AbstractLacconotus LeConte, the sole Nearctic representative of the eurypine Mycteridae, is revised, based on morphological features of adults. The following syn. n. is proposed: Lacconotus pallidus Van Dyke, 1928 = Lacconotus pinicola Horn, 1879. The former is a light-colored form with a southern California distribution. A subgen. n.,Alcconotus, is described for Lacconotus pinicola, producing the following comb. n.: Lacconotus (Alcconotus) pinicola (Horn). A lectotype is designated for Lacconotus pinicola. A key separating the two subgenera and species is provided, as are photographs and illustrations of salient structures of adults, and maps showing collection localities. Lacconotus punctatus is newly recorded in Alabama, Arkansas, Massachusetts, Oklahoma, Texas, and Wisconsin; Lacconotus pinicola is newly recorded in Arizona and Utah in the USA, and Baja California Norte in Mexico. Phenology information shows a north-to-south gradation in occurrence time. PageBreak being in New and Old World tropical regions (Mycterus Clairville (Mycterinae), three species of Hemipeplus Latreille (Hemipeplinae), and two species of Eurypinae. The latter are represented by Lacconotus punctatus LeConte and Lacconotus pinicola Horn . The distribution of the family in North America is decidedly either western or eastern, i.e., there are no species represented in the interior of the continent, and no single species bridges this distributional gap  are the most diverse with 26 genera and 160 species recognized worldwide, the greatest diversity regions . The Neaonal gap .Hemipeplus)  e.g., , to Melas) e.g., , and Pyts) e.g., . In a phLacconotus was undertaken for several reasons. For some time, it was recognized  that the eastern and western species of the genus were rather dissimilar structurally; recent collections of specimens, especially through Michael Caterino’s “California Beetle Project”, have added much more material for study; and, it is the first author’s goal to revise all genera of world Mycteridae, including the many presently poorly known genera of Eurypinae.This study of Physcius fasciatus Pic, Physiomorphus spp., Phaeogala rufa Abdullah) or in palm leaf axils or dead foliage (e.g. Eurypus spp.). With the exception of Stilpnonotus spp., eurypine larvae have flattened bodies and well-developed, complex urogomphal plates, typical of larvae that move subcortically. As with many other groups of Tenebrionoidea, and Coleoptera generally, relatively little is known of the specific habits of eurypine Mycteridae. Larvae have been described for only a few species; these descriptions  and indicated that larvae of this species occur under bark of poplar (Populus) and fir (Abies). Lacconotus pallidus (= Lacconotus pinicola), from under bark of dead oak (Quercus sp.).The biology of Lacconotus punctatus and Lacconotus pinicola, below.Other specific details pertinent to natural history, derived from label data or other sources, are given under PageBreak® digital camera fitted to a Leica MZ95 stereoscope. Approximately 30 separate photographs were taken for each specimen/structure; these were imported into Combine ZP . Information added by the authors for clarity is enclosed in square brackets ().Label data on type specimens are recorded Abbreviations of collections ].; Lacconotus pallidusLacconotus pinicola), 177; Lacconotus pinicola, all in MCZC).—lectotype (here designated), female, labeled: “Veta Pass 27.6 Col / 592 / [red] Type 7976 / [handwritten] Lacconotus pinicola (Schwz) / J.L. LeConte Collection / LECTOTYPE ♀ Lacconotus pinicola.—holotype, male (CAS type # 2585), labeled “Mt. Wilson Cal. 6.13.3 / 7701 / Van Dyke Collection / Holotype [along left margin of label covered in red ink] ♂ Lacconotus pallidus Van Dyke". ALLOTYPE, female, labeled "Carmel, Monterey Co VI-4-1916 Cal. / Van Dyke Collection / Allotype [along left margin of label covered in red ink] ♀ Lacconotus pallidus Van Dyke". Four PARATYPES. Male, labeled "Carmel, Monterey Co VI-4-1916 Cal. / Van Dyke Collection / Paratype [along left margin of label] ♂ Lacconotus pallidus Van Dyke". Male, labeled "ParaisoSpgsCal V.28 1924 L.S. Slevin / L.S. Slevin Collection / Paratype [along left margin of label] Lacconotus pallidus Van Dyke". Female, labeled "ParaisoSprings V.31 1916 Cal. / L.S. Slevin Collection / Paratype [along left margin of label] Lacconotus pallidusPageBreak Van Dyke". Female, labeled "Paraiso Springs V.29 1916 Cal. / CHAMISAL / L.S. Slevin Collection / Paratype [along left margin of label] Lacconotus pallidus Van Dyke". (Lacconotus (Alcconotus) pinicola may be distinguished from Lacconotus punctatus by the following features: body color ranging from testaceous to dark brown, uniform dorsally  with the following: TL 4.8–7.5 mm; GEW 1.5–2.3 mm; TL/GEW 2.8–3.4. Dorsal body surface uniformly testaceous to dark brown or piceous  based on its lighter color, relatively narrower pronotum, shorter relative length of the elytra, and deeper punctation. However, upon examination of the type series and other specimens, we have determined that the only feature of Van Dyke’s that withstands scrutiny is the habitus color. As well, more detailed examination has revealed that the male and female genitalia and male sex patch are virtually identical between Lacconotus pinicola and Lacconotus pallidus. One feature, mentioned by Lacconotus pallidus in southern California. We herein consider Lacconotus pallidus a pale “form” of Lacconotus pinicola.PageBreakLacconotus (Alcconotus) pinicola is newly recorded from Arizona, Utah, and Baja California Norte in Mexico (see Appendix A). Published records of Lacconotus pinicola are from British Columbia , including one specimen which was recorded as emerging from a dead Pinus ponderosa branch. It has also been found on Engelmann spruce (Picea engelmanni Parry ex Engelm.), scrub oak (Quercus turbinella Greene), and cherry (Prunus sp.). Specimens have been collected at UV lights, with malaise and flight-intercept traps, and by beating vegetation. Lacconotus pinicola has been illustrated by A number of specimens examined were found on ponderosa pine (


D. hansenii, D. maramus, C. famata, C. zeylanoides and H. burtonii species, and previously selected for their ability to grow in dry-cured ham-like substrates, were screened for antagonistic activity against a toxigenic strain of P. nordicum and inhibition of ochratoxin A (OTA) biosynthesis. On average, yeast inhibitory activity was lowered by increasing fungal inoculum and enhanced by NaCl presence. In the assay conditions, H. burtonii and C. zeylanoides were the most effective, both in inhibiting P. nordicum growth and OTA production. D. hansenii was the species with the lowest inhibitory activity, especially in the absence of salt. OTA production dropped from the range < LOD − 5000 ppb in P. nordicum control plates to the range < LOD − 200 ppb in yeast-added plates. OTA production increased in the presence of NaCl in P. nordicum control plates, while salt enhanced inhibition against OTA production in yeast-added plates.Twelve yeast strains isolated from the surface of Italian typical dry-cured hams, belonging to  The mycete populations (yeasts and moulds) colonizing the surface layers of dry-cured meat products during maturation have been regarded as positively contributing to the chemosensory properties of final outcome, by means of oxidation prevention and generation of volatile compounds enhancing aroma ,2. In thP. nordicum, a toxigenic contaminant of protein-rich food [P. nordicum isolates proved to be important producers of ochratoxin A (OTA), a mycotoxin classified by the International Agency for the Research on Cancer (IARC) as a possible human carcinogen, suspected to be involved in nephropathies and urothelial tract tumours [P. nordicum [P. nordicum inside dry-curing plants. Among moulds isolated from dry-cured meat facilities and products, Penicillia were largely dominant, and the contamination levels have been found to be associated to the environmental conditions of ripening rooms ,5, geogrich food , was isoich food . Several tumours . OTA wasnordicum , includinordicum ,14. CurrP. nordicum contamination based on microbial antagonists can be investigated as a possible way to improve food safety without affecting sensory quality and properties of typical dry-cured meat products. As a rule, during manufacturing a wild yeast population spontaneously grows on dry-cured ham surface, achieving different counts according to processing stage and type [Debaryomyces, Candida and less frequently to Cryptococcus, Rhodotorula and Rhodosporidium [Candida zeylanoydes [w and salt typical of dry-cured ham surface [Biological control of and type ,16. Yeasporidium ,16,17. Nlanoydes  and prov surface . Recentl surface ,20. The  surface ,22, coff surface , fruits  surface  or ferme surface , cheese  surface . Recent  surface ,27. in vitro the ability of selected yeasts isolated from dry-cured ham surface to compete with P. nordicum and to inhibit OTA accumulation in the perspective of using them as surface starter cultures. The aim of this work is to evaluate n = 12) were selected from a total of 270 isolated from dry-cured ham during the maturing process [Tested yeast strains ( process  and storP. nordicum and ability to grow in a dry-cured ham-like substrate [Candida zeylanoides (n = 2), Hyphopichia burtonii (n = 2), Debaryomyces hansenii (n = 2), Debaryomyces maramus (n = 3) and Candida famata (n = 3), by means of the Biolog system . Furthermore, yeast cells from the pure cultures of the above-mentioned strains underwent genetic identification by sequencing the D1/D2 domain of 26rRNA encoding gene [C. zeylanoides and H. burtonii species were confirmed by genetic system, while the remaining strains were genotyped as D. hansenii. The yeast strains, selected in a previous study for their potential competition against ubstrate , were puing gene . The strYeast cultures were grown in 10 mL Malt Extract Broth  at 25 °C for 48 h. Suspensions were prepared by inoculating 25 mL of YPD broth with a loop full of cells and incubating on a rotary shaker (180 rpm) at 28 °C for 24 h. The density of yeast cultures was determined spectrophotometrically by measuring their optical density at 600 nm. P. nordicum isolated from cured pork meat in a previous research [An ochratoxin A producing strain of research  and idenresearch , was use2, 104, 106 conidia/mL using a hemocytometer. The strain has been grown on Petri dishes (9 cm diameter) with Czapek Yeast extract Agar  and incubated at 25 °C for 7 days. At the end of incubation, 10 mL of sterilized water was added to the culture surface and it was gently scraped to remove most of the conidia for the inoculum preparation. The obtained conidial suspension was adjusted to 10et al. [The assay was carried out by means of the test described by Bleve et al.  modified8 cells to obtain a thick, continuous layer on plate surface. This suspension was distributed into Petri plates containing 15 mL of YPD agar media. Then, three 10 μL portions for each P. nordicum suspension corresponding to 102, 104, 106 conidia/mL, were separately spotted on each plate and incubated at 20 °C. The inocula of P. nordicum were carried out at the same time (co–cultured) and four days after the yeast-media preparation (delayed). Two replicate experiments for each condition were performed. Culture media (YPD and YPD +3% NaCl) plates not inoculated with yeasts were included as control.Experiments were performed on YPD agar medium adjusted to pH 6, with and without the addition of sodium chloride (3%). A top agar was prepared by mixing 6 mL of YPD with 0.7% agar and 1 mL of yeast suspension containing 10et al. [Fungal growth was expressed as the average measure (mm) of two orthogonal diameters/colony after 14 days of incubation. The inhibitory activity was calculated by the Equation (1) reported by Lima et al. :P. nordicum were analyzed for OTA content after 14 days of incubation. OTA was measured according to the method of Bragulat et al. [et al. [®, 250 × 2.1 mm, 3 µm) on a Agilent 1100 chromatographic system  under isocratic condition at room temperature, with an aqueous NH3/NH4Cl :CH3CN = 85:15 v/v mobile phase; the flow was 0.2 mL/min and the injected volume was 20 µL. The FLD detection was obtained by means of an Agilent 1100 Fluorescence Detector  . OTA has been expressed as ng/g medium. The limit of detection (LOD) and the limit of quantification (LOQ) in plates were 0.2 ppb and 0.6 ppb respectively.Control and yeast-added plates inoculated with t et al. , modifie [et al. , analyzi8 cells in the case of test plates and with 1 mL of physiological solution in the case of control plates. Plates were incubated at 20 °C for 14 days and stored at 20 °C before OTA analysis.Culture media (YPD and YPD +3% NaCl) were spiked with 500 ppb OTA/plate; then, the top agar was added with 1 mL of yeast suspension containing 10vs. P. nordicum were estimated by means of General Linear Model (GLM) procedure for the main effects “yeast species”, “salt presence”, “fungal inoculum level” and their two-way interactions; the Bonferroni t-test was used to statistically separate LSM when P < 0.05. Percentile distribution of OTA amount in yeast-added- and control plates inoculated with P. nordicum, was analyzed using non parametric test (χ2 test), according to yeast species.Data analysis was carried out using the SPSS software package . Data of yeast inhibitory activity and OTA production were inspected for normal distribution using the procedures Frequencies  and Kolmogorov-Smirnov test. Least Square Means (LSM) of yeast inhibitory activity w values, NaCl content, pH and nutrients of dry-cured ham surface, and exerting in vitro competition potential against P. nordicum [The biocontrol test was performed using 12 yeast strains selected among a far exceeding number isolated from dry-cured hams . Yeast snordicum . D. hansenii, D. maramus, C. zeylanoides, C. famata, H. burtonii), at least two strains were included in the biocontrol assays, and tested strains caused a visible growth reduction of P. nordicum after 14 days of incubation, as displayed in P. nordicum inoculum were related to colony diameters, because the larger colonies at 14 days of incubation corresponded to higher inocula. For each phenotypic species (P. nordicum was not influenced by yeast co- or delayed inoculation (data not shown). A different inhibitory activity against P. nordicum was found among yeast species (C. zeylanoides and H. burtonii showed the highest inhibition (86% and 88% respectively) and D. hansenii the lowest one (58%).Inhibitory activity against  species : C. zeylP. nordicum inoculum: the growth of colonies from the lowest inoculum concentration (102 conidia/mL) was the most inhibited by antagonistic yeasts; similar growth was observed in colonies grown 104 and 106 conidia/mL as inoculum.Assayed yeast inhibitory activity was affected by the concentration of P < 0.05) the inhibitory activity of yeasts against P. nordicum, and a significant interaction between salt and yeast species has been found . As a consequence, the influence of salt over yeast inhibitory activity changed according to yeast species, as displayed in P. nordicum was quantified both in yeast-added and control plates after 14 days of incubation  were created and named high (H), intermediate (M) and low (L) with reference to OTA content range . P < 0.001): control plates were mostly allocated to H group, the yeast-added ones to L group, while the frequencies of control and treated plates in M group were similar.A comparison was carried out between control and yeast-added plates to compare percentage distributions between groups (χ2 statistic). The yeast inhibitory activity over OTA production was significant  C. zeylanoides differed from C. famata and D. hansenii; (iii) D. maramus differed from D. hansenii. The effect of yeast species over OTA distribution in H, M and L groups is reported in Ochratoxin A values detected in control and yeast-treated plates were split with reference to salt presence in YPD medium . In contOTA analysis in spiked control and yeast-added plates had the same % recovery, showing that yeast presence did not affect OTA extraction from the growing medium . The microbial community growing on dehydrated surface of dry-cured ham during the maturing phase is dominated by several species of yeasts and moulds ,34; the in vitro against an OTA-producing P. nordicum strain [8 cfu/mL) may restrict the availability of nutrients and sites for colonization, essential for the germination of mould spores [H. burtonii and C. zeylanoides the most, and D. hansenii the least effective in inhibiting P. nordicum growth. Information on the antagonistic activities of yeasts isolated from dry-cured meat products against toxigenic fungi is scarce : recent m strain . Althougd spores . SignifiD. hansenii is the predominant species in dry-cured ham isolates during processing [D. hansenii in dry-cured meat products is most probably due to its moderately halophilic properties, accounting for its optimal growth at 3–5% salt [D. hansenii strains isolated from dry-cured meat products were able to growth up to 15% NaCl if compared to C. zeylanoides [D. hansenii against P. nordicum could be predictive of less effectiveness of this yeast as biocontrol agent. ocessing ,17, but –5% salt . A recenlanoides . HoweverC. zeylanoides was found to decrease from early to late processing steps [in vitro tested strains of C. zeylanoides highly inhibited P. nordicum, under dry-curing plant conditions , C. zeylanoides, having been found to fail in maintaining high populations in matured dry-cured ham, could have less ecological fitness than D. hansenii. Furthermore, C. zeylanoides was included among emerging pathogenic yeasts [The occurrence of ng steps ,17: in Ing steps . So, evec yeasts  and consHyphopichia burtonii, referred to as “yeast-like fungi”, was isolated from dry-cured hams after surface fat application during maturation. The yeast was probably introduced by the rice flour mixed with the spreadable pork fat (sugna) used at mid maturing to prevent excessive dehydration of ham surface [P. verrucosum and the growth of P. roquefortii [P. nordicum showed in the present study. Otherwise, the attribution of H. burtonii to dry-cured ham native yeasts is questionable and its potential as biocontrol agent against P. nordicum should be tested in ripening plant conditions, before sugna application.  surface . It was uefortii ,38, and Debaryomyces maramus and C. famata (the anamorphic form of D. hansenii) were isolated throughout the processing of Parma ham and other traditional Greek and Spanish dry-cured products [D. hansenii and C. zeylanoides. In the case of orange fruits, a C. famata population (108 cfu/mL) gave a successful biocontrol toward Penicillium digitatum [C. famata exerted lower inhibition against P. nordicum than H. burtonii and C. zeylanoides, and an higher inhibition than D. hansenii. Penicillium contamination in cured meat production facilities and products was found to be affected by a large variability [P. nordicum corresponding to 102, 104, 106 conidia/mL. Yeast inhibitory activity was affected by fungal concentration: at the lowest inoculum level, yeast biocontrol was more effective (P < 0.05), showing that the concentration of the mould is a key factor for yeast effectiveness. As a consequence, in future in vivo applications of antagonistic yeasts for dry-cured ham biocontrol, it will be important to keep the population of contaminating moulds as low as possible, in agreement with processing procedures for high quality dry-cured meat products aimed at this specific target [P. nordicum, capable of growing and producing OTA in dry-cured pork samples in the presence of 5% NaCl and 0.90 aw [Debaryomyces spp., mostly in the case of D. hansenii. products ,39,40, eigitatum  in the piability : accordic target . Among yc target ,19, a pr 0.90 aw . A signiD. hansenii gave, on average, the least inhibition among tested yeasts, but, thanks to its halophilic aptitude [D. hansenii was able to synthesize toxic proteins with killer capacity against sensitive strains, but the lethal activity of these toxins was demonstrated or increased only in the presence of NaCl [The tested strains of aptitude , may be  of NaCl ,45. OtheP. nordicum was in pure culture (control plates) or cultured with yeast strains (yeast-added plates). The temperature of the biocontrol test (20 °C) was previously found suitable for OTA high production in dry-cured pork model systems [P. nordicum correlated with the sharp reduction of OTA , the nutrients not required for fungal growth could be used for OTA biosynthesis. Ochratoxin A frequency distribution into groups of equal frequency (33.3%) L, M and H changed according to yeast presence and species. Treated plates corresponded to low (L) or intermediate (M) OTA category production, while control plates fell mostly in high (H) category. In agreement with inhibitory activity values reported in D. hansenii gave OTA value frequency different from H. burtonii, C. zeylanoides and D. maramus, C. famata differed from H. burtonii and C. zeylanoides and D. maramus from H. burtonii. These results suggested that the same mechanism of biocontrol was effective against both P. nordicum growth and OTA production; otherwise, other studies reported that although the growth of spoilage fungi is inhibited by biocontrol of antagonistic yeasts, their metabolic activities are not necessarily reduced [P. nordicum growth in yeast-added plates . The inhibition was effective against both fungal growth and OTA production, and was significantly influenced by yeast species, level of fungal contamination and NaCl presence in culture medium. This study demonstrated that selected strains of yeasts isolated from dry-cured ham, exerted D. hansenii, but other species exerted a more efficient control on P. nordicum growth and OTA synthesis. The antagonistic activity of tested yeasts was negatively affected by increasing levels of fungal contamination. Assayed yeasts could be effective biocontrol agents for dry-cured meat products, if capable of quickly growing and long lasting. In this respect, future works will be aimed to test the efficacy of different yeasts species in ripening plants, to optimize the inoculum distribution and to achieve suitable conditions for yeast stable and efficient colonization of product surface.NaCl promoted a significant increase of biocontrol activity, favoring the most halophilic yeast strains such as


Plasmodium falciparum malaria, which can lead to poor maternal and fetal outcomes. Few data exist on the epidemiology of severe P. falciparum malaria in pregnant women.Pregnant women are more susceptible to severe P. falciparum malaria among pregnant women at the Kassala and Medani maternity hospitals, which are located in areas of unstable malaria transmission, in eastern and central Sudan, respectively. Pre-tested questionnaires were used to gather socio-demographic, clinical and obstetrical data. Suitable tests were performed for clinical and biochemical investigations.A hospital-based study was carried out to assess the pattern of severe P. falciparum malaria. These were hypoglycaemia , severe anaemia , hypotension , jaundice , cerebral malaria , repeated convulsions , hyperparasitaemia  and more than one manifestation . While the mean (SD) presenting temperature was significantly lower for women presenting with hypoglycaemia , other clinical and biochemical characteristics were not significantly different among women with different manifestations of severe P. falciparum malaria.Among 222 pregnant women diagnosed with malaria at the two hospitals, 40 (18.0%) women at mean (SD) gestational age of 29.3 (6.7) weeks fulfilled one or more of the WHO criteria for severe Preventive measures for pregnant women such as insecticide-treated bednets and chemoprophylaxis may be beneficial in areas of unstable malaria transmission. Early detection and prompt treatment of severe malaria, especially in pregnant women with hypoglycaemia, are needed. P. falciparum malaria , other clinical and biochemical characteristics were not significantly different among women with different manifestations of severe P. falciparum malaria, Table While the mean (SD) presenting temperature was significantly lower in women presenting with hypoglycaemia [38.2(0.6) versus 38.8(0.7) °C, P. falciparum malaria observed during pregnancy in this epidemiological setting. With the exception of lower temperatures, other clinical and biochemical criteria were not different among women with different criteria of severe P. falciparum malaria. Previously, severe anaemia and jaundice had been observed as the presenting manifestations of severe P. falciparum malaria in pregnant women in central and eastern Sudan [P. falciparum malaria [The main findings of the present study were that hypoglycaemia and severe anaemia were the predominant presenting manifestations of severe rn Sudan ,14. In nrn Sudan . Generalrn Sudan ,7. The grn Sudan ,17. It hrn Sudan . Thus, trn Sudan . None ofrn Sudan . Therefo malaria . Hyperin malaria ,21. Intr malaria . Compare malaria . PatientP. falciparum malaria. Thus, in such situation these patients would have received blood transfusion and artemisinins combination therapy rather than quinine treatment.In the present study, 12 30%) and 6 (15%) patients presented with severe anaemia and cerebral malaria, respectively. Cerebral malaria and severe malarial anaemia are two major syndromes causing malaria-related mortality . The pat0% and 6 P. falciparum malaria there are syndromes associated with mortality rates that are lower (e.g. severe anaemia) and higher  [P. falciparum malaria, which is higher than in non-pregnant adults [P. falciparum malaria and healthy controls.There were no maternal deaths in this study, early diagnosis; prompt effective treatment could explain this observation. These women would appear to represent a milder spectrum of disease e.g. severe anaemia and hypotension. It have been shown that, within the broad definition of severe cidosis) . We prevcidosis) ,30. Matet adults . The othPreventive measures for pregnant women such as insecticide-treated bednets and chemoprophylaxis may be beneficial in areas of unstable malaria transmission. Early detection and prompt treatment of severe malaria, especially in pregnant women with hypogylcaemia, are needed.The authors declare that they have no competing interests.AAA and EME carried out the study and participated in the statistical analysis and procedures. MMM carried out the biochemical tests. IA and MIE coordinated and participated in the design of the study, statistical analysis and the drafting of the manuscript. All the authors read and approved the final version.


