Penicillium roqueforti is a filamentous fungus used for making blue cheeses worldwide. It also occurs as a food spoiler and in silage and wood. Previous studies have revealed a strong population genetic structure, with specific traits associated with the different populations. Here, we used a large strain collection from worldwide cheeses published recently to investigate the genetic structure of P. roqueforti.P. roqueforti that was consistent with previous studies, with two main genetic clusters . In addition, we detected a finer genetic subdivision that corresponded to the environment and to protected designation of origin (PDO), namely the Roquefort PDO. We indeed found evidence for eight genetic clusters, one of the cluster including only strains from other environments than cheeses, and another cluster encompassing only strains from the Roquefort PDO. The W-C- and W+C+ cheese clusters were not the most closely related ones, suggesting that there may have been two independent domestication events of P. roqueforti for making blue cheeses.We found a genetic population structure in P. roqueforti.The additional population structure revealed here may be relevant for cheese-makers and for understanding the history of domestication in  Cheese making by early Neolithic farmers was a major advance in food processing, allowing to preserve milk in a non-perishable, transportable form, and making milk more digestible for adults as cheese contains much less lactose than fresh milk . EarliesPenicillium roqueforti. Originally, P. roqueforti was not inoculated during blue cheese production but contaminated the milk spontaneously with spores from the environment. Since the end of the 18th century, P. roqueforti asexual spores (conidia) are inoculated into the cheese curd [in vitro from monospore isolations [For making the variety of blue cheeses worldwide, such as the very famous French Roquefort, English Stilton, Spanish Cabrales, Danish Danablue or Italian Gorgonzola, industrials use specific strains of the fungal ascomycete species, ese curd ,5 at theese curd ,5. Then,olations ,5. This Penicillium roqueforti also occurs as a common spoilage agent in food , and even in refrigerated stored food due to its capacity to tolerate cold temperatures, low oxygen concentrations, alkali and weak acid preservatives. Penicillium roqueforti is also found in other environments such as silage or occasionally in wood, but its natural ecological niche is still unknown [P. roqueforti [Wallaby (W) and CheesyTer (C), shared with several other Penicillium species isolated from cheese environment, such as P. camemberti, the fungus used to make Brie and Camembert cheeses [Wallaby and CheesyTer genomic islands seemed to encompass crucial metabolic genes providing competitive advantage and a better use of the cheese substrate, as revealed by experiments showing that strains carrying Wallaby and CheesyTer  had a higher growth rate on cheese medium than strains without them , and a lower growth rate on poor medium [i.e. cheese, but also wood, silage, and none of them carried either Wallaby or CheesyTer. unknown –8. Previqueforti –11, revequeforti . The fir cheeses ,13. The P. roqueforti genetic clusters still belong to a single species, as supported by the genealogical concordance phylogenetic species recognition (GCPSR) criterion [More recently, population genetics analyses on a larger collection of strains showed that this second cluster of W-C- strains showed further genetic subdivision, separating strains collected in cheese from those collected in other environments ; these triterion  and by iriterion . Howeverriterion .P. roqueforti, with possibly different genetic clusters of cheese-making strains used for different types of cheeses, harboring specific morphologic or metabolic traits, would be both of high applied interest and of fundamental importance, for understanding the process of domestication. Here we aimed at investigating whether a finer genetic subdivision could be detected within P. roqueforti, and in particular according to the type of cheeses. To this goal, we used the recently published large collection of 240 P. roqueforti strains isolated from worldwide cheeses and from other environments [Identifying a finer genetic subdivision within ronments  and lookronments , a goal P. roqueforti strains previously analysed [e.g., Roquefort, Gorgonzola, Stilton, Cabrales, Blue Gouda, Danish blue, Cheddar blue), 28 strains were isolated from other environments  and two isolates were of unknown origin [We used the set of 240 analysed , that han origin .Wallaby (W) and CheesyTer (C).All the 240 strains had been genotyped previously  using thK = 1 to K = 10, using admixture models and 500 000 MCMC iterations, after a burn in of 50 000 steps. The output was processed using CLUMPP v1.1.2 [deltaK statistics [http://taylor0.biology.ucla.edu/structureHarvester/), to identify the K value corresponding to the strongest structure. A discriminant analysis of principal components (DAPC) was also computed using the adegenet package [i.e., K = 3 and K = 8. FST and Fisher’s exact tests of population differentiation were computed using Genepop on the web [http://www.splitstree.org/).Individual-based Bayesian clustering method implemented in STRUCTURE 2.3.3  was usedP v1.1.2 , to idenP v1.1.2 . We compatistics  via the atistics   but also from other environments such as silage or wood (N = 28). The population structure inferred showed genetic subdivision with well-delimited clusters up to K = 8 , from strains lacking both . At K = 3, the W-C- strains were split into two well-delimited clusters, one with only cheese strains and a second with strains from various environments  were further split into two clusters, one including mainly strains isolated from cheeses belonging to the Roquefort protected designation of origin (PDO), and the other one including mainly non-cheese strains . At K = 6, this non-cheese cluster split into two clusters without obvious segregation according to environment of collection. At K = 7, one of the two W+C+ cheese cluster split into two clusters, one including mainly strains isolated from Gorgonzola cheeses. Finally, at K = 8, strains isolated from the Roquefort PDO were further split. From K = 9 and above, no further well-delimited cluster could be identified.We reinvestigated available genetic data of 240 strains of to K = 8 . The deldata set , separatronments , as showK = 8 thus represents the finest genuine genetic structure detectable in our dataset .The clusters . Fisher’P. roqueforti for making blue cheeses. The orange cluster, including W-C- strains isolated from silage, wood, food spoiler and even cheese, appeared closest to the W+C+ cheese strains along this axis, while the W-C- cheese strains were farther left. The green and grey clusters including W-C- strains from mostly silage or spoiled food, were grouped close one to each other, separated from the W-C- cheese cluster by the second axis.The discriminant analysis of principal component (DAPC), assuming no particular model such as panmixia, also discriminated eight genetic clusters that mostly corresponded to the clusters found with the Bayesian clustering method implemented in STRUCTURE . The thrThe Splitstree also discriminated the same eight genetic clusters and inferred similar genetic relationships among them as the DAPC . The SplP. roqueforti. Overall, we detected in P. roqueforti a genetic structure consistent with previous studies [FST showed that the differentiation was genuine. The additional population structure revealed here may be relevant for cheese-makers, as phenotypic differences have been reported between the previously identified clusters in P. roqueforti [Here, we used the strain collection from worldwide cheeses published recently , as well studies –11, althqueforti . SpecifiP. roqueforti. Indeed, the DAPC and the Splitstree both show that the W+C+ and W-C- cheese clusters are not the most closely related, which could not be seen in previous studies considering only the three main genetic clusters. These relationships suggest two different independent origins of the strains used in cheese production. These two cheese main groups correspond to the W+C+ and W-C- cheese clusters, respectively, and harbour different growth rates [The finer population structure revealed here compared to previous studies may also allow better understanding the history of domestication in th rates  and diffth rates . This maWallaby and CheesyTer. The strains did not appear intermediated between the W+C+ and W-C- strains in any analysis, rendering the hypothesis of hybridization unlikely. These strains may instead have acquired the two genomic islands recently by horizontal gene transfers. The mechanisms involved are still unknown but horizontal gene transfers among Penicillium strains seem frequent [Interestingly, the yellow W-C- cheese cluster harbored a few strains carrying the frequent . They cofrequent .P. roqueforti than previous studies thus bring new, interesting results, improving our understanding of the history of domestication. It should be noted, however, that the environments other than cheeses from where strains could be collected were mostly anthropogenic, i.e., silage and spoiled food, and may not correspond to the exact wild population of origin. Strains collected as food spoiler could even correspond to feral individuals. This may be the case in particular for the few strains clustering within cheese clusters. However, the finding that several well-delimited genetic clusters correspond to non-cheese strains, together with the fact that they include strains from wood, suggest that most of the strains from spoiled food and silage represent genetically isolated populations, and not only feral strains. Indeed, if food spoiler strains were feral cheese strains, they would cluster within our “cheese clusters”, which was not the case.Our analyses exploring finer genetic subdivision within S1 Table(XLSX)Click here for additional data file.


