E. coli (268), S. aureus (61), P. aeruginosa (64) or K. pneumoniae (98). A group of 300 complication-free chronically HBV-infected patients were controls. We found that HBV DNA levels were elevated in patients with each of the bacterial infections . ALT and HBeAg were strong determinants of high HBV DNA concentration. Patterns of determinants varied in infections by Gram-positive and Gram-negative bacteria. Patients with HBV DNA ≥ 2000 IU/mL had higher rates of all four concomitant bacterial infections . All types of strains isolated from HBV-positive patients showed less resistance to tested antimicrobials. The HBV DNA serum concentrations were inversely correlated to the number of ineffective antimicrobials in E. coli, P. aeruginosa and K. pneumoniae infections , but not S. aureus (P = 0.194). In conclusion, bacterial infections are associated with a high level of HBV replication, which, in turn, has a significant positive impact on bacterial resistance to antimicrobials. These correlations vary between Gram-negative and Gram-positive bacteria.Bacterial infections are common in patients suffering viral hepatitis and critical for prognosis. However, any correlation between HBV and concomitant bacterial infections is not well characterized. A retrospective study was conducted from Jan 2012 to Jan 2014 on 1333 hospitalized patients infected with bacteria. Among them, 491 HBV-infected patients were co-infected with  Hepatitis B virus (HBV) is one of the leading infectious diseases in the world in terms of the number of sufferers and the clinical significance, particularly in China2467Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae are all common human pathogens in infections associated with ascites, a serious complication of end-stage liver disorders. Liver abscess are commonly caused by S. aureus, P. aeruginosa and, especially, K. pneumoniae9K. pneumoniae (hvKP) strain can even lead to liver abscess in healthy young adults11P. aeruginosa is one of the most common causes of bacteremia in liver transplant recipientsE. coli infection has been reported to make a significant contribution to the development of primary biliary cirrhosis (PBS)Bacterial infections are commonly seen in patients with hepatitis, including HBV-induced hepatitis. For example, in vitroE. coli, S. aureus, P. aeruginosa or K. pneumoniae, and analyzed potential correlations between HBV infection and different common concomitant bacterial infections.Although a previous study gave some hints about potential effects of HBV on bacteria E. coli, S. aureus, P. aeruginosa or K. pneumoniae from Jan 2012 to Jan 2014. Beijing You’an Hospital is a tertiary major hospital specializing in liver disorders and infectious diseases. A total of 1333 patients were identified through bacterial cultures. Patients infected with more than one type of bacteria were excluded. Patients were also excluded if they were co-infected with hepatitis C virus (HCV) or human immunodeficiency virus (HIV). Inclusion criteria for the present study were: detectable hepatitis B surface antigen (HBsAg), age ≥18 years, and at least one positive culture for E. coli, S. aureus, P. aeruginosa or K. pneumoniae. Culture samples included blood, urine, stool, catheter, abscess puncture fluids, ascites, sputum, and bile. All blood samples were drawn upon admission of the patients to the hospital and were sent to the central laboratory for blood tests (including HBV-related blood tests) and bacterial cultures. Clinical and laboratory data were gathered and analyzed. Demographic and clinical characteristics, including age, sex, fibrosis stage, alanine transaminase (ALT) and gamma-glutamyl transaminase (GGT) serum levels, body mass index (BMI), platelet count, the presence or absence of diabetes or liver cancer, and HBV-related markers such as HBV DNA concentration, HBsAg titer, HBeAg, etc., were extracted from clinical databases. Liver fibrosis was classified according to Ishak’s score. An age-matched, sex-matched and bacterial infection-free group of 300 chronically HBV-infected patients were selected as controls.A retrospective study was conducted in Beijing You’an Hospital  on consecutive hospitalized patients with a positive culture for Beijing You’an Hospital Ethics Committee has approved this study and all relevant experiments. Approval covered the retrospective analysis of all patients and control subjects. All subjects gave written informed consent upon admission for their information to be stored and used for research. All experiments were performed in accordance with the human experimentation guidelines of the PR China, which were followed in the conduct of this clinical research.E. coli, 210 (61 isolated from HBV(+) patients) S. aureus, 256 (64 isolated from HBV(+) patients) P. aeruginosa, and 249 (98 isolated from HBV(+) patients) K. pneumoniae strains had been frozen and stored at −80 °C. Susceptibility testing ) to amikacin, amoxicillin-clavulanate, ampicillin, ampicillin-sulbactam, aztreonam, ceftizoxime, cefepime, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, levofloxacin, piperacillin, tetracycline, SMZ-TMP, chloramphenicol, imipenem, meropenem and piperacillin-tazobactam was performed on each Gram-negative strain . Extended spectrum β-lactamase (ESBL) production was also determined by the Phoenix 100 system. Susceptibility testing to amoxicillin, ciprofloxacin, clindamycin, gentamicin, linezolid, oxacillin, penicillin, dalfopristin, rifampicin, tetracycline, Trimethoprim-sulfamethoxazole (SMZ-TMP), ampicillin, vancomycin, erythromycin, amikacin, furadantin, tobramycin, teicoplanin, and trimethoprim was preformed on every Gram-positive strain (S. aureus).The 618 (268 isolated from HBV(+) patients) Serum HBV DNA levels were quantified by a COBAS Amplicor HBV Monitor  with a detection limit of 20 IU/mL. HBsAg titer was determined with the Architect HBs-Antigen QT assay  based on an automated chemiluminescent microparticle immunoassay, following the manufacturer’s recommendations. The Architect HBs-Antigen QT assay measures a range of HBsAg from 0.05 to 250 IU/mL. Samples with higher HBsAg titer required dilution to bring them into the range of the calibration curve.t-test and the Wilcoxon rank-sum test were used for analysis of continuous variables. Continuous variables were assessed for normality and are presented as the mean ± SEM. Continuous variables were compared with Spearman’s rho correlation analysis. Categorical variables were compared with the chi-square test or Fisher’s exact test. P values of 0.05/n (n = the number of comparisons) are considered statistically significant based on the Bonferroni correction for multiple comparisons. A statistical trend was defined as a P value < 0.1 but >0.05/n. Logistic regression was used to analyze risk factors for HBV DNA concentration. All variables with a P value < 0.05 were included in the multivariate model. Forward selection with use of the likelihood-ratio test was used to select the final multivariate model for determinants of HBV DNA concentration.SPSS software (version 15.0) was used for data analysis. Student’s A total of 491 patients were selected for the current study according to the criteria described above. Baseline characteristics of these patients are summarized in E. coli (P = 0.002), P. aeruginosa (P = 0.008), K. pneumoniae (P = 0.003), S. aureus (P = 0.010)). Therefore, we performed uni- and multivariate logistic regression analysis of potential determinants of HBV DNA levels in patients with different co-infections. In all four bacterial infections, ALT and HBeAg were strong determinants of high HBV DNA concentration in both uni- and multivariate analysis. Liver cancer was significant only for Gram-negative bacterial infections (P. aeruginosa and K. pneumoniae) in both uni- and multivariate analysis. The combinations of determinants of HBV DNA concentration were almost the same in patients co-infected with HBV and E. coli or K. pneumoniae, where fibrosis, ALT, BMI and HBeAg were independent determinants. Liver cancer was an independent determinant in K. pneumoniae infection (P = 0.048) but not in E. coli infection (P = 0.071). Meanwhile, in patients co-infected with S. aureus, only ALT and HBeAg were independently correlated to serum HBV DNA levels. HBsAg serum level was not associated with HBV DNA concentration in patients with any bacterial infection  patients. P. aeruginosa strains were less resistant to 11 antibiotics, K. pneumoniae strains to 13, and S. aureus strains to 11. All Gram-negative strains were sensitive to carbapenems (imipenem and meropenem) and chloramphenicol, and resistant to ampicillin. No difference in resistance to piperacillin was observed between strains isolated from patients with or without HBV infections. The percentages of strains expressing extended spectrum β-lactamase (ESBL) among strains isolated from HBV(+) patients and HBV(−) patients were compared. The rates were lower in all three types of Gram-negative isolates from HBV(+) patients compared to the isolates from HBV(−) patients  , but this was not the case for S. aureus infection (P = 0.194) , but not S. aureus (P = 0.228).To compare the changes in drug resistance of strains from patients with or without HBV infection, we collected all strains isolated from HBV(−) patients admitted in the same study period and compared their resistance to 19 antimicrobials with that of strains isolated from HBV(+) patients . All fouctively) . The HBV= 0.194) . HBeAg pin vitro, leading to the possibility that high HBV DNA concentration may contribute to control of bacterial infectionIn the present study, we investigated the crosstalk between HBV infection and common concomitant bacterial infections in 491 patients with HBV and bacterial co-infections. A previous study indicated that a peptide extracted from HBV demonstrated potent antimicrobial activity S. aureus strains showed differences from those infected with the other bacteria in terms of the correlation between HBV DNA serum concentration and the number of ineffective antimicrobials  patients showed less resistance to the antimicrobials we tested compared to bacteria isolated from HBV(−) patients. The resistance to antimicrobials appeared to be inversely correlated to HBV DNA serum concentration. The reason for this correlation remains unknown. Direct evidence on the crosstalk between HBV and bacterial infections is very rare in previous studies. However, both viral and bacterial infections are controlled by the host immune system. HBV infection has a great impact on the immune system and may compromise its ability to contain concomitant bacterial infections, leading to less chance of emerging new resistance to antimicrobials. Further studies are necessary to confirm this hypothesis. Patients infected with crobials . Antimicin vitro investigation, or interventional clinical trials. Second, the study population in this work is not representative for all phases of HBV infection. All patients involved were admitted to hospital with significant clinical conditions, and those with mild chronic HBV infections are underrepresented. Third, the numbers of HBV-infected patients co-infected with each bacterium are not large enough to draw final conclusions, especially for the HBeAg negative patients. Larger studies are required to fully address all questions in this study.Our study has several limitations. First, clinical association studies cannot indicate a causal relationship. Therefore, the regulatory relationship between HBV infection and concomitant bacterial infections remains to be revealed. Our study provides the basis for the design of further In summary, we found that higher HBV DNA serum levels were observed in patients with concomitant bacterial co-infections if compared to the levels in those without. The HBV DNA serum level was inversely correlated to bacterial strains’ resistance to antimicrobials. However, further studies are warranted to understand the mechanisms behind this crosstalk.How to cite this article: Li, W. et al. Clinical correlation between HBV infection and concomitant bacterial infections. Sci. Rep.5, 15413; doi: 10.1038/srep15413 (2015).


