Olfaction and gustation underlie behaviors that are crucial for insect fitness, such as host and mate selection. The detection of semiochemicals is mediated via proteins from large and rapidly evolving chemosensory gene families; however, the links between a species’ ecology and the diversification of these genes remain poorly understood. Hence, we annotated the chemosensory genes from genomes of select wood-boring coleopterans, and compared the gene repertoires from stenophagous species with those from polyphagous species.Dendroctonus ponderosae), and 47 ORs, 30 GRs, 31 IRs, 4 SNMPs, 12 OBPs, and 14 CSPs in the emerald ash borer (Agrilus planipennis). Four SNMPs and 17 CSPs were annotated in the polyphagous wood-borer Anoplophora glabripennis. The gene repertoires in the stenophagous D. ponderosae and A. planipennis are reduced compared with those in the polyphagous A. glabripennis and T. castaneum, which is largely manifested through small gene lineage expansions and entire lineage losses. Alternative splicing of GR genes was limited in D. ponderosae and apparently absent in A. planipennis, which also seems to have lost one carbon dioxide receptor (GR1). A. planipennis has two SNMPs, which are related to SNMP3 in T. castaneum. D. ponderosae has two alternatively spliced OBP genes, a novel OBP “tetramer”, and as many as eleven IR75 members. Simple orthology was generally rare in beetles; however, we found one clade with orthologues of putative bitter-taste GRs (named the “GR215 clade”), and conservation of IR60a from Drosophila melanogaster.We annotated 86 odorant receptors (ORs), 60 gustatory receptors (GRs), 57 ionotropic receptors (IRs), 4 sensory neuron membrane proteins (SNMPs), 36 odorant binding proteins (OBPs), and 11 chemosensory proteins (CSPs) in the mountain pine beetle  contains supplementary material, which is available to authorized users. Dendroctonus ponderosae Hopkins (Curculionidae), the emerald ash borer Agrilus planipennis Fairmaire (Buprestidae), and the Asian longhorn beetle Anoplophora glabripennis Motschulsky (Cerambycidae). The aim was to investigate how their host breadth (stenophagy vs. polyphagy) may correlate with the diversification of genomic repertoires of chemosensory genes, and at the same time reveal genes that can be targeted for improved control of these forest pests [Interpreting chemical information in the environment is of paramount importance for the fitness of animals. In insects, the chemical senses –olfaction and taste– underlie the ability to find mates, food, and oviposition sites, and to avoid harmful situations and non-host habitats . The chest pests .Orco, which is present in all insect genomes except for those of the most basal lineages [Most insect chemosensory genes encode membrane-bound receptors from three divergent gene families, which generally are expressed in primary sensory neurons. The odorant receptors (ORs) represent the principal means of sensing volatile chemicals from a distance, including pheromones and odorants from plants and microbes . The reclineages , 12. Thelineages , where Olineages –16. The lineages , 18 belolineages , 20. Thelineages , 22. Thelineages , 24. Thelineages , 25, 26,lineages –29. Memblineages .Snmp1 and Snmp2 genes) are found in most insect genomes [Snmp3) has been identified in Coleoptera [Drosophila melanogaster and moths [Proteins from additional gene families play various other roles in insect chemosensation. Genes encoding the sensory neuron membrane proteins (SNMPs), which are related to scavenger proteins of the CD36 family, are expressed in certain OSNs that express OR genes . Two rep genomes , and a tleoptera . SNMP1 ind moths , 34–36, nd moths , 38, whind moths –42. Finand moths , 44, andnd moths , 45, 46.Tribolium castaneum [A. glabripennis [Leptinotarsa decemlineata [T. castaneum [The links between chemosensory gene evolution and ecological specialization are poorly understood, although it has been hypothesized that traits, such as host breadth and social behavior, may correlate with the diversification of these gene families . Analyseastaneum , 55, theripennis , and themlineata . The CSPastaneum , 57.D. ponderosae, and the emerald ash borer A. planipennis. Proteins encoded by these genes were phylogenetically analyzed with those from the polyphagous species T. castaneum and A. glabripennis, with the latter species being particularly relevant because it also exhibits a wood-boring lifestyle, and it has been recorded from > 100 host plants [T. castaneum feeds on a large variety of seeds and other dried foods. D. ponderosae is a devastating North American bark beetle, able to kill a number of pine (Pinus) species over landscape-scales through mass attacks coordinated by an aggregation pheromone [A. planipennis is native to Eastern Asia, but has become an invasive pest in North America, threatening the existence of several ash (Fraxinus) species [D. ponderosae, the chemical ecology of this species is less understood, although odor-mediated attraction to hosts and conspecifics has been shown [D. ponderosae [A. planipennis [A. planipennis), i.e., they include a comparatively small number of identified chemosensory genes, lack expected orthologues, and include many partial gene models. A recent study included the genomic annotations of the ORs of these two species in a large-scale analysis [To test the hypothesis that variation in host range may be reflected in chemosensory gene repertoires, we annotated these genes in the genomes of two stenophagous wood-boring and phloem-feeding pests of the Coleoptera: the mountain pine beetle t plants . In contheromone , 60. As heromone . A. plan species . Compareen shown –65. Prevnderosae  and A. pnipennis . Howeveranalysis , but we D. ponderosae (“Dpon”) identified a total of 111 chemosensory genes, including 49 ORs, 2 GRs, 15 IRs, 3 SNMPs, 31 OBPs, and 11 CSPs [DponOr45) was the result of a transcript chimera; one IR gene (previous DponIr56e.1) showed no homology to insect IRs; two IR gene fragments (previous DponIr21a.2 and 56e.2) were dropped because they were revealed to belong to the same genes as two other previously reported partial IR genes; one IR gene (previous DponIr93a.2), four OBP genes , and one CSP gene (previous DponCsp5) were assembly isoforms or alleles of other genes  that was complete in the transcriptome study was incomplete in the genome assemblies; hence the original model was retained.A previous study of the antennal transcriptome of  11 CSPs . Our preA. planipennis (“Apla”), 24 chemosensory genes  were previously identified from an antennal transcriptome [AplaObp4 and AplaObp8) were discarded because they were assembly isoforms; two IR genes were identified as separate fragments of AplaIr25a (isotig01857-ApIR and G3QO8C008JMTAX_ ApIr); and both existing short AplaGR gene models were found to lack homology to insect GRs. Several of the original gene models were renamed, especially in D. ponderosae  to follOrco and 7 putative pseudogenes, were annotated in the genome of D. ponderosae, of which 63 OR genes were completed to full-length. Except for DponOR53INT (195 amino acids), all partial but putatively functional DponORs are above 350 amino acids in length, with the majority only missing a short N-terminal exon (named A1) that could not be confidently identified due to absence of transcriptomic support. As has been observed with the OR genes in other insect genomes, a large proportion of the genes in both species occur in tandem arrays on scaffolds  with mutually exclusive N-terminal A1-A2 exons assembled consecutively with seemingly shared C-terminal B-E exons. The A. planipennis genome contains 47 OR genes, including Orco and one pseudogene. In this species, 31 of the ORs were completed to full-length models, with partial OR genes encoding protein sequences between 174 and 393 amino acids. In both species, the number of introns in full-length OR genes varies between four and seven, whereas Orco is interrupted by ten introns in both species , and we can here afford to present and discuss the results for these particular species in more detail. Our phylogeny , whereas the largest expansion in A. planipennis contained 18 ORs (AplaOR16–33 in Group 2B). Well-supported orthologous relationships were only found for AglaOR55/DponOR57–59, AglaOR38/DponOR10–11, and TcasOR73FIX/DponOR56.The DponORs and AplaORs were recently included in phylogenetic analyses that covered ORs from ten coleopteran genomes, and which allowed for classification and revision of nine higher-order monophyletic OR subfamilies  across the Coleoptera . SeveralD. ponderosae, we annotated 60 GR transcripts (including 57 full-length models and one pseudogene) that are encoded by 49 genes. Seven of these genes were regarded to exhibit alternative splicing, each producing either two or four splice variants , with no evidence of alternative splicing. The putative receptors for carbon dioxide and sugars contain several introns, whereas the majority of the remaining putative bitter-taste GR genes contain only one or two introns in both species. However, several of these GRs, especially in D. ponderosae, contained one to four additional introns  are present in D. ponderosae, whereas no evidence of GR1 was recovered from the genome assembly of A. planipennis, nor from the available raw sequence reads  that was placed within the clade of conserved fructose receptors, which is dominated by a lineage expansion in T. castaneum. Most of the remaining GRs (putative bitter-taste GRs) of all four species in the analysis grouped in small to large species-specific expansions, which in many cases comprise large suites of alternatively spliced proteins, especially from T. castaneum and A. glabripennis. Among the putative bitter-taste GRs, only a single clade was represented by one orthologue from each of the four species. This clade was highly supported  and named the “GR215 clade” based on the GR representative from T. castaneum , including two pseudogenes. The number of IRs in A. planipennis was 31 (22 full-length models), including one pseudogene. Members of the conserved antennal IR8a, IR21a, IR25a, IR40a, IR41a, IR68a, IR76b, and IR93a were identified in both species  also has one IR that grouped with IR60a from D. melanogaster (“Dmel”) with high support (0.93), suggesting that this IR is conserved in beetles. Hence, the IRs from this group identified in the present study were named DponIR60a and AplaIR60a, whereas the orthologues from T. castaneum, L. decemlineata, and A. glabripennis retained their original names . The remaining divergent IRs from D. ponderosae and A. planipennis generally grouped in species-specific lineage expansions of various sizes, with only a few IRs being individually placed. Whereas the antennal IR genes are known to contain several and often very large introns, the number of introns in the divergent IRs was low , which is fewer than in other species considered polyphagous (Table . Two of the DponOBP genes (DponObp37 and DponObp38) were exclusive to the male assembly, suggesting that they are located on the neoY chromosome. OBPs are classified into different groups based on the number of conserved cysteine (C) residues and their phylogenetic relationships [D. ponderosae and A. planipennis contain one Plus-C member each, similar to other beetles .Our genome annotations revealed 36 OBPs in ionships . The “clionships . The insles Fig. . In contDponObp6 and DponObp7) showed evidence of alternative splicing, supported by transcriptomic data [DponObp7; Minus C-group) or six shared exons  , encoding 500 amino acids. Apart from a short N-terminal exon housing the signal peptide, this gene contains four similarly sized exons, each presenting the conserved Minus-C motif. This extraordinary Minus-C “tetramer” model was supported by previous transcriptomic data from this species [Tomicus yunnanensis; accession: GFJU01117056.1) and the red palm weevil , retrieved from the Transcriptome Shotgun Assembly (TSA) collection at NCBI. The first three Minus-C exons of DponObp4 are separated by short, approx. 