Spodoptera exigua (Lepidoptera: Noctuidae), is a key insect pest of edible vegetables around the world and it is resistant to insecticide of different classes. Insecticides that are effective to this pest and selective to predator stinkbugs are required for the integrated management of S. exigua.The beet armyworm, Podisus maculiventris (Heteroptera: Pentatomidae) through different bioassays.The toxicity of four commercial insecticide formulations azadirachtin + pyrethrin, spinosad, pyrethrin and chlorantraniliprole was tested on the target pest and their side effect were evaluated on the spined soldier bug, S. exigua than to the predator P. maculiventris but opposite results were obtained for pyrethrin and azadirachtin + pyrethrin in contact toxicity bioassay. Chlorantraniliprole was the most toxic to S. exigua in oral toxicity bioassay, followed by spinosad, pyrethrin and azadirachtin + pyrethrin. Spinosad in oral toxicity bioassay was the most toxic to P. maculiventris, followed by pyrethrin, azadirachtin + pyrethrin and chlorantraniliprole. Spinosad caused irritability to the predator while pyrethrin to the pest. The insecticide repellency was not observed over the tested insect species. The synthetic insecticide chlorantraniliprole was less toxic than the natural pyrethrin, azadirachtin + pyrethrin and spinosad to the predator.Spinosad and chlorantraniliprole were more toxic to P. maculiventris to controlling S. exigua in integrated pest management (IPM) programs.This work provides useful information on the combination of commercial insecticides with the predator  Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), native to Southeast Asia, is an important insect pest of edible vegetables in several regions of the world  × 100  × 100 . The selk] × 100 . The relcticides . To meas overlap . Differesion 9.2 .S. exigua, P. maculiventris and T. molitor in the USA. The laboratory studies did not involve endangered or protected species.No specific permits are required to rear S. exigua, followed by the spinosad, pyrethrin and azadirachtin + pyrethrin, with relative toxicity of 1.00, 10.99, 16.75 and 28.19, respectively. No P. maculiventris mortality was observed with chlorantraniliprole; therefore, the LC50 of this treatment was not calculated. Pyrethrin and azadirachtin + pyrethrin were more toxic than the spinosad to P. maculiventris by contact with treated glass vials. Azadirachtin + pyrethrin, spinosad and, mainly, chlorantraniliprole were more toxic to the pest than to the predator while pyrethrin toxicity to both the pest and predator was similar . Nevertheless, behavior of avoidance to insecticides by S. exigua and P. maculiventris was detected. Behavioral avoidance to insecticide-treated surfaces was recognized through its two components–insecticide repellence  and insecticide irritability . The pyrethrin and spinosad reduced the time spent by S. exigua  and by P. maculiventris  in the treated half of the dish, respectively. The pest remained longer in the treated area with spinosad  (P. maculiventris and S. exigua in treated or untreated parts of the dishes were similar (The time proportion spent by  = 0.03) . Typical similar .S. litura and its predator P. maculiventris. Three bioassays were carried out evaluating the direct contact to insect body or ingestion through artificial diet and drinking water of insecticides on the mortality and the behavior of the pest and its natural enemy. Spinosad and chlorantraniliprole were less toxic to P. maculiventris than to the pest, oppositely resulted for pyrethrin and azadirachtin + pyrethrin in contact toxicity bioassay. Spodoptera exigua was highly affected negatively by chlorantraniliprole in oral toxicity bioassay, followed by spinosad, pyrethrin and azadirachtin + pyrethrin. In oral toxicity bioassay, spinosad was the most toxic to P. maculiventris, followed by pyrethrin, azadirachtin + pyrethrin and chlorantraniliprole. The natural insecticides pyrethrin, azadirachtin + pyrethrin and spinosad were more toxic than the synthetic chlorantraniliprole to the predator.Our study evaluated the effect of pesticides used in organic farm and a synthetic formulation on Bracon nigricans (Szépligeti) (Hymenoptera: Braconidae) adults [Podisus nigrispinus  and Supputius cincticeps (Stål) (Heteroptera: Pentatomidae), 72 h after to dry residues exposure [Bombus impatiens Cresson (Hymenoptera: Apidae) [Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) [Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae) [Galendromus