It is strongly desirable to understand the mechanism of metabolic regulation for catabolite regulation to improve the performance of fermentation.Most bacteria can use various compounds as carbon sources. These carbon sources can be either co-metabolized or sequentially metabolized, where the latter phenomenon typically occurs as catabolite repression. From the practical application point of view of utilizing lignocellulose for the production of biofuels etc., it is strongly desirable to ferment all sugars obtained by hydrolysis from lignocellulosic materials, where simultaneous consumption of sugars would benefit the formation of bioproducts. However, most organisms consume glucose prior to consumption of other carbon sources, and exhibit diauxic growth. It has been shown by fermentation experiments that simultaneous consumption of sugars can be attained by -1 using wild type Escherichia coli. The result indicates that the transcript levels of global regulators such as crp, cra, mlc and rpoS decreased, while those of fadR, iclR, soxR/S increased as the dilution rate increased. These affected the metabolic pathway genes, which in turn affected fermentation result where the specific glucose uptake rate, the specific acetate formation rate, and the specific CO2 evolution rate (CER) were increased as the dilution rate was increased. This was confirmed by the 13C-flux analysis. In order to make clear the catabolite regulation, the effect of crp gene knockout (Δcrp) and crp enhancement (+crp) as well as mlc, mgsA, pgi and ptsG gene knockout on the metabolism was then investigated by the continuous culture at the dilution rate of 0.2 h-1 and by some batch cultures. In the case of Δcrp  mutant, TCA cycle and glyoxylate were repressed, which caused acetate accumulation. In the case of + crpmutant, glycolysis, TCA cycle, and gluconeogenesis were activated, and simultaneous consumption of multiple carbon sources can be attained, but the glucose consumption rate became less due to repression of ptsG and ptsH by the activation of Mlc. Simultaneous consumption of multiple carbon sources could be attained by mgsA, pgi, and ptsG mutants due to increase in crp as well as cyaA, while glucose consumption rate became lower.In order to make clear the catabolic regulation mechanism, several continuous cultures were conducted at different dilution rates of 0.2, 0.4, 0.6 and 0.7 hE. coli, and its crp, mlc, ptsG, pgi, and mgsA gene knockout mutants. The results indicate that catabolite repression can be relaxed and crp as well as cyaA can be increased by +, mgsA, pgicrp, and ptsG mutants, and thus simultaneous consumption of multiple carbon sources including glucose can be made, whereas the glucose uptake rate became lower as compared to wild type due to inactivation of ptsG in all the mutants considered.The transcriptional catabolite regulation mechanism was made clear for the wild type  Biomass samples were taken after another two residence times, where 200 mL of culture broth was taken for the measurement of biomass and extracellular metabolite concentrations, and the remaining 800 mL of the culture broth was processed for gas chromatography-mass spectrometry (GC-MS) analysis. The biomass sample was kept on ice for 2-3 min, and the sample was centrifuged at 6,000 rpm at 2°C for 15 min [The labeling experiments were started after the steady state was confirmed. The unlabeled  feeding medium was replaced by an identical medium containing 3.2 g/l of unlabeled glucose, 0.4 g/l [U-r 15 min . The celr 15 min .Preparation of biomass hydrolysates and recording of the GC-MS spectra  were made as described previously ,28. The The detailed flux calculation is given previously . Briefly2 can contribute in generating ATP via oxidative phosphorylation. The pathways involved in electron transport and oxidative phosphorylation have variable stoichiometry due to the use of different dehydrogenases and cytochromes. Then the specific ATP production