M. intracellulare was the dominant species (40.5%), followed by M. abscessus (28.4%). 109 patients, with 185 total isolates, had full clinical records available for review. 84.4% (38/45), 85.7% (24/28%) and 63.6% (7/11) of patients with isolation of M. intracellulare, M. abscessus and M. kansasii, respectively, were categorized as definite NTM disease. Whereas all the 10 patients with isolation of M. gordonae were defined as unlikely NTM disease. The majority of NTMs isolates yielded from respiratory specimens in Beijing Chest Hospital were clinically significant, and M. intracellulare and M. abscessus was the dominated species of NTM lung disease. NTM lung infections demonstrated some specific chest radiograph characteristics.The clinical relevance of non-tuberculous mycobacteria (NTM) has been reported to be different dramatically by species or by regions, however, no such evaluation has been performed in China.A retrospective study was performed in Beijing Chest Hospital. All the NTM strains isolated from respiratory specimens in the past 5 years, and patients’ clinical records (symptoms and radiographic information etc.) were investigated. The clinical relevance was evaluated according to the criteria recommended by the American Thoracic society. Totally 232 NTM strains were recruited, among them,  Mycobacterium tuberculosis789In recent years, the frequency of nontuberculous mycobacteria (NTM) isolation from respiratory specimens, as well as the number of NTM infection patient, has increased rapidly in many countries1234In China, tuberculosis remains a serious public health problem and as a consequence, patients with positive outcomes of acid-fast bacilli (AFB) smear test or with isolation of mycobacteria by culture for sputum specimens have been routinely diagnosed as pulmonary tuberculosis and been administrated with anti-tuberculosis drugsWe performed an institution-based observational study for NTM lung disease in Beijing Chest Hospital , and investigated the clinical, microbiological and radiographic information of possible NTM infection patients from 2010 to 2015. The purpose of the current study was to determine the isolation frequency of different NTM species from respiratory specimen over a 5-year period, and to evaluate their clinical significance.All NTM strains isolated from respiratory specimens in Beijing Chest Hospital over a 5-year period from May 2010 to May 2015 were retrospectively analyzed. The medical records of patients from whom NTM strains isolated were retrospectively reviewed. Informed consent was obtained from all subjects included in the study. The protocols and procedures for the protection of human subjects were approved by the Ethics Committee of Beijing Chest Hospital. Furthermore, all the methods were carried out in accordance with the approved guidelines.Sputum and bronchial washing specimens were decontaminated using the N-acetyl-L-cysteine 2% NaOH method. Löwenstein-Jensen medium culture or liquid culture using MGIT 960 system , or both methods were performed to recover mycobacteria. Mycobacterial growth in PNB-containing medium was used as a presumptive test for NTM screening.rpoB and hsp65 genes. Preparation of the PCR amplification and DNA sequencing were done as described previouslyNTM isolates were identified to the species level by sequence alignment of 16S rDNA, 16-23S rRNA gene internal transcribed spacer (ITS), M. gordonae and M. terrae isolations, well-known environmental contaminants were considered unlikely in relation to the lung diseases regardless of the other manifestations14Patient with clinical record containing symptom, radiographic and microbiological information was considered clinically evaluable and classified as having definite, probable, or unlikely NTM lung disease according to the diagnostic criteria for NTM lung disease issued by American Thoracic Society(ATS) in 2007Two TB specialists simultaneously analyzed the radiographic features of chest computed tomography (CT) scan. The definite NTM lung disease was classified into three forms, according to CT findings. The upper lobe cavitary form was defined as a combination of cavities, consolidation, and pleural thickening in the upper lobes, regardless of reticulonodular opacities. The nodular bronchiectatic form was defined as unilateral or bilateral bronchiectasis and nodular changes without visible cavities in the upper lobes. For nodular bronchiectatic form, the implication of lingular segment or the right middle lobe was investigated. Patient’s disease remained unclassified if it belonged neither to the upper lobe cavitary nor the nodular bronchiectatic form. In the unclassifiable form, multifocal lobular or segmental consolidations usually comprised the predominant findingt-tests respectively. P values of less than 0.05 were considered to indicate statistical significance.All statistical analyses were performed using SPSS22.0 software . Comparisons of categorical variables and numerical variables were performed using the Pearson Chi-squared tests or M. intracellulare , M. abscessus , M. kansasii , M. fortuitum , M. avium , M. gordonae , M. szulgai , M. terrae, M. simiae , M. parascrofulaceum  and M. neoaurum . 226 strains were isolated from sputa, 5 strains from bronchial washings and 1 strain from lung tissue.From May 2010 to May 2015, a total of 232 NTM isolates were reported from respiratory specimens which included M. gordonae was isolated from sputum sample of one patient who acquired M. abscessus-associated lung disease during antibiotic treatment. This strain was excluded from further analysis. The implicated NTM organisms are shown in Among those 232 isolates, 109 patients which accounted for 185 NTM isolates had full clinical records available for review. All patients were free of human immunodeficiency virus infection. Patients were then grouped into the three disease categories based on criteria cited above. Of the 109 patients, 72 (66.1%) had definite diseases, 27 (24.8%) had probable disease, and 10 (9.2%) had unlikely disease. M. intracellulare (84.4%), and M. abscessus (85.7%) were finally categorized as definite disease, while all the isolations of M. intracellulare, M. abscessus, M. kansasi, M. kansasii, M. avium and M. szulgai were defined as clinically significant. On the other hand, none of the 10 patients, with M. gordonae isolation, were considered to have relation with disease.NTM isolation demonstrated extremely high clinical significance among the 109 enrolled patients as 99 (90.8%) were defined as definite or probable disease. The absolute majority of patients with isolation of Characteristics of the patients in current study are summarized in M. intracellulare lung disease had more tendency to infect the lingular segment and the right middle lobe compared with M. abscessus lung disease (72.7% (16/22) vs 26.3% (5/19), p < 0.05).Among 72 patients with definite NTM disease, 29 had the upper lobe cavitary form , 2, 3, 4M. intracellulare and M. avium) are the most common NTM isolates. On the other hand, M. avium is predominant in North- and South America, M. intracellulare is most frequently isolated in Australia-Queensland, South Africa and some provinces in China62021M. intracellulare was the most common species of NTM isolation, followed by M. abscessus in Beijing Chest Hospital which is located in north of China.NTM have been implicated in an increasingly large proportion of pulmonary disease throughout the world, in both immunocompetent and immunocompromised hosts161718M. intracellulare was the most common NTM species (40.5%) isolated from clinical respiratory specimens, and it was also the most common pathogen (52.8%) of NTM lung disease in our institution. Although in some areas M. intracellulare is less likely to cause NTM lung diseaseM. intracellulare is the predominant bacteria for NTM lung disease in Beijing Chest Hospital. (3) M. abscessus was the second most commonly isolated species (28.4%) and represented the second most common pathogen (33.3%). Since M. intracellulare and M. abscessus lung diseases had the most poor prognosis among all the NTM lung diseases, according to our outcomes, the NTM lung disease treatment will be very challenging in China as those two species account for the majority of NTM lung diseases13It is believed that NTM disease is under-reported in the tuberculosis-endemic countries. The main reason could be the high burden of tuberculosis which attracts bulk of the attention of clinicians. In our knowledge, this is the first research to document the clinical significance of NTM isolates from respiratory specimens in China, a high TB burden country. The major findings of this study are as following: (1) NTM strains isolated from respiratory specimens demonstrated very good relation with lung infections (90.9%). (2) M. kansasii is the second most common (approximately 20%) type of NTM pulmonary disease in Japan and USA25M. kansasii is the most frequently identified isolate, accounting for 45% of all NTM isolatesAlthough the incidence of pulmonary disease caused by M. gordonae in this study, which warns clinicians to interpret NTM recovery by MGIT960 systems with cautions, especially for M. gordonae.Among the definite, probable and unlikely NTM lung disease, there were no significant differences in the sex, mean age, BMI, presence of underlying disease and symptoms, but fewer patients with unlikely NTM lung disease were sputum AFB smear positive. It is possible that unlikely NTM lung diseases have less bacterial load, however with a more sensitive method, such as liquid culture, the chances to recover it can increase. 8 out of the 10 unlikely NTM infection cases had isolations recovered by MGIT960 only, which was consistent with this assumption. At a hospital in USA, the dramatic increase of NTM isolation was attributed to the more sensitive liquid culture technique, which led to less clinical significance of NTM isolationsM. fortuitum. A study in Japan also found that among 26 patients with a minimum of two M. fortuitum isolations, none showed clinical aggravation during the follow-up periodCompared with reports from other countries1415M. intracellulare lung diseases were more likely to be implicated in the lingular segment and the right middle lobe in contrast with M. abscessus lung diseases.NTM lung infection may have some specific image characteristics for a CT scan. Among patients with definite NTM lung disease, fewer cases have unclassifiable form for radiographic features compared with patients with probable and unlikely NTM disease. Based on our research, cases without typical nodular bronchiectatic and upper lobe cavitary forms are less likely to be related to NTM lung disease. The position of nodular bronchiectatic lesion demonstrated species-specific characteristics. M. intracellulare and M. abscessus. NTM lung infections frequently demonstrated the upper lobe cavitary form or nodular bronchiectatic form for chest radiograph examination. However, as NTM only accounted for 2.6% of all the mycobacterial isolations in our hospital according to previous workIn conclusion, a substantial proportion (90.9%) of patients, from whom NTM isolates were recovered, exhibited definite or probable NTM lung disease in our institution. The most common etiologies of NTM lung disease included How to cite this article: Duan, H. et al. Clinical Significance of Nontuberculous Mycobacteria Isolated From Respiratory Specimens in a Chinese Tuberculosis Tertiary Care Center. Sci. Rep.6, 36299; doi: 10.1038/srep36299 