. This linkage, which to our knowledge has yet to be identified in other organisms, provides insight into the regulation of sex-specific olfactory development. Silencing of dsx during pupal development was found to disrupt expression of two female ORs, 62 and 123 , which also encodes a terminal transcription factor in the sex determination pathway that is spliced in a sex-specific manner [Drosophila brain, as well as a saturating clonal analysis of Fru-positive neurons. They noted that the proportion of cells in the D. melanogaster brain that expresses Dsx is smaller and partially overlaps with Fru, an interesting observation given that Neville et al. [Drosophila Dsx and Fru may act together, either in a physical complex or through co-regulation of target genes, to control sex-specific neural development. It will therefore be interesting to functionally assess the roles of Fru during neural development in A. aegypti.To date, analysis of the development of sexual dimorphism in the olfactory system has largely centered on analysis of the roles of c manner . Cacheroc manner  performee et al.  suggesteDrosophila researchers have begun to link Dsx and Fru to specific sexually dimorphic neural physiologies, neural circuitries, and behaviors, and so another challenge will be to further develop genetic technologies with the goal of being able to perform comparably technical analyses in mosquitoes. For example, in D. melanogaster, the activation of a set of Fru-positive olfactory receptor neurons (ORNS) that express OR67d, which responds to male pheromone cis-vaccenyl acetate, was found to inhibit male courtship of other males and induce female receptivity to other males. The ORNs expressing 67d converge to a single glomerulus, DA1, in both the sexes, but the projections from the DA1 glomerulus to the protocerebrum were found to be sexually dimorphic, suggesting that differential behaviors induced by this pheromone result from sex-specific neural circuitries [fru isoforms function as a bidirectional switch to activate different behaviors in males and females. In another study, reduction of Ecdysone receptor-A in FruM-positive neurons, which is associated with an increase in male-male courtship activity, was found to result in significant reduction in the size of two antennal lobe glomeruli, suggesting that EcR-A is required for establishment of male-specific neuronal architecture in the D. melanogaster olfactory system [OR expression, changes in the overall neural circuitry responding to the odorant may induce dimorphic behaviors in males and females, and it will be interesting to examine this in A. aegypti in the future.cuitries . Kohl etcuitries  further As discussed by Brady et al. , the surdsx during female pupal development significantly reduces adult A. aegypti female lifespan , overexpression of dsxF in adults dramatically reduced the life span of both males and females. Overexpression of the male isoform of fru in males or females yielded similar results. Interestingly an RNAi line targeting fru reduced lifespan in D. melanogaster females only. Shen et al. [D. melanogaster. Thus, as discussed above, any impact that Dsx might have on Yp expression could underlie the decreased longevity of A. aegypti dsx-targeted females. Finally, silencing dsx in A. aegypti was shown to result in decreased expression of p53 [p53 in the D. melanogaster female nervous system results in increased life span [p53 expression following dsx silencing may contribute to the decreased lifespan observed in dsx-targeted A. aegypti females.The results of this study demonstrate that targeting lifespan . The genlifespan . While on et al.  suggesten et al.  demonstrn of p53 . Overexpife span . It is tA. aegypti pupal nervous system. The present investigation extended these initial findings through assessment of the effects of developmental siRNA-mediated dsx silencing in adult females. Targeting of dsx resulted in decreased size of the female wing and proboscis  or chitosan/siRNA nanoparticle feedings  of control, dsx-KD A, or dsx-KD B siRNA. 24 hr APF brains  and antennae (B1-3), as well as fourth larval instar brains (D1-3) and antennae (E1-3) are shown. Corresponding mean gray values from 19 or more tissue samples  compiled from two replicate experiments are shown at right . In comparison to control tissues, significantly lower values were detected for dsx-KD A and dsx-KD B tissues (P<0.0001***). Error bars denote standard deviations. Percentage decreases in mean gray values are indicated for dsx-KD A or dsx-KD B treatments in each chart.Silencing of (PDF)Click here for additional data file.S1 TableThe method of siRNA delivery  is noted for each phenotype assessed in the investigation.(PDF)Click here for additional data file.S2 TableOR genes. The position of each sequence relative to the open reading frame of each OR gene is indicated.5’ flanking Dsx consensus binding sequences were identified upstream of the indicated (PDF)Click here for additional data file.S3 TableOR transcript signals detected in 24 adult female antennae (compiled from two replicate experiments) following pupal microinjection of control, dsx-KD A, or dsx-KD B siRNA. Significantly lower values were detected for dsx-KD A or dsx-KD B vs. control animals (P<0.0001***). SD = standard deviation.Mean gray values for the indicated (PDF)Click here for additional data file.


