Rickettsia felis  is the causative agent of an emerging flea-borne rickettsiosis with worldwide occurrence. Originally described from the cat flea, Ctenocephalides felis, recent reports have identified R. felis from other flea species, as well as other insects and ticks. This diverse host range for R. felis may indicate an underlying genetic variability associated with host-specific strains. Accordingly, to determine a potential genetic basis for host specialization, we sequenced the genome of R. felis str. LSU-Lb, which is an obligate mutualist of the parthenogenic booklouse Liposcelis bostrychophila (Insecta: Psocoptera). We also sequenced the genome of R. felis str. LSU, the second genome sequence for cat flea-associated strains , which are presumably facultative parasites of fleas. Phylogenomics analysis revealed R. felis str. LSU-Lb diverged from the flea-associated strains. Unexpectedly, R. felis str. LSU was found to be divergent from R. felis str. URRWXCal2, despite sharing similar hosts. Although all three R. felis genomes contain the pRF plasmid, R. felis str. LSU-Lb carries an additional unique plasmid, pLbaR (plasmid of L. bostrychophila associated Rickettsia), nearly half of which encodes a unique 23-gene integrative conjugative element. Remarkably, pLbaR also encodes a repeats-in-toxin-like type I secretion system and associated toxin, heretofore unknown from other Rickettsiales genomes, which likely originated from lateral gene transfer with another obligate intracellular parasite of arthropods, Cardinium (Bacteroidetes). Collectively, our study reveals unexpected genomic diversity across three R. felis strains and identifies several diversifying factors that differentiate facultative parasites of fleas from obligate mutualists of booklice. Alphaproteobacteria, all known species of Rickettsiales  are Gram-negative obligate intracellular bacteria associated with a wide range of eukaryotes (Rickettsia (Rickettsiaceae) is an extraordinarily diverse assemblage of species known from plants, amoebae, ciliates, arthropods, annelids, leeches, and vertebrates  . Accordingly, the potential exists that genetic diversity between R. felis isolates contributes to the observed variation in host fitness effects as well as the divergent lifecycles of these bacteria.Among the more recently recognized emerging rickettsial agents is kettsiae  and the kettsiae . Since ies felis , R. felipathogen . Althoug genomes , little coptera) . In its varially . Clearanvarially  and adulvarially  results ed fleas . Thus, RR. felis host specialization, we sequenced the genomes of two strains: 1) R. felis (str. LSU-Lb), which is 100% present within an all-female L. bostrychophila colony  versus nonhematophagous (booklice) arthropod hosts, provides novel insight into the molecular basis for rickettsial virulence and horizontal transmission. Our work also underscores the potential of the mobilome for orchestrating Rickettsia host adaptations.In an effort to determine whether genetic variability underlays a colony , and 2) s colony . These gC. felis . SurprisRickettsia felis str. LSU (R. felis str. LSU-Lb (C. felis (cat fleas) and L. bostrychophila (booklice), respectively, were propagated in the tick-derived ISE6 cell line in modified L15B growth medium  and rinsed with NaCl solution (0.85 w/v). Concentrated bacteria was resuspended in 200 μM of manufacturer’s PBS buffer and total DNA was extracted using the PureLink Genomic DNA mini Kit (Invitrogen) as described by the manufacturer’s instructions.str. LSU  and R. f. LSU-Lb , which wh medium . Cultureh medium . BrieflyR. felis str. LSU and R. felis str. LSU-Lb. Ion Xpress Plus Fragment Library Kit (Life Technologies) was used to prepare high-quality fragment libraries from total gDNA for sequencing on the Ion Personal Genome Machine (PGM) System. This kit includes: Ion Shear Plus Reagents for enzymatic fragmentation of genomic DNA and Ion Plus Fragment Library Kit for library preparations from the enzymatically fragmented DNA . Template-positive Ion Sphere Particles (ISPs) containing clonally amplified DNA were produced using the Ion OneTouch 200 Template Kit v2 (for 200 base-read libraries) with the Ion OneTouch Instrument. The Ion OneTouch ES instrument was used to enrich ISPs intended for the Ion PGM System using the Ion PGM 200 Sequencing Kit and an Ion 316 sequencing chip. A total sequence output of Q20 quality, derived from the predicted per-base quality scores and corresponding to an error rate of 1%, is approximately 600 Mb for the Ion 316 chip.The Ion Torrent Next Generation Sequencing technology was utilized for sequencing the genomes of R. felis str. LSU and R. felis str. LSU-Lb genomes, single nucleotide polymorphisms (SNPs) were determined as follows. Raw sequence reads from R. felis str. LSU and R. felis str. LSU-Lb libraries were independently mapped to the R. felis str. URRWXCal2 genome  using the long-read option of the Burrows–Wheeler Aligner (BWA) v0.5.9 and a minimum score threshold of 200. Variants were called with mpileup and bcftools, part of the samtools v0.1.19 package, using the following settings: A maximum allowed depth of 10,000 reads per position (-d); indels called only when the average per-sample depth does not exceed 1,000 (-L); a minimum base quality of 7 (-Q); a coefficient for modeling homopolymer errors of 50 (-h); a gap-opening sequencing error probability of 10 (-o); a gap extension error probability of 17 (-e); and a minimum of four gapped reads required to consider indel candidates (-m). Genomic context  and substitution impact  were determined using the R. felis str. URRWXCal2 chromosome (GenBank ID CP000053) and plasmid pRF (CP000054) annotations.For R. felis str. LSU reads, as well as 15 selected SNPs predicted from the R. felis str. LSU-Lb reads, were selected for sequencing. Genomic and plasmid DNA was extracted using Qiagen DNEasy Tissue Kit . Primers for each variant were designed from flanking regions on contigs using Primer3Plus -amplified using FastStart PCR Master Mix (Roche) 50 ng of DNA template, and 300 μM of each primer under standard thermocycling conditions on a PTC-200 PCR machine . The amplified PCR products were gel purified using QIAquick Gel Extraction Kit (QIAGEN) and bidirectionally sequenced with corresponding primers on an ABI PRISM 3130 Automated sequencer (Applied Biosystems) at the GeneLab core facility in the School of Veterinary Medicine at LSU. In several cases where direct sequencing failed, purified PCR products were cloned into pCR2.1-TOPO  according to manufacturer’s instructions. Plasmid inserts were sequenced by the LSU Gene Lab using the M13 forward (-20) and M13 reverse (-24) primer pairs.For gene variant confirmation, all 23 SNPs predicted from the mer3Plus  and syntR. felis str. URRWXCal2 (CP000053 and CP000054) using ABACAS 1.3.1, and overlapping contigs merged into supercontigs. Additional contigs from R. felis str. LSU that matched to Wolbachia spp. were similarly ordered against Wolbachia endosymbiont of Cimex lectularius (AP013028), a close relative identified by 16S rDNA sequence analysis (data not shown). The assemblies and related information for the R. felis str. LSU and R. felis str. LSU-Lb genomes were deposited in GenBank and assigned the Bioproject IDs PRJNA258182 and PRJNA258188, respectively.Raw reads from each strain were subsequently assembled de novo to the level of contig using Mira v3.4.1.1 under default settings, except the number of passes was set to 4, “skim each pass” was enabled, and minimum read length was set to 50. Homology searches (vs. NCBI’s NR database) were used to partition the contigs into rickettsial and nonrickettsial sequences. Additionally, reads from each LSU strain were mapped to full-length 16S rDNA sequences in the GreenGenes database from May 2013 (v. 13_5) using the long-read option of BWA v0.5.9, and binned by taxonomy to assess the microbial diversity in each sample. Rickettsial sequences were subsequently ordered against R. felis str. URRWXCal2, and 2) the Rapid Annotation using Subsystem Technology (RAST) v2.0 server  The ab initio gene prediction program fgenesb , trained0 server . The fgesupplementary table S2, Supplementary Material online); 2) the chromosomes of R. felis strains URRWXCal2 (NC_007109), LSU, and LSU-Lb; 3) 20 Rickettsia plasmids ; and 4) the pRF plasmid sequences of R. felis strains URRWXCal2 (NC_007110), LSU, “AUS-Lb” (GQ329881), and LSU-Lb. AUS-Lb refers to an unnamed R. felis strain collected from L. bostrychophila in the Darling Downs region of Queensland, Australia  were selected for analysis. Multiple sequence alignment of each OG was performed using MUSCLE (default parameters) , with reR. felis genomes alone was carried out to identify a core genome and determine the composition of the accessory genome. To provide consistency in gene predictions across all three assemblies, we implemented the bacterial gene prediction program fgenesb , providing a more robust determination of the divergence pattern of all three R. felis strains from their closest relative (R. akari). A phylogeny was estimated under maximum likelihood using RAxML v.7.2.8  with additional booklouse-associated strains. Despite no other sequenced genomes from booklouse-associated R. felis strains being available for comparison, we utilized several conserved genes  sequenced from various L. bostrychophila-associated R. felis strains from geographically isolated regions   . NucleotR. felis pRF plasmid sequences was carried out to evaluate the conservation of genes. The 68 CDS of pRF from R. felis str. URRWXCal2 were used as a reference, with OGs from all strains mapped on a circular plot using Circos , was utilized for phylogeny estimation. Rickettsia DALP proteins are extremely variable across homologs except for the C-terminal regions, which encode a DnaA_N domain (pfam11638) typical of chromosome and plasmid replication initiation proteins. The OG including DALP sequences was combined with a sequence from Orientia tsutsugamushi encoding a DnaA_N domain (YP_001937581) that was closest to Rickettsia DALP sequences in BLASTP analyses. All sequences were aligned using MUSCLE v3.6 (default parameters). A phylogeny was estimated under maximum likelihood using RAxML v.7.2.8  database was used, coupled with a search against the Conserved Domains Database . Alignments were concatenated into one data set and used in phylogeny estimation with RAxML as described above. An additional protein (DUF1016) encoded within the RAGE was combined with homologs retrieved from BLASTP searches against several taxon-specific databases: 1) “Rickettsiales,” 2) “Alphaproteobacteria ,” 3) “Proteobacteria (minus Alphaproteobacteria),” 4) “Bacteria (minus Proteobacteria),” and 5) “minus Bacteria.” The top 20–50 (query-dependent) subjects from each search resulting in significant (>40 bits) alignments were all compiled, aligned using MUSCLE v3.6 (default parameters), and analyzed with RAxML as described above.Several proteins encoded on c6440 were selected for further analysis. Within a Rickettsiales amplified genetic element (RAGE), 14 proteins encoding