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Euphrasia (Orobanchaceae), a group with multiple ploidy levels, frequent self-fertilization, young species divergence and widespread hybridization. We use a phylogenetic approach to investigate the colonization history of British Euphrasia, followed by a DNA barcoding survey and population genetic analyses to reveal the causes of shared sequence variation. Phylogenetic analysis shows Euphrasia have colonized Britain from mainland Europe on multiple occasions. DNA barcoding reveals that no British Euphrasia species has a consistent diagnostic sequence profile, and instead, plastid haplotypes are either widespread across species, or are population specific. The partitioning of nuclear genetic variation suggests differences in ploidy act as a barrier to gene exchange, while the divergence between diploid and tetraploid ITS sequences supports the polyploids being allotetraploid in origin. Overall, these results show that even when lacking species-level resolution, analyses of DNA barcoding data can reveal evolutionary patterns in taxonomically complex genera.DNA barcoding is emerging as a useful tool not only for species identification but also for studying evolutionary and ecological processes. Although plant DNA barcodes do not always provide species-level resolution, the generation of large DNA barcode data sets can provide insights into the mechanisms underlying the generation of species diversity. Here, we study evolutionary processes in taxonomically complex British  Euphrasia, and while we find no species with a diagnostic sequence profile, we do see evolutionary patterns, such as divergence between two different species groups with different chromosome numbers. This shows DNA barcoding can be useful for studying evolutionary patterns in complex plant groups.The sequencing of short standardized pieces of DNA (DNA barcodes) is commonly used to tell species apart. This approach is thought to be of limited value in complex plant groups where species have evolved recently. We use DNA barcoding in taxonomically complex British  Overall, these results are used to improve our understanding of the evolution of a complex regional plant assembly, and to test the efficacy of DNA barcoding for studying species-level variation in a taxonomically complex group.Previous broad-scale surveys of Euphrasia species are all annual, selfing or mixed-mating small herbaceous plants, which occur in a range of habitats including coastal turf, chalk downland, mountain ridges and heather moorland , which has been suggested to be incorporated into the core DNA barcode  following the manufacturer’s protocol, but with an extended incubation of 1 h at 65 °C. These DNA samples were added to existing DNA extractions of 68 individuals from We performed PCRs in 10 μL reactions, with DNA amplification and PCR conditions for each primer given in We assembled, manually edited and aligned sequences using Geneious v. 8 . We scored indels as unordered binary characters and appended them to the matrices. We used gap coding as implemented in Gapcoder , with inEuphrasia samples from https://treebase.org).We used Bayesian phylogenetic analyses in MrBayes v. 3.1.2  to inferθ (per site), Tajima’s D and divergence between ploidy levels (DXY).We examined patterns of sequence variation using a range of population genetic methods. We investigated the amount of sequence diversity across species using descriptive statistics, and then tested the cohesiveness of taxa using analysis of molecular variance (AMOVA) and related methods. Analyses were performed separately on ITS2 and a concatenated matrix of either all sampled plastid loci, or just the core DNA barcode loci. For plastid data, haplotypes were determined from nucleotide substitutions and indels of the aligned sequences. Basic population genetic statistics were performed in Arlequin Version 3.0 , and thiFCT) for a given number of genetic clusters . We considered the best grouping to have the highest FCT value after 100 repetitions. This analysis investigated interspecific differentiation, thus only used species samples, excluding hybrids.Genetic divergence among sampling localities was explored with spatial analysis of molecular variance SAMOVA; . Spatialhttp://www.fluxus-engineering.com/), treating gaps as single evolutionary events.The relationships between haplotypes were inferred by constructing median-joining networks . The only non-diploid in the clade is one individual of tetraploid British E. ostenfeldii, which appears to be correctly identified and thus may have captured the diploid ITS variant through hybridization. Overall, terminal branches of the tree are short, indicative of limited divergence. The only exception was the long branch of E. disperma from New Zealand, a result seen in previous Bayesian analyses , trnL-trnF  and atpB-rbcL . The plastid tree (E. borneensis (Borneo) and E. fedtschenkoana (Tian Shan). This clade received moderate support in our analysis (pp = 0.85). While partially informative of broad-scale relationships, most terminal branches were extremely short, and gave no information on interspecific relationships.The final concatenated plastid alignment was 1692 bp in length, for a total of 82 tid tree  recovereE. scottica) presented double peaks, and were excluded from analyses. Overall diversity across taxa was modest, with a nucleotide diversity (π) of 2.3 %, and θ (per site) of 0.01781. There were 33 nucleotide substitutions and one indel, from which we called 23 alleles.The final ITS2 alignment contained 130 individuals representative of all 19 British taxa, and was 380 bp in length. Only two samples  obligate coastal ‘E. fharaidensis’. Overall, 86 % of taxa had one of six widespread alleles. There were also a large number of rare variants, with over two-thirds restricted to a single population  was not significantly different from zero.The ITS2 data revealed strong partitioning by ploidy. Of the 23 alleles, three  were restricted to diploids, and 19 to tetraploids, with only one allele (H2) shared across ploidy levels . H2 was E. vigursii and E. rivularis, possessed allele H1, which is common to other diploid taxa, or population-specific variants , but no tetraploid-specific allelic variation. The two sampled diploid–tetraploid hybrids  possessed the full range of alleles: H2, which is common across ploidy levels, tetraploid-specific H3 and diploid-specific H1. Most (9/12) tetraploid hybrid populations had alleles shared with their putative parents, while the other populations had unique alleles.The putative hybrid species, FCT value in the SAMOVA was when K = 2 K = 3, SAMOVA distinguished clusters corresponding to the two ploidy groups, and a third group of hybrid species derived from inter-ploidy mating. Analysis of molecular variance also supported the strong division by ploidy, with 88.2 % of variation attributed to ploidy differences (P < 0.001). A high proportion of variation was also partitioned by taxa (63.2 %), and regions (25 %), though this may be inflated by limited sampling within species.The highest DXY = 0.051 (5.1 %). The phylogenetic network revealed broad clusters that largely corresponded to diploids and tetraploids, separated by many mutations , surrounded by other widespread tetraploid variants , and singleton variants.Summary statistics and a network analysis revealed substantial divergence between diploid and tetraploid variants. An average of 18.3 site differences were found between unique diploid and tetraploid alleles, with divergence measured as utations . The diprbcL in 48 samples revealed no polymorphism and was excluded from subsequent analyses. The final matK alignment was 844 bp with one indel, and revealed 17 haplotypes. Our supplemental locus, the rpl32-trnL region, was 630 bp with nine indels. The final concatenated alignment of variable plastid loci (matK + rpl32-trnL) was 1474 bp for 130 samples, with 2.7 % segregating sites (40 sites) across 38 haplotypes. Nucleotide diversity was exceptionally low with π = 0.3 %, and θ (per site) was similarly low at 0.00381. Tajima’s D was not significantly different from zero (−0.27).Initial sequencing of E. arctica (and its hybrids), E. foulaensis and E. micrantha (and its hybrids), while H10 is also shared across three species in Scotland. Results from just the barcoding locus matK were consistent with the concatenated alignment of variable plastid loci [see Similar to ITS, most plastid haplotypes were individual or population specific , with only four haplotypes being widespread . As such, clade divergence, and much of the speciation present within British Euphrasia, long pre-dates recent glacial divergence and the origin of young British endemic taxa. Overall, this suggests that British Euphrasia diversity has been assembled from a diverse pool of European and amphi-Atlantic taxa. Evidence for recurrent rounds of colonization from continental Europe have also been seen in phylogeographic studies of various other taxa, in particular tree species such as Fagus sylvatica , and where widespread are either shared across species within a ploidy level (for ITS) or across ploidy levels and species (plastid DNA). Patterns of shared sequences are often surprising, including between geographically disparate populations 100+ km apart, and between ecologically specialized taxa that seldom co-occur in the wild.Taxonomically complex Euphrasia, which generally show similar patterns of mutation to autotrophic taxa . P.M.H. and M.R. acknowledge funding from the Scottish Government’s Rural and Environment Science and Analytical Services Division (RESAS). Research by G.G. is supported by the Research Council of Norway: Projects N248799 and N257642; and Norwegian Taxonomy Initiative: Project N70184215. Research by A.D.T. is supported by a Natural Environment Research Council (NERC) Fellowship NE/L011336/1.A.D.T and P.M.H. conceived and designed the study. A.D.T., G.G., N.d.V. and C.M. provided samples. C.M. identified the specimens. X.W. and M.R. performed the sequencing. X.W., G.G., M.R. and A.D.T analysed the data. A.D.T. and X.W. wrote the manuscript, with comments from all other authors.None declared.The following additional information is available in the online version of this article—Table S1. Voucher information and population details of British Euphrasia samples used for UK DNA barcoding. The cpDNA regions include matK and rpl32-trnL, with rbcL generated for a subset of taxa but excluded from the final analysis (see main text). The columns headed ‘cpDNA’ and ‘ITS 2’ indicate whether the sample was successfully sequenced (1) or not (0). Taxa with an asterisk were also included in the global phylogenetic analysis.Table S2. PCR conditions and primer sequences for regions sequenced in this study. Loci used for DNA barcoding were matK, rbcL and ITS2, supplemented with rpl32-trnL. Regions used for phylogenetic analysis were atpB-rbcL, trnL intron, trnL-trnF and ITS.Table S3. Spatial analysis of molecular variation (SAMOVA) of ITS sequence data across British Euphrasia populations.Table S4. Plastid haplotype frequencies across British Euphrasia species.Table S5. Spatial analysis of molecular variation (SAMOVA) of plastid sequence data across British Euphrasia populations.Table S6. The distribution of matK haplotypes for populations of British Euphrasia. The cpDNA (n) column indicates the sample sizes.Table S7. Hierarchical analysis of molecular variance (AMOVA) for matK sequenced in British Euphrasia populations.Supplementary TablesClick here for additional data file.