Plutella xylostella (L.), is a destructive pest that attacks cruciferous crops worldwide. Immune responses are important for interactions between insects and pathogens and information on these underpins the development of strategies for biocontrol-based pest management. Little, however, is known about immune genes and their regulation patterns in P. xylostella. A total of 149 immune-related genes in 20 gene families were identified through comparison of P. xylostella genome with the genomes of other insects. Complete and conserved Toll, IMD and JAK-STAT signaling pathways were found in P. xylostella. Genes involved in pathogen recognition were expanded and more diversified than genes associated with intracellular signal transduction. Gene expression profiles showed that the IMD pathway may regulate expression of antimicrobial peptide (AMP) genes in the midgut, and be related to an observed down-regulation of AMPs in experimental lines of insecticide-resistant P. xylostella. A bacterial feeding study demonstrated that P. xylostella could activate different AMPs in response to bacterial infection. This study has established a framework of comprehensive expression profiles that highlight cues for immune regulation in a major pest. Our work provides a foundation for further studies on the functions of P. xylostella immune genes and mechanisms of innate immunity.The diamondback moth,  Drosophila melanogaster. In this model organism, genes encoding AMPs are activated by NF-κB transcription factors in response to infection through the Toll and immune deficiency (IMD) signaling pathways. Other immune pathways in insects are the JNK and JAK-STAT pathways, which participate in cell stress or wound response45The innate immune system, which consists of cellular and humoral responses, is the first line of defense against bacterial and fungal infections in insects2D. melanogaster (Order: Diptera) and Bombyx mori and Manduca sexta, two representatives of Order: Lepidoptera. They showed, for example, that while some genetic components of the Toll signaling pathway may be similar between Lepidoptera and Diptera, some other genes differ. Gaps remain in some of these pathways and underline the need for further studies to better understand the differences between the Toll and IMD signalling pathways in these two insect orders. With greater availability of genetic tools and published genome sequences in Lepidoptera, it is possible to acquire a better understanding of the genetics of the immune system in other insect species such as P. xylostella, a notorious pest attacking many economically important food and oil crops in Brassicaceae.The innate immune system is known to be highly conserved in the animal kingdom and evolutionarily very stable. It is, however, suggested that as insects are exposed to a multiplicity of continuously evolving pathogens, genes involved in signaling pathways may be under selection, leading to diversification8P. xylostella immunity have mainly focused on individual immune genes and aspects such as production and characterization of cecropinCotesia vestalis) on midgut proteinase activity, inhibition of phenoloxidase activity by metabolites of entomopathogenic nematodes (Xenorhabdus nematophila), and immune suppression by the parasitoid Cotesia plutellae polydnaviral gene1112P. xylostella immunity at the molecular level are poorly understood.Recent studies of P. xylostella, has emerged as an important field to facilitate the development of effective biological control tactics. Comparative analyses of immune genes at the genome wide level in insects is the first step to define the candidate genes and functional networks associated with immune regulation. Thus, the aim of this study was to identify the immune signaling pathways, along with the pathogen recognition proteins and immune effectors, from the recently sequenced P. xylostella genomeP. xylostella genome (data not shown), the detailed information on these components will be addressed in other paper. The expression patterns of immune genes that identified in this study were examined in P. xylostella at various