Dendroctonus valens LeConte , is a destructive invasive pest of conifers which has become the second most important forest pest nationwide in China. Dendroctonus valens is known to use host odors and aggregation pheromones, as well as non-host volatiles, in host location and mass-attack modulation, and thus antennal olfaction is of the utmost importance for the beetles’ survival and fitness. However, information on the genes underlying olfaction has been lacking in D. valens. Here, we report the antennal transcriptome of D. valens from next-generation sequencing, with the goal of identifying the olfaction gene repertoire that is involved in D. valens odor-processing.The red turpentine beetle (RTB), S-transferases, and aldehyde dehydrogenase. Predicted protein sequences were compared with counterparts in Tribolium castaneum, Megacyllene caryae, Ips typographus, Dendroctonus ponderosae, and Agrilus planipennis.We obtained 51 million reads that were assembled into 61,889 genes, including 39,831 contigs and 22,058 unigenes. In total, we identified 68 novel putative odorant reception genes, including 21 transcripts encoding for putative odorant binding proteins (OBP), six chemosensory proteins (CSP), four sensory neuron membrane proteins (SNMP), 22 odorant receptors (OR), four gustatory receptors (GR), three ionotropic receptors (IR), and eight ionotropic glutamate receptors. We also identified 155 odorant/xenobiotic degradation enzymes from the antennal transcriptome, putatively identified to be involved in olfaction processes including cytochrome P450s, glutathione-D. valens. The genes reported here provide a significant addition to the pool of identified olfactory genes in Coleoptera, which might represent novel targets for insect management. The results from our study also will assist with evolutionary analyses of coleopteran olfaction.The antennal transcriptome described here represents the first study of the repertoire of odor processing genes in  A sophisticated olfactory system is crucial to insects for survival and reproduction ,2. AntenOBPs are small, hydrophilic proteins that are secreted by the accessory cells and accumulate in the sensillar lymph ,3. The sORs have seven transmembrane domains with inverted membrane topology ,11 and aDrosophila [IRs are relatives of ionotropic glutamate receptors (IGluRs) with atypical binding domains that are conserved across proteosome lineages; IRs are far more ancient than ORs . IRs werosophila . The IR osophila . The antosophila .Antheraea polyphemus (Saturniidae), [Drosophila [SNMPs were first found in the dendritic membrane of sex pheromone-sensing olfactory neurons (ORNs) of the wild silk moth, niidae),  and are niidae), –25. SNMPosophila . SNMP1 aosophila . Insectsosophila –29.Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae), is a secondary pest of pines in its native range in North and Central America. Dendroctonus valens was introduced into China in the early 1980s, where it aggressively kills pine species native to China [D. valens in its native range is clearly different from that in China [D. valens in 1999, it has infected over 5,000,000 ha of pine forest, and more than 10 million Pinus tabuliformis Carr. as well as other pine species such as Pinus bungeana Zucc [D. valens is the second most important forest pest nationwide in China [D. valens fitness because the beetle uses aggregation pheromones, as well as host and non-host volatiles in intra- and inter-specific communication [D. valens. Investigating the repertoire of odor processing genes involved in D. valens olfaction could provide valuable insights into the molecular mechanisms of insect olfaction, and also identify possible molecular targets that could be manipulated for D. valens control.The red turpentine beetle (RTB), to China –32. The in China . Since tana Zucc ,33–35 anin China  and its nication –50. HoweTribolium castaneum has been sequenced, our knowledge of the molecular basis of odorant reception in Coleoptera, the largest insect order, remains relatively limited. Recently, the advent of RNA-Seq approaches (next generation sequencing techniques) triggered an exponential growth in our knowledge of insect olfaction. Genes involved in odor processing in Coleoptera have been identified from insects whose genomes have not been sequenced, such as Ips typographus, Dendroctonus ponderosae [Agrilus planipennis [Although the genome of nderosae , and Agrnipennis . AdditioD. valens to identify the olfactory gene repertoire involved in D. valens’ odor-processing. In this study, we conducted a transcriptome analysis of antennae of adult beetles, and identified 68 putative chemosensory transcripts comprising 21 OBPs, six CSPs, four SNMPs, 22 ORs, four GRs, three IRs, and eight ionotropic glutamate receptors. We also obtained 155 odorant/xenobiotic degradation enzymes from the antennal transcriptome, putatively identified to be involved in olfaction processes including cytochrome P450s, glutathione-S-transferases, and aldehyde dehydrogenase.Here, we employed a transcriptome approach based on next-generation sequencing of antennae of D. valens antennaapp:addword:antenna sample by the Illumina HiSeq 2000 platform, which were assembled into 39,831 contigs with a mean length of 409 bp and an N50 length of 814 bp. After clustering and redundancy filtering, we finally acquired 22,058 unigenes  with a mean length of 828 bp and a N50 length of 1360 bp. Of the 22,058 unigenes, those with a sequence length of more than 500 bp accounted for 48.79% of the transcriptome assembly  were obtained from a assembly . All the−5. The species distribution of the best match result for each sequence is shown in T. castaneum genes, comprising 69.10%. Interestingly, there was only an 18.29% similarity of D. valens antennal transcripts to those of the congener D. ponderosae. However, this result does not mean that the identified transcripts in D. valens share more similarity with T. castaneum transcripts than D. ponderosae transcripts. Instead, it is likely due to the availability of more T. castaneum than D. ponderosae sequences in the NCBI protein database used in the homology analysis. The protein number of T. castaneum in the NCBI database is 36,478, 1.2 times higher than that of D. ponderosae . With more protein sequences annotated from D. ponderosae, we believe that more sequences in D. valens will align to those of D. ponderosae.Among 22,058 transcripts, 15,531 (70.40%) were matched by the BlastX homology search to entries in the NCBI non-redundant (nr) protein database with a cut-off E-value of 10D. valens antennae  could be annotated based on sequence homology. Because one transcript could align to more than one biological process, the 8,743 transcripts were assigned to the biological process category , cellular component category , and molecular function category . The major GO terms associated with molecular function were binding (41.09%) and catalytic activity (38.19%), which potentially reflects the metabolic processes of the antennal tissue . Cellulaantennae . Under tantennae .D. valens focused on gene families associated with olfactory processing. By homology analysis, we identified 68 putative odor-reception genes, including 21 OBPs, six CSPs, four SNMPs, 22 ORs, four GRs, three IRs, and eight IGluRs. As well, 155 putative odorant/xenobiotic degradation enzymes were found , but more than I. typographus (15). Sequence analysis identified 18 unigenes with a full length ORF (Open Reading Frame) with predicted signal peptide sequences  had a full length ORF  had at least 87% similarity with corresponding ORs of D. ponderosae , T. castaneum, Megacyllene caryae, and Agrilus planipennis. Eleven DvalORs were excluded from analysis due to insufficiently long amino acid sequences.A phylogenetic tree of ORs based on the neighbor-joining method was constructed using protein sequences of ORs from D. valens were clustered with at least one bark beetle orthologous gene in the phylogenetic tree except for two ORs . Among them, five ORs from D. valens were clustered with one D. ponderosae orthologous gene.ORs are divided into several subgroups of various size and content in the phylogenetic tree . Almost T. castaneum and M. caryae. Receptor groups 4–6 contained ORs only from T. castaneum. Group 7 contained 11 ORs from M. caryae, but no ORs from T. castaneum or A. planipennis. The results showed eight of 11 ORs from D. valens were present within group 7. These eight DvalORs were further divided into two subgroups: group 7a and 7b. Subgroup 7b was exclusive to bark beetle species, including ORs from D. valens, I. typographus, and D. ponderosae. In three groups , we found most of the candidate genes were grouped together with orthologs in D. ponderosae and I. typographus. The amino acid sequences of all the 11 ORs are listed in The numbering of coleopteran OR subfamilies has been standardized in previous studies ,51, and D. ponderosae  database at NCBI using BlastX revealed 97% identity with the Orco homolog of D. valens, which is in agreement with several other recent antennal transcriptomic studies [Four GR candidates (GR1-4) were identified in  studies ,5,26. OnD. valens, none of which represented full-length genes. Among them, SNMP1 and SNMP1a are orthologs of SNMP1. DvalSNMP1 and DvalSNMP1a were very similar to the DponSNMP1 and DponSNMP1a published in GenBank, with 94% and 95% identity, respectively  relationships among the SNMPs (-5), and showed 85% identity with a sensory neuron membrane protein of D. ponderosae (GenBank accession number: AFI45067.1). The information for all four SNMPs is listed in We also found four SNMPs  in the aectively . The genhe SNMPs . DvalSNMD. valens. IR1 and IR3 have high sequence similarity to the ionotropic receptor 8a of D. ponderosae (GenBank accession number: AGI05169.1) with amino acid identities of 87%, and 97%, respectively . These genes are likely involved in olfaction processes. Among the odor/xenobiotics degradation genes, the number of esterase genes (66) was the highest, followed by cytochrome P450 genes (49). Cytochrome P450s are thought to participate in detoxification, odor processing, and neuro/development functions in insects [D. valens antennae are involved in odor/xenobiotic degradation, including glutathione S-transferases , aldehyde dehydrogenase (14), epoxide hydrolases (four), and several antioxidants -3-carene), as well as non-host volatiles -3-hexen-1-ol, and (E)-2-hexen-1-ol) during host location, and uses aggregation pheromones and congeneric kairomones  in intra- and interspecific communication [D. valens’s biology and provide a foundation to further unravel the molecular mechanisms underlying olfaction in D. valens. Functional studies will be required to ascertain the specific role of these genes in D. valens olfaction. Furthermore, critical molecular targets of important semiochemicals such as (+)-3-carene, trans-verbenol, and frontalin could be identified via computational and molecular methods, such as homology modeling, molecular docking, and RNA interference (RNAi). By collating chemical and ecological knowledge with the putatively identified genes in this study, we can begin to elucidate the olfactory mechanisms of D. valens.The antennal transcriptome described here represents the first study on the repertoire of odor processing genes of the destructive and invasive red turpentine beetle, nication –50. The T. castaneum [I. typographus and D. ponderosae [A. plannipennis [D. ponderosae [A. plannipennis [Prior to this study, coleopteran species’ odorant reception genes had been characterized from the genome of astaneum , the antnderosae , and thenipennis . Howevernderosae , and sevnipennis . Odorantnipennis ,62. In tnipennis ,5,26. ThD. valens