P = 0.02), while GG genotype carriers had a higher risk of HCC  than those with CC and CG genotypes in healthy group. Rs187238 GG genotype increased the risk from CHB to LC status , meanwhile the trend also existed by controlling confounding factors . Haplotype Crs187238Trs1946518 moderately decreased the risk of CHB carriers developing into HCC  after adjustment. In conclusion, IL-18 rs187238 GG genotype may increase the risk of HCC in healthy population and the risk of LC in CHB carriers.IL-18 polymorphisms influence the transcriptional activity of the IL-18 gene and associated with various diseases. However, their relationships with hepatitis B virus-related liver diseases had not reached a consensus. So we conducted this case-control study with a view to clarifying the association. We included four groups: healthy controls, chronic hepatitis B virus (CHB) carriers, liver cirrhosis (LC) and hepatocellular carcinoma (HCC) groups with each group of 250 persons. Odd ratios (ORs) and 95% confidence intervals (95%CIs) with or without adjustment were calculated. Haplotype analysis was also performed. The results showed people carrying rs187238 CG genotype had a lower risk of LC (CG vs. CC: OR = 0.59, 95%CI = 0.38–0.91,  From 1990 to 2013, ranking of viral hepatitis on leading death causes worldwide rose from the tenth to seventh . Genomic+ T cells depending on its cytokine milieu [+ T cells can contribute directly to disease pathogenesis and inhibit viral replication during HBV infection [Interleukin-18 (IL-18), firstly described as interferon (IFN)-gamma inducing factor, is one of the members of the IL-1 cytokine superfamily . IL-18 me milieu . CD4+ T nfection . Cytotoxnfection . IL-18 pnfection . All thenfection . Previounfection –19. Rs19nfection . Hass SLnfection . Li Y etnfection .Several researches have studied the impact of the two polymorphisms on HBV-induced liver diseases, but no consistent conclusions had been achieved –26. The P = 0.002) and diabetes history (P = 0.021), while they had no meaningful difference in smoking history (P = 0.100) and family history (P = 0.647). Furthermore, patients carrying CHB related liver diseases had higher laboratory parameters than healthy controls, including T-Bil, ALT and AST levels (P <0.001), which are evidences of liver injury [The included subjects were equally categorized into four groups with 250 participants, healthy controls, CHB, CHB-positive LC and CHB-positive HCC Table . The aver injury .P = 0.142; rs1946518: P = 0.90). A LC patient and five HCC patients were failed to detect 187328 genotype, and three patients with HCC was failed to detect rs1946518 polymorphism. For rs187238 polymorphism, as displayed in Table The alleles and genotype distributions of IL-18 polymorphisms were shown in Table P = 0.02). The genotype GG at187238 was more frequent in LC patients than CHB patients (2% vs. 0.8%) and logistic regression analyses showed a higher LC risk in CHB carriers with rs187238 GG genotype than individuals with CG or CC genotypes . However, after controlling for gender, age, smoking and alcohol consumption, the result indicated no significant difference in rs187238 polymorphism between LC and health controls  were analyzed. As shown in Table rs187238Trs1946518 moderately decreased the risk of CHB carriers developing into HCC  were suggested to associate with different expression of IL-18 by causing differences in transcription factor binding . Rs18723Recently, the association between the two polymorphisms (rs187238 and rs1946518 polymorphisms) and HBV-related liver diseases were frequently researched. Lau HK et al. included 559 healthy controls and 342 HCC patients to investigate the relationship and finally found rs187238 GC and CC genotypes not only increased the risk of HCC, but also was responsible for vascular invasion and enhanced the prognosis in HBV-related HCC . On the Different from the previous studies, we enrolled 1000 people who were at different stages from health to HCC. The four groups did differ significantly in alcohol history and diabetes history. For LC patients carrying HBV, heavy alcohol consumption is a independent increased risk of HCC . Lin CW Our results should be interpreted with caution because of several limitations. Firstly, though we recruited 1000 samples in this study, the sample size of each group was relative small which may restrict its detail subgroup analysis by the clinical index. Secondly, considering we just controlled four factors , other factors including environmental background, treatment protocols and living habits may cause some bias. Thirdly, all participants were all from Shaanxi Province, China, which may not stand for all the Chinese population. Lastly, the fundamental experiments should be further conducted to validate our results and explore the possible mechanism.In conclusion, our research revealed that rs187238 GG genotype increased the risk of HCC in healthy population and the risk of LC in CHB carriers. However, rs1946518 polymorphism had no impact on CHB-related liver diseases. These results indicated that IL-18 rs187238 polymorphism may be involved in the progression of CHB-related diseases in Northwest Chinese population.This study was approved by the Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University . The research protocol was completed according to the approved guidelines.For the current analysis, we established a case-control study of 250 healthy controls, 250 CHB carriers, 250 LC patients and 250 HCC patients. HBV infection was detected by ELISA method that requiring positivity of serum HBsAg and serum negativity for anti-HCV. LC patients and HCC patients were confirmed by pathological diagnosed. All the subjects were from the Second Affiliated Hospital of Xi’an Jiaotong University, and Xijing Hospital of Fourth Military Medical University, Xi’an, Shaanxi Province, China. Every participant was interviewed in-person including questions on age, demographics, ethnicity, height and weight, smoking and drinking habits, and medical history. Blood samples were collected after interview.The samples were centrifugated and stored in −80°C freezers for long-term storage. The genomic DNA was extracted and concentrated by the method described in our previous studies , 49. Twoχ2 test and the degree of freedom was 1 in the cases and controls. The significant difference in allele and genotype frequencies between cases and controls was determined by Pearson's χ2 test. ORs and 95%CIs were calculated with and without adjustment for gender, age, smoking and drinking. We evaluated the risk in the dominant model (AA+ Aa vs. aa), the recessive model (aa vs. Aa+AA), and the allele model (a vs. A) respectively . A two-sided P-value < 0.05 was considered statistically significant in all the tests.All the statistical analyses were completed using the SPSS software package . HWE was examined by comparing expected and observed frequencies using Alrlquin 3.1 program . The genotype frequencies of observed values were compared with expected values obtained from HWE theory . Haplotype analysis was performed by PHASE v2.1 software. The calculation was performed by


Agrotis segetum (Lepidoptera: Noctuidae) in Northeast China. More specifically, pollen grains were dislodged from 2566 A. segetum long-distance migrants captured on Beihuang Island (Bohai Sea) and identified to many (plant) species level. Pollen belonged to 26 families of plants, including Fagaceae, Oleaceae, Leguminosae, Asteraceae, Pinaceae and Rosaceae, including common species such as Citrus sinensis, Olea europaea, Ligustrum lucidum, Robinia pseudoacacia, Castanopsis echinocarpa, Melia azedarach and Castanea henryi. As the above plants are indigenous to southern climes, we deduce that A. segetum forage on plants in those locales prior to engaging in northward spring migration. Our work validates the use of DNA-assisted approaches in lepidopteran pollination ecology research and provides unique and valuable information on the adult feeding range and geographical origin of A. segetum. Our findings also enable targeted (area-wide) pest management interventions or guide the future isolation of volatile attractants.Pollen grains are regularly used as markers to determine an insect’s movement patterns or host (plant) feeding behavior, yet conventional morphology-based pollen grain analysis  encounters a number of important limitations. In the present study, we combine conventional analytical approaches with DNA meta-barcoding to identify pollen grains attached to migrating adults of the turnip moth,  Plant-pollinator interactions can reveal co-evolutionary processes in both animal and plant communities . There aAside from revealing plant-pollinator linkages, pollen can equally serve as a natural marking material to determine an insect’s host plant range and its related geographical origin or movement patterns in time or space ,9. PollePollen identification is done with the aid of a light microscope (LM) or scanning electron microscope (SEM) and guided by pollen identification keys, micrographs and reference collections. Though LM-based pollen identification is constrained by its low level of resolution, the drawback to using SEM for pollen analyses is the regular absence of reference micrographs and its increased cost and time requirements ,12. FurtHeliconius and Laparus have evolved adaptations to pollen-feeding [Helicoverpa zea in Arkansas [Agrotis ipsilon and Pseudaletia unipuncta in Iowa and Missouri [H. zea and Trichoplusia ni collected in Oklahoma [Molecular-guided palynology equally carries promise to illuminate aspects of the ecology of Lepidoptera. Although most lepidopteran species are herbivorous during the larval stage , their a-feeding , the maj-feeding ,26. AlsoArkansas , AgrotisMissouri  and H. zOklahoma . In the Agrotis segetum Denis and Schiffermaller (Lepidoptera: Noctuidae), is a pest of wide geographic distribution and agricultural importance [A. segetum undertake seasonal migration [A. segetum moths. Our work validates the use of DNA-assisted palynology in aerobiology and lepidopteran ecological research and has important implications for future (area-wide) pest management of A. segetum and other migratory lepidopteran pests.The turnip moth, portance ,30,31. Pigration . HoweverA. segetum, of which 17.03% had adhering pollen grains on the proboscis. For the majority of individuals which adhered pollen by body , pollen grains of one single plant species adhered to the adult body, while the remainder of individuals harbored grains of multiple species. We recorded a total of 40 pollen grains from at least 26 families. Using a combination of DNA sequences and pollen morphology, 12 of the 40 samples were identified to species level: Castanea mollissima Blume, Pterocarya rhoifolia Siebold et Zucc., Olea europaea L., Amorpha fruticosa Linn., Ligustrum lucidum Ait., Robinia pseudoacacia L., Castanopsis