Anopheles gambiae sensu lato is attributable to the differential biological traits of the sibling species, with their heterogeneous geographical distribution, behavioral dissimilarities and divergent population dynamics. These differences are critical to their roles in malaria transmission. Studies were, therefore, undertaken on the spatial distribution of these species and malaria prevalence rates in Bayelsa State, September, 2008-August 2010.Much of the confusing ecophenotypic plasticity of Anopheles was separated morphologically and the sibling species PCR- identified. Simultaneously, malaria prevalence rates were calculated from data obtained by the examination of blood smears from consenting individuals at hospitals/clinics.Mosquito sampling was in 7 towns/villages in 7 Local Government Areas (LGAs) in 3 eco-vegetational zones: Fresh Water Swamp Forest (FWSF): Sagbama, Yenagoa, Kolokuma-Opokuma LGAs; Brackish Water Swamp Forest (BWSF): Ogbia, Ekeremor, Southern Ijaw LGAs; Mangrove Water Forest (MWF): Nembe LGA. Adults were collected twice quarterly by the Pyrethrum Spray Catch (PSC) technique. An. gambiae s.s. was dominant across the 3-eco-vegetational zones. Spatial distribution analyses by cell count and nearest neighbor techniques indicated a tendency to clustering of species. An. gambiae s.s. and An. arabiensis clustered in Ekeremor LGA while these 2 species and An. melas aggregated in Nembe. The gonotrophic  status examination revealed that 34.3, 23.5, 23.1 and 18.4% of the population were fed, unfed, gravid and half gravid respectively. The highest malaria prevalence rates were obtained at Kolokuma-Opokuma and Nembe LGAs. Variation in prevalence rates among LGAs was significant . The highest prevalence rate was in the age group, 30-39 yrs, while the lowest prevalence was in the 0-9 yrs group.An. gambiae s.s. either in allopatry or sympatry across eco-vegetational zones. In areas where the sibling species clustered, they probably formed nidi for transmission. Socio-economic conditions might have contributed to reduced prevalence in Yenagoa, State Capital.High malaria prevalence rates were associated with  An. gambiae s.l. is the differential biological traits of the sibling species, their heterogeneous geographical distributions, behavioral dissimilarities and divergent population dynamics [Plasmodium parasites and transmitted by female Anopheles mosquitoes. In Africa, the most efficient vectors are the Anopheles gambiae complex and Anopheles funestus group. An. gambiae is a complex of seven sibling species varying in their vectorial ability and ecological niche [An. gambiae s.s., An. arabiensis, An. quadrianulatus A, An. quadrianulatus B; the salt water-breeding An. melas and An. merus, and An. bwambae found in hot springs in Uganda. The differences in the biology of the sibling species of An. gambiae s.l. have highlighted the need for mapping their spatial distribution and malaria prevalence patterns in order to enhance effective implementation of integrated control approaches [An. gambiae complex and Plasmodium falciparum malaria in Bayelsa State, Nigeria.Much of the confusing eco-phenotypic plasticity of dynamics . These ddynamics -5. Humanal niche ,7. The sproaches . Maps haproaches . The preThe study was conducted in 7 Local Government Areas (LGAs), Bayelsa State, Nigeria. Bayelsa State is located  in the lower Delta plain formed during the Holocene of the quaternary period by the accumulation of sedimentary deposits . The vegBefore the commencement of the study, consent was obtained from the Ministry of Health, Bayelsa State, through the Primary Health Care (PHC) department, the village and household heads.Sampling of the study population involved successive selection of new participants who presented at the Out-Patient Department of the 7 selected General Hospitals/ Clinics  in each LGA until a sample size as described in Daniel  was obtaEDTA bottles were labeled following entry into the routine register with data on sex, occupation, and location of participants. A 2 ml-volume of intravenous blood was collected from each individual and transferred to a labeled EDTA bottle. Grease-free slides were labeled using patients’ details from the EDTA bottles. An aliquot of the blood was measured with a 1 ml-micropipette and dropped on a labeled slide to prepare thick and thin blood films following WHO standard procedures . PreparaCollection of mosquitoes was undertaken in 7 villages/towns in the 7 LGAs. Their co-ordinates were obtained by geographic positioning system (GPS). The villages were randomly selected based on accessibility and availability of supporting staff. Selection of houses was based on their similarity in architectural designs. Six houses were used in each town/ village; these houses were utilized throughout the study. There were 1–2 rooms in each house.An. gambiae s.l. adults were preserved dried in Eppendorf tubes containing desiccated silica gel for molecular characterization.Adult mosquitoes were collected by the Pyrethrum Spray Catch (PSC) method ), 0600-0Extraction method had been extensively discussed .Plasmodium falciparum malaria prevalence rates and PCR- identified An. gambiae complex from each site. Spatial maps were displayed and classified using a specific identifier of Arc View Spatial Extension [A scanned administrative map (1:500.000) of the State was geo-referenced and digitized using Arc View GIS software  ,18. Sepaxtension .The cell count and K-nearest neighbor analyses in Dave and Uriel  were adaCulex quinquefasciatus (46.5%), Anopheles gambiae s.l. (31.3%), Aedes aegypti (13.8%), Anopheles funestus (6.1%) Anopheles nili (2.3%) were identified during the survey. Differences in species abundance were significant .Five culicid species  were identified. An. gambiae s.s. was dominant across eco-vegetational zones. Detailed data appear in Ebenezer et al.[Three species   were sympatric in Nembe  in Yenagoa, Ekeremor Sagbama and Ogbia LGAs; Mesoendemic (PR ≥ 40% ≤50%) in Southern Ijaw LGA and Hyperendemic (PR > 50%) in Nembe and Kolokuma-Opokuma LGAs  Table . An. game Figure . The gone Figure . Variatis Figure . The higAn gambiae sibling species were similar to results obtained by Sogoba et al.[An. gambiae complex had been documented [Anopheles gambiae s.l. population was virile, with approximately 35% fed and about 40% half gravid or gravid.The clustered spatial patterns among the ba et al. and probba et al.. Sympatrcumented . These ccumented . The AnoBreman  providedAn. gambiae sibling species that formed nidi for transmission [An. gambiae s.s. was abundant and allopatric could be attributed to the high density and vectorial competence of the efficient An. gambiae s.s. as a malaria vector [An. gambiae s.s. was allopatric. Although the literature indicates that annual deaths from malaria are mainly in infants and young children [The low malaria prevalence rates in Yenagoa LGA, the State capital and the only semi-urban location in the study area was probably due to the higher living standard  ,25. One smission . The higa vector ,16,20,26children ,28; thesAn. gambiae s.s. either in allopatry or sympatry across eco-vegetational zones. In areas where the sibling species clustered, they probably formed nidi for transmission. Socio-economic conditions might have contributed to reduced transmission in Yenagoa, State Capital.High malaria prevalence rates were associated with The authors declare that they have no competing interests.Anopheles gambiae s.l. PIA was the Co-Supervisor of AE’s doctoral project. SNO was the Principal Supervisor of AE’s doctoral project and corresponding author. TC facilitated field collections at the communities in Bayelsa State. All authors read and approved the final version of the manuscript.AE was the doctoral student responsible for the fieldwork and laboratory analyses. MAEN designed the molecular component of the doctoral proposal and supervised the morphological identification of


Pityophthorus pubescens (Marsham) (Coleoptera: Curculionidae: Scolytinae) to the potential aggregation pheromone component, (E)-(+)-pityol, and its racemic form, (E)-(±)-pityol, in different Pinus spp. L.  stands in the Basque Country (Northern Spain). Both (E)-(+)-pityol and (E)-(±)-pityol equally attracted more males than females, and they were all significantly different from the blank controls. Data about P. pubescens gallery systems in naturally infested P. radiata D. Don branches are also provided. The presence of one male and one female was the most common gallery habitation found in debarked twigs. Current results suggest that P. pubescens may be monogynous and females might mediate the aggregation of mating partners.Field studies were conducted to determine the flight response of the twig beetle  Fusarium circinatum (= F. subglutinans f.sp. pini) (Niremberg and O'Donnell) , has spread throughout the world. It is well reported that several insect species, mainly bark beetles, are capable of transmitting the pathogen (Pityophthorus Eichhoff (Coleoptera: Curculionidae: Scolytinae) have been traditionally considered as a secondary bark beetles group, due to their life habits and development in branches of dead and decaying trees  in the Basque Country (northern Spain)  have been associated with F. circinatum.Pitch canker disease, caused by the fungal agent pathogen . Twig beng trees . Howeverlifornia  and P. pP. pubescens. According to previous research, it is considered as the only species of the genus inhabiting P. radiata stands of the Basque Country , and it breeds in a broad number of Pinus L.  species -(-)--verbenone) . However, there is a lack of knowledge about the biology and chemical ecology of the species.Little is known about the biology of  species . It seemR, 5S)-2-(1-hydroxy-1-methylethyl)-5-methyltetrahydrofuran [hereafter (E)-(+)-pityol] as an aggregation pheromone component has been presented by López et al. (2011). Results obtained via effluvia collection revealed that both sexes emit (E)-pityol, although neither enantiomeric composition nor differences in emissions between sexes were elucidated. Moreover, behavioral assays in Y-tube olfactometer showed a stronger attractive effect on males . Taking into account these previous results, the main objective of our study was to determine the response of P. pubescens to (E)-(+)-pityol and the racemic mixture in different Pinus spp. stands of the Basque Country, in order to provide insight into the aggregation behavior of the species, and in the second instance, to infer the breeding behavior of the species through the observation of gallery systems.Evidence of (2E)-(+)-pityol  and the racemic mixture (99%) were