components of an F-like type IV secretion system (F-T4SS), were combined with homologs from other rickettsial RAGEs and an F-T4SS from “ORFs 38–41 of c6440 were predicted to encode an operon of a large toxin and adjacent repeats-in-toxin (RTX)-like type I secretion system (T1SS). Two proteins homologous to ABC transporters of RTX-like T1SSs were combined with homologs retrieved from BLASTP analyses as described for DUF1016, with a similar protocol for phylogeny estimation. Finally, the large toxin encoded immediately upstream of the RTX-like T1SS (ORF 38) was analyzed using BLASTP searches against the NR database. The Conserved Domains Database was used to identify modular domains at the N- and C-termini, which were collectively used to characterize the protein as a putative toxin.R. felis strains was estimated prior to genome assembly. Relative to strain URRWXCal2, 23 and 92 SNPs were calculated for strains LSU and LSU-Lb, respectively . Inspection of the genomic positions of SNPs revealed different patterns of divergence within strains LSU and LSU-Lb relative to str. URRWXCal2. For strain LSU-Lb, 15 SNPs were detected in repeat regions, either within or near Rickettsia palindromic elements (RPEs). RPEs are short (∼100–150 nt), selfish elements that insert in intergenic regions and functional RNAs, as well as CDS, wherein ORFs are not disrupted . Notable exceptions include SNPs in genes encoding two putative effector proteins , a member of the proliferated ProP osmoregulatory transporters, and two proteins on the pRF plasmid.Polymorphism across ectively . Estimatisrupted . Under a genomes . In the R. felis str. LSU, half of the estimated SNPs occurred in either one intergenic region or within the ORF encoding an NACHT domain-containing ATPase (pfam05729) that is pseudogenized in all other Rickettsia genomes . Of the other half of estimated SNPs, five were found in four genes that we previously determined to be commonly associated with mobile elements  and ANK (CDI29634) domains. Like R. felis str. LSU-Lb, an SNP was determined in the gene encoding the autotransporter Sca7, although it introduced a frameshift. As Sca7 proteins are not conserved across Rickettsia genomes  more prevalent in genes that are derived from lateral gene transfer (LGT). Rickettsia felis str. LSU-Lb is more divergent when compared with strain URRWXCal2, with SNPs found predominantly in intergenic regions, repeat regions, and conserved Rickettsia genes. The greater divergence in R. felis str. LSU-Lb is more reflective of genetic drift, likely due to its presumed obligate symbiosis with L. bostrychophila.Collectively, the number of SNPs estimated from the sequencing reads generated for R. felis str. LSU sequencing run yielded 3,036,844 reads which assembled into 10,578 total contigs ; 7,827 contigs  with a top BLASTn match to Rickettsia were subsequently ordered and combined into 21 chromosomal and 1 plasmid sequence. In similar fashion, the R. felis str. LSU-Lb run yielded 5,463,632 reads which assembled into 59,374 total contigs ; 5,266 Rickettsia-like contigs  were ordered and combined into 35 chromosomal and 8 plasmid sequences. An additional 52,299 nt sequence, suspected to be a novel plasmid, was identified as a single contig within the initial LSU-Lb assembly and removed prior to ordering. The assembled genomes of R. felis strains LSU and LSU-Lb are similar to the previously published R. felis str. URRWXCal2, despite neither assembly resulting in a closed chromosome , the numbers of predicted ORFs are variable in the LSU  and LSU-Lb  genomes. As described below, these predictions were manually evaluated separately for the chromosomes and pRF plasmids, improving the total numbers of predicted ORFs across all three genomes. The identification of split ORFs that inflated automated ORF predictions, coupled with lack of single contigs despite high coverage for both LSU and LSU-Lb genomes, suggests that the high repeat content of R. felis genomes is difficult to resolve with current, short-read sequencing technology.The romosome . Both LSromosome . RelativWolbachia species are known to exist in the LSU C. felis colony  previously reported from the LSU colony . Additionally, we identified 438 contigs that contained genes encoding proteins with top BLASTP hits to Wolbachia spp. The characterization of this genome sequence will be reported elsewhere.Two divergent s colony , an unchin fleas . Infecti species . Previouelis LSU . Thus, tR. felis str. LSU-Lb genome; however, one 52.3-kb contig was built that contained mostly plasmid-like genes, yet the profile of these genes, as well as the estimated size of the replicon, did not match plasmid pRF. This putative plasmid is discussed extensively below.No additional microbial genomes were detected in assembling the R. felis strains, together with R. akari and R. australis, within the TRG rickettsiae (A). In response to earlier reports classifying R. felis as belonging to either TG . Collectively, robust genome-based phylogeny estimation unambiguously supports the derivation of three Rickettsia groups  from the poorly understood AG rickettsiae, consistent with unique host specialization and genome characteristics that define each distinct lineage . Unexpectedly, far more divergence separated the flea-associated strains relative to the SNP analysis; this is partially a consequence of our somewhat conservative SNP calling routine but also may be a reflection of the shorter reads and poorer assembly of R. felis str. LSU relative to R. felis str. URRWXCal2, which was assembled into one chromosome . Nonetheless, compared with R. akari, it is evident that all three R. felis strains collectively comprise a species that is clearly distinct from other members of TRG rickettsiae (∼4.5% aa divergence).Consistent with the results from SNP analysis, phylogeny estimation recovered the split of  strains B. Unexperomosome . Estimat. LSU-Lb C. NonethR. felis genomes resulted in a core genome encoding 1,261 proteins (D). Despite this, only roughly half (46.8%) of these conserved proteins are of uniform length across all three genomes, reflecting the unequal distribution of RPEs and other insertion elements, as well as minor discrepancies in gene start sites . Still, a manual assessment of generated OGs revealed that, relative to R. felis str. URRWXCal2, split and truncated proteins from str. LSU (n = 347) and str. LSU-Lb (n = 109) grossly contributed to overestimation of the accessory genome . This likely reflects the shorter reads generated by next-generation sequencing, in conjunction with the high repeat density of R. felis genomes, preventing a portion of genes to assemble properly. Once adjusted for, the accessory genome revealed similar numbers for proteins unique to each genome, as well as proteins shared by any two combinations (D).OGs of proteins generated across all three proteins D. Despitinations D.R. felis str. URRWXCal2 and the multicontig assemblies of strains LSU and str. LSU-Lb revealed substantial colinearity across all three genomes . In conjunction with the minimal accessory genome, minor deviations in synteny can be attributed to differential numbers of transposases and repeat regions, which are probable factors preventing closure of the LSU and str. LSU-Lb genomes. It is likely that such duplicate transposases and other insertion sequences have mediated several homologous recombination events resulting in minor synteny breaks across these genomes; such has been observed in Rickettsia  conserved in the LSU and str. LSU-Lb genomes (data not shown).An approximation of synteny conservation across the closed genome of ckettsia  and otheckettsia  genomes Rickettsia genes  were utilized for comparing R. felis flea-associated strains  with R. felis str. LSU-Lb and other L. bostrychophila-associated strains . Importantly, these other booklouse-associated strains are from regions  geographically isolated from the LSU L. bostrychophila colony (sca4 was invariant), all but one (in sca5) united R. felis str. LSU-Lb with the flea-associated genomes to the exclusion of the other L. bostrychophila-associated R. felis strains (E), indicating a potential host shift to L. bostrychophila that occurred independent of the geographically isolated L. bostrychophila-associated R. felis strains.Five highly conserved a colony . Surpris strains E, indicaR. felis carry the pRF plasmid, which was originally identified in strain URRWXCal2  but high synteny . A core set of 54 CDS is present across all four plasmids, yet many genes are either split or truncated in one or more plasmids. Only 15 CDS are conserved in length across all four plasmids . One of these, pRF_56, was originally annotated as a hyaluronidase, an enzyme thought to facilitate pathogen spread through host tissues. Characterization of a similar protein from Streptococcus pyogenes did not reveal a hyaluronidase function, but rather beta-N-acetylglucosaminidase activity, with the ability to remove beta-O-linked N-acetylglucosamine from mammalian glycoproteins , as well as on the chromosomes of Rickettsia bellii str. RML369-C (YP_538411), Rickettsia endosymbiont of Ixodes scapularis (REIS) (EER21570), and R. monacensis str. IrR/Munich (CDI29587). Remarkably, a homolog (CDG49776) is also encoded on the chromosome of the obligate intracellular bacterium Cardinium cBtQ1 (Bacteroidetes), a facultative endosymbiont of the whitefly Bemisia tabaci . Despite this, a phylogeny of conserved pRF genes grouped the L. bostrychophila-associated strains together relative to the pRF plasmids of strains LSU and URRWXCal2 (data not shown). Consistent with this, a phylogeny estimation of DALPs, the sole protein encoded across all sequenced Rickettsia plasmids as revealed by our OG generation, grouped strains LSU-Lb and “AUS-Lb” and strains LSU and URRWXCal2 as sister clades (B). Thus, despite considerable decay in gene content across pRF plasmids, these extrachromosomal elements share a common origin and have diverged in tandem with their respective chromosomes.Two gene fusions were identified in pRF of r clades B. Thus, R. felis str. LSU-Lb assembly revealed a single contig comprised many plasmid-like genes. This contig, hereafter referred to as plasmid of L. bostrychophila associated Rickettsia (pLbaR), encodes 43 predicted proteins, including a ParA gene typically present on Rickettsia plasmids . Multiple attempts to circularize this putative plasmid through PCR were unsuccessful, potentially due to transposases flanking the large contig. Despite this, the contig size (52.3 kb) and %GC (33) of pLbaR are typical of other Rickettsia plasmids, and 29 predicted proteins were grouped into OGs in a clustering analysis across 20 Rickettsia plasmids (B). Importantly, the DALP encoded on pLbaR is most closely related to the DALP from plasmid pRPR (Rickettsia peacockii), which is substantially divergent from pRF plasmids (Rickettsia plasmid (B).Inspection of the plasmids B. Importplasmids , support plasmid B.A). RAGEs are extensively proliferated and highly pseudogenized in the genomes of the Scrub Typhus agent, Orientia tsutsugamiushi . Thus, R. felis str. LSU-Lb is the second Rickettsia species harboring a plasmid with an RAGE, lending further insight on the dynamic nature of these fascinating mobile elements.Remarkably, ORFs 2–23 of pLbaR encode an integrative conjugative element, named RAGE, known from the genomes of some rickettsial species . The profig. 