development life stages, in different tissues and in susceptible and insecticide-resistant lines. Finally, P. xylostella immune response to bacterial pathogens was studied.Understanding the genetics and regulatory mechanisms of immune systems of insect pests, such as P. xylostella genome, and classified into 20 families and 4 functional groups: immune recognition, signaling pathways, immune effectors, and others. The immune recognition genes included PGRPs, βGRPs, galectins, C-type lectins, fibrinogen-related proteins (FREPs), and scavenger receptors (SCRs). The signaling pathways genes were further classified into the pathway-related categories of TOLL, IMD, JNK and JAK-STAT. The immune effectors comprised prophenoloxidase (PPO), thioester-containing proteins (TEPs), and AMPs. Others included the enzyme superoxide dismutase (SOD), catalases and peroxidases that participate in detoxification of reactive oxygen species (ROS)  . The totP. xylostella, fewer than the 13 for D. melanogaster, 11 in A. gambiae, and 12 in B. mori  contains a trans-membrane region that may enable signal transmission into the cytoplasm and function in the IMD pathway. The domain architecture analysis  specifically for β-galactoside sugara genome , and phy B. mori . The dom B. mori  suggestsP. xylostella genome  B. mori . This mi insects . The dom insects  suggestsP. xylostella genome  to regulate AMP gene expressionP. xylostella TRAF6 needs to be further validated because D. melanogaster TRAF6 is not implicated in immune signaling16P. xylostella might be due either to the homologue search method based on sequence similarity or incomplete genome information. It is also possible that the function of MyD88 is substituted by other adaptors. Further experiments would be needed to identify the cause.Spätzle (SPZ) is a ligand of the Toll receptor, and forms a complex with Toll receptor resulting in activation of the Toll signaling pathway3536entified . Common  B. mori , suggest species . The dom species , indicatP. xylostella genome. Interestingly, TAK1 is not only a downstream component of IMD, but also a protein kinase that triggers the JNK pathway to activate gene expression in response to cell stress or wound56P. xylostella genome. The common 1:1 orthologs of genes in the IMD and JNK pathways of P. xylostella and other insects indicated that these pathways were complete and conserved , one Janus kinase (Hopscotch), and one transcription activator (STAT) have been identified in the JAK-STAT pathway36P. xylostella genome. In addition, only one TEP, which is under the control of the JAK-STAT pathwayP. xylostella genome compared to other insect species  into a less toxic product, hydrogen peroxide (H2O2). H2O2 is converted to water and oxygen by catalases. Peroxidase also scavenges H2O2 and converts it to hydroperoxide4849D. melanogaster, with one family (Cu-Zn SOD) located in the cytosol and the other (Mn-Fe SOD) in the mitochondria. Seven SOD genes were identified in P. xylostella genome compared with 4 in D. melanogaster, 5 in A. gambiae and 6 in B. mori. Five of the 7 SODs in P. xylostella were Cu-Zn SOD, and the other two were Mn-Fe SODs, while three Cu-Zn SOD and one Mn-Fe SOD are present in D. melanogasterP. xylostella, 20 in D. melanogaster, 26 in A. gambiae and 23 in B. mori. Thirteen catalase genes were identified in P. xylostella, significantly more than the 2 in D. melanogaster, 1 in A. gambiae, and 7 in B. mori. Previous studies have shown that plants attacked by insects may increase ROS production, resulting in oxidative damage to the insect midgutP. xylostella genome is likely to reflect their roles in ROS detoxification and the wider co-evolution of this herbivore with cruciferous plants.Reactive oxygen species contribute to defense against invading microbial pathogens, but over-production can also harm host cells. Thus, ROS production must be tightly controlled. The concentration and conversion of ROS can be regulated by superoxide dismutases (SODs), peroxidases and catalases. SODs convert superoxide radical  were strongly expressed in the 4th-instar larval midgut, while PGRP-SA (Px015207) was expressed at a higher level in the 4th-instar larval head. Most βGRP genes were highly expressed in the heads of both larvae and adults. In the midgut, only three βGRP genes  were strongly expressed  compared to the susceptible strain (SS)  , which wain (SS) . Genes iP. xylostella AMPs could be induced by bacterial infection , but not active against Gram-positive bacteria (Arthrobacter globiformis and Bacillus thuringiensis), another studyM. sexta is active against Gram-positive bacteria (Bacillus cereus) but almost inactive against Gram-negative bacteria (E. coli). These studies suggest that gloverin in different insects may have species-specific functions in defense against pathogens.Results from the bacteria-feeding experiment suggest that nfection . After fP. xylostella genome, including components of the conserved immune signaling pathways , pathogen recognition and immune effectors. Based on the identified genes  and beta-1,3-glucan from bacteria and fungi, and can trigger the Toll pathwayM. sexta larval plasma, β-1,3-glucanase-related protein may stimulate prophenoloxidase activation72P. xylostella genome suggests that βGRPs may be involved in effective defense against Gram-negative bacteria and fungi. These results also suggest that while recognition proteins in the P. xylostella immune system may be diverse, the intracellular signal transduction genes may be more conserved. Previous studies in D. melanogasterA. gambiaeB. moriP. xylostella. In contrast with βGRPs, only seven AMP genes in three families were identified in P. xylostella genome , but the number of C. jejuni declined during pupal development, coinciding with the increased expression of AMPs, indicating effective innate immunity at the pupal stageP. xylostella may be key to defense against pathogens and cell stress in this important developmental stage. On the other hand, since the head is the center for nervous system and feeding in insects with many important sense organs to perceive various environmental signalsGene expression profiles showed that most immune genes were highly expressed at the pupal stage and in the head tissue. Previous work showed that the bacterial pathogen P. xylostella immune genes studied here were identified based on bioinformatics analysis. Their in vivo functions still need to be validated by molecular studies, such as gain-of-function or loss-of-function analysis. Nevertheless, through this study, we have generated information about the genetic composition and regulation of the immune system of an important agricultural pest. Our work will help drive future studies focusing on identifying molecular functions and mechanisms of immune genes and pathways of P. xylostella and other arthropods.All the D. melanogaster and Anopheles gambiae were downloaded from the immunology database at http://cegg.unige.ch/Insecta/immunodb. The Bombyx mori and Tribolium castaneum immune genes were edited in previous studies35P. xylostella genome database −6 was performed to collect putative immune genes from P. xylostella genome, and the default E-value of 1.0 was used to search for antimicrobial peptides (AMPs) that are 12-50 amino acids long and readily filtered out by high stringency conditions. As some AMPs, such as gloverin and moricin, could only be found in Lepidoptera, the relevant sequences from D. plexippus, M. sexta and B. mori were downloaded from the NCBI GenBank as queries to search for AMPs in P. xylostella database. The selected immune-related P. xylostella genes were then manually edited by comparing with P. xylostella transcriptome database ( http://iae.fafu.edu.cn/DBM/) and the Fgenesh program ( http://linux1.softberry.com/berry.phtml). Finally, the edited sequences were manually confirmed against the NCBI curated and conserved domains (CDD) database by blastX. Domain architecture was analyzed by the SMART ( http://smart.embl-heidelberg.de/), NCBI CDD database ( http://www.ncbi.nlm.nih.gov/Structure/cdd/docs/cdd_search.html) and PROSITE ( http://au.expasy.org/prosite/). Transmembrane domains were analyzed by TMHMM server v. 2.0 ( http://www.cbs.dtu.dk/services/TMHMM/). Signal