60 bp introns, whereas the final exon is separated by a 1.26 kb intron. To investigate how such a large OBP may have originated, we individually aligned the four Minus-C exons of DponOBP4 together with a subset of Minus-C OBPs, i.e., those encompassed under the most recent node shared by the individual DponOBP4 exons. The resulting phylogeny grouped DponOBP4 exons 2 and 3 together with moderate support, while exon 4 was positioned in a sister clade. Exon 5 was widely separated from the other exons but without support  in A. planipennis, and 17 (16 full-length models) in A. glabripennis  in D. ponderosae and A. planipennis, and compared their gene repertoires with those in polyphagous species, in particular T. castaneum and A. glabripennis, with the latter species being a wood-borer recorded from > 100 host trees [A. glabripennis, the six main insect chemosensory gene families have now been identified from genomes of four species of Coleoptera [L. decemlineata [. Hence, our study facilitates analysis of the molecular evolution of chemosensation in the largest order of insects, Coleoptera, and we can now begin to address questions of how beetle ecology relates to the diversification of these crucial gene families.We annotated the chemosensory gene families from the genomes of the two stenophagous wood-borers st trees . Our annst trees , 67, witleoptera , 56, whemlineata . Hence, Pinus specialist D. ponderosae and the Fraxinus specialist A. planipennis are lower than in T. castaneum and A. glabripennis , with especially large differences observed among the ORs, GRs, and IRs, followed by the OBPs, especially in A. planipennis. The smaller gene numbers in stenophagous species are largely explained by reductions in the extent and number of paralogous radiations in the two specialized wood-borers. Their lower numbers of GRs are at least partly manifested through low frequency of alternative splicing in both D. ponderosae and A. planipennis. In fact, none of the GR genes in the latter species showed indications of alternative splicing, which is unique among the coleopterans investigated so far. In D. ponderosae, seven GR genes are putatively alternatively spliced, but with only two or four splice variants per gene, comprising 18% of the total GRs. Though only 13% of the GRs of T. castaneum are splice variants, 30 splice variants are encoded by the single gene TcasGr214 [A. glabripennis [AglaGr99). The same percentage is found in the oligophagous Colorado potato beetle, L. decemlineata, which feeds on a variety of solanaceous plants, with up to 13 splice variants encoded by one gene (LdecGr48) [L. decemlineata also has fewer chemoreceptors, especially a highly reduced IR repertoire, and the size of its OR repertoire is similar to that in D. ponderosae. D. ponderosae and A. planipennis also have proportionally fewer pseudogenes than A. glabripennis and T. castaneum, especially among the GRs. This suggests that the smaller complements of chemoreceptors are due to reduced gene gain via duplication rather than increased gene loss via pseudogenization.The number of chemosensory genes varies between insect taxa due to different rates of gene gain (via tandem duplication) and loss (via pseudogenization and deletion). This variation is thought to relate to specific lifestyles and adaptations to the environment, and may ultimately result in speciation , 51, 70.casGr214 . In A. gripennis , 39% of decGr48) . HoweverA. planipennis has well-developed eyes, and visual stimuli are important for mate and host attraction [A. planipennis is part of the relatively distant infraorder Elateriformia, whereas the other species belong to the “higher beetles”, Cucujiformia [A. glabripennis and the oligophagous chrysomelid L. decemlineata belong to the same superfamily (Chrysomeloidea), which is sister to the superfamily of the stenophagous D. ponderosae (Curculionoidea) [Culex quinquefasciatus mosquito is thought to be attributed to its broader host range  as compared to the more host-specific mosquitos Anopheles gambiae and Aedes aegypti [D. sechellia is losing OR and GR genes at a faster pace than its generalist congeners D. melanogaster and D. simulans [. On the other hand, the sizes of the chemoreceptor gene repertoires are relatively conserved across sixteen species of Anopheles species irrespective of host range, suggesting that correlations between chemoreceptor repertoires and host range are not always evident, at least not among congeners [While the observations across these five beetle species suggest a correlation between the number of chemosensory genes and diet breadth, additional species need to be sampled to draw general conclusions, because other ecological factors and the species phylogeny may also play a role. For instance, traction , 71, whijiformia . Indeed,jiformia –75. Howejiformia . Additioonoidea) . Hence,  aegypti . Moreovesimulans . On the ongeners .D. ponderosae and A. planipennis are restricted to the three receptor gene families, and simple orthologous relationships among receptors were generally rare. In the present comparison with the polyphagous species, the positions in the trees of the expansions appear to be “arbitrary”, but may possibly reflect differences in ecological traits, such as feeding from conifers (D. ponderosae), angiosperms (A. planipennis and A. glabripennis), or stored products (T. castaneum). However, an analysis of the ORs from ten beetle genomes suggests that the distribution of ORs across the tree is at least partly dictated by the coleopteran phylogeny, with more similar distributions observed in closely compared to distantly related taxa [D. ponderosae are found within subfamily 7, followed by 5A and 1. Interestingly, D. ponderosae appears to have lost ORs from several subfamilies that are largely conserved across the Coleoptera. Especially noteworthy are subfamilies 3 and 5B, with the former housing relatively large proportions of the Apla-, Agla-, Tcas-, and LdecORs, and the latter containing one or a few ORs from most beetles investigated so far [D. ponderosae are related to its ecology. In contrast, A. planipennis has no ORs in subfamilies 7, 5A, or 1, and most of its ORs belong to subfamily 2B, which is highly reduced in D. ponderosae. However, a noteworthy common denominator in the two stenophagous wood-borers is their lack of ORs in subfamily 4, which is unique among the beetles investigated so far [Larger gene-lineage expansions in ted taxa . This inted taxa , 79, 80.ted taxa , 81, 82,d so far . Functiod so far . WhetherA. planipennis assembly or from the raw sequence data. While drosophilids lack GR2 and still detect carbon dioxide [A. planipennis has lost this ability, or if the AplaGr1 gene simply was missed in the sequencing of A. planipennis. Both genomes contained genes for six putative sugar receptors (GR4–9) and one fructose receptor (GR10), which is fewer than in the two polyphagous species and might relate to their narrow host ranges. Reduction in candidate sugar receptor lineages was previously observed in the gall-midge Mayetiola destructor (Diptera), which is specialized on wheat and a few other grasses [T. castaneum), and functional studies of these receptors are warranted to shed light on this conservation. Notably, A. planipennis is missing GRs from a major clade of our tree, suggesting a loss of GRs in this species, or that these GRs originated in more recent coleopteran lineages.Conserved GRs for carbon dioxide were found in both stenophagous wood-borers, but GR1 was not recovered from the  dioxide , it is u grasses . SimilarD. ponderosae and A. planipennis. Duplications have occurred for DponIr41a and AplaIr76b, which is also the case in several species of Lepidoptera [DmelIr60a in all beetle species. This orthologue is also conserved in moths and caddisflies, demonstrating a broad occurrence of this receptor in insects [D. ponderosae encoded more IR75 members (11) than any other beetle genome. IR75 paralogs have been implicated in olfaction and/or taste in moths based on expression patterns [D. melanogaster [D. ponderosae.Although we identified fewer IRs in the stenophagous compared to the polyphagous species, the reduction was not as large as for the ORs and GRs. All members of the conserved olfactory antennal IRs  and membidoptera . Further insects . Surprispatterns , and undnogaster . FunctioA. planipennis that are related to TcasSNMP3 [. The absence of SNMP3 in several beetle species suggests that its taxonomic occurrence is restricted. T. castaneum has a total of six SNMPs, and is only exceeded by M. destructor in terms of genomic content of SNMP genes [We revealed four SNMPs in each of the three wood-boring species, which included two members in casSNMP3 . The absMP genes . While SMP genes , the rolMP genes , SNMP2, MP genes .D. ponderosae and A. planipennis, and the OBP family is especially reduced in both species. In contrast to the receptors, several orthologous clades with proteins from all four species were present in both gene families, suggesting that the evolutionary forces acting on these genes are different as compared to those acting on the receptor genes. The DponOBP family also exhibited alternative splicing and a “tetramer” gene form, neither of which have been previously reported from insects. Two genes (DponObp6a/b and DponObp7a/b) alternated a first exon that encoded the signal peptide, a hydrophobic motif that allows excretion of the protein from the cell. Previous transcriptome data indicate that DponObp6 is antenna-specific, whereas DponObp7 is expressed in a variety of tissues, but not in the antenna , with reductions in stenophagous beetle species largely explained by limited lineage expansions in most gene families , entire lineage losses, and less alternative splicing of GR genes. The reductions in gene numbers may be further augmented in st pests .D. ponderosae (Curculionidae), A. planipennis (Buprestidae), and A. glabripennis (Cerambycidae) were conducted to identify chemosensory genes, using query sequences from I. typographus, D. ponderosae, T. castaneum, A. glabripennis, and L. decemlineata [D. ponderosae was conducted using primarily the published assembly of the female genome (accession APGL01000000) [A. planipennis were annotated from a public genome assembly (PRJNA230921) made available through the i5K project, and were used with permission. The CSP and SNMP genes from A. glabripennis were annotated using the published genome assembly (GCA_000390285.2) [e-value of cut-off at 10− 1 was used in the BLAST searches except for the putative bitter-taste GRs and divergent IRs where a cut-off at 3  was used to ensure that divergent sequences were not missed. HMMER 3.1b1  with Pfam profiles PBP_GOBP (PF01395) and OS-D (PF03392) were also used to identify potential OBPs and CSPs from the NCBI REFSEQ sequences. The protein sequences of all identified genes were then blasted against each respective genome until the annotation of all novel hits was completed. Gene annotations and determination of exon/intron structure were performed manually using Geneious software package 7.1.9.  