occidentalis (Nesbitt) (Acari: Phytoseiidae) [Macrolophus pygmaeus (Rambur) (Hemiptera: Miridae) caused by chlorantraniliprole was lower than 25% and, therefore, classified as harmless [M. pygmaeus feeding on the plant, but did not affect the behavior of this insect. In addition, chlorantraniliprole did not affect the M. pygmaeus predation rate [Amphiareus constrictus (Stål), Blaptostethus pallescens Poppius and to the minute pirate bug, Orius tristicolor (White) (Hemiptera: Anthocoridae) than to the tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and reduced the walking activity of the pest [The insecticides spinosad and chlorantraniliprole were more toxic to the pest than to the predator in this study, a condition essential for integrated pest management (IPM) programs ,38. Spin) adults . The chlexposure . This in Apidae)  and highsopidae) ; the solconidae)  and the seiidae) . The morharmless . This inion rate . Chloranthe pest . The higthe pest ,46.S. exigua in this study agrees with reports for cruciferous lepidopteran-pests, including the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), the small white, Pieris rapae (L.) (Lepidoptera: Pieridae) and the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) [P. maculiventris by ingestion in treated water than by contact with residues in the glass vial. A total of 71% of reports showed a lethal effect of spinosad for predatory insects in the laboratory and it is also toxic for some pollinators [The spinosad toxicity by contact or ingestion against ctuidae) . The spictuidae) . Howeverlinators . Howeverlinators .P. maculiventris than to S. exigua in this study agrees with their knock-down effect on other insects [Azadirachta indica A. Juss. (Meliaceae) with widespread use against insect-pests [Chrysoperla externa (Hagen) and Ceraeochrysa cubana (Hagen) (Neuroptera: Chrysopidae) mortality by azadirachtin was high (100%) [P. maculiventris [Harmonia axyridis  (Coleoptera: Coccinellidae), from 3rd instar to adulthood. The azadiractin also increased the larval stage of H. axyridis when applied on 1st and 3rd instar nymphs of this predator [The higher toxicity of insecticides used in organic farm (pyrethrin and azadiractin + pyrethrin) to  insects . The azact-pests . Howeverct-pests ,50,51. Th (100%)  and thisiventris . This copredator . Compounpredator .S. exigua in the treated portion of the dish. A similar response, observed for P. maculiventris to spinosad, may be an adaptive behavior that reduces pest and predator exposure to toxic residues of this compound [S. exigua with the spinosad in the treated portion of the dish may indicate an arrestant effect of this compound, as reported for the European chafer, Amphimallon majale (Razoumowsky) (Coleoptera: Scarabaeidae) larvae with imidacloprid [P. nigrispinus [The effect of the insecticides pyrethrin and spinosad on insect behavior in this study was expected because they are neurotoxic compounds whose sub-lethal impacts affect nerve interactions in these organisms ,54,55. Tcompound . Pesticicompound –59. The acloprid . This nerispinus  and pollrispinus .S. exigua and P. maculiventris could improve IPM programs involving the use of P. maculiventris as natural enemy. The Entrust and especially the Coragen were more toxic to the pest than to the predator. However, Entrust, via ingestion of treated distilled water, was highly toxic to the predator, which could be negatively affected by this pesticide in the field by feeding on treated preys. The PyGanic Crop Protection EC 5.0 II and Azera were more toxic to this predator than to the pest. Coragen was less toxic than the botanicals PyGanic Crop Protection EC 5.0 II and Azera, and the microbial Entrust for the predator. Prior inclusion of biopesticides in IPM programs, their risk and the sub-lethal effects on non-target organisms should be assessed. The biopesticides Azera, Entrust and PyGanic Crop Protection EC 5.0 II are not recommended as insecticide options in areas with the predator P. maculiventris. Other natural enemy species which coexist with this predator in a same crop could be also affected negatively by these insecticides. The synthetic, Coragen showed potential selectivity for P. maculiventris. However, these results were obtained in laboratory conditions and the effect of this insecticide on pests and natural enemies is suggested to be tested in greenhouse and field. These tests are suggested to be conducted