transcriptome analyzed here, 70.41% of 22,058 transcripts had homologous matches in GenBank with the cutoff value of 10−5. Among these transcripts, 39.6% of the genes were annotated on one or more GO terms by GO analyses. More than 60% of the transcripts had no GO terms, similar to what was found with Manduca sexta [I. typographus and D. ponderosae [A. planipennis [D. valens transcripts that may potentially represent novel genes.In the nipennis  This resD. valens antennal transcriptome. Even though the number of putative OBP-coding genes in D. valens is much lower than that of D. ponderosa (31), the majority of putative OBPs showed a high similarity to those of D. ponderosa, and most of the OBPs of D. valens clustered together with DponOBPs. CSPs are another more conserved class of small binding proteins which can bind pheromone compounds [D. valens antennal transcriptome. Among the six candidate CSPs, four sequences were clustered together with DponCSPs. Dendroctonus valens and D. ponderosae are sympatric in North America, and the sequence similarity of olfactory-related orthologous genes is very high between the two species, suggesting that the OBPs and CSPs of D. valens may have similar forms of expression and function as those of D. ponderosae. It was interesting that nearly one third of the transcripts which encoded putative OBPs and CSPs in D. ponderosae were identified only in non-sensory tissues rather than the antennal tissues [OBPs play an important role in odor processing by insects, facilitating the transport of odorant molecules through the sensillar lymph and serving as the liaison between the external environment and ORs ,4. In thompounds –9. Here  tissues ,4, suggeZ)-11-octadecenyl acetate  in Drosophila melanogaster and (Z)-11-hexadecenal (a lipid-derived pheromone) in Heliothis virescens, and suggested that SNMP acts in concert with odorant receptors to capture pheromone molecules on the surface of olfactory dendrites, particularly pheromones with hydrophobic tails [D. valens, four SNMPs were identified. Because bark beetle pheromones lack the hydrophobic tails of D. melanogaster and Heliothis virescens pheromones, we assume that SNMPs may have an alternate functional role. The expression of antennal SNMPs in D. valens, similar to I. typographus and D. ponderosae, suggests that SNMPs may be involved in pheromone detection by bark beetles.SNMPs are membrane proteins observed to associate with chemosensory neurons in insects . There aic tails . In the D. valens. The number of IR genes in D. valens is much lower than that of D. ponderosae (15). We could not exclude the possibility that some of transcripts were missed in our antennal transcriptome, because of the lower sequencing depth of D. valens than that of D. ponderosae. Sequence alignments showed that the putative D. valens IRs have high similarity with the known ionotropic receptor of D. ponderosae, DponIR8a. DponIR8a belongs to the group of IR8a, which is a broadly expressed co-receptor and necessary for odor responses[D. melanogaster and it may form heteromers with another variant ionotropic receptor [The IR family contains two major groups, the conserved antennal IRs involved in olfaction and species-specific divergent IRs that are expressed in other tissues including gustatory organs . We idenderosae 1. We coulreceptor .D. valens antennal transcriptome. This number is much lower than for two other bark beetles . The lower number of receptors in D. valens may be due to technical reasons. Despite the number difference in the putative OR-coding genes, the majority of DvalORs grouped together with orthologs in D. ponderosae and I. typographus. In agreement with previous results, we found distinct expansions of OR lineages in bark beetles, and the subgroup 7b was exclusive to three bark beetle species in the OR phylogenetic tree [D. valens antennal transcriptome. Further function studies on DvalOR1 and DvalOR2 may help in deciphering their role in D. valens olfaction.ORs belong to a large multigene family encoding seven transmembrane domain proteins ,11, and tic tree ,5. In adD. valens, four GR candidates (GR1-4) were identified. In the phylogenetic tree, Gr1, Gr 2, and Gr4 in D. valens grouped together with ItypGR3 and DponGR3, which may function as carbon dioxide receptors, and DvalGr3 was clustered with ItypGR6, tentatively assigned as a trehalose receptor [B. mori [Helicoverpa armigera [D. melanogaster [D. valens GRs play important roles in the detection of carbon dioxide, sugars, or bitter compounds. Future studies on GR expression patterns of D. valens in chemosensory and non-chemosensory tissues  will be helpful in understanding the actual roles of GRs in D. valens.GRs are mostly expressed in gustatory receptor neurons in taste organs and are involved in detection of sugars, bitter compounds, and contact pheromones ,5. In th[B. mori  and Harmarmigera —heat sennogaster —and carbnogaster . It is tD. valens antennal transcriptome. This is the first report of genes involved in odor degradation in D. valens. These genes, including esterases, cytochrome P450 genes, glutathione S-transferases, aldehyde dehydrogenase, epoxide hydrolases, and antioxidants, are likely involved in odor degradation. For insects, the olfactory system must not only precisely detect conspecific semiochemicals, but also rapidly inactivate the chemical signal once the message is conveyed to allow for continued reception of signals[A. polyphemus (ApolSE), which could effectively degrade the acetate component of the pheromone blend [M. sexta (GST-msolf1) has been shown to play a key role in sex pheromone detection by inactivating the aldehyde component of the sex pheromone blend [D. ponderosae, degrades several pine host monoterpenes [D. valens, suggesting that these P450 genes may be involved in detoxification or odor degradation of these monoterpenes [D. valens antennal transcriptome, but their functional roles remain to be determined. Understanding the molecular mechanism(s) of signal inactivation is important in fundamental biology and may lead to novel molecular targets for insect pest control [It is generally accepted that insect semiochemicals require specific odorant degrading enzymes based on the functional group(s) present on each semiochemical. In our study, a high number of odor/xenobiotic degradation genes were found in the f signals. Mountinf signals,61,69–78f signals. At presne blend ,60. A glne blend . Cytochrne blend . DponCYPterpenes . Here, w control .Dendroctonus valens. The genes reported here provide valuable insight into the molecular mechanisms of insect olfaction, and also represent possible novel targets for D. valens control or management. In addition, the results from our study provide the basis for a deeper understanding of coleopteran olfaction in the context of complex behavior, and information for comparative and functional genomic analyses of related species.This study reports the first antennal transcriptome analysis in D. valens demonstrated close similarity in sequence alignments to those of two other two bark beetles. The phylogenetic trees showed that the majority of the odor processing genes of D. valens cluster with the analogous genes in D. ponderosae. In addition, we also found a distinct species-specific expansion of OR lineages in our results. Dendroctonus valens and D. ponderosae are sympatric in their host range in North America, and the two species live in similar coniferous habitats and share a number of biologically relevant compounds, including host compounds, pheromone compounds and non-host volatiles  and a specific organ (antennae). As the first step towards understanding their functions, a comprehensive examination of the chemosensory gene expression patterns in the context of different tissues and life stages in D. valens could provide important information on the functions of the chemosensory genes. Following that, functional studies on the chemosensory genes will help to depict the molecular mechanisms underlying D. valens olfactory detection from an evolutionary viewpoint.The insect olfactory gene repertoire may be larger than currently understood. For example, nearly one third of the transcripts encoding putative OBPs and CSPs in l tissue ,5, suggeDendroctonus valens were collected by Lindgren funnel traps baited with a kairomone lure (3-carene) in P. tabuliformis plantations at the Tunlanchuan Forest Farm , Gujiao, Shanxi Province, China. Forest Pest Control Station of Shanxi Province issued the permit for the fieldAdults of D. valens were identified using the morphological characteristics of front and elytral declivity [collections . About one thousand newly emerged adults  were collected. All eclivity  and sexeeclivity . In totacDNA library construction and Illumina sequencing of the samples were performed at Beijing Genomics Institute Shenzhen, China . BrieflyDendroctonus valens were sequenced on the Illumina HiSeq 2000 platform. Transcriptome de novo assembly was carried out using the short reads assembling program Trinity-2014 [The cDNA library constructed from the antennae of ity-2014 . Trinity−5 to find similarities to the unigenes or contigs of other species. Unigenes larger than 150 bp were first aligned by BlastX to protein databases, including Nr, Swiss-Prot, KEGG and COG , retrieving proteins with the highest sequence similarity with the given unigenes along with their protein functional annotations. Then, we used the Blast2GO program [http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The signal peptides of the protein sequences were predicted using SignalP 4.0 [The unigenes or contigs were matched by a BlastX homology search to the entries in the NCBI non-redundant (nr) protein database with a cut-off E-value of 10 program  to get G program . The ORFnalP 4.0 .D. valens and other insect species .The sequences of 60 odorant reception genes  of S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Quambalaria cyanescens, from the peritoneal fluid of a man after he switched from continuous ambulatory peritoneal dialysis to nocturnal intermittent peritoneal dialysis. Based on the morphological examination and multigene phylogeny, the clinical isolate was confirmed as Q. cyanescens. This pathogen exhibited low sensitivity to all tested echinocandins and 5-flucytosine. Interestingly, morphological characterization revealed that Q. cyanescens UM 1095 produced different pigments at low temperatures (25°C and 30°C) on various culture media. It is important to monitor the emergence of this rare fungus as a potential human pathogen in the tropics. This study provides insight into Q. cyanescens UM 1095 phenotype profiles using a Biolog phenotypic microarray (PM). Of the 760 nutrient sources tested, Q. cyanescens UM 1095 utilized 42 compounds, and the fungus can adapt to a broad range of osmotic and acidic environments. To our knowledge, this is the first report of the isolation of Q. cyanescens from peritoneal fluid, revealing this rare fungus as a potential human pathogen that may be misidentified using conventional methods. The detailed morphological, molecular and phenotypic characterization of Q. cyanescens UM 1095 provides the basis for future studies on its biology, lifestyle, and potential pathogenicity.Peritonitis is the leading complication of peritoneal dialysis, which is primarily caused by bacteria rather than fungi. Peritonitis is responsible for approximately 18% of the infection-related mortality in peritoneal dialysis patients. In this paper, we report the isolation of a rare fungus,  Quambalaria. de Beer et al. [Ramularia and Sporothrix to propose a new genus, Quambalaria, to accommodate Ramularia pitereka and Sporothrix eucalypti. The first Q. cyanescens