echinocarpa Miq., Citrus sinensis Blanco, Melia azedarach L., Elaeagnus umbellata Thunb., Chenopodium album L. and Adenophora trachelioides Maxim  or family level . The respective success rates for species-level and genus-level identification using a combination of pollen morphology, DNA meta-barcoding and distribution data were 30% and 70%. The exclusive use of DNA barcoding had a respective success rate of 12.5% and 70% and the reliance upon pollen morphology yielded correct results in a respective 15% and 75% of cases.Over the course of the study, we collected a respective total of 1380 and 1186 male and female adult es Maxim  and 16 oA. segetum adults that were captured and analyzed for pollen grains, variable annual percentages of pollen-carrying individuals were recorded . Overall, 437 pollen grains representing 26 families, 18 genera and 12 species were found in the samples   14–2017) , with 2514–2017) .A. segetum migrant individuals did not differ among successive stages of the year-long migration season . Meanwhile, the number of pollen taxa obtained from A. segetum during May and June was significantly higher than during other stages of the migration season   .A. segetum. In early-season, pollen grains from Pinaceae (31.9%), Leguminosae (10.34%) and Oleaceae (9.48%) were most commonly recorded  or Monocotyledons . Also, the proportion of herbaceous plants was significantly higher than woody plants  (Pollen grains that were collected from migrant < 0.007) .A. segetum.Pollen grains have been commonly used as a natural marker to reveal foraging patterns or host plant associations for different types of pollinators ,4,24. InA. segetum adults carry pollen grains from 24 families within the angiosperms and two different gymnosperm families. This is in line with other records of angiosperm plants as common (adult feeding) host plants for Noctuid moths [A. segetum adults may also visit gymnosperm plants and eat their pollen and/or nectar. This result was similar to that for A. ipsilon [A. ipsilon [Mythimna separate [Heliconius ethilla, in which females consistently carry the largest pollen load [Heliconius populations would not require that a male’s due to daily spermatophore production, so females carry the largest pollen load than that of males [A. segetum adults visit herbaceous plants more often than woody plants, which differs from A. ipsilon [M. separate [H. zea, S. exigua. [A. segetum preference for certain host plants and their relative contribution to population dynamics.More specifically, we showed that migrating id moths ,28,33,34len load , howeverseparate ,37. Poll exigua. ,39. For H. zea, A. ipsilon and P. unipuncta adults in northern parts of the United States carried exotic pollens from Texas, suggestive of a migration range that surpasses several hundred km [A. segetum is equally a long-distance migratory pest [C. sinensis, M. azedarach, O. europaea, L. lucidum, R. pseudoacacia, C. echinocarpa and C. henryi) revealed how A. segetum adults captured in Beihuang island could have migrated over distances >800 km [Aside from revealing plant-pollinator linkages, palynology can also help determine an insect’s migration routes, geographical origin, habitat association and the diversity of (adult) food sources ,12,40. Pndred km ,27. Prevory pest . Our det >800 km . Early-sH. zea and other noctuid moths [Meanwhile, the above could equally explain why spring-collected moths had significantly more adhering pollen grains as compared to summer or autumn migrants. Similar patterns were observed for id moths ,28,33,34id moths ,28. HoweA. segetum migrant individuals, with increased importance of Pinaceae, Brassicaceae and Asteraceae during early-, mid- and late-season, respectively. These temporal shifts in the relative importance of particular plant taxa for A. segetum populations can be related to climatic conditions and geographic location influencing presence of particular plants and species-specific flowering or pollen-shedding regimes. While the greatest diversity of flowering plants occurs during the spring and most Rosaceae bloom during this season, flowering Asteraceae are relatively more important during the fall. Though Leguminosae, Brassicaceae and Asteraceae had been identified as primary foraging resources for A. segetum [A. segetum.The relative importance of a particular (plant) taxon as a foraging resource can be inferred through the relative frequency of its adhering pollen grains on field-caught individuals . Our wor segetum ,46, our  segetum , our worA. segetum moths were effective pollinators of C. sinensis, M. azedarach, O. europaea, L. lucidum, R. pseudoacacia, C. echinocarpa, C. henryi and other plants. The flowers of these plants may contain specific attractant volatile components and the identification of these volatiles may allow us to use of floral attractant for biological control of A. segetum.Nectar-feeding moths can be attracted by the odors of their floral hosts ,48. FlorrbcL + matK as the plant barcode [matK is difficult to amplify using universal primer sets and its discrimination power differs between taxonomic groups [rbcL and ITS2 to identify pollen grains in our study. Yet, pollen DNA barcoding for pollination study applications still remains at the proof-of-concept stage [A. segetum in a range of plant species such as C. sinensis, M. azedarach, O. europaea, L. lucidum, R. pseudoacacia, C. echinocarpa and C. henryi. Meanwhile, our molecular-assisted pollen grain analysis provided evidence for the northward spring migration of A. segetum. This research provides the basis for a more in-depth assessment of the relationship between A. segetum food plant choice and host plant choice by comparing the identity of pollen grains on A. segetum moths’ exterior body versus its crop. Furthermore, our work also enables an advanced assessment of the relative importance of pollen and nectar as food resources, particularly in light of the moth’s (long-distance) migratory behavior. Lastly, our study permits the targeting of (area-wide) management interventions in areas and habitats where migratory A. segetum populations originate.Conventional palynology lacks discriminatory power, is time-consuming and requires advanced skills, thus drastically limiting the number of samples that can be studied ,19,52. T barcode , while o barcode ,55. As mpt stage . Furtherpt stage  and incopt stage . Some scpt stage  and othept stage . Our compt stage . Our resA. segetum long-distance migrants was carried out on Beihuang island , which is located in the center of the Bohai Strait at a distance of ≈60 km from the mainland to the south and ≈40 km to the north. And the location of this island has been drawn in Guo et al. study [A. segetum moths were collected using a vertical-pointing searchlight trap . The searchlight trap was switched on at sunset and switched off at sunrise on all nights from April to October 2014–2017, with the exception of days that exhibited power cuts or when heavy rain occurred. Moths were collected with a nylon net bag (60 mesh) beneath the trap, which was changed manually every 2 h each night. Twenty moths  were removed from bags every morning. Captured moths were placed individually into 2 mL tubes and frozen until microscopic examination.Collection of l. study . A. segeA. segetum adult proboscis was dissected and examined at 200× magnification using a stereomicroscope . To prevent contamination, a piece of paper towel (9 cm × 9 cm) was placed on the microscope slide and changed with each new sample and all forceps were rinsed with ethyl alcohol after each sample. Pollen grains were isolated from the proboscis, mounted on aluminum stubs, dried in a fume hood and subsequently sputter-coated with gold palladium using a Hummer vacuum coating machine. After coating, each specimen was photographed using a Hitachi S-4800 SEM .Pollen was usually found on the proboscis and occasionally on the eye or other parts of the body ,27. To dFor DNA extraction from a single pollen grain, we modified the extraction method described by Chen et al. . Under aThe partial region of chloroplast gene was amplified using universal primers. The nucleotide sequences (5′ to 3′) of the primers as were as follows: primers rbcla forward (ATGTCACCACAAACAGAAAC) and reverse (TCGCATGTACCTGCAGTAGC) ; primersPCR amplification products were separated by electrophoresis on a 1.5% agarose gel containing ethidium bromide and then visualized and photographed under ultraviolet trans-illumination prior to purification. PCR products were purified with a Gel Extraction Kit  and sub-cloned into pEASY-T3 Cloning Vector  and the inserts were sequenced with standard M13 primers .http://www.geo.arizona.edu/palynology/sem/semuofa.html) and palynological database . Next, species were verified according to their DNA sequences. The species identity of sequences generated in this study was determined using the Basic Local Alignment Search Tool  method [SEM photographs are undistorted by the optical interference encountered in light microscopy and thus enable a sound assessment of pollen grain surface sculpture. The pollen grain’s morphological features were identified using modern palynological textbooks and atlases, including of pollen flora of China , pollen ) method ,67. The ) method . Pollen A. segetum during different migration stages was analyzed using a one-way analysis of variance (ANOVA) in conjunction with Tukey’s honestly significant difference (HSD) test. Differences in the annual mean frequencies of pollen deposits on female and male A. segetum moths were analyzed by Student’s t-test. Differences in annual frequencies of pollen adherence on female and male A. segetum moths and the characteristics of pollen source plants were all compared by using a Chi-square test. All statistical analyses were performed by using SPSS 20.0  and all proportion data were logit transformed before being analyzed.The rates and the taxa of pollen adhering to A. segetum and a broad range of (host plant) species in China. Furthermore, our innovative use of conventional morphology-based pollen identification and DNA metabarcoding permits a rapid, (relatively) straightforward and low-cost identification of a multitude of pollen taxa attached to Noctuid insect body. Our results advance our understanding of the trophic relationship between a (long-distance) migratory Noctuid moth and its host plants over large geographical scales and lays the basis for effective, targeted management of globally-importance agricultural pest.Palynology or pollen grain identification is useful in studying insect migration and pollination ecology and in shedding light upon different aspects of plant-insect interactions. In this study, we gain a profound understanding of the interactions between the turnip moth