or CLC Main Workbench 8.1 . Scaffold locations and additional annotation details for all genes are reported in Additional file Exhaustive tBLASTn searches against the genome assemblies of mlineata , 56, 66.1000000) . The curD. ponderosae and A. planipennis was named GR10 in both species. The naming scheme for the remaining, putative bitter-taste GRs, followed the two criteria described for the ORs with consecutive numbering starting at GR11. The consecutive numbering of the divergent IRs started at IR101 to avoid implying orthology to unrelated IRs in D. melanogaster. Conserved antennal IRs and all SNMPs were named according to their orthologous relationships with proteins in D. melanogaster and T. castaneum [D. ponderosae and A. planipennis [A. glabripennis annotated here were named consecutively according to scaffold positions. Exons of OR genes were named exon A, B, C, D, and E following ancestral splicing patterns [Following established nomenclature guidelines, two criteria were considered for the naming of the ORs, most GRs, and the ‘divergent’ IRs. First, genes in tandem arrays on scaffolds were given consecutive numbers. Secondly, preliminary trees were constructed to assign numbers to genes following their phylogenetic position, which for tandemly duplicated genes corresponded to their scaffold locations . Hence,astaneum , 31, 33.nipennis , 67 thatpatterns , where tpatterns ). No speT. castaneum retained their PAR suffix as previously reported). Gene models split across scaffolds were given a JOI suffix, which were confirmed by transcriptomic data when available [D. ponderosae, we found indications of alternative splicing with mutually-exclusive N-terminal exons assembled consecutively with one or several seemingly shared C-terminal exons. Potential splice variants were named as e.g., GR#a, GR#b, GR#c etc. by convention [Models of chemosensory genes were frequently incomplete due to assembly gaps, large introns, or small and/or highly divergent terminal exons. Hence, we added suffixes to these gene names according to established protocols , 88. Provailable , 69. Modnvention . Highly A. glabripennis and T. castaneum were excluded from the phylogenetic analyses of all chemosensory gene families.Multiple sequence alignments of protein sequences were performed using MAFFT v 7.017 , implemeD. ponderosae and A. planipennis were analyzed together with those from A. glabripennis [T. castaneum (Tenebrionidae). The OR set used for the latter species was an updated version of the original dataset [A. glabripennis were retrieved from McKenna et al. [T. castaneum GRs from Tribolium genome sequencing consortium [L. decemlineata (Chrysomelidae) [D. melanogaster (Diptera) [D. melanogaster. Sequences of SNMPs from T. castaneum were retrieved from Dippel et al. [D. melanogaster SNMPs from GenBank (accession numbers: ABW70129.1 and NP_650953.1). Croquemort proteins, which are non-SNMP members of the CD36 family, from D. melanogaster and T. castaneum (accession numbers NP_787957.1 and XP_008192356.1), were included to root the tree. The OBP and CSP analyses included sequences from D. ponderosae, A. planipennis, A. glabripennis, and T. castaneum. OBPs from A. glabripennis were retrieved from Wang et al. [T. castaneum from Dippel et al. [The OR sequences from ripennis  and T. c dataset , which i dataset . Partial dataset , were eaa et al. , and T. nsortium . Due to melidae)  to facilmelidae) . We alsoDiptera)  to suppol et al. , and D. Additional file 1:Table S1. Amino acid sequences and annotation details of the chemosensory genes identified in the genomes of Anoplophora glabripennis, Agrilus planipennis, and Dendroctonus ponderosae. Information on genomic location, gene structure, and protein length is presented alongside annotation notes where relevant. (XLSX 38 kb)Additional file 2:Table S2. Gene name correspondence and revisions made to original models annotated from transcriptomes of Dendroctonus ponderosae and Agrilus planipennis. (XLSX 19 kb)Additional file 3:Figure S1.Left panel: Unrooted phylogeny of select Minus-C odorant binding proteins (OBPs) to indicate the evolutionary relationships of the four main exons of DponOBP4. Included are OBPs from Dendroctonus ponderosae , Agrilus planipennis , Anoplophora glabripennis , and Tribolium castaneum . The tree is based on a trimmed MAFFT alignment, and constructed using FastTree. Numbers at nodes are local support values, calculated using the Shimodaira-Hasegawa (SH) test implemented in FastTree. Exact SH values are only shown if > 0.7, whereas SH values for all branches are indicated by the colored circles; support increases with the size and brightness of the circles. The sources of sequence data and explanation of protein suffixes are detailed in the Materials and Methods section. Right panel: Amino acid identity matrix of the four major exons of DponOBP4, calculated using Geneious software based on a MAFFT alignment. (JPG 719 kb)Additional file 4:Anoplophora glabripennis, Agrilus planipennis, and Dendroctonus ponderosae. (TXT 157 kb)Translated amino acid sequences of all chemosensory genes annotated in the present study from


AbstractPionothele Purcell, 1902 comprises two nominal species known only from South Africa. We describe here a new species, Pionothelegobabebsp. n., from Namibia. This new species is currently only known from a very restricted area in the Namib Desert of western Namibia.The mygalomorph spider genus  Pionothele Purcell, 1902 is a poorly known taxon comprising only two species described from southwestern South Africa. In P.straminea Purcell, 1902 and described a second, new species P.capensis Zonstein, 2016. Similarities between female specimens of Pionothele and those in the genus SpiroctenusPionothele may be more widespread and diverse than is currently known. We describe herein a new species, Pionothelegobabeb sp. n., from the Namib Desert in central western Namibia; the type locality at the Gobabeb Research & Training Center is about120 km southeast of the Atlantic coastal city of Walvis Bay. The description of this new species extends the distribution of Pionothele significantly northward, indicating that the genus may contain considerable undescribed diversity, particularly in the intervening areas.The nemesiid genus Habitat and ecology. Fifteen males were collected in pitfall traps after a rain event at Gobabeb; specimens were observed along interdune and gravel plain transects – two of six habitats monitored by long-term pitfall trapping (Pionothele have been collected from dune ecosystems (or close proximity thereof).trapping . GobabebSpecies concept applied. This new species of Pionothele is delineated using a traditional morphological species concept wherein species are defined as those populations with qualitative phenotypic characteristics that differ in a discrete manner from other populations or groups.Institutional and quantitative morphological abbreviations used in this paper are defined as follows:BME Bohart Museum of Entomology, Davis, California.NMNNational Museum of Namibia, Windhoek, NamibiaCASCalifornia Academy of Sciences, San Francisco, California.The following features are explicitly defined and illustrated in ANTd number of teeth on the anterior margin of cheliceral fang furrow.Cl, Cw carapace length and width. Carapace length taken along the midline dorsal-most posterior position to the anterofrontal edge of the carapace (chelicerae are not included in length). Carapace width taken at the widest point.AME, ALE, PME, PLE anterior median, anterior lateral, posterior median, and posterior lateral eyes, respectively.LBl, LBw labium length and width taken from the longest and widest points, respectively.PTl, PTw male palpal tibia length and width.Bl palpal bulb length from embolus tip to the bulb base, taken in the ventral plane at its longest point.PTLs, TBs number of female prolateral patella and tibial spines leg III.STRl, STRw sternum length and width. Sternum length from the base of the labium to its most posterior point. Width taken across the widest point, usually between legs II and III.PLS posterior lateral spinneretTSrd, TSp, TSr number of tibiaI spines on the distal most retrolateral, prolateral, and midline retrolateral positions.ITC inferior tarsal clawsensuFormat, descriptors, and morphological features measured/examined follows closely TM, Richmond, VA) where images were recorded at multiple focal planes and then assembled into a single focused image using Helicon Focus . The female genital region was removed from the abdominal wall and tissues dissolved using trypsin; spermathecae were examined and photographed in the manner described above. Following Digital images of specimens were made using a BKPlus Digital Imaging System  using WGS84 map datum.http://zoobank.org/638FB63E-DB51-4FB5-85AF-C04E81D3DBD7urn:lsid:nmbe.ch:spiderfam:0007http://zoobank.org/4B5E1D34-582C-4259-BAE5-D5FE6AF68BEEurn:lsid:nmbe.ch:spidergen:00127Pionothele Purcell, 1902: 380 . – Taxon classificationAnimaliaAraneaeNemesiidaehttp://zoobank.org/87176CD8-22EB-4428-A293-80D16646EFD2http://species-id.net/wiki/Pionothele_gobabebBME) and additional male paratypes  from the Erongo Region, Namibia, in vicinity of Gobabeb Research & Training Center, along D1983 and Kuiseb River, – 23.56984 15.03984, coll. by J. Bond and T. Lamb 27.ix.2013.Male holotype . As such we do not describe the female as a paratype so as not to confuse the type locality or the identity of the species if the female specimen is eventually discovered to be a different species – acknowledging that mygalomorph spiders are known to be highly endemic with considerable species crypsis (see Bond & Stockman 2008). Nevertheless, we are reasonably confident that these specimens are conspecifics given similarities in morphology , habitat, and an explicit morphological species concept (applied herein).BME. Single female specimen (NMB012_001) from the Erongo Region, Namibia, in vicinity of Intersection C39 and Huab River, – 20.36035 14.19186898, coll. J. Bond 19.ix.2013, deposited in BME.Male specimens (12) collected in pitfall trips in vicinity of the type locality at Gobabeb, deposited in the Known only from the Erongo Region, Namibia.