AbstractPholidopterafrivaldszkyi in south-western Russia occurring far beyond the accepted area of the species distribution in the Carpathian-Balkan region were performed. Using the mitochondrial DNA cytochrome c oxidase subunit I gene fragment, we found a very low level of genetic diversity in these populations. Phylogeographic reconstruction did not support recent introduction events but rather historical range fragmentation. The grouping of the Russian and Romanian haplotypes in a distinct phylogenetic clade suggests that the pre-glacial range of P.frivaldszkyi had extended towards the Ponto-Caspian region, with considerable gene flow between different refugia. However, post-glacial northward expansion of the species from supposed Caucasus refugia contributed most likely to the current disjunct distribution of this relict-like bush-cricket.Phylogenetic analysis and assessment of the species status of mostly isolated populations of  Pholidopterafrivaldszkyi , is a regionally rare and endangered grassland-dwelling Orthoptera (Tettigoniidae) species with a decreasing population trend  were collected at seven sites (3–6 ind. per site) from the Kursk , Tambov  and Penza (Krutec) regions in Russia  gene from each sampled individual followed the protocol described in PageBreakthat was previously determined as the best-fit substitution site evolutionary model for the organism studied (ML) analysis and Bayesian inference. For construction of an ML tree we used the MEGA5.2 software , respectively, in order to use them as outgroup species in rooted trees.A total of 26 adult individuals of  studied . We recosoftware , for whisoftware . Markov PageBreakP.frivaldszkyi in Russia. Analysis of the 778 bp long sequences of mtDNA COI gene in 26 individuals revealed only two (0.3%) variable sites with three unique haplotypes (GenBank accession numbers KY554960–KY554962), which may indicate a strong demographic decline  are considered as typical habitat for kyi e.g. ; thus, o species . However species  rejects  species .P.frivaldszkyi also extended towards the Ponto-Caspian region, similarly to the ranges of many other recent Orthoptera species (e.g. PageBreakPageBreakMediterranean and Pontic refugia during the last ice-age (P.frivaldszkyi. This situation could therefore attract even more attention on the evolution and ecology (e.g. relict habitats, The most plausible evolutionary explanation of the observed phylogeographic pattern is range contraction during the last glacial period and subsequent post-glacial species expansion. Traces of a genetic connection between the eastern Romanian and Russian populations suggest that the pre-glacial range of ies e.g. . After ties e.g. . Such poies e.g.  and relaies e.g. . On the ies e.g.  is proba ice-age . Althoug ice-age  as forme ice-age , the gen ice-age . Thus, s


Biomphalaria pfeifferi is highly compatible with the widespread human-infecting blood fluke Schistosoma mansoni and transmits more cases of this parasite to people than any other snail species. For these reasons, B. pfeifferi is the world’s most important vector snail for S. mansoni, yet we know relatively little at the molecular level regarding the interactions between B. pfeifferi and S. mansoni from early-stage sporocyst transformation to the development of cercariae.B. pfeifferi to S. mansoni as it occurs in nature by undertaking Illumina dual RNA-Seq on uninfected control B. pfeifferi and three intramolluscan developmental stages  using field-derived west Kenyan specimens. A high-quality, well-annotated de novo B. pfeifferi transcriptome was assembled from over a half billion non-S. mansoni paired-end reads. Reads associated with potential symbionts were noted. Some infected snails yielded fewer normalized S. mansoni reads and showed different patterns of transcriptional response than others, an indication that the ability of field-derived snails to support and respond to infection is variable. Alterations in transcripts associated with reproduction were noted, including for the oviposition-related hormone ovipostatin and enzymes involved in metabolism of bioactive amines like dopamine or serotonin. Shedding snails exhibited responses consistent with the need for tissue repair. Both generalized stress and immune factors immune factors  exhibited complex transcriptional responses in this compatible host-parasite system.We sought to capture a portrait of the response of B. pfeifferi in transmission of S. mansoni, including with an emphasis on more natural, field-derived specimens. We have identified B. pfeifferi targets particularly responsive during infection that enable further dissection of the functional role of these candidate molecules.This study provides for the first time a large sequence data set to help in interpreting the important vector role of the neglected snail  Biomphalaria pfeifferi is the world’s most important snail vector for the widespread human-infecting blood fluke Schistosoma mansoni. Despite this, we know relatively little about the biology of this highly compatible African snail host of S. mansoni, especially for specimens from the field. Using an Illumina-based dual-seq approach, we captured a portrait of the transcriptional responses of Kenyan snails that were either uninfected with S. mansoni, or that harbored 1-day, 3-day, or cercariae-producing infections. Responses to infection were influenced both by the extent of schistosome gene expression and infection duration. We note and discuss several alterations in transcriptional activity in immune, stress and reproduction related genes in infected snails and the B. pfeifferi symbionts detected. Several host genes were highly up-regulated following infection and these might comprise excellent candidates for disruption to diminish compatibility. This study provides for the first time a large sequence dataset to help in interpreting the important vector role of B. pfeifferi in transmission of S. mansoni, including with an emphasis on more natural, field-derived specimens. Schistosomiasis is one of the world’s most prevalent neglected tropical diseases with over 218 million people worldwide requiring preventive chemotherapy in 2015, 92% of those occurring in 41 countries in Africa –6. ThereSchistosoma mansoni. Biomphalaria pfeifferi is one of 18 Biomphalaria species known to transmit S. mansoni. Biomphalaria pfeifferi has a broad geographic distribution in sub-Saharan Africa where the majority of cases of S. mansoni occur and exhibits a high degree of susceptibility to S. mansoni [B. pfeifferi typically shows high infection rates (50%+) following exposure to S. mansoni from locations throughout Africa, but even to isolates originating from the Americas [B. pfeifferi is the world’s most important intermediate host for S. mansoni. Understanding the role of B. pfeifferi in human schistosomiasis transmission becomes more critical because expanding agriculture and water development schemes [S. mansoni as well.Among the most important schistosome species infecting humans and the one with the broadest geographical range is  mansoni –16. For Americas . For the schemes  and clim schemes  threatenB. pfeifferi’s importance in transmission of S. mansoni, it is surprising we lack even the most basic information at the molecular level about its interactions with, and responses to, S. mansoni. Such responses could be particularly interesting in the case of B. pfeifferi because it differs from other major S. mansoni-transmitting snail species in that it is a strong preferential selfing species, a characteristic potentially resulting in low genetic diversity within populations [B. pfeifferi reflects the simple fact that it is often difficult to maintain this species in the laboratory, in contrast to the Neotropical snail B. glabrata which has been the standard model laboratory snail host for S. mansoni for decades [Biomphalaria glabrata surely remains an important intermediate host of S. mansoni in the Neotropics, but given that the vast majority of S. mansoni cases occur in sub-Saharan Africa, it is critical that we extend more attention to the relevant African snail, B. pfeifferi.Given ulations –23. Our  decades . BiomphaBiomphalaria snails and their interactions with S. mansoni and other trematodes including echinostomes. All of these studies have been undertaken with B. glabrata and have been amply reviewed and discussed [Biomphalaria glabrata genome has now been published [Biomphalaria species that are responsible for transmitting the most S. mansoni infections by far have been largely ignored with respect to application of modern high-throughput sequence-based tools.The advent of genomics approaches including high throughput sequencing techniques have lead over the past decade to several studies of iscussed –36. In aublished . IronicaB. glabrata began with studies of expressed sequence tags [B. glabrata has the capacity for more diverse immune responsiveness than previously known, including production of diversified molecules like FREPs (fibrinogen-related proteins) [et al. [B. glabrata during the intramolluscan development of both S. mansoni and Echinostoma paraensei, and showed snail defense-related transcripts were generally down-regulated starting shortly after infection. A later generation array including ~31,000 ESTs from B. glabrata provided new insights into how the APO or amebocyte-producing organ of B. glabrata responds to immune challenge [B. glabrata transcriptional responses of the molluscicide niclosamide that is commonly used for snail control operations [Projects going beyond the study of individual genes or gene families of nce tags –40, ORESnce tags ,42, and nce tags ,44. Thesroteins) ,45,46. H [et al.  examinedhallenge , and to erations .B. glabrata and S. mansoni have focused on genetic linkage studies to identify chromosome regions of interest that contain genes influencing resistance to infection [B. glabrata gene products shown to influence susceptibility to S. mansoni [Additional recent studies of the interactions between nfection ,50,51. F mansoni –32,52.et al. [B. glabrata, and identified 1,685 genes that exhibited differential expression after immune challenge. More recent studies employing RNA-Seq have identified B. glabrata genes associated with a state of heightened innate immunity [B. glabrata strains differing in their susceptibility to S. mansoni [B. glabrata and to a lesser extent for Oncomelania hupensis [Schistosoma japonicum, to date there have been no equivalent studies published for B. pfeifferi, or for other schistosome-transmitting planorbid snails, including species of Bulinus, several of which transmit members of the Schistosoma haematobium species group in Africa, southern Europe and southwest Asia.Relevant to the present study, Deleury et al.  publisheimmunity  or with  mansoni . Despitehupensis ,56, the B. pfeifferi, and of B. pfeifferi infected with S. mansoni for 1 or 3 days, or with naturally acquired cercariae-shedding or “patent” infections. The intramolluscan transcriptional responses of S. mansoni will be the subject of a separate paper. The challenge of parsing S. mansoni sequences from the aggregate of reads obtained from infected B. pfeifferi has been aided by availability of the S. mansoni genome [S. mansoni [With this in mind, we have undertaken an Illumina RNA-Seq study of i genome  and stag mansoni –60.B. pfeifferi, an important neglected vector species that has hitherto been understudied. Included among the snail genes highlighted are several that relate to stress, immune or reproductive functions, or that may be key players in influencing the noteworthy widespread ability of this snail to support schistosomiasis transmission.Our view of schistosome-snail encounters has also been largely formed by studies of lab-reared