Phytophthora, Pythium and downy mildew species, cause devastating diseases on a wide range of host species and have a significant impact on agriculture. Here, we report comparative analyses on the genomes of thirteen straminipilous species, including eleven plant pathogenic oomycetes, to explore common features linked to their pathogenic lifestyle. We report the sequencing, assembly, and annotation of six Pythium genomes and comparison with other stramenopiles including photosynthetic diatoms, and other plant pathogenic oomycetes such as Phytophthora species, Hyaloperonospora arabidopsidis, and Pythium ultimum var. ultimum. Novel features of the oomycete genomes include an expansion of genes encoding secreted effectors and plant cell wall degrading enzymes in Phytophthora species and an over-representation of genes involved in proteolytic degradation and signal transduction in Pythium species. A complete lack of classical RxLR effectors was observed in the seven surveyed Pythium genomes along with an overall reduction of pathogenesis-related gene families in H. arabidopsidis. Comparative analyses revealed fewer genes encoding enzymes involved in carbohydrate metabolism in Pythium species and H. arabidopsidis as compared to Phytophthora species, suggesting variation in virulence mechanisms within plant pathogenic oomycete species. Shared features between the oomycetes and diatoms revealed common mechanisms of intracellular signaling and transportation. Our analyses demonstrate the value of comparative genome analyses for exploring the evolution of pathogenesis and survival mechanisms in the oomycetes. The comparative analyses of seven Pythium species with the closely related oomycetes, Phytophthora species and H. arabidopsidis, and distantly related diatoms provide insight into genes that underlie virulence.The kingdom Stramenopile includes diatoms, brown algae, and oomycetes. Plant pathogenic oomycetes, including  Straminipila ( = stramenopile) and are more closely related to organisms in aquatic environments such as brown algae and diatoms. There is continued discussion on the higher level nomenclature of Straminipila within the kingdom ChromistaAphanomycesPeronosporaPhytophthoraPlasmoparaPseudoperonosporaPythiumOomycetes are a diverse group of organisms that morphologically resemble Fungi, yet are members of the Pythium species belongs to the peronosporalean lineage that includes hemibiotrophic Phytophthora species and the obligate biotrophic Hyaloperonospora species , Py. arrhenomanes (ATCC 12531 = CBS 324.62), Py. irregulare (DAOM BR486 = CBS 250.28), Py. iwayamai (DAOM 242034 = CBS 132417), Py. ultimum var. sporangiiferum (DAOM BR650 = CBS 219.65), and Py. vexans (DAOM BR484 = CBS 119.80); Pythium , 6.6 to 14.4 Gb of purity-filtered (PF) reads were generated by the Illumina GAII/IIx sequencer . Assembled genomes of six Pythium species yielded 3,685 to 11,542 contigs with an N50 contig length ranging from 9.8 to 37.4 Kb  H. arabidopsidis  Pythium species, ranging from 1,495 to 1,767 bp  and Ph. infestans (1.7 introns per gene). Average exon length in the six newly sequenced Pythium genomes ranged from 312 to 630 bp, consistent with Py. ultimum var. ultimum (498 bp). Similar to Py. ultimum var. ultimumPythium species contain an InterPro protein domain, comparable to that observed with Phytophthora species (55 to 66%) Annotation of the six 1,767 bp . AnalysiPy. arrhenomanes, Py. irregulare, Py. iwayamai, and Py. vexans. For these four species, a single pooled cDNA library was constructed for each species using RNA isolated from mycelium grown under five different growth conditions  and sequenced using the Illumina GAII. The total number of purity filtered reads ranged from 21.7 to 31.7 million reads per library with 82–88% of reads mapping to the cognate genome ) of e genome  indicati. vexans . A gene Pythium proteome, we clustered orthologs and close paralogs in seven predicted Pythium proteomes . First, comparisons were made between core Pythium genes and the rest of the genes from each species. The core Pythium genes were enriched in genes involved in pathogenesis and signaling processes including elicitin (IPR002200), necrosis-inducing (IPR008701), peptidase C1A (IPR000668), protease inhibitor ), and HECT ubiquitin ligase (IPR000569) whereas genes involved in transport activities  are underrepresented (P<0.05) (To gain insight into the unique features of the core (P<0.05) .Py. vexans-specific genes . The Py. aphanidermatum-specific genes were highly enriched (P<0.01) for aspartic peptidase (IPR021109), endoglucanase (IPR009009), cutinase (IPR000675), and pectate lyase (IPR002022) domains. The highly represented domains in Py. arrhenomanes were protease inhibitor ), cutinase (IPR000675), necrosis inducing (IPR008701), and pectate lyase (IPR002022). Similarly, pathogenesis related domains such as peptidase (IPR006026) and proteinase inhibitor I25 (cystatin (IPR018073)) were highly represented (P<0.05) in Py. irregulare-specific genes. The leucine-rich repeat (LRR) containing domain (IPR001611), carbonic anhydrase (IPR018338), and chitinase II (IPR011583) were over-represented (P<0.05) in Py. iwayamai-specific genes  and nucleotide-sugar transporter-related (IPR007271)) domains were highly over-represented in ic genes . A numbePythium species were identified using the CAZymes Analysis Toolkit (CAT) Pythium genomes revealed that these organisms exhibit substantial variation in number of CAZymes (Pythium genomes.Carbohydrate-active enzymes (CAZymes) are involved in the biosynthesis and degradation of diverse glycoconjugates, oligosaccharides, and polysaccharides  CAZymes . The CE Py. aphanidermatum and Py. arrhenomanes, respectively, but not in the other Pythium genomes  compared to all Phytophthora species (166–216 members) yet strikingly larger than the repertoire of the biotroph H. arabidopsidis (72 members) and the diatoms (31–32 members) (Pythium genomes with PL the least represented (3 families). We observed that in general Pythium species have a highly reduced set of secreted CAZymes when compared to Phytophthora species, which underwent gene expansion Pythium-CAZymes is reported in a companion paper .Interestingly, we identified eight and six cutinase-encoding genes (CE5 family) in  genomes  suggestimembers)  in agreePythium species, like many oomycete pathogens, secrete effector proteins as well as degradative enzymes that alter host physiology and facilitate colonization. Indeed, the genomes of Ph. infestans, Ph. ramorum, Ph. sojae, Py. ultimum var. ultimum, and H. arabidopsidis contain large complex families of effector genes that encode secreted proteins which have been implicated in pathogenesis Pythium proteomes were predicted using SignalP v3.0 Pythium species -like proteins with CBM, and expanded families of cell wall degrading enzymes. Overall, depending on the species, 63–78% of the predicted secreted proteins in Pythium species surveyed have expression support ( species . Genes eecretome . Of the  support .Pythium secretome (P<0.001) relative to the rest of the predicted proteome from Pythium (Pythium secretome are elicitin (IPR002200), glycoside hydrolases (IPR000322), glycosyl transferase (IPR001296), peptidase C1A (IPR000668), EGF-like domain (IPR006209), and pectate lyase (IPR004898) (P<0.001)  .Pythium species-specific secretome, we compared the frequency of occurrence of PFAM domains and GO terms in the species-specific secretomes relative to the rest of the proteome from that particular species using Chi-square tests. The Py. aphanidermatum-specific secretomes were highly enriched for hydrolase activity (GO:0004553) including cutin hydrolase activity (GO:0050525) and carbohydrate metabolic process (GO:0005975) (P<0.05) (Py. arrhenomanes-specific secretomes were highly enriched for cellulose catabolic process (GO:0030245), hydrolase activity (GO:0004553), and pectin lyase activity (GO:0047490). The Py. iwayamai-specific secretomes were highly enriched for peptidase activity (GO:0008233), transmembrane transport (GO:0055085), and nucleic acid binding (GO:0003676). The most represented GO terms in Py. ultimum var. sporangiiferum-specific secretome were cysteine-type peptidase activity (GO:0008234), cellulose binding (GO:0030248), and isomerase activity (GO:0016853). The transmembrane transport (GO:0055085) as well as sugar binding and sugar modification activities  were most enriched in Py. vexans-specific secretome while pectate lyase (GO:0030570), proteolysis (GO0006508), and glycosyl bonds hydrolase activity (GO:0016798) were the most enriched GO terms in the Py. irregulare-specific secretome. Enrichment of hydrolase, pectate lyase activity and cell wall modification process in species-specific secretome indicates that degrading host cell wall is one of the major functions of Pythium secretome as illustrated for other oomycete pathogens To document the protein family domains and biological functions enriched in the (P<0.05) . SimilarP<0.05) in the species-specific secretomes of Py. aphanidermatum  may play important role in counteracting the physiological impact of host defense compounds Py. iwayamai along with several peptidase domains . The Py. irregulare-specific secretome was highly enriched for peptidase A1 (IPR001461), pectinesterase (IPR000070), NPP1 domain (IPR008701), serine protease inhibitor ). The protein domains specifically enriched in different Pythium species  highlight the differences between these groups of plant pathogens in terms of their pathogenicity and host preference.Similarly, the protein family domains including cutinase (IPR000675) and glycoside hydrolase (IPR000743) that hydrolyze glycosidic bonds, and peptidase A1 (IPR001461) were highly enriched (dermatum . In addiPhytophthora species encode large numbers (350 to 563) of potential effector proteins that are implicated in pathogenesis Py. ultimum var. ultimum genome Pythium species.The genomes of the three Pythium species sequenced in this study. Consistent with previous analyses of the Py. ultimum var. ultimum genome Pythium species sequenced. Our results suggest that in all seven of the Pythium species surveyed, RxLR effectors are absent signifying substantial differences in virulence and the interaction of Pythium species with plant hosts as compared to Phytophthora and Hyaloperonospora species  within the secretome of Ph. infestans, Ph. sojae, and all seven Pythium species  from all Pythium species revealed a conserved LxLYLAR/K motif followed by conserved WL motif  that are presumed to enter host cytoplasm WL motif  that is  species . The intStraminipila includes phytopathogenic oomycetes and autotrophic diatoms. A phylogenetic tree constructed using the Bayesian analyses of nuclear large subunit of rDNA from 14 stramenopiles shows five broad clades: the Phytophthora species with Hyaloperonospora and Py. vexans, the Pythium species with globose sporangia, the Pythium species with filamentous sporangia, the diatom group with T. pseudonana and P. tricornutum, and a separate clade of S. parasitica (The