isolate (strain CBS 357.73) was recovered from human skin and described as Sporothrix cyanescens in 1973 [et al. [Cerinosterus cyanescens). Based on a molecular analysis of partial large subunit (LSU)-rDNA, a later study revealed that C. cyanescens was closely related to Microstroma juglandis but differed from other species within the genus Cerinosterus [et al. [Fugomyces and renamed C. cyanescens as Fugomyces cyanescens. The phylogenetic analysis of the internal transcribed spacer (ITS) region in conjunction with the LSU sequences and ultrastructural analysis data reclassified Fugomyces cyanescens in the new family Quambalariaceae as Q. cyanescens [Q. pitereka, Q. eucalypti and Q. cyanescens are distinct species [Quambalaria contains five species, including Q. cyanescens, Q. pitereka, Q eucalypti, Q. coyrecup and Q. simpsonii [Quambalariaceae is a new family of fungi belonging to the order Microstromatales in the class Exobasidiomycetes . The famr et al.  first de in 1973 . In 1987nosterus . Sigler  [et al.  then estQ. cyanescens is a hyaline basidiomycete isolated from a broad range of ecological niches, including air, soil, and insect larvae as well as in association with diverse plant sources in various countries [Q. cyanescens is frequently associated with bark beetles feeding on numerous plants [Corymbia and Eucalyptus species [Q. cyanescens produces the antibiotic sesquiterpene globulol [Quambalaria species (closely related to Q. cyanescens) exhibited potent antimicrobial activities against various human and plant pathogens [et al. [Q. cyanescens.ountries ,2,6,8. Qs plants . This fu species ,6. Notabglobulol . The cruathogens ,12. Stod [et al.  describeQ. cyanescens is a rare human basidiomycetous pathogen [Q. cyanescens was also reported as an environmental contaminant associated with pseudoepidemic nosocomial pneumonia [pathogen ,14,15. Tneumonia .Q. cyanescens isolated from the peritoneal fluid of a patient after nocturnal intermittent peritoneal dialysis. The characteristic morphological features and molecular data identified the fungal strain as Q. cyanescens UM 1095. The present study provides insight into Q. cyanescens UM 1095 phenotype profiles, including its nutrient utilization pattern and sensitivity to osmolytes, ions and acidic environments, using the Phenotypic MicroArray (PM) system.We report the first case of Approval for this study was obtained from the Medical Ethics Committee of the University Malaya Medical Centre (reference number: 20157–1519), and written consent was obtained .A 49-year-old male was found to have end-stage renal disease (ESRD) due to hypertensive nephrosclerosis and admitted to the nephrology ward of the Division of Nephrology of UMMC for hemodialysis in April 2014. On 8 December 2014, the patient received continuous ambulatory peritoneal dialysis (CAPD) after Tenckhoff catheter insertion on 21 November 2014. He was recruited to an Automated Peritoneal Dialysis (APD) trial. His APD regime was a two-hour cycle for five cycles per night, using two bags of 5 L 2.5% dialysate. He was stable on CAPD treatment until he changed to nocturnal intermittent peritoneal dialysis (NIPD) treatment using automated cycler machine on 19 December 2014. During NIPD treatment, cloudy peritoneal effluence was observed, but the patient had no symptoms and signs of peritoneal irritation, including fever, abdominal pain, diarrhea or vomiting. He was treated empirically as peritoneal peritonitis and was started on intra-peritoneal (IP) antibiotics, i.e. ceftazidime  and Cefazolin . Peritoneal fluid examination showed the presence of 560/μL neutrophils (95% polymorph) and confirmed the diagnosis of peritoneal peritonitis. He demonstrated initial response with reducing peritoneal fluid white cell count from 560/μL to 240/μL. Nonetheless, the peritoneal fluid culture revealed the growth of yeast-like organism and the patient started to receive antifungal therapy (IV fluconazole). He denied any contact with animals and had no recent travel. His Tenckhoff catheter was subsequently removed on 14 January 2015 due to the unresolved peritonitis. He completed 14 days of IV fluconazole and was discharged well. He received CAPD treatment two months after the resolution of peritonitis.Q. cyanescens UM 1095 was isolated from the peritoneal dialysis fluid of a dialysis patient with suspected peritonitis after switching from continuous ambulatory peritoneal dialysis to nocturnal intermittent peritoneal dialysis in the Division of Nephrology, University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. The morphological and cultural features of Q. cyanescens UM 1095 were examined on Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), corn meal agar (CMA) and V8 agar incubated at 25°C, 30°C and 35°C for three days, with alternate-day examination for fungal growth. The macroscopic examination was performed to study the colonial characteristics, including the color of the pigment, texture, and topography. A slide culture was carried out as previously described with slight modifications [ications . The sliin vitro antifungal susceptibility of Q. cyanescens UM 1095 was examined using the Sensititre YeastOne  with a broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 guidelines. The isolate was tested against nine antifungal agents , and the minimum inhibitory concentrations (MICs) were examined after 24-hour incubation.The Quambalaria species were compiled for multilocus phylogenetic analysis , the small subunit of the ribosomal RNA gene (SSU) and the D1/D2 domain of the 26S rRNA gene were used as targets for the molecular identification of the fungal isolate. Total DNA extraction, PCR amplification, and sequencing were performed as described previously ,15. The analysis . MicrostM-Coffee . IndividQ. cyanescens UM 1095 was evaluated using Biolog PM analysis . Ten MicroPlate panels (PM1 to PM10), which included carbon, nitrogen, phosphorus, sulfur, nutrient supplements, peptide nitrogen, osmolytes and pH sources, were used in this study. The metabolic profile of Q. cyanescens UM 1095 was grown on SDA at 35°C for 24 hours. The cell suspension was extracted by rolling a sterile, wetted cotton swab over the colonies and resuspended in 15 mL IFY-0 inoculating fluid . The uniform cell suspension was adjusted to 62% transmission at OD590 nm using a turbidimeter .PM1-10 inoculating fluids were prepared according to the manufacturer’s protocol . PM1-10 Q. cyanescens UM 1095 were deposited in GenBank with accession numbers KT186106, KT186108, and KT186107, respectively.The ITS, SSU and D1/D2 domain nucleotide sequences of Q. cyanescens UM 1095 white colonies on SDA were sparse at 25°C but became dense at 30°C and 35°C  were ovoid, ellipsoid or cylindrical shape with a sympodial conidiogenesis , 583 bp (D1/D2 domain) and 1010 bp (SSU), respectively (Q. cyanescens UM 1095 was found to be 100% (617/617 bp) identical to the ITS of Q. cyanescens strain AUMC 6293 and the SSU was 100% (1010/1010 bp) identical to the SSU of Q. cyanescens strain CBS 876.73. However, the D1/D2 domain of Q. cyanescens UM 1095 was 99% (582/583 bp) identical to the D1/D2 domain of Q. simpsonii culture-collection CBS:124773.The preliminary morphological identification of the ectively . By querQuambalaria species. The SSU region was excluded in the multilocus phylogenetic analysis due to the limited number of Quambalaria SSU sequences in the NCBI database. As shown in Quambalaria species are well separated. Multilocus phylogenetic analysis revealed that UM 1095 was clustered together with Q. cyanescens strain 11PU348  and all tested echinocandins, including anidulafungin (MIC > 8 μg/mL), caspofungin (MIC > 8 μg/mL) and micafungin (MIC > 8 μg/mL). Q. cyanescens UM 1095 is most likely susceptible to amphotericin B (MIC = 1 μg/mL) and fluconazole (MIC = 0.5 μg/mL), itraconazole (MIC = 0.03 μg/mL) and posaconazole (MIC = 0.015 μg/mL).Q. cyanescens UM 1095 nutritional phenotypic profiles using Biolog PM analysis. Biolog PM metabolic panels (PMs 1–8) contain approximately 200 assays of carbon source metabolism, 400 assays of nitrogen source metabolism, 100 assays of phosphorus and sulfur source metabolism, and 100 assays of biosynthetic pathways  that were used in this study consisted of approximately 100 assays of ion effects and osmolarity, 100 assays of pH effects and pH control with deaminases and decarboxylases , sodium benzoate pH 5.2 (20–100 mM) and sodium nitrite  the patient denied to be contacted with any animals ii.) no other bacteria or fungi were isolated from the Tenckhoff catheter tip, iii.) the white blood cell count in the cloudy peritoneal effluent of the patient was abnormally high, and iv.) no other organism was isolated from the peritoneal fluid. However, at this stage of knowledge, the pathogenic role of Q. cyanescens in peritonitis remains unknown, although it is a low-virulence human pathogen [Peritonitis is the main complication of peritoneal dialysis and is mostly caused by bacterial pathogens. An estimated 18% of the infection-related mortality in peritoneal dialysis patients is attributed to peritonitis . By comportality ,24. Varireported ,25–27. Nreported . Peritonreported . Hamai e [et al.  reportedtic cats . In the patients , lymphompatients . However [et al.  proposedQ. cyanescens UM 1095 based on basic microbiological examination. Gram staining showed no bacteria but only fungal elements. On Brilliance Candida agar, we obtained dark blue and yeast-like colonial morphology and identified the isolate as a Candida species, as in a previous study [Rhodotorula mucilaginosa, which did not correspond to the culture characteristics. This failure of accurate identification of our isolate identity was most likely due to the absence of a Quambalaria species database in the identification system. When standard assimilation tests fail to determine the isolate identity, species-level identification was achieved through specific culture characteristics and molecular characterization. Subsequent colonial morphological examination and multilocus phylogenetic analysis identified the clinical isolate as Q. cyanescens. In contrast to a previous study [Quambalaria genus but also clustered our isolate with Q. cyanescens.At the initial stage, we failed to identify us study . The APIus study , multiloQ. cyanescens UM 1095 had the same resistance profile as Q. cyanescens 11PU348, which exhibited high MICs to three compounds of the echinocandin class of drugs and 5-flucytosine. Echinocandin resistance is considered rare, although a few studies have shown echinocandin resistance in Candida albicans, C. parapsilosis, C. tropicalis, C. guilliermondii and C. kefyr [Candida species is associated with the mutations in two hot spot regions of FKS genes, which encode the major subunit of the 1,3-D-glucan synthase complex [Q. cyanescens UM 1095 is unknown.The main aim of peritonitis treatment is to resolve inflammation by eradicating the causative pathogen(s). The early and rapid identification of causative pathogen(s) is important with the advent of clinical management to ensure good clinical outcomes and to reduce the unnecessary overuse of antimicrobial agents. In this study, we found that C. kefyr –31. In t complex –35. HoweQ. cyanescens UM 1095 has a high capacity to utilize a number of simple sugars and complex carbohydrates such as pectin and glycerol. This high variability may explain why Q. cyanescens is the only species of the Quambalaria genus that is found in virtually all habitats, including plants, insects, and humans. However, we observed that Q. cyanescens UM 1095 utilizes a narrow spectrum of nitrogen, phosphorus, sulfur and peptide nitrogen sources. The most striking finding was the high metabolic versatility