kindly synthesized by Prof. W. Francke , following the methodology described in Mori and Puapoomchareon (Both (mchareon .Pinus spp. mature stands (ca. 30–35 years) of similar area in the Basque Country during 2011. The first trapping assay was conducted from 1 May to 30 June 2011, whereas the second one took place from 15 July to 15 September. This temporal frame was set due to the flight periods of P. pubescens in these months (S. López unpublished data). Three eight-unit Lindgren multiple funnel traps , 106 cm height, were placed in each stand. Sampling localities and their geographic coordinates are detailed in Table 1. Each trap was hung between two adjacent trees, with the top of the trap at 2 m above the ground and the distance between traps at least 75 m. One trap was set as a blank control, whereas the other two traps were baited either with (E)-(+)-pityol or the racemic mixture. Releasing devices consisted of one 1.5-mL capped plastic microcentrifuge vial  containing 2.5 µl of the attractant. After sealing them, caps were pierced with a number 2, heated insect pin (www.panreac.com) were added to each trap cup in order to kill and preserve captured insects. Insects caught were removed every week during the two months , taken to the laboratory, and identified under a Leica MZ95 stereo microscope  with the aid of identification keys , Basque Country. Every week, from the beginning of May until the end of June, 40 naturally pruned branches were randomly picked up from the ground. Branches were dissected with a scalpel under a stereo microscope. Photographs were taken with a DFC300 (Leica Microsystems) camera attached to the stereo microscope.Naturally-pruned branches (ca. 10 cm length and 5 mm diameter) were collected from a 30–40 year-old Pinus species and subjected to a two-way ANOVA analysis (with sex and pheromone taken as factors), followed by Tukey post-hoc tests at a significance level of α = 0.05. A square root transformation was used to normalize the data and correct the heteroscedasticity. All the analyses were performed with the statistical software SPSS 2004 SYSTAT statistical package Field-trapping mean catches of males and females were compared independently for each P. pubescens, no other bark beetle species were found in multiple funnel traps. A total of 9,121 P. pubescens were caught during field trapping from May to June, whereas 8,241 were trapped from July to September. Mean sex ratio, expressed as the percentage of males caught, for each sampling period and Pinus spp. stand is shown in Table 2. Twoway ANOVA revealed significant differences within sexes in all Pinus spp. stands, both in May–June  and July–September . In all these cases male catches were significantly higher. Significant differences were found between (E)-pityol baited traps and the blank control, although there were no differences between (E)-{+)-pityol and the racemic mixture in both sexes and experiments .Apart from P. pubescens, 108 (73.46%) contained both a male and female, whereas 24 (16.32%) contained only one female. In addition, seven males (4.76%) were found alone in a gallery, four galleries (2.72%) were found to have two males and one female, three had two males and two females (2.04%), and only one had one male and two females (0.68%). It is remarkable that all beetles initiating galleries  were found until mid-May. On the contrary, galleries containing more than a single P. pubescens were collected at later dates. 102 galleries with a pair of beetles of both sexes had been excavated in the inner bark, with parental galleries showing a longitudinal pattern . However, six galleries containing a pair were observed in the pith of relatively fresh branches . Pair-occupied parental galleries showed a mean length of 6.57 ± 2.90 mm (range 2.28–14.19 mm).Of 147 examined galleries that contained at least one E)-(+)-pityol and the racemic mixture on P. pubescens in field trapping. (E)-(+)-pityol is considered as a component of the aggregation pheromone of some species of the genus Pityophthorus. It was first isolated from male P. pityographus -(+)-pityol is also known to be the female-produced sex pheromone of cone pine beetles, Conophthorus resinosae (C. coniperda (C. ponderosae (E)-(+)-pityol. In current work, attraction mediated by (E)-(+)-pityol and the racemic mixture did not differ, with both attracting significantly more male P. pubescens than females.Here, we provide evidence of the attractive effect of -(+)esinosae , C. conioniperda , and C. nderosae . In all E)pityol appears to be inactive to Pityophthorus spp. (P. pityographus did not response to the mixture of racemic grandisol and (E)-(-)-pityol, whereas an attractive effect was observed when replacing (E)-(-)-pityol with (E)-(+)-pityol (P. setosus nor P. carmeli were attracted to (E)-(-)-pityol (E)-(-)-pityol did not interrupt the response of P. setosus to (E)-(+)-pityol (E)-(+)-pityol and the racemic mixture  led to focus our work on these two compounds.In contrast, the negative enantiomer of -pityol . Neither)-pityol , and (E))-pityol . Thus, tPityophthorus spp. complex, are characterized as harem polygynous, according to their mating system -pityol or the racemic mixture represented more than the 80% of the total catches in almost all Pinus spp. stands. These results are consistent with Dallara et al. -(+)-pityol and -(-)-7-methyl-1,6-dioxaspiro(4.5)decane (E)-(-)conophthorin) mixture, whereas in monogynous P. setosus, attraction to (E)-(+)-pityol alone was significantly higher in males than in females. Moreover, male P. pityographus, another polygynous species, is only attracted by the combination of cis-1-(2-hydroxyethyl)-1-methyl-2-(1-methyl-ethenyl)-cyclobutane ((±)-grandisol) and (E)-(+)-pityol -pityol . In factE)-pityol would attract males and a single male subsequently would enter the gallery initiated by each female. However, since both sexes appear to emit (E)-pityol, the biological implication of male-produced (E)-pityol remains still unclear. In Conophthorus spp., males emit (E)-(-)-conopthorin in order to repel and warn rival males (P. pubescens would emit (E)-pityol as a warning signal for conspecific males, since we have demonstrated that (E)-pityol elicits a significant attractive effect on males. Thus, male-produced (E)-pityol requires further studies to disclose its biological activity.According to our results, we propose the following host-attacking and mating sequence. Pioneering females would perform with the suitable-host seeking proccess and start the gallery construction. Then, the emission of  was found in 56 breeding galleries. Female P. parviscolyti were found feeding on larvae and eggs of P. pubescens. Phoretomorphic females were also observed attached to the first pair of coxae of both sexes of P. pubescens.In those gallery systems in which a pair of opposite conspecifics was present, eggs were observed. They had been deposited in small individual chambers on both sides of the mother gallery, and they were greyish, with oblong shape, 0.354 mm average length , and 0.545 mm average width . Larvae were also present in some galleries, but no measures or biological data were taken. The mite species E)-(+)-pityol or the more readily available and economical racemic mixture as a useful tool for monitoring populations of P. pubescens in Pinus spp. stands. In addition, it could even be used as a potential mate-finding disruptor in field due to its strong attractive effect on males. This kind of strategy was previously used with C. coniperda, with promising results (C. coniperda (P. pubescens would be feasible.In light of our results, we suggest using ( results . There aoniperda , and it


Lactobacillus casei, L. paracasei, and L. rhamnosus form a closely related taxonomic group (Lactobacillus casei group) within the facultatively heterofermentative lactobacilli. Here, we report the complete genome sequences of L. paracasei JCM 8130 and L. casei ATCC 393, and the draft genome sequence of L. paracasei COM0101, all of which were isolated from daily products. Furthermore, we re-annotated the genome of L. rhamnosus ATCC 53103 , which we have previously reported. We confirmed that ATCC 393 is distinct from other strains previously described as L. paracasei. The core genome of 10 completely sequenced strains of the L. casei group comprised 1,682 protein-coding genes. Although extensive genome-wide synteny was found among the L. casei group, the genomes of ATCC 53103, JCM 8130, and ATCC 393 contained genomic islands compared with L. paracasei ATCC 334. Several genomic islands, including carbohydrate utilization gene clusters, were found at the same loci in the chromosomes of the L. casei group. The spaCBA pilus gene cluster, which was first identified in GG, was also found in other strains of the L. casei group, but several L. paracasei strains including COM0101 contained truncated spaC gene. ATCC 53103 encoded a higher number of proteins involved in carbohydrate utilization compared with intestinal lactobacilli, and extracellular adhesion proteins, several of which are absent in other strains of the L. casei group. In addition to previously fully sequenced L. rhamnosus and L. paracasei strains, the complete genome sequences of L. casei will provide valuable insights into the evolution of the L. casei group. Lactobacillus is the largest group of the family Lactobacteriaceae and contains more than 130 species. The species Lactobacillus casei, L. paracasei, and L. rhamnosus are phylogenetically and phenotypically closely related and are regarded together as the Lactobacillus casei group within the facultatively heterofermentative lactobacilli L. casei, L. paracasei, and L. rhamnosus have for long been used as probiotics in a wide range of different products marketed in many countries. L. casei and L. paracasei have also been isolated from a variety of environmental habitats, including raw and fermented dairy  and plant materials . They are used as acid-producing starter cultures in milk fermentation as adjunct cultures for intensification and for acceleration of flavor development in bacterial-ripened cheeses. They are commonly the dominant species of nonstarter lactic acid bacteria in ripening cheese.The genus L. casei group, the genomes of five L. paracasei strains  and three L. rhamnosus strains  have been fully sequenced to date L. rhamnosus ATCC 53103 L. rhamnosus GG, the original strain of L. rhamnosus ATCC 53103, was isolated from a healthy human intestinal flora, and is one of the most widely used and well-documented probiotics, which confer a health benefit on the host when administered in adequate amounts L. rhamnosus GG can shorten the duration of