rasitica .Pythium (7 species), Phytophthora (3 species), H. arabidopsidis, and diatoms (2 species) were clustered revealing the stramenopile-core , Pythium/Phytophthora-specific (including clusters specific to Phytophthora and Pythium), Hpa-specific (clusters specific to H. arabidopsidis) and diatom-specific clusters  relative to the non-core genes (genes other than core genes) from stramenopiles  were highly enriched with protein domains possibly related to pathogenesis including necrosis inducing (IPR008701), elicitin (IPR002200), glycoside hydrolases , and peptidases  (P<0.05) as compared to the rest of the genes from Pythium and Phytophthora. For several groups of over-represented domains in the Pythium-Phytophthora-specific genes, a direct or indirect role in host-pathogen interaction and/or plant pathogen lifestyle has already been hypothesized or demonstrated Pythium-Phytophthora-specific clusters, suggestive of a role in protection of the pathogen against host-encoded defense-related proteases as shown in other oomycete pathogens H. arabidopsidis, the H. arabidopsidis-specific genes  showed under-representation of domains related to transport (IPR011547), host-targeted degradative enzyme (IPR021067), elicitin (IPR002200), and necrosis-inducing protein (IPR008701) (P<0.05)  . It is pPythium genome sequences permits the first detailed investigation of genome evolution within the genus and comparison with that of other straminopiles. By comparison with Py. ultimum var. ultimum, we identified syntenic regions across stramenopiles and analyzed rearrangements in gene order. Previous analyses of synteny between selected regions of Py. ultimum var. ultimum and Phytophthora species showed very similar ortholog content in broad regions but a high level of rearrangement in local gene order Pythium species, H. arabidopsidis and the diatom T. pseudonana. Due to the fragmented nature of the assemblies in all of the genomes, we identified contigs or scaffolds with a minimum of five genes and identified syntenic blocks in comparison to the Py. ultimum var. ultimum genome using MCscan Py. ultimum var. ultimum genome. The comparison of Pythium genomes shows a varying degree of synteny between Py. ultimum var. ultimum and six other Pythium species. Among all Pythium species, Py. ultimum var. ultimum was most syntenic with Py. ultimum var. sporangiiferum and least syntenic with Py. arrhenomanes and Py. iwayamai followed by Py. aphanidermatum  with Ph. infestans (supercontig 1.2), H. arabidopsidis  and T. pseudonana (chromosome 3). As expected, the gene order was highly conserved among Pythium species. The level of synteny revealed by our analyses extends the previously reported synteny between Py. ultimum var. ultimum and Phytophthora species Pythium species but also between other stramenopiles. Within oomycetes, the conservation of synteny between species recapitulates the phylogeny shown in Pythium species) than with distantly related species (e.g. H. arabidopsidis and T. pseudonana).In order to examine the conservation of gene order across three oomycete genera, we compared The genome sequences of 13 stramenopiles enabled genome-wide comparison of gene repertoires within and between phytopathogenic oomycetes and non-pathogenic diatoms. Our comparative analyses of stramenopiles indicate that developmental innovations in oomycete pathogens involve secretion of a large number of effector molecules, proteolytic enzymes, and cell wall hydrolyzing enzymes. However, expansion of a suite of genes encoding effectors and proteolytic enzymes reflect specific adaptations to trophic lifestyle.Phytophthora species show expansion and diversification of protein families associated with plant infection such as some glycoside hydrolases, ABC transporters and in particular, oomycete pathogenesis related genes. In contrast to the biotrophic H. arabidopsidis, which exhibits dramatic reductions in genes encoding RxLR effectors and other secreted pathogenicity proteins, cell wall hydrolytic enzymes and transporters, the non-biotrophic group (Phytophthora and Pythium) seems to have a large suite of pathogenicity related genes, as a result of expansion of effector families in Phytophthora and proteolytic enzymes in Pythium  machine  and the genomic DNA was extracted following a protocol modified from Moller et al.Py. arrhenomanes (ATCC 12531 = CBS 324.62), Py. irregulare (DAOM BR486 = CBS 250.28), Py. iwayamai (DAOM 242034 = CBS 132417), Py. ultimum var. sporangiiferum (DAOM BR650 = CBS 219.65), and Py. vexans (DAOM BR484 = CBS 119.80) using the Illumina Genomic DNA Sample kit . The libraries were paired-end sequenced (76 or 120 bp) on the Illumina Genome Analyzer II/IIx sequencer. The purity-filtered (PF) reads were first trimmed and then quality filtered using custom Perl scripts to remove reads with low quality bases (<Q20) (Approximately 1 g of freshly harvested mycelium from s (<Q20) .The trimmed and quality filtered reads were then assembled using Velvet v0.7.63 Py. aphanidermatum (DAOM BR444 = CBS 132490), a single-end and a paired-end (3 Kb) library was created and sequenced using the Genome Sequencer FLX instrument following the manufacturer’s protocol . The paired-end library was sequenced using the Titanium chemistry. The single-end library yielded 1,299,108 reads (507 Mb) with an average length of 390 bp. The paired-end library yielded 380,566 reads (137.5 Mb) with an average length of 361 bp. 256,098 of these reads were indentified as a member of a valid read pair. The reads were assembled with the Newbler assembler v2.3 For Pythium specific repeat library constructed previously Phytophthora matrix and SNAP Pythium ultimum Genome Database (http://pythium.plantbiology.msu.edu/). All the oomycete ESTs in dbEST The assembled genomes were annotated using the MAKER v2.03 The phylogeny of 14 stramenopiles was created by using 123 ITS rDNA sequences obtained from GenBank. Nucleotide sequences were aligned by ClustalW Orthologous and close paralogous genes were identified using OrthoMCL v1.4 Pythium secretome. Sequences that were predicted to contain transmembrane domains or organelle-targeting signals were omitted from the secretome. The clustering of secreted protein was done using OrthoMCL v1.4 Signal peptides were predicted using SignalP v3.0 Pythium, stramenopile-core and taxa-specific gene families were calculated as the number of occurrences over the total number of protein domain or GO hits among secreted versus non-secreted proteins. Significance of enrichment/depletion is assessed by a Chi-square test with Bonferroni correction for multiple testing. Only protein domains with enrichment p-value≤0.05 and GO with enrichment p-value≤0.05 were considered.InterProScan Pythium, Phytophthora, H. arabidopsidis and diatom genomes were automatically annotated using the CAZymes Analysis Toolkit – CAT The carbohydrate-active enzyme coding genes of et al.Pythium genome sequences to identify proteins with an amino-terminal signal peptide based on SignalP prediction using SignalP v3.0 H. arabidopsidis genome which is reported to have 134 candidate RxLR effectors was used as a positive control. Second, an HMM profile of the RxLR domain was constructed by manually aligning the RxLR domains of the 53 RxLR effectors from Phytophthora species and H. parasitica. The resulting alignment was fed to the hmmbuild program (HMMer software) H. arabidopsidis genome. Furthermore, the whole Pythium proteome was also searched with the HMM. Third, a comprehensive database of RxLR effector proteins from Phytophthora species H. arabidopsidisA. laibachiiPythium were identified by BLASTP search against the RxLR effector database at E-value cutoff of 10−5. Fourth, a string searches was performed for the RxLR, RxLx and Rx[LMFY][HKR] motif within the amino terminus of each six frame translation of Pythium genomes, 30 to 150 residues from the signal peptide.The candidate RxLR effectors were identified using the approach described by Win Pythium genomes for candidate YxSL[RK] effectors using a HMM profile of the putative YxSL[RK] motif, a novel effector motif identified first in Py. ultimum var. ultimum, constructed using 57 genes containing YxSL[RK] motif from three Phytophthora species and Aphanomyces eutieches. Using the YxSL[RK] motifs from Py. ultimum var. ultimum as a control, we identified an initial set of 123 proteins containing the YxSL[RK] motif in 7 Pythium species. Using the same HMM profile we were able to identify 21 additional proteins with the YxSL[RK] motif in the Ph. infestans and Ph. sojae genomes. After searching against the HMM profile and multiple alignment of the proteins we selected a set of 141 proteins, which includes 120 candidate effectors from seven Pythium species and 21 from two Phytophthora species, with YxSL[RK] motif situated between 30 to 150 amino acids positions.We computationally screened the six Ph. infestans and Py. ultimum var. ultimum CRN sequences was performed to identify proteins with putative LFLAK-like domains. The candidate CRN sequences from Ph. infestans and Pythium species were used to construct an HMM profile and the CRN sequences from Py. ultimum var. ultimum were used as a control. Two criteria were used to identify candidate LxLYLAR/K proteins. First, the conserved motif should be preceded by a signal peptide and followed by WL motif. Second, the conserved motif should be located between 40 to 65 amino acids after first methionine. Using the HMM profile, we identified additional candidate effectors with an LxLYLAR/K domain. To validate our computational methods, the same HMM profile was used to identify the CRN effectors from H. arabidopsidis genome which is reported to have 20 candidate CRN effectors For the CRN effectors, a BLASTP search against 21 well-defined amino-terminal domains from Pythium species . Initially, plugs of 10% V8 agar containing Pythium species were incubated for 1 day in yeast extract broth  at 25°C with shaking (200 rpm). Approximately 50 mg of hyphae growing out of the plugs were then transferred to flasks containing media for the various expression assays, with the exception of Py. iwayamai, mycelium was grown under the following conditions: 1, nutrient-rich YEB medium for 3 days at 25°C with shaking (200 rpm); 2, and nutrient-starved Plich medium  for 10 days at 25°C in standing culture, as previously described Py. iwayamai was isolated from a cool temperature site and therefore incubated at 10°C and was not exposed to above mentioned conditions. Condition 5 was not included, because Py. iwayamai did not resist the heat stress at 35°C. For each condition listed above, mycelium was harvested, macerated in liquid nitrogen and RNA was extracted as described One pooled cDNA library was constructed for each of four For each library, the purity filtered reads from the Illumina Genome Analyzer II pipeline were mapped using Tophat v1.1.4 Pythium genomes, Ph. infestans, H. arabidopsidis, and T. pseudonana were compared to each other via an all-by-all BLASTP Pythium, Ph. infestans, H. arabidopsidis, and