Helicobacter pylori (H. pylori) infection and eradication therapy have been known to influence gastric ghrelin and leptin secretion, which may lead to weight gain. However, the exact relationship between plasma ghrelin/leptin levels and H. pylori infection has remained controversial. The aim of this study was to investigate plasma ghrelin and leptin levels in H. pylori-positive and -negative patients, to compare the two levels of the hormones before and after H. pylori eradication, and to examine the correlation between body mass index (BMI) and active ghrelin or leptin levels, as well as that between atrophic pattern and active ghrelin or leptin levels.H. pylori-positive patients who underwent upper gastrointestinal endoscopy, 46 diagnosed as having peptic ulcer and 26 as atrophic gastritis, were enrolled. Control samples were obtained from 15 healthy H. pylori-negative volunteers. The extent of atrophic change of the gastric mucosa was assessed endoscopically. Body weight was measured and blood was collected before and 12 weeks after H. pylori eradication therapy. Blood samples were taken between 8 and 10 AM after an overnight fast.Seventy-two H. pylori-positive patients than in H. pylori-negative patients. In particular, plasma active ghrelin levels were significantly lower in patients with gastritis compared with patients with peptic ulcer. Plasma ghrelin levels decreased after H. pylori eradication in both peptic ulcer and gastritis patients, while plasma leptin levels increased only in peptic ulcer patients. Plasma leptin levels and BMI were positively correlated, and active ghrelin levels and atrophic pattern were weakly negatively correlated in peptic ulcer patients.Plasma ghrelin levels were significantly lower in H. pylori infection and eradication therapy may affect circulating ghrelin/leptin levels. This finding suggests a relationship between gastric mucosal injury induced by H. pylori infection and changes in plasma ghrelin and leptin levels. Helicobacter pylori (H. pylori), a Gram-negative, spiral-shaped bacterium that colonizes the stomach, is a major cause of atrophic and chronic gastritis, peptic ulcers, and gastric malignant lesions such as mucosa-associated lymphoid tissue lymphoma and adenocarcinoma [H. pylori reduces the relapse rate of peptic ulcer [H. pylori infection and obesity [arcinoma –3. Eradiic ulcer  and the ic ulcer . However obesity , 7.H. pylori colonization [Appetite and energy expenditure are regulated mainly by two hormones, ghrelin and leptin, produced in the gastric mucosa, which may be modified by nization . Ghrelinnization . Ghrelinnization . Active nization . Activatnization . Desacylnization . It is gnization , 15, desnization . It has nization –19. Thisnization , 20.H. pylori infection and eradication. A study by Nwokolo et al., first reporting on the possible relationship between ghrelin and the effect of H. pylori eradication, showed that cure of H. pylori increased plasma ghrelin levels in healthy asymptomatic subjects, which in turn may lead to increased appetite and weight gain [H. pylori eradication [H. pylori infection and/or eradication therapy had no effect on ghrelin levels [H. pylori infection, and gastric leptin expression was reduced after H. pylori eradication, but serum leptin levels did not change significantly after cure of H. pylori infection [H. pylori and production of these hormones are regulated by the H. pylori strain [H. pylori infection [H. pylori eradication may interface with plasma ghrelin and leptin to affect body weight has remained unknown.A number of studies have reported the relationship between plasma ghrelin/leptin levels and the effects of ght gain . In contdication , 23. Nonn levels , 25 and n levels . In addinfection . It has i strain , the extnfection , the durH. pylori-positive and -negative patients, and the levels of the two hormones were compared before and after H. pylori eradication. Furthermore, the correlations between body mass index (BMI) and active ghrelin or leptin levels and between atrophic pattern and active ghrelin or leptin levels were also examined.Therefore, in the present study, plasma ghrelin and leptin levels were measured in H. pylori-positive patients referred for upper gastrointestinal endoscopy at Mie Prefectural General Medical Center, Yokkaichi, Japan, between November 2011 and October 2014. The subjects’ diagnoses were duodenal and gastric ulcer in 46 and atrophic gastritis in 26. Control samples were obtained from 15 healthy H. pylori-negative volunteers. All patients and controls received an explanation of the procedures and possible risks associated with the study and gave their written, informed consent to participate. This study was performed in conformity with the Declaration of Helsinki and was approved by our institutional ethics committee . The exclusion criteria were pregnancy, BMI >30 kg/m2, diabetes mellitus, cachectic state including advanced cancer, systemic infection, thyroid and liver disease, renal impairment, use of medications effective against H. pylori during the preceding 3 months, history of eradication therapy before the study, and history of previous gastric surgery.The study subjects were 72 H. pylori-positive patients received triple therapy with lansoprazole 30 mg, amoxicillin 750 mg, and clarithromycin 200 mg twice per day for 7 days after the endoscopic examination. Body weight was measured and blood was collected before and 12 weeks after the treatment. Blood samples were taken between 8 and 10 AM after an overnight fast, transferred into BD™ P800 tubes  containing spray-dried K2EDTA anticoagulant and a proprietary cocktail of protease, esterase, and dipeptidyl peptidase 4 (DPP-4) inhibitors. The collected samples were centrifuged at 1100–1300 × g for 10 min, and plasma was separated and stored at -80 °C until assay.Endoscopic diagnosis of gastric mucosal atrophy was performed using the Kimura-Takemoto classification of atrophic pattern . AccordiH. pylori infection were performed by RUT  before treatment. The success of the H. pylori eradication therapy was assessed by ImmunoCard STAT!®HpSA®Stool antigen test  12 weeks after the cessation of therapy.Endoscopic examination and determination of ELISA kits were used for the measurement of active serum ghrelin , des-acyl serum ghrelin (SCETI K.K.), and leptin . Serum levels of both ghrelin and leptin were measured and calculated according to the manufacturer instructions. Ghrelin levels are expressed as fmol/mL and leptin levels as ng/ml.P values less than 0.05 were considered significant.Continuous variables were compared using the Kruskal-Wallis test or the Mann-Whitney test (two-sided), and categorical variables were compared using Fisher’s exact test. Paired measures of plasma ghrelin and leptin levels were analyzed using the Wilcoxon signed-rank test. Ghrelin/leptin levels and BMI or atrophic pattern were assessed for correlations with Pearson correlation coefficients. All statistical analyses were performed with IBM SPSS software Ver. 22. Data are expressed as mean ± SD. H. pylori-positive patients (46 peptic ulcer and 26 atrophic gastritis) and 15 healthy H. pylori-negative volunteers were studied. There were no significant differences in BMI between each pair of groups. They included 20 current smokers and 31 alcohol drinkers. There were no differences in laboratory data (data not shown). H. pylori eradication was successful in 39 of 46 peptic ulcer patients and 18 of 26 atrophic gastritis patients who received eradication therapy.The characteristics of the subjects are shown in Table H. pylori-negative control patients than in H. pylori-positive patients (both peptic ulcer and gastritis)  Fig. . With reis) Fig. . Since pH. pylori eradication in both peptic ulcer and gastritis groups, regardless of its success (data not shown).There were no significant changes in BMI in after H. pylori eradication, altered levels of circulating ghrelin and leptin were observed. In peptic ulcer patients, although not significant in all cases, ghrelin levels decreased after H. pylori eradication   Plasma active and desacyl ghrelin levels decreased after H. pylori eradication in both peptic ulcer and gastritis patients, while plasma leptin levels increased only in peptic ulcer patients. (iii) Plasma leptin levels and BMI were positively correlated, and the active ghrelin levels and atrophic pattern were weakly negatively correlated in peptic ulcer patients.In the current study, there were three major findings. (i) Plasma ghrelin levels were significantly lower in H. pylori-positive patients than in H. pylori-negative control volunteers. It seems logical that H. pylori-positive patients with atrophic gastric mucosa have low plasma ghrelin levels, considering the negative correlation between active ghrelin levels and atrophic pattern, as also proposed by Kawashima et al [H. pylori-positive patients with type I strain (expressing the virulence factors of cytotoxin-associated gene-A and Vacuolating cytotoxin A) have lower circulating ghrelin levels than those with the less virulent type II strain (expressing no virulence factors) [H. pylori-positive patients than in H. pylori-negative patients.In common with previous studies , 31–33, ma et al . In addifactors) . The popfactors) , hence tH. pylori infection reduced circulating ghrelin levels, presumably by destroying ghrelin-producing cells, and that acute gastric mucosal injury such as peptic ulcer increased circulating ghrelin levels because of stress related to ghrelin secretion.Furthermore, consistent with Suzuki et al. and IsomH. pylori eradication on circulating ghrelin/leptin levels has remained controversial. Nweneka et al. reviewed previous studies on circulating ghrelin levels before and after H. pylori eradication in a meta-analysis [H. pylori eradication [H. pylori eradication did not have any significant effect on circulating ghrelin levels. On the other hand, Bocian et al. showed that H. pylori eradication was associated with increased circulating leptin levels and decreased ghrelin levels, thereby resulting in an increased BMI in many studies [H. pylori eradication in both peptic ulcer and gastritis patients, and plasma leptin levels increased in peptic ulcer patients. A possible mechanism for decreased levels of plasma ghrelin after H. pylori eradication is as follows: before eradication, plasma ghrelin is released transiently into blood due to gastric mucosal oxidative stress and injury induced by H. pylori infection, and after eradication, the release of plasma ghrelin decreases in accordance with improvement of gastric mucosal injury. The decreased ghrelin levels in the present study are possibly attributed to the relatively short term of observation and it can be further assumed that the ghrelin levels would return to normal or even higher in a longer-term follow-up observation.The effect of analysis . Some stdication , 31, 39,dication , 29, 40 dication , 23. The studies . Consist studies , the preH. pylori eradication, presumably, the suppressed appetite of peptic ulcer patients