S. aureus and non-influenza viruses. Clinical data evidenced that rhinovirus infection may increase the S. aureus carriage load in humans and its spread. In children, respiratory syncytial virus infection is associated with S. aureus carriage. The mechanisms by which some non-influenza respiratory viruses predispose host cells to S. aureus superinfection can be summarized in three categories: i) modifying expression levels of cellular patterns involved in S. aureus adhesion and/or internalization, ii) inducing S. aureus invasion of epithelial cells due to the disruption of tight junctions, and iii) decreasing S. aureus clearance by altering the immune response. The comprehension of pathways involved in S. aureus-respiratory virus interactions may help developing new strategies of preventive and curative therapy.Viral infections of the respiratory tract can be complicated by bacterial superinfection, resulting in a significantly longer duration of illness and even a fatal outcome. In this review, we focused on interactions between  The development of upper and lower respiratory tract infections is determined by the interaction between one micro-organism and the host immune response. More recently, the interaction between the resident microbiota and incoming pathogens may also participate in the development of respiratory tract infections.Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae that compose a significant part of the respiratory tract microbiota [It has been postulated for a long time that viral infection of the respiratory tract indirectly predispose to bacterial superinfection by disruption of the respiratory mucosal epithelium , or passcrobiota .S. aureus or S. pneumoniae [S. aureus and non-influenza respiratory viruses were not reviewed recently.The role of interactions between viruses and bacteria in the pathogenesis of respiratory infections have been extensively studied in the literature and notably those between influenza viruses and eumoniae –10. HoweS. aureus and non-influenza viral pathogens in the respiratory tract, with a focus on the mechanisms by which these interactions are potentially mediated.The aim of this report is to describe the current knowledge on possible interactions between S. aureus is a commensal bacterium of the skin and mucosa, colonizing 15 to 36% of the whole population [S. aureus carriage in humans remains the nose [S. aureus is however a Janus-faced bacterium and beyond its commensal status, it is also a life-threatening pathogen. It is in fact considered to be one of the leading causes of nosocomial and community-acquired bacterial infections [pulation ,12. The the nose , pharynxthe nose ,14, vagithe nose . S. aurefections . In addifections , it is afections ,19.S. aureus is also an important pathogen in lung infection, mostly implicated in hospital-acquired pneumonia [S. aureus expresses a wide repertoire of surface proteins that recognize cellular adhesive molecules and it is therefore able to adhere to and internalize into lung epithelial cells, which protects the bacteria from the host immune system and facilitate chronic infection [In vitro experiments have demonstrated that the activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathway in infected pulmonary epithelial cells results in inflammation enhancement via IL-8 expression; furthermore, intracellular S. aureus can lead, after an initial lag period, to the apoptosis of these cells. [S. aureus associated with the apoptosis of lung cells were shown to be pore-forming toxins, namely Panton-Valentine leukocidin (PVL) and alpha-hemolysin [neumonia . S. aurenfection . In vitre cells. ,22 In neemolysin ,24.S. aureus is frequently involved in secondary bacterial pneumonia occurring during seasonal influenza outbreaks [S. aureus was the most common pathogen, accounting for 27% of the cases in both critically ill children and adults [S. aureus co-infection was associated with significantly higher morbidity and mortality [S. aureus and the secondary staphylococcal pneumonia is still unclear. However, recent evidence from in vitro and in vivo tests showed that host physiologic changes induced by influenza virus can lead to the transition from asymptomatic colonization to invasive disease [S. aureus nasopharyngeal carriage has been found to be associated with some other respiratory viruses like rhinovirus and respiratory syncytial virus (RSV).Besides, utbreaks . During d adults ,26. S. aS. aureus. Several studies showed that natural or experimental rhinovirus infection in S. aureus nasal carriers leads to increased S. aureus airborne dispersal, especially when sneezing is a part of the syndrome [S. aureus from staphylococcal carriers to their environment and the transmission of the bacterium between humans. A recent study showed that rhinovirus infection is associated with changes in the upper respiratory tract microbiota [H. parainfluenzae, Neisseria subflava and S. aureus, and returned to their baseline level after the infection was cleared [S. aureus nasal load by 39% compared to baseline bacterial load [Rhinovirus is the second respiratory virus that has been most frequently reported to interact with syndrome –33. Thescrobiota . In this cleared . It has ial load . These fS. aureus, H. influenzae, M. catarrhalis and S. pneumoniae are the most common pathogens colonizing the nasopharynx and the lower airways [A prospective microbiological analysis showed that 40% of children with severe RSV bronchiolitis had a bacterial co-infection in their lower airways and were at increased risk for bacterial pneumonia . Bacteri airways ,36.S. aureus was not found among the most abundant bacteria. However, the difference of S. aureus abundance was significantly higher in RSV than in rhinovirus-infected infants [H. influenzae and S. pneumoniae, but negatively associated with S. aureus nasopharyngeal colonization [S. aureus was shown to colonize 77% of RSV-infected patients with a positive association between RSV and S. aureus nasopharyngeal carriage [A recent prospective study investigated the differences in the nasopharyngeal microbiome during acute respiratory tract infections due to human rhinovirus or RSV in 135 infants aged less than 6 months . By cont infants . Anothernization . In anotcarriage . The conS. pneumoniae [S. aureus is clearly more than a passive phenomenon during RSV infection and further studies are needed to elucidate the interactions between these 2 pathogens.To date, most of the studies dedicated to the impact of RSV infection on the bacterial colonization of the upper respiratory airways identified a synergistic interaction between RSV and eumoniae ,42. NeveS. aureus colonization is important and should not be neglected as this may worsen the disease outcome and even be fatal in some cases [There is no doubt that the impact of infection with respiratory non-influenza viruses on me cases . The recS. aureus adhesion and internalization within non-professional phagocytic cells through at least two distinct mechanisms: binding of bacteria to the membrane-associated hemagglutinin of influenza-infected cells, and binding of bacteria to free virions, followed by internalization of virus-coated bacteria into non-infected cells [S. aureus co-infection promotes influenza virus replication and pathogenicity; indeed, extracellular bacteria secrete staphylokinase that facilitates the binding of influenza virus to the host cells, whereas intracellular S. aureus inhibits influenza virus-induced type I IFN signaling through impaired signal transducers and activators of transcription (STAT1 and STAT2) dimerization [S. aureus and influenza virus seem to be deeply understood, the interactions between S. aureus and other respiratory viruses were less investigated. S. aureus and non-influenza respiratory viruses.The high incidence of staphylococcal superinfection during pandemic and seasonal influenza and the important mortality risk associated to this condition promoted the in-depth investigation of the molecular and immunologic mechanisms that are involved in the bidirectional synergism between both pathogens. Influenza virus is known to promote staphylococcal superinfection by alteration of the host immune system via increased production of pro-inflammatory cytokines and interferons (IFNs), impairment of phagocytic cell functions and suppression of type 17 immunity ,10. In aed cells . Besidesrization ,46. WhilRhinovirus, the most common cause of upper respiratory tract infections (URTI), primarily targets the nasal and nasopharyngeal epithelial cells . The innin vitro in epithelial cells of the upper and lower airways. In 1981, Selinger, Reed and McLaren developed an in vitro model for studying bacterial adherence to virus-infected epithelial cells [S. aureus was significantly higher in rhinovirus-infected cells compared to uninfected cells. Only recently, various in vitro studies have shown that inflammation due to rhinovirus infection increased cellular patterns that facilitate the adhesion and internalization of S. aureus within host cells [Several potential mechanisms through which rhinovirus increases susceptibility to bacterial infection have been demonstrated al cells . They rest cells –56.S. aureus in the presence of epithelial cells [S. aureus cell wall-anchored Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) that bind type I motif of fibronectin through a tandem beta-zipper interaction [S. aureus and the host cell, and the complex formed by FnBPA/B, fibronectin and α5β1 integrin leads to S. aureus internalization inside the epithelial cell [S. aureus epithelial cells adhesion [In primary human nasal epithelial cells, it has been demonstrated that rhinovirus infection up-regulates the expression of cellular fibronectin ,56. The al cells . The fiberaction . The α5βeraction . Fibroneial cell . Then thadhesion ,56.S. aureus to epithelial cells is also mediated by ICAM-1 [S. aureus uptake by the epithelial cells [The intercellular adhesion molecule 1 (ICAM-1), which is the receptor of the major group of rhinoviruses , have bey ICAM-1 ,54. Bothal cells . The up-al cells –63.S. aureus, was found to bind ICAM-1 [S. aureus to ICAM-1, whose expression is enhanced by rhinovirus infection, is mediated by EAP.Interestingly, the extracellular adherence protein (EAP), an adhesin naturally secreted by d ICAM-1 . EAP beld ICAM-1 , adhesiod ICAM-1 –68, and d ICAM-1 ,69–72. Id ICAM-1 ,65,73–75S. aureus and its epithelium invasion [H. influenzae and Pseudomonas aeruginosa [The mucosal barrier of the nasal cavity is the first site of exposure to inhaled respiratory pathogens and plays an important role in host defenses in terms of innate immunity. Its integrity is regulated in large part by tight junctions of epithelial cells , which iinvasion . The rhiruginosa . This prS. aureus have been shown to be bi-directional: whereas rhinovirus promotes S. aureus adhesion and internalization within host cells, S. aureus enhances the rhinovirus replication [S. aureus secretes several toxins including pore-forming toxins, toxic shock syndrome toxin and enterotoxins [In vitro, staphylococcal enterotoxins A and B (SEA and SEB) were shown to enhance the rhinovirus replication in A549 epithelial cells in a dose-dependent manner; however, they were found able to enhance neither ICAM-1 expression nor Il-1β, IL-6 and IL-8 secretion [Interactions between rhinovirus and lication . S. aurerotoxins . In vitrecretion . In contecretion –85. TherS. aureus. The different mechanisms described above are summarized in All these experimental data provide evidence of bi-directional synergism between rhinovirus and S. aureus and this virus [S. aureus co-infection via at least two mechanisms: enhancing the adhesion of S. aureus to the RSV-infected cells [S. aureus [RSV is a worldwide seasonal virus that affects mainly young children. RSV disease manifestations can vary from mild URTI to severe pneumonia or bronchiolitis, which can lead to hospitalization and serious complications like respiratory failure . Despiteis virus –90. RSV ed cells ,89, and . aureus ,90.In vitro studies had shown that RSV-infected epithelial cells bind more S. aureus than uninfected cells [S. aureus than cells expressing low concentrations of these antigens [S. aureus adhesion to host cells remain unknown.ed cells ,89. Studed cells . In addiantigens ,89. Whilantigens ,89. Thisantigens . NeverthS. aureus [S. aureus infection and decrease the bacterial clearance [Pro-inflammatory cytokines and type I IFNs are known to be secreted by RSV-infected epithelial cells after viral sensing by different PRRs, mainly through NFκB and Interferon Regulation Factors (IRFs) pathways . In addi. aureus . This in. aureus . IFN-γ wlearance ,94.S. aureus and other pathogenic bacteria like S. pneumoniae and P. aeruginosa from the lungs of mice following secondary bacterial infection [S. aureus following RSV infection.Murine models had been used to demonstrate that, despite the increased number of inflammatory cells, RSV decreases the clearance of nfection . It was S. aureus interactions with rhinovirus or RSV have been actively, yet insufficiently, investigated. However, bacterial interactions with other respiratory viruses are poorly studied and only partial studies are available. For example, parainfluenza virus has been shown to enhance the ability of non-typeable H. influenzae and S. pneumoniae to adhere to human respiratory epithelial cells [S. aureus in humans. Only one in vitro study conducted on bovine embryonic lung cells investigated the effect of bovine parainfluenza virus infection on adherence of several bacterial agents, including S. aureus, and found no virus-specific effect on any of the tested bacteria [S. pneumoniae resulted in an increased susceptibility to infection with human metapneumovirus. Nevertheless, this was not the case for cells pre-incubated with H. influenzae, M. catarrhalis or S. aureus [S. aureus and coronavirus has been demonstrated in vivo in a swine model. Lipoteichoic acid from S. aureus increased the susceptibility to coronavirus infection in pigs via increased secretion of pro-inflammatory cytokines IL-6, IL-12, IL-23 and IFN-γ[S. aureus colonization and other respiratory viruses complicates the understanding of potential interactions between these pathogens.al cells . Howeverbacteria . Besides. aureus . On the and IFN-γ. To dateS. aureus and influenza virus have been deeply studied, the interactions between this bacterium and other common respiratory viruses were only partially investigated [S. aureus carriage and non-influenza respiratory virus infections, as well as deeper insights into mechanisms of interactions between these different pathogens. The evaluation of the concrete risk of switch from commensal S. aureus colonization to invasive disease during viral respiratory tract infections may help us to propose efficient strategies of decolonization when needed. Furthermore, the comprehension of the involved molecular pathways may help to develop new strategies of preventive and curative treatments.While clinical and mechanistic aspects of the synergism between stigated . LiteratCDHR3: Cadherin-Related Family Member 3; cFn: cellular Fibronectin; dsRNA: double-stranded RNA; ENA78: Epithelial Neutrophil Activating peptide-78; FnBP: Fibronectin Binding Protein; HRV: Human Rhinovirus; ICAM-1: InterCellular Adhesion Molecule 1; IFN: Interferon; IL: Interleukin; IRFs: Interferon Regulator Factors; IκB: NFκB Inhibitor; LDLR: Low-Density Lipoprotein Receptor; MDA5: Melanoma Differentiation Associated protein-5; NFκB: Nuclear Factor kappa-light-chain-enhancer of activated B cells; NKG2D: Natural Killer Group 2D receptor; Nod2: Nucleotide-binding oligomerization domain-containing protein 2; NOX: membrane-bound NADPH OXidase; Rac1: Ras-related C3 botulinum toxin substrate 1; RANTES: Regulated on Activation, Normal T cell Expressed and Secreted chemokine; RIG-I: Retinoic acid Inducible Gene-1; ROS: Reactive Oxygen Species; S. aureus: Staphylococcus aureus; SEA: Staphylococcal Enterotoxin A; SEB: Staphylococcal Enterotoxin B; ssRNA: single-stranded RNA; TLR: Toll-Like receptor; ZO-1: Zona Occludens 1.