T. pseudonana genomes. Each syntenic block is represented as a link whose ends represent the syntenic regions from other species.All protein coding genes from the seven Py. aphanidermatum, AKXY00000000 for Py. arrhenomanes, AKXZ00000000 for Py. irregulare, AKYA00000000 for Py. iwayamai, AKYB00000000 for Py. ultimum var. sporangiiferum, and AKYC00000000 for Py. vexans. The genome assemblies, transcript sequences, and protein sequences are also available for download and BLAST searching at Pythium Genome Database (PGD) website . Also available for download at the PGD are the annotation files in GFF3 format and the functional annotation of the gene models (http://pythium.plantbiology.msu.edu/download.shtml). The genome assembly and annotation files are also available for download from the Dryad Digital Repository (http://dx.doi.org/10.5061/dryad.h748p). The WGS reads are available in the NCBI Short Read Archive (SRA) under the accession SRP006957. The RNA-Seq Reads are available the NCBI SRA under the accession SRP006964.The whole genome shotgun projects have been deposited at DDBJ/EMBL/GenBank under the accession numbers: AKXX00000000 for Figure S1Shared clusters of secreted proteins in Pythium. The Venn diagram shows the distribution of secreted protein clusters among Pythium species. The putative secreted proteins from seven Pythium species were predicted by using SignalP v3.0 (TIF)Click here for additional data file.Table S1Summary of sequence reads generated for five Pythium species. Sequencing was done using the Illumina Genome Analyzer (GA) II or IIx platform. PF, purity-filtered; Par, Pythium arrhenomanes; Pir, Pythium irregulare; Piw, Pythium iwayamai; Puls, Pythium ultimum var. sporangiiferum; Pve, Pythium vexans.(XLS)Click here for additional data file.Table S2Assembly parameters for five Pythium species. Sequenced reads were assembled using Velvet v0.7.63 Pythium arrhenomanes; Pir, Pythium irregulare; Piw, Pythium iwayamai; Puls, Pythium ultimum var. sporangiiferum; Pve, Pythium vexans.(XLS)Click here for additional data file.Table S3Assembly and annotation statistics of thirteen stramenopile genomes. Assembly and annotation statistics were compared to data published by Levesque et al. for Pythium ultimum var. ultimumet al. for Phytophthora infestanset al. for Phytophthora ramorum and Phytophthora sojaeet al. for Hyaloperonospora arabidopsidiset al. for Thalassiosira pseudonanaet al. for Phaeodactylum tricornutumPythium aphanidermatum; Par, Pythium arrhenomanes; Pir, Pythium irregulare; Piw, Pythium iwayamai; Puls, Pythium ultimum var. sporangiiferum; Pult, Pythium ultimum var. ultimum; Pve, Pythium vexans; Phin, Phytophthora infestans; Phrm, Phytophthora ramorum; Phsj, Phytophthora sojae; Hpa, Hyaloperonospora arabidopsidis; Thps, Thalassiosira pseudonana; Phtr, Phaeodactylum tricornutum.(XLS)Click here for additional data file.Table S4Whole transcriptome sequencing data for four Pythium species. Sequence reads from each species were mapped to the respective genome using TopHat v1.1.4 (XLS)Click here for additional data file.Table S5Protein domains enriched or depleted in core Pythium and Pythium species-specific clusters. Fold change numbers are color coded as black for enriched and red for depleted domains. Only protein domains significantly enriched or depleted are shown.(XLS)Click here for additional data file.Table S6Sizes of carbohydrate-active enzyme (CAZyme) classes in the stramenopile genomes. The CAZymes coding genes were automatically annotated using the CAZymes Analysis Toolkit – CAT Pythium aphanidermatum; Par, Pythium arrhenomanes; Pir, Pythium irregulare; Piw, Pythium iwayamai; Puls, Pythium ultimum var. sporangiiferum; Pult, Pythium ultimum var. ultimum; Pve, Pythium vexans; Phin, Phytophthora infestans; Phrm, Phytophthora ramorum; Phsj, Phytophthora sojae; Hpa, Hyaloperonospora arabidopsidis; Thps, Thalassiosira pseudonana; Phtr, Phaeodactylum tricornutum.(XLS)Click here for additional data file.Table S7Quantitative comparison of pathogenicity-related proteins in stramenopiles. Genes were predicted for all datasets using PFAM v23.0 prediction and BLASTP searches. Results were compared to published genome datasets for Pythium ultimum var. ultimumPhytophthora species Hyaloperonospora arabidopsidisThalassiosira pseudonanaPythium ultimum var. ultimum were taken from Lévesque et al.Pythium aphanidermatum; Par, Pythium arrhemonanes; Pir, Pythium irregulare; Piw, Pythium iwayamai; Puls, Pythium ultimum var. sporangiiferum; Pult, Pythium ultimum var. ultimum; Pve, Pythium vexans; Phin, Phytophthora infestans; Phrm, Phytophthora ramorum; Phsj, Phytophthora sojae; Hpa, Hyaloperonospora arabidopsidis; Thps, Thalassiosira pseudonana.(XLS)Click here for additional data file.Table S8Number and percentage of secreted proteins from stramenopiles. The table shows the number of secreted protein and the percentage of proteins to be secreted from 13 stramenopiles. Also shown is the number and percentage of secreted proteins with transcript support. The secreted proteins were identified by SignalP v3.0 Pythium ultimum var. ultimum were taken from Lévesque et al.(XLS)Click here for additional data file.Table S9Gene ontology (GO) molecular function and biological process categories enriched in core Pythium and species-specific secretome. The table shows the enrichment fold in core Pythium and specific-specific secretome as compared to the non-secretome. Only GO terms significantly enriched in secretome are shown.(XLS)Click here for additional data file.Table S10Protein domains enriched in core Pythium and species-specific secretome. The table shows the enrichment fold in core Pythium and species-specific secretome as compared to the non-secretome. Only domains significantly enriched in secretome are shown.(XLS)Click here for additional data file.Table S11Protein domains enriched or depleted in stramenopile-core, diatom, Hpa and Pythium/Phytophthora-specific gene families. The table shows the enriched or depleted protein domains in taxa-specific and stramenopile-core gene families as compared to rest of the genomes. Fold change numbers are color coded as black for enriched and red for depleted domains. Only Protein domains significantly enriched or depleted are shown. Hpa, Hyaloperonospora arabidopsidis.(XLS)Click here for additional data file.Table S12Number of genes syntenic to Pythium ultimum var. ultimum. Syntenic genes were identified through reciprocal best matches between gene models and block identification using MCscan (XLS)Click here for additional data file.


Motivation: Stable isotope-labelling experiments have recentlygained increasing popularity in metabolomics studies, providing unique insights into thedynamics of metabolic fluxes, beyond the steady-state information gathered by routine massspectrometry. However, most liquid chromatography–mass spectrometry data analysissoftware lacks features that enable automated annotation and relative quantification oflabelled metabolite peaks. Here, we describe mzMatch–ISO, a newextension to the metabolomics analysis pipeline mzMatch.R.Results: Targeted and untargeted isotope profiling using mzMatch–ISOprovides a convenient visual summary of the quality and quantity of labelling for everymetabolite through four types of diagnostic plots that show (i) the chromatograms of theisotope peaks of each compound in each sample group; (ii) the ratio of mono-isotopic andlabelled peaks indicating the fraction of labelling; (iii) the average peak area ofmono-isotopic and labelled peaks in each sample group; and (iv) the trendin the relative amount of labelling in a predetermined isotopomer. To aid furtherstatistical analyses, the values used for generating these plots are also provided as atab-delimited file. We demonstrate the power and versatility ofmzMatch–ISO by analysing a 13C-labelled metabolome datasetfrom trypanosomal parasites.Availability: mzMatch.R and mzMatch–ISO are available free of chargefrom http://mzmatch.sourceforge.netand can be used on Linux and Windows platforms running the latest version of R.Contact:rainer.breitling@manchester.ac.uk.Supplementary information:Supplementary data are available at Bioinformaticsonline Supplementary Fig. S1) (Liquid chromatography–mass spectrometry (LC–MS) is a technique that combinesthe physical separation capabilities of liquid chromatography with the highly sensitive massdetection properties of mass spectrometry. Metabolomics studies use LC–MS for theglobal detection and relative quantification of metabolites in complex biological samples.Recently, LC–MS has been applied to trace the metabolism of stable isotope-labelledmetabolic precursors in biological systems as a function of time (www.thermo.com), MassHunter (www.chem.agilent.com) and MarkerLynx(www.waters.com). Although they are all capable ofidentifying and quantifying metabolites of interest in unlabelled data, features that enablethe extraction and relative quantification of isotope peaks from labelled data eitherrequire manual intervention or are non-existent. Furthermore, software that can handlelabelled MS data, such as MetExtract . This list can also contain characteristic adducts orfragments of metabolites of interest where appropriate. Automated targeted and untargetedisotope profiling can be performed using the mzMatch–ISO functionsPeakML.Isotope.TargettedIsotopes and PeakML.Isotope.UntargettedIsotopes, respectively.The latter can be used for profiling global label distribution by looking for the isotopesof all identified peaks in a PeakML file; or all compounds in databases such as the KyotoEncyclopedia of Genes and Genomes (KEGG) (http://mzmatch.sourceforge.net/isotopes-targetted.php.Isotope profiling using mzMatch–ISO requires the LC–MS raw data (.mzXML) filesto be preprocessed by extracting the peaks using XCMS highlights the capabilities of mzMatch–ISO in demonstrating a complex biologicalphenomenon.The automated untargeted isotope annotation and relative quantification capabilities ofmzMatch–ISO are demonstrated by the analysis of LC–MS data from procylic formmzMatch–ISO provides an efficient and user-friendly output for the analysis andcompact visualization of isotope-labelled metabolomics datasets without the need forspecialist bioinformatics skills, allowing rapid, precise and meaningful biologicalinterpretation. The algorithm can be implemented directly in R, or from the IDEOM graphicaluser interface, to facilitate follow-up statistical processing, analyses and re-plotting ofthe results.Funding: A.C. was funded by a Scottish Universities LifeSciences Alliance (SULSA) grant to R.B. Funding for A.J. was provided by aNetherlands Organisation for Scientific Research NWO-Vidigrant to R.B. D.J.C. was supported by an Australian National Health andMedical Research Council postdoctoral training fellowship. F.A. wassupported by SysMO, NWO-Vidi and SULSA. M.P.B. was supported by the Wellcome Trust throughThe Wellcome Trust Centre for Molecular Parasitology, whichis supported by core funding from the Wellcome Trust[085349].Conflict of Interest: none declared.