improved with the healing of the ulcer, which led to increased food intake and increased plasma leptin levels as a consequence. In contrast, gastritis patients did not show a reduced appetite in the first place. That is probably the reason they exhibited changes neither in dietary behavior, nor in the concomitant plasma leptin levels. We suspect that there is a complex relationship among leptin secretion, adipose tissue mass, and gastric mucosal injury.The increase of plasma leptin levels after eradication in peptic ulcer patients needs explanation. Plasma leptin levels and BMI were positively correlated, while there were no significant changes in BMI after eradication in the present study. Previous studies suggested that short-term fasting caused a decline in circulating leptin concentration much greater than the change in adipose mass , 42. RegH.pylori eradication were measured in relation to peptic ulcer and gastritis. The most important findings are that our study showed the levels of the hormones changed independently and that the changes differed in peptic ulcer and gastritis patients.Remarkably, in the present study, both ghrelin and leptin levels before/after The present study had certain limitations: lack of histological evaluation such as ghrelin/leptin expression in the stomach, small sample size, and short-duration of follow-up. Therefore, we believe further research including histological evaluation will definitely improve our understanding in a future long-term study.H. pylori-positive patients than in H. pylori-negative patients. Furthermore, plasma active and desacyl ghrelin levels decreased and plasma leptin levels increased after H. pylori eradication. However, the precise mechanism remains an open question for research. Further study will be necessary to elucidate the exact effect of H. pylori eradication on plasma ghrelin/leptin levels, which may lead to body weight gain and the ultimate development of lifestyle-related diseases.In conclusion, the results of the current study showed that plasma ghrelin levels were significantly lower in


Ambrosia artemisiifolia L., (common ragweed), is an annual invasive and highly troublesome plant species originating from North America that has become widespread across Europe. New sets of genomic and expressed sequence tag (EST) based simple sequence repeats (SSRs) markers were developed in this species using three approaches. After validation, 13 genomic SSRs and 13 EST-SSRs were retained and used to characterize the genetic diversity and population genetic structure of Ambrosia artemisiifolia populations from the native (North America) and invasive (Europe) ranges of the species. Analysing the mating system based on maternal families did not reveal any departure from complete allogamy and excess homozygosity was mostly due the presence of null alleles. High genetic diversity and patterns of genetic structure in Europe suggest two main introduction events followed by secondary colonization events. Cross-species transferability of the newly developed markers to other invasive species of the Ambrosia genus was assessed. Sixty-five percent and 75% of markers, respectively, were transferable from A. artemisiifolia to Ambrosia psilostachya and Ambrosia tenuifolia. 40% were transferable to Ambrosia trifida, this latter species being seemingly more phylogenetically distantly related to A. artemisiifolia than the former two. Inferring recent demographic history and contemporary evolutionary processes are major goals in the field of population genetics. Climate change, human disturbances of natural habitats and human-aided dispersal can cause dramatic shifts in the distributions of natural species, and biological invasions are increasingly prevalent worldwide. Analyzing the genetic diversity and population genetic structure of native and introduced populations of an invasive species allows recovering pathways of invasion and identifying founding events and/or admixture events among invasive populations. All these processes affect the demographic success and future expansion of the invasive species and determine its potential for adaption to new environmental conditions. Their understanding is invaluable for devising appropriate management strategies ,2.The molecular markers used for population genetics studies are currently essentially of two kinds: genome-wide Single Nucleotide Polymorphisms (SNPs) identified by next-generation-sequencing-based techniques such as Restriction site Associated DNA sequencing (RAD-seq) or genotyping-by-sequencing , and SimAmbrosia in the Asteraceae family includes at least 51 species collectively known as “ragweeds” and mainly distributed in North America [A. artemisiifolia L., A. trifida L., A. psilostachya D.C. and A. tenuifolia Spreng.) occur in Europe but are native from America [A. artemisiifolia is an annual herb mostly known as a successful invasive and a highly allergenic plant causing severe rhinitis and asthma [th century by the import of contaminated grain and forage [A. artemisiifolia has colonized different types of habitats such as railways, riversides and wastelands, as well as cultivated fields where it is now a noxious weed competing with several summer crops [A. artemisiifolia, reliable and polymorphic molecular markers are needed. To date, only a few gSSR markers have been developed from French A. artemisiifolia populations [A. artemisiifolia [The genus  America . Four di America ,14. A. ad asthma ,16. It hd forage . A. arteer crops . To inveulations ,19. Thesulations –25, Nortulations  and Chinulations . In addiulations –26. Excesiifolia –23.A. artemisiifolia following three different approaches , (b) investigate the genetic diversity, population structure and mating system of A. artemisiifolia using populations sampled in North America and Europe, and (c) assess marker transferability to A. trifida, A. psilostachya and A. tenuifolia.The present study had three purposes: (a) develop new nuclear SSR markers for A. artemisiifolia individuals were sampled from 11 populations spanning the invasive range in Europe and 5 populations in North America  [A. psilostachya and A. tenuifolia have the same basic chromosome number as A. artemisiifolia but variable ploidy levels [A total of 321  America , Fig 1. ribed in . All thr2n = 36, , 29,30).2n = 36, , we coun2n = 36,  in 10 ply levels , 29, 32.A. artemisiifolia individuals was isolated using the DNeasy Plant Mini Kit  and processed by GenoScreen, . A SSR enriched DNA library was obtained as described in [For the SSR-enriched gDNA library approach, total gDNA from 8 ribed in . BrieflyA. artemisiifolia individual was extracted using the DNeasy Plant Mini Kit . Sequencing was performed at GENTYANE . An Illumina paired-end shotgun library was prepared by shearing 2 μg DNA using a Covaris M220 ultrasonicator  and following the standard Illumina TruSeq DNA Library Kit protocol . Sequencing was conducted on the Illumina MiSeq with 250 bp paired-end reads.For the Illumina gDNA sequencing approach, total gDNA from one A. artemisiifolia transcriptome 454 sequence data were downloaded from Genbank Sequence Read Archive [For the EST public data use approach, the two existing sets of  Archive . They coFor each of the three sequence datasets, stringent sequence quality control and filtering were performed using the ShortRead package in the Bioconductor software . BrieflyA. artemisiifolia individuals from three populations . PCRs were performed in 10-μL as previously described [SSRs were identified with QDD version 3.1 . Only 2-escribed . CyclingA. artemisiifolia were used to genotype 384 individuals, including 321 A. artemisiifolia (16 populations), 20 A. trifida (two populations), 22 A. psilostachya (one population) and 21 A. tenuifolia (one population) individuals  or unexpectedly low . We used data from A. artemisiifolia and the software Bayescan [All SSR loci were screened for evidence of selection based on an FBayescan . This prBayescan , we usedA. artemisiifolia was investigated using five gSSR markers  in six additional French populations sampled in 2014 and located within a few kilometres around population GEN13.03 , expected heterozygosity (HS) and the genetic differentiation (FST) were calculated using Fstat [FST values was based on 1000 bootstrap resampling over loci. Inbreeding coefficient (FIS) were estimated with INEST 2.1 [FIS = 0) based on the Deviance Information Criterion (DIC). Genetic diversity and differentiation parameters for A. artemisiifolia were calculated over all populations, over North American populations and over European populations.The allelic richness per locus and per population using a rarefaction method (ng Fstat . SignifiNEST 2.1  using a K) best fitting the data. The length of the burn-in period was 100,000 runs followed by 500,000 Markov Chain Monte Carlo. Ten iterations were performed for each value of K from 1 to 15. K was determined graphically based on log likelihood values as previously described [K. Finally, Clumpp 1.1.2 [Population structure was assessed using Structure 2.2 . The admescribed  using thescribed . In addiescribed  was usedFST values based on the F-model [FST values, and two models that used either latitude or longitude as explanatory variables. In addition to the study of genetic divergence patterns, we investigated the signature of recent bottleneck events using the Wilcoxon test for excess expected heterozygosity implemented in INEST2.1 and based on the method of Cornuet and Luikart [Genetic divergence is likely to vary across populations because of differences in population effective sizes and local migration rates. This is especially the case when recent founder effects have occurred, such as during range expansions. Patterns of genetic divergence were estimated for the invasive range (Europe) by calculating population-specific  F-model . We used F-model  implemen Luikart . AnalyseA. artemisiifolia  than other markers, but a very high within-population genetic diversity (Hs = 0.776), suggesting balancing selection at or near the locus. This locus was therefore discarded for further analyses.Genetic polymorphism was assessed using 16 ividuals . After cividuals  was unamividuals . SSR86 sversus 4.748) but the difference was not significant  .All 26 retained loci were polymorphic and revealed high levels of genetic diversity . The fre= 0.778) . The meaA. artemisiifolia and large numbers of pollen donor parents.Thirty-six maternal progenies sampled from six French populations were analysed with five gSSRs to estimate mating system parameters. In addition, direct evidence for the presence of null alleles was sought by considering the progenies from maternal plants apparently homozygous at one locus. If a null allele was present, the maternal plant would actually be heterozygous . Its progeny would thus contain some plants apparently homozygous for alleles different from the maternal one, but