3gamiushi , yet usu. bellii  and Rickassiliae . Aside fassiliae . AlthougB). This underscores the common origin of these elements, despite R. felis and REIS being well diverged from one another in the estimated phylogeny of Rickettsiaceae (A). Relative to most chromosomal encoded RAGEs, the plasmid-encoded RAGEs of REIS lack a toxin-encoding gene between the genes coding for TraU and TraW; on pLbaR, a unique gene encoding a protein DUF1016 is found in this position. DUF1016 proteins belong to a diverse family of restriction endonucleases . Remarkably, the DUF1016 protein of pLbaR grouped with a homolog from Legionella longbeachae (WP_003636576) in a separate clade well diverged from that containing the alphaproteobacterial DUF1016 proteins. This LGT underscores the modular nature of RAGEs  fused into a single protein B. This utsiaceae A. Relatiucleases . BLASTP of RAGEs .Rickettsia RAGEs further bolsters the close relatedness of the pLbaR and pREIS3 RAGEs, and indicates the three plasmid encoded RAGEs are divergent from the chromosomal encoded RAGEs (C). Relative to the F-T4SS of “Candidatus Protochlamydia amoebophila” UWE25, the plasmid encoded RAGEs branch off even before an RAGE encoded in the O. tsutsugamushi Ikeda genome, illustrating that multiple invasions of divergent RAGEs have occurred within Rickettsia genomes throughout evolution. The presence of RAGEs on plasmids from two Rickettsia species indicates that plasmids are the likely vehicles by which RAGEs spread across Rickettsia genomes. Their incorporation into Rickettsia chromosomes indicates that RAGE invasions are an active process shaping the composition of rickettsial genomes, equipping recipients with many genes important for survival within eukaryotic cells  (A). In Vibrio RTX-T1SSs, secretion of the large MARTX toxin requires two ABC transporters, RtxB and RtxE (B). Remarkably, BLASTP analysis of ORFs 39–41 revealed highest similarity to a Cardinium cBtQ1 T1SS operon that is duplicated and thought to have arisen from an LGT event with Vibrio spp.  A. In Viband RtxE , and it and RtxE . Importaand RtxE , which cand RtxE B. Remarkrio spp. . Compare genomes  would seC). Given that the conserved rickettsial AprD-like transporters belong to the Group 3 transporter clade, this is the first report of an RTX-like T1SS from any species of Rickettsiales. Within the Group 2 transporter clade, the duplicate ABC transporters of RTX and RTX-like T1SSs form separate clades (RtxB and RtxE), and assemblage within each clade indicates substantial LGT across divergent obligate intracellular bacteria and other Gammaproteobacteria, including species of Vibrio. Aside from R. felis and Cardinium cBtQ1, three gammaproteobacterial endosymbionts of insects  have acquired RTX-like T1SSs, despite reductive genomes due to obligate intracellular lifestyles. As RTX-T1SSs and associated MARTX toxins are known from Vibrio plasmids . Given that large RTX toxins are often encoded nearby the RTX T1SS genes, we investigated whether pLbaR_38 had characteristics of RTX toxins. RTX proteins are highly modular and extraordinarily diverse in protein domain composition across species, with a wide range of ascribed functions . Tox-SGS domains are associated with recombination hot spot (RHS) proteins of insects and crustaceans, with domain architecture indicative of an origin from bacterial polymorphic toxin systems  encoding a large protein of 3,241 aa, hereafter referred to as pLbaR_38 A. Given unctions . Neverthunctions . pLbaR_3unctions , 2) an iunctions , and 3)  systems . Large b systems  as well  systems . AccordiCardinium cBtQ1 genome (A), consistent with the close relatedness of the RTX-like T1SSs of these two species. The internal sequence of this RHS-like toxin also shares similarity with proteins encoded in the genomes of some Wolbachia spp, very few Rickettsia spp., Diplorickettsia massiliensis, and Orientia tsutsugamishi . Although none of these proteins has characterized functions, it is clear that diverse sequences have recombined into the large ORF that encodes pLbaR_38. Furthermore, minimal regions within pLbaR_38 and the two RHS-like toxins of Cardinium cBtQ1 have low similarity with RTX-like toxins encoded in the genomes of “Candidatus Hamiltonella defensa” and “Candidatus Regiella insecticola” (data not shown), consistent with the similar RTX-like T1SSs common to all of these genomes. Interestingly, a nearby ORF on pLbaR encoding a hypothetical protein of 614 aa (pLbaR_36) is similar (22% ID) to a protein (NP_966396) encoded in the genome of the Wolbachia endosymbiont of Drosophila melanogaster (wMel). Curiously, in the wMel genome, NP_966396 is directly adjacent to an ORF encoding a protein (NP_966397) with similarity to pLbaR_38, indicating that a multigene transfer has likely occurred between wMel and pLbaR. This is also reflected by the ANK repeats encoded by pLbaR_38, which are most similar to dozens of ANK-repeat domain containing proteins encoded within many Wolbachia genomes, some of which also encode an OTU-like cysteine protease domain like pLbaR_38 (data not shown). Collectively, pLbaR_38 is a highly modular protein containing eukaryotic-like domains and regions similar to proteins encoded in other intracellular bacterial genomes. Its close proximity to an RTX-like T1SS suggests a potential mechanism for secretion of this large toxin by R. felis LSU-Lb into its booklouse host.Remarkably, pLbaR_38 shares significant similarity with two RHS-like proteins encoded in the 1 genome A, consisRickettsia spp. and arthropod hosts, and also the checkpoint at which species diverge into pathogens or symbionts, are ill defined. Flea-borne transmission of R. felis has proved challenging to characterize  versus horizontal transmission of rickettsial species is believed to play an important role in determining whether mutualistic or parasitic interactions evolve ; therefoacterize  as both acterize  and vertacterize  transmisea feces , possiblR. felis  and potential host specialization (LSU-Lb) have resulted in genetic divergence . Unexpectedly, assembly of the R. felis str. LSU genome revealed more divergence not witnessed by initial SNP estimation, though manual assessment of gene predictions revealed many split genes and gene fragments that appear to be artifacts of sequencing and assembly. Such artifacts were far less observed for R. felis str. LSU-Lb. One possible reason for the difficulty encountered with the R. felis str. LSU sequencing and assembly is the presence of many Wolbachia reads in the generated data. Another is a higher instance of repeat regions and encoded transposases in R. felis str. LSU relative to R. felis str. LSU-Lb. Notwithstanding, future sequencing efforts, ideally utilizing a combination of short- and long-read sequencing methods, will be needed to resolve the incomplete genes models within both R. felis str. LSU and R. felis str. LSU-Lb genomes, as well as to evaluate the few missing genes that are present in the Rickettsia core genome and/or the R. felis str. URRWXCal2 genome.Consistent with the SNP analysis, analyses of the assembled genomes suggest that  strains B and C. R. felis genome  and dynamic accessory genome (D). Relative to R. felis str. URRWXCal2, both LSU and LSU-Lb genomes lacked 21 genes, which are all probable pseudogenes (average size of 80 aa) relative to other Rickettsia homologs. Similar profiles were found for genes shared by R. felis str. URRWXCal2 and either LSU or LSU-Lb strain, indicating a pseudogene-laden accessory genome characteristic of rickettsial reductive genome evolution  does not show a pattern consistent with strict divergence between flea- and booklouse-associated strains (A). Since its discovery in 2005 as the first plasmid from an obligate intracellular bacterium , PolC-like DNA helicases, stringent response hydrolases and synthetases, and proteins containing histidine kinase and tetratricopeptide domains, many other genes with diverse functions are known to piggyback on these elements XK phosphodiesterase superfamily  in the chophila . Rickettelements . Adding erfamily . Bacterisistance . The clockettsia . The preCardinium endosymbiont cBtQ1 genome encodes highly similar homologs to all of these proteins. Gene exchanges between Rickettsia spp. and Cardinium endosymbionts of diverse arthropod species have previously been reported. Originally described from pseudogenes of R. peacockii , with wCle provisioning biotin to its insect host in an established obligate mutualism   . Thus, iconidae) .Cardinium endosymbiont cBtQ1 and R. felis str. LSU-Lb suggests another means for the putative secretion of their RHS proteins. The RTX-like T1SSs encoded in both genomes would serve to facilitate secretion of such large substrates, especially given that T6SS substrates are typically smaller in size than large RHS proteins , it is imperative to determine whether these strains also carry plasmid pLbaR, or contain different factors that are associated with independent colonization(s) of L. bostrychophila.Although the type VI secretion system (T6SS) has been implicated in the secretion of some bacterial RHS proteins , the lacproteins . Collectthropods . Given t strains E, it is R. felis has been difficult to sort in the traditional TG/SFG rickettsiae classification scheme, consistent with it possessing attributes of both groups. Based on 1) its phylogenetic position , 2) its predominant association with insects, and 3) substantial LGT of pRF plasmid genes with species of AG rickettsiae, R. felis was grouped together with R. akari and R. australis in the TRG rickettsiae , which is an endoparasitoid of the leafminer Liriomyza trifolii (Diptera: Agromyzidae), an uncharacterized Rickettsia species has been shown to induce thelytokous parthenogenesis . Interestingly, other members of this lineage have been reported from the Birch Catkin Bug, Kleidocerys resedae (Hemiptera: Lygaeidae)  (Relative to TG (body lice and fleas) and SFG (ticks) rickettsiae, a broader host range has been reported for TRG rickettsiae  . Althougboensis” . For theogenesis . A BLASTgaeidae) , as wellionidae) . The effR. felis infecting L. bostrychophila, reproductive parasitism has evolved into obligate mutualism, supported by 1) a paucity of males known in nature, 2) the near 100% rickettsial infection rate, and 3) no viable males after eliminating rickettsia (E), the lack of males in our LSU L. bostrychophila colony, coupled with a 100% infection rate, suggests R. felis str. LSU-Lb is also an obligate mutualist, though this needs to be substantiated. It is unknown how exactly R. felis contributes to parthenogenesis, other than having a probable role in oocyte development , R. felis is found in a variety of tissues throughout L. bostrychophila, including single cell mycetocytes and an organ-forming mycetome .