rate may be estimated by the following equation:The ATP production is made either by substrate level phosphorylation and oxidative phosphorylation, where the reducing power of NADH and FADHNADH OPand OPFADH2 is the specific ATP production rates via oxidative phosphorylation, and those may be estimated bywhere and(P/O) and (P/O)are the P/O ratios for NADH and FADH2, respectively.where (P/O) to be 2 and (P/O)' to be 1.Here, we set vNADH may also be estimated by:The specific NADPH production rate 3PG: 3-Phosphoglyceric acid; 6PG: 6-Phosphogluconolactone; AKG: 2-Keto-D-gluconate; AcCoA: Acetyl-CoA; AcP: Acetylphosphate; ASP: Aspartate; ADP: Adenosine diphosphate; ATP: Adenosine-5'-triphosphate; AMP: Adenosine monophosphate; cAMP: Cyclic AMP; CIT: Citrate; DHAP: Dihydroxyacetone phosphate; E4P: Erythrose 4-phosphate; F1,6-BP: Fructose 1,6-bisphosphate; F6P: Fructose 6-phosphate; FUM: Fumarate; G6P: Glucose-6-phosphate; GAP: Glyceraldehyde 3-phosphate; GLX: Glyoxylate; ICIT: Isocitrate; NAD/NADH: Nicotinamide adenine dinucleotide; NADP/NADPH: Nicotinamide adenine dinucleotide phosphate; MAL: Malate; OAA: Oxaloacetate; P: Phosphate; PEP: Phosphoenolpyruvate; PYR: Pyruvate; R5P: Ribulose 5-phosphate; Ru5P: Ribose 5-phosphate; S7P: Sedoheptulose 7-phosphate; SUCCoA: Succinyl-CoA; SUC: Succinate; X5P: Xylulose 5-phosphate.Ack: Acetate kinase; AKGDH: 2-Keto-D-gluconate dehydrogenase; 6PGDH: 6-Phosphogluconate dehydrogenase; EI: Eyme I; EIIA: Ezyme IIA; EIICB: Ezyme IIB; EI-P: Posphorylated state of EI; EIIA-P: Posphorylated state of EIIA; EIICB-P: Posphorylated state of EIICB; G6PDH: Glucose-6-phosphate dehydrogenase; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; ICDH: Isocitrate dehydrogenase; HPr: Histidine-containing protein; HPr-P: Phorylated state of HPr; Mdh: Malate dehydrogenase; Mez: Malic enzyme; Pck: Phosphoenolpyruvate carboxykinase; PDH: Pyruvate dehydrogenase; Pfk: Phosphofructokinase; Pyk: Pyruvate kinase; SDH: Succinate dehydrogenase; UQ: Ubiquinone;aceA: Isocitrate lyase gene; aceB: Malate synthase A gene; aceE, F: Pyruvate dehydrogenase gene; ackA: Acetate kinase gene; acnA/B: Aconitase gene; acs: Acetyl-coenzyme A synthetase; arcA/B: Anoxic redox control protein; atpA: ATP synthase; cra: Catabolite repressor/activator; crp: Cyclic AMP receptor; crr: Catabolite repressor; cyaA: adenylate cyclase gene; cyoABCDE: Cytochrome o oxidase gene; cydAB: Cytochrome d oxidase gene; edd: 6-Phosphogluconate dehydrogenase gene; eda: 2-Keto-3-deoxy-6-phosphogluconate aldolase gene; fadR: Fatty acid metabolism regulator; fnr: Fumarate/nitrate reduction transcriptional regulator; fumA, B, C: Fumarase gene; fur: Fe-responsive gene regulator; gltA: Citrate synthase gene; gnd: 6-phosphogluconate dehydrogenase gene; icdA: Isocitrate dehydrogenase A gene; iclR: Isocitrate lyase regulator; lpdA: Dihydrolipoamide dehydrogenase gene; mdh: Malate dehydrogenase gene; mgsA: Methylglyoxal synthase gene; mlc: DNA-binding transcriptional repressor; ndh: NADH dehydrogenase gene; nuoABCEFGHIJKLMN: NADH:ubiquinone oxidoreductase; pckA: Phosphoenolpyruvate carboxykinase gene; pfkA: 6-Phosphofructokinase I gene;pgi: Glucose-6-phosphate isomerase gene; pgk: Phosphoglycerate kinase gene; ppc: Phosphoenolpyruvate carboxylase gene; ppsA: Phosphoenolpyruvate synthase gene; pta: Phosphate acetyltransferase gene; ptsG, ptsHI: Pts gene; pykA, F: Pyruvate kinase gene; rpoS: RNA polymerase sigma factor; sdhC: Succinate dehydrogenase; sodA: Speroxide dismutase; soxR/S: Superoxide stress regulon; sdhABCD: Succinate dehydrogenase gene; sucA: 2-Oxoglutarate decarboxylase; sucB: Dihydrolipoamide succinyltransferase; sucC: Succinyl-CoA ligase gene; sucD: Succinyl-CoA synthetase gene; talAB: Transaldolase gene; tktAB: Transketolase gene; tpiA: Triose phosphate isometase; ubiE: Uuinone/menaquinone biosynthesis methyltransferase gene; yfid: Autonomous glycyl radical cofactor; zwf: Glucose 6-phosphate dehydrogenase gene.The authors declare that they have no competing interests.