Bacillus megaterium bacteriophage Slash is described here. Several key features related to morphogenesis, replication/recombination, DNA metabolism, and lysis are described. Slash also encodes a homolog of SleB, a germination-specific cell wall amidase.The complete annotated genome sequence of  Bacillus megaterium is a ubiquitous, spore-forming, Gram-positive bacterium . Transmission electron microscopy was performed at the Microscopy and Imaging Center at Texas A&M University.Bacteriophage Slash was obtained from a soil sample collected in College Station, TX. Phage DNA was sequenced using 454 pyrosequencing at the Emory GRA Genome Center . Trimmed FLX Titanium reads were assembled to a single contig at 111-fold coverage using the Newbler assembler version 2.5.3 (454 Life Sciences), with the default settings. PCR confirmed the completed contig. The genes were initially predicted using GeneMarkS  genome with a G+C content of 35.6% and a coding density of 90.1%. Thirty-two genes were assigned functions by BLASTp and InterPro analyses (https://cpt.tamu.edu/cpt-software/releases/pause/) revealed that Slash has a direct terminal repeat of 567 bp. The genes encoding DNA metabolism enzymes, including recombinase, DnaB-like helicase, DNA polymerase III (epsilon subunit), DNA primase, Holliday junction resolvase protein, and a RecB-like exonuclease, were identified. Slash also carries DNA biosynthesis genes . Four putative sigma/transcription factors were also identified. Four homing endonucleases with unknown sequence specificities were identified, including two with an AP2 domain. The AP2 domain is a DNA binding domain that was originally identified in plant transcription factors  was identified. Another muralytic enzyme  is encoded by a gene outside of the lysis cassette.Several morphogenesis genes were identified, encoding tape measure protein, head-to-tail joining protein, tailspike protein, and the terminase large subunit. Although the large terminase lacks homology to the terminases of known packaging strategies, an analysis of the raw sequencing data using the Pause method ( factors . A lysisGene 103 encodes a LysM/SCP domain-containing protein. The LysM domain is a peptidoglycan binding domain that is found in a variety of endolysins and bacterial pathogenesis factors . ProteinKF669661.The genome sequence of phage Slash was contributed to GenBank as accession no.


Polyparasitism is still widespread in rural communities of the developing world. However, the epidemiology of polyparasitism and implications for morbidity are poorly understood. We studied patterns of multiple species parasite infection in two rural communities of Côte d’Ivoire, including associations and interactions between infection, clinical indicators and self-reported morbidity.Plasmodium infection, using a suite of quality-controlled diagnostic methods. Additionally, participants were examined clinically and we measured haemoglobin level, height, weight and mid-upper arm circumference to determine nutritional status. An anamnestic questionnaire was administered to assess people’s recent history of diseases and symptoms, while a household questionnaire was administered to heads of household to collect socioeconomic data. Multivariate logistic regression models were applied for assessment of possible associations between parasitic (co-)infections and morbidity outcomes.Between August and September 2011, two purposely selected rural communities in southern Côte d’Ivoire were screened for helminth, intestinal protozoa and Plasmodium falciparum diagnosed as the predominant species, followed by Schistosoma haematobium, Schistosoma mansoni and hookworm. There were considerable differences in polyparasitic infection profiles among the two settings. Clinical morbidity such as anaemia, splenomegaly and malnutrition was mainly found in young age groups, while in adults, self-reported morbidity dominated. High parasitaemia of P. falciparum was significantly associated with several clinical manifestations such as anaemia, splenomegaly and fever, while light-intensity helminth infections seemed to have beneficial effects, particularly for co-infected individuals.912/1,095 (83.3%) study participants had complete parasitological data and 852 individuals were considered for in-depth analysis. The rate of polyparasitism was high, with P. falciparum endemicity. Interactions between helminth infections and P. falciparum burden  are evident and must be taken into account to design future interventions.Clinical morbidity is disturbingly high in young age groups in rural communities of Côte d’Ivoire and mainly related to very high  Plasmodium spp. include anaemia and splenomegaly associated with erythrocyte death and splenic sequestration, respectively[Schistosoma mansoni and Schistosoma haematobium) are rarely fatal, but cause long-term chronic morbidity[Schistosoma spp. infections may cause tissue damage, and hence have been associated with organ pathology mainly driven by migrating parasite eggs in the human body.Hundreds of millions of people in the developing world are at risk of parasitic diseases, such as malaria and neglected tropical diseases (NTDs)-3. Amongectively,10. Helmorbidity,12. Thisorbidity. Nutritiorbidity. SchistoPlasmodium and helminths, other studies found high anaemia rates in individuals with single species P. falciparum infections[Schistosoma japonicum co-infections in the Philippines. In another study carried out in Senegal, light-intensity infections of S. haematobium were associated with lower malaria parasitaemia in children, but the opposite was found in Kenyan children where high-intensity infections of both parasites were positively associated[To date, most research on parasitic disease-related morbidity focused on single species infections, whilst the health impact due to polyparasitism remains poorly understood. For coufections-21. Intefections showed ssociated,24. ThusHealth effects from multiple species infections are complex due to associations between parasites and possible synergism/antagonism on disease outcome. Additionally, associations are further complicated due to a diversity of proximal and distal risk factors (e.g. socioeconomic status and poor nutrition), as well as demographic, exposure and immunological factors. The aim of the study presented here was to deepen the understanding of the epidemiology of polyparasitism and its implications for morbidity. Residents from two purposely selected communities in Côte d’Ivoire were examined with a suite of diagnostic methods, interviewed with a pre-tested questionnaire and subjected to detailed clinical examinations.The study protocol was approved by the institutional research commission of the Swiss Tropical and Public Health Institute  and received clearance from the ethics committees of Basel  and Côte d’Ivoire (reference no. 09-2011/MSHP/CNER-P). District health authorities and village chiefs were informed about the objectives, procedures and potential risks and benefits of the study. Written informed consent was obtained from each individual (and parents/guardians of children aged below 18 years), emphasising that participation is entirely voluntary and that participants can withdraw from the study at any time without further obligation.Schistosoma infection received praziquantel (40 mg/kg). Participants with clinical malaria ) were given artemisinin-based combination therapy (artesunate-amodiaquine for adults and artemether-lumefantrine for children) and paracetamol against fever. An anti-anaemic treatment in severely anaemic individuals with no malaria symptoms was provided. All treatment regimens were offered free of charge. Data were coded and confidentially treated.At the end of the survey, albendazole (400 mg for participants >2 years and 200 mg for children aged 1-2 years) against soil-transmitted helminthiasis irrespective of infection status was administered. Individuals with a S. haematobium and hookworm[S. mansoni and soil-transmitted helminth infections[We purposely selected two rural communities in south and south-central Côte d’Ivoire based on different helminthiases endemicity profiles; Sahoua and Ancihookworm-29, and fections,26. UsinS. haematobium[In Sahoua, detailed demographic data  were readily available from the Taabo HDSS database. In Ancien Carrefour, a demographic survey was carried out with the assistance of four designated local people to identify all households and to collect demographic information. Additionally, socioeconomic data and behavioural aspects regarding parasitic diseases were gathered at the unit of the household, by administering a pre-tested questionnaire. Finallyematobium. Hence, Pre-packed plastic bags containing one stool container , labelled with the name and unique identifiers for each participant, were distributed among all households. Household members were asked to return filled containers the next morning. Participants were invited for a finger-prick blood sample that was subjected to an RDT for malaria  and thick and thin blood films prepared on microscope slides.S. haematobium, urine samples were subjected to a filtration method[S. haematobium (urine filters) and S. mansoni, Ascaris lumbricoides, Trichuris trichiura and hookworm eggs (Kato-Katz thick smears) were counted and recorded separately. Thick and thin blood films were stained with Giemsa.Stool (and urine) specimens and thick and thin blood films were transferred to nearby laboratories. The stool samples were processed as follows. First, a small portion of stool (1-2 g) was fixed in sodium acetate-acetic acid-formalin (SAF). Second,n method. Kato-KaFor quality control, 10% of the slides were randomly selected and re-examined by a senior microscopist. In case of discordant results, the respective slides were re-examined and the results discussed among the concerned technicians until agreement was found. If results from a specific technician had an error rate above 10%, all slides on that day were re-read.Entamoeba histolytica/E. dispar, Entamoeba hartmanni, Entamoeba coli, Endolimax nana, Iodamoeba bütschlii, Giardia intestinalis, Chilomastix mesnili and Blastocystis hominis. Helminth eggs were also recorded for each species separately[Plasmodium species identification under a microscope, following standardised, quality-controlled procedures[The SAF-fixed stool specimens and the Giemsa-stained blood films were forwarded to a laboratory in Abidjan. SAF-fixed stool samples were subjected to an ether-concentration technique and the spectrum of intestinal protozoa investigated included parately. Giemsa-ocedures.Disease-related morbidity was classified into two types; clinical manifestations and self-reported morbidity. Clinical manifestations were assessed during a medical examination by two experienced clinicians. These included liver and spleen enlargement determined by palpation and in case of splenomegaly graded according to the Hackett’s scale, anaemiaData were double-entered and cross-checked in EpiInfo version 3.5.3 . Statistical analysis was performed in Stata version 10.1 . A two-sample analytical approach was employed. The first sample consisted of individuals who had complete parasitological datasets, which was used for basic parasitological and polyparasitism frequency analysis. The second sample included those individuals who, additionally, had complete socioeconomic data and clinical measurements, thus allowed investigating the relationship between co-infection, socioeconomic status and morbidity using multivariate regression models. Two different age cut-offs were used; (i) <5, 5-9, 10-14, 15-24, 25-39 and ≥40 years in sample 1 and (ii) <5, 5-9, 10-18, 19-39 and ≥40 years in sample 2. The rationale to use slightly different age groups was to account for common parasite peak prevalences in sample 1,17 and hSchistosoma spp. and soil-transmitted helminth infections were grouped according to guidelines of the World Health Organization (WHO)[et al.