in a scenario involving multiple exposure routes (e.g. pesticide residue on plants and/or treated prey) and the combination of the pesticide fator with other stressors (e.g. temperature fluctuations).The knowledge on risk associated with pesticides toward natural enemies is one of the key for IPM programs. The results obtained in the present study with the toxicity of the NOP compliant biopesticides Azera, Entrust and PyGanic Crop Protection EC 5.0 II, used in organic farm, and a synthetic formulation, Coragen, against S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S7 File(PDF)Click here for additional data file.S8 File(PDF)Click here for additional data file.S9 File(PDF)Click here for additional data file.S10 File(PDF)Click here for additional data file.S11 File(PDF)Click here for additional data file.S12 File(PDF)Click here for additional data file.S13 File(PDF)Click here for additional data file.S14 File(PDF)Click here for additional data file.S15 File(PDF)Click here for additional data file.S16 File(PDF)Click here for additional data file.S17 File(XLS)Click here for additional data file.


Mycobacterium tuberculosis complex (MTC) targets. Porotic hyperostosis and cribra orbitalia were found in 10 and 13 individuals, respectively. Maternal ancestry analysis revealed Native American mtDNA haplogroups A and C1 in three individuals. Three samples showed osteological evidence suggestive of TB. katG and mtp40 sequences were detected in three individuals, indicating probable TB infection by two MTC lineages. Tuberculosis infection in the Tenetehara-Guajajara population since the 18th century points to a panorama of the disease resulting, most probably, from European contact. However, the important contribution of African slaves in the population of Maranhão State, could be also considered as a source of the disease. This study provides new data on TB during the Brazilian Colonial Period. This is the first report integrating paleopathological and paleogenetic data for the study of TB in Brazil.Tuberculosis (TB) has been described in Native American populations prior to the arrival of European explorers, and in Brazilian populations dating from the Colonial Period. There are no studies demonstrating TB infection in native Brazilians, and the history and epidemiological scenario of TB in Brazil is still unknown. The aim of this study was to verify the presence of TB infection among the native Tenetehara-Guajajara population from Maranhão State, Brazil, 210 ± 40 years ago. A Tenetehara-Guajajara skeleton collection was submitted to paleopathological analysis, and rib bone samples (n = 17) were used for paleogenetic analysis based on  Mycobacterium tuberculosis complex (MTC), which comprises Mycobacterium tuberculosis sensu stricto, M. africanum, M. canettii, M. bovis, M. caprae, M. microti, M. pinnipedi, and M. mungi [Mycobacterium tuberculosis complex emerged about 70,000 years ago and accompanied migrations of anatomically modern humans out of Africa [M. tuberculosis sensu stricto and other animal strains [Tuberculosis is an infectious disease caused by bacteria of the M. mungi ,2. Mycobf Africa . Previouf Africa ,5, and t strains .Mycobacterium tuberculosis complex infection caused by ancient strains most closely related to those from sea lions was recently found in Peruvian mummies [Paleopathological evidence described in pre-Columbian populations of Peru, Chile ,8, and V mummies . In Braz mummies , as well mummies . There aWe used paleogenetic analysis to investigate presence of TB infection in Native American Tenetehara-Guajajara skeletal remains dating from 210 ± 40 years ago, post-contact with Europeans and Africans. Study of a post-contact native group with bone lesions suggestive of TB allowed investigation of biological and cultural influences on disease processes in those Brazilian communities.Setor de Antropologia Biológica-SABMN) of the National Museum of the Federal University of Rio de Janeiro . Rib samples (n = 17), SABMN00699-SABMN00707, SABMN00709-SABMN00711, SABMN00713-SABMN00715, and SABMN00717- SABMN00718, were supplied for the SABMN/MN/UFRJ for analysis at the paleogenetic laboratory of LABTRIP/IOC/Fiocruz. All necessary permits were obtained for the study, which complied with all relevant regulations.The Tenetehara-Guajajara skeletal remains are held in the collection of the Biological Anthropology Section , and SABMN00717 is from 140 ± 30 BP  [In 1945, when the isolated Tenetehara no longer