+ crpmutant and prepared other single gene knockout mutants. YQ discussed on the result obtained, and participated in preparing the manuscript, KS considered experimental design, analyzed the result, and prepared manuscript. All authors read and approved the final manuscript.RY carried out fermentation, GC-MS and RT-PCR experiments, assayed, made statistical analysis, YH carried out fermentation experiments, DS carried out fermentation and GC-MS experiments, KN constructed Global regulators and their regulated genes.Click here for fileThe effect of dilution rate on the metabolic flux distribution. All fluxes are given as absolute values (mmol/g/h) and as values relative to the specific glucose consumption rate (in parentheses), values in < > indicate exchange coefficients.Click here for fileThe effect of dilution rate (specific growth rate) on the specific ATP production rate (3a) and specific NADPH production rate (3b). Filled square symbols are from the present study, open triangle symbols are from [are from , while oare from .Click here for fileBatch fermentation result of using a mixture of glucose and xylose as a carbon source. (a) wild type, (b) crp+ mutant.Click here for fileContinuous fermentation result of using a mixture of glucose and xylose as a carbon source. (a) wild type, (b) + crpmutant.Click here for fileptsG mutantContinuous fermentation result of using a mixture of glucose and xylose as a carbon source for .Click here for filecrpBatch fermentation result of using glucose as a carbon source for Δ.Click here for filemlc mutantBatch fermentation result of using glucose as a carbon source for .Click here for file


AbstractDeinodryinus veltenisp. n. is described from middle Eocene Baltic amber. The species differs from other fossil Palaearctic species of Deinodryinus Perkins owing to the shape of the antenna , the large compound eyes, and the distal part of the stigmal vein much longer than the proximal part. A revision and a key to the fossil Palaearctic species of Deinodryinus Perkins, 1907 is presented. Dryinidae  are parasitoids of Auchenorrhyncha , from Baltic amber, and Deinodryinus? aptianus Olmi, Rasnitsyn & Guglielmino, 2010, a compression fossil from Early Cretaceous marl of the Khurilt rock unit (Mongolia). The latter species is tentatively placed within Deinodryinus given that it is uncertain whether the attribution of this Early Cretaceous species to a modern genus is justified. Insufficient characters were preserved in Deinodryinus? aptianus to support its placement in a new generic taxon.rrhyncha , 2007. T species : DeinodrDeinodryinus from Baltic amber, and the taxon is described herein.Recently we have discovered an additional new fossil species of The descriptions follow the terminology used by , 1999. TDeinodryinus? aptianus and Deinodryinus areolatus, respectively by The redescriptions of The material studied in the present paper is deposited in the following institutions:PIN A.A. Borissiak Palaeontological Institute,Russian Academy of Sciences, Moscow (Russia).SNMS Staatliches Museum für Naturkunde Stuttgart, Abt. Paläontologie–Sektion Bernstein, Stuttgart (Germany).GenusPerkins, 1907http://species-id.net/wiki/DeinodryinusDeinodryinusDeinodryinus paradoxus Perkins, 1907, designated by  Perkins, 1907: 45. Type species: TrisanteonTrisanteon hirticornis , monotypic and original designation. Kieffer, 1913: 300 ; palpal formula 6/3; forewing usually with distal part of stigmal vein longer than proximal part, less frequently as long as, or shorter than proximal part; forewing usually with pterostigma four or more than four times as long as broad; antennal hairs usually much longer than breadth of segments, less frequently shorter than breadth of segments; vertex frequently with two strong oblique keels connecting posterior ocelli to occipital carina; paramere without dorsal process, usually with more or less large inner branch wrapping penis, less