infectious diarrhea, reduce antibiotic-associated symptoms, and alleviate food allergy and atopic dermatitis in children In the L. casei ATCC 393 and L. paracasei JCM 8130 , which were isolated from a cheese and milk product, respectively, and the draft genome sequence of L. paracasei COM0101 isolated from a commercial fermented milk product. Furthermore, we re-annotated the genome of L. rhamnosus ATCC 53103. We then compared sequenced genomes of the L. casei group to gain a broader view of the genetic variability within the group. Comparison of the genome sequences of strains isolated from the human gut and dairy products can provide valuable insights into the lifestyle adaptation of the L. casei group.In this paper, we present the complete genome sequences of L. paracasei JCM 8130 and L. casei ATCC 393 were obtained from the Japan Collection of Microorganisms (JCM) and the American Type Culture Collection (ATCC), respectively. In this study, ten strains of putative L. paracasei isolated from the fermented milk product Yakult  exhibited the same pattern by random amplification of polymorphic DNA fingerprinting L. paracasei strain designated as COM0101 for sequencing. L. paracasei JCM 8130, L. casei ATCC 393, and L. paracasei COM0101 were cultured in MRS  broth (Difco) at 37°C for 24 h, and the genomic DNAs were isolated and purified as previously described L. paracasei JCM 8130, L. casei ATCC 393, and L. paracasei COM0101 were determined by the whole-genome shotgun strategy using Sanger sequencing (3730xl DNA sequencers) and 454 pyrosequencing (GS-FLX sequencers). We generated 19,200  and 284,003  sequences from the L. paracasei JCM 8130 genome, 28,416  sequences from the L. casei ATCC 393 genome, and 131,707  sequences from the L. paracasei COM0101 genome. The 454 pyrosequencing reads were assembled using the Newbler assembler software. A hybrid assembly of 454 and Sanger reads was performed using the Phred-Phrap-Consed program. Gap closing and re-sequencing of low-quality regions were conducted by Sanger sequencing to obtain the high-quality finished sequence. The overall accuracy of the finished sequence was estimated to have an error rate of <1 per 10,000 bases (Phrap score of ≥40). The deep sequencing dataset of L. paracasei JCM 8130 and L. paracasei COM0101 are deposited in the DDBJ/GenBank/EMBL Sequence Read Archive under the accession numbers DRA000955 and DRA000956, respectively.The genome sequences of Lactobacillus rRNA sequences as queries. Protein domains were identified using HMMER with the Pfam database. Orthology across whole genomes has been determined using BLASTP reciprocal best hits in all-against-all comparisons of amino acid sequences. Two sequences were identified as highly conserved orthologs if their BLAST score ratio is more than 0.8. When two genome sequences were compared using BLASTN, non-matching regions were predicted as genomic islands. The presence of an N-terminal signal peptide sequence was predicted using the SignalP L. rhamnosus ATCC 21052 (accession no. AFZY01000000), L. rhamnosus HN001 (ABWJ00000000), L. rhamnosus LMS2-1 (ACIZ00000000), L. paracasei 8700∶2 (ABQV00000000), and L. casei (zeae) KCTC 3804 (BACQ01000000) were obtained from GenBank.An initial set of predicted protein-coding genes was identified using Glimmer 3.0 L. paracasei JCM 8130, L. casei ATCC 393, and L. rhamnosus ATCC 53103 are deposited in the DDBJ/GenBank/EMBL database under the accession numbers AP012541–AP012543, AP012544–AP012546, and AP011548, respectively. The draft genome sequence of COM0101 has been deposited in public database under the accession numbers BAGT01000001–BAGT01000184.The complete genome sequences of L. rhamnosus ATCC 53103, which we previously reported in the short paper L. paracasei JCM 8130 and L. casei ATCC 393. The genome of L. paracasei JCM 8130 consists of a circular chromosome of 2,995,875 bp and two plasmids, and that of L. casei ATCC 393 consists of a circular chromosome of 2,924,929 bp and two plasmids  of ATCC 393 shared 14 genes, such as beta-galactosidase and cystathionine beta-synthase, with a 65-kb plasmid (accession no. FM179324) of L. rhamnosus Lc 705 (L. paracasei COM0101 that consists of 184 contigs (>500 bp) with a total length of 3,003,364 bp. The COM0101 genome contained 2,767 predicted protein-coding genes. One of the highly redundant contigs contained a gene for plasmid replication protein that showed 100% amino acid identity with that of L. paracasei strains, indicating that the COM0101 genome probably has at least one plasmid. Their chromosome sizes (2.9–3.0 megabases [Mb]) were among the largest group in the Lactobacillus genomes, with an average size of 1.8–2.0 Mb  KCTC 3804. A three-way comparison between the genomes of L. casei ATCC 393, L. rhamnosus ATCC 53103, and L. paracasei ATCC 334 using the BLAST score ratio analysis L. casei ATCC 393 showing a high score for L. rhamnosus ATCC 53103 than those showing a high score for L. paracasei ATCC 334  showed that 1,715 protein-coding genes were common to 17 sequenced L. casei strains L. casei group, likely inherited from their common ancestor. All dispensable protein-coding genes, which were found in one or more but not all the 10 completely sequenced strains of the L. casei group, were functionally classified based on the clusters of orthologous groups from the NCBI COGs database, and the gene repertoires were compared . We next classified all protein-coding genes of L. rhamnosus ATCC 53103 and sequenced intestinal lactobacilli on the basis of the COGs database , which contained genes encoding the two-component sensor and regulator, four bacteriocin immunity proteins, ATP-binding cassette (ABC) transporter with the proteolytic domain, and small peptides. The cluster was conserved in the genomes of the L. casei group, but in the corresponding region of L. casei ATCC 393, a gene for bacteriocin ABC transporter was interrupted by transposase (LBCZ_2129 to LBCZ_2133) and genes for immunity proteins were absent, suggesting that L. casei ATCC 393 may not be able to produce bacteriocin.Bacteriocins are small antimicrobial peptides produced widely by lactic acid bacteria. The cas genes, are known to constitute a defense system against the propagation of phages and plasmids; these were observed in the genomes of a number of lactic acid bacteria L. rhamnosus ATCC 53103 contained a CRISPR region  and four CRISPR-associated genes (LRGG_2116 to LRGG_2119). The 36-bp-long sequence was present 25 times and separated by 30-bp unique spacer sequences. It has been reported that two distinct types  of CRISPR loci were identified in the L. casei genomes L. casei strains BD-II, BL23, LC2W, and Zhang also have an Lsal1-type CRISPR region at the same locus on the chromosome, suggesting that the ancestral strain of the L. casei group had encoded a CRISPR region. However, the 36-bp repeat sequence of the four L. casei strains differs by two bases from that of L. rhamnosus ATCC 53103, and the number of the repeat sequences was different (17–22) among these strains. COM0101 has the orthologs of the four CRISPR-associated genes, indicating that COM0101 also may have a CRISPR region. In contrast, L. paracasei JCM 8130, L. casei ATCC 393, L. rhamnosus Lc 705, and L. rhamnosus ATCC 8530 had no CRISPR, suggesting that these strains may have lost a CRISPR region during adaptation to their environment where phage detection is not essential.CRISPRs, along with their associated L. casei group . It has been reported that the comparison of the genomes of L. paracasei ATCC 334 and BL23 revealed 12 and 19 GIs (>5 kb) in ATCC 334 and BL23, respectively L. rhamnosus ATCC 53103 that were not conserved in L. paracasei ATCC 334 (a cheese isolate) (L. rhamnosus ATCC 53103 included six carbohydrate utilization gene clusters (regions −1 to −6), four of which were completely or partially present in L. paracasei BL23, whose ecological origin is unclear. This result supports the previous findings that cheese isolates, including L. paracasei ATCC 334, have undergone significant gene decay, including loss of many genes involved in carbohydrate utilization L. paracasei ATCC 334 contains a lower number of genes related to carbohydrate transport and metabolism compared with the other sequenced L. paracasei strains  , Fig. 1. strains . In probL. paracasei ATCC 334, 15 and 24 GIs were found in L. paracasei JCM 8130 and L. casei ATCC 393, respectively -type transporter systems, glycosyl hydrolases, transcriptional regulators, and other carbohydrate-related proteins  is reminiscent of the many clusters found in Bifidobacterium longumThe six carbohydrate utilization gene clusters of proteins . L. rhamL. rhamnosus ATCC 53103 overlapped with all the hypervariable regions among the sequenced L. rhamnosus strains . Three of the six hypervariable regions were prophage-like regions . The other three regions corresponded to regions containing carbohydrate utilization gene clusters , indicating that L. rhamnosus strains show flexibility in sugar utilization. Two of the five PTS-type transporter systems in region-5 and two in region-6 were missing in Lc 705, ATCC 8530, and LMS2-1 strains  in L. rhamnosus ATCC 53103  clearly showed that the L. paracasei strain isolated from Yakult produced no pilus structures by an immunoelectron microscopy using immunogold staining L. rhamnosus GG to Caco-2 cells and intestinal mucus was approximately 10 times that of strain Shirota, which was obtained from Yakult L. rhamnosus GG encodes the intact SpaCBA and L. paracasei COM0101 encodes truncated SpaC. Furthermore, L. paracasei JCM 8130, L. paracasei BD-II, and L. paracasei LC2W also contained truncated spaC gene Click here for additional data file.Figure S2Genetic relationships among L. paracasei strains as defined by multilocus sequence typing. (A) Concatenated sequences of five MLST loci  were analyzed as described previously (B) Venn diagram comparing the gene inventories of four L. paracasei strains. Data resulted from reciprocal BLASTP analysis. The numbers of shared and unique genes are shown.(EPS)Click here for additional data file.Figure S3COG classification of dispensable protein-coding genes of the L. casei group.(EPS)Click here for additional data file.Figure S4Synteny between the chromosomes in the L. casei group. Each plot point represents reciprocal best matches by BLASTP comparisons between orthologs.(EPS)Click here for additional data file.Figure S5The spaCBA pili cluster arrangement. Genes and their orientations are depicted with arrows.(EPS)Click here for additional data file.Table S1General genomic features of strains sequenced in this study.(PDF)Click here for additional data file.