existed, Dr. Pedro Lima travelled to the Pindaré-Gurupi River villages to study the bio-anthropology and health of the community . During rrected) .The original series of remains was numbered from SABMN00699 to SABMN00719 in the book of the Biological Anthropology Section (MN/UFRJ). Skeletons were examined to i) provide general descriptions ; ii) idein situ. Ribs were chosen for analysis because of their direct association with lung disease and acceptable state of preservation.Most of the major bones were available for examination, although the small and spongy bones were lost, because of taphonomic processes in the humid tropical location of the cemetery. Teeth were still articulated and were examined Criteria for bone samples to be submitted to aDNA analysis were i) rib samples only from adult/subadult skeletons of both sexes; ii) only one rib from each individual, either left or right; and iii) specimens with pathological signs were not used for the paleogenetic analysis.Precautions were taken to avoid contamination by modern DNA and cross-contamination, including use of protective clothing, gloves, head covering, masks, and sterile instruments and equipment. We implemented standard aDNA procedures to avoid aDNA degradation, contamination from modern DNA, and cross-contamination during the paleogenetic analysis as described –33. The ™ 1000. Sediment samples removed from the first cleaning of bones were used as controls for the archaeological site and submitted to MTC paleogenetic procedures.Seventeen ribs were provided by the biological anthropology section (MN/UFRJ) for paleogenetic analysis . ExogenoM. tuberculosis T92 (ATCC27294T), M. tuberculosis H37Rv (ATCC27294T), M. bovis BCG (ATCC19210T) and M. africanum T85 (ATCC25420T). The pPCRs were purified by a GFX PCR DNA and Gel Band Purification kit  and directly sequenced using an ABI BigDye Terminator kit (Applied Biosystems) according to the manufacturer’s protocol, with analysis in both directions, on an ABI 3730 (Applied Biosystems) automated sequencer. BioEdit v. 7.0.4 , and Lasergene Seqman v. 7.0.0  were used for editing and sequence analysis. Sequencing and sequence analysis were performed to confirm MTC molecular targets. Probes were labeled by chemiluminescence using Gene Images Alkphos Direct Labeling and Detection Systems (Amersham) as described elsewhere [Dotblot and aDNA hybridization procedures were conducted as described elsewhere . Human Dlsewhere .Taq DNA Polymerase High Fidelity (Invitrogen). Additional MTC genotyping markers were applied, as described (Taq DNA Polymerase High Fidelity (Invitrogen), and whole genomic amplification (WGA) (®-T and pGEM®-T Easy Vector Systems kit (Promega) following manufacturer’s instructions, with least three clones produced. Sequencing and sequence analysis were performed as described above. MTC sequences were submitted to GenBank.To confirm MTC infection and exclude false positive or environmental bacteria contamination, PCRs using MTC targets IS6110 and IS1081 were conducted on archaeological samples, following conditions and primers described , 36, usiescribed . These mon (WGA) . Reconst5’-CACATCAACTGCAACTCCAAA-3’ and H16422: 5’-ATTGATTTCACGGAGGATGG-3’, designed in this study using PRIMER3 (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/). PCR procedures, using Platinum Taq DNA Polymerase High Fidelity (Invitrogen), were replicated at least twice and pPCRs directly sequenced or/and cloned. Cloning, sequencing, and sequence analysis were performed as described above. Cambridge Reference Sequence  of the mtDNA was used as target to determine human ancestry. Four primer pairs were used: L16070/H16259 , L16209/Skeletal remains of 17 adults/subadults of the Tenetehara collection were analyzed, comprising eight males, six females, and three undetermined . The lonpiranha teeth, with both angles of the four upper incisors cut off with a blade [The rounded mongoloid skull was consistent with the description of most Native Amazonian communities, and dental loss and caries were observed in most individuals. Intentional dental modification was described in contemporary Tenetehara-Guajajara by Lima  and conf a blade , were pr a blade ,44, this a blade .cribra orbitalia indicated trauma and underweight affecting even the young individuals, consistent with people facing extreme challenges to survival. Some developmental anomalies, such as butterfly vertebrae, and rib anomalies were observed  with rib periosteal reactions