[Classes of intensity for on (WHO). Intestion (WHO). The sevon (WHO),40. The on (WHO). Indicat[et al. and wereA household-based asset approach was used to determine participants’ socioeconomic status, which allowed stratifying individuals into economic groups . This approach has been successfully applied and a detailed description is given in a previous study conducted in Côte d’Ivoire. As a me2 and Fisher’s exact test were applied, as appropriate, while for the mean number of concurrent infections two non-parametric tests  were used to account for skewed distributions. Comparison for Hb levels by Plasmodium-helminth co-infection categories was executed with one-way ANOVA or Kruskal-Wallis test in case of unequal variances.For frequency statistics to compare for infection and morbidity rates between different strata, χTo estimate associations between parasitic infections and morbidity indicators, multivariate logistic regression models were used. In a first step, bivariate associations were assessed and parasite species and morbidity indicators not showing any significant relationships were removed from further analyses. In each model (poly-) parasitic infection status or infection intensity served as covariates, depending on better fit of the model. A forward stepwise elimination approach was applied for each model, including covariates at a significance level of 0.15. All models were adjusted by age group, sex and socioeconomic status. Significant relationships between an outcome (infection with a parasite or a specific morbidity indicator) and explanatory variables (infection status or intensity of infection) were expressed as adjusted odds ratios (ORs) with corresponding 95% confidence intervals (CIs). Regression analysis for clinical outcomes was performed for children/adolescents aged <18 years and adults aged ≥18 years separately, accounting for age-specific morbidity and parasitic infection patterns.P. falciparum-malnutrition comorbidity; (ii) P. falciparum-hookworm co-infection; and (iii) P. falciparum-S. haematobium co-infection. Considering the high prevalence of P. falciparum in this age range and that morbidity depends on parasitaemia, P. falciparum infections with a parasitaemia of >500 parasites/μl of blood were used instead, while malnutrition only included moderate and severe cases. For the adult models, co-infection with P. falciparum-S. mansoni was of main interest as well as hookworm-S. mansoni co-infection. All significant associations were presented for the four co-infection categories: (i) none of the two conditions; (ii) mono-infection species 1; (iii) mono-infection species 2; and (iv) co-infection with both species and were expressed as OR with 95% CI.Co-infection and co-morbidity pairs of the multivariate regression models for children/adolescents comprised (i) 2 = 6.79, degrees of freedom (d.f.) = 5, p = 0.236; sample 2: χ2 = 4.50, d.f. = 4, p = 0.343).Overall, 1,095 out of a total of 1,370 inhabitants in the two communities participated in the survey, resulting in an overall compliance of 79.9%. Considering inter-village differences, the study participation was higher in Ancien Carrefour (88.0%) compared to Sahoua (75.3%). Figure S. haematobium prevalence in Sahoua was 25.9%. S. mansoni was much more prevalent in Ancien Carrefour than in Sahoua . Concerning infection rates with soil-transmitted helminths, similar patterns were found in both communities; A. lumbricoides and T. trichiura infections were rarely found, hookworm being the predominant species with prevalences of 32.4% and 26.8% in Ancien Carrefour and Sahoua, respectively. Most of the soil-transmitted helminth infections were of light intensity. The endemicity profiles of the two settings differed not only for Schistosoma, but also for intestinal protozoa infections. In Sahoua a higher affection by intestinal protozoa was found in terms of prevalence and intensity of infection. Overall, the three most common intestinal protozoa species were E. coli, B. hominis and E. nana, with prevalences of 33.2%, 28.7% and 22.3%, respectively. The known pathogenic protozoan species G. intestinalis and E. histolytica/E. dispar were detected in 105 (11.5%) and 86 (9.4%) participants, respectively. In both localities three different Plasmodium species were identified; P. falciparum was the predominant species , whilst P. malariae and P. ovale were detected in 33 (3.6%) and 3 (0.3%) of the participants, respectively.The S. mansoni  than females.Males were significantly more often infected with hookworm  and Schistosoma species , hookworm  and three intestinal protozoan species . S. haematobium, S. mansoni and hookworm infections showed peak prevalences in the age groups of 10-14 years , 15-24 years  and 25-39 years , respectively. The pathogenic intestinal protozoon species G. intestinalis was more often found in young children (peak in the age group of 5-9 years with 22% infected), while the prevalence of non-pathogenic intestinal protozoa increased with age . Malaria parasites were significantly more often found in younger individuals, and the three individuals identified to harbour P. ovale all belonged to the youngest age group of under-fives.Several parasites showed significant associations with age, such as both S. mansoni were significantly associated with a lower socioeconomic status and more prevalent in participants from poorer households .Hookworm and P. falciparum , the major co-infections identified were concurrent infections with P. falciparum and helminths, particularly hookworm (18.1%), S. haematobium (10.0%) and S. mansoni (8.9%). Co-infection patterns differed with age group. In younger age groups, where infections with helminths were less common, concurrent infections with P. falciparum and pathogenic intestinal protozoa like G. intestinalis and E. histolytica/E. dispar added up to the polyparasitic burden with a peak prevalence of 19.2% and 9.0%, respectively, in children aged 5-9 years.Polyparasitism was common; on average, a study participant harboured 2.5 concurrent parasitic infections. The maximum number of parasite species found in the same host was nine. The median number of infections significantly differed between age groups  illustrated in Figure P. falciparum and S. mansoni with adjusted ORs of 2.03 and 2.14, respectively. All 78 (66.7%) S. mansoni-positive individuals who were co-infected with P. falciparum, exclusively had low P. falciparum parasitaemia with <500 parasites/μl blood (data not shown). S. mansoni infection shared a strong positive association with hookworm infection  and vice versa . Most intestinal protozoa species showed significant positive associations between each other. E. coli, for example, was associated with E. histolytica/E. dispar (OR = 3.19), E. nana (OR = 4.68) and I. bütschlii (OR = 5.93) . Significant associations from multivariate regression models between a particular parasite species and any other parasite, sex, age group and socioeconomic status are presented in Additional fileThe findings from the multivariate regression analysis revealed a significant positive association in both ways for vs. 28.0% and 42.4% vs. 28.7%, respectively). Anaemia did not differ between boys and girls, but was associated with female sex in adulthood (age >18 years). 19% of all study participants showed any sign of moderate or severe nature for malnutrition  . In adolescents and adults aged 16-39 years, individuals with S. mansoni mono-infection were found to have significantly higher Hb values  compared to individuals with neither infection  or with P. falciparum mono-infection . This effect from helminth infections on Hb was found in male individuals only. Malnutrition was negatively associated with both Schistosoma species. The association between pallor and S. mansoni confirmed the negative direction also observed in the association between anaemia and light-intensity infections of this helminth species.Individuals presenting light-intensity infection with S. mansoni (particularly light-intensity infections) seemed to have beneficial effects on clinical outcomes like anaemia and malnutrition, a significant positive association was found with several symptoms involving the digestive tract such as diarrhoea, abdominal pain and blood in the stool . Self-reported blood in urine and blood in the stool were both positively associated with S. haematobium infection. Eggs of S. haematobium are expected to be excreted in urine, but five out of the 126 (4%) individuals identified with S. haematobium were found to excrete eggs also in the stool.Although S. haematobium-infected individuals (p = 0.002).Self-reported chronic morbidity was negatively associated with the highest category of concurrent infections with pathogenic parasites , harbouring three or more pathogenic species (p = 0.022), but the association was of positive direction for P. falciparum (>500 parasites/μl of blood) in combination with malnutrition added up to the risk of having clinical manifestations such as anaemia and splenomegaly in children <18 years of age. The relationship of P. falciparum (>500 parasites/μl of blood)-S. haematobium co-infection was not of an additive nature but all three infection categories showed significantly higher ORs for splenomegaly among children.Table P. falciparum infection since co-infection with hookworm and a P. falciparum parasitaemia >500 parasites/μl of blood was not associated with anaemia. In adults, an antagonistic relationship was found for P. falciparum-S. mansoni co-infections; individuals co-infected had significantly lower ORs for several morbid sequelae such as anaemia, pallor and recent history of chronic disease.The pattern of negative associations between helminth infections and several morbidity outcomes found in the single-species models were confirmed in the co-infection models. Children infected with hookworm only had significantly lower ORs for anaemia. Additionally, hookworm had an antagonistic effect in relation to S. mansoni infection among adults, on the other hand, was found to be significantly positively associated with self-reported abdominal pain in combination with other parasites, such as P. falciparum and hookworm.S. haematobium infections were of high intensity (≥50 eggs/10 ml of urine). The second study location, Ancien Carrefour, has not been subjected to regular deworming before. We found hookworm and S. mansoni prevalences of 32.4% and 28.4%, respectively, indicating a moderate-risk community[Plasmodium infection was common in both settings. Consequently, polyparasitism was prevalent, even though varying in parasite species composition, which is in line with earlier studies conducted in rural areas of Côte d’Ivoire[S. mansoni shows important intra-stool and day-to-day variation, and hence multiple Kato-Katz thick smears are necessary to increase diagnostic sensitivity[Plasmodium and intestinal protozoa. Helminth infections were most common in the school-aged and adult populations. Peak prevalences varied from species to species; for instance children aged 10-14 years for S. haematobium, adolescents and young adults for S. mansoni and adults for hookworm. These findings are consistent with previous observations[Since 2010, the population of Sahoua, located at the northern edge of the Taabo HDSS in south-central Côte d’Ivoire, has benefitted from multiple rounds of deworming, targeting schistosomiasis, soil-transmitted helminthiasis and lymphatic filariasis, which are all endemic in this zone,45. Howeommunity. Plasmodd’Ivoire-18. In fsitivity-49. Despsitivity. The extrvations,17,52.S. mansoni[Several significant associations between parasite species are worth highlighting; some of which have been discussed before, particularly the co-infection of hookworm and . mansoni-55. It i. mansoni,43,56. T. mansoni. This isS. mansoni infection seemed to have a beneficial effect on anaemia in case of light-intensity infection, but was at the same time associated with a number of gastro-intestinal symptoms with higher intensity. Furthermore, anaemia was significantly associated with high-intensity infections of P. falciparum, E. histolytica/E. dispar in all individuals and S. haematobium in children aged <9 years (data not shown). These results are in line with findings from earlier studies on disease outcome in relation to P. falciparum parasitaemia and intensity of S. haematobium[As parasite species varied for different age groups, so did clinical manifestations , which mainly occurred in children. In adults, morbidity patterns were mainly driven by chronic, yet subtle but still disabling morbidities, as assessed by self-reported symptoms and recent history of chronic disease. The transition from acute and clinical morbidity to more chronic conditions in older age is explained by the constant exposure to these parasites in highly endemic areas, and the slowly acquired protective immunity,59. Our ematobium,51 and jematobium,61. Two ematobium, but stiematobium. Additioematobium,60.S. mansoni). This confirms earlier studies carried out in comparable rural settings elsewhere in West Africa[S. japonicum and T. trichiura were associated with higher odds of anaemia with a synergy index (SI) of 2.9. As a consequence of existing interactions between parasites, treatment campaigns against specific diseases should be conducted with caution and should consider local patterns of co-infection with other species since treating one parasite may exacerbate the consequences to another one. Furthermore, co-infection may influence the effect and efficacy of drugs or vaccines[Schistosoma infection[Another interesting finding of this study is the indication of protective effects on anaemia in individuals co-infected with helminths .Click here for filePrevalence of clinical morbidity and indicators for malnutrition by sex and age group. This file can be viewed with: Adobe Acrobat reader (url:http://get.adobe.com/uk/reader/).Click here for file