Corythucha ciliata (Say) is a highly invasive pest insect that feeds on sycamore trees (Platanus spp.) worldwide. The interaction between Platanus species and this insect pest has not yet been studied at the molecular level. Therefore, a recent study was conducted to compare the gene expression and metabolite profiles of Platanus acerifolia leaves in response to C. ciliata feeding damage after 24 and 48 h. We employed high throughput RNA sequencing (RNA- seq) to identify a total of 2,828 significantly differentially expressed genes (DEGs) after C. ciliata feeding. In addition, 303 unigenes were found to be up-regulated at both time points. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that monoterpenoid biosynthesis, the linoleic acid metabolism pathway, and alpha- linolenic acid metabolism were the most prominent pathways among the DEGs. Further analysis of the metabolite profiles showed that nine metabolites were significantly different before and after C. ciliata damage. In addition, we analyzed DEGs detected in the P. acerifolia and C. ciliata interaction using Mapman. The terpene synthase gene family was also identified. We suggest that the results obtained from DEGs and metabolite analysis can provide important information for the identification of genes involved in the P. acerifolia–C. ciliata interaction, which might be necessary for controlling C. ciliata efficiently.The sycamore lace bug,  Platanus L. is an important genus of large trees commonly known as sycamore or plane trees. Platanus species are widely planted all over China as shade trees due to their large spreading crown, and also for their rapid growth and pruning resistance. The sycamore lace bug, Corythucha ciliata  (Hemiptera: Tingidae), which is native to the central and eastern parts of North America, was subsequently introduced into Europe and Asia [C. ciliata specifically damages members of the Platanus genus, including Platanus occidentalis, P. acerifolia, and P. orientalis. The most important relationship between P. acerifolia and C. ciliata is that P. acerifolia is the most widely planted Platanus species in China [C. ciliata was first discovered in Changshan, China in 2002. So far, C. ciliata has been observed to cause damage to P. acerifolia trees in Hunan, Hubei, Shanghai, Zhejiang, Jiangsu, Shandong, Henan, Chongqing, Guizhou, and Beijing in China [C. ciliata infestation can cause growth disruption, weakness, and even death in P. acerifolia, and it has seriously affected landscaping in many Chinese cities. Currently, the management of C. ciliata still mainly depends on chemical insecticides [P. acerifolia is a widely planted street tree in urban areas, so the use of pesticides does not only cause serious environmental pollution but could harm human health. Therefore, to understand the interaction mechanism between P. acerifolia and C. ciliata would provide a great help to accelerate the development of an effective environmentally friendly pest management strategy to manipulate the populations of C. ciliata which is also problematic for other Asian and European countries. ata Say, 832 . The control group was represented by leaves not infested by insects. Also, 12 cDNA libraries were constructed for RNA sequencing. The sequencing data is stored in the NCBI Sequence Read Archive ; under BioProject ID number PRJNA484863 and accession numbers SRR8631807 to SRR8631818. High throughput DNA sequencing generated 7.08 × 109 to 8.66 × 109 raw bases per sample, of which >93% had Q-scores between 20 and 30  between the feeding treatments vs. the controls at each time point. A p-adjust value ≤0.05 and |log2 fold change |≥1 were set as the thresholds to detect significant DEGs. A total of 2790 DEGs was identified between the 24 h treatment and the control  term enrichment. For the 24 h treatment, the significantly enriched GO terms in the DEGs, 159 GO terms were significantly enriched, the ten most significant terms were: oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen (GO:0016702), dioxygenase activity (GO:0051213), monocarboxylic acid biosynthetic process (GO:0072330), lipid biosynthetic process (GO:0008610), terpene synthase activity (GO:0010333), fatty acid metabolic process (GO:0006631), organic acid biosynthetic process (GO:0016053), carboxylic acid biosynthetic process (GO:0046394), monooxygenase activity (GO:0004497), heme binding (GO:0020037) (To investigate the functions of the DEGs detected in the 0020037) . In the 0020037) . Also, f0020037) .C. ciliata damage. “Monoterpenoid biosynthesis”, “Linoleic acid metabolism” and “alpha-Linolenic acid metabolism” were prominent pathways between the treatments and controls in response to C. ciliata feeding damage after 24 and 48 h , Mitogen-Activated Protein Kinases (MAPK), Pathogenesis-Related (PRs), and PR-proteins, is shown in P. acerifolia–C. ciliata interaction and their MapMan functional categories is given in To reveal the role of DEGs in the plant response to P. acerifolia leaves. We identified 14 putative terpene synthase (TPS) genes from our RNA-seq dataset. Among them, seven showed significantly up-regulated expression after both 24 and 48 h of C. ciliata feeding. These up- regulated TPS genes may be involved in enhancing resistance to C. ciliata by producing volatiles that either repel this pest or attract its natural enemies. To examine the phylogenetic relationships among these TPS proteins and those from several other species, we constructed an unrooted neighbor-joining phylogenetic tree. The TPS genes were classified into five different families (clades) named TPS-b to TPS-g  ; these included glycoside, cinnamate, indole- 3- acetamide, L-tryptophan, epigallocatechin, L-fucitol and three amino acids    and Partis (PCA) A analyse serine) B. Flavon serine) . In addi serine) . These gTo validate the unigene TPM values obtained by RNA-seq, the expression of 32 unigenes was tested using qRT-PCR assays. The primers were listed in C. ciliata, is an important insect pest that has been causing damage to P. acerifolia trees in recent years [C. ciliata usually feeds on the undersides of P. acerifolia leaves, and the leaves develop white spots at the beginning of feeding at the upper side that can cause the leaves to appear chlorotic. The damage caused by this insect pest can reduce leaf photosynthesis, leading to a decline in some physiological and biochemical indexes such as leaf photosynthetic rate, stomatal conductance, transpiration rate, chloroplast pigments and soluble sugars. C. ciliata can only complete its life cycle on the sycamore plant. Based on the importance of the host plant and the limited host specificity of this pest, studying the molecular mechanisms behind the P. acerifolia– C. ciliata interaction is important for understanding the expression of resistance in Platanus species to this insect pest. The sycamore lace bug, nt years . C. ciliC. ciliata-induced changes to the leaf transcriptome of P. acerifolia. By analyzing gene expression profiles at two different time points, we identified 1907 and 921 unigenes that were significantly up-regulated after 24 and 48 h of feeding, respectively. Previous studies have shown that insect stress can induce massive changes at the transcriptional level in some plants ,11,12. TC. ciliata feeding. These 303 genes are the key core genes expressed during the interaction between P. acerifolia and C. ciliata. Combining the GO, KEGG enrichment analysis, and MapMan results, we speculated that JA-related pathways and terpenoids play an important role in this plant–insect interaction system. The plant hormone JA plays vital roles in regulating the plant response to various insect stresses [C. ciliata feeding damage. This indicates that in P. acerifolia, JA signal transduction may act through a mechanism similar to that in other plants. At the same time, in addition to JA pathway-related genes, we also identified related functional genes, especially genes that encode peroxidases and a series of transcription factors. The identification of these genes will provide the basis for the subsequent development of insect-resistant Platanus trees through genetic screening and possibly through genetic transformation.A total of 303 genes were up-regulated in leaves after both 24 and 48 h of stresses . In our P. acerifolia damaged by C. ciliata [E)-4,8-dimethyl-1, 3,7-nonatriene, and trans-β-caryophyllene were detected in undamaged leaves. In addition, another five chemicals, α-thujene, sabinene, myrcene, cis-3-hexenyl acetate, and γ-terpinene, were induced in leaves after feeding by C. ciliata. Among these volatile chemicals, cis-3-hexen-1-ol and 1,8-cineole were found to significantly attract C. ciliata, whereas trans- β-caryophyllene repelled this insect pest [The controlled production of plant volatiles plays an important role in the indirect plant defense response against insects . Followi ciliata . Four chect pest . Our stuP. acerifolia and C. ciliata. We also identified the candidate key enzymes involved in the biosynthesis of this compound by analyzing our leaf transcriptome dataset. Functional studies on the proteins encoded by these candidate genes need to be performed to determine their roles in epigallocatechin biosynthesis. A biological study should be carried out to observe the effects of epigallocatechin on feeding, development, and oviposition by C. ciliata.Plant secondary metabolites also play important roles in plant-insect interactions . InsectsP. acerifolia and C. ciliata using transcriptome sequencing and metabolite analysis. The high-throughput RNA sequencing technology used in this study provides a comprehensive look at the transcriptome and does not rely on the limited genomic resources presently available for Platanus species. It is worth noting that this study was performed on detached leaves and this could not fully represent the response of leaves on the tree under natural conditions. In the future, similar studies using leaves under natural conditions are needed to further confirm the key candidate genes and compounds found in the study. Additionally, further functional research on DGEs identified here should be carried out in order to clarify their specific roles in plant–insect interactions, thereby enabling the development of insect-resistant sycamore trees and providing a basis for the development of strategies to protect sycamore trees and to manage C. ciliata infestations.Our study examined the intrinsic mechanisms underlying the interaction between Corythucha ciliata nymphs were collected from P. acerifolia trees planted at the Beijing Forestry University in Haidian District, Beijing. The nymphs were raised indoors on fresh P. acerifolia leaves at room temperature (25 °C ± 2 °C), a relative humidity of 50%–70%, and a 16 h day/8 h night photoperiod. The P. acerifolia leaves used in the experiments were collected from the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences in Haidian District, Beijing. Only healthy and untreated leaves were selected for the experiments. The leaves were removed from the tree by cutting the base of the petiole with a sterile scalpel, and they were inserted into narrow glass test tubes containing 10 mL sterile water. After the insertion of the leaf, the petioles were sealed with absorbent cotton and placed in a 2 L glass beaker. One-hundred newly-emerged C. ciliata adults were placed on each leaf. The experiments were carried out in artificial climate chambers . The measurements in three biological replicates at each of two different time points after infestation of C. ciliata to P. acerifolia leaves (24 and 48 h) were performed. The control group was represented by leaves not infested by insects. After the feeding periods were finished, the insects on the leaves were removed with a soft brush. RNA was extracted from the leaves for RNA-sequencing and qRT-PCR assays.http://www.idtdna.com/primerquest/Home/Index). The cDNA was synthesized by Prime Script RT Reagent Kit  . The qRT-PCR was performed using GoTaq 2-Step qRT-PCR System  and on the ABI Prism1 7500 . qRT-PCR was performed with the follow PCRcycling condition: 95 °C for 10 s, followed by 40 cycles of 95 °C for 30 s and 60 °C for 1 min. Two housekeeping genes, actin (TRINITY_DN78316_c0_g1) and Alpha tubulin (TRINITY_DN106409_c0_g1), were selected to use as reference genes for normalization of gene expression in the qRT-PCR analysis. The relative expression levels of each gene were evaluated by the comparative2-ΔΔCt.Total RNA was isolated from plant tissues (leaves) using the Trizol reagent , and RNA-seq libraries were prepared according to the manufacturer’s protocol . The libraries were then sequenced on an Illumina Hiseq 2500 instrument  to produce 150 bp paired-end reads. qRT-PCR assays were conducted according to our previously described method . Primershttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All downstream analyses were based on high quality, clean sequencing read data. De novo assembly of the transcriptome was performed with Trinity 2.4.0 with default parameter [http://www.ncbi.nlm.nih.gov/) by blastx with a threshold of E <0.00001. RSEM was used to determine the expression levels for unigenes by calculating TPM [http://bioinformatics.psb.ugent.be/webtools/Venn). GO and KEGG enrichment analyses were then performed on the differentially expressed unigenes using Goatools [P.acerifolia transcriptome assembly using the HMMER package [We first used fastp  to removarameter . All assting TPM . The difting TPM . Venn diGoatools  and KOBAGoatools , respectGoatools  for a gr package  with a t package  with 100https://www.metaboanalyst.ca) [The leaf samples treatment method is the same as for RNA-seq except for six biological replicates; leaf metabolites were extracted using a previously described method . Brieflylyst.ca) . The metlyst.ca) . The met