did not show positive results for MTC aDNA hybridization. Seven specimens positive for MTC hybridization demonstrated porotic hyperostosis and rbitalia .mpt40 and katG targets in three individuals. PCR exhibited absence of amplification for all molecular targets in negative controls, even after application of RP and WGA.All positive pPCR including those of unexpected length were submitted to nucleotide sequencing. The MTC sequencing that provided low quality or no sequences were excluded. Nucleotide sequencing confirmed PCR results for Taq DNA polymerase (qiagen.com). However, RP and WGA could produce chimeras in non-homogenous aDNA extracts. Forst and Brown [RP and WGA increased the aDNA concentration  and probnd Brown  attestednd Brown  verifiednd Brown ,60. The mtp40 sequence fragment (katG target (GenBank: MF773496-97) from SABMN00709 and SABMN00710 , M. canettii CIPT140070017 (GenBank: FO203510), and M. microti 12 (GenBank: CP010333). mtp40 is absent in some MTC strains, including M. bovis, M. caprae, and some M. tuberculosis [M. tuberculosis and M. africanum [M. bovis. Fletcher et al. [mtp40 gene in three 18th century Hungarian mummies, excluding M. bovis infection. The SABMN00711 individual was positive for IS aDNA hybridization, but no TB or nonspecific bone lesions were observed. The present study confirmed infection by pathogenic MTC Mycobacteria.The ition 35  identicaition 35 . Maximumrculosis . Neverthfricanum ,62. The r et al.  detectedkatG sequences from samples SABMN00709 and SAMMN00710 showed the SNP A74G that discriminates M. canetti and G86A strains from all M. tuberculosis and M. canetti sequences. G86A SNP is present in M. africanum, M. bovis, M. caprae, and M. microti strains and corresponds to the ACT (Thr) 203 codon described (in reverse position) by Fletcher et al. [katG 203 target has been used to identify MTC subspecies. Huard et al [katG 203 present in PGG1a, including M. africanum subtype Ia, M. microti, M. caprae, and M. bovis. Other PPGs with ACC katG 203 include 1b: M. canettii, M. tuberculosis, M. africanum subtype Ib; 2: M. tuberculosis; 3: M. tuberculosis. Fletcher et al [katG analysis. In the present study, MTC strain PPG 1a was identified, suggesting M. africanum subtype Ia or M. bovis infection in samples SABMN00709 and SABMN00710, rather than M. tuberculosis strains. Both these individuals were negative for IS aDNA hybridization, and only SABMN00710 had both porotic hyperostosis and cribra orbitalia, bone evidence of poor health.The r et al. . The katrd et al  classifier et al  genotypemtp40 results pointed to M. tuberculosis or M. africanum strains, while katG ruled out M. tuberculosis strains, suggesting M. africanum and M. bovis members. This multiple infection possibility reflects the post-contact scenario blending cultures and epidemiological backgrounds of Europeans and Africans.Tuberculosis genotyping revealed two MTC species/lineages affecting the Tenetehara-Guajajara population. The M. africanum has not become established outside of West Africa. They point out that, even with the massive migration to the Americas during slave trade, the diseased either did not survive the journey, or, if it did, was outcompeted in the New World by M. tuberculosis [M. africanum for ethnic West Africans was also suggested by the authors as a reason it did not become established in Native Americans or European explorers. The results presented here may show some level of M. africanum strain infection.Jong et al.  discusserculosis . Host prkatG and mtp40 segments. This was not unexpected, due to the highly degraded aDNA. Hybridization is reported to be more sensitive than PCR in aDNA detection [cribra orbitalia manifestations.A previous study discussed the lack of specificity of IS6110 and IS1081 targets . Our attetection , especiaetection  that areFinding anatomically normal bones positive for MTC aDNA has been previously described, including in skull, femur, and ribs ,18. RollServiço de Proteção ao Índio—SPI) and other Brazilian government agencies concerned with indigenous health, confirm the endemic conditions of the Tenetehara-Guajajara in the 20th century. Records describe malaria, smallpox, and syphilis, as well as TB, among other endemic and epidemic diseases. The impoverished living standards of the Tenetehara-Guajajara population following the Colonial Period contributed to health issues faced in the past century. Some of their groups succeeded in escaping slavery and extermination, migrating upstream in the Pindaré-Gurupi basin, and remaining isolated in poor conditions for almost a century.No detailed medical documents can be found for the Colonial Period or for Jesuits and travelers during the Empire Period in Maranhão State. Data from the former Native Protection Service Click here for additional data file.S1 FigNomenclature of clones: The first number correspond to mtDNA PCR target (cl1-cl4) and the second to the number of clones generated. Target 1: primers L16070/H16259; 2: L16209/H16410; 3: L16234/H16422; 4: L16268/H16498.(TIF)Click here for additional data file.


Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic respiratory disease in swine resulting in enormous economic losses. To identify the components that contribute to virulence and unveil those biological processes potentially related to attenuation, we used isobaric tags for relative and absolute quantification technology (iTRAQ) to compare the protein profiles of the virulent M. hyopneumoniae strain 168 and its attenuated highly passaged strain 168L. We identified 489 proteins in total, 70 of which showing significant differences in level of expression between the two strains. Remarkably, proteins participating in inositol phosphate metabolism were significantly downregulated in the virulent strain, while some proteins involved in nucleoside metabolism were upregulated. We also mined a series of novel promising virulence-associated factors in our study compared with those in previous reports, such as some moonlighting adhesins, transporters, lipoate-protein ligase, and ribonuclease and several hypothetical proteins with conserved functional domains, deserving further research. Our survey constitutes an iTRAQ-based comparative proteomic analysis of a virulent M. hyopneumoniae strain and its attenuated strain originating from a single parent with a well-characterized genetic background and lays the groundwork for future work to mine for potential virulence factors and identify candidate vaccine proteins. M. hyopneumoniae is the causative agent of porcine enzootic pneumonia, which is a worldwide epidemic that can cause enormous economic losses as a result of retarded growth in pigs and the cost of disease control . Th. Th M. hDR of 1% . 50.5% oDR of 1% .Of the 489 proteins identified, 273 proteins were annotated into 9 groups based on the KEGG categories. The categories with the highest abundance were translation (70 proteins), carbohydrate metabolism (45 proteins), replication and repair (41 proteins), nucleotide metabolism (40 proteins), and membrane transport (40 proteins) . MeanwhiWe detected and quantified 489 proteins, of which 35 were more abundant in the Mhp168 strain and 35 in the Mhp168L strain, based on having a log2 ratio ≥ 0.585 or ≤-0.585 and a statistically significant differences at p<0.05 .Gene Ontology (GO) enrichment analysis was then performed for the differentially expressed proteins for the categories “molecular function” (F), “cellular components” (C), and “biological process” (P) .  FigureTo examine which biological pathways were apparently altered, we carried out a pathway analysis. Enriched pathways were grouped into 2 categories with P value <0.05 . The two M. hyopneumoniae. All these three proteins are cytosolic enzymes, lacking transmembrane domains or traditional signal sequences  whose gene was also absent in its genome. Nevertheless, Ferrarini et al. [ M. hyopneumoniae might function as IolJ from other organisms and confirmed that M. hyopneumoniae had been able to utilize myo-inositol from the culture medium.Among all the sequenced mycoplasma species,lization . A previyorhinis . In our i et al.  hypothes in vivo. Considering the fact that myo-inositol has been one stable readily abundant component in the culture medium containing swine serum, we may assume that M. hyopneumoniae retains the ability of degrading myo-inositol in vivo, then the capability is increased with passage in vitro. The vaccine strain might have developed an enhanced ability to cope with differences in environmental carbon sources. However, the myo-inositol utilization gene cluster existed in both strains, and there were no genetic variations in the coding sequences or intergenic regions of those genes [The increments of those protein levels might facilitate the catabolism of myo-inositol to produce dihydroxyacetone-phosphate (DHAP) and acetyl-coenzyme A (CoA). DHAP can enter glycolysis while acetyl CoA can be widely used in