Paramecium tetraurelia and all studied Tetrahymena species. This raises the question of whether the high mitochondrial G+C content observed in P. tetraurelia is a characteristic property of Paramecium mtDNA, or whether it is an exception of the ciliate mitochondrial genomes known so far. To test this question, we determined the mitochondrial genome sequence of Paramecium caudatum and compared the gene content and sequence properties to the closely related P. tetraurelia.Despite the fact that the organization of the ciliate mitochondrial genome is exceptional, only few ciliate mitochondrial genomes have been sequenced until today. All ciliate mitochondrial genomes are linear. They are 40 kb to 47 kb long and contain some 50 tightly packed genes without introns. Earlier studies documented that the mitochondrial guanine + cytosine contents are very different between P. caudatum mitochondrial genome was significantly lower than that of P. tetraurelia (22.4% vs. 41.2%). This difference in the mitochondrial nucleotide composition was accompanied by significantly different codon usage patterns in both species, i.e. within P. caudatum clearly A/T ending codons dominated, whereas for P. tetraurelia the synonymous codons were more balanced with a higher number of G/C ending codons. Further analyses indicated that the nucleotide composition of most members of the genus Paramecium resembles that of P. caudatum and that the shift observed in P. tetraurelia is restricted to the P. aurelia species complex.The guanine + cytosine content of the P. caudatum mitochondrial genome, exemplified by the effective number of codons, is more similar to the distantly related T. pyriformis and other single-celled eukaryotes such as Chlamydomonas, than to the closely related P. tetraurelia. These differences in base composition and codon usage bias were, however, not reflected in the amino acid composition. Most probably, the observed picture is best explained by a hitherto unknown  mechanism that increased the guanine + cytosine content in P. tetraurelia mtDNA on the one hand, and strong purifying selection on the ancestral amino acid composition on the other hand. These contradicting forces are counterbalanced by a considerably altered codon usage pattern.Surprisingly, the codon usage bias in the  Tetrahymena: T. pyriformis , excluding only the terminal repeat regions that usually flank the linear ciliate mtDNA. The overall nucleotide composition was 9.9% G, 42.7% A, 34.9% T, and 12.5% C, the G+C content thereby being significantly lower than that of P. tetraurelia mtDNA (22.4% vs. 41.2%). The coding part of the genome was 41,091 bp (94.1%), the protein-coding part 36,585 bp (83.8%) in length. We determined 25 protein-coding genes with known function, 17 additional ORFs with unknown function, the small and large subunits of the ribosomal RNA genes and three transfer RNA genes . However, the intergenic regions adjacent to the cox1 and cob genes were considerably longer , but much lower than that of P. tetraurelia (43.2%). This difference was even more pronounced at 3rd codon positions, with 56.5% G or C ending codons in P. tetraurelia and only 12.8% and 11.9% G or C ending codons in P. caudatum and T. pyriformis, respectively. The G+C content of the noncoding regions (intergenic spacers) was slightly lower compared to protein-coding genes in all species, but the relative differences between species were similar  from 13 more Paramecium species. A phylogenetic tree of Paramecium based on sequences of the cox1 gene is shown in Figure rd codon positions of the cox1 gene revealed high G+C values (45-50%) in species of the P. aurelia complex and a low G+C content (3-17%) in the remaining paramecia  the most frequent synonymous codon ended in A or T (and these are the termination codons). Furthermore, both the number of rare codons and the effective number of codons (Nc) indicate a more balanced usage of synonymous codons in P. tetraurelia compared to the other ciliates  differed significantly between ia Table . Only ines Table , 3. GeneP. tetraurelia , P. biaurelia [GenBank:FN421325], P. tetraurelia [GenBank:FN421326], P. novaurelia [GenBank:FN421327], P. jenningsi [GenBank:FN421328], P. schewiakoffi [GenBank:AM072773]), P. multimicronucleatum [GenBank:AM072766], P. calkinsi [GenBank:FN421329], P. nephridiatum [GenBank:FN421331], P. duboscqui [GenBank:FN421332], P. polycaryum [GenBank:FN421330], P. putrinum [GenBank:FN421333], P. bursaria [GenBank:FN421334].The ® protocol as described in [P. caudatum was amplified in overlapping fragments of 3-15 kb length using Phusion™ high fidelity DNA polymerase (Finnzymes OY). PCR primers were designed from alignments of conserved gene regions from P. tetraurelia [GenBank:NC001324] and T. pyriformis [GenBank:NC000862].DNA extraction followed a Chelexribed in . The neaXbaI (Fermentas) to obtain smaller fragments. Restriction fragments up to 3 kb were A-tailed using a Taq DNA polymerase (Fermentas), cloned into pGEM-T Vector (Promega), and sequenced with universal vector primers. Longer fragments were completed by primer walking. Peripheral regions of the linear mtDNA were amplified via step-out PCR . The tRNAscan-SE server [Nc) was estimated with the program CodonW v. 1.3 http://codonw.sourceforge.net// and the number of rare codons was calculated according to [Nc describes to what degree the codon usage in a genome is biased. It is a number between 20 and 61 where 20 means only one codon is used for each amino acid and values approaching 61 suggest equal usage of synonymous codons [Sequences were manually checked and assembled using BioEdit v. 7.0.5.3  and thenE server  was emplE server  was usedE server . NucleotE server . The effrding to . Nc descs codons .DB conducted the experiments and performed all necessary analyses, TUB wrote the grant proposal and assisted in the design of the experiments. Both authors wrote the manuscript and approved the final version.ParameciumCodon usage and open reading frames in . Effective number of codons Nc in protein-coding genes and putative ORFs of Paramecium tetraurelia and P. caudatum mtDNA.Click here for fileAmino acid composition of the mitochondrial genome. Amino acid composition in the mitochondrial genomes of Paramecium caudatum, P. aurelia and Tetrahymena pyriformis .Click here for file


Here, genome-wide analyses of Oryza sativa and Arabidopsis thaliana revealed apparently divergent patterns of miRNA gene origins. A large proportion of miRNA genes in O. sativa were TE-related and MITE-related miRNAs in particular, whereas the fraction of these miRNA genes much decreased in A. thaliana. Our results show that the majority of TE-related and pseudogene-related miRNA genes have originated through inverted duplication instead of segmental or tandem duplication events. Based on the presented findings, we hypothesize and illustrate the four likely molecular mechanisms to de novo generate novel miRNA genes from TEs and pseudogenes. Our rice genome analysis demonstrates that non-MITEs and MITEs mediated inverted duplications have played different roles in de novo generating miRNA genes. It is confirmed that the previously proposed inverted duplication model may give explanations for non-MITEs mediated duplication events. However, many other miRNA genes, known from the earlier proposed model, were rather arisen from MITE transpositions into target genes to yield binding sites. We further investigated evolutionary processes spawned from de novo generated to maturely-formed miRNA genes and their regulatory systems. We found that miRNAs increase the tunability of some gene regulatory systems with low gene copy numbers. The results also suggest that gene balance effects may have largely contributed to the evolution of miRNA regulatory systems.The origin and evolution of microRNA (miRNA) genes, which are of significance in tuning and buffering gene expressions in a number of critical cellular processes, have long attracted evolutionary biologists. However, genome-wide perspectives on their origins, potential mechanisms of their  Small RNA molecules of about 20–30 nucleotides have emerged as significant regulators of gene expression and genome stability Arabidopsis thaliana further made known that many of them arose from the whole genome duplications (WGDs), tandem duplications and segmental duplications followed by their dispersal and diversification, somewhat similar to the behavior of protein-coding genes A. thaliana showed that their origin could not be fully explained by the above-mentioned hypothesis, and thus these researchers even proposed that some miRNA genes might have been derived from random sequences and spontaneously formed from foldback sequences In recent years, great efforts have been devoted towards the investigation of the origin of miRNA genes and consequently several modes have been proposed. In plants, for example, based on the observation that some miRNA precursors had extended similarity beyond miRNA sequences with target genes, Allen and colleagues de novo generation of miRNA genes have been proposed, by and large, little has been explored, to any great depth, regarding mechanisms of their origins. As for either the inverted duplication model or the hypothesis of “miRNA genes derived from TEs or pseudogenes”, for instance, we are unable to elucidate the mystery of the origin and particularly precise mechanisms behind, let alone the discrepancy and relationships between different modes of origin and those de novo origin mechanisms in particular. Undoubtedly, a comprehensive understanding towards evolutionary history of de novo generated miRNA genes and their regulatory systems remains to be understood.To date, the origin of miRNA genes has been limitedly investigated under one or another model organism alone by using a subset of them. Even motivated by recent advances in the whole genome sequencing as well as deep sequencing of small RNA libraries, a genome-wide picture of how miRNA genes originate is still too far away to be outlined for any organism, to say nothing of profound insights into how miRNA genes of different origins diversify. While hypotheses for Oryza sativa and A. thaliana, and comprehensively investigated and compared modes of miRNA gene origins  and inverted duplication-generated , respectively. In comparison, the proportions of miRNA genes both originated from TEs  and generated through inverted duplication events  in A. thaliana were significantly lower than those in O. sativa .According to their modes of origin, we classified the whole dataset of miRNA genes identified in the  subset. . In bothO. sativa (38.39%) was at least two-fold higher than in A. thaliana (15.82%). Of total sequences of 43,137 and 24,016 bp miRNA genes in O. sativa and A. thaliana, therefore, 16,560 and 3,799 bp should overlap with TEs in a random state. In fact, however, overlapped sequences were 16,048 bp (96.91%) and 612 bp (16.11%) in O. sativa and A. thaliana, respectively, indicating that TE content overlapped with miRNA genes in O. sativa was significantly larger than in A. thaliana . Furthermore, TEs related to miRNA genes and their binding sites were mainly miniature inverted-repeat transposable elements (MITEs) in O. sativa. Of TEs related to miRNA genes and their targets in the O. sativa genome, DNA/Stowaways accounted for approximately 67.34% and 82.43%, respectively. These proportions were much higher than the fraction of DNA/Stowaways on average, comprising 17.33% of the detected TEs in the O. sativa genome on the whole . In A. thaliana, however, TEs related to miRNA genes and their targets were predominantly other TEs, such as DNA/MuDR, DNA/hAT, LTR/Gypsy and RC/Helitron  was older than those related to miRNA targets . Nevertheless, average age of TEs  related to miRNA genes was younger than those related to miRNA targets  in A. thaliana. We found that miRNA genes generated from different origin modes possessed diverse features, evidenced by their function, sequence, structure, distribution, expression, regulation, conservation and polymorphisms . In A. thaliana, intersection number of pseudogene-related and inverted duplication was larger than the status at random (P = 0.0301). Furthermore, in both plant species, intersection number of TE-related and tandem duplication, TE-related and segmental duplication, pseudogene-related and tandem duplication, pseudogene-related and segmental duplication was much smaller than the status at random (P<10−4).In this study, two approaches were used to better illustrate relationships among different modes of miRNA gene origins. We first attempted to ensure whether intersection number of these datasets was smaller or larger than the status at random. For this purpose, we performed Monte Carlo simulations to estimate intersection number at random. As