Transcriptome sequencing has become a method of choice for evolutionary studies in microbial eukaryotes due to low cost and minimal sample requirements. Transcriptome data has been extensively used in phylogenomic studies to infer ancient evolutionary histories. However, its utility in studying cryptic species diversity is not well explored. An empirical investigation was conducted to test the applicability of transcriptome data in resolving two major types of discordances at lower taxonomic levels. These include cases where species have the same morphology but different genetics (cryptic species) and species of different morphologies but have the same genetics. We built a species comparison bioinformatic pipeline that takes into account the nature of transcriptome data in amoeboid microbes exemplifying such discordances.Our analyses of known or suspected cryptic species yielded consistent results regardless of the methods of culturing, RNA collection or sequencing. Over 95% of the single copy genes analyzed in samples of the same species sequenced using different methods and cryptic species had intra- and interspecific divergences below 2%. Only a minority of groups (2.91–4.87%) had high distances exceeding 2% in these taxa, which was likely caused by low data quality. This pattern was also observed in suspected genetically similar species with different morphologies. Transcriptome data consistently delineated all taxa above species level, including cryptically diverse species. Using our approach we were able to resolve cryptic species problems, uncover misidentification and discover new species. We also identified several potential barcode markers with varying evolutionary rates that can be used in lineages with different evolutionary histories.Our findings demonstrate that transcriptome data is appropriate for understanding cryptic species diversity in microbial eukaryotes.The online version of this article (10.1186/s12862-018-1283-1) contains supplementary material, which is available to authorized users. The vast majority of eukaryotic diversity is microbial, but many aspects of their behavior and biodiversity remain poorly understood –3. Our uThe problem of cryptic species in taxonomy has been known since Linnaean time. The full extent of the challenges it posed to taxonomy and other related fields such as conservation biology, agriculture and diagnostic medicine was realized with the advent of molecular techniques –24. CommDNA sequencing of single or few markers, selected for their DNA barcode potential, has played an instrumental role in uncovering hidden diversity in living organisms , 29–31. Since genomes contain the history of an organism, an ideal solution to resolving cryptic species diversity is to analyze whole genome data. However, this endeavor is not feasible due to the associated high cost as well as limited understanding of microbial genomes. Low cost alternative HTS approaches are allowing the generation of tens of thousands of genes from under-sampled microbes . While mCochliopodium and Endostelium representing diverse groups within one of the major subclades (Discosea) of Amoebozoa. Our transcriptomic comparative analysis revealed a consistent pattern of inter- and intra-specific divergences among known or suspected cryptic species. We also identified several barcode markers with varying evolutionary rates that can be used in microbial eukaryotes. Our findings demonstrate the appropriateness of transcriptome data for cryptic species diversity studies.In this study we used transcriptome data to address cryptic species diversity problems in microbial eukaryotes. We developed a bioinformatics pipeline suited to handle transcriptome data for comparative study taking into account the nature of sequence quality and paralogy. We analyzed up to 30,000 transcripts (contigs) per sample in amoeboid microbes exemplifying the two major discordances of molecular and morphological data. These lineages including a thecamoebida isolate, Dictyostelium discoideum and Acanthamoeba castellanii using OrthoVenn [We built a bioinformatic pipeline for comparative analysis of genomic and transcriptomic data from multiple species see Fig. . This pirthoVenn  Fig. 1)DictyosteOur transcriptomic comparative analysis reveals a similar pattern of inter- and intra-specific divergences among known (named species with distinct morphology) or suspected cryptic species, species indistinguishable with genetic or morphological data Table . When isCochliopodium, C. minus (CCAP 1537/1A) and C. pentatrifurcatum (ATCC 30935), were originally described as separate species based on morphological analysis [C. minutoidum, a closely related species, which is clearly distinct in the barcoding markers [C. minus, 20,630 C. pentatrifurcatum, and 8561 C. minutoidum contigs, we extracted 12,767 orthologous subgroups, of which 10,829 had at least one sequence from both C. pentatrifurcatum and C. minus. This set of groups was filtered to keep only those that were classified as single-copy and which contained at least one identified eukaryotic sequence  contained sequences that were no more than 2% divergent between the two species  with C. minutoidum, no groups were below the 2% divergence cutoff . No contig groups less than 2% divergent were found , we noticed that many sequences from our new isolate were very similar or identical to sequences published by Kang et al.  for theiWe used criteria including pattern of sequence divergence (COI-like), ubiquity, nature of paralogy and evolutionary conservation to choose potential barcode markers among transcriptome contigs analyzed. A total of 660 clusters were present in at least 2 of the amoebozoan clades analyzed, of which 217 were present in all three clades  and require several trials to amplify single gene products since most of the universal primers designed for multicellular eukaryotes fail to work in most microbes . For thiOne of the anticipated challenges in comparative transcriptomic study in cryptic or closely related species is that the results might vary based on the physiological state of an organism at the time of RNA collection . MoreoveDistance calculations of matched groups from whole transcriptome data show an interesting pattern among isolates of the same species originating from different labs, as well as genetically similar (suspected cryptic) species. Divergences exceeding 2% between these isolates and species accounted for comparable proportions of the contigs  and similar morphologies with different genetics (Endostelium).Amoeboid eukaryotes belonging to the supergroup Amoebozoa include diverse lineages that are largely understudied. In the last 3 years the supergroup has seen an explosion of transcriptome data mostly used to study deep relationships within the supergroup , 46, 55.Cochliopodium is a genus of lens-shaped amoeba in which taxonomy has greatly relied upon the morphology of flexible microscales present in the cell coat [Cochliopodium with dramatically different scale morphology have identical COI and SSU-rDNA gene sequences [Cochliopodium pentatrifurcatum and C. minus) are the same species despite their drastic difference in scale morphology [Cochliopodium is made of proteinaceous material and is encoded in the genome [. Some Cochliopodium species have even been observed to possess two types of scales in one individual . Similarly, Amoebae belonging to the genus Korotnevella have also been reported to sometimes express more than one type of scale [Cochliopodium isolates (C. pentatrifurcatum and C. minus) originated from different localities but were grown under similar culturing conditions in our laboratory, so environmental factors are unlikely to explain the difference in scale morphology in these species. Further investigation is needed to examine the factors affecting expression of different scale morphologies in Cochliopodium and other amoebae. Given the overwhelming genetic evidence and the unreliability of scale morphology in the genus, we recommend the synonymization of Cochliopodium pentatrifurcatum to C. minus based on taxonomic priority. Similarly, using the same approach we discovered that two undescribed isolates of amoebae belonging to clade Thecamoebida from our lab and the publication of Kang et al. [ell coat , 57. Recequences , 35. Thirphology , 45. Scae genome . It is nof scale . The Cocg et al.  are consEndostelium. Kang et al. [‘E. zonatum LINKS’ in their phylogenomic study. Comparison of this isolate with our and their E. zonatum PRA-191 showed that the two isolates (LINKS and PRA-191) are very divergent (average 19.5%), far beyond the species delimitation thresholds used in any organism , Cochliopodium minus (CCAP 1537/1A), and Endostelium zonatum (PRA-191) from previous studies [Endostelium zonatum PRA-191 (SRX2163157), Endostelium zonatum LINKS (SRX2691243), and Thecamoebida isolate RHP1–1 (SRX2691210) from Kang et al. [Cochliopodium minutoidum (CCAP 1537/7) and a new Thecamoebida isolate designated as UK-YT1 . New transcriptome data were collected using the same protocol, for total RNA, as in [Transcriptomes of  studies , 60 wereg et al.  were alshttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to inspect reads from Kang et al. [http://transdecoder.sf.net) was then run on the eukaryotic and unidentified contigs from each transcriptome to trim non-coding regions and collect the resulting CDS’s, which were then used for the subsequent transcriptome-wide comparisons.FastQC . The reiopodium , 29. Thiiopodium  to matchCochliopodium (C. pentatrifurcatum ATCC 30935 vs. C. minus CCAP 1537/1A (transcriptomes from two independent samples each) vs. C. minutoidum CCAP 1537/7), Endostelium , and Thum LINKS  isolatesDictyostelium discoideum and Acanthamoeba castellanii. This set of 2054 genes was extracted from comparing the genomes with OrthoVenn [Distances within OSGs generated by the pipeline were organized into three partitions; all OSGs, only single-copy OSGs (i.e. HGs which needed no subdividing) containing identified eukaryotic contigs, and only OSGs from the above which matched a set of 2054 single-copy genes shared between the genomes of rthoVenn , a web aC. pentatrifurcatum, C. minus, and E. zonatum PRA-191 were less than 2%, where they exist. OSGs from each comparison were combined based on their matching OrthoVenn cluster, and clusters were kept only if at least two genera  were represented. Clusters with sequences from all analyzed transcriptomes were examined by BLAST and accession number of genome sequences were used to determine gene identity.Potential barcodes were selected from the single-copy, OrthoVenn cluster-matching OSGs if their intraspecific distances in Additional file 1:Figure S1. Histogram of average distances within ortholog groups, including single- and multi-copy identified and unidentified groups, from genus Cochliopodium. A. C. pentatrifurcatum intraspecific variation. B. C. minus intraspecific variation. C. C. pentatrifurcatum-C. minus inter-specific distance. D. C. pentatrifurcatum-C. minutoidum inter-specific distance. (PDF 88 kb)Additional file 2:Figure S2. Histogram of average distances within genome-matching and nonmatching single-copy eukaryotic ortholog groups from genus Cochliopodium. A. C. pentatrifurcatum intraspecific variation. B. C. minus intraspecific variation. C. C. pentatrifurcatum-C. minus inter-specific distance. D. C. pentatrifurcatum-C. minutoidum interspecific distance. (PDF 88 kb)Additional file 3:Figure S3. Histogram of average distances within ortholog groups, including single- and multi-copy identified and unidentified groups, from genus Endostelium. A. E. zonatum PRA-191 transcriptomes sequenced in different labs [E. zonatum PRA-191 sequenced by Kang et al. [‘E. zonatum’ LINKS sequenced by Kang et al. [ent labs , 46. B. g et al.  vs. ‘E. g et al. . . B. E. ood 2017  vs. ‘E. g et al. .


Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, are believed to harbour similar potencies to provoke hepatobiliary diseases in their definitive hosts, although their populations have substantially different ecogeographical aspects including habitat, preferred hosts, population structure. Lack of O. felineus genomic data is an obstacle to the development of comparative molecular biological approaches necessary to obtain new knowledge about the biology of Opisthorchiidae trematodes, to identify essential pathways linked to parasite-host interaction, to predict genes that contribute to liver fluke pathogenesis and for the effective prevention and control of the disease.The three epidemiologically important Opisthorchiidae liver flukes O. felineus and its gene repertoire accompanied by a comparative analysis with that of O. viverrini and Clonorchis sinensis. We observed both noticeably high heterozygosity of the sequenced individual and substantial genetic diversity in a pooled sample. This indicates that potency of O. felineus population for rapid adaptive response to control and preventive measures of opisthorchiasis is higher than in O. viverrini and C. sinensis. We also have found that all three species are characterized by more intensive involvement of trans-splicing in RNA processing compared to other trematodes.Here we present the first draft genome assembly of All revealed peculiarities of structural organization of genomes are of extreme importance for a proper description of genes and their products in these parasitic species. This should be taken into account both in academic and applied research of epidemiologically important liver flukes. Further comparative genomics studies of liver flukes and non-carcinogenic flatworms allow for generation of well-grounded hypotheses on the mechanisms underlying development of cholangiocarcinoma associated with opisthorchiasis and clonorchiasis as well as species-specific mechanisms of these diseases.The online version of this article (10.1186/s12864-019-5752-8) contains supplementary material, which is available to authorized users. Opisthorchis felineus  is a member of the triad of epidemiologically important fish-borne liver trematodes, which also includes O. viverrini  and Clonorchis sinensis . These liver flukes are known to cause serious human diseases affecting bile ducts and the gall bladder. Liver fluke infection is recognized as the major risk factor of cholangiocarcinoma , we triO. felineus and C. sinensis. The expression of these three genes in O. felineus exceeded the expression of the homologs in C. sinensis more than 20–100-fold. Another group of differentially expressed genes were CAP domain genes . Their expression in O. felineus was 50–200-fold higher as compared with the homologous C. sinensis genes. The group of differentially expressed genes for the pair O. felineus–O. viverrini also includes the genes encoding ribosomal proteins L19 (two genes out of two in the genome), papain family cysteine proteases (2 genes out of 25) and glyceraldehyde 3-phosphate dehydrogenase (one out of three).Kunitz-type inhibitors (3 out of 14 genes in the genome) were observed among the genes differentially expressed in Interestingly, products of majority of the differentially expressed genes found in our study contain domains characteristic for helminth-secreted proteins. In particular, CAP protein family , papain family cysteine proteases and Kunitz-type inhibitors are among the most represented compounds of secretome across 44 helminth species . The proO. felineus and other carcinogenic trematodes, S. haematobium and O. viverrini [The detoxification system of the liver fluke is of special interest, since its components are promising pharmacological targets –32. Detoiverrini , 34.Schmidtea mediterranea), whereas the parasitic species  most likely have only one cytochrome P450 [O. felineus CYP is involved in the metabolism of exogenous substrates, is important for survival of adult individuals, and represents a promising target for anthelminthic therapy [O. felineus detoxification phase I is also represented by the genes encoding aldo-keto reductases, aldehyde dehydrogenases, and alcohol dehydrogenases  are among these proteins. CYPs family in parasitic and free-living flatworms is drastically different: the free-living species have dozens of diverged CYP genes  and glutathione-S-transferase (GST). The Haemonchus contortus genome [C. elegans genome [O. felineus genome as well as in the genomes of other Opisthorchiidae and Schistosomatidae. In addition, we did not find any arylamine N-acetyltransferases (PF00797), which are also common phase II enzymes in vertebrates. Thus, the detoxification phase II of parasitic flatworms has some features distinguishing it from the corresponding systems of the other organisms, including the host.UGTs, common phase II xenobiotic metabolism enzymes in vertebrates, enhance hydrophilicity and availability of substrates to efflux transporters. The UGT superfamily comprises two families (UGT1 and UGT2) and over 20 isozymes. UGTs play a significant role in drug resistance of helminths . Currents genome  and 72 Us genome . We failO. felineus genome contains 23 genes encoding ABC transporters. Four of ABC genes are homologous to human P-glycoprotein , ABCC1 , and ABCG2  , 39. TheO. felineus genome. Most eukaryotes have only one NPC2 gene. Earlier, 25 genes coding for NPC2-like proteins have been detected in the O. viverrini and C. sinensis genomes [O. felineus metacercariae and adult has shown that 43 of the observed 48 NPC2 proteins are differentially expressed in metacercariae rather than in adult worm  encode the proteins that are potentially involved in intracellular and extracellular transport of sterols. We have discovered 48 NPC2 genes in the  genomes . The fun genomes . Such diO. viverrini granulin (Ov-GRN-1) stimulates proliferation of cholangiocytes [O. felineus granulins are yet vague.Granulins, the growth factors secreted by liver flukes, are involved in the development of cholangiocarcinoma . As has giocytes . The proOv-GRN-4 gene in the O. viverrini genome is fragmented  display high interspecific conservation but still are considerably different from both GRN-1/GRN-4 and one another  activates proliferation of hemocytes, thereby providing resistance to S. mansoni infection [Four genes (GRN-1–GRN-4) coding for single domain granulins have been detected in the three studied opisthorchiid genomes as well as one gene of a multidomain progranulin, PGRN Fig. . The gented Fig. a, possibnfection .Fig. 8GrO. felineus genome size is approximately 684 Mbp, being slightly longer as compared with С. sinensis and almost the same as the O. viverrini genome; and all three genomes have very similar content and diversity of repetitive elements. The total number of the predicted O. felineus protein-coding genes is approximately by one-third smaller as compared with O. viverrini and C. sinensis [O. felineus genome or bona fide loss of genes. Actually, the obtained O. felineus gene annotation showed even superior indicators of completeness in BUSCO evaluation  sequences by Pitaksakulrat et al. [O. felineus, C. sinensis and O. viverrini obtained by analysis of concatenated amino acid sequences from mitochondrial protein-coding genes by Wang et al. [The phylogenetic relationships between the genera Opisthorchis and Clonorchis is controversal . Kang etiverrini . Similara et al. , cox1, nt et al. . The samg et al. . and Liug et al. .C. sinensis and O. viverrini species grouping together and O. felineus representing a sister group. Our results are in agreement with the phylogenetic tree estimated using paramyosin gene (Pm-int9) [In our work analysis of 1563 protein families suggested the phylogenetic tree topology with Pm-int9) , mitochoPm-int9) .O. felineus and C. sinensis are closely related and do not support separation of C. sinensis from the genus Opisthorchis. Presumably, C. sinensis occupies an intermediate position between O. felineus and O. viverrini. The geographic vector of distribution of opisthorchiasis and clonorchiasis foci also favors this hypothesis. The opisthorchiasis caused by O. felineus has been recorded in many European countries; however, the most intense foci of this disease are in North Asia, namely, in Russia and Kazakhstan [O. viverrini opisthorchiasis are in the Southeast Asia, namely, in Thailand, Laos, Vietnam, and Cambodia [Taken together, the results of analysis of the synteny between three opisthorchiid species and of their phylogenetic relationships demonstrate that zakhstan . Clonorczakhstan . The maiCambodia .Schistosoma mansoni [O. felineus, e.g. the resolution of polycistronic gene products.We have found that all three studied opisthorchiidae species are characterized by much more extensive involvement of trans-splicing in RNA processing compared to the most well-studied trematode,  mansoni . Since tArabidopsis thaliana, Drosophila melanogaster, and C. elegans [The bimodality of intron length distributions has been observed also in many eukaryotic species. Two classes of introns, with a peak of short introns or microintrons and a much flatter peak of longer introns ranging up to thousands of base pairs, are present in humans,  elegans . Some Ci elegans . Two pea elegans  and Mono elegans . HoweverO. felineus annotation only. While expression levels of a number of genes showed remarkable differences between the species, the overall expression profiles were highly consistent across the compared species, suggesting a high similarity of all biological pathways in adult liver flukes that colonize the bile ducts of mammals.There are some difficulties in comparative analysis of gene content and expression between three liver flukes, since the existing genome assemblies are draft, and the corresponding gene annotations are far from being complete and compatible. However, we have made an effort to generate a congruent set of orthologous expression units based on O. felineus genomic data is an obstacle to the development of comparative molecular biological approaches necessary to obtain new knowledge about the biology of epidemiologically important Opisthorchiidae trematodes, to identify essential pathways linked to parasite-host interaction, to predict genes that contribute to liver fluke pathogenesis and for the effective prevention and control of the disease. Here we present the first draft genome assembly of O. felineus and its gene repertoire accompanied by a comparative analysis with that of O. viverrini and Clonorchis sinensis.Lack of O. felineus genome we provide, and comparative transcriptomics data will help support the development of novel drugs and vaccines for the treatment and prevention of liver fluke infection.This study contribute to comparative genomics of flatworms, molecular mechanisms of RNA processing in helminths and evolutionary history of Opisthorchiidae trematodes. The availability of http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm. The animals were kept and treated according to the protocols approved by the Committee on the Ethics of Animal Experiments with the Institute of Cytology and Genetics .All of the procedures were in compliance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for animal experiments O. felineus metacercariae were collected from naturally infected fish (Leuciscus idus) caught in the Ob River near Novosibirsk (Western Siberia) and routinely extracted [Mesocricetus