macromolecular biosynthesis and energy production to support cell growth and proliferation. The presence of inositol in mammalian hosts' bloodstream  and the se genes . It may M. hyopneumoniae cannot synthesize purines and pyrimidines de novo. However, this organism is able to take up exogenous nucleobases and nucleosides, as well as those produced internally by DNA and RNA degradation, and then synthesize nucleotides through salvage pathways and interconversions [versions . In the versions . We founversions . Thus, t M. hyopneumoniae offence and defense, meriting more attention. To predict putative novel virulence factors and search for potential drug/vaccine targets, we used VirulentPred to perform the in silico analysis for the 35 upexpressed proteins in the virulent strain 168. The server categorized 18 proteins to be associated with virulence. Out of these, 10 proteins are ones with known functions, and the others are termed as “hypothetical” or “uncharacterized”. As a complement to the bioinformatics analyses, we also searched manually the 35 proteins and/or their paralogues for the references that support their virulence-related activities in M. hyopneumoniae, or in other pathogens. Then additional 8 proteins classified as “nonvirulent” were recruited into the group of putative virulence proteins. The results are summarized in Mycoplasmas contain a range of virulence factors in their pathogenic machinery, including enzymes, transporters, transcriptional regulators, lipoproteins . Among tLipoproteins. Three lipoproteins were identified in this study. MHP168_392 and MHP168_393 were downexpressed and MHP168_418 were upexpressed in the virulent strain 168. Most lipoproteins are surface exposed and important components of mycoplasma membranes, and some in M. hyopneumoniae have been identified to play important roles in pathogenesis [ M. hyopneumoniae 232, has been identified as a species-specific, highly immunogenic membrane-associated protein [ M. hyopneumoniae 232 (mhp379) is a surface-exposed exonuclease with probable function in importing nucleic acid precursors [ in vitro [ M. hyopneumoniae usually cannot employ the similar mechanisms used by other mycoplasma species to generate antigenic diversity through genetic variation [ogenesis –49. Mhp3 protein . The homecursors . Furtherin vitro . M. hyopariation . Nonetheariation . The ideAdhesion-Related Proteins. The adherence of M. hyopneumoniae to ciliated respiratory epithelium is mainly mediated by the membrane protein P97 [ M. flocculare and M. hyopneumoniae [ M. hyopneumoniae genome [tein P97 . The fineumoniae . A previeumoniae . Howevereumoniae . P102 ace genome . The seqe genome . Thus, a M. hyopneumoniae [In our proteomic data, we detected several upregulated moonlighting proteins in strain 168 that could be used to invade host cells: elongation factor Tu , 31, enoeumoniae . Fibroneeumoniae . Those m M. hyopneumoniae in part. Significantly expressed protein O-sialoglycoprotein endopeptidase in strain 168 also regulates the invasion of pathogen via degrading sialoglycosylated host-cell proteins and mediating adhesion to host cells , 58. TheTransporters. Transporter proteins play important roles in bacteria, transporting various molecules to support survival and growth in different niches [2+ ion transporter (MgtE) is a highly Mg2+-selective channel gated by Mg2+, transporting substrates across the cytoplasmic membrane by utilizing the electrochemical gradient [t niches . Cation-t niches –62. Mg2+gradient . Both ofgradient . Inactivgradient . ABC traEnzymes. Two lipoate-protein ligase A (LplA-1 and LplA) were upregulated in the pathogenic strain 168. LplA ligates exogenous lipoic acid to lipoyl domains of certain metabolic enzymes complexes involved in oxidative metabolism [ L. monocytogenes was damaged specifically in the host and virulence was 1/300th as that of wild-type in animals [ M. hyopneumoniae.tabolism . Previou animals . PossiblEnzymes PrsA have been treated as potential targets for therapeutic and vaccine candidates according to previous reports . CysteinTranscriptional Regulator. In strain 168, we found the expression of ribonuclease III (Rnc) was significantly elevated. This ribonuclease is known to be assisting in pathogenesis of other bacteria via regulating the synthesis of virulence factors through RNase III-dependent posttranscriptional manner [ M. hyopneumoniae, therefore contributing to high virulence. l manner , 70; itsUncharacterized/Hypothetical