of Q. cyanescens UM 1095 to grow and adapt to a broad range of osmotic environments. This adaptability may be an important contributing factor to its pervasive ability to grow in diverse unfavorable environments. As previously noted [Hortaea werneckii, a causative agent of tinea nigra. However, Kogej et al. [Saccharomyces cerevisiae. In the present study, PM analysis showed that Q. cyanescens UM 1095 is capable of growing well in NaCl concentrations of 1–3%. The ability of Q. cyanescens UM 1095 to utilize various osmolytes showed that the fungus has a good osmoadaptation system to tolerate osmotic stress, which merits further study.All microbial pathogens require a variety of nutrient sources, including carbon, nitrogen, phosphorus and sulfur, for growth and proliferation. Here, we performed comprehensive phenotypic characterization and metabolic profiling of this rare clinical isolate using Biolog PM to provide a better understanding of its nutritional requirements, which are likely needed for it to grow. A closer analysis of the Biolog data revealed that ly noted , 3% NaClj et al.  showed tQ. cyanescens UM 1095. This is the first reported isolation of Q. cyanescens from peritoneal fluid. Based on the macroscopic examination on agar, we observed that the clinical isolate exhibits a mycelium-yeast dimorphism characteristic, which is typical of Q. cyanescens. Multilocus phylogenetic analysis revealed that Q. cyanescens UM 1095 is tightly clustered with the Q. cyanescens strain 11PU348, which was also isolated from humans. In this study, Biolog PM was used to characterize the phenotypic profile of Q. cyanescens UM 1095. Comprehensive phenotypic characterization and metabolic profiling of this rare species enable the design of interventions to prevent the proliferation of this potential pathogen. The Biolog PM may be a effective diagnostic tool, especially when combined with multilocus phylogenetic analysis. We hope that the in-depth analysis of the Q. cyanescens UM 1095 phenotypic profile will help to decipher its basic biology and provide a valuable platform for future fungal research.In this study, we isolated a rare clinical fungal pathogen: S1 Fig(PDF)Click here for additional data file.S2 FigM1: 100 bp ladder (i-DNA Biotechnology); M2: Lambda DNA/HindIII Marker (Fermentas).(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Aspergillus flavus and A. parasiticus. Aflatoxins can contaminate cottonseed under conducive preharvest and postharvest conditions. United States federal regulations restrict the use of aflatoxin contaminated cottonseed at >20 ppb for animal feed. Several strategies have been proposed for controlling aflatoxin contamination, and much success has been achieved by the application of an atoxigenic strain of A. flavus in cotton, peanut and maize fields. Development of cultivars resistant to aflatoxin through overexpression of resistance associated genes and/or knocking down aflatoxin biosynthesis of A. flavus will be an effective strategy for controlling aflatoxin contamination in cotton. In this study, genome-wide transcriptome profiling was performed to identify differentially expressed genes in response to infection with both toxigenic and atoxigenic strains of A. flavus on cotton (Gossypium hirsutum L.) pericarp and seed. The genes involved in antifungal response, oxidative burst, transcription factors, defense signaling pathways and stress response were highly differentially expressed in pericarp and seed tissues in response to A. flavus infection. The cell-wall modifying genes and genes involved in the production of antimicrobial substances were more active in pericarp as compared to seed. The genes involved in auxin and cytokinin signaling were also induced. Most of the genes involved in defense response in cotton were highly induced in pericarp than in seed. The global gene expression analysis in response to fungal invasion in cotton will serve as a source for identifying biomarkers for breeding, potential candidate genes for transgenic manipulation, and will help in understanding complex plant-fungal interaction for future downstream research.Aflatoxins are toxic and potent carcinogenic metabolites produced from the fungi  Aspergillus flavus and A. parasiticus , . Th. ThAsperasiticus . The priA. flavus, it is highly important to take necessary steps to manage aflatoxin contamination in cotton. Both pre- and post-harvest strategies have been used to lessen the aflatoxin contamination in cotton and other crops. Pre-harvest strategies include control of insect pests and proper irrigation to manage aflatoxin contamination. The post-harvest strategies include control of storage conditions that are less favorable to fungal growth, and detoxification of aflatoxin from contaminated seeds and grains . F. FA. flamination , 8], .A. flavus is characterized with the features of a necrotrophic fungal pathogen . A. A16]. Afections , 19]. H. HA. flad cotton , 20],  us usG. raidex.jsp)  and Bowtdex.jsp)  with numA. flavus infection in comparison to non-inoculated control was validated by reverse-transcription PCR using gene specific primers  following the method described earlier . T. T32]. T earlier .. The average quality of the reads in the libraries after filtering was >33. There was negligible amount of adapter/primer contamination in the reads. Altogether, 907,357,848 high quality reads were obtained from all six libraries, which totaled to 90.73 Gbp sequencing data . Out of high quality filtered reads, 59.6% mapped to the D genome of cotton (Gossypium raimondii), whereas 75.6% mapped to the A genome (G. arboreum) across all six libraries. The higher percentage of alignment of the reads to the A genome could be due to its higher genome size as compared to the D genome.Sequencing of the six libraries generated 911,040,814 reads of 100 bp long resulting in a total of 91.1 Gbp sequence data  from NIP library to a gene coding for auxin transport protein of Theobroma cacao (TCM_019010) in the NCBI database demonstrated that the transcript was not chimeric which could have occurred due to repetitive regions in the genes. These results strongly supported a high quality transcriptome assembly of cotton.The high quality reads from each of the six libraries from different experimental conditions were assembled independently into transcripts with a length more than 200 bp. The lengthwise distribution of transcripts of six independent libraries of cotton under different experimental conditions is shown in nscripts . HoweverG. arboreum. In total, 19,750 un-annotated unique transcripts matched with G. arboreum protein sequences. Thus, we annotated total 494,559 transcripts (84.31%) of the cotton transcriptome. The transcripts which did not match to known genes may represent novel genes or genes that may have diverged from their homologs or noncoding RNAs ,  an anTPI wan peanut .HRGPs), phytoalexins and lignin-like substances as a resistance mechanism in response to fungal pathogen infection , . Tnfection , 47]..HRGPs), LOX) genes of the LOX biosynthesis pathway were differentially expressed in pericarp tissue under A. flavus infection. The LOX2 gene was up-regulated under infection by both the strains in pericarp, whereas, the LOX1 (Gorai.004G241400) was specifically up-regulated under the atoxigenic strain infection in pericarp. The LOX3 gene was down-regulated in the pericarp tissue under infection by both strains . However, the LOX genes did not show any change in response to A. flavus infection in seed. Most volatile compounds and hydroperoxy fatty acids are the products of the LOX biosynthesis pathway , , , 48], , . SA-induynthesis , 49], w, wAOS) aynthesis , was up-ve genes . The tra pathway  was downn plants . MPK4 wa pathway , was up-athogens . In the bidopsis  was highRPK) represent PRRs that are involved in the perception of pathogen signal and trigger inducible defense  . T. TFig 4Bes (ROS) , 55]. U. UFig 4B and ROS , 56]. T. TFig 4B and ROS , [56]. T oxidase  was diffathogens , [56]. Tn plants .Aux/IAA, Gretchenhagen-3 (GH3) family, Auxin response factor (ARF) and small auxin-up RNA (SAUR) genes , , . Aux regR) genes , [58], . I. IAux/IAransport . The overansport .Arabidopsis . A. AArabidGhWRKY3 is known to be induced under wounding and fungal infection in cotton . T. T64]. Tbidopsis . Overexp cinerea . A transbidopsis .A. flavus infection. The five bZIP TFs, bZIP14, GIA1, bZIP67, bZIP44 and bZIP42, were specifically induced under the atoxigenic strain infection, whereas bZIP1 and bZIP5 were induced under the toxigenic strain infection . The bZIP TFs were not induced by the fungus in seed; bZIP70 and bZIP21 were down-regulated under the atoxigenic strain infection in seed.The role of bZIP family TFs in biotic stress responses of plants is well established . The bZIMYB TFs were also differentially expressed under A. flavus infection in cotton. More MYB genes were up-regulated in pericarp as compared to seed . MYB93, MYB63, MYB102, MYB105, MYB102 and MYB36 were induced under both the atoxigenic and the toxigenic strains infection in pericarp, whereas MYB62 and MYB2 were induced in seed . The gene expression pattern of MYB TFs has been studied in Arabidopsis in response to wounding and pathogen infection , , , 66]. R. R66]. Red genes  These reLEA) storage protein was shown to be induced in response to A. flavus infection in cotton . T. T22]. Tn cotton . In thisrocesses . The exp of HSFs . There awounding . Most ofnfection . The strnfection . Glyoxalhirsutum . The expeactions , 69] we weLEA) s. Under molecular function category, most of the responses were highly enriched in pericarp as compared to seed. In pericarp, transcription regulator activity (GO:0030528), transcription factor activity (GO:0003700), transmembrane transporter activity (GO:0022857) and enzyme inhibitor activity (GO:0004857) were enriched under the atoxigenic strain infection in comparison with the toxigenic strain infection (. Peroxidase activity (GO:0004601), pectate lyase activity (GO:0030570), antioxidant activity (GO: 0016209) and hydrolase activity (GO:0004553) were enriched under the toxigenic strain infection in pericarp as compared to the atoxigenic strain infection , response to wounding (GO:0009611), response to external stimulus (GO:0009605), response to chemical stimulus (GO:0042221) and flavonoid biosynthesis (GO:0009813) were highly represented under the atoxigenic strain infection in pericarp. On the other hand, response to biotic stimulus (GO:0009607), response to stress (GO:0006950), regulation of defense response (GO:0031347), response to chitin (GO:0010200), defense response to fungus (GO:0050832) and signal transduction (GO:0007165) were enriched under the toxigenic strain infection in seed  metabolism was observed under the toxigenic strain infection in seed followed by the atoxigenic infection in pericarp  in pericarp and seed are shown in bstances , 42], w, wFig 7.bstances . The alkasmonate . This suesponses . The alp pathway , 74]. T. TFig 7.esponses . The bioS1 Fig).Validity of the next generation sequence data was confirmed by reverse-transcription PCR of 10 genes belonging to different functional categories with fold change expression of 5-fold or above (from sequence data) under experimental conditions relative to non-inoculated control. The results showed significant up-regulation of their mRNA accumulation under infection by the atoxigenic or the toxigenic strain infection in a tissue-dependent manner Click here for additional data file.S1 TableSheet 1, Nomenclature of genes and primer sequences used for expression analysis through RT-PCR; sheet 2, All differentially expressed genes discussed in the manuscript and used for heatmap in (XLSX)Click here for additional data file.