auratus) were purchased from the Animal Breeding Facility with the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences. Five hamsters aged 6 to 8 weeks were orally infected with 75 O. felineus metacercariae. Euthanasia was performed using carbon dioxide, and all efforts were made to minimize suffering. The animals were maintained in their home cage while CO2 was induced at a flow 10% per minute. Adult flukes were recovered from the hepatobiliary tract of hamsters three months after the infection, pooled and thoroughly washed with saline.Two thousands of xtracted , 61. Terhttps://www.ncbi.nlm.nih.gov/sra in the research: ERR604978-ERR604981, SRR189060 for C. sinensis; SRR497632, SRR497633 for O viverrini; ERR576952, ERR576954, ERR576956, ERR576958, ERR576968 for F hepatica; ERR1328228-ERR1328233, ERR1328243, ERR1328260-ERR1328267 for S mansoni. Genome assemblies and gene annotations were taken from WormBase ParaSite (version WBPS6) [O. viverrini) [C. sinensis) [S. mansoni) [E. multilocularis) [M. lignano) [F. hepatica (PRJEB6687) [To analyze transcriptomic data Sequence Read Archives (SRA) were taken from NCBI n WBPS6) , 63: PRJverrini) , PRJDA72inensis) , PRJEA36mansoni) , PRJEB12cularis) , PRJNA28lignano) , F. hepaJEB6687) .O. felineus worm was fragmented in a Covaris S2  sonicator to an average fragment size of 200–500 bp and the sizes selected on Pepin Prep  for the insert were 180 and 270 bp. Construction of genome libraries and all subsequent manipulations were performed using paired-end library preparation kit in accordance with the manufacturer’s protocols . Genome libraries were suitable for paired-end reading. Sequencing was performed on Illumina Genome Analyzer II (GAII) and HiSeq1500  platforms. For mate-pair libraries, the gDNA was isolated from the pool of 50 randomly chosen worms, and Nextera Mate Pair Sample Preparation Kit  was used according to manufacturer’s protocols. Three different insert size libraries of 3, 5, 8, and 10 kb were sequenced on Illumina HiSeq 1500 .Genomic DNA (gDNA) was isolated from using proteinase K digestion following phenol–chloroform extraction. For short insert libraries, the gDNA isolated from a randomly chosen single adult TRIzol® reagent was used for total RNA extraction from the pooled adult (randomly chosen 50 adult worms) and the pooled metacercaria (1000 worms) homogenized tissue using standard protocol . The RNA concentration in samples was measured using BioAnalyzer 2100 . The mRNA-seq libraries (cDNA libraries) were prepared with mRNA-Seq Sample Preparation kit (Illumina) according to the manufacturer’s instructions. The cDNA libraries were sequenced in an Illumina Genome Analyzer II (GAII) DNA Sequencing Platform .Raw sequencing data were preprocessed  were used as technical replicates. Initial calling was done by HaplotypeCaller module in -ERC(GVCF) mode, and the results were subjected to GenotypeGVCFs module for joint genotyping. Indels were hard-filtered according to the GATK best practices recommendations. Hard filtering of the resulting SNPs was performed using the stricter criteria QD < 5.0, FS > 40.0, MQ < 35.0, MQRankSum < − 2.5, SOR > 2.5, ReadPosRankSum < − 4.0, and DP < 150 or DP < 90 . An intersection of the two technically repeated variant annotations was finally taken . Further analysis of the heterozygosity rate was limited to genomic intervals with fragment coverage lying between 0.75 and 1.25 of the median coverage in order to reduce the impact of potential assembly and mapping errors (collapsed or duplicated sequences). Heterozygous variants falling into these intervals were counted and divided by the total length of the intervals.The examined genomes were compared using the scheme implemented by Young et al. . GenomicO. felineus, O. viverrini, C. sinensis, and S. mansoni were compared in a pairwise manner. The sequences were aligned with the help of MUMmer v. 3.23 [O. felineus, O. viverrini, and C. sinensis scaffolds with a length over 1000 bp were aligned at the level of amino acid sequences by the promer program  to S. mansoni chromosome 1 (Smp.Chr_1), used as a reference. The syntenic blocks were identified in the constructed alignments with SyMap v. 4.2 [O. viverrini and C. sinensis scaffolds were aligned at the amino acid sequence level (the promer program with the above listed parameters) to SJOL01009849 sequence of O. felineus, which was the longest one (3238 362 bp). Third, the O. viverrini and C. sinensis scaffold sequences with a length over 1000 bp were aligned to the O. felineus scaffolds using the nucmer program . Then 10 O. felineus scaffolds that had the largest coverage by O. viverrini and C. sinensis sequences according to the nucleotide alignment were selected. These sequences were analyzed using the SyMap program together with the homologous opisthorchiid scaffolds. The analysis was conducted for both the sequences with unmasked repeats and the genome sequences with repeats masked with N symbols.The genomic sequences of  v. 3.23 . First, p v. 4.2 . Second,http://genome.sfu.ca/cgi-bin/orthoclusterdb/runortho.cgi).Additionally we used OrthoCluster software to calculate syntenic blocks using order and direction between sets of orthologous protein coding genes . Unlike O. viverrini (AN: SRR1821044), C. sinensis (AN: SRR096372), and F. hepatica (AN: ERR576947) were obtained from the Sequence Read Archive database . RepeatModeler 1.0.8 [Two methods were applied to de novo identify repetitive elements in both raw sequencing data and already assembled scaffolds/contigs. Tedna 1.2.1  was useder 1.0.8  was appler 1.0.8  databaseF. hepatica were also merged with the general RepeatMasker library, combined, and used to map the repeats on F. hepatica genome assembly only.The repeat sequences were clustered by similarity using cd-hit  with a cThe dynamics of transposable elements in opisthorchiid genomes were estimated by taking CpG adjusted Kimura 2P distance to adjust for multiple substitutions of repeats masked by RepeatMasker to their consensus sequences from the library using perl scripts  available from the RepeatMasker package .O. felineus genome were predicted using a combination of ab initio gene finding algorithms, O. felineus transcriptome assemblies, and cross-species alignments of transcript and protein sequences from other trematodes , we also performed genome-guided transcriptome assembly for these species using cufflinks [In order to explore the properties of transcriptomes in ufflinks  softwareO. felineus protein products was done using KOBAS and KAAS web-services. Gene set enrichment analyses (GSEA) were performed with the use of GOstat [The protein-coding genes were annotated by running InterProScan  against f GOstat  R packagThe SL RNA sequence was determined from the overrepresented 5-prime sequences of Trinity de novo assembled transcripts and further verified by overrepresentation of its forward strand in both reads of paired-end RNA-seq libraries as well as by homology to the known flatworm SL RNAs. To determine the sites of trans-splicing in the target transcripts, a fraction of SL RNA containing reads was extracted from RNA-seq data using cutadapt , requiriTo reduce a potential bias in further estimates of splicing efficiency, all RNA-seq reads were subject to the second round of “hard” SL trimming: up to 1-bp match to SL sequence was cut out but only if it was supported by a valid site of SL splicing determined before Fig. . The effThe available and newly obtained RNA-seq data was preprocessed by cutadapt v1.18  in orderO. felineus metacercariae and adult worms  was analyzed using full O. felineus gene annotation and default DESeq2 [The differential expression between t DESeq2  workflowO. felineus and two other opisthorchiids. The predicted cDNAs of O. felineus were aligned to the O. viverrini and C. sinensis genomes using Spaln v2.3.0 [O. felineus genome. Finally, only reciprocal-best pairs that cover each other by > 90% were retained. Viverrini and C.A comparative analysis of gene expression across three opisthorchiid species is complicated by pronounced inconsistencies between their existing gene annotations based on draft genome assemblies. To solve this problem, we identified the gene fragments exactly common for n v2.3.0  splice-aO. felineus biological replicates). A threshold-based Wald test was used for statistical testing of more than fourfold difference between the species, with controlling 1% false-discovery rate by BH algorithm.The obtained annotations of ‘orthologous’ coding regions of genes detected for each species were used to prepare gene-wise count tables from RNA-seq data on adult stages. Raw counts were normalized to the actual length of the region in the corresponding species to exclude the bias arising from slight interspecies fluctuations (< 10%) of the latter. RLE  method was used to normalize the data to sequencing depth. DESeq2  R packagO. viverrini, C. sinensis, F. hepatica, and S. mansoni, were used to identify orthologous clusters. Proteinortho v. 5.15 [O. felineus, F. hepatica and S. mansoni gene models suggested various mRNA splicing variants and, correspondingly, several amino acid sequences for some genes. The produced mRNA-level orthology tables were subsequently merged into gene-level orthologies. This procedure was found to be slightly more sensitive compared to the simple approach of selecting the longest amino acid sequence per gene.The protein sequences of four trematodes, namely,  v. 5.15  with ‘-sPhylogenetic trees were constructed based on the amino acid sequences, represented by one sequence for each organism in the clusters of orthologous groups; 1563 groups we used for analysis. Amino acid sequences were aligned in each group of orthologous genes using AQUA v. 1.1  with defAdditional file 1:Figure S1. Genome assembly pipeline. Figure S2. Gene annotation pipeline. Figure S3. Genome-wide synteny between O. felineus, O. viverrini and С. sinensis.Figure S4. Repetitive elements in O. felineus, C. sinensis, O. viverrini and F. hepatica genomes. Figure S5. Stage-specific expression of detoxification genes estimated on O. felineus transcriptome data. (PDF 438 kb)Additional file 2:Table S1. Sequenced libraries of O. felineus.Table S2. Functionally annotated genes of O. felineus.Table S3.F. hepatica transposable elements and their classification. 3.2. O. felineus transposable elements and their classification. 3.3. O. viverrini transposable elements and their classification. 3.4. C. sinensis transposable elements and their classification. Table S4. Genome-wide synteny between O. felineus, O. viverrini and С. sinensis. Table S5. Summary statistics of the gene annotations produced by a number of prediction approaches used for the annotation of O. felineus genome assembly. Table S6. Differential gene expression in O. felineus metacercariae and adult stage. Data are presented as FPKM values (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) with p-value adjusted < 0.05. Table S7. Orthologous genes detected by ProteinOrtho software with the aid of syntenic information. Table S8. Differentially expressed genes in adult O. felineus and O. viverrini . Table S9. Differentially expressed genes in adult O. felineus and C. sinensis . Table S10. Statistics on completeness of genome annotations, as reported by BUSCO software using Eukaryota (EU) & Metazoa (MZ) sets of orthologs. Table S11. Evaluation report of O. felineus genome assembly produced by REAPR software. (XLSX 4591 kb)