Pantoea calida, a recently described environmental Enterobacteriaceae organism, has not yet been associated with human infection.P. calida. After pituitary adenoma resection, a 52-year-old Caucasian woman developed febrile meningitis confirmed by cerebrospinal fluid analysis. P. calida was grown in pure culture from this fluid and was firmly identified with partial rpoB gene sequencing. She was cured by a 14-day course of meropenem.We report a case of postoperative meningitis caused by P. calida must be added to the list of opportunistic Enterobacteriaceae pathogens responsible for postsurgical meningitis. It is easily identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Pantoea calida has been recently described as a species of Enterobacteriaceae after its seminal isolation and culture from powdered infant formula [P. calida from a cerebrospinal fluid (CSF) specimen and identified it as the cause of postsurgical meningitis. formula . It has A 52-year-old Caucasian woman had a medical history of hypertension, smoking and alcoholism. She presented with binocular diplopia. A computed tomography scan showed an intrasellar lesion with invasion of her right cavernous sinus that was further confirmed by brain magnetic resonance imaging. An endoscopic endonasal biopsy was performed and histopathology showed a nonspecific inflammation and subacute hemorrhagic alteration of her nasal mucosa. An antibiotic prophylaxis with cefazolin 2gr was given. A transnasal surgical excision was performed, and no leak of CSF was observed after the surgery. Histopathology confirmed a single prolactin pituitary adenoma. Five days after the surgery, she presented a fever (38.6°C) and a meningeal syndrome with consciousness disorder, neck stiffness and a Glasgow Coma Scale evaluated at 13 (E4V4M5) but without focal neurological deficits. The CSF collected by lumbar puncture showed hypoglycorrhachia , an elevated protein level (5.88g/L) and 4500 leukocytes/mL including 98% polymorphonuclear leukocytes. There was no detectable organism on direct microscopic examination after Gram staining. Treatment combining intravenous vancomycin 1gr twice a day and intravenous meropenem 2gr three times a day was started for presumptive postsurgical meningitis. Her clinical condition rapidly improved, with apyrexia and a net regression of the meningeal syndrome within 2 days. Five days after the onset of symptoms, vancomycin was stopped and meropenem 6gr per day was continued for a total of 14 days. At 90-day follow-up she was afebrile, healthy and without any sign of meningitis.P. calida with identification score of 2.26, 2.17 and 2.17 for the three spots deposited on the MALDI-TOF-MS plate. Furthermore partial rpoB gene sequencing performed as previously reported [P. calida with 99.88% sequence similarity with the reference sequence (GenBank GQ892191). Antimicrobial susceptibility was performed by diffusion method in agar and incubated aerobically for 24 hours at 37°C. The isolate was resistant to ampicillin  >8mg/L), amoxicillin-clavulanate , and cephalothin . It was susceptible to ceftriaxone , imipenem , ciprofloxacin , gentamicin (MIC<1mg/L) and co-trimoxazole (MIC<1mg/L).The CSF was inoculated on Polyvitex  and Columbia agar with 5% sheep blood and incubated at 37°C under a 5% carbon dioxide atmosphere. After 48-hour incubation, small whitish round shiny domed colonies were observed on both culture media. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) performed as previously described ,3 yieldeP. calida was isolated from the CSF of a patient with postoperative meningitis [rpoB gene sequencing which relies on the analysis of unique nucleotidic signatures [Here, ningitis . The isoningitis ,3 relyingnatures . These tP. calida on the culture of CSF, as well as the fact that it was the first identification of this microorganism in our laboratory, secure the conclusion that P. calida was responsible for this postoperative meningitis. Accordingly, the patient was rapidly cured after an effective antibiotic treatment was administrated. In this case, the source of infection remained unknown. Indeed, P. calida has been documented only in powdered infant formula [Cronobacter species (formerly Enterobacter sakazakii) causing health-care-associated meningitis in neonates [The absence of any bacteria other than  formula  and our neonates  is also neonates ,9.P. calida is closely related to members of the Enterobacteriaceae genus Erwinia, Tatumella, Kluyvera, Citrobacter and Cronobacter, which also comprise opportunistic pathogens causing meningitis, mainly in newborns [Erwinia and Tatumella organisms have not been associated with central nervous system infection, Kluyvera meningitis has been reported in a newborn [Citrobacter bacteria are opportunistic pathogens seldom reported as causing meningitis in neonates [Citrobacter koseri meningitis has been reported after free-diving [Cronobacter species along with Enterobacter species have been reported as causes of health-care-associated meningitis [Indeed, newborns . Whereas newborn . Also Cineonates , childreneonates  and adulneonates . Recentle-diving . Lastly,ningitis .P. calida should be added to the list of Enterobacteriaceae pathogens responsible for infectious postoperative meningitis. Sources different from powdered infant formula should be investigated. P. calida meningitis can be cured by appropriate antibiotic treatment. This new pathogen can easily be identified by using MALDI-TOF-MS [I-TOF-MS ,3.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CSF: Cerebrospinal fluid; MALDI-TOF-MS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MIC: Minimal inhibitory concentration.The authors declare that they have no competing interests.NC, RN, SDLR, and PHR took care of the patient. SF and MD took care of the microbiology of the specimens. SF, NC, RN, and SDLR wrote the case. PHR and MD evaluated the draft and suggested revisions. All authors read and approved the final manuscript.


Diffuse sclerosing osteomyelitis of the manidble ( DSMO) is a form of chronic non-bacterial osteitis (CNO) and predominantly affects patients < 18 years. It can result in cosmetic disfigurement. No uniformly effective treatment exists. Intravenous pamidronate (IV-PAM) has been reported to be effective in multifocal CNO.To describe clinical and radiologic outcome of a DSMO patient following treatment with IV-PAM.A 4-year old Caucasian female was diagnosed with DSMO and prospectively followed from 2007-2014. She presented with a 20-month history of facial asymmetry and painful bony expansion of the left hemimandible. CT-scan revealed bone expansion suggestive of fibrous dysplasia, but two consecutive bone biopsies revealed inflammatory cells only. Infectious osteomyeltis was also suspected, but cultures were negative and IV and oral antibiotics were of no benefit. Focal MRI revealed bone marrow and soft tissue edema with periosteal reaction, consistent with DSMO. Subsequent whole-body MRI revealed no other bony lesions. Naproxen was of no benefit and she was started on monthly 1-day IV-PAM infusions . The response to treatment was assessed according to visual analogue score for pain , sequential MRIs and clinical photos.She received 8 monthly IV-PAM infusions. After 1st dose, VAS decreased from 10 to 0. MRI documented resolution of abnormal signal at 5 months with gradual mandibular remodelling. Clinical photos confirmed resolution of facial asymmeter over 5 years with sustained improvement at 7 years. She had a minor MRI confirmed flare at 67 months which responded to Naproxen monotherapy. She remains asymptomatic at 84 month follow-up.DSMO is challenging to diagnose and treat due to its rareness and lack of uniformly effective treatment. IV-PAM was effective in this young refractory patient and resulted in resolution of pain, mandibular remodelling and cosmetic improvement.P. Miettunen: grant / research support from: Novartis. A. Rice: none declared. X. C. Wei: none declared. W. Yu: none declared.