shown in O. sativa and A. thaliana. Here, approaches of pattern recognition were applied to test the similarity of miRNA genes with respect to modes of their origin across different datasets. If the classification accuracy between two datasets is relatively high, one might expect that similarities of miRNA gene features of these two datasets are relatively low, and vice versa. As shown in O. sativa, as well as pseudogene-related and inverted duplication in A. thaliana, were relatively high. However, similarities of miRNA gene features between TE-related and tandem duplication, TE-related and segmental duplication, pseudogene-related and tandem duplication, as well as pseudogene-related and segmental duplication, were relatively low. We further analyzed the other two types of miRNA genes as below: first, miRNA genes with its target sites overlapped with TEs; and second, TE-related miRNA genes with its target sites overlapped with the same TEs. It is our findings that Monte Carlo simulation and pattern recognition of these miRNA genes were consistent with the above-analyzed TE-related miRNA genes  were related to two copies of inverted non-MITEs with >80% sequence similarity. Whereas, among miRNA genes related to MITEs, there were only one  associated with pairs of inverted MITEs with >80% sequence similarity (P<10−3). We then compared features of non-MITE-related miRNA genes with MITE-related ones and found that some of them were different . The first MITE was overlapped with MIR806h  was at least two-fold higher than in A. thaliana (15.82%), leading to that TE composition overlapped with miRNA genes in O. sativa was significantly larger than in A. thaliana. In addition, as a modern monocotyledonous plant, O. sativa undergoes both natural and artificial selections and might encompass remarkably high rate of genomic changes, such as rapid nucleotide substitutions O. sativa but only one in A. thalianaThe origins of miRNA genes have attracted extensive attention in recent years, and a variety of hypotheses have been continuously proposed de novo generated lies at the core of understanding the origin of miRNA genes. By means of genome-wide analysis of miRNA genes in A. thaliana, Allen and colleagues first put forward the inverted duplication hypothesis, proposing that these miRNA genes were generated from inverted duplication events of one of their target genes A. thaliana and O. sativa, it was inverted duplication instead of segmental or tandem duplication events that mainly gave rise to the origin of TE-related and pseudogene-related miRNA genes. Based on the above-described data, we hypothesize the four possible mechanisms, which may be used to explain de novo generated novel miRNA genes from TEs and pseudogenes in flowering plants . Although miRBase has extensively been accepted as a standard for miRNA annotation, some released miRNAs might be incorrectly annotated and not assessed with sufficient stringent criteria prior to their addition to the database. To eliminate these potentially non-authentic miRNAs, we applied the MIRcheck program (http://web.wi.mit.edu/bartel/pub/software.html) to select miRNAs which have ≤4 mismatches, ≤2 continuous mismatches in both miRNA and putative miRNA* sequences, ≤2 asymmetrically unpaired nucleotides in miRNA sequences O. sativa and A. thaliana genomes, 290 and 142 satisfied this criteria. Genome sequences, annotation data as well as alternative splicing data of the O. sativa ssp. japonica, O. sativa ssp. indica and A. thaliana were collected from the release 6.0 of The Institute of Genome Research (TIGR) database (ftp://ftp.tigr.org), Beijing Genomics Institute (BGI) Rice Information System (http://rice.genomics.org.cn/rice/index2.jsp) and the release 8 of The Arabidopsis Information Resources (TAIR) database (http://www.arabidopsis.org/), respectively. All TE-related genes and non-coding genes were excluded in the further analysis. For genes with alternative splice forms, we merely selected the transcripts with the longest sequences in length. Binding sites of miRNAs on protein-coding genes of O. sativa and A. thaliana were identified by using psRNATarget server (http://bioinfo3.noble.org.psRNATarget/) with default parameters O. sativa and A. thaliana were taken from the Pseudogene.org database (http://www.pseudogene.org/databases.html).miRNA sequences of O. sativa and A. thaliana genomes, were identified by using the RepeatMasker program (http://www.repeatmasker.org) with default parameters. miRNA genes overlapped with TEs or with at least 50% coverage with TEs were regarded as TE-related miRNA gene. Similarly, miRNA genes overlapped with pseudogenes or with at least 50% coverage with pseudogenes were considered as pseudogene-related miRNA gene. Inverted duplication-generated miRNA genes were identified by using the approaches as below: (1) pre-miRNA sequences were subject to FASTA searches against cDNA sequences; (2) cases that reverse complemented pre-miRNA sequence matched with cDNA sequence with E-value<0.05, identity-value>0.8 and miRNA sequence lies in the matched regions were selected. Tandem duplication generated miRNA genes were identified in quest of contiguous miRNA genes. In this study, we only included the pair of miRNA genes with a distance <15000 bp within the same family or their BLASTN E-value of pre-miRNA <e-10. Segmental duplications which generated miRNA gene pairs were identified by using DAGchainer program (ftp://ftp.tigr.org/pub/software/DAGchainer/) with parameters “-s –l”. They primarily include self-comparisons to ignore tandem duplication alignments. We used protein pairs with BLASTP E-value<e-10 and miRNA gene pairs within the same family or their BLASTN E-value of pre-miRNA <e-10 as the input data when running DAGchainer program.Repetitive sequences, including TEs and simple sequence repeats (SSRs) in both http://omiclab.genetics.ac.cn/GOEAST/). Pre-miRNA secondary structures were predicted by using Vienna RNA Package (http://rna.tbi.univie.ac.at/). Promoters (TATA box) and enhancers of miRNA genes were identified from upstream 1 kb regions of pre-miRNAs by using the software TSSP (http://linux1.softberry.com/berry.phtml). siRNA sequences of O. sativa and A. thaliana were taken from the Cereal small RNA database  and the Arabidopsis Small RNA Project (ASRP) database (http://asrp.cgrb.oregonstate.edu/), respectively. When we calculated siRNAs encoding miRNA precursors O. sativa, A. thaliana, Triticum aestivum, Zea mays, Brassica napus, Medicago truncatula, Poplar trichocarpa, Vitis vinifera, Monodelphis domaestica, Glycine max, Selaginella moellendorffii, Pinus taeda, Physcomitrella patens and Sorghum bicolor. miRNA expression data of A. thaliana, comprising of three tissues by 454 sequencing, were downloaded from ASRP database. For each miRNA, the mean of these three values were estimated and used. DNA methylation data of A. thaliana were downloaded from Annoj . We adopted data of polymorphic regions in A. thaliana, which composed of 20 datasets with resequencing microarrays Gene Ontology (GO) enrichment analyses of miRNA target genes were implemented by using the software GOEAST (−0.32(1+exp)−L/1000)) if L<150 a.a., where n = 6 and L is the length of the alignable region) and (2) the length of alignable region between the two sequences is ≥80% of the longer protein. Gene expression data from 40 cell types of O. sativa were downloaded from Yale Virtual Center for Cellular Expression Profiling of Rice . Meanwhile, For A. thaliana, we obtained gene expression data from 79 tissues at Weigel world, (http://www.weigelworld.org/resources/resources/microarray/AtGenExpress).The mean of expression intensity was used as its expression level for each gene. For miRNAs with multiple target genes, however, average values of gene expression intensity, copy number and alternative splice number were collectively estimated. Segmental duplication blocks were simply identified by using DAGchainer with parameters the same as in the above-described analysis. Two paralogous genes were considered as tandem duplication when they are separated by fewer than 20 genes.To obtain detailed information of gene copy number, the protein sequence of each query gene was used to search against all other proteins using BLASTP (E<e-10). For proteins belonging to the same family, we used two criteria as below P-value merely when such a number was larger than N. Otherwise, the frequency of intersection number larger than N was used as empirical P-value.To illustrate relationships among different miRNA gene origin modes, two methods were used here. First, random intersection number and statistical significance of each two datasets of miRNA genes with different origin modes were deduced by means of 100,000 Monte Carlo simulations. Briefly speaking, given M miRNA genes as the whole set of miRNA genes, assume that m of these M miRNA genes belong to dataset 1 and n of these M miRNA genes belong to dataset 2, and there are N miRNA genes belong to both of the two datasets. We randomly sampled m and n miRNA genes without replacement from the whole set of miRNA genes,respectively, and then counted intersection number of these two miRNA gene sets. We repeated the sampling in a number as large as 100,000 times in this study, and then took average intersection number as random one. The frequency of intersection number smaller than N was taken as an empirical http://www.ailab.si/orange/). For each pair of miRNA gene datasets from different origin modes, six discriminative models were built by using six different classifiers, including Naive Bayes, k Nearest Neighbours, Decision Tree, C4.5, Support Vector Machine (SVM) and Random Forest. Once a discriminative model was built, it can be used to determine a miRNA gene's origin. The accuracy estimation was performed for the discriminative model by using 5-fold cross-validation method. Original dataset was randomly divided into five equally sized subsets and then in i-th iteration (i = 1..5) i-th subset was used for testing the prediction accuracy of the model that has been built on other remaining subsets. The average accuracy of these tests was the final accuracy of this model. The average accuracy of six discriminative models was the final accuracy of every two datasets.Second, we built discriminative models with the above-described features of miRNA genes to distinguish miRNA genes from their different origin modes implemented with Orange software Click here for additional data file.Figure S2Schematic figure of osa-MIR806h. (A) osa-MIR806h and flanking regions in the japonica genome and ortholous regions in the indica genome. Light blue boxes represent gene, red box represent MITE, and dash lines represent homologous relationships between genes in the japonica and indica genomes. (B) Predicted fold-back structure of osa-MIR806h precursor. Red line represents mature miRNA sequence.(PDF)Click here for additional data file.Figure S3Schematic figure of osa-MIR446 and its target. (A) osa-MIR446's target on LOC_Os08g05620 in the japonica genome and ortholous regions in the indica genome. (B) FASTA alignments of osa-MIR446 and LOC_Os08g05620. Red regions represent MITE, while blue regions represent mature miRNA sequence and its target sequence. (C) Predicted fold-back structure of osa-MIR446 precursors. Red line represents mature miRNA sequence.(PDF)Click here for additional data file.Figure S4Phylogenetic analyses of MITEs overlapped with some miRNA genes and their targets. (A) MITEs overlapped with osa-MIR806h, targets of osa-MIR806h and other osa-MIR806 family members. (B) MITEs overlapped with osa-MIR446, its target on LOC_Os08g05620 and other genes. Phylogenetic trees were built through Neighbor-Joining method by using ClustalW and Phylip with default parameters.(PDF)Click here for additional data file.Table S1O. sativa and A. thaliana.Summary of miRNA genes with different modes of origin in (XLS)Click here for additional data file.Table S2O. sativa and A. thaliana genomes.Statistics for TEs overlapped with miRNA genes, TEs overlapped with miRNA targets and TEs distributed in the (DOC)Click here for additional data file.Table S3O. sativa and A. thaliana.Difference among miRNA genes with different modes of origin in (XLS)Click here for additional data file.Table S4Relationships of miRNA genes with different origins.(DOC)Click here for additional data file.Table S5A list of 58 selected miRNA genes.(XLS)Click here for additional data file.Table S6O. sativa.Difference between miRNA genes overlapped with non-MITEs and MITEs in (DOC)Click here for additional data file.Table S7de novo generated accorded with model (C).Summary of miRNA genes (XLS)Click here for additional data file.Table S8de novo generated accorded with model (D).Summary of miRNA genes (XLS)Click here for additional data file.