actually carrying one maternal null allele and one paternal detectable allele. Evidence for the presence of null alleles was obtained for all five markers. Depending on the marker considered, from 25% (3 out of 12) to 35% (8 out of 23) of the progenies from plants scored as homozygotes contained non-matching genotypes. Mating system parameters were estimated after excluding these progenies. The maternal inbreeding coefficient was non-significant (F = 0). Multi-locus outcrossing rates (tm) were high and not significantly lower than 1 in all populations . The ratFIS values were on average higher in the native range than in the invasive range (ST in the native range was low (0.042) but significant. FST in the invasive range was higher (0.071) and also significant. The difference in FST values between the two areas was significant based on 99% bootstrap confidence intervals.We compared patterns of genetic diversity between the native range (North America) and the invasive range (Europe). Allelic richness within population and mean expected heterozygosity within population were slightly higher in North America than in Europe . Howeverve range , althougK, whereas the Evanno method (49) suggested that the most likely number of genetic clusters was six  in comparison to the null model with no explanatory factor (posterior probability: 0.28) or the model including longitude (posterior probability: 0.04). Population-specific FST values increased with latitude  was greater in the invasive range. A similar trend had also been observed previously [FST values may arise simply because only a small area of the native range was sampled (five American populations) in our study and in [Genetic diversity within population was similar in North America (the native range) and in Europe, but genetic differentiation among populations  could simply reflect the elevated genetic divergence of some populations. Indeed, Structure analyses are known to be biased towards inferring extra genetic clusters when some populations have undergone strong recent genetic drift; in that case, and contrary to the assumptions of the admixture model, not all genetic clusters are ancestral sources for the present populations [The main pattern of population structure we observed in Europe was a west-east gradient. Differentiation between the western and eastern parts of the European invasive range had previously been observed  and attrulations . Populatulations . This, tulations . PopulatA. artemisiifolia  were transferable to A. psilostachya and A. tenuifolia, whereas only 40% were transferable to A. trifida. The genus Ambrosia is composed of many, not clearly delineated species [A. artemisiifolia, A. psilostachya and A. tenuifolia as related species belonging to one same group, while A. trifida was classified in a separate group [A. artemisiifolia, A. psilostachya and A. tenuifolia but n = 12 for A. trifida, [A. artemisiifolia and A. psilostachya was suggested [Most SSR markers developed for  species  for whicte group . This istrifida, ,29,30,32trifida, . The sucA. psilostachya and A. tenuifolia are perennial species that reproduce both sexually and clonally [A. artemisiifolia and A. trifida, being less widespread and invasive, our SSR markers will be useful for assessing vegetative versus sexual reproduction, as well as for identifying colonization sources and relatedness among populations. However, given that several ploidy levels were reported in these species [A. artemisiifolia are transferred to A. psilostachya and A. tenuifolia. A. trifida is a noxious annual weed, very widespread in its native area [A. artemisiifolia and the low success rate of markers transferability, we recommend that additional SSR markers are specifically developed for A. trifida.clonally , 32, 62. species , 29, 32,ive area  and intrive area ,65. DespA. artemisiifolia, providing useful new resources for genetic studies of this highly noxious invasive weed. All markers were highly polymorphic. EST-SSRs revealed as many alleles as gSSRs and yielded similar genetic diversity estimates. The genetic patterns revealed for a set of American and European populations confirmed results from previous studies by showing a high within-population genetic diversity in both the native and invasive ranges. A geographical gradient of genetic variation in Europe was consistent with at least two major colonization events in Western and Eastern Europe, respectively. Secondary founding events were identified, especially in Central Europe. In addition, we settled a former controversy by demonstrating that inbreeding observed within populations is attributable mostly to the presence of null alleles rather than to selfing. Last, most SSRs were transferable to three other Ambrosia species. These SSRs can readily be used for studying key aspects of the biology and population dynamics of the two species most closely related to A. artemisiifolia (A. psilostachya and A. tenuifolia).Large sets of genomic SSRs and EST-SSRs were developed and validated in S1 FigThe red arrows indicate the chromosomes. Gx100.(TIF)Click here for additional data file.S2 FigResults of Bayescan FSToutlier analysis on 14 gSSR (a) and 13 EST-SSR loci (b). The vertical bars correspond to threshold P-values of 0.05 (solid line) and 0.01 (dashed line) for the neutral model. (a) Data from all 16 populations. (b) Data from 11 European populations.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 TableA. artemisiifolia.Loci in bold represent the markers selected in (DOCX)Click here for additional data file.S2 TableA. artemisiifolia.Loci in bold represent the markers selected in (DOCX)Click here for additional data file.S3 TableA. artemisiifolia.Loci in bold represent the markers selected in (DOCX)Click here for additional data file.S4 TableN: number of individuals genotyped, A: average allelic richness after rarefaction, HO observed heterozygosity, HS expected heterozygosity, FIS inbreeding coefficient estimated taking into account the presence of null alleles. * FIS estimates significantly greater from zero (using the Bayesian model comparison based on Deviance Information Criterion implemented in INEST2.1).(DOCX)Click here for additional data file.


Melanotus cribricollis, feed on bamboo shoots and roots, causing serious damage to bamboo in Southern China. However, there is currently no effective control measure to limit the population of this underground pest. Previously, a new entomopathogenic fungal strain isolated from M. cribricollis larvae cadavers named Metarhizium pingshaense WP08 showed high pathogenic efficacy indoors, indicated that the fungus could be used as a bio-control measure. So far, the genetic backgrounds of both M. cribricollis and M. pingshaense WP08 were blank. Here, we analyzed the whole transcriptome of M. cribricollis larvae, infected with M. pingshaense WP08 or not, using high-throughput next generation sequencing technology. In addition, the transcriptome sequencing of M. pingshaense WP08 was also performed for data separation of those two non-model species. The reliability of the RNA-Seq data was also validated through qRT-PCR experiment. The de novo assembly, functional annotation, sequence comparison of four insect species, and analysis of DEGs, enriched pathways, GO terms and immune related candidate genes were operated. The results indicated that, multiple defense mechanisms of M. cribricollis larvae are initiated to protect against the more serious negative effects caused by fungal infection. To our knowledge, this was the first report of transcriptome analysis of Melanotus spp. infected with a fungus, and it could provide insights to further explore insect–fungi interaction mechanisms.Larvae of  Melanotus cribricollis; Coleoptera: Elateridae) pose danger to bamboos, especially the Phyllostachys spp./[Fe], catalase-B, peroxidase-like protein, peroxiredoxin, oxidoreductase, etc , β-1,3-glucan recognition protein (β-GRP), peptidoglycan receptor proteins (PGRP), lysozyme, glutathione S transferase (GST), cytochrome P450 and dehydrogenase/reductase SDR family proteins. Apart from those, many other immune related genes were also identified in the ase, etc . Here, tase, etc .M. cribricollis with GNBP homologs from different insects. All GNBP sequences from M. cribricollis clustered together and with the GNBPs from other Coleopteran insects, which was consistent with the traditional classification  were up-regulated. The most obvious differences were observed at 7 d post-infection, especially for those two putative GNBP3 unigenes, c56746.graph_c0  and c54949.graph_c0 , . Phylogefication . Additiofication .M. cribricollis larvae are the preeminent threat to sustainable development of bamboo forestry, especially the Phyllostachys spp. [Metarhizium spp. is a promising entomopathogenic fungi that can be used for the biological control of M. cribricollis. A comparison analysis of RNA-Seq data between infected and uninfected M. cribricollis larvae reported here, will advance our understanding of the complexity of insect immune systems in the defense against fungal invasion.Currently, hys spp. . MetarhiAn indoor bioassay was conducted to determine the time point of sample collection. The result suggested that 1 d and 3 d may represent the early phase when fungi started invasion, and 5–7 d could be critical time points likely representing the initiation of the intense interaction between insect and fungi, followed by the moribund stage of bamboo wireworms. Here, the 1 d and 3 d sampling points were chose as representative of the start of infection, 5 d and 7 d were chose as the initiation of fierce interaction, and 4 d was selected as an additional sampling point to make sure there was no omission of critically antagonistic responses for further data mining .de novo assembly result suggested that abundant and high quality data was acquired by high-throughput sequencing technology in this study. The high mapped ratios of both M. cribricollis and CK-Metar indicated the sequencing and assembly data were credible for further analysis. The low ratio of M. pingshaense WP08 from the infected group was in accordance with the very small percentage of fungi tissue in M. cribricollis larvae ” and “peroxisome (ko04146)”) were ubiquitous, suggesting that these two were likely general regulatory tactics involved in anti-fungi physiological processes. Additionally, some (“Fc gamma R-mediated phagocytosis (ko04666)” and “phagosome (ko04145)”) were specifically enriched at 7 d, which might correspond with the specific process of infection. The discovery of enriched pathways related to sugars, amino acids and organic acids metabolism suggested that fungal infection disturbs the normal growth and development of the insect host . As to GM. cribricollis, unigenes especially the DGEs, involved in several important immune related BP terms were analyzed. These unigenes were annotated based on orthologous genes