Fascioliasis is an emerging and important chronic parasitic disease caused by two trematode liver fluke species: Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica) infecting several herbivorous mammals including cattle, goats, sheep, and humans. We report a 9-year-old girl who suffered from F. hepatica infection and underwent right hepatectomy because of increasing abdominal pain resistant to anthelmintic chemotherapy. When anthelmintic drug treatment is not effective and abdominal pain persists, surgical resection including hepatectomy should be kept in mind for resistant F. hepatica infection. Humans may ingest the infective larvae (metacercariae) by eating contaminated aquatic vegetables , by drinking contaminated water, or by washing vegetables and kitchen utensils with metacercariae-carrying water. After ingestion, the metacercariae pass into the peritoneal cavity, then enter the liver parenchyma (hepatic phase). They migrate through the liver parenchyma to enter the bile ducts, where they mature and release eggs (biliary phase). This process often lasts for 1 to 3 months.[1] Here we report a 9-year-old girl who suffered from F. hepatica infection and underwent right hepatectomy.A 9-year-old girl of Ankara, the capital of Turkey, presented with a complaint of abdominal pain, fever, and vomiting for one week. She had weight loss of about 2 kilograms in last two months. On examination right upper quadrant was mildly tender. A lesion in right lobe of liver was detected on ultrasound abdomen and she was hospitalized with a diagnosis of liver abscess. Complete blood count showed hemoglobin 11.6gm/dl, leukocyte count 11.3x109/L and platelet count of 385x109/L. Peripheral blood smear revealed 40% eosinophilia, 16% lymphocyte and 44% neutrophils. C-reactive protein was 10.3 mg/dl . Liver function tests showed aspartate aminotransferase 66 U/L  and alanine aminotransferase 154 U/L . Bilirubin levels, alkaline phosphatase, and gamma-glutamyl transferase were within normal limits. On ultrasound there was a linear hypoechogenic lesion sized 98 mm x 50 mm in right lobe of the liver in anterior part. There was also a multilobular hypodense 70 mm x50 mm x 78 mm sized lesion on segments 5, 6, and 7 on MRI. Serum immunoglobulin (Ig) E was slightly elevated.Cephoperazone-sulbactam treatment was initiated for liver abscess. Serological analyses by ELISA and immunofluorescent antibody tests (IFAT) was negative for hydatid cyst. Indirect hemagglutination (IHA) revealed positivity for F. hepatica at a titration of 1/2560. Triclabendazole treatment was started at a dose of 10 mg/kg once a day and a second dose was given after ten days. Cephoperazone-sulbactam treatment was continued for 14 days and patient was discharged.One month later, repeat USG revealed slight progression in size of the lesion (83 mm x 47 mm). She was again hospitalized because of abdominal pain and fever. Meropenem treatment (60 mg/kg/day) was started and given for one month. Triclabendazole treatment was given again at the same dosage. Liver trucut biopsy was performed for etiology. There was necrotic tissue and eosinophilic abscesses on biopsy. F. hepatica IHA decreased to 1/1280 after two months. She was discharged, but no resolution was observed again after one month follow-up. She needed multiple admissions for abdominal pain.Surgical intervention was planned after consultation with surgery department and a right hepatectomy, including the segments 4, 5, 6, 7, and 8, was performed . Microscopically, a cross-section of a partially degenerated F. hepatica was seen within the necrotic tunnel which was formed as the parasite travels through the liver parenchyma. The tunnel was composed of necrotic debris-like material, inflammatory cells, with many surface spicules of the parasite scattered around . At follow-up, the patient recovered completely without any complaints or symptoms with IHA test negative for F. hepatica.Hepatectomy due to F. hepatica infection have been reported in adult patients. Yen et al reported a 36-year-old woman patient treated with right hepatectomy for a mass in liver. F. hepatica infection was found onhistopathology.[2] In the index case, the lesion in the liver was very large-sized but we suspected parasitic infection preoperatively. No resolution was achieved after anthelmintic drug treatment.Definitive diagnosis of fascioliasis depends on serologic tests and/or demonstration of F. hepatica eggs in stool and/or bile samples. CT scan can depict two types, one consisting of an abscess-like lesion with single or multiple hypodense nodular areas, while the second consists of tunnel-like branching hypodense areas that are better delineated after contrast injection and is highly suggestive of fascioliasis in an appropriate clinical setting. Eosinophilia, positive IHA test for F. hepatica, and CT/US findings of our patient lead to the diagnosis of F. hepatica infection. Fascioliasis can be prevented with public education and environmental precautions such as avoiding consumption of contaminated water and plants. Prognosis is excellent with appropriate treatment.Source of Support: NilConflict of Interest: None declared


Giardia duodenalis trophozoites have included comparisons of the electrophoretic mobility of enzymes and of chromosomes, and sequencing of genes encoding β-giardin, triose phosphate isomerase, the small subunit of ribosomal RNA and glutamate dehydrogenase. To date, G. duodenalis organisms have been sub-classified into eight genetic assemblages (designated A–H). Genotyping of G. duodenalis organisms isolated from various hosts has shown that assemblages A and B infect the largest range of host species, and appear to be the main (or possibly only) G. duodenalis assemblages that undeniably infect human subjects. In at least some cases of assemblage A or B infection in wild mammals, there is suggestive evidence that the infection had resulted from environmental contamination by G. duodenalis cysts of human origin.Techniques for sub-classifying morphologically identical  Giardia organisms, designated Giardia duodenalis  [G. duodenalis is the only Giardia species that causes human infection; other currently recognised species in this genus include the following (hosts are mentioned in parentheses): Giardia muris (rodents) [G. microti  [G. psittaci (budgerigars) [G. ardeae (great blue herons) [G. agilis (amphibians) [Morphologically similar or identical lamblia) , can infrodents) , G. micruskrats) , G. psiterigars) , G. arde herons)  and G. ahibians) .G. duodenalis organisms. Early work of this type involved examination of the electrophoretic mobility of G. duodenalis enzymes [G. duodenalis, designated assemblages A and B [G. duodenalis emerged from study of the electrophoretic mobility of Giardia chromosomes [G. duodenalis DNA, and restriction fragment length polymorphism (RFLP) analysis and sequencing of the resulting PCR products, added further insight into the heterogeneity of the organism, confirming the existence of assemblages A and B, and – in conjunction with data from enzyme electrophoresis – delineating six additional assemblages (C–H) [From the 1980s onwards, increasingly precise methods have been developed to sub-classify morphologically identical  enzymes . In the  A and B . Additioomosomes . Polymeres (C–H) , 34–37.Giardia duodenalis genes (genetic loci) used for genotyping the organisms include genes encoding β-giardin (bg), triose phosphate isomerase (tpi), the small subunit of ribosomal RNA (ssu) and glutamate dehydrogenase (gdh) [Giardia duodenalis assemblages have been shown to be either relatively specific to certain hosts (assemblages C–H) or essentially unrestricted in terms of the species of host that they can infect  [G. duodenalis exclusively to one or other assemblage. This point is pertinent to G. duodenalis, and non-human hosts for the respective assemblages [se (gdh) . Giardiablage A) , a situaemblages .G. duodenalis cysts excreted by animals), is limited. One study from the United Kingdom suggested that contact with farm animals  and with pets  was a risk factor for giardiasis in human subjects [G. duodenalis can be transmitted between dogs and human subjects was obtained from a study in a tea-growing community in northeast India [G. duodenalis infection in human subjects and in dog(s) occupying the same household. For one such household, genetic identity between G. duodenalis in a dog and in human subject(s) was reported [G. duodenalis might have been either, or both, dog-to-human or human-to-dog. One caveat that applies to genetic studies of G. duodenalis that rely on faecal cysts as the starting material for molecular analysis is whether the presence of such cysts necessarily reflects infection, rather than resulting merely from coprophagy of faecal material containing cysts, and passage of these cysts through an animal’s gastrointestinal tract without causing infection [Unambiguous direct evidence that human giardiasis can be an example of a zoonosis, i.e. a human infection acquired from non-human hosts under “natural” conditions  [Giardia infection can occur under experimental conditions. It is, however, unclear whether the result of the experiment just described constitutes evidence for the zoonotic transmissibility of G. duodenalis, under “normal field conditions”.Dogs have been infected with organism . There ihed rat) . This woG. duodenalis organisms obtained from human subjects with Giardia infection has shown that assemblages A and B appear to be the only ones that undeniably cause human infections [G. duodenalis organisms that have genetic markers characteristic of non-A, non-B, assemblages [G. duodenalis concurrently have been described [G. duodenalis.Genotyping of fections , althougemblages , 49. Mixescribed , 18, 28.G. duodenalis organisms can show sequence differences between different copies of the same gene  [Individual ygosity) , 7.G. duodenalis has focussed on actual or presumed animal-to-human transmission, there is increasing evidence that Giardia cysts of human origin can contaminate the environment and infect wild mammals  [G. duodenalis genetic assemblages has provided a level of precision and specificity that was lacking when essentially the only tool was morphological examination of trophozoites. For example, excretion of assemblage B cysts by Australian sea lions, and relative proximity of colonies of these animals to human settlements at coastal sites, speaks to the probability of initial infection of the animals by cysts of human origin [G. duodenalis infection in freely ranging gorillas may reflect human-to-gorilla transmission of the parasite [Although much of the literature on inter-species transmission of ubjects) . The abin origin . Similarparasite , 21.G. duodenalis infection in Canadian communities during the 1990s, one ceased and the other diminished after, in each case, a beaver excreting G. duodenalis cysts was removed from the water source supplying the respective community [G. duodenalis cysts excreted by the beavers. The respective studies predated current knowledge of G. duodenalis assemblages. Archival material (Giardia organisms) from these outbreaks was, however, available for study by modern molecular techniques some two decades later [G. duodenalis, whereas the water contained G. duodenalis of assemblage B, and assemblages A and B were isolated from the infected human subjects. In the other outbreak, the beaver was found to be infected with assemblage B, whereas the infected human subjects included one with assemblage A infection [Giardia infection in beavers and in human subjects merely involves beaver-to-human transmission of the parasite, have yielded to a more nuanced appreciation of environmental contamination by human-derived G. duodenalis cysts that may infect beavers [Of two outbreaks of human ommunity . These oes later . Using snfection . Consequ beavers .


Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snail's potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different.Nuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail,  Biomphalaria glabrata is an intermediate host for several digenean trematode parasitic worms, including Schistosoma mansoni, the causative agent of human intestinal schistosomiasis. Human schistosomiasis is the most widespread trematode infection affecting around 200 million people, leading to a chronic debilitating disease and up to 200,000 deaths per year, across 75 developing countries [B. glabrata has been the focus for much research, including several gene discovery projects [B. glabrata genome [http://129.24.144.93/blast_bg/2index.html). The progress made in developing these resources has also provided the potential for the snail to become a new model organism for other purposes, including testing and identification of endocrine disrupting chemicals (EDCs) and for understanding fundamental biology, including endocrinology.The tropical freshwater snail ountries . Due to projects . Tools hprojects ; a 1.2K projects ,9). Thisa genome  . Somal risks . Therefoal risks .One class of transcription factor involved in regulating endocrine function in vertebrates are the nuclear receptors (NRs). NRs regulate and coordinate multiple processes by integrating internal and external signals, thereby maintaining homeostasis  ThesCrassostrea gigas is unresponsive to estrogen [C. gigas also did not identify an AR [Molluscs are affected by EDCs and one of the most cited examples is that of the marine pollutant tributyltin (TBT), found in antifouling paint, which is responsible for imposex in at least 195 species of gastropods worldwide . TBTestrogen , The preestrogen , the enzestrogen . The preestrogen ,42, but estrogen  since hoestrogen . A recenfy an AR .B. glabrata [B. glabrata it is particularly timely to search for the NRs of B. glabrata. The genome of the owl limpet, L. gigantea [L. gigantea genome portal and by amplification of products from B. glabrata mRNA. We assess the conservation, divergence, and uniqueness of gastropod NRs in comparison with the previously characterised receptors of a vertebrate (Homo sapiens), insect (Drosophila melanogaster), nematode worm (Caenorhabditis elegans) (reviewed in [S. mansoni)  and theiewed in ).B. glabrata and L. gigantea at National Centre for Biotechnology Information (NCBI) using TBlastX with default parameters [B. glabrata genome (version 4.01: http://129.24.144.93/blast_bg/2index.html) (TBlastX expect limit:1e-04) and assembled L. gigantea transcripts from the filtered gene models in the JGI Genome Portal (http://genome.jgi-psf.org/Lotgi1/Lotgi1.info.html) using BLASTN (expect limit: 1e-99). This was followed by a wider search using NR gene sequences spanning all the groups present in humans and D. melanogaster. A secondary search using the identified L. gigantea and B. glabrata NR genes against both the gastropod genomes using a low expect threshold value identified additional family members, some of which were distinct from human and fly NRs. Query sequences were filtered for low complexity regions. Identified contigs from B. glabrata were downloaded and processed through GENSCAN [http://genes.mit.edu/GENSCAN.html) using default parameters, to identify predicted coding regions, intron-exon boundaries, and peptides. Predicted peptides were assessed using BLASTP (expect limit: 1e-10) against the non-redundant GenBank database to check for the presence of the NR domains (LBD/DBD). The identified nucleotide sequences from both molluscs containing NR domains were analysed for redundancy using clustering analysis with sequence overlap cut-off set at 0.5 and segment coverage cut-off at 0.25 in Seqtools (8.4ver) (http://www.seqtools.dk/).Human AR (GenBank: EAX05380) and ERα (GenBank: AAI28575) gene sequences were used to search for expressed sequence tags (ESTs) in rameters . These ED. melanogaster NRs according to the recognised nomenclature [B. glabrata too short to be classified were left out of the subsequent analysis. Where there was no vertebrate or invertebrate homologue, subfamily classification was made, with a number representing the subfamily, a capital letter for the group, and a number for the individual gene.The identified genes were named based on the phylogenetic analysis and sequence similarity of the full-length sequence to previously characterized human and nclature . Small sB. glabrata snails using TRI-reagent  according to the manufacturer’s protocol and treated with DNAse to eliminate contaminating genomic DNA. Total RNA was extracted from embryonic samples using the RNeasy Fibrous tissue mini kit , which included a DNAse treatment as part of the protocol. Quantification and purity of each RNA sample were determined by spectrophotometry , and the RNA integrity was visually checked by agarose gel electrophoresis. 4μg of total RNA from adult snails and 1.5μg total RNA from embryonic samples was reverse transcribed in a 20μl reaction using the Superscript III cDNA synthesis kit  with 5 μM of a custom oligo (dTAP) primer (TGACTCGAGTCGACATCGAT20) following the manufacturer’s instructions. Residual RNA was removed by adding 1μl of RNase H (2 U/μl) to the reaction and incubating it at 37°C for 20 min. RT-PCR with 18S primers (18S-F: CGCCCGTCGCTACTATCG and 18S-R: ACGCCAGACCGAGACCAA) [Total RNA was isolated from whole homogenized adult AGACCAA)  verifiedhttp://bioinfo.ut.ee/primer3-0.4.0/), to amplify fragments from DBD and/or LBD for each B. glabrata NR to confirm transcription and sequence. 25μl PCRs contained 2μl cDNA (diluted 1 in 20), 1 X PCR Buffer, 2.5mM MgCl2, 0.2mM dNTPs, 0.5μM forward and reverse primers and 1.25U Taq DNA polymerase . Reaction conditions were 2 min at 95°C followed by 35 cycles of 30 sec at 95°C, 30 sec at 55–64°C (optimised for each primer pair (Specific PCR primers  were desmer pair ) 1 min 3B. glabrata were translated using proteomics tools at EXPASY (http://www.expasy.org/). The predicted peptide sequences from both B. glabrata and L. gigantea were analysed using PFAM domain analysis [H. sapiens), fruit fly (D. melanogaster) nematode (C. elegans) and parasitic trematode (S. mansoni) NRs  was useoni) NRs . The NR gans NRs  was disrPhylogenetic reconstruction was performed using Maximum Likelihood analysis with Bayesian inference and MEGA 6.06. Bayesian inference was conducted with MrBayes version 3.1.2  using thB. glabrata, L. gigantea and nuclear receptors from selected species. Maximum parsimony with heuristic searches, branch swapping set to tree-bisection-reconnection, topological constraints not enforced, and 1000 bootstrap replicates were performed using MEGA6.06 [Maximum parsimony was also used to provide additional phylogenetic support for classification, naming, and the phylogenetic relationships observed between MEGA6.06 . An apprMEGA6.06 .B. glabrata and a total of 33 from L. gigantea consisting of 7 newly identified NRs and confirming 26 sequences which had been previously identified from L. gigantea in previous studies [D. melanogaster NRs and with the identified mollusc NRs found no further NR homologs. Representative genes for each of the major nuclear receptor groups were detected, suggesting the assembled genome for B. glabrata provides a good representation of total gene content with physical genome coverage of approximately 27.5X (Genbank: APKA00000000.1). The numbers found are comparable to the 48 NRs reported in the human genome [D. melanogaster [Apis mellifera [Tribolium castaneum [C. elegans [S. mansoni [C. elegans is due to extensive proliferation of one gene (HNF4) within the nematode phylum [C. gigas, a marine bivalve, [Thirty nine NR sequences were identified from the genome of  studies ,48,49  for which only partial sequences were obtained from the genome were not included for further phylogenetic analysis, since it was not possible to ascertain if they are multiple partial genes or parts of the same transcript. In addition to this BgTLX, BgNRU4 and LgTR were not analysed further as these were also incomplete sequences. A separate analysis was made of 2DBD genes from B. glabrata and L. gigantea with other previously identified 2DBD sequences from S. mansoni and other species as the 2DBD region could not be aligned with the other NRs.Each predicted nuclear receptor was evaluated for expression in or genes , 31 fromia pulex . All ampNR genes . Three BB. glabrata and 30 for L. gigantea) were aligned with S. mansoni, D. melanogaster, C. elegans and H. sapiens NR sequences  is the same in all trees, close to NR0A subfamily within the NR1 group; however the NR0 subfamily was derived to accommodate nuclear receptors that lack either the DBD or the LBD and these 3 B. glabrata NRs contain both domains , 5B (DHR39) 1J and the 2DBD NRs were found in several of the invertebrate species. Representation of these latter groups in L. gigantea and B. glabrata shows that these nuclear receptors pre-date the ecdysozoan/lophotrochozoan split.The identified genes have been named in agreement with the unified nomenclature for NRs  based onilateria ,25. The B. glabrata and L. gigantea , appears to activate transcription constitutively in the absence of a ligand [B. glabrata and L. gigantea (M. cornarietis [C. gigas (Genbank: EKC20050), Mizuhopecten yessoensis (GenBank: BAN84542.1) and M. edulis [The nuclear receptors that bind to steroids, such as androgens, estrogens and progestogens, are responsible for the long-term effects of steroid hormones on reproduction, behaviour, immunity, stress responses and development. The Group 3 steroid receptor family includes the estrogen related receptor (ERR), as well as the estrogen receptor (ER) and group 3C subfamily with the androgen, progesterone, glucocorticoid and mineralocorticoid receptors. Steroid receptors were originally thought to be a vertebrate specific gene family but the identification of the genes with a clear homology to human ERR in trichoplax  and an aC. gigas , Mytiluss edulis  and Chla farreri  and gastifornica , Lymnaeaa ollula , Marisa narietis , Thais clavigera , Nucellalapillus , Bithynitaculata  and the vulgaris . The phyvulgaris ,75,80. Svulgaris ,74,81,82a ligand ,37,39,40narietis , C. giga. edulis . ERRs ha. edulis .C. gigas [We identified no convincing homologues in either of the two molluscs for the AR or for any other members of group 3C , which aC. gigas  stronglyC. gigas , its exiC. gigas ,85), ratC. gigas –88, althin vitro suggest alternative pathways must exist for spermatogenesis/oogenesis in molluscs and other nuclear receptors have been proposed as initiating these pathways for reproductive processes, some of which exist in vertebrates and which may be particularly important in invertebrates. Although vertebrates have subsequently evolved further processes involving steroid hormones, the presence of orthologous NRs involved in other pathways in molluscs offers the potential opportunity to study these conserved networks in an invertebrate.The absence of a molluscan AR and the constitutive expression of the ER B. glabrata, [L. gigantea [L. stagnalis [D. melanogaster ultraspiracle (USP) RXR homologue basal to the clade  which grouped with the human PPAR genes  has been previously identified in molluscs including labrata, , L. gigagigantea  and L. stagnalis . We founhe clade . BgRXR hlapillus ; RXR is lapillus . It has lapillus  and may lapillus . Converglapillus . We idenAR genes ; howeverAR genes .L. gigantea and other invertebrates [S. mansoni THR was able to heterodimerise with the RXR. The molluscan THRs cluster with the human THRα/β  play key roles in growth development and metabolism in vertebrates. In invertebrates exogenous (food) sourced THs have been suggested to be involved in signalling [The RXR also binds with the thyroid hormone receptor . Both gastropods contain a THR homologue , which htebrates . Wu et an THRα/β . The pregnalling .Ultraspiracle (USP), the fly orthologue of the RXR , has beiewed in . EcR homtagnalis , but itstagnalis . Interesinfected . The EcRstigated  revealinrocesses .L. stagnalis (GenBank: GU932671), the rock shell, T. clavigera [C. gigas [N. lapillus [N. lapillus did not bind to all-trans retinoic acid or any other retinoid tested nor was it able to activate the