frequently with reduced inner branch; tibial spurs 1/1/2.Female: macropterous or micropterous; palpal formula 6/3; in macropterous specimens forewing usually with distal part of stigmal vein longer than proximalWorldwide.Cicadellidae  (100–115 mybp).Female with antenna filiform and compound eye small .Female: macropterous; length 6.2 mm; length of main regions: head: 0.87 mm; antennae: 3.06 mm; mesosoma: 1.37 mm; prothorax: 0.87 mm; mePageBreaksothorax + metathorax + propodeum: 1.37 mm; metasoma: 3.12 mm. Antenna filiform . Palpi not visible. Compound eye small http://species-id.net/wiki/Deinodryinus_areolatusElectrodryinus areolatus N. Deinodryinus areolatus (N. Ponomarenko): Deinodryinus areolatus (N. Ponomarenko): Deinodryinus areolatus (N. Ponomarenko): Type: Holotype, female, Eocene Baltic amber (40–45 mybp).Female with antenna clavate and compound eye large ; distal Female: macropterous; length 4.5 mm. Head black, except anterior region of face brown; clypeus testaceous, except central brown spot; mandible testaceous, except teeth and proximal region brown; antenna testaceous; mesosoma and metasoma black; legs brown-testaceous. Antenna 10-segmented, clavate, densely hairy, less than three times as long as head (157:63); antennal segments in following proportions: 17:10:30:22:18:15:11:11:10:13. Clypeus with anterior margin weakly emarginated. Antennal torulus distinctly separated from epistomal sulcus. Mandible with four teeth progressing larger from anterior one to posterior. Compound eye apparently bare, normally protruding. Subocular sulcus present. Occipital carina complete. Temple prominent. Posterior ocelli hardly visible, not touching occipital carina. Palpal formula 6/3. Pronotum not crossed by transverse impressions; pronotal tubercle reaching tegula; posterior margin of pronotum longer than anterior margin. Thoracic structure similar to that of extant Deinodryinus. Scutum shiny, finely punctate, longer than pronotum (20:15). Notauli complete, posteriorly separated. Propodeum reticulate rugose, with areolae very broad; dorsal surface approximately as long as posterior surface; posterior surface very steep, not distincly visible. Petiole distinct. Forewing hyaline, without dark transverse bands, with normal venation of Anteoninae; pterostigma narrow, more than four times as long as broad (40:8); marginal cell open; distal part of stigmal vein about PageBreakas long as proximal part (18:17); stigmal vein not S-shaped. Forewing with usual three basal cells clearly enclosed by pigmented veins . Shape of wings usual for Deinodryinus. Protrochanter not slender, without proximal slender stalk, slightly longer than broad (10:6). Segment 3 of protarsus produced into hook; segment 1 of protarsus slightly shorter than segment 4 (15:17). Forelegs chelate. Chela without rudimentary claw. Arolium much shorter than enlarged claw (7:32). Enlarged claw without subapical tooth and other teeth. Segment 5 of protarsus about as long as enlarged claw, with lamellae hardly visible. Tibial spurs 1/1/2.Male: unknown.Unknown.In the holotype the sculpture of the vertex, face, and pronotum is hardly visible; the scutellum and metanotum are not visible.Guglielmino & Olmisp. n.urn:lsid:zoobank.org:act:AD16D59C-D1EF-4DF9-8ACC-F1E55C42DAA2http://species-id.net/wiki/Deinodryinus_velteniPageBreakThe species is named after Mr. Jürgen Velten .Type: Holotype, female, Eocene Baltic amber (40–45 mybp) (SMSN).Female with antenna clavate and compound eye large ; distal Female: macropterous; length 4.0 mm. Colour apparently brown-black, except palpi testaceous. Antenna 10-segmented, clavate, short, covered with dense and short hairs, thickened distally; antennal rhinaria absent; antennal segments in following proportions: 5:7:8:13:10:9:8:6:6:9; antenna much shorter than body, approximately three times as long as head : 75:25. Head only partly visible, slightly convex, dull, apparently granulated and hairless; occiput excavated; compound