Bacillus subtilis is a ubiquitous Gram-positive model organism. Here, we describe the complete genome of B. subtilus myophage Grass. Aside from genes encoding core proteins pertinent to the life cycle of the phage, Grass has several interesting features, including an FtsK/SpoIIIE protein. Bacillus subtilis is a soil-dwelling, endospore-forming, Gram-positive bacterium .Grass was isolated from a soil sample collected in Bethlehem, PA. Phage DNA was sequenced using 454 pyrosequencing at the Emory GRA Genome Center . The trimmed FLX Titanium reads were assembled to a single contig at 128.6-fold coverage using the Newbler assembler version 2.5.3 (454 Life Sciences) with the default settings. The contigs were confirmed to be complete by PCR. Genes were predicted using GeneMarkS (https://cpt.tamu.edu/cpt-software/releases/pause/) method to show that the terminal repeat is 4,208 bp in length.Grass contains a 152,440-bp unique genome with a coding density of 89.1% and a G+C content of 42.3%. In the unit genome, 242 coding sequences were identified, of which 92 were novel hypothetical genes and 95 were conserved hypothetical genes. The genome encodes 3 tRNAs and contains 38 rho-independent terminators. Electron microscopy imaging shows that Grass is a myophage. The TerL showed homology to the TerLs of phages with long terminal repeats. The raw sequencing data were processed using the Pause  were also found.Genes encoding proteins pertaining to replication and recombination, DNA biosynthesis, packaging, morphogenesis, and lysis were identified. Genes for replication and modification proteins include UvsW helicase, DnaB-like helicase, primase, polymerase, RecA, and several DNA-binding proteins and nucleases. Genes encoding DNA biosynthesis proteins include dUTPase, dihydrofolate reductase, thymidylate synthase, nucleotidyltransferase, ribonucleotide diphosphate reductase subunits alpha and beta, and a ribonucleotide reductase stimulatory protein. The large terminase and portal proteins were also annotated. Genes encoding phage morphogenesis proteins were identified . A holin (class II with two transmembrane domains in an N-in C-in topology) and endolysin (Bacillus phage SPO1. How these proteins are involved in phage replication and assembly is unknown.Interesting features in the Grass genome are genes encoding an FtsK/SpoIIIE protein, an RtcB family protein, and a ParM protein. SpoIIIE is an ATPase common in sporulating bacteria that pumps DNA into the forespore . RtcB isKF669652 to GenBank.The genome sequence of phage Grass was contributed as accession no.


Bartonella vinsonii subsp. arupensis in pre-enriched blood of 4 patients from Thailand. Nucleotide sequences for transfer-messenger RNA gene, citrate synthase gene, and the 16S–23S rRNA internal transcribed spacer were identical or closely related to those for the strain that has been considered pathogenic since initially isolated from a human in Wyoming, USA.We identified  Bartonella bacilliformis, B. henselae, and B. quintana are well-known human pathogens, and several other Bartonella species, including B. elizabethae, B. tamiae, B. vinsonii subsp. arupensis, have been associated with various clinical manifestations in humans , a strictly North American rodent species frequently found across a wide geographic area, including Wyoming. Similar strains of B. vinsonii subsp. arupensis have not been found in other animals in North America, suggesting that deer mice are natural hosts of this bacterium  was enrolled as an afebrile patient. The 4 patients had some common clinical symptoms, such as headache, myalgias, dizziness, and fatigue. In addition, 3 of the patients had elevated levels of liver enzymes. All patients reported trapping or killing rats or seeing rats inside or around their houses during the 2 weeks before onset of symptoms, and all patients owned a dog and/or cat .Bartonella species, we tested blood clots from each patient for the presence of the bacterium. For testing, we used a liquid growth medium (Bartonella α-Proteobacteria growth medium [BAPGM]) as a pre-enrichment step (Bartonella detection (2 for 7 days.To determine if the patients were infected with –Bartonella-specific regions in the transfer-messenger RNA (ssrA) gene, the citrate synthase (gltA) gene, and the 16S–23S rRNA internal transcribed spacer, respectively. Positive controls (B. doshiae) and negative controls were included within each PCR run to evaluate the presence of appropriately sized amplicons and contamination, respectively. Amplicons were recovered from PCRs by gel purification (QIAGEN) and sequenced in both directions.DNA was extracted from this pre-enriched medium by using the QIAamp DNA Mini Kit . DNA was analyzed following published protocols (B. vinsonii subsp. arupensis. The gltA sequences (338 bp) from all 4 patient samples were identical to those for previously reported variants from a febrile patient in Thailand (GenBank accession no. GQ200857) and from stray dogs in Thailand (GenBank accession no. FJ946836). These gltA sequences were 0.8% divergent from the type strain of B. vinsonii subsp. arupensis (GenBank accession no. AF214557).Sequences for the 3 targets were similar to those for the type strain of ssrA sequences (251 bp) revealed 2 similar variants. One variant was identical to the type strain of B. vinsonii subsp. arupensis (GenBank accession no. JN029783) and was identified in samples from 3 of the patients . The other variant (GenBank accession no. JN394654), from patient 45-00025, was 2.8% divergent from the type strain of B. vinsonii subsp. arupensis.The B. vinsonii subsp. arupensis (GenBank accession no. AF312504). The other variant (GenBank accession no. JN402328), from patient 45-00250, was 0.9% divergent from the type strain of B. vinsonii subsp. arupensis (GenBank accession no. AF312504).In addition, two 16S–23S rRNA internal transcribed spacer variants were identified. One variant (GenBank accession no. JN402327), identified in samples from 3 patients , was 0.4% divergent from the type strain of Bartonella species, B. henselae and B. tamiae, have been reported in association with human diseases  and mechanism(s) of transmission of this bacterium to humans remain unclear, the previous finding of B. vinsonii subsp. arupensis among stray dogs in Thailand (B. vinsonii subsp. arupensis. Further investigation is needed in this regard.In the current study, sequence data from 3 genetic targets provide additional evidence to confirm infection with Yersinia pestis, was spread by infected rats on ships traveling from Asia to other continents. Bartonella elizabethae and other rat-associated Bartonella species that originated in the Old World have been similarly translocated to the New World and other regions (,B. vinsonii subsp. arupensis in humans in France, Russia, and Nepal, suggest that the role of this bacterium as a human pathogen may be more geographically widespread than previously believed.It is known that diseases of humans and animals can translocate across the globe. For example, bubonic plague, caused by Bartonella species across broad geographic regions and even between hosts. In addition, our finding of B. vinsonii subsp. arupensis in patients in Thailand suggests that this pathogen may be responsible for some undiagnosed cases of febrile illness and other types of illnesses in Thailand and possibly other countries in Asia.Given the postulated New World origin of this pathogen, its ecology might serve as a model system to examine a possible translocation of


Leishmania infection in animal models, and salivary components have been postulated as vaccine candidates against leishmaniasis. In the sand fly Phlebotomus papatasi, natural sugar-sources modulate the activity of proteins involved in meal digestion, and possibly influence vectorial capacity. However, only a handful of studies have assessed the variability of salivary components in sand flies, focusing on the effects of environmental factors in natural habitats. In order to better understand such interactions, we compared the expression profiles of nine P. papatasi salivary gland genes of specimens inhabiting different ecological habitats in Egypt and Jordan and throughout the sand fly season in each habitat.Sand fly saliva can drive the outcome of The majority of investigated genes were up-regulated in specimens from Swaymeh late in the season, when the availability of sugar sources is reduced due to water deprivation. On the other hand, these genes were not up-regulated in specimens collected from Aswan, an irrigated area less susceptible to drought effects.Expression plasticity of genes involved with vectorial capacity in disease vectors may play an important epidemiological role in the establishment of diseases in natural habitats. Phlebotomus papatasi [Many studies have demonstrated an environmental role in gene expression. Differential gene expression can be caused by biotic  or abiotic factors . Effects of the environment on gene expression are referred to as gene-by-environment-interactions, and the response displayed by organisms to such environmental change, phenotypic plasticity . Althougpapatasi -9, how tpapatasi .Phlebotomus papatasi is the primary vector of Leishmania major in Northern Africa and the Middle East [Prosopis farcta, Capparis spinosa, Ricinus communis, Solanum nigrum, and Rochia indica are some of the most attractive plants for P. papatasi [P. papatasi, as demonstrated by the finding that 50% of the field collected flies had ingested this carbohydrate [Atriplex halimus [L. major reservoir host, the fat sand rat Psammomys obesus [dle East ,12. The dle East  and dispdle East . In addiP. papatasi females collected in non-irrigated areas in the Jordan Valley are older than 8 days [Previous studies have demonstrated that sugar appears to influence many aspects of sand fly physiology, including longevity ,14. Whern 8 days , in irrin 8 days . Accordin 8 days .Leishmania development is not yet fully understood. Recent studies have contributed to our understanding of some of these complex interactions. Although a sugar-rich diet is associated with a greater number of gonotrophic cycles for P. papatasi , thus, increasing the chance for Leishmania transmission [R. communis, C. spinosa, and S. luteum can actually decrease the number of Leishmania in P. papatasi by 45% [Leishmania load in the gut of sand flies was found when they fed on Malva nicaeensis compared to A. halimus [The interaction of sugars with various aspects of sand fly physiology and i by 45% . In addi halimus ,7.P. papatasi [P. papatasi prefers feeding on branches of the Syrian mesquite P. farcta collected from a humid habitat rather than on branches of the same plant collected from a dry and salty soil [The quality of the sugar meals is also believed to influence sugar-feeding behavior of papatasi . Such eflty soil .P. papatasi and L. longipalpis s.l. are not expressed after a sugar meal [PperTryp1 and 1, a chymotrypsin, PperChym3, are up-regulated when P. perniciosus is sugar-fed [L. longipalpis s.l. salivary protein content is also increased after a sugar-meal [The expression of some sand fly genes involved in digestion and nutrient acquisition are also modulated by sugar-meals. Chitinases of the sand flies gar meal , whereasugar-fed . Likewisgar-meal .L. longipalpis s.l. a 6 KDa peptide named maxadilan is responsible for the vasodilatory effect [P. papatasi, this is accomplished by adenosine and 5'AMP [Saliva components have been identified for several insect species including sand flies -22. Likend 5'AMP . A diverLeishmania transmission as it is necessary for successful blood feeding. Interestingly, P. papatasi saliva exacerbates L. major infection in mice [SP15) present in the sialome of P. papatasi [Sand fly saliva is essential for the success of  in mice ; however in mice , suggestpapatasi , though papatasi ,25-27. BP. papatasi have been identified, and those encompass the products of 12 genes [P. papatasi vectorial capacity are differentially modulated in distinct ecological habitats [P. papatasi salivary gland genes across specimens collected in distinct ecotopes and obtained during different periods during the sand fly season. Our results indicate that the pattern of salivary gland gene expression exhibited is more associated with the distinct environmental conditions presented in natural habitats than with the geographic origins of the specimens. Additionally, the data presented support the notion that expression plasticity of sand fly salivary gland genes exhibited in distinct ecological