snails and schistosomes. RNA-Seq offers a way to bridge and expand upon these traditional views by revealing the detailed molecular and cellular mechanisms taking place in genetically diverse hosts and parasites. This is the first Illumina study performed on samples of both field-derived vector snails and their corresponding schistosome parasites, adding a unique perspective to our understanding of schistosome transmission “in the wild” in endemic regions. This approach also serves to remind us that the snails targeted for infection by schistosome miracidia in the field are best considered as holobionts with potentially complex sets of symbiotic associates ,62. FinaSchistosoma mansoni eggs that were hatched to obtain miracidia used to infect some of the Biomphalaria pfeifferi snails used in this study. Fecal samples were obtained and pooled from five S. mansoni-positive primary school children aged 6–12 years from Obuon primary school in Asao, Nyakach area, Nyanza Province, western Kenya . Written and signed consent was given by parents/guardians for all children. The KEMRI Ethics Review Committee (SSC No. 2373) and the UNM Institution Review Board (IRB 821021–1) approved all aspects of this project involving human subjects. All children found positive for S. mansoni were treated with praziquantel following standard protocols. Details of recruitment and participation of human subjects for fecal collection are described in Mutuku et al. [We enrolled human subjects who provided fecal samples containing u et al. . This prBiomphalaria pfeifferi used in Illumina sequencing were collected from Kasabong stream in Asembo Village, Nyanza Province, western Kenya  in November 2013. Snails were transferred to our field lab at The Centre for Global Health Research (CGHR) at Kisian, western Kenya. Snails sized 6-9mm in shell diameter were placed into 24-well culture plates and exposed to natural light to check for the shedding of digenetic trematode cercariae, including cercariae of S. mansoni [S. mansoni cercariae and non-shedding snails (controls) were separated and held for one day in aerated aquaria containing dechlorinated tap water and boiled leaf lettuce. After cleaning shells with 70% EtOH, whole shedding and control snails were placed individually into 1.5ml tubes with 1ml of TRIzol  and stored at -80°C until extraction.Snails shedding Biomphalaria pfeifferi confirmed to be uninfected were exposed to S. mansoni using standard methods to hatch the parasite eggs [ite eggs . Snails B. pfeifferi obtained from two 454 GS FLX  runs and six Illumina-sequenced B. pfeifferi exposed to molluscicide, all field-derived from Kenya . For each biological treatment , total RNA was purified separately from three individual snails  using the TRIzol protocol provided by the manufacturer . RNA samples were further purified using the PureLink RNA Mini Kit . Genomic DNA contamination was removed with RNase-free DNase I  at 37°C for 10 minutes. This combination method based on the two RNA extraction assays had been developed in our lab and proved to produce a high quality of RNA from snail samples . RNA quaComplementary DNA (cDNA) synthesis and Illumina Hi-Seq sequencing was performed at the National Center for Genome Resources (NCGR) in Santa Fe, NM. Most liquid handling was performed by a Sciclone G3 Automated Liquid Handling Workstation  with Multi TEC Control . Synthesis of cDNA and library preparation was prepared using Illumina TruSeq protocol according to the manufacturer’s instructions . Complementary DNA libraries were paired-end sequenced (2x 50 base reads) on a HiSeq2000 instrument .http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).Sequencing adapters, nucleotides with a Phred quality score <20 within a sliding window of 4bp, and non-complex reads were removed using Trimmomatic v.0.3 . Raw reaS. mansoni genome (GeneDB: S. mansoni v5.0) using STAR v.2.5 2-pass method [S. mansoni. However, the reads contributing to the positive percentage values for the controls are ones that we have found to be shared with either B. glabrata or another organism such that they represent a background level of sequence similarity obtained by chance. Although partial mapping of reads may occur, none appear to be expressed S. mansoni transcripts. None of the unexposed control reads mapping to the S. mansoni genome are unequivocally S. mansoni. By contrast, S. mansoni-exposed snails  all expressed bona fide S. mansoni genes. Only in 1d, 3d, and shedding snails were transcripts clearly distinctive to S. mansoni found, such as venom allergen proteins  , tegument allergen-like proteins (Accession: P14202), and cercarial stage-specific proteins (Accession: ABS87642.1), verifying the presence of a S. mansoni infection. This explanation also serves to verify that individual snails (such as 1dR2) with low S. mansoni percentages were indeed infected, such that they could be expected to be responsive to infection. Therefore, relatively low S. mansoni genome mapping, especially for shedding-R3, should not be interpreted that the infection was not successful, but rather as an indication of the transcriptional activity.To reduce assembly of chimeric transcripts, we created a novel pipeline to separate reads of related organisms when only one organism has a sequenced genome while also allowing for recovery of shared reads . First, s method  or Tophas method   using STAR. Reads that first mapped to S. mansoni and then also to B. glabrata were determined to be shared reads and added to the reads destined for B. pfeifferi transcriptome de novo assembly.Reads that mapped to S. mansoni genome. Pseudo-mate reads were created to allow maximum read usage in all stages of analysis (details and script available at https://github.com/lijingbu/RNA-Seq-Tools). This tool, pseudoFastqMate.pl, creates pseudo mate reads for single reads in a fastq file by generating a string of N’s the same length and quality score as its mate read. Reads entirely made up with Ns were ignored during the mapping process and have no impact on the final alignment and read counts.One issue encountered was to deal with both paired- and single-end reads resulting from initial quality filtering and from discordant or single-mate mapping to the S. mansoni, were assembled using Trinity v2.2 RNA-Seq de novo assembler [de novo and B. glabrata genome-guided assemblies were employed to maximize the chances of recovering unique B. pfeifferi transcripts. The de novo assemblies were concatenated and redundancy reduced using the EvidentialGene tr2aacds pipeline [In silico translation of the transcriptome was done using TransDecoder v3.0 (https://transdecoder.github.io) [Unaligned paired and unpaired reads, determined not to solely belong to ssembler ,67. Trinpipeline . Evidentthub.io)  to extraBiomphalaria pfeifferi CDS were annotated based on their closest homologs and predicted functional domains in the following databases and tools: BLASTp with NCBI non-redundant protein database , BLASTn with NCBI nucleotide database , Gene Ontology [B. pfeifferi CDS were further scrutinized against molluscan transcripts and proteins identified in the literature.Ontology , KEGG [7Ontology . For queB. pfeifferi and non-S. mansoni origin. Screening for the presence of third party symbionts was one of our motivations for investigating field-derived snails in the first place. We performed the de novo assembly pipeline without first removing non-snail or non-schistosome sequences to get a more complete view of the complex environment in which S. mansoni development takes place. CDS coverage, sequence identity, and E-value of BLASTn, BLASTp, and MEGABLAST results were all taken into consideration when determining organism identification. The BLASTn and MEGABLAST against the NCBI nucleotide database had minimum sequence identity of 70% and E-value <10−06 and the BLASTp against the NCBI protein database had a minimum sequence identity of 30% and E-value <10−06. Query coverage (qcov) was also calculated in all BLASTs. When different BLASTs disagreed in their taxonomic assignment, the hit with highest percent query coverage, highest sequence identity, and lowest E-value was chosen, in that order. Although minimum parameters were set, nearly all CDS BLAST hits exceeded these bounds. BLASTp hits tended to have better quality hits because nucleotide sequences from the NCBI nucleotide database often contained non-coding regions that our CDS lack. CDS designated as “undetermined” had hits that did not meet minimum BLAST parameters. CDS that had a non-molluscan BLAST hit but still mapped to the B. glabrata genome  were considered “shared” sequences.As a consequence of sequencing field-collected specimens, we expected some reads to be of non-B. pfeifferi and non-S. mansoni CDS were categorized into 14 broad taxonomic groups: Mollusca, Amoebozoa, SAR, Viruses, Plantae, Fungi, Bacteria, Rotifera, Platyhelminthes, Arthropoda, Annelida, Nematoda, Chordata, and Miscellaneous. Potential trematode CDS were further filtered to require a minimum of 70% query coverage. Genomes and CDS of specific symbionts of interest (if publicly available) were interrogated using BLASTn .Non-Biomphalaria immunobiology have focused on molecules with TLR or immunoglobulin domains, we undertook an analysis of these groups of molecules. Biomphalaria pfeifferi CDS with a BLASTp or BLASTn annotation as a toll-like receptor (TLR), were further screened for toll/interleukin-1 receptor (TIR), leucine-rich repeats (LRR), and transmembrane regions with InterProScan5 and TMHMM (Transmembrane helix prediction based on hidden Markov model) [Given that a number of previous studies of v model) . CDS ideB. pfeifferi transcriptome assembly and assess similar transcripts across related species, B. pfeifferi predicted ORFs were compared to other molluscan peptides  using BLASTp . ORFs with 100 or more amino acids were extracted from each transcriptome. To maximize sensitivity for retaining ORFs that may have functional significance, predicted ORFs were scanned for homology to known proteins in the Uniref90 database with a subsequent search using PFAM and hmmer3 to identify protein domains.To estimate the completeness of our S. mansoni were mapped to EvidentialGene-generated B. pfeifferi CDS with Bowtie2 [Properly paired reads not filtered as  Bowtie2 . Read ab Bowtie2 . Pairwis Bowtie2 . TranscrS. mansoni read counts (suggestive of less extensive parasite activity and/or more effective host limitation of parasite development). We hypothesized that the snail response is influenced by the extent of S. mansoni representation, as assessed by examining normalized parasite read counts from each infected snail. In addition to doing “3 controls vs. 3 infected” (3v3) comparisons, for each time point we also examined “3 control vs. 2 infected” (3v2) comparisons where the two snails harbored higher S. mansoni read counts to identify CDS whose responses were associated with S. mansoni abundance. We also performed “3 control vs. 1 infected” (3v1) comparisons where the one infected snail was the one with low S. mansoni read counts. The overall DE results include all CDS that were differentially expressed in any of the three comparisons, the results for each comparison being separately singled out and enumerated.As noted above, field-collected specimens of both snails and schistosomes are naturally more genetically diverse than lab-reared counterparts, so variation in response among infected snails might be expected. In fact, by chance, for each of the time points studied, one of the 3 infected snails examined differed notably from the other two in having fewer normalized cDNA was synthesized from 5μg of total RNA from the original samples by the SuperScript II First-Strand Synthesis Kit for RT-PCR (Invitrogen) in