transcription of reporter genes in response to stimulation by retinoids [One retinoic acid receptor (RAR) was identified in each gastropod species. RARs have previously been identified in the gastropod lavigera , the bivC. gigas  and recelapillus . In vertlapillus . Howeveretinoids . More woB. glabrata and L. gigantea, (designated as BgTR and LgTR) that in mice have been shown to control spermatogenesis [We also identified homologues of Group 2 C/D vertebrate testicular receptor 4 (TR4) in ogenesis  and follogenesis . As an oogenesis ). Thus dtailless (tll) gene of D. melanogaster is involved in embryonic CNS and larval eye development [Tlx gene has also been found to be a key component of retinal development and vision [B. glabrata we identified homologues of photoreceptor cell-specific nuclear receptor (PNR), dissatisfaction (DSF), TLX and FAX1, while in L. gigantea, we identified a further NR2E gene . This sequence is located immediately after the DBD and plays an important role in high affinity interactions of the receptor with DNA [Drosophila HR39 is essential for sexual development, required in females both to activate spermathecal secretion and repress male-specific courtship genes, and controls the expression of specific cytochrome P450 genes [Germ cell nuclear factor (GCNF) homologues (Group 6) have previously been reported in bilaterians (reviewed in ) and we C. gigas  which di, Dm_HR4 . During l stages . In Grouwith DNA . These G50 genes . The conB. glabrata as well as the two 2DBD-NRs previously reported from L. gigantea [S. mansoni [We identified two 2DBD-NRs in gigantea . We comp mansoni  as well  mansoni .S. mansoni may open up avenues to further characterize host-parasite interactions and potentially to interfere with schistosome development within the host. Parasites are known to interfere with the snail reproductive system, since one effect of trematode infection in snails is parasitic castration . The iniewed in . This man snails  is potenn snails . Both orgnalling ,119.In addition to discovering the intricacies of the molecular interactions between host and parasite with a view to disrupting the development of the schistosome, the other key strategy for schistosomiasis control is to reduce the numbers and/or distribution of the intermediate host snails. Currently molluscicides, such as the commercially available niclosamide-based Bayluscide, are effectively employed in schistosomiasis control programs; however the problems of resistance and toxicity to other organisms means that the search for alternative, more selective, compounds is on-going. The capacity of nuclear receptors to bind small ligands, including exogenous substances such as natural products and synthetic chemicals, makes them potential targets for molluscicides .B. glabrata could provide a simple model system, not only to study the development and diversification of endocrine systems, but also to investigate and test gene function and response to external stimuli. The identification of many vertebrate-like NRs in B. glabrata could make it a suitable model candidate to investigate functional relationships of individual receptors. The ultimate choice of model organisms lies not only with the biology of the organism, but also on its tractability for study and manipulation. Aside from the genome, the full range of genomic tools available for B. glabrata, such as RNAi [B. glabrata a good candidate. However, the advent of next generation sequencing makes transcriptome study from non-model organisms a real possibility and therefore other mollusc species may ultimately make better model systems. In particular, this approach could be used to identify inputs from signal transduction pathways, potential hormone metabolic genes, co-activators, co-repressors and other unknown factors that may impinge upon receptor activity. Based on the absence of Group3C NRs, it can be concluded that B. glabrata is an inappropriate model for mammalian steroid hormone function mediated via NRs, as the genes for several major steroid hormone receptors are not present. However since steroid hormones have also been shown to act via non-genomic mechanisms in vertebrates, using membrane bound receptors (mPR and GPR30) [There is a long tradition in biology of examining biochemical processes in simplified models to elucidate mechanisms in more complex organisms. We have demonstrated that there are some NR groups with a single representative in the mollusc species’ examined instead of the multiple genes from humans. For example in the NR1 subfamily, both molluscs have single homologues of several significant NRs, such as the retinoic acid receptor (RAR), THR and ROR. For these groups,  as RNAi ,121, BAC as RNAi  and micr as RNAi ,122,123 d GPR30)  there isB. glabrata and 33 in L. gigantea representing all seven principal vertebrate nuclear receptor groups. Molluscan endocrinology is poorly understood, so further study to determine the functionality of these identified NRs promises new insights, especially concerning the many unanswered questions regarding the effects of steroids and other EDCs on molluscs. The importance of the snail as an intermediate host for schistosomes, also justifies further investigation into the function of such genes. The absence of vertebrate NRs such as VDR, CAR and PXR, as well as steroid hormone NRs, AR/PR/GR/MR for which we found no orthologues in the two mollusc species examined in this study, indicates that several significant signalling pathways are absent in gastropods. Nevertheless, we have identified an array of NRs common to both vertebrates/molluscs and molluscs/flies. These results add weight to Thummel’s speculation about the convergent regulation of NRs in vertebrates and invertebrates [We identified 39 nuclear receptor genes in tebrates . Elucidatebrates . The potS1 FigB. glabrata, L. gigantea, H. sapiens, D. melanogaster, C. elegans and S. mansoni respectively.The NRs from six different species were subjected to phylogenetic comparisons using Bayesian inference. The Bayesian tree (midpoint rooted) with posterior probability values is shown. Notations Bg, Lg, Hs, Dm, Ce and Sm in association with receptor names denote sequences from (PDF)Click here for additional data file.S2 FigB. glabrata, L. gigantea, H. sapiens, D. melanogaster, S, mansoni and C. elegans, respectively.The nuclear receptors from six different species were subjected to phylogenetic comparison using maximum likelihood method using Jones-Taylor-Thornton (JTT) substitution model. Node labels indicate bootstrap values. Notations Bg, Lg, Hs, Dm, Sm and Ce in association with the receptor name denote sequences from (PDF)Click here for additional data file.S3 FigB. glabrata, L. gigantea, H. sapiens, D. melanogaster, S. mansoni and C. elegans, respectively.The nuclear receptors from six different species were subjected to phylogenetic comparison using maximum parsimony. Node labels indicate bootstrap values. Notations Bg, Lg, Hs, Dm, Sm and Ce in association with the receptor name denote sequences from (PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 FileB. glabrata, L. gigantea, H. sapiens, D. melanogaster, C. elegans, and S. mansoni respectively.Notations Bg, Lg, Hs, Dm, Ce and Sm in association with receptor names denote sequences from (TXT)Click here for additional data file.S1 TableForward and reverse primer sequences with their product size (as submitted to GenBank), their corresponding GenBank accession numbers and their optimised annealing temperatures.(DOCX)Click here for additional data file.S2 TableL. gigantea NRs, including the EST description, GenBank accession number and E-value.Detailed summary of all the top BLAST expressed sequence tag (EST) hits for (PDF)Click here for additional data file.


Heterorhabditis are capable of selectively seeking, infecting and killing their insect-hosts in the soil. The infective juvenile (IJ) stage of the Heterorhabditis nematodes is analogous to Caenorhabditis elegans dauer juvenile stage, which remains in ‘arrested development’ till it finds and infects a new insect-host in the soil. H. indica is the most prevalent species of Heterorhabditis in India. To understand the genes and molecular processes that govern the biology of the IJ stage, and to create a resource to facilitate functional genomics and genetic exploration, we sequenced the transcriptome of H. indica IJs.Nematodes are the most numerous animals in the soil. Insect parasitic nematodes of the genus H. indica transcripts showed higher orthology with parasitic nematodes as compared to free living nematodes. In-silico expression analysis showed 30 % of transcripts expressing with ≥100 FPKM value. All the four canonical dauer formation pathways like cGMP-PKG, insulin, dafachronic acid and TGF-β were active in the IJ stage. Several other signaling pathways were highly represented in the transcriptome. Twenty-four orthologs of C. elegans RNAi pathway effector genes were discovered in H. indica, including nrde-3 that is reported for the first time in any of the parasitic nematodes. An ortholog of C. elegans tol-1 was also identified. Further, 272 kinases belonging to 137 groups, and several previously unidentified members of important gene classes were identified.The de-novo sequence assembly using Velvet-Oases pipeline resulted in 13,593 unique transcripts at N50 of 1,371 bp, of which 53 % were annotated by blastx. H. indica IJs for the first time. The transcripts showed high similarity with the parasitic nematodes, M. hapla, and A. suum as opposed to C. elegans, a species to which H. indica is more closely related. The high representation of transcripts from several signaling pathways in the IJs indicates that despite being a developmentally arrested stage; IJs are a hotbed of signaling and are actively interacting with their environment.We generated high-quality transcriptome sequence data from The online version of this article (doi:10.1186/s12864-016-2510-z) contains supplementary material, which is available to authorized users. Steinernema, and Heterorhabditis. These EPNs symbiotically associate with gram-negative gammaproteobacteria, Xenorhabdus, and Photorhabdus, respectively [Nematodes are the most abundant metazoans on earth and show remarkable diversity in their ecological and feeding habits . Althougectively . Becauseectively –6. The Eectively –10, nemaectively  and ecolectively , 13.Heterorhabditis spp. is a developmentally arrested stage analogous to the dauer stage of the C. elegans [C. elegans [The infective juvenile (IJ) stage of the  elegans , and inf elegans . IJs are elegans , 17. IJs elegans . They ca elegans , 20, and elegans –23. A si elegans –26.Heterorhabditis bacteriophora TTO1-M31e strain has been sequenced [H. bacteriophora GPS-11 strain [H. bacteriophora TTO1-M31e were published earlier [C. elegans and C. briggsae [Pristionchus [Strongyloides stercoralis [Ostertagia ostertagi [Heterorhabditis IJ ‘recovery’ is not adequate to decipher the various molecular and physiological pathways specific to these IJs [Genomic tools and technologies have allowed the researchers to uncover the amazing biology of nematodes –29. The equenced  and is a1 strain  and tran earlier . Large abriggsae –38, insetionchus , 40, anircoralis  and Ostestertagi  and manystertagi –48. Howehese IJs , 49. AddH. indica was the first species of this genus recorded from India [H. indica is the most predominant species of Heterorhabditid nematode in India and is found in almost all the geographical parts of the country. Therefore, H. indica is naturally suitable for incorporation in insect biological control programs in India. In the present study, the transcriptomic analysis of the IJ stage of H. indica was carried out to understand the molecular processes and pathways active at this stage, and to create a resource for further functional genomics and genetic investigations.om India . Since tH. indica using the Illumina GAIIx platform yielded about 51.2 million reads of 100 base read-lengths generating 64x coverage. After quality filtering, 42.3 million high-quality reads totalling 4.2 gigabases of data were obtained. The de-novo sequence assembly was carried out by Velvet at different k-mer lengths (51–93 with step size of 4) with minimum contig length of 200. The optimal assembly was attained at