Background: The aim of this study was to determine the prevalence of virulence-associated genes and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) analysis of Campylobacter spp. isolated from children with diarrhea in Iran. Methods: A total of 200 stool specimens were obtained from children under 5 years during July 2012 to July 2013. Detection of C. jejuni and C. coli was performed by standard biochemical and molecular methods. The presence of virulence-associated genes and genetic diversity of isolates was examined using PCR and ERIC-PCR analyses. Results: A total of 12 (6%) Campylobacter spp. were isolated from patients including 10 (4.5%) C. jejuni and 2 (1.5%) C.coli. The flaA, cadF and ciaB genes were present in 100% of isolates, while no plasmid of virB11 gene was present in their genome. The prevalence of invasion-associated marker was 100% among C. coli and was not detected in C. jejuni isolates. The distribution of both pldA and the genes associated with cytolethal distending toxin (CDT) was 58.3% in C. jejuni isolates. Seven distinct ERIC-PCR profiles were distinguished in three clusters using ERIC-PCR analysis. Genotyping analysis showed a relative correlation with geographic location of patients and virulence gene content of isolates. Conclusion: To our knowledge, this is the first molecular survey of Campylobacter spp. in Iran concerning genotyping and virulence gene content of both C. jejuni and C. coli. ERIC-PCR revealed appropriate discriminatory power for clustering C. jejuni isolates with identical virulence gene content. However, more studies are needed to clearly understand the pathogenesis properties of specific genotypes.  Campylobacter spp. especially C. jejuni and C. coli are considered as potential etiological agents that caused many undiagnosed cases of acute diarrhea in children in developing countries including Iran [C. jejuni and C. coli virulence and also their diversity that leads to the practical applications to elucidate Campylobacter colonization and the control of this organism. Recurrent exposure to these organisms might raise level of specific immunity correlated with age in developing countries. Therefore, children younger than 5 years of age are mainly affected by these organisms [ing Iran -3. The prganisms . Campylobacter spp. are not applied in routine diagnostic programs of clinical laboratories in most developing countries. The pathogenicity of Campylobacter species is dependent on their ability to bind to the human intestinal cells and CadF protein. This protein, which is encoded by cadF gene, is responsible for Campylobacter binding to extracellular matrix of human intestinal cells [flaA, encodes a flagella protein which mediates motility, colonization, and invasion of gastrointestinal tract and it is essential for establishing human infection. Above organisms are fastidious and need nutrient-rich-based medium and microaerobic atmosphere . This real cells . AnotherciaB (an invasion protein), virB11 (the IV secretory system), and pldA (an outer membrane phospholipase A) genes encode proteins associated with increased bacterial invasion on cultured epithelial cells; however, their exact roles in invasion have remained to be elucidated [cdtA, cdtB, and cdtC. In epidemiology of infectious diseases, bacterial typing is of great value in source tracking studies. To analyze the genetic relatedness of C. jejuni, several molecular typing methods based on PCR have been developed. enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) has been shown high discriminatory power, good legibility, and ease of use in most epidemiological investigations. The ucidated . CytoletC. jejuni and C. coli isolated from children with diarrhea. CDT is one of the most characterized virulence factors in Campylobacter spp. Pathogenesis induces cell cycle arrest in G2 phase and promotes DNA damage together with apoptotic death in human monocytic cells; therefore, its presence is supposed to be associated with the severity of the disease.In this study, we aimed to determine the prevalence of virulence-associated genes and ERIC-PCR analysis of Study design and data collection procedure. The study was started after obtaining ethical approval from Research and Publication ethics office of Tarbiat Modares University . A total of 200 stool specimens were collected from children with acute diarrhea attended to two major Children’s Hospital Medical Center in Tehran from July 2012 to July 2013. All children under five years of age were included in this study. Children with persistent diarrhea or previous treatment with antibiotics in the last 5 days were excluded from the study. Acute diarrhea was defined as diarrhea which takes about 14 days or less. Demographic data were collected by a co-worker resident in hospital. All samples were transported to the laboratory in a modified Cary-Blair transport medium . Identification of C. jejuni and C. coli was performed by the standard culture, Gram staining, and conventional biochemical tests and confirmed by molecular methods [Confirmation of presumptive Campylobacter species by duplex-PCR. Samples were incubated at 42°C for 48-72 h in microaerophilic conditions onto modified charcoal cefoperazone deoxycholate agar medium  sulfate, 0.25 g/L sodium pyruvate, and 15 g/L agar, pH 7.4 ± 0.2 at 25º) plus campylobacter CCDA selective supplement . DNA templates were extracted by boiling method [Campylobacter spp. was performed by PCR amplification of cadF gene. A duple-PCR was applied for simultaneous detection of hipO and asp genes, specific to C. jejuni and C. coli, respectively [2, 0.1 μM each primer, 1 unit of Taq DNA polymerase, and deionized water. The primer sequences and their designation are shown in C. jejuni ATCC 29428 and C. coli ATCC 43478 were used as reference strains. ectively . PCR wasDetection of virulence/invasion-associated genes. The presence of virulence/invasion-associated genes, including invasion-associated marker (iam), pldA, and ciaB, (responsible for Campylobacter invasion and attachment), virB11 (involved in Campylobacter virulence), and CDT were investigated using specific primers which specifically amplify within the coding region of each gene. The distribution of flaA gene (responsible for Campylobacter attachment) was examined by primers specifically designed according to the flaA locus sequence of C. jejuni strain (GenBank accession no. AF050186.1). Due to the species-allele-specification of CDT sequence, two separate primer pairs were used which were selected according to cdt locus sequence of C. jejuni and C. coli standard strains. Enterobacterial repetitive intergenic consensus PCR. ERIC-PCR assay was performed according to the method introduced by Versalovic and colleagues [2, 0.2 μM each primer, and 1 unit Taq DNA polymerase. The reaction was placed in a DNA thermal cycler . ERIC-PCR patterns were analyzed based on the Dice similarity coefficient using GelClust software [lleagues . The prisoftware .Campylobacter spp., including 10 (4.5%) C. jejuni and 2 (1.5%) C. coli were isolated from 12 (6%) samples. The study population was made up of 200 children with acute diarrhea, including 110 (55%) male and 90 (45%) female with the mean age of 27.4 months (2.3 years). Among 200 stool samples, Duplex-PCR assay. The cadF gene was positive in 12 (100%) suspected cultures, and an amplification band of 400 bp was obtained for cadF+ isolates indicative of Campylobacter spp. Duplex-PCR for species identification indicated the presence of 10 (4.5%) C. jejuni and 2 (1.5%) C. coli isolates, respectively  involved in CDT production, and pldA was found in 7 (58.3%) of C. jejuni isolates corresponding to amplification bands of 631, 714, 524, and 913 bp, respectively. However, no CDT encoding genes were found among C. coli isolates. The plasmid virB11 gene was also absent among our Campylobacter spp. isolates. The prevalence of iam was 100% among C. coli, while no amplification was obtained for C. jejuni isolates  was reported to be 80-100% in different studies concerning Campylobacter spp. infections in children with moderate to severe diarrhea [ciaB and flaA are both involved in maximal invasion of human intestinal cells. This result can justify broadly existence of these gens in clinical Campylobacter spp. The prevalence of virulence-associated genes  was 58.3% in C. jejuni isolates, while none of the genes were detected among C. coli isolates. The CDT toxin induces cell cycle arrest in G2 phase and promotes DNA damage; therefore, its presence is supposed to be associated with the severity of the disease in C. jejuni. However, variations which may occur within cdt gene sequences and may affect their detection through amplification methods should not be ignored during interpretation of negative results in C. coli. Moreover, a direct correlation was observed in this study between detection of pldA gene and the presence of white and red blood cells in stool of patients, which may be due to contribution of pldA gene product in pathological changes in intestinal epithelium. In agreement with our results, Rizal and colleagues [cdt and pldA genes among 50% and 55% of C. jejuni isolates, respectively, while none of the genes were detected within their C. coli isolates. Seven distinct ERIC-PCR profiles were distinguished from 10 C. jejuni isolates which were located in three clusters by ERIC-PCR. Cluster analysis showed that all isolates (no. 4) within cluster I were isolated from patients who lived in the south of Tehran. Except one isolate, all others within cluster II (no. 3) have also isolated from the patients in a similar geographical location in Tehran (east and center). Moreover, all of the isolates in cluster I and II revealed identical virulence gene content. Two isolates within cluster III were isolated from west of Tehran, but no clear correlation could be determined between ERIC-PCR profile and virulence genes content of isolates within this cluster. Sahilah and colleagues [C. jejuni isolates. However, Wardak et al. [C. jejuni and C. coli into two clusters. The distribution of both lleagues  reportedlleagues  reportedk et al.  showed tC. coli isolates which raises the question that to how extent is the typeability power of ERIC-PCR for C. coli strains. This emphasizes that more studies are needed to clearly understand the ability and role of this typing method in C. coli epidemiological studies. However, it is noteworthy that ERIC-PCR analysis has been proved as a well-documented molecular tool in epidemio-logical studies of C. jejuni strains. ERIC-PCR was unable to type our C. jejuni and C. Coli genotypes in Iran. Nevertheless, further studies are needed to more clearly understand the correlation between virulence-associated genes and specific genotypes of C. jejuni and C. coli clinical isolates.To our knowledge, this is the first molecular survey of