a 20μl reaction using random hexamers. Manufacturer directions were followed for the reaction profile. An additional 80μl of molecular grade water was added to the cDNA for a final volume of 100μl. qPCR target primer sequences were generated in Primer3 software  and detaB. pfeifferi following infection with S. mansoni, we analyzed the transcriptome from Illumina sequencing of infected snails at 1-day (1d), 3-day (3d), and from shedding snails using three biological replicates each  that includes isoforms. From nucleotide sequence length histograms, we calculated that more than half of the CDS were between 300–499 nucleotides with 6.7% > = 1500 nucleotides  genes, 27 of which encode complete TLRs [B. pfeifferi transcriptome had 190 CDS annotated as a homolog to a B. glabrata TLR  domain and 118 have at least one LRR (leucine-rich repeat) domain. In total, we found 48 complete B. pfeifferi TLRs  and 142 partial homologs to B. glabrata TLRs  in our transcriptional study. Others may certainly exist in the genome of B. pfeifferi.Pattern recognition receptors like TLRs and VIgLs  are key components of the innate immune response and their involvement in the cumented ,77. The ete TLRs . Our B. rata TLR . Note thB. glabrata genome [B. pfeifferi transcriptome, at least in part. Our BLAST annotations identified 249 B. pfeifferi CDS homologous to B. glabrata FREPs and 12 of these were verified to be full-length FREP homologs  polypeptides shared between the two species   . We founthan 90% . To furtgigantea ), two bia fucata ) and Craea gigas ), and onculoides )  of these were determined to be of non-mollusc origin   ; S2 Filede novo assembly, there are some familiar snail symbionts listed in Chaetogaster annelids, 2) Trichodina ciliates, and 3) Capsaspora owczarzaki [Among the eukaryotic sequences retrieved from generation of the czarzaki  and 4) mczarzaki –89  that uses B. pfeifferi from East Africa as a first intermediate host [Lastly, even after the initial screening and removal of n origin . Two indth reads . We sequphalaria . Most ofate host . Like coS. mansoni in the dual transcriptome as measured by read counts is variable among the three replicates for each development time sampled in shedding snails  replicates isolating either the two snails that contained higher S. mansoni read counts  or the one snail with the fewest S. mansoni read counts of each triplicate time point (3v1). With respect to the overall response patterns of snails that yielded either high or low numbers of S. mansoni reads, in most cases, for both up- and down-regulated CDS, the majority of significantly differentially expressed CDS fell into the 3v3 comparison category  clearly showed an additional allotment of down-regulated features. For the other two time points, the snails with high and low S. mansoni read counts did separate from one another, and especially noteworthy is the relatively small proportion of down-regulated features in the 3v1 comparisons.The extent of representation of g snails . Normalicategory , indicatB. pfeifferi. Multidimensional scaling (MDS) plots show that for each of the three groups of infected snails, overall transcript expression of the experimental groups is distinct from the control groups , proportionately more features were up-regulated than for snails with high S. mansoni read counts. In two out of three comparisons, snails with low read counts for S. mansoni had fewer down-regulated genes than snails with high levels of S. mansoni read counts.With respect to transcripts involved in reproduction and potentially associated with glabrata . We alsoglabrata ; Fig 10.Quantitative RT-PCR (qPCR) was used to validate differential expression trends by quantifying mRNA transcripts of four single-copy genes (3 up-regulated and 1 down-regulated) that highlight varying expression patterns in 1d, 3d, and shedding snails. Overall expression patterns are similar between the qPCR and Illumina DE results  with theThis paper represents a novel pipeline for dual RNA-Seq studies where the genome of just one of the interacting partners, the parasite in this case, is available. It also highlights an advantage of using field specimens in RNA-Seq studies to reinforce the notion that individual snails are actually holobionts, and the symbiont species they carry with them may play a role in influencing susceptibility to schistosome infection or in modulating disease transmission. Also, variance among the individual snails within the groups examined presented challenges for traditional bioinformatics analyses but also revealed the heterogeneity that realistically exists among naturally diverse snails and schistosomes as they encounter one another in real-life settings in the field. We must also note that the identity and functional role for many of the CDS remain unknown thus posing rich opportunities for study for the future.B. pfeifferi-S. mansoni system studied here is noteworthy for the high degree of susceptibility shown by the snail to infection [S. mansoni is characteristic of B. pfeifferi throughout its range [S. mansoni or known to be shedding S. mansoni cercariae contained transcripts contributed by S. mansoni. The extent of representation of S. mansoni in the dual transcriptome is variable among the replicates for each time sampled  is suggestive of an association. Neorickettsia has been found from non-human schistosomes [Neorickettsia in human-infecting schistosomes. For example, the Neorickettsia might be associated with metacercariae of other digeneans that are commonly found encysted in B. pfeifferi from natural habitats.The recovery of a few sequences of the digenean-inhabiting stosomes  but furtChaetogaster annelids which mostly colonize the external soft surfaces of freshwater snails and are known to ingest digenean miracidia and cercariae [Trichodina ciliates known to live on the soft surfaces of snails but with poorly characterized influence on their snail hosts [Capsaspora owczarzaki, a Filasterean amoeba-like symbiont known from Biomphalaria glabrata [B. pfeifferi considering microsporidians are known from both Biomphalaria and Bulinus [Richtersius coronifer and two from the Illumina de novo assembly similar to Ramazzottius varieornatus. Control-R1 had read counts >10 for the two R. coronifer CDS and 1d-R3 had read counts >10 for a R. varieornatus CDS. It is not unprecedented to find tardigrades associated with snails. Fox and García-Moll [Echiniscus molluscorum in the feces of land snails from Puerto Rico. Although the tardigrade may have been ingested along with food, the authors did not rule out the possibility that E. molluscorum may be a symbiont of the snail.With respect to eukaryotes, CDS representing the following groups were recovered: 1) ercariae –105; 2) il hosts ; 3) Capsglabrata ,108; 4)  Bulinus ; 5) Perk Bulinus ,111; 6) cía-Moll  identifiRibeiroia, representatives of which occur in East Africa and are known to infect Biomphalaria there [Ribeiroia sporocysts and/or rediae, though the extent of this infection must have been minimal as the transcriptomics response of this snail was not unusual compared to the other control snails. It may also have been infected with Ribeiroia metacercariae which are most familiarly known to infect amphibians or fish [Biomphalaria spp. from Kenya . The other snail, 3d-R2, yielded confirmed amphistome sequences, probably from the commonly recovered species Calicophoron sukari [S. mansoni and an amphistome, reflective of real-life circumstances in the habitat of origin where this amphistome species is the most common digenean to infect B. pfeifferi [S. mansoni reads recovered from this snail relative to 3d-R1 and 3d-R3. It has also been noted that B. pfeifferi ingests amphistome metacercariae  which are abundant on the submerged vegetation in the habitat from which the snail was collected, so this may be an alternative explanation for the presence of amphistome reads in 3d-R2. The peculiar nature of infection in this snail further justifies our rationale for including it in the separate analyses (3v1) described in the results.It was not surprising that two of our snails yielded several reads mapping to sequences from other digeneans. The first, control-R3, returned sequences consistent with ia there . It seem or fish ,114, butn sukari , so it mfeifferi . This coB. pfeifferi were up-regulated .S. mansoni reads recovered. However, at 1d and 3d, snails with fewer S. mansoni reads had higher proportions of up-regulated features than did snails with higher numbers of S. mansoni reads. Furthermore, for both 3d and shedding snails, snails with low S. mansoni read counts had smaller proportions of down-regulated features. These patterns are suggestive that up-regulated host responses might limit S. mansoni gene expression and that snails with less parasite gene expression may be less vulnerable to gene down-regulation, but care in interpretation is required as alternative explanations may exist. For example, as noted above, replicate 3d-R2 also contained an amphistome infection. Negative interactions among the two digeneans which are known to occur from experimental studies  may account for the limited number of S. mansoni reads.Many host CDS responded uniformly across individual snails regardless of the number of B. glabrata snails following exposure to molluscicides [B. glabrata snails elaborated more complex up-regulated responses following exposure to molluscicides [B. pfeifferi did following exposure to S. mansoni. Complex patterns in stress response gene families were also noted for 3d and shedding snails. It is noteworthy that exposure to S. mansoni, a specific extrinsic biotic stressor, also provokes components of a generalized stress response in B. pfeifferi and B. glabrata [At 1d, up-regulated responses, as exemplified by CDS for phospholipases, endoglucanases, and several proteases and protease inhibitors, were usually less pronounced than at 3d, suggesting it takes a few days to mobilize responses. Notable at 1d were down-regulation of CDS that might lower hemoglobin levels, and influence feeding behavior and heart beat rate. Infected snails exhibited complex mixed responses with respect to mucins, multidrug resistance proteins, glutathione-S-transferases and cytochrome P450 family members. Cytochrome P450s are part of the stress response shown by scicides  and to bscicides . For heascicides  than B. glabrata ,116.et al. [Snails with 3 day infections had the highest number of up-regulated CDS. Some of the features down-regulated at 1d were again down at 3d. Additionally, one CDS (aryl hydrocarbon receptor) associated with controlling circadian rhythm  was downet al. , are invet al. .B. glabrata exposed to S. mansoni [The overall responses of shedding snails were surprising in not being more dramatically altered relative to controls than they were. This is because snails with more advanced schistosome infections (28+ day infections) experience several noteworthy physiological changes, including altered feeding behavior, decreased locomotory activity, increased heartbeat rate –121,123  mansoni .Down-regulated levels of features potentially helping to explain reduced growth rates ,126, redB. pfeifferi infected with S. mansoni, suffer parasitic castration, marked by a sharp or complete reduction in production of eggs [B. pfeifferi, egg-laying begins to decline 7–10 days following exposure to S. mansoni and is complete in most snails by 14 days. The time course and extent of castration are influenced by the age of the snail at the time of exposure and by the dose of miracidia received [Snails infected with the proliferating larval stages of digenetic trematodes, including  of eggs ,125,130.received . In somereceived .B. pfeifferi  involved in neuro-endocrine control of reproduction ,133. We feifferi ; Fig 9 ag-laying . Furtherglabrata  and the ma duryi .Lymnaea stagnalis in inhibiting hormones involved in stimulating egg-laying or the albumen gland [B. glabrata infected with S. mansoni [B. pfeifferi. Kynurenine 3-monooxygenase-like transcripts were up-regulated in all snails with 3 day infections. By degrading tryptophan, this enzyme may limit concentrations of serotonin.Although infections of 1 or 3 days duration are too young to manifest castrating effects, up-regulation of some features with possible inhibitory effect on reproduction were noted at these times. Several features were also down-regulated at 1 day, including ovipostatin 2, a type 1 serotonin receptor (relevant because of serotonin’s ability to stimulate egg-laying), and schistosomin. Schistosomin has been implicated in en gland . A role  mansoni  and we sAplysia [B. pfeifferi, especially given its preference for self-fertilization. We found evidence only for the expression of temptin, which was up-regulated at 3d, but otherwise was not differentially expressed. Likewise, only temptin was isolated in proteins released from B. glabrata [B. glabrata [It was of interest to learn if the water-borne pheromones  that favor aggregation in Aplysia  were expglabrata  and egg-glabrata . It has glabrata .S. mansoni-infected snails [S. mansoni reads, and thus may mark an early phase in initiation of castration by diverting tyrosine to production of melanin as opposed to dopamine. Transcription of enzymes involved in dopamine metabolism are strongly affected in S. mansoni-infected snails. Tyrosinase-1 is also discussed in the next section regarding its potential involvement in defense responses.Our results with shedding snails are most pertinent with respect to parasitic castration. Several reproduction-related neuropeptides, including caudal dorsal cell hormone, and neuropeptides associated with production of egg and egg mass fluids such as snail yolk ferritin (vitellogenin), galactogen synthesis, lipopolysaccharide binding protein/bacterial permeability-increasing proteins (LBP/BPI) or aplysianin/achacin-like protein  were notd snails . This maBiomphalaria (we found 6 different versions in B. pfeifferi), with ovipostatin 5 being the most prominent responder in shedding snails. In L. stagnalis, ovipostatin is passed in seminal fluid from one individual to another during mating and inhibits oviposition in the recipient [B. pfeifferi is predominantly a self-fertilizer [L. stagnalis [S. mansoni reads.There are numerous ovipostatins produced in ecipient . Althougrtilizer , oviposttagnalis  and thoutagnalis . Notablyet al. [B. glabrata, either from control snails or snails at 12 days post infection with S. mansoni. They noted many reproductive neuropeptides, such as egg laying hormone 2, at significantly reduced levels at 12d compared to controls. They also reported an increase in some neuropeptides including FMRFamide, luqin, NKY, and sCAP in infected snail CNS. Based on predicted protein interaction networks, Wang et al. [S. mansoni miracidia peptides so may be a key player in regulating parasite-induced changes in host physiology. A homolog to the B. glabrata LAP2 was present in our transcriptome but was not differentially expressed in any sample. When comparing our results to those of Wang et al. [B. pfeifferi, rather than B. glabrata. Our methods may bias against detection of changes in expression of potentially rare neuropeptide transcripts, but cast a wider net for potential downstream effects of castration, so provides a valuable complementary view to the approach taken by Wang et al. [Wang et al.  recentlyg et al.  suggesteg et al. , it shoug et al. .B. glabrata studies), ficolins [S. mansoni reads, up-regulated responses were observed for a number of additional immune features. Cu,Zn SOD is of particular interest because previous work has implicated high expression of certain alleles of Cu,Zn SOD with resistance to S. mansoni in the 13-16-R1 strain of B. glabrata, because of Cu,Zn SOD’s involvement in converting superoxide anion to schistosomicidal hydrogen peroxide [et al. [B. glabrata to either S. mansoni or E. paraensei.At 1d ; Fig 10,ficolins , and chificolins ,48. For peroxide –146. Our [et al.  who noteet al. [B. glabrata to S. mansoni or E. paraensei, so much so either might be considered as markers of infection. Although a FREP2 homolog was consistently up-regulated following exposure of B. pfeifferi to infection, a FREP4 homolog was not expressed in B. pfeifferi at any of the time points we examined.Hanington et al.  also fouS. mansoni reads were macrophage expressed gene-1, known to be up-regulated in both abalone following bacterial infection [B. glabrata in early exposure to S. mansoni [S. mansoni reads. Hemocytin, a homolog for an insect humoral lectin with a role in hemocyte nodule formation [S. mansoni-infected snails at all three time points, especially at 3d when it was increased over 8-fold in expression. For both 1 and 3d, hemocytin expression was highest in those snails with fewer S. mansoni reads. FREP3, previously implicated in resistance to S. mansoni in B. glabrata [B. pfeifferi system. It was modestly up-regulated only at 1d, in the snail with fewest S. mansoni reads.In contrast, among CDS more up-regulated in the 1 day infected snail with a low proportion of nfection  and from mansoni . Hemocytormation , was conglabrata , was minB. glabrata amebocyte-producing organs to fucoidan, a fucosyl-rich PAMP chosen to mimic the surface of S. mansoni sporocysts [B. glabrata exposed to S. mansoni [B. glabrata resistant to S. mansoni exhibits higher levels of a TLR on its immune cells, and exposure to S. mansoni significantly enhances their expression, whereas compatible snails show little response following exposure to infection [B. pfeifferi showed no conspicuously up-regulated TLR genes at 1d, and we found no B. pfeifferi TLR with strong homology to the TLR reported by Pila et al. [et al. [et al. [B. glabrata in early exposure to S. mansoni. Down-regulation of cytidine deaminase might therefore be associated with lower responsiveness to S. mansoni infections, particularly early in infection .Down-regulated immune features at 1d were relatively few but prominent among them were FREP12 and its precursors, toll-like receptor 8 and cytidine deaminase. FREP12 down-regulation has also been noted upon exposure of orocysts , and in  mansoni . A strainfection . Our B. a et al. , but the [et al.  associat [et al.  observedS. mansoni reads had high levels of several transcripts including for aplysianin-like proteins and FREP 5. Aplysianin, first described from Aplysia, is an L-amino oxidase that has tumoricidal and bactericidal effects [B. glabrata [B. glabrata [B. glabrata in response to successfully developing S. mansoni or Echinostoma paraensei [The responses of putative immune factors were most extensive in snails at 3d and this is not surprising as this is a critical stage in the early establishment of the parasite. Several CDS mentioned with respect to the 1d response were again noted at 3d. Snails with more  effects , and a dglabrata . Aplysiaglabrata . FREP 5 araensei .S. mansoni reads at 3d revealed a different group of up-regulated transcripts, with hemocytin again being prominent. Also notable were distinctive CDS potentially involved in hemocyte aggregation [B. glabrata to LPS [The snail with relatively few regation , FREP 7,a to LPS  and to ba to LPS . Down-reS. mansoni infection within B. pfeifferi  were found to be up-regulated in 1d and 3d (mostly up-regulated in 3d). The possible role of GIMAPs in immunity has not been realized in protostomes until it was shown that several GIMAPs were up-regulated in the amebocyte organ of  stimuli . This fipoptosis .Immune-related responses for shedding snails were surprising for being mostly up-regulated , with onS. mansoni reads had distinctly higher responses for aplysianin-A-like, beta-1,3-glucan binding protein-like, and FREPS 2, 7 and 14. By contrast, the snail with a low percentage of S. mansoni reads expressed higher levels of macrophage-expressed gene, chitinase-3-like-protein, a distinct CDS with a leucine rich repeat and immunoglobulin domain, and TLR 3.Among those up-regulated features, shedding snails with high levels of B. pfeifferi transcripts. Most did not show strong patterns of up- or down regulation in this compatible species following exposure to S. mansoni, but zinc metalloproteinase/disintegrin-like was down-regulated at 1d and zinc metalloproteinase nas-13- and -14-like showed some up-regulation in shedding snails. Probable serine carboxypeptidases (versions 1–5) revealed a mixed pattern of expression at 3d, but were mostly up-regulated, whereas probable serine carboxypeptidase CPVL was down-regulated in shedding snails. Granulin, a growth factor that drives the proliferation of immune cells was up-regulated at both 1d and 3d [Features highlighted in recent genetic linkage studies ,50,51 ind and 3d ,33.B. glabrata for which isolates or inbred lines are known that are resistant to S. mansoni, B. pfeifferi is a species typically discussed in the context of its high compatibility with many S. mansoni isolates. Although particular lineages of B. pfeifferi may certainly come to light that exhibit strong incompatibility, key factors that dictate compatibility might best be sought not among the putative immune factors that characterize the B. glabrata response, but among those genes that exhibit the strongest transcriptional responses, up or down, to S. mansoni exposure  to form a ceramide, an important lipid signaling molecule that has been reported in Crassostrea gigas [Although many B. pfeifferi CDS that were highly altered in their expression are unknowns and thus represent intriguing subjects for future research, some did have homologs in the database and could also represent outstanding future targets for manipulation to discourage ea gigas . A transea gigas . Howeverea gigas .B. pfeifferi, one that is probably responsible for transmission of more S. mansoni to people than any other Biomphalaria species. We have deliberately chosen to emphasize the study of field-derived B. pfeifferi and S. mansoni to provide a more realistic view of the context in which they live, and how they interact in the wild, including with third party symbionts. Our approach has revealed that the extent of S. mansoni transcriptional activity varies among snails and this is reflected in different transcriptional responses of the snails, suggestive of diverse trajectories in what is typically a highly compatible host-parasite model. We have highlighted several snail features warranting further study with respect to their roles in potentially supporting or enabling parasite development, that might limit the extent of development, and that might play a role in the diminished egg production typically shown by snails with shedding S. mansoni infections. Another generation of research exploiting the power of techniques like CRISPR-Cas, when it becomes available for snails, will enable further dissection of the functional role of these candidate molecules. A further challenge will then be to determine how the responses of compatible snails, or perhaps of the schistosome parasites within, can be exploited, ideally to prevent or suppress in a highly specific manner the development of schistosome parasites in snails.In conclusion, provided here is a de novo assembled transcriptional database based on over half a billion paired-end reads for an understudied schistosome vector, S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigB. pfeifferi transcriptome predicted by TransDecoder.Percentages next to organisms show the percent of proteins present in our (TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.