k-mer 83 which resulted in 18,710 contigs with 909 bp N50 , Ancylostoma ceylanicum (741 hits), Haemonchus contortus (558 hits), Loa loa (466 hits), Brugia malayi (397 hits), Wucheria bancroftii (357 hits), C. elegans (269 hits), C. brenneri (193 hits), Heterodera glycines (167 hits), C. remanei (153 hits), C. briggsae (141 hits), H. avenae (67 hits), M. incognita (35 hits), Bursaphelenchus xylophilus (13 hits)  downloaded from the wormbase (http://parasite.wormbase.org/ftp.html). The blastx resulted in 2,745 protein hits  Fig. . Due to C. elegans, 3,103 of C. briggsae, 3,171 of C. remanei, 2,164 of P. pacificus and 346 of H. bacteriophora  and Protein K(c)lusters (PRK) databases. The results of the analysis are presented in Additional file in silico quantitation of transcripts was done by mapping the reads from individual libraries to the non-redundant set of 13,593 transcripts using TopHat, and transcript abundance were calculated using Cufflinks. The FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values for all the transcripts are given in Additional file To get an estimate of transcript abundance, C. elegans, C. briggsae, B. malayi, Loa loa and Trichinella spiralis pathways. The analysis of KEGG pathways represented by the abundant transcripts revealed that, among others, at FPKM ≥ 1,000, the various signaling pathways like PI3K-Akt, Hippo, HIF-signaling pathway, Rap, MAPK, calcium, sphingolipid, cGMP-PKG, insulin signaling pathway were represented by at least one or more protein  kinases were classified into 187 groups, and we found that 137 kinase groups were common between C. elegans and H. indica, whereas, 50 kinase groups were not found in H. indica. The details of kinase groups common between C. elegans, and H. indica are given in Table H. indica but present in C. elegans are listed in Additional file The kinome analysis was done to identify the protein kinases important in signal transduction in all the above mentioned signaling pathways that regulate metabolism, cell cycle, growth and development, and responses to environmental stimuli. As against 438 kinases reported from  elegans , we deteA total of 2,374 secreted proteins were predicted  revealed 2,968 sequences showing the presence of 3,635 SSRs. Out of the 2,968 sequences, 465 sequences contained more than one SSRs and 209 SSRs were present in compound formation , which is responsible for nuclear translocation of RNAi triggers in C. elegans, is recorded for the first time in any parasitic nematode.nematode . We coulH. indica transcriptome was analysed for presence of members of functionally important gene classes like neuropeptides (FMRFamide-related peptides (flp), non-insulin, non-FMRFamide-related neuropeptide-like proteins (nlp), uncoordinate (unc), dauer formation (daf), fatty acid and retinol binding protein (far), nuclear hormone receptor (nhr), C-type lectin domain containing proteins (lec), lysozymes (lys) and lethal (let) gene classes at two stringency levels of 25 and 30 % sequence similarity and query coverage. The results are presented in Table C. elegans tol-1 in the transcriptome of H. indica IJs .Additionally, the H. indica IJs resulted in 13,593 unique, high-quality transcripts at N50 value of 1,371 bp. Further, 6,320 out of 13,593 (53 %) transcripts could be annotated by blastx against nr database. Most of the blastx hits showed similarity with A. suum and not H. bacteriophora which is a closely related species. This anomaly may be attributed to the absence of H. bacteriophora sequences from nr database. Standalone blast identified 2,745 hits with H. bacteriophora.The transcriptome sequencing and assembly of C. elegans dauer larvae and analogous developmentally arrested 3rd stage larvae (L3i) of parasitic nematodes [C. elegans, a developmentally arrested dauer stage is formed during conditions of low food abundance, high temperature [daf  genes identified in C. elegans that are involved in formation and regulation of dauer stages are placed into four dauer pathways-a cyclic guanosine monophosphate (cGMP) signaling pathway, an insulin/IGF-1-like signaling (IIS) pathway regulated by insulin-like peptide (ILP) ligands, a dauer transforming growth factor-β (TGF-β) pathway regulated by the Ce-DAF-7 ligand, and a nuclear hormone receptor (NHR) regulated by a class of steroid ligands known as dafachronic acids (DAs) [S. stercoralis revealed that out of four pathways involved in dauer formation, two were conserved while two were not, suggesting their conserved and novel modes of developmental regulation [daf-1  . Epistatds (DAs) , 41. Anagulation . Our resC. elegans [Romanomermis [EPN IJs are not known to feed, but they utilize the lipids and glycogen energy reserves stored in the body for their survival. We found genes involved in various pathways like fatty acid degradation, glycolysis, and glyoxalate in the IJ transcriptome. All these three pathways catabolize energy reserves such as fatty acids and glucose and generate ATPs that are utilized for the IJ survival. Glyoxalate pathway has been known to be important for dauer stages of  elegans  and has nomermis .H. indica IJs essential for nematode survival under stressed conditions and various other activities  far genes, 98 nhr, nine lec, 15 let but no lys gene class members  genes proved the role of insulin signaling in IJ formation and maintenance in H. indica. Neuropeptides like flp and nlp are involved in environmental sensing by the nematode. In the flp gene class, flp-1, flp-3, flp-5, flp-12, flp-17 and flp-18 were the prominent members. In the recent years, flp genes are emerging as important targets for nematode management, and it has been shown that disruption of flp gene expression impaired nematode parasite’s ability to locate its host [H. indica, like nlp-4, has no known homologs in other nematode species [nlp-18 in C. elegans encodes four predicted neuropeptide-like proteins; and is expressed in a variety of neurons, spermatheca, the rectal gland, and the intestine [lec gene class were identified in H. indica including lec-6. lec-6 encodes a 'proto' type galectin  containing a single carbohydrate recognition domain and is suggested to be important for cell adhesion and aggregation, proliferation, or programmed cell death in C. elegans [H. indica, members of the lectin protein family might possibly be involved in recognition of the symbiont bacteria. Similarly, tol-1 found expressing in H. indica IJs has been reported to be involved in behavioral responses to the pathogenic microbes by promoting the development of sensory neurons that monitor microbial metabolism and are required for a pathogen-avoidance behavior in C. elegans [tol-1 could be involved in the maintenance of a specific symbiotic relationship between Heterorhabditis nematodes with Photorhabdus bacterium, but this hypothesis would need further testing.The sequence divergence between  signals –84. Simimotility –88. Idenits host –95. Othe species , 96, 97, elegans –101. Lik elegans . Hence, H. indica. After using cd-hit-est and filtering out <300 bp transcripts, we have identified 13,592 unique transcripts in H. indica infective juveniles. 18.6 % of the proteins were similar to an animal parasite A. suum. We found that similar to C. elegans, all the four dauer formation pathways-cGMP-PKG signaling pathway, insulin signaling pathway, dafachronic acid pathway, and TGF-β were conserved in H. indica and were active in the IJ stage of the nematode. Several important signaling pathways were found active in the IJs indicating that despite being a developmentally arrested stage, IJs are a hotbed of signaling and are actively interacting with their environment. Similarly, glycolysis and fatty acid degradation pathways were highly active in IJs indicating a breakdown of food reserves required for survival. Twenty-four orthologs of C. elegans RNAi pathway effector genes were found in H. indica IJ transcriptome, including nrde-3 that has been identified in any of the parasitic worms for the first time. Using a low stringency approach, we have identified several additional members of important gene classes in H. indica. Our results and analysis lay down the groundwork for further functional genomic investigations on these gene classes in Heterorhabditis nematodes.Here we presented a transcriptomic insight into the infective juvenile stage of the EPN, Heterorhabditis indica nematodes were isolated from the soil collected from Ghaziabad district, UP, India by using greater wax moth Galleria melonella as a bait. The nematodes were maintained in the laboratory on Galleria using standard procedures.The http://www.ebi.ac.uk/ena) for public access . The assembled nucleotide and protein sequences are also available for blast and download at http://insilico.iari.res.in/hindica/. The assembled data is included with the manuscript as Additional file Total RNA was extracted from the frozen IJs using Nucleospin RNA isolation kit  according to the manufacturer’s instructions. Extracted RNA was assessed for quality and quantity using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA with an RNA integrity number (RIN) of 8.0 was used for mRNA purification. mRNA was purified from 1 mg of intact total RNA using oligodT beads (Illumina® TruSeq® RNA Sample Preparation Kit v2). The purified mRNA was fragmented at elevated temperature (90 °C) in the presence of divalent cations and reverse transcribed with Superscript II Reverse Transcriptase (Invitrogen Life Technologies) by priming with random hexamers. Second strand cDNA was synthesized in the presence of DNA polymerase I and RNaseH. The cDNA was cleaned using AgencourtAmpure XP SPRI beads (Beckman-Coulter). Illumina adapters were ligated to the cDNA molecules after end repair and the addition of an ‘A’ base followed by SPRI clean-up. The resultant cDNA library was amplified using PCR for the enrichment of adapter-ligated fragments, quantified using a Nanodrop spectrophotometer (Thermo Scientific) and validated for quality with a Bioanalyzer (Agilent Technologies). It was then sequenced on the Illumina Hiseq 2000 platform at SciGenom Next-Gen sequencing facility, Cochin, India. Both the raw and assembled sequence data generated has been deposited in the European Nucleotide Archive (ENA) database (Paired orphan sequence reads obtained from IJs were used for assembly of the transcriptome . The lowhttp://bioinformatics.ysu.edu/tools/OrfPredictor.html) [http://www.nematodes.org/nembase4/). In addition, blastx was performed to identify homologues at ≥30 % query coverage and ≥50 % sequence identity and e-value 1e–5 in other databases including RefSeq (PRK), SWISSPROT [C. elegans, C. remanei, C. briggsae, M. hapla, M. incognita, H. bacteriophora, Pristionchus pacificus, Brugia malayi, S. ratti, Trichinella spiralis and A. suum. Blastp hits with e-value scores 1e–5 and query coverage above 50 % were considered as annotated homologous proteins and python script was employed for filtering reciprocal best hits. KEGG orthologs were identified using the KEGG Automated Annotation Server (KAAS) using nematode database. iPATH server was used for mapping it to KEGG reference pathway [C. elegans RNAi pathway genes were also identified in the H. indica transcriptome by performing tblastx with e-value ≤ 1e–5.ORFPredictor web server (or.html)  was usedor.html) , homologor.html) , the Nator.html)  and NEMAWISSPROT , EuropeaWISSPROT , DNA DatWISSPROT , ProteinWISSPROT  and ProtWISSPROT . NematodWISSPROT  databaseWISSPROT . To stud pathway . The gen pathway . The resThe high-quality reads were mapped to the non-redundant assembled transcripts using TopHat v-2.0.9. –119. Asshttp://pgrc.ipk-gatersleben.de/misa) with at least 10 repeats for mono-, 6 repeats for di-, and 5 repeats for tri-, tetra-, penta- and hexanucleotide for simple SSRs.Potentially secreted peptides were identified using the SignalP 4.1 software  from thehttp://insilico.iari.res.in/hindica/. The assembled sequences are also supplied as an Additional file The data supporting the results of this research paper are included within this article and its additional files. The raw and assembled sequence data has been deposited in the Eueopean Nucleotide Archive (ENA) database for public access . The assembled nucleotide and protein sequences are available for blast and download at