from three other insect species, D. melanogaster, T. castaneum and D. ponderosae and comprised of complete gene sequences, fragments, even allelic or splice variants of the same gene. The results revealed some important immune defense or stress response unigenes with different functions; here, we mainly illuminate the putative encoding genes of gram negative binding proteins, β-1,3-glucan recognition protein, peptidoglycan receptor, lysozyme, glutathione S transferase, cytochrome P450, dehydrogenase/reductase SDR family.To fully understand the cellular and humoral immune responses of D. melanogaster [T. castaneum after being parasite [Migratoria manilensis) and mosquitoe mutants (Dirofilaria immitis) [Meligethes aeneus) in response to bacterial injection [Galleria mellonella [Drosophila OregonR adult males with septic injury [T. castaneum, with LE and LD not markedly up-regulated after LPS injection [M. cribricollis larvae, and classified into PGRP-SB, PGRP-LF and PGRPs. Based on the result, we found that responsiveness of the GNBP3 gene , PGRP-LF gene  and PGRP3 gene (c55093.graph_c0) were the most distinguished, indicating that these encoding PRRs were more sensitive to fungal infection and play key roles in protecting the insect host. Furthermore, obvious differences were noticed at 7 d compared with the control, suggested that M. cribricollis larvae may start intense resistance at 7 d against M. pingshaense WP08 infection (Insect innate immune responses are initiated by recognition patterns between pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs), consequently activating corresponding signal transduction pathways  and finally induce the expression of anti-microbial proteins (AMPs) or other defense molecules –22. Insenogaster  and signparasite . Experimimmitis) , 33–34. immitis) , 35. Curimmitis) , 35. Thenjection . Six putc injury , and sevnjection , 32. In nfection .HaLyz) in Helicoverpa armigera was reported to be up-regulated after injection of E. coli or Beauveria bassiana but showed different expression patterns [G. mellonella, lysozyme expression was observed even in naïve larvae revealing its importance in early stage immune response to inhibit Candida albicans infection [Spodoptera spp. expressed lysozyme at higher levels after M. anisopliae infection, especially at moribund stage [Paracoccidioides brasiliensis transition could be inhibited by higher inflammatory concentrations of lysozyme but not lower physiologic concentrations [M. cribricollis defense response against M. pingshaense WP08. However, the mechanisms responsible for fungistatic and/or fungicidal activity of lysozymes are not clear [Lysozymes are polypeptides which exhibit antibacterial and antifungal activities ). The lepatterns . Similarnfection . Larval nd stage . Interesnd stage ,41; Paraot clear ; more woPlutella xylostella larvae infected by Isaria fumosorosea [B. bassiana [GSTs are major enzymes involved in the detoxification process of endogenous and xenobiotic toxins in multiple organisms . It is aosorosea . Hou CX bassiana . In thisCulex quinquefasciatus [M. pingshaense WP08. However, the stimuli-responsive activity of P450 was not quite obvious in general, only four DEGs  were up-/down-regulated (M. cribricollis larvae was not predominant before 7d.Cytochrome P450 (CYPs) are mixed function oxidases and among them cytochrome P450 monooxygenases constitute a large and complex superfamily of heme-thiolate proteins, with representatives in all living organisms . P450s aasciatus . P450 prasciatus –53. Someegulated  Tables, Dehydrogenases/reductases (SDR) play an important role in various pathogen/biochemical pathways, including intermediary metabolism and biotransformation of xenobiotics, and only 20% of them are well characterized –55. SDRsM. cribricollis larvae against M. pingshaense WP08 was investigated using the Illumina next generation sequencing technology. Our results indicated that M. cribricollis larvae could initiate additional defense mechanisms while the fungi could induce serious negative effects that increase with an increase in post-infection time. The fierce infection did not stop until the larvae died. This study is the first to report the genetic background of M. cribricollis both uninfected and infected by fungi. This result may help the further exploration of insect–fungi interaction mechanisms, and might benefit bio-control strategies combined with molecular biological methods to control this devastating pest.In conclusion, the immune system of S1 Fig(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 Table(XLS)Click here for additional data file.S8 Table(XLS)Click here for additional data file.S9 Table(XLS)Click here for additional data file.S10 Table(XLS)Click here for additional data file.


Anaplasma platys, the causative agent of infectious canine cyclic thrombocytopenia, is a tick-borne pathogen that also has been implicated as potentially zoonotic. To provide molecular evidence on the multiple infections of A. platys variants in Philippine dogs.A. platys from infected dogs in the Philippines were molecularly characterized. For screening, 25 dogs suspected to have canine anaplasmosis were tested using a 16S rRNA-based nested polymerase chain reaction (PCR). Infection was confirmed by sequencing of positive amplicons. Second round PCR targeting a longer 16S rRNA fragment was subsequently performed on the first round PCR amplicons of the positive samples. Further characterization using the heat-shock operon (groEL) gene was also performed on the A. platys-positive samples.DNA fragments of A. platys sequences. On the other hand, 36 groEL clone sequences were obtained and found to be 85.1-99.8% identical with each other and 85.0-88.9% identical to the closest previously registered A. platys sequences. Four dogs were found coinfected with 2-3 groEL variant sequences. Phylogenetic analyses suggest that the detected A. platys in the Philippines may represent unique variants.10 16S rRNA sequences were obtained and found 99.6-100% identical to each other and 99.6-99.7% identical to the closest registered A. platys variants were detected in Philippine dogs. Coinfection of different A. platys variants in dogs was also demonstrated. The present study may indicate the potential genetic diversity of A. platys in the country. Anaplasma platys is a Gram-negative intracellular bacteria of the family Anaplasmataceae [mataceae  that hasmataceae -4. In thmataceae  and dogsmataceae . Nearby mataceae , China [mataceae , Korea [mataceae , Japan [mataceae , and Taimataceae  already A. platys based on the heat-shock operon gene (groEL) are limited. This particular gene has been used to identify genotypes of a related Anaplasma species. Pathogenicity of Anaplasma sp. maybe related to its genotype [groEL in assessing clinical signs or effects to the animal has been limited.Characterizations of genotype , althougA. platys detection in the Philippines are available. The present study aimed to detect and molecularly characterize A. platys based on the 16S rRNA and groEL genes from infected dogs in the country. The obtained sequences were further subjected to phylogenetic analyses using different methods.Only few reports on The study was performed in accordance with the Institutional Animal Care and Use Committee guidelines of Southwestern University, Cebu and the Animal Welfare Act of the Philippines (RA8485), and with the approval of the attending veterinarians and proprietor of the veterinary establishment.Ehrlichia canis using an Immunocomb®  commercial test kit [DNA samples were obtained from blood of 25 dogs that were presented at the GPY Veterinare Animale Group of Veterinary Clinics, Cebu, Philippines. These dogs were thrombocytopenic and exhibited clinical signs suggestive of canine anaplasmosis  and weretest kit . DNA wasEhrlichia/Anaplasma spp. Those found positive with the EHR16SD/EHR16SR screening PCR were further analyzed. Using the first round fD1/Rp2 amplicons, hemi-nested PCRs were performed utilizing the primer pairs fD1/EHR16SR and EHR16SD/Rp2 targeting a total of approximately 1400-1600 bp. A final volume of 10 and 25 µl was set for the first and final round PCRs using a similar method, respectively [A. bovis [For the 16S rRNA PCR, DNA samples were tested using a previously described method  with sliectively . The neggroEL-based nested PCR assay. In the first round, primers EEgro1F and EEgro2R [DNA samples which were found positive during the screening PCR were further analyzed using a  EEgro2R  were use EEgro2R . For the EEgro2R . Step-dogroEL sequences were aligned as previously described [Final amplicons were purified using a Gel Extraction Kit  and were cloned using a TA cloning kit . DNA sequencing was performed as described previously . Obtaineescribed  to obtaiescribed . All seqhttp://tree.bio.ed.ac.uk/software/figtree/).All sequences were manually trimmed to include only the sequence of interest. Percent identities were computed, and multiple sequence alignments were performed as previously described . AnalyseA. platys sequences from the Philippines and Thailand. From the A. platys-positive samples, further characterization using the groEL resulted to 36 clone sequences (KP027333-KP027368), which were 85.1-99.8% identical with each other and 85.0-88.9% identical to the closest registered A. platys sequences.The screening PCR revealed 10 positive dogs. Obtained sequences (KP006398-KP006406) after alignment (1467 bp) were 99.6-100% identical to each other and 99.6-99.7% identical to the closest registered groEL variant sequences. To the best of our knowledge, several studies have reported multiple infections of different tick-borne pathogens in dogs [A. platys in dog have an effect on the clinical expression of the disease.From the positive dogs, four were found coinfected with 2-3  in dogs ,21, but  in dogs , furtherA. platys subclade supported by moderate to high bootstrap values . Phylogenetic analyses’ results suggest that the detected A. platys in the Philippines may represent unique variants as the obtained sequences branched separately under the A. platys clade. These results can indicate the potential genetic diversity of A. platys in the country. This study supports the usefulness of groEL in investigating genetic diversities in Anaplasma species. On the other hand, further studies on the multiple infection of several A. platys variants in dogs that may also be coinfected with E. canis [Identities of the newly obtained sequences to registered E. canis ,30, whicA. platys variants were detected in Philippine dogs. Coinfection of different A. platys variants in dogs was also demonstrated. The present study may indicate the potential genetic diversity of A. platys in the country.APY and RHDY conceptualized the study and analyzed and wrote the manuscript. RRV contributed in the data analysis. NY and HI gave valuable insights and support in the conduct of the study.