Aspergillus species is a very important subject. Mass spectral fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is generally employed for the rapid identification of fungal isolates. However, the results are based on simple mass spectral pattern-matching, with no peak assignment and no taxonomic input. We propose here a ribosomal subunit protein (RSP) typing technique using MALDI-TOF MS for the identification and discrimination of Aspergillus species. The results are concluded to be phylogenetic in that they reflect the molecular evolution of housekeeping RSPs.Accurate identification of Aspergillus species were first verified and compared to compile a reliable biomarker list for the identification of Aspergillus species. In this process, we revealed that many amino acid sequences of RSPs  registered in the public protein databases needed to be corrected or newly added. The verified RSPs were allocated to RSP types based on their mass. Peak assignments of RSPs of each sample strain as observed by MALDI-TOF MS were then performed to set RSP type profiles, which were then further processed by means of cluster analysis. The resulting dendrogram based on RSP types showed a relatively good concordance with the tree based on β-tubulin gene sequences. RSP typing was able to further discriminate the strains belonging to Aspergillus section Fumigati.The amino acid sequences of RSPs of genome-sequenced strains of Aspergillus species, even for species within section Fumigati. The discrimination power of RSP typing appears to be comparable to conventional β-tubulin gene analysis. This method would therefore be suitable for species identification and discrimination at the strain to species level. Because RSP typing can characterize the strains within section Fumigati, this method has potential as a powerful and reliable tool in the field of clinical microbiology.The RSP typing method could be applied to identify The online version of this article (doi:10.1186/s12866-017-1009-3) contains supplementary material, which is available to authorized users. Aspergillus is a saprophytic genus found in diverse environments [A. fumigatus, are causative agents of aspergillosis [Aspergillus species is a very important subject, especially in the field of clinical mycology.ronments . Some spgillosis . Becausegillosis , accuratA. fumigatus strains. Multi-locus sequence typing (MLST), focusing on seven types of gene fragments, has also been applied to characterize A. fumigatus strains [The identification of fungal species has up to now been based on the morphological characteristics of colonies and filaments as observed by microscopy . However strains . These DA. fumigatus isolates has been reported using this method [Aspergillus isolates at the species and strain level [(i) the general finding that the proteome is very dynamic in living cells and hence protein pattern expectedly could be subject to changes in response to growth conditions,(ii) doubts whether differences and similarities in mass spectral patterns are completely consistent with the established taxonomy,(iii) a lack of comprehensive databases covering all clinically relevant species.In the field of medical microbiology, much attention has been paid recently to the mass spectrometric technique of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOF MS) as a tool for the rapid identification of fungal isolates . MALDI–Ts method , 13. Sevin level –16. Howein level  has poinFurthermore, in the author’s opinion, the reported mass spectra of fungal samples sometimes show too few peaks when sample preparation is performed using the recommended protocol proposed by the manufacturers.+ ions) and their masses are distributed in the range of ca. 4 - 30 kDa, RSPs can be easily observed in MALDI mass spectra [To overcome these problems relating to mass spectral fingerprinting, we have proposed a method using ribosomal proteins as biomarkers for microorganism analysis by MALDI–TOF MS –25. Ribo spectra . We have spectra –25. The  spectra . Our proA. fumigatus strains as a model [in silico inspection by sequence homology analysis and MALDI–TOF MS measurements. Post-translational modifications such as acetylation and methylation could also be verified. In this way, the expressed masses of RSPs observed under 16,000 Da could finally be confirmed.The aim of our project is to extend the RSP-based method to the identification of eukaryotic fungi. As the first step, we have investigated the actual state of information of RSPs of fungi registered in public protein databases through the characterization of ribosomal protein fractions extracted from genome-sequenced  a model . In our  a model , we reveAspergillus species to establish biomarker references for the reliable identification of Aspergillus species. First, verification and correction of the amino acid sequences of RSPs and confirmation of post-translational modifications common to all sample strains were performed to accurately determine the expressed mass, as described in our previous paper [Aspergillus species. The selected RSPs of each strain were categorized into “RSP types” based on their mass and used to construct a dendrogram. The resulting dendrogram was compared with that arrived at using the DNA-based method; the reliability of the species identification and discrimination of this method was then assessed.As the next step, this paper describes the results of comparable characterization of RSPs of eleven us paper . RSPs wiA. fumigatus strains were verified to compile the reference mass list of expressed RSPs. We noted that more than half of the amino acid sequences in the public databases, such as UniProtKB [in silico inspection using sequence homology analysis and verification of actual expressed masses of RSPs by MALDI–TOF MS measurements. In this study, by applying this strategy, the amino acid sequences of RSPs of ten genome-sequenced strains of Aspergillus species were further verified and compared to build a reliable biomarker list for the identification of Aspergillus species. The sample strains used in this study are summarized in Table A. fumigatus strains. The genome sequence of A. viridinutans is not yet published, but the authors have annotated the RSP sequences manually. Finally, 26 kinds of RSPs whose molecular weights were under 16,000 Da were selected that are common to those already verified for A. fumigatus strains [A. fumigatus, for which the mass spectra were reported in ref. [A. fumigatus A1163 and Af293 have been reported in ref [In our previous paper , the aminiProtKB  and the niProtKB  were inc strains . Support in ref. ). Additi in ref.  to preved in ref ).Table 1A. viridinutans, annotated by the authors, was not added to this list). Depending on the species, about 10 - 60% RSPs needed to be corrected or newly added. The source of sequence errors in prokaryotic bacteria was chiefly due to misidentification of the start codon [Aspergillus fungi seemed to be due to misidentification of the exon/intron structure, resulting in incorrect CDS as well as an incorrect stop codon caused by frame shift. Because this type of error is unique to prokaryotes, a similar problem in the annotations of RSP genes might have occurred in other fungi.Table rt codon . The maiA. fumigatus [Aspergillus species. In addition, S27 showed a common +28 Da shift, suggesting two methylations. Although this modification has, to our knowledge, not been reported before, two methylations of S27 were concluded to be common modifications in Aspergillus species.The post-translational modifications were then confirmed by referring to already-reported modifications in eukaryotic RSPs. Details of the assignments of each modification are described in the Supporting Information . Interestingly, these five species form a cluster in the dendrograms based on both the RSP type  of common eight RSPs in section Fumigati are also shared with A. clavatus.lin Fig. . RSP typlin Fig.  also shoA. niger, A. kawachii, A. flavus, A. oryzae, and A. nidulans are distinct from the cluster of section Fumigati. The RSP types of these species show little in common with section Fumigati species: only L39 of A. oryzae and A. flavus was common. Of these species, pairs of ‘A. niger and A. kawachii’ and ‘A. flavus and A. oryzae’ are known to be very closely interrelated: A. flavus and A. oryzae are assumed to be ecotypes of the same species because they have only 350 unique genes, even when comparing the total genome sequences [A. niger and A. kawachii and also 100% of RSPs agreed between A. flavus and A. oryzae. The ratio of conformity between species thus appears to be reflected in the relatedness between species.The positions of equences . In the Aspergillus fungi [As discussed above, the dendrogram based on the RSP types shows relatively good concordance with the tree based on β-tubulin gene sequences Fig.  and