habitats may have epidemiological consequences and may affect the immunogenicity of a sand fly salivary protein-based vaccine.The most highly expressed proteins in the sialome from 12 genes ; however12 genes , and the12 genes . Recentlhabitats . Here, wPhlebotomus papatasi used in this study were either obtained from field collections or from a colony (Israeli strain - PPIS) maintained at the University of Notre Dame. These PPIS specimens are from a colony originally established in the mid 1970's that went through several bottlenecks, the most current of which was in July 2007. Thus, the PPIS colony displays very low levels of genetic polymorphism. For field samples, sand flies were collected at 3 locations: Aswan , in a village adjacent to the Nile River (Baharif - Southern Egypt); Northern Sinai , in a Bedouin village (Om Shikhan - Northeastern Egypt); and Swaymeh , near the Dead Sea, in Jordan.Phoenix dactylifera), mangoes (Mangifera indica), wheat (Triticum aestivum), corn (Zea mays), and clover (Trifolium spp.) under artificial irrigation. The human population is approximately 400 and the village is stocked with domestic animals including cattle, dogs, and goats. Daily temperatures typically range from 24°C to 45°C, and it seldom rains in this locality. This site was chosen for our study because of the large number of sand flies present in the area as observed by U.S. Naval Medical Research Unit No. 3 (NAMRU-3) researchers over the previous 15 years, for the absence of Leishmania-infected flies [The collection site in Aswan, Baharif village, is located on the east margin of the Nile. This village is typically cultivated with date palms  El Ruafa Dam, on Wadi El Arish. Uncultivated areas around the reservoir are variably covered by low desert brush, with minating . ClimatiAtriplex halimus and Suaeda spp.) as the natural flora. Swaymeh also is an endemic area for L. major.Swaymeh, Jordan, is an area of low elevation at approximately 350 m below sea level. The climate is considered Saharan Mediterranean with temperatures ranging from a minimum of 8-12°C in the winter and a maximum of 35-40°C in the summer. Mean rainfall is <50 mm, with all rain fall ocurring from November-April. Area soil is mostly sandy or sandy hammada with granite fragments and saline, with tropical and halophytic vegetation (chenopods such as P. papatasi represents approximately 95% of the Phlebotomus species [2 (dry ice) (for trappings done in Aswan and North Sinai), or non-baited (Swaymeh). Sand flies were transferred from collection bags and maintained alive until dissected. Flies were euthanized in water and detergent just prior to dissection. P. papatasi were identified by microscopic examination of female spermateca according to Lane [Whenever possible, sand fly trappings were carried out three times a year, early (June), middle (August) and late (September) for years 2006 and 2007. While in Aswan and Swaymeh we performed 3 trappings ; for the sites in North Sinai only 2 trappings took place: early and mid 07 in North Sinai. For each of the 3 locations  species ,32. Sand to Lane ; their hhttp://www.fao.org/giews/english/windisp/data.htm and clipped using the boundaries of Egypt and Jordan.The Normalized Difference Vegetation Index (NDVI) was used as a measure of healthy vegetation cover . NDVI ishttp://earlywarning.usgs.gov/fews/africa/index.php and Jordan http://www.eeaa.gov.eg/English/main/about.asp. The digital elevation maps also were clipped using the boundaries of Egypt and Jordan.Digital elevation data also were downloaded for Egypt P. papatasi individually using the RNAeasy Mini Kit  according to instructions and stored at -80°C.The RNA was extracted from the dissected tissues  of all the 20 primer and 0.5 μM dNTPs (10 mM), incubated at 65°C for 5 minutes (min) and kept in ice for at least 1 min; 4 μl 5 × SuperScript™ III Reverse Transcriptase First-Strand Buffer, 5 mM DTT (0.1 M), 20 Units of RNase OUT, and 200 Units of SuperScript™ III Reverse Transcriptase (200 u/μl) were added to the reaction. The mixture was incubated for one hour at 50°C and stored at -20°C.cDNAs were synthesized using Invitrogen reagents , following the manufacturer's instructions. In brief, 12 μl RNA from each sample were added with 2.5 μM Oligo (dT)RT-PCR reactions were set up with 10 μl SYBR Green reagent , 0.6 μl each forward and reverse primer , 0.5 μl each cDNA sample, and 8.3 μl Ultra Pure DNase/RNase-Free Water (Invitrogen). Reactions were analyzed in 96-well plate format using a 7900 HT Fast Real Time PCR System (Applied Biosystems) under the following conditions: initial incubation at 50°C for 2 min and 95°C for 10 min; followed by 40 cycles of 95°C for 15 sec, 55°C for 1 min; ending with a dissociation step of 95°C for 15 sec, 55°C for 15 sec, and 95°C for 15 sec.P. papatasi salivary gland genes assayed for expression in this study are SP12, SP14, SP28, SP29, SP30, SP32, SP36, SP42, and SP44. The primers used in the RT-PCR reactions were published elsewhere [P. papatasi specimens from each field catch were used for individual RT-PCR reactions, and the expression profile for all 9 cDNAs was assessed using the same 20 field-caught samples. Each reaction was repeated four times for each gene (twice in two different plates) for a total of 80 reactions per sand fly trapping. Since the expression analyses of nine genes in eight different sand fly trappings were performed, a total of 5760 RT-PCR reactions were performed during the completion of this study.The 9 lsewhere . A total-ΔΔCT method [-ΔΔCT, where ΔΔCT = ΔCT(sample) - ΔCT, ΔCT = ΔCT(sample) -ΔCT, CT = cycle at which a statistically significant increase in the emission intensity over the background. The calibrator was represented by the average expression (mean ΔCT) of the seven non-fed samples (PPIS) dissected 24 hours after emerging [Differential expression results for each salivary protein gene were displayed as fold changes over a control, using the 2T method . The folemerging . Fold chStatistical analyses were carried out using the software GraphPad Prism v. 5.01 . The statistical tests used were the non-parametric Kruskal-Wallis tests, for comparisons among more than two data sets, and/or Mann-Whitney tests, for pairwise comparisons between data sets when the results for Kruskal-Wallis test were statistically significant, or for comparisons when only two data sets were present. The level of significance was adjusted for multiple comparisons using Bonferroni's correction. Differences were considered statistically significant at α = 0.0006. The values of fold change in Tables P. papatasi sand flies were collected from three distinct geographic locations. Samples were collected in Aswan, Southern Egypt, as well as in the Northern Sinai Peninsula, Northeastern Egypt. P. papatasi were also collected in Swaymeh, Jordan. The elevation of the Egyptial collection sites were 117 m, 141 m above sea level for Aswan and North Sinai, respectively . Two types of analyses were carried out. For the seasonal analyses, gene expression levels were compared between specimens collected at different time points within each habitat  were differentially regulated through the season  displayed significantly different levels of expression between the populations of Aswan and North Sinai (Table SP28 and SP30) and one (SP32) in individuals from Swaymeh (Table SP30) was up-regulated in flies from Aswan. Likewise, between Swaymeh and North Sinai one gene was differentially expressed: SP32 was up-regulated in Swaymeh  were up-regulated in Swaymeh in comparison with Aswan , mid (August) and late (September). Our study involved the assessment by real time quantitative PCR of 160 individuals and was based on comparisons made to colonized, water-fed only flies. Significant differences in expression levels were found between distinct ecotopes and seasonal periods.In addition to genetic variability, expression level differences could influence vaccine efficacy. It recently has been suggested that salivary component expression level differences between laboratory-reared and field-caught sand flies may interfere with the sand fly saliva-mediated protection against  in mice ,52. Our L. longipalpis s.l. [L. longipalpis s.l. collected from different locations [L. longipalpis s.l., P. papatasi is not a species complex. Nevertheless, several studies have shown physiological, behavioral, and genetic differences between P. papatasi populations from different geographic localities [P. papatasi Israeli strain was higher than sand flies from India or Egypt for infection with L. major. Likewise, comparing P. papatasi colonies originating from three different populations in Egypt, Hanafi et al. [L. major infection than flies from Aswan or the Nile Delta. Additionally, the feeding rate on mice by P. papatasi from Aswan was lower than the rate obtained for the other two populations [P. papatasi from several colonized and natural populations based on polymorphisms on the Cytochrome b (cyt b) haplotypes demonstrated moderate genetic differentiation between populations from Egypt and the Middle East [P. papatasi Internal Transcribed Spacer 2 (ITS2) and NAD dehydrogenase subunit 4 gene (ND4) indicated absence of genetic structuring across the P. papatasi geographical range [P. papatasi populations, gene expression can present a stronger correlation with ecological habitat than with genetic distance [P. papatasi salivary gland gene expression from field-collected sand flies needs to be thoroughly assessed so that differences observed in flies collected in different ecological habitats can be correlated with the geographic origin of the populations, with seasonal factors present in each habitat, or with both.Expression differences of a sand fly salivary gland gene were noted in pis s.l. . Such diocations . Howevercalities -59. Studi et al.  indicateulations . In addidle East . Contrasal range . Despitedistance . TherefoP. papatasi salivary gland gene expression are driven by factors associated with the geographic origin of the populations or by environmental factors (or perhaps both) we performed two types of gene expression comparisons: geographic analyses between specimens collected in different ecological habitats during the same period of the season, and seasonal analyses between specimens collected in the same habitat at different periods of the season. From our analyses, three types of results may be expected: (1) a population displaying higher or lower levels of expression of a given gene for the three geographic comparisons made, but without seasonal differences, indicating that only factors associated with the geographic origin of the populations were responsible for the differences observed in regards to the expression of that specific gene; (2) a population exhibiting higher (or lower) expression medians of a specific gene in the three geographic comparisons as well as in the seasonal analysis, suggesting that both geographic origin-related and seasonal factors drove the expression of that gene; and (3) a population displaying only significant seasonal differences in the expression of a gene, indicating that seasonal environmental factors played a major role in controlling the expression of that gene.To assess whether differences in P. papatasi populations from different ecological habitats. Although most of the salivary gland genes from P. papatasi collected early in the season exhibited similar levels of expression  are higher than in flies from an irrigated area (Aswan) late in the season, when drought may affect sugar content of plants [Our geographic analyses indicate that salivary gland genes display expression variability between on Table , and onlf plants .P. papatasi populations from different habitats can exhibit differences in the percentages of gravid or engorged females, as observed in the Jordan Valley [Phenology studies demonstrated that n Valley ,32. Howen Valley .P. papatasi glycosidase activity patterns presented elsewhere [P. papatasi salivary gland genes  are also differentially regulated during the season  cases is also higher at the Swaymeh site [P. papatasi salivary gland genes analyzed were up-regulated late in the season. As the incubation period for CL caused by L. major ranges between two to eight weeks [P. papatasi specimens through the season in natural habitats may contribute to the increase in CL cases.Schlein and Jacobson  demonstrted area . Salivarate host ,24,25. Ameh site  as compaht weeks ,65, thesLeishmania may be effected by the amount of exposure the host receives [The use of saliva or salivary