peptide was analyzed by SignalP3.0 .The sequences of immune genes in P. xylostella, D. plexippus, B. mori, D. melanogaster, A. gambiae, and T. castaneum. Multiple sequence alignments were performed with ClustalW and neighbor-joining method was used to construct phylogenetic trees by MEGA5.0 with 1000 bootstrap replicates.The phylogenetic relationships of immune genes were compared among six insect species: P. xylostella transcriptome database st, 2nd, 3rd and 4th instar larvae; P: Pupae; Ma: Male adults; Fa: Female adults) and tissue-  specific patterns of expression were then visualized using Java TreeView.RNA sequencing of different strains, developmental stages and tissues were performed previously in our laboratory15P. xylostella  was collected in July 2004 from a vegetable field in Fuzhou , Fujian province, southeastern China. All necessary permits were obtained from the Institute of Plant Protection at the Fujian Academy of Agricultural Sciences. Two insecticide resistant lines, chlorpyrifos- and fipronil-resistant lines (CRL and FRL), were selected previously50) for CRL and FRL were 574-fold  and 72-fold (16.85 mg L−1 vs. 0.23 mg L−1) higher than the SS line, respectively. The three lines were reared on radish seedlings at 25 ± 2 °C, 70-80% RH and a 16 h light/8 h dark photoperiod without exposure to insecticides. Adults were fed with 10% honey solution and held in 500 mL plastic bottles for mating and oviposition. The newly hatched larvae were able to pass through holes on the bottom of bottles and drop onto radish plants underneath. Individuals at various developmental stages were collected for quantitative real-time PCR (qRT-PCR) analysis. The insects used for expression analysis of pathogen-induced immune gene were reared on radish seedlings to the third instar. The Gram-negative bacteria Enterobacter sp. (GenBank Accession Number: JQ396388) and Serratia sp. (JQ396393), and the Gram-positive bacterium Enterococcus sp. (KC150018) which were isolated from P. xylostella midgut by our lab were cultured in Luria Bertani (LB) media . The 3rd-instar larvae were placed in Petri dishes and starved for 12 hours. Cabbage leaves, dipped in a suspension of Enterobacter sp., Serratia sp. or Enterococcus sp. at OD600  =  1.0, were then added to the Petri dishes as diet. Cabbage leaves dipped in sterilized double distilled water were used as controls. P. xylostella larvae were allowed to feed on the cabbage leaves for 12 hours, and then collected for further analysis.A susceptible strain of P. xylostella eggs (~200), the 3rd-instar larvae, 4th-instar larvae, pupae, and adults  were collected from the SS strain, samples were surface-sterilized with 75% ethanol for 60 sec, and then rinsed with DEPC water. The 3rd-instar larvae from CRL and FRL lines, as well as SS fed with the bacteria-dipped cabbage leaves were also collected (10 larvae per group). Total RNA was extracted from the whole bodies of each group using TRIzol  Co., Ltd. ). cDNA was synthesized from 2 μg total RNA according to the instructions of GoScriptTM Reverse Transcription System .To investigate the changes in gene expression profiles during various life stages, newly laid P. xylostella transcriptome, and evaluate immune response to bacterial infection. Primers for the tested genes are listed in TM thermal cycler with cycling parameters as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles of 10 sec at 95 °C and 30 sec at 58 °C. To determine whether gene expressions in different lines, various stages or in response to bacterial infection were significantly different, data were analyzed by one-way ANOVA followed by LSD post hoc test using IBM SPSS Statistics 19.qRT-PCR was performed to validate the expression profiles based on X.X. and M.Y. designed the research; X.X., M.X., L.Y., H.L. and J.L. performed the research; X.X., M.Y., X.Y., S.B., G.M.G. and L.V. wrote and revised the manuscript.How to cite this article: Xia, X. et al. Genome-wide characterization and expression profiling of immune genes in the diamondback moth, Plutella xylostella (L.). Sci. Rep.5, 09877; doi: 10.1038/srep09877 (2015).