withus fungi . This reA. fumigatus and other species within section Fumigati, accurate discrimination of these strains is very important [Fumigati, so misidentifications often occur. To overcome this problem, Yaguchi et al. have characterized the species within section Fumigati by molecular phylogenetic analysis using multiple genes [Fumigati. To reveal the effectiveness of this method, the strains of A. fumigatus, A. lentulus, A. viridinutans, A. felis, A. pseudoviridinutans, A. wyomingensis, A. udagawae, and N. fischeri belonging to section Fumigati were characterized.Because sensitivity to antifungal agents differs between mportant . Howeverle genes . On the Table The distribution of the RSP types within species tended to be consistent, and the variation was assumed to be small. This allowed the typing of sample strains to be conducted using the mass list of the genome-sequenced strain. Fig. A. lentulus strains completely match those of the type strain. Because A. lentulus was originally regarded as a sibling species of A. fumigatus, it has proved difficult to discriminate them morphologically [A. fumigatus, which can be used for discrimination between A. fumigatus and A. lentulus. Interestingly, N. fischeri is very close to the A. lentulus cluster, in which only L42 was different from that of A. lentulus. This species is close to the A. fumigatus cluster in the case of the β-tubulin gene as shown in Fig. The RSP types of ogically . Severalogically , 36; howA. udagawae strains form a relatively clear-cut cluster. Although some RSP peaks were not detected clearly, moderate concordance with the reference mass of the type strain was confirmed. Five RSPs  are further matched completely to those of A. fumigatus, whereas three RSPs  are totally different from the other species. These RSPs can therefore be used to discriminate A. udagawae from other species. The three strains  were formerly reported as variant isolates of A. fumigatus (or Aspergillus sp.) but were re-identified, using multiple genes , as A. udagawae [A. udagawae cluster, supporting this re-identification.RSP typing has demonstrated that udagawae . In RSP A. lentulus and A. udagawae, former A. viridinutans strains  do not form an obvious cluster. Interestingly, the type strain of A. viridinutans (IFM 47045T) is located separately from other former A. viridinutans strains. The diversity of A. viridinutans has already been reported, and this species was divided into some species [A. pseudoviridinutans, A. felis and A. wyomingensis, respectively. In each species, the high conformity of RSPs was indicated.In contrast to the high conformity of RSPs in  species –39. The Fumigati, the reliability of identification results increases on increasing the numbers of evaluation points . This gives MALDI-TOF MS great potential, since many proteomic peaks that assist with the identification of fungal species are processed. Our proposed RSP typing represents the next generation of mass spectral identification/discrimination of fungal strains, in that it supersedes current mass spectral fingerprinting, which is simple pattern-matching without peak assignment.In the context of the ongoing conversion, on the basis of fungal taxonomy, from morphology to molecular phylogeny, molecular biological methods have been adopted for the identification and discrimination of fungal strains. To avoid misidentification of closely-related species, especially within section Aspergillus RSPs. The sequences and expressed mass of RSPs of other fungal species can now be easily verified and corrected by homology analysis using the sequence list summarized in Supporting Information Additonal file 2: Table SI-2.The merits of RSP typing are (1) it requires no commercial database and (2) it can be used to perform phylogenetic analysis. As for the first point, RSP typing requires reference to the RSP biomarker lists, via the internet, constructed from the public protein databases. At this time, of course, commercial mass spectral databases are more substantial than available RSP information. However, as whole genome-sequencing of fungal species progresses, information on RSPs is expected to expand exponentially in the near future. Although we initially encountered a confused situation as concerns the protein information registered on the public protein databases, we have successfully corrected the errors in the amino acid sequences and the names of representative Fumigati, this method is potentially a powerful and reliable tool in the field of clinical microbiology.The second benefit of this method is valuable, because the identification results have a phylogenetic rationale: they relate to a combination of more than a dozen housekeeping proteins. This method can eliminate the influence of growth and experimental conditions, if only the RSP peaks are observed. RSPs are one of the most expressed proteins, and RSP fractions are easily collected by cell-grinding and ultracentrifugation. The discriminatory power of the RSP typing appears to be comparable with the conventional β-tubulin gene analysis. This method would therefore be suitable for species identification and discrimination at strain to species level. Because RSP typing can characterize the strains within section The strains of the 14 species used in this study are summarized in Table +, m/z 2932.6) and myoglobin  as references.Sample preparation, apparatus, and MALDI–TOF MS data acquisition methods were similar to those described in our previous papers –25. The A. viridinutans IFM 54703T, sequenced by Chiba University’s Medical Mycology Research Center, was not registered on the public databases at this time, in-house draft genome sequence data were used. The annotated RSP sequences of A. viridinutans have been deposited at DDBJ under the accession numbers LC213039-LC213063. The amino acid sequences of L39 and S21 of A. niger were not determined from shotgun sequences in the database. Therefore, these sequences were determined and have been deposited at DDBJ under the accession numbers LC255002 and LC215003. The sequence mass of each RSP was predicted using a Compute pI/Mw tool on the ExPASy proteomics server [+ ion. Detailed construction procedure of the ribosomal protein biomarker list together with peak assignments are shown in Supporting Information Additional file The amino acid sequence of each RSP was obtained from UniProtKB . Becauses server . After ts server  .The partial DNA sequence of β-tubulin was obtained from the UniProtKB and the alignment of the sequences was performed using ClustalW  softwareAdditional file 1: Table SI-1.The accession number (TrEMBL) of ribosomal protein biomarkers of genome-sequenced strains used in this study. (DOCX 22 kb)Additional file 2: Table SI-2.Corrected amino acid sequences and relating information of RSPs. (DOCX 80 kb)Additional file 3:Figure SI-1. Mass spectra of RSPs of N. fischeri NRRL 181T. Figure SI-2. Mass spectra of RSPs of A. lentulus IFM 54703T. Figure SI-3. Mass spectra of RSPs of A. viridinutans IFM 47045T. Figure SI-4. Mass spectra of RSPs of A. udagawae IFM 46973T. Figure SI-5. Mass spectra of RSPs of A. clavatus NRRL 1NT. Figure SI-6. Mass spectra of RSPs of A. niger CBS 513.88. Figure SI-7. Mass spectra of RSPs of A. kawachii IFO 4308. Figure SI-8. Mass spectra of RSPs of A. flavus NRRL 3357. Figure SI-9. Mass spectra of RSPs of A. oryzae RIB 40. Figure SI-10. Mass spectra of RSPs of A. nidulans FGSC A4. (PPTX 472 kb)Mass spectra of genome sequenced sample strains used in this study. Additional file 4:Figure SI-11. Peak shift (+42 Da) of S24 with acetylation. (a) A. fumigatus A1163, (b) A. viridinutans IFM 47045T, (c) A. clavatus NRRL 1NT, (d) A. niger CBS 513.88, (e) A. flavus NRRL 3357, and (f) A. nidulans FGSC A4. Figure SI-12. Methylation of L42. (1) Peak shift of +14 Da from sequence mass in L42 of the Aspergillus species; (a) A. fumigatus A1163, (b) N. fischeri NRRL 181T, (c) A. lentulus IFM 54703T, (d) A. viridinutans IFM 47045T, (e) A. udagawae IFM 46973T, (f) A. clavatus NRRL 1NT, (g) A. niger CBS 513.88, (h) A. flavus NRRL 3357, and (i) A. nidulans FGSC A4. (2) Amino acid sequences around Lys-55. Figure SI-13. Dihydroxylation of S23. (1) Peak shift of +32 Da from sequence mass in L23 of Aspergillus species; (a) A. fumigatus A1163, (b) N. fischeri NRRL 181T, (c) A. niger CBS 513.88, (d) A. flavus NRRL 3357, and (e) A. nidulans FGSC A4. (2) Amino acid sequences around Pro-64. Figure SI-14. Peak shift (+28 Da) of S27 with dimethylation. (a) A. fumigatus A1163, (b) A. niger CBS 513.88, (c) A. flavus NRRL 3357, and (d) A. nidulans FGSC A4. (DOCX 187 kb)Post-translational modifications. Additional file 5: Figure SI-15.Figure SI-16. Detailed construction procedure of the ribosomal protein biomarker list together with peak assignments. (PPTX 61 kb)Detailed experimental protocols. Detailed sample preparation procedures.