components in a multi-component vaccination strategy is a viable option ,24,25. Hreceives .P. papatasi vectorial capacity to transmit L. major, a parasite responsible for cases of cutaneous leishmaniasis, is evident in field caught specimens, as demonstrated in this work. Moreover, more than half of the salivary gland genes are up-regulated at the end of the sand fly season, when availability of sugar is scarce and disease transmission is increased. Therefore, gene-by-environment interactions can also be an important factor in transmission of pathogens in natural habitats.The genetic plasticity of genes involved with IVCA participated in the sand fly collection, performed the real time PCR reactions, analyzed the data, and drafted the manuscript. MAM conceived and coordinated the project and participated in project design, sand fly collections, dissection of salivary glands, and manuscript drafting. MRO participated in sand fly collections, dissection of salivary glands, drafting of the manuscript, and designing of the project. DFH and MAS oversaw operations and sand fly collections in Egypt and Jordan, respectively, including acquiring appropriate permissions and permits when necessary. HAH coordinated all sand fly collections in Egypt and RM and SK coordinated collections in Jordan. EYF and SSE-H participated in all sand fly collections in Egypt and advised on collecting sites. MW and GS maintained the sand fly colony. DAP acquired and formatted the environmental and landscape data. All authors have read and approved the final manuscript.The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.The study protocol was approved by the U.S. Naval Medical Research Unit No. 3 Institutional Review Board in compliance with all applicable Federal regulations governing the protection of human subjects. IRB # 193, DoD # NAMRU3.2006.0011.One of the co-authors is a military service member; some of the other co-authors are employees of the U.S. Government. This work was prepared as part of our official duties. Title 17 U.S.C. §105 provides that 'Copyright protection under this title is not available for any work of the United States Government'. Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties.SP12 expressionFigure S1 - . SP12 expression profiles were assessed as fold changes (Y axis) over the control non-sugar fed, and colony-maintained P. papatasi using the 2-ΔΔCT method. Seasonal analyses are displayed in (A), (B) and (C), representing the populations of Aswan, Swaymeh and North Sinai, respectively. P. papatasi were collected at different periods during the sand fly activity season. Graphs (D), (E) and (F) display the geographic comparisons between expression profiles of P. papatasi collected in June (early in the season) and August (middle) 2007, and September (late) 2006, respectively. Horizontal bars represent the expression mean values between the samples and each sample represents an individual fly. Asterisk (*) indicates statistically significant differences (p < 0.0006) between every two groups analyzed. Aswan (black), Swaymeh (dark gray) and North Sinai (light gray) color schemes are shown. Triangle, circle and square represent expression levels of sand flies collected early, in the middle and late in the season, respectively.Click here for fileSP14 expressionFigure S2 - . SP14 expression profiles were assessed as in Figure S1. Seasonal analyses are displayed in (A), (B) and (C), representing the populations of Aswan, Swaymeh and North Sinai, respectively. P. papatasi were collected at different periods during the sand fly activity season. Graphs (D), (E) and (F) display the geographic comparisons between expression profiles of P. papatasi collected in June (early in the season) and August (middle) 2007, and September (late) 2006, respectively. Horizontal bars represent the expression mean values between the samples, and each sample represents an individual fly. Asterisk (*) indicates statistically significant differences (p < 0.0006) between every two groups analyzed. Aswan (black), Swaymeh (dark gray) and North Sinai (light gray) color schemes are shown. Triangle, circle and square represent expression levels of sand flies collected early, in the middle and late in the season, respectively.Click here for fileSP28 expressionFigure S3 - . SP28 expression profiles were assessed as in Figure S1. Seasonal analyses are displayed in (A), (B) and (C), representing the populations of Aswan, Swaymeh and North Sinai, respectively. P. papatasi were collected at different periods during the sand fly activity season. Graphs (D), (E) and (F) display the geographic comparisons between expression profiles of P. papatasi collected in June (early in the season) and August (middle) 2007, and September (late) 2006, respectively. Horizontal bars represent the expression mean values between the samples and each sample represents an individual fly. Asterisk (*) indicates statistically significant differences (p < 0.0006) between every two groups analyzed. Aswan (black), Swaymeh (dark gray) and North Sinai (light gray) color schemes are shown. Triangle, circle and square represent expression levels of sand flies collected early, in the middle and late in the season, respectively.Click here for fileSP29 expressionFigure S4 - . SP29 expression profiles were assessed as in Figure S1. Seasonal analyses are displayed in (A), (B) and (C), representing the populations of Aswan, Swaymeh and North Sinai, respectively. P. papatasi were collected at different periods during the sand fly activity season. Graphs (D), (E) and (F) display the geographic comparisons between expression profiles of P. papatasi collected in June (early in the season) and August (middle) 2007, and September (late) 2006, respectively. Horizontal bars represent the expression mean values between the samples and each sample represents an individual fly. Asterisk (*) indicates statistically significant differences (p < 0.0006) between every two groups analyzed. Aswan (black), Swaymeh (dark gray) and North Sinai (light gray) color schemes are shown. Triangle, circle and square represent expression levels of sand flies collected early, in the middle and late in the season, respectively.Click here for fileSP30 expressionFigure S5 - . SP30 expression profiles were assessed as in Figure S1. Seasonal analyses are displayed in (A), (B) and (C), representing the populations of Aswan, Swaymeh and North Sinai, respectively. P. papatasi were collected at different periods during the sand fly activity season. Graphs (D), (E) and (F) display the geographic comparisons between expression profiles of P. papatasi collected in June (early in the season) and August (middle) 2007, and September (late) 2006, respectively. Horizontal bars represent the expression mean values between the samples and each sample represents an individual fly. Asterisk (*) indicates statistically significant differences (p < 0.0006) between every two groups analyzed. Aswan (black), Swaymeh (dark gray) and North Sinai (light gray) color schemes are shown. Triangle, circle and square represent expression levels of sand flies collected early, in the middle and late in the season, respectively.Click here for fileSP32 expressionFigure S6 - . SP32 expression profiles were assessed as in Figure S1. Seasonal analyses are displayed in (A), (B) and (C), representing the populations of Aswan, Swaymeh and North Sinai, respectively. P. papatasi were collected at different periods during the sand fly activity season. Graphs (D), (E) and (F) display the geographic comparisons between expression profiles of P. papatasi collected in June (early in the season) and August (middle) 2007, and September (late) 2006, respectively. Horizontal bars represent the expression mean values between the samples and each sample represents an individual fly. Asterisk (*) indicates statistically significant differences (p < 0.0006) between every two groups analyzed. Aswan (black), Swaymeh (dark gray) and North Sinai (light gray) color schemes are shown. Triangle, circle and square represent expression levels of sand flies collected early, in the middle and late in the season, respectively.Click here for fileSP36 expressionFigure S7 - . SP36 expression profiles were assessed as in Figure S1. Seasonal analyses are displayed in (A), (B) and (C), representing the populations of Aswan, Swaymeh and North Sinai, respectively. P. papatasi were collected at different periods during the sand fly activity season. Graphs (D), (E) and (F) display the geographic comparisons between expression profiles of P. papatasi collected in June (early in the season) and August (middle) 2007, and September (late) 2006, respectively. Horizontal bars represent the expression median values between the samples and each sample represents an individual fly. Asterisk (*) indicates statistically significant differences (p < 0.0006) between every two groups analyzed. Aswan (black), Swaymeh (dark gray) and North Sinai (light gray) color schemes are shown. Triangle, circle and square represent expression levels of sand flies collected early, in the middle and late in the season, respectively.Click here for fileSP42 expressionFigure S8 - . SP42 expression profiles were assessed as in Figure S1. Seasonal analyses are displayed in (A), (B) and (C), representing the populations of Aswan, Swaymeh and North Sinai, respectively. P. papatasi were collected at different periods during the sand fly activity season. Graphs (D), (E) and (F) display the geographic comparisons between expression profiles of P. papatasi collected in June (early in the season) and August (middle) 2007, and September (late) 2006, respectively. Horizontal bars represent the expression mean values between the samples and each sample represents an individual fly. Asterisk (*) indicates statistically significant differences (p < 0.0006) between every two groups analyzed. Aswan (black), Swaymeh (dark gray) and North Sinai (light gray) color schemes are shown. Triangle, circle and square represent expression levels of sand flies collected early, in the middle and late in the season, respectively.Click here for fileSP44 expressionFigure S9 - . SP44 expression profiles were assessed as in Figure S1. Seasonal analyses are displayed in (A), (B) and (C), representing the populations of Aswan, Swaymeh and North Sinai, respectively. P. papatasi were collected at different periods during the sand fly activity season. Graphs (D), (E) and (F) display the geographic comparisons between expression profiles of P. papatasi collected in June (early in the season) and August (middle) 2007, and September (late) 2006, respectively. Horizontal bars represent the expression mean values between the samples and each sample represents an individual fly. Asterisk (*) indicates statistically significant differences (p < 0.0006) between every two groups analyzed. Aswan (black), Swaymeh (dark gray) and North Sinai (light gray) color schemes are shown. Triangle, circle and square represent expression levels of sand flies collected early, in the middle and late in the season, respectively.Click here for fileP. papatasi salivary gland gene expressionTable S1 - . Median gene expression levels and ranges for all 9 salivary gland genes from each location and collection date are presented.Click here for file


Several studies show that nutrition delivery is insufficient, resulting in large energy deficits during the ICU stay : the proA prospective interventional study over three periods  in the mixed ICU of a university teaching hospital. Inclusion: patients requiring >72 hours of ICU. Intervention was a two-step quality program after baseline analysis: first, implementation of feeding guidelines; and second, additional presence of an ICU dietitian. Variables: anthropometry, severity scores, energy delivery and balances , feeding route, length of stay, and mortality.P < 0.001). The feeding technique changed: use of EN increased from A to B (stable in C); combined and PN increased progressively. Oral intakes were uniformly low . Hospital mortality paralleled severity in periods B and C. The hospital stay was shorter in period C (P = 0.048). See Table In total, 604 admissions and 6,073 days were analyzed. Patients in period A were less sick  than those of periods B and C. Energy delivery and balance increased gradually: impact was particularly marked in the cumulated energy balance on day 7 P < 0.00. The feeA bottom-up protocol improved nutritional support. The ICU dietitian further improved the process , achieving better early energy balance.