Purpose. Helicobacter pylori is a common gastric disease-inducing pathogen. Although an increasing number of recent studies have shown that H. pylori is a risk factor for liver disease, the potential association between H. pylori infection and chronic hepatitis C still remains controversial. The aim of our meta-analysis was to evaluate a potential association between H. pylori infection and chronic hepatitis C. Methods. We searched the PubMed, Embase, CNKI, Web of Science, and the Cochrane Central Register of Controlled Trials (CENTRAL) databases between January 1, 1994, and May 1, 2015. Results. This study included a total of 1449 patients with chronic hepatitis C and 2377 control cases. The prevalence of H. pylori was significantly higher in patients with chronic hepatitis C than in those without chronic hepatitis C. The pooled odds ratio was 2.93. In a subgroup analysis, the odds ratios were 4.48 for hepatitis C virus- (HCV-) related cirrhosis and 5.45 for hepatocellular carcinoma. Conclusion. Our study found a strong association between H. pylori and chronic hepatitis C, particularly during the HCV progression stage; thus, we recommend active screening for H. pylori in patients with chronic hepatitis C. Helicobacter pylori (H. pylori) is a Gram-negative spiral-shaped bacteria that colonizes the gastric mucosa and can induce chronic gastritis, gastric ulcers, and gastric malignancy [ H. pylori [ H. pylori infection [ H. pylori infection correlates significantly with socioeconomic conditions and the age at infection. In addition, all H. pylori-infected individuals subsequently develop gastritis [lignancy . Worldwi. pylori . Both denfection . The ratastritis .Hepatitis C virus (HCV) was first recognized in 1989. To date, nearly 180 million people worldwide have been infected with HCV. If not well controlled, chronic hepatitis C (CHC) can progress into cirrhosis and ultimately into hepatocellular carcinoma (HCC) , 6, whic H. pylori may be a risk factor for the development of cirrhosis and HCC in patients with CHC [ H. pylori might not contribute to the mechanism of HCV-related HCC [ H. pylori and CHC remains controversial, and it is unclear whether H. pylori is associated with CHC and the progression of HCV-related cirrhosis and HCC. In this study, we aimed to confirm the associations between H. pylori and CHC and to further assess the relationship of H. pylori with HCV-related cirrhosis and HCC through a meta-analysis. Therefore, the results of this study might propose clinical medicine approaches for patients with CHC who might develop cirrhosis or HCC.Increasing evidence suggests thatwith CHC –10; howeated HCC , 12. TheWe searched PubMed, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), and Cochrane Central Register of Controlled Trials (CENTRAL) databases from January 1, 1994, to May 1, 2015. We used keywords or subject headings to search for “hepatitis C” and “helicobacter pylori or helicobacter species.” There were no language restrictions. We also screened bibliographies of selected original studies, review articles, and relevant conference abstracts. H. pylori-related morbidity between patients with CHC and a control group with nonhepatic disease. (2) Concrete numbers of cases and controls and a H. pylori positivity rate were included. (3) The study groups exhibited definite HCV positivity, whereas the control groups were HCV-negative. (4) H. pylori was detected in all study groups through serological or PCR testing. (5) Studies presenting information exclusively about patients undergoing liver transplantation, viral hepatitis , human immunodeficiency virus, acute HCV, autoimmune liver disease, nonalcoholic fatty liver disease (NAFLD), and other types of hepatitis were excluded.Citations were merged in Endnote version X7  to facilitate management. Two reviewers (J. Wang and Y.-X. Zheng) independently applied the inclusion criteria to all retrieved studies. Disagreements between reviewers were resolved by consensus. For each eligible study, the following criteria were set and reviewed independently by the reviewers: (1) Eligible study designs were randomized controlled trials (RCTs), case-control studies, or cross-sectional studies comparing H. pylori detection method, the number of cases and controls, the prevalence of H. pylori infection in cases and controls, and numbers of HCC, cirrhosis, and noncirrhosis cases among patients with CHC. Twelve studies, all case-control studies, were included after consensus was achieved between the two reviewers. The observational study quality was assessed using the Newcastle-Ottawa Scale (NOS) [For each eligible study, the following data elements were selected: first author, publication year, country,le (NOS) , 14. ThiI2 statistics. If significant heterogeneity  was found, the random-effect model was used for the analysis; if the heterogeneity was considered to be insignificant , on the other hand, the fixed-effect model was used. Studies with substantial heterogeneity (I2 > 50%) were considered unsuitable for the meta-analysis. Subgroup and sensitivity analyses were used to discover the source of heterogeneity. Confounding factors were segregated for further analysis.RevMan 5.2  was used to perform the meta-analysis. We used TSA, version 0.9 , to assess the required information size. Data were combined when appropriate and displayed in forest plots. Risk ratios (RRs) were used to assess risk in cohort studies; odds ratios (ORs) were provided for case-control studies and were regarded as approximate RRs in this meta-analysis. Study heterogeneity was tested using the chi-square test and Twelve case-control studies were included from among a total of 159 studies identified through online database searches. Most ineligible studies were excluded on the basis of information in the title or abstract. The selection and analysis process is shown in a flow diagram in P = 0.05) was determined, indicating a 2.93-fold higher H. pylori positive rate among patients with CHC than among healthy controls. Whereas all 12 studies exhibited significant heterogeneity  in the total analysis, we divided the included studies into two subgroups based on the H. pylori detection method. We found that the subtotal pooled OR of the PCR test subgroup was obviously higher than that of the serological test subgroup (4.78 versus 2.89) and the I2 values of both subgroups nearly 50% . The major source of interstudy heterogeneity might, therefore, have originated from other confounding factors. As shown in the study characteristics . According to the above information, patients with CHC appear to have a nearly 2.59-fold higher H. pylori positivity rate when compared to healthy controls. However, disease stage was the main factor influencing the H. pylori positive rate among patients with CHC. Therefore, further analysis of the extracted data according to HCV progression stage is needed.In the meta-analysis of 12 studies , a pooleeristics , three a H. pylori positivity rate among patients with CHC without cirrhosis was 1.97-fold higher than that of the healthy controls . The H. pylori positivity rate among patients with CHC having cirrhosis was 4.48-fold higher  than that of the control group, and the comparable rate among patients with HCV-related HCC was 5.45-fold higher . All I2 values for the above analyses were <50% , indicating that the studies exhibited low heterogeneity with respect to each other in the three meta-analyses. The extracted data were suitable for analysis, and the results were reliable because of the deep analysis enabled by disease stage ranking.To attenuate the influence of the HCV disease stage on the meta-analysis, we further generalized the extracted data from included studies to a noncirrhosis group, cirrhosis group, and HCC group, all of which were compared with the control group. Forest plots of all meta-analysis are shown in Z-curves to assess relationships with conventional significance boundaries (Z = ±1.96), the required information size, and the trial sequential monitoring boundaries. In our trial sequential analysis, the type I error risk was set at α = 0.05 with a power of 0.80. Relative risk reduction (RRR) was defined by the average incidence of included studies at 10%. Our determined required information size was 5407. In fact, the included number was 3826. From Z-curve did not reach the required information size (RIS), it crossed the trial sequential monitoring boundary and conventional boundary, which show the result was true-positive.Trial sequential analysis (TSA) combines conventional meta-analysis methodology with repeating significance testing methods applied to accumulating data in clinical trials. TSA uses cumulative A funnel plot is used to assess publication bias for outcomes >10. We included 12 studies in this meta-analysis. This evaluation is shown in H. pylori infection is a major global health issue. In addition, the World Health Organization has recognized H. pylori as a group 1 carcinogen contributing to gastric cancer since 1994 [ H. pylori and liver disease, particularly CHC [ H. pylori infection and CHC has been reported, it remains controversial. Several studies have shown a strong correlation between H. pylori infection and CHC and HCV-related disease. In contrast, other studies maintained that H. pylori infection does not correlate with CHC development. We conducted this study to assess the association between the H. pylori infection rate and CHC.nce 1994 . Furtherarly CHC . Althoug H. pylori among patients with CHC relative to controls. In other words, H. pylori infection is strongly associated with CHC. In addition, TSA provided firm evidence to support our results. Otherwise, the rate of H. pylori infection would continue to increase with CHC progression. This could implicate H. pylori as a risk factor for CHC progression. The role of H. pylori in CHC and CHC development were supported by considerable evidence. Sakr et al. also reported that the histopathological changes were more severe in patients with H. pyloripositive HCV than in patients with HCV alone [ H. pylori could increase sustained virological responses in patients with CHC [ in vitro demonstrated a cytopathic effect of H. pylori on hepatocytes [The result of our meta-analysis indicates a higher prevalence ofCV alone . Furtherwith CHC . Moreoveatocytes , 32. The H. pylori incidence rate (95% CI: 3.49–5.74) among patients with HCV-related cirrhosis relative to the control group, which strongly indicates a correlation between late-stage cirrhosis and H. pylori infection. However, two [ H. pylori infection. Furthermore, El-Masry