Babesia and Theileria spp. isolates in China. Similar to 18S rDNA, the 40S ribosomal protein S8 gene, RPS8, including both coding and non-coding regions is a useful and novel genetic marker for defining species boundaries and for inferring phylogenies because it tends to have little intra-specific variation but considerable inter-specific difference. However, more samples are needed to verify the usefulness of the RPS8 (coding and non-coding regions) gene as a marker for the phylogenetic position and detection of most Babesia and Theileria species, particularly for some closely related species.Piroplasmosis is a serious debilitating and sometimes fatal disease. Phylogenetic relationships within piroplasmida are complex and remain unclear. We compared the intron–exon structure and DNA sequences of the RPS8 gene from  Babesia and Theileria, are tick-transmitted protozoa that are highly pathogenic to ruminants, horses, pigs, dogs, cats and cattle, and in some cases, even to humans. In the vertebrate hosts, the infection usually causes fever, anemia and haemoglubinuria, and in severe cases, death [The piroplasms, comprising mainly the genera s, death . AnimalsT. equi and B. microti [Babesia and Theileria spp., previous studies only relied on 18S rDNA gene [Babesia species in China [Babesia and Theileria spp., is limited, which precludes its use despite its advantage for use as a molecular marker for lower-level phylogeny [B. microti and T. equi but its usefulness as an everyday tool to classify Apicomplexan parasites is limited by availability of complete genome sequences and requirement for a large number of permutations [Babesia and Theileria species. There are some controversial species placements such as  microti -6. In th microti . HoweverDNA gene . The rDNDNA gene , the 18Sin China . Internain China , howeverin China ,13. Infohylogeny ,14,15. GT. equi [Babesia and Theileria isolates from China for phylogenetic analysis in this study. Structure and sequence signatures in ribosomal RNA and proteins are defining characteristics of the three domains of life and instrumental in constructing the modern phylogeny . Based oT. equi . Among tBabesia and six Theileria spp. in China , whereas within Babesia and Theileria species (intraspecific variability) the identity was approximately 89% to 99.7% respectively (data not shown). The nucleotide sequences of the RPS8 (coding and non-coding regions) genes of Babesia and Theileria species in this study have been deposited in the GenBank database under accession Nos. JN400408 to JN4004028 and Nos. JX402859 to JX402860. The nucleotide sequences of the 18S rDNA genes of several Theileria species in this study have been deposited in the GenBank database under accession Nos. KF559355 to KF559357. The nucleotide sequences of the RPS8 (coding and non-coding regions) genes of B. bovis USA isolate (NW_001820855), B. microti USA isolate (FO082874), T. annulata Ankara strain (NC_011099), T. parva Muguga strain (NC_007345), T. orientalis Shintoku strain (AP011947), T. equi Florida strain (ACOU00000000) were drawn from the GenBank database.The complete sequences of the RPS8 (coding and non-coding regions) gene were obtained from twenty-three isolates representing seven in China . The pri species . The ideBabesia and Theileria species, the RPS8 non-coding regions are highly divergent. Using the Tamura–Nei model of sequence evolution, d values (Genetic Distance) were calculated independently across coding and non-coding region sequences (Data not shown). Across Babesia and Theileria species, d ranges were 0.07903 - 0.47956 (coding) and 0.12511- 1.14484 (non-coding regions). The d-values for non-coding regions are on average 2-fold greater than those for coding regions. Furthermore, the degree of variation in the RPS8 (coding and non-coding regions) dataset was considerable when observing pairwise differences between sequences. Among the eight Babesia and seven Theielria species sequenced, the average p-distances were 0.45, while for the 18S rDNA the average p-distance is 0.083, indicating the RPS8 (coding and non-coding regions) being more variable than the 18S rDNA (Data not shown). The ribosome, with its conserved central role in protein synthesis, has long constituted a prime subject for phylogenetic analysis . The codTheileria species, there was less resolution in the ML analysis given that the RPS8 gene is highly variable. The datasets with significant heterogeneity result in the long-branch attraction and poor resolution at relatively deeper nodes when using the ML approach [Phylogenetic trees based on the 18S rDNA and RPS8 (coding and non-coding regions) genes were constructed by the best-fit model of Bayesian and Maximum likelihood (ML) analysis  and 3. TBabesia and Theileria were polyphyletic with four major clades being defined . Clade II consisted of only three representatives of a single species, T. equi/B. equi , for which the phylogenetic status is controversial [Theileria spp. in the 18S rDNA tree (PP = 1.00), furthermore, there are poorly supported as a sister taxon to other Theileria spp. with ML (BP = 16) analysis of RPS8. However, there was relatively moderate statistical support for T. equi as a sister taxon to other Theileria spp. in the Bayesian analysis (PP = 0.91). Its position relative to Clade I and Clade III comprising the majority of Babesia sp. was unresolved, indicating the unclear evolutionary position of T. equi [B. microti was placed at the root of piroplasms, thereby, separating it from the Babesia and Theileria clades  in the 18S rDNA tree as well as there was moderate statistical support  in the RPS8 tree. These data indicate that 18S rDNA and RPS8 reliably distinguishes the deeper branches among some Babesia species. Within B. ovata isolates, the phylogenetic relationship between the B. ovata Zhangjiachuan isolate and the Wenchuan isolate is reliable in the RPS8 tree  as showed in the 18S rDNA tree . The RPS8 gene seems to be equal to the 18S rDNA in recognizing close lineages among some Babesia and Theileria species. Overall topology of the two trees showed similar major branching orders, beginning with the outgroup B. bovis and other two Babesia species (Babesia sp. Xinjiang-2005 and Babesia sp. Kashi2) were poorly statistically supported  but the latter two Babesia species are closely related with high statistical support , which was not reconciled with morphological and biological data in previous studies (B. bovis and the other two Babesia species (Babesia sp. Xinjiang-2005 and Babesia sp. Kashi2)  and unreliable sister-taxon relationship between Babesia sp. Xinjiang-2005 and Babesia sp. Kashi2   . This isTheileria species, clade I in the 18S rDNA tree was composed of two subclades, one subclade comprising T. annulata isolates and T. parva, with the other subclade consisting of T. uilenbergi, T. luwenshuni, T. sinensis isolates and T. sergenti. The phylogenetic status of two subclades resolved with strong statistical support . Furthermore, T. annulata and T. parva were closely related with a strong statistical support . T. luwenshuni formed a separate branch that is distantly related to T. sergenti and T. sinensis with statistical support  . In the RPS8 tree, T. luwenshuni formed a separate branch with strong statistical support  [For , BP=84) . T. uile, BP=76) ,26. Babesia species than the RPS8 gene. However, the RPS8 gene seems to be equal to the 18S rDNA in recognizing lineages among some closely related Babesia and Theileria species. In short, 18S rDNA as a slow-evolving marker more reliably distinguishes deeper branches among some T. equi to other Theileria spp. The clade of T. equi was strongly supported as sister taxon to other Theileria spp. with ML (BP=97) analysis of the combined dataset of the two markers. However, the phylogenetic tree constructed based on the combined dataset of the two markers is not reliable with poor statistical support (Data not shown). A phylogenetic tree was reconstructed based on the combined dataset of the two markers. The resulting tree is presented in This study was approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. All sheep and calves, were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People's Republic of China.Babesia and Theileria-free area and maintained in an isolated stable. All sheep and calves were splenectomized and treated with antibiotics to promote the healing of wounds one month before the study. During this time, blood films were taken weekly from the ears of the sheep and calves to be examined by Giemsa stain for the presence of haemoprotozoan parasites. The experimental animals were tested by PCR with the universal primers for Babesia and Theileria species based on the 18S rDNA sequences prior to use to ensure that they were free of hemoparasites. After collecting blood containing parasites, the experimental animals were treated with anti-piroplasmosis drug and they were rehabilitated.All sheep and calves were purchased from a Babesia bovis (Shanxian and Lushi) [B. bigemina (Kunming and Lushi) [B. major (Yili) [B. ovata (Wenchuan and Lushi and Zhangjiachuan) [Babesia sp. Kashi2 (Kashi) [B. motasi (Ningxian and Tianzhu and Lintan) [Babesia sp Xingjiang-2005 (Kashi) [Theileria annulata (Xingjiang and Ningxia and Shanmenxia) [T. sergenti (Lushi) [T. sinensis (Weiyuan and Lintan and Lintao) [T. uilenbergi (Longde) [T. luwenshuni (Ningxian) [T. equi (Zhaoyuan) [T. annulata (Ankara strain) [T. orientalis (Shintoku strain) [T. equi (USDA strain) [T. parva (Muguga strain) [B. microti (RI isolate) [Toxoplasma gondii (ME49 strain) [B. bovis (T2Bo strain) [The isolates used in this study are listed in d Lushi) , B. biged Lushi) , B. major (Yili) , B. ovatiachuan) ,31,32, B (Kashi) , B. mota Lintan) ,34, Babe (Kashi) , Theilernmenxia) ,35, T. s (Lushi) , T. sine Lintao) ,38, T. u(Longde) , T. luweingxian) , T. equihaoyuan) . The ref strain) , T. orie strain) , T. equi strain) , B. micrisolate) , Toxopla strain) , B. bovi strain) .Babesia and Theileria isolates. Daily post-infection rectal temperatures were measured to monitor for disease and blood smears were examined to monitor for presence of piroplasms. When the parasitemia reached more than 5% of whole blood, blood was collected into heparinised tubes. Parasite DNA was isolated using a genomic DNA Purification Kit  according to the manufacturer’s instructions. The amount of DNA isolated was assessed spectro-photometrically. Control DNA was isolated from venous blood of uninfected sheep and calves.Sheep and calves were infected intravenously with 15 ml of cryopreserved infected blood stock of these Babesia microti by conventional PCR using forward primer 5’- ATGGGTATT(A/C)TCA(G/T/)C(A)GT(C/G)GAC(T)AG-3’ and reverse primer 5’- GCGTTTCTTCTTA(G)TCCATACG -3’. The reaction mixture consisted of 10×PCR buffer, 1.5 mM MgCl2, 200 mM each deoxynucleotide triphosphate, 40 pmol each primer, 1.5 U of Taq polymerase (Takara) and approximately 10 ng of DNA, in a final volume of 50 ml. Each PCR consisted of 35 cycles of denaturation at 94°C for 45 s, annealing at 52°C for 60 s, and extension at 72°C for 60 s; an initial denaturation step consisting of incubation at 94°C for 5 min and a final extension step consisting of incubation at 72°C for 10 min was also included. After PCR amplification, the PCR fragment was cloned into the pGEM-T Easy vector (Promega) according to the manufacturer’s recommendations, At least two positive clones from each sample were sequenced by using ABI PRISMTM 377XL DNA sequencer (TaKaRa). All new data has been deposited in GenBank.The partial RPS8 gene was amplified from the genomic DNA extracted from each sample except Toxoplasma gondii was used for all loci. ML analyses were conducted with PhyML [Both ML and Bayesian approaches were used to evaluate each of the individual loci separately. The outgroup taxon th PhyML  as impleth PhyML , using tth PhyML . Akaike th PhyML .Toxoplasma gondii was used as an outlier group. The phylogenetic trees were constructed based on the combined data of 18S rDNA and RPS8 (coding and non-coding regions) gene sequences from Theileria and Babesia species determined in our laboratory or obtained from the GenBank database.The sequence data were also examined using Maximum likelihood analyses in PhyML . The comhttp://genes.mit.edu/GENSCAN.html). The RPS8 deduced amino acid sequence was analyzed with the Expert Protein Analysis System (http://us.expasy.org/). Theoretical molecular weights and isoelectric point (pI) were determined using Peptide-Mass (http://us.expasy.org/tools/peptide-mass.html), and prediction of potential phosphorylation sites was carried out at the NetPhos website. Identification of other motifs was conducted using the Motif scan program (http://hits.isb-sib.ch/cgi-bin/PFSCAN).The program Genescan for predicting the locations and exon-intron structures of genes in genomic sequences from a variety of organisms (