Burkholderia sp. strain UYPR1.413 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Parapiptadenia rigida collected at the Angico plantation, Mandiyu, Uruguay, in December 2006. A survey of symbionts of P. rigida in Uruguay demonstrated that this species is nodulated predominantly by Burkholderia microsymbionts. Moreover, Burkholderia sp. strain UYPR1.413 is a highly efficient nitrogen fixing symbiont with this host. Currently, the only other sequenced isolate to fix with this host is Cupriavidus sp. UYPR2.512. Therefore, Burkholderia sp. strain UYPR1.413 was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the GEBA-RNB project. Here we describe the features of Burkholderia sp. strain UYPR1.413, together with sequence and annotation. The 10,373,764 bp high-quality permanent draft genome is arranged in 336 scaffolds of 342 contigs, contains 9759 protein-coding genes and 77 RNA-only encoding genes. Fabaceae family, and carry out the Biological Nitrogen Fixation (BNF) process. BNF is catalyzed by the rhizobial nitrogenase complex, whereby N2is reduced to ammonium.Rhizobia are soil bacteria that have acquired the ability to establish symbiotic associations with plants, mainly from the Azorhizobium , Bradyrhizobium , Ensifer , Mesorhizobium  and Rhizobium ). In 2001 symbiotic nitrogen fixing bacteria belonging to the group of Betaproteobacteria  were reported as root nodule bacteria, introducing the term of Alpha and Beta-rhizobia to differentiate both groups of rhizobia [Beta-rhizobia identified so far belong to only two genera: Burkholderia  and Cupriavidus  and the association seem to be mainly with plants from the Mimosoideae subfamily [Mimosa genus is preferentially nodulated by Beta-rhizobia [Beta-rhizobia species have been described belonging to the Burkholderia  genus  but only two in the Cupriavidus  genus (C. taiwanensis  and C. necator ) [Well-known and studied rhizobia are those belonging to the α-proteobacteria (eg. ubfamily . Additiorhizobia . Differeecator ) –17]..AzorhizoBurkholderia  sp. UYPR1.413 strain has been isolated from a root nodule of Parapiptadenia rigida  (Benth.) Brenan found in an angico plantation in Artigas, Uruguay [P. rigida belongs to the Mimosoideae subfamily and is a woody species, which can reach 30 m in height and a diameter of 60 to 80 cm [Parapiptadenia in the Americas of which only P. rigida is present in Uruguay. A survey of symbionts of P. rigida in Uruguay demonstrated that this species is nodulated by rhizobia belonging to the genera Burkholderia , Cupriavidus  and Rhizobium , of which the Burkholderia  microsymbionts predominated [Burkholderia  sp. UYPR1.413 strain belongs to a group of microsymbionts that were able to nodulate and fix nitrogen with P. rigida[Burkholderia  sp. UYPR1.413 high-quality permanent draft genome sequence and its annotation. Uruguay . P. rigito 80 cm . The wooto 80 cm . There aominated . BurkholP. rigida. In thisBurkholderia  sp. strain UYPR1.413 is a motile, Gram-negative, non-spore-forming rod  project at the U.S. Department of Energy, Joint Genome Institute (JGI) for projects of relevance to agency missions . The genBurkholderia  sp. strain UYPR1.413 was grown to mid logarithmic phase in TY rich media [ch media  on a gyrch media .Burkholderia  sp. UYPR1.413 was generated at the DOE Joint genome Institute (JGI) using state of the art technology [The draft genome of chnology . An Illuchnology . All rawchnology  (2) 1–3 chnology  (3) Illuchnology . ParametGenes were identified using Prodigal , as partThe genome is 10,373,764 nucleotides with 62.28 % GC content  nodulate South African papilionoid species, in contrast to the other nine  that are able to nodulate mimosoid species. A comparison of the mimosoid nodulating strains reveals that UYPR1.413 has the largest genome (10.4 Mbp), with the highest KOG count (1670) and the lowest GC (65.28 %) percentage in this group. All 13 of these genomes share the nitrogenase-RXN MetaCyc pathway catalyzed by a multiprotein nitrogenase complex. However, only Burkholderia  sp. UYPR1.413 has been shown to fix effectively with Parapiptadenia rigida . The genome attributes of Burkholderia  sp. UYPR1.413 will therefore be important for ongoing molecular analysis of the plant microbe interactions required for the establishment of leguminous tree symbioses with this host.me group . This trme group . Phyloge species . GlasshoYPR2.512 . There aGEBA-RNB: Genomic Encyclopedia of Bacteria and Archaea-Root Nodule BacteriaJGI: Joint Genome InstituteTY: Trypton YeastCTAB: Cetyl trimethyl ammonium bromideWSM: Western Australian Soil MicrobiologyBNF: Biological Nitrogen FixationThe authors declare that they have no competing interests.EF supplied the strain and background information for this project, PVB supplied DNA to JGI, TR performed all imaging, SDM and WR drafted the paper, JH provided financial support and all other authors were involved in sequencing the genome and editing the final manuscript. All authors read and approved the final manuscript.