et al. reported an increasing incidence of H. pylori as the Child-Pugh class increased [ H. pylori infection and the Child-Pugh class in patients with HCV-related cirrhosis.Moreover, the subgroup analysis demonstrated a 4.48-fold higherver, two , 19 of tncreased . Given t H. pylori and HCC. Many clinical observational studies have reported the detection of H. pylori in abnormal liver tissues, particularly cancerous tissues [ H. pylori and HCC. The patients with HCC in that study had diverse etiologies. Only two of the articles included in that study were related to HCV-positive HCC [Finally, the OR was 5.45-fold higher in the HCC group than the control group. This finding obviously indicates a strong link between tissues . A meta-tive HCC . TherefoOur study had some potential limitations. Firstly, the final results of this meta-analysis were greatly affected by the limitations of the included publications. Although we attempted to identify all related studies, it is possible that some escaped our attention. Furthermore, some studies with negative results might not have been published, which would cause unavoidable publication bias. In addition, all articles identified in our search were in the English and Chinese languages, which may have caused language bias. Regarding the control group source, most included studies used blood donors as controls, although some studies used hospital samples. This might have also generated bias. H. pylori detection methods differed among the included studies, a factor that might have led to different positivity rates. Some of the included studies detected and diagnosed H. pylori infection via the serology method, whereas others used PCR-based methods. Usually, the serology method is less sensitive and specific [ H. pylori and H. hepaticus belong to the Helicobacter genus [ H. pylori might share genetic sequences with other Helicobacter species such as H. hepaticus. Therefore, diagnosis of H. pylori infection via PCR alone is not sufficient.Secondly, thespecific . Meanwhier genus . H. pylo H. pylori infection [ H. pylori-infected individuals older than 40 years had a high risk of severe HCV-related disease [ H. pylori infection with respect to ages. Moreover, the patients in our study resided in different countries, including China, Italy, France, Egypt, Sweden, and Japan. Therefore, the H. pylori incidence might have varied with respect to socioeconomic differences. This might have caused the misestimating of the odds ratios.Thirdly, nearly all included articles were case-control studies. The quality of this type of study is lower than that of randomized clinical trials. In addition, both age and socioeconomic status have been considered high-risk factors fornfection . El-Masr disease . However H. pylori infection and CHC. In particular, it also revealed strong correlations of H. pylori infection with HCV-related cirrhosis and HCV-related HCC. We suggest the importance of H. pylori screening of patients with chronic hepatitis, particularly those with HCV-related cirrhosis and/or HCC. Obviously, given the limitations of the included studies, the role of H. pylori as a risk factor in the progression of CHC remains unclear. We recommend conducting additional randomized controlled trials and prospective studies to clarify the effect of this pathogen on CHC development.In conclusion, our meta-analysis demonstrated a positive association between


Auxenochlorella protothecoides and the non-photosynthetic, obligate heterotroph Prototheca wickerhamii. Remarkably, the plastid genome of A. protothecoides is only slightly larger than that of P. wickerhamii, making it among the smallest plastid genomes yet observed from photosynthetic green algae. Even more surprising, both algae have almost identical plastid genomic architectures and gene compositions (with the exception of genes involved in photosynthesis), implying that they are closely related. This close relationship was further supported by phylogenetic and substitution rate analyses, which suggest that the lineages giving rise to A. protothecoides and P. wickerhamii diverged from one another around six million years ago.The forfeiting of photosynthetic capabilities has occurred independently many times throughout eukaryotic evolution. But almost all non-photosynthetic plants and algae still retain a colorless plastid and an associated genome, which performs fundamental processes apart from photosynthesis. Unfortunately, little is known about the forces leading to photosynthetic loss; this is largely because there is a lack of data from transitional species. Here, we compare the plastid genomes of two “transitional” green algae: the photosynthetic, mixotrophic  Prototheca, Helicosporidium, Polytoma, and Polytomella.There is a diversity of feeding strategies across the tree of life. Photoautotrophs, for instance, produce organic materials through photosynthesis and, thus, do not require exogenous organic matter. Heterotrophs, alternatively, survive on organic components from the environment. In eukaryotes, photosynthesis occurs in the chloroplast, which evolved circa 1.5 billion years ago through the endosymbiosis of a cyanobacterium by a unicellular, heterotrophic protist13435789Helicosporidium sp. ATCC 50920, which is a parasite of various invertebratesHelicosporidium plastid DNA (ptDNA) is highly reduced (<40 kb), having lost various genes related to photosynthesis, and is similar in structure and content to the plastid genomes of apicomplexan parasites, such as Plasmodium falciparumHelicosporidium are from the non-photosynthetic trebouxiophyte genus ProtothecaPrototheca wickerhamiiHelicosporidium and various photosynthetic trebouxiophytes. The closest known photosynthetic relative of Prototheca species is Auxenochlorella protothecoides15161719A. protothecoides cells are switched to heterotrophic conditions, their plastids can degenerate, resulting in the suppression and eventual elimination of photosynthesis21A. protothecoides could provide insights into the loss of photosynthesis.Currently, the only complete plastid genome sequence available from non-photosynthetic green algae is that of the trebouxiophyte A. protothecoides and P. wickerhamii. Both genomes show a surprising amount of similarities, including severe ptDNA contraction and similar gene orders and gene contents, photosynthesis-related genes notwithstanding. Our phylogenetic inferences and other genomic analyses confirmed that A. protothecoides and P. wickerhamii are indeed closely related, with a recent divergence time of about six million years. Together, our results provide interesting clues about the loss of photosynthesis and the evolution of obligate heterotrophy within green algae.Here, in the hopes of better understanding the shift from a photoautotrophic to heterotrophic lifestyle, we report and compare the plastid genome sequences of A. protothecoides plastid genome is an 84.58 kb, AT-rich (69.2%), circular-mapping molecule , circular, AT-rich (68.8%), and compact (28.8% non-coding) (A. protothecoides and P. wickerhamii ptDNAs largely results from being no introns and relatively little intergenic DNA (P. wickerhamii (discussed below). Further contributing to the ptDNA streamlining in A. protothecoides and P. wickerhamii is the lack of plastid inverted repeat elements. The absence of these elements, however, is a reoccurring theme throughout the Trebouxiophyceae (The molecule . It has -coding) . These t-coding)  and gree-coding) . The genenic DNA . Moreoveophyceae .A. protothecoides and P. wickerhamii are not uncommon for green algae, especially non-photosynthetic species. In fact Helicosporidium sp. has one of the smallest ptDNAs ever observedA. protothecoides, the P. wickerhamii ptDNA is still larger and more expanded than that of Helicosporidium sp.  related to P. wickerhamii. The two algae are separated by relative short branch lengths, suggesting that they diverged from one another recently in evolutionary history. These results are consistent well with the phylogenies based on 18 s rRNA  and are in fact similar to those observed between other closely related algal strains or species2728P. wickerhamii ptDNA and its 17 relatives. We found that the overall similarities between the plastid genomes of P. wickerhamii and A. protothecoides are highest (The rate of synonymous substitution (Ks) can be used to estimate the divergent time among speciesis 0.816 . If we a highest  in all tPrototheca lineage following the loss of photosynthesis. Among 109 genes in the ptDNA of A. protothecoides ptDNA, 70 are also present in that of P. wickerhamii, meaning 39 genes were lost from the lineage of P. wickerhamii following its divergence from that of A. protothecoides  , with ambiguous functions but likely connected to photosynthesis3132P. wickerhamii ptDNA, as are cemA and ccsA, which encode a plastid envelope membrane proteinL(GAG)), trnS(GGA) and trnT(GGU)) have also been eliminated from the P. wickerhamii plastid. Significant gene content differences were also observed between P. wickerhamii and Helicosporidium sp.   and S5. dium sp.  and S4, P. wickerhamii, we analyzed the junctions flanking deleted genes and gene clusters relative to A. protothecoides. In total, 17 regions containing missing genes were identified (labeled breakpoint BP 1 to BP17), ranging from <0.5 kb to >7.5 kb  was purchased from the Culture Collection of Algae at the University of Göttingen, Germany (SAG) and cultured in malt peptone medium containing 10 g/L malt extract (Sigma) and 2.5 g/L proteose-peptone (Sigma).The P. wickerhamii plastid genome was obtained using Sanger chemistry and assembled with the SeqMan program from the DNASTAR Lasergene package. The sequencing primers were designed based on the partially sequenced genome A. protothecoides and P. wickerhamii plastid genomes have been deposited into GenBank under the access numbers KC843975 (A. protothecoides) and KJ001761 (P. wickerhamii).Both the C. variabilis NC64A and Helicosporidium plastid genomes were adjusted for comparison correspondingly. C. variabilis NC64A was reversed and started with atpI, while Helicosporidium sp. started with rpl12.The pairwise alignments were performed by Blastn  in bl2seq 2.2.23For mutation rate analyses, we did pairwise alignments using GeneWise for each orthologous geneE-value threshold of 1e-7), the blastp alignments were combined and filtered using SOLAR 0.9.6 , and the clustering was performed with Hcluster_sg 0.5.0 .We used the TreeFam methodologytufA, rpoC1, rps7, rps8, rps11, rps12, rps19, rpl2, rpl5, rpl14, rpl16, rpl20) from 26 Chlorophyta taxa were defined. The amino acid sequences were aligned with MUSCLE 3.7A total of 12 single-copy TreeFam protein-encoding gene clusters .