Virulence-Related Proteins. According to the previously published genome data, 268 of 695 (~39%) M. hyopneumoniae proteins were not assigned functions and annotated as “uncharacterized” or “hypothetical” proteins [proteins . In manyproteins , 71, whi M. hyopneumoniae or novel drug/vaccine targets. The VirulentPred results showed that all the overrepresented proteins in strain 168 among the differentially expressed uncharacterized/hypothetical proteins are predicted to be associated with virulence. Thus the protein sequences were submitted to BlastP, Pfam, and InterPro web servers for putative function annotation. In our study, 45 hypothetical proteins and 78 uncharacterized proteins were identified. Among them, 11 uncharacterized/hypothetical proteins displayed differential expression levels, 8 were overrepresented, and 3 were underrepresented in strain 168. It is reasonable for us to suppose that the differentially expressed proteins of unknown function might give a clue for pathogenic mechanisms of M. hyopneumoniae. This protein was annotated to be a Cof-like hydrolase, while might function as the phosphatase Cof in E.coli, catalyzing the hydrolysis of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate to 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate [Proteins ADQ90691, ADQ90727, and ADQ90524 were annotated as hydrolase enzymes. Members of this class are involved in various significant biological processes, including virulence mechanisms , 72. ADQhosphate .+-dependent deacetylation. As the deacetylase of M. fermentans is expressed inside mammalian cells, it inhibits cell proliferation but promotes their antioxidation and antistarvation capacities, and alters gene expression, affecting physiological functions and the corresponding signal transduction pathways in host cells [ M. hyopneumoniae is an extracellular pathogen, thus it remains unclear whether ADQ90530 in M. hyopneumoniae functions similarly with secreted deacetylase in intracellular pathogen M. fermentans. Elucidating this issue will require further investigation.The hypothetical protein ADQ90530 was categorized as a sirtuin , which is responsible for NADst cells . The lonProtein ADQ90824 was predicted to be an N-6 adenine-specific DNA methylase, which controls methylation at adenine residues of important biological processes. The methylation process plays an important role in bacterial pathogenesis by regulating the synthesis of virulence factors, and DNA adenine methylases serve as promising antimicrobials and vaccines targets , 75.Leucine-rich repeats (LRRs) are found to be present in a number of proteins with diverse functions, including cell-adhesion molecules, virulence factors, and extracellular matrix-binding glycoproteins, and function in signal transduction, cell-adhesion, and protein-protein interactions , 77. ProUncharacterized protein ADQ90859 belongs to the mycoides cluster lipoprotein, LppA/p72 family; members of this protein family are predicted lipoproteins with a typical prokaryotic signal peptidase II processing and lipid attachment site . Paralog4folate + NAD+ <==> 5, 10-methylene-H4folate + CO2 + NH3 + NADH + H+, and H-protein shuttles some of the intermediate products [ Francisella tularensis isolated from mouse spleens compared with in vitro grown controls, suggesting that H-protein may play an important role in the metabolic fitness of Francisella tularensis [ M. hyopneumoniae; we assumed hypothetical protein ADQ90910 also play a part in the adaptive response of pathogen to in vivo environment.Hypothetical protein ADQ90910 was classified as one member (H-protein) of the glycine cleavage system composed of four proteins: the T-, P-, L-, and H-protein. This system catalyzes the reversible reaction: Glycine + Hproducts . H-protelarensis . A similThe “DUF31” domain that has no known function was found in uncharacterized protein ADQ90827, and none of the domains was predicted in hypothetical protein ADQ90324. The function of them cannot be identified but is predicted to be associated with virulence. M. hyopneumoniae. The components that play roles in these critical mechanisms are natural targets for specific drugs. These potential virulence factors are usually immunogenic and can be treated as vaccine candidates [In conclusion, this survey identified mycoplasmal proteins and unveiled biological processes potentially related to the difference in virulence between the pathogenic and attenuated strains ofndidates , 83. Our