Neural circuits that control copulation in male flies have been identified. Related research article Pavlou HJ, Lin AC, Neville MC, Nojima T, Diao F, Chen BE, White BH, Goodwin SF. 2016. Neural circuitry coordinating male copulation. eLife5:e20713. doi: 10.7554/eLife.20713raison d’etre of life on Earth, and sex in the animal kingdom can take many forms. Some organisms – including worms and snails – are hermaphroditic and can self-fertilise to produce offspring .A number of outstanding questions remain. For example, does an equivalent circuit control the genitalia of female flies during copulation? Moreover, how do the results of Pavlou et al. link to recent studies that explored the neural circuitry controlling the persistence and motivation of male flies during copulation ? And how


Gymnocypris przewalskii is an ideal model system to study highland adaptation of fish, because it evolved specific genetic and phenotypic characteristics to adapt to chronic cold and alkaline environments in Lake Qinghai. MicroRNAs (miRNAs) are small noncoding RNAs that regulating gene expression post-transcriptionally in a wide range of biological processes. In this study, we focus on the role of miRNAs in adaptation of G. przewalskii to extreme conditions in Lake Qinghai. We generate the first miRNAome of G. przewalskii in Schizothoracinae fish. Using several genomic resources, we inferred 341 conserved miRNAs belonged to 152 miRNA families and 43 novel miRNAs in G. przewalskii, and also identified 15 teleost-specific miRNAs. Using a large scale of conserved miRNAs, we constructed a high-confidence phylogenetic tree between teleost and mammals than mitochondria and nuclear genes. In addition, we found that several miRNA family (e.g. miR-10 and let-7) members highly expressed in G. przewalskii, which may function in multiple biological processes. Finally, we predicted a total of 34,258 miRNA targets genes. Conserved miRNAs target genes participating in signal transduction, cell differentiation and biosynthetic process, and showed signature of functional constraint. While novel miRNAs in a species displayed species-specific targets and involved in ion binding, transport and oxidoreductase activity, may affect the expression patterns of targets with signature of gene family expansion or positive selection under extreme environment. Taken together, this study demonstrated that miRNAs may involve into roles of adaptation of G. przewalskii to highland aquatic environment, and also provide insights into miRNA regulatory network in Schizothoracinae fish as a case study.Tibetan naked carp  MicroRNAs (miRNAs) are a family of endogenous RNAs that regulated gene expression pattern at post-transcriptional level. The miRNAs are usually 22 nucleotides (nt) long with a seed region (2–8 nt) that binds to the 3’ untranslated region (UTR) of a targeted mRNA and repressed the protein synthesis (plant: mRNA degradation). The miRNA have diverse functions, including developmental, metabolic and physiological processes ,2. RecenGymnocypris przewalskii (Teleostei: Cyprinidae) is one of the best-studied Schizothoracinae fish species in TP, making it an ideal model to study evolutionary biology. Many studies have revealed that G. przewalskii provides many key insights into genetic basis of speciation and adaptation [G. przewalskii survives in both its natural habitant Lake Qinghai  and connective rivers of freshwater environment in the spawning migration. Besides high PH aquatic environment, G. przewalskii had evolved to surmount chronic cold and hypoxia environment in Lake Qinghai [G. przewalskii is considered as an exceptional model to investigate the genetic mechanisms of aquatic wildlife adaptation to extreme environments in the TP.Schizothoracinae fishes are the predominant fish fauna in the Tibetan Plateau (TP), which have more than 60 species widely distributed throughout TP altitudes ,9. Theseaptation –16. Unli Qinghai ,17,18. WG. przewalskii [G. przewalskii to extreme aquatic environment in Lake Qinghai. Past evidences revealed that miRNAs could contribute to speciation and environmental adaptation in fish species [G. przewalskii and multiple bioinformatics analyses to uncover function of miRNAs in high altitude adaptation.Recent studies have identified key genes contributing to speciation and adaptation of ewalskii ,16,19, b species ,20. TherG. przewalskii that were collected in Lake Qinghai . At 192 hour post fertilization (hpf), all the larvae of G. przewalskii were hatched out. Four G. przewalskii individuals (four replicates) were collected and then anaesthetized with MS-222 , finally frozen in liquid nitrogen prior to total RNA extraction for RNA library preparation followed by small RNA sequencing.This study was based on wild-caught https://www.ncbi.nlm.nih.gov/sra).Total RNAs were isolated using TRIzol  and then assessed the quality and quantity of each RNA sample with an Agilent 2100 bioanalyzer . Only RNA samples with high purity (OD260/280 between 1.8 and 2.2) and high integrity  were used to construct the small RNA library [http://blast.ncbi.nlm.nih.gov/) and Rfam database (http://www.sanger.ac.uk/software/Rfam). The number and frequency of total and unique sequences of miRNA, tRNA, tRNA, rRNA, snoRNA and intro were also respectively calculated. Only the miRNAs in unique sequence were considered as candidates for miRNA annotation. Therefore, the putative miRNAs ranging from 15 to 30 nt were searched against known precursor/mature miRNAs in miRBase release 20 database [Danio rerio, Oryzias latipes, Tetraodon nigroviridis, Pan troglodytes, Bos taurus, Gallus gallus, Homo sapiens, Rattus norvegicus) to identify conserved miRNAs. Considering the difference among species, alignments of the putative miRNAs to miRNA precursor/mature miRNA of above selected animal species in miRBase allowed for no more than three mismatches.Small RNA-seq raw reads were treated by following procedures: (1) trimming contaminating  tags and small tags); (2) removing poor-quality reads and resulting in clean read ; (3) reads with same sequence were named unique sequence. Numbers and lengths of total and unique sequences were calculated, respectively. Then all the sequences were annotated by searching against NCBI Genbank noncoding (Nr) database  was used to predict putative novel miRNA precursors against the zebrafish genome with the following criteria: (1) candidates are unannotated sequences which match the reference genome, aligning to intronic regions or to antisense exon regions; (2) sequences and structures (stem-loop structures) of the genes which satisfy the criteria of hairpin miRNA formation, and that the mature miRNAs are present in one arm of hairpin precursors; (3) mature miRNA strand and its complementary strand (miRNA*) should contain 2-nt 3’-overhangs; (4) hairpin precursors do not contain large internal loops or bulges; (5) secondary structures of hairpins have minimum free energy (MFE) less than −18 kcal/mol [G. przewalskii. Finally, we analyze and compared the bias of each nucleotide position and the first nucleotide of above identified conserved and novel miRNAs in G. przewalskii.A draft genome assembly of kcal/mol ; (6) theG. przewalskii and other 8 animal species, and was finally illustrated by heatmap with ggplot2 package in R software [After aligning miRNAs and their precursors to above 8 animal species miRNAs deposited in miRBase using Clustal X and MEGA 5.0 ,25, onlysoftware .G. przewalskii and other 8 animal species were treated as Dollo type characters and assigned equal weight [et al. [All identified conserved miRNA families of l weight . Then, pl weight  implemenl weight . Bayesial weight  with par [et al. .G. przewalskii according to previous method [To evaluate and calculate miRNA abundance in each replicate libraries, small RNA reads were mapped into identified conserved miRNA of s method . FirstlyIn silico analysis of miRNA target was predicted in published mRNA resources [www.targetscan.org), based on the complementary region between miRNAs and mRNAs and the thermodynamic stability of miRNA-mRNA duplex. All the miRNA target mRNAs were calculated and clustered by Gene Ontology (GO) terms using Blast2GO software [esources ,14 usingsoftware .Total 60,037,248 raw reads and 47,030,422 (78.34%) clean reads were generated by small RNA sequencing . Then clG. przewalskii and other 8 animal species . We suggested that this may because the incomplete annotation of teleost genomes. Moreover, we identified 15 teleost-specific miRNAs, in consistent with recent studies [We found high miRNA sequence similarity between  animals . The nucver carp ,38. The ver carp ,35,38,39 teleost ,38,39. H studies ,41. For The miRNA showed higher level of conservation than protein-coding sequence, which had been confirmed by previous studies ,31,40,42G. przewalskii, therefore, these highly expressed miR-10 family members may play important roles in sex differentiation at early stage. This finding was also consisted of previous study in development of tilapia gonads [G. przewalskii. We also found that let-7 family had the largest number of family members in G. przewalskii. Past evidences revealed that let-7 have diverse functions involved into different biological processes, including cell proliferation, development, growth and immune [G. przewalskii. Furthermore, we also identified a number of species-specific miRNAs highly expressed in G. przewalskii. To date, functions of many specific miRNAs are still not well-understood. Therefore, whether the functions of species-specific miRNAs are related to organism adaptation to specific environments, these issues remain as interesting questions for further confirm.In general, abundant miRNAs play fundamental and broader regulatory functions ,40,45. Wd immune . These fG. przewalskii [G. pachycheilus and G. przewalskii [G. przewalskii inhabited Lake Qinghai in an extremely saline and alkaline, hypoxic and chronic cold environment, its gene regulatory network may adapt these different ecological niches during long-term evolution. We speculated that these novel miRNAs may affect the expression patterns of these genes with signature of gene family expansion or positive selection, and be potential drivers of ultimately speciation of G. przewalskii. However, this hypothesis needs to be further confirmed by comprehensive verification works in future.Most of the novel miRNAs in a species displayed species-specific targets ,45. In tewalskii . Anotherewalskii ,49. The ewalskii . As the G. przewalskii would provide insights for future integrated studies focusing on determining potential roles of miRNAs in different ecological niches adaptation and speciation in Schizothoracinae fish on the Tibetan Plateau.In summary, this case study in S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.