eye normally bulging; ocelli partly visible; ocellar triangle apparently equilaterPageBreakal; temple distinct. Pronotum long, crossed by anterior strong transverse impression, with posterior disc, without posterior collar; pronotum apparently almost glabrous, shiny, slightly shorter than head (22:25); pronotal disc flat posteriorly, much longer than anterior collar; pronotal tubercle reaching tegula. Scutum dull, apparently glabrous, granulated, slightly shorter than pronotum (19:22). Notauli complete, posteriorly separated; minimum distance between notauli approximately as long as antennal segment 2. Scutellum very humped . Pterostigma shape similar to that of extant Deinodryinus. Marginal cell open. Stigmal vein not S-shaped, with distal part much longer than proximal part (20:14); stigmal vein forming an angle between proximal and distal parts. Forewing with usual three basal cells clearly enclosed by pigmented veins . Hindwing apparently slightly darkened. Hindwing shape usual for Anteoninae. Foreleg segments in following proportions: 30 (coxa): 7 (trochanter): 43 (femur): 29 (tibia): 7 (tarsomere 1): 3 (tarsomere 2): 5 (tarsomere 3): 11 (tarsomere 4): 24 (tarsomere 5). Foreleg chelate. Enlarged claw slightly shorter than tarsomere 5 (22:24). Protrochanter short, slightly longer than broad (7:5). Protrochanter shape similar to that of Anteoninae. Tarsomeres 2 and 3 of protarsus produced into a hook. Rudimentary claw absent. Arolium much shorter than enlarged claw (8:22). Distal apex of enlarged claw apparently pointed. Tarsomere 5 of protarsus with numerous lamellae on inner margin and distal apex. Midleg segments in following proportions: 12 (coxa): 8 (trochanter): 26 (femur): 24 (tibia): 22 (tarsomere 1): 9 (tarsomere 2): 6 (tarsomere 3): 4 (tarsomere 4): 6 (tarsomere 5). Hindleg segments in following proportions: 19 (coxa): 5 (trochanter): 33 (femur): 32 (tibia): 23 (tarsomere 1): 10 (tarsomere 2): 8 (tarsomere 3): 4 (tarsomere 4): 6 (tarsomere 5). Petiole shape and length usual for Anteoninae. Palpal formula 6/3. Shape, length and breadth of wings usual for Anteoninae. Shape and morphology of body usual for Anteoninae. Tibial spurs 1/1/2.y humped , much shy humped , shorterMale: unknown.Unknown.PageBreakIn the holotype the clypeus, mandibular teeth, frontal line, occipital carina, POL, OL, OOL, OPL, TL, and posterior surface of the propodeum are not visible; the sculpture of the pronotum, scutellum, metanotum, and mesopleura is not distinct; the enlarged claw is only partly visible because of a closed chela so that it is not possible to see if there are subapical teeth and lamellae; and tarsomere 5 of the protarsus is only partly visible so that it is impossible to count the lamellae and to see if there are one or two rows of lamellae.Deinodryinus is present in all zoogeographical regions: six species are Palaearctic; 22 Afrotropical; nine Oriental; three Nearctic; 110 Neotropical; and two Australian. The only known fossil species have been found in the Palaearctic region, and all have been summarized in the present paper. Because of its geographic distribution, Deinodryinus is considered a ‘Pangean’ genus  and Deinodryinus hispanicus ], and one in the Nearctic region [Deinodryinus atriventris ]. The presence of fossil species in Baltic amber and Early Cretaceous Mongolia marl perhaps indicates that these areas had relatively warm climates in the past, conclusions supported by other faunal and floral elements in these deposits, and that the genus had a much wider distribution.With 152 species, the genus n’ genus . The genDeinodryinus areolatus and Deinodryinus velteni do not exhibit significant differences from extant species of the genus. This is common with many fossil dryinids. By contrast, Deinodryinus? aptianus, visible only in ventral aspect, with legs partly missing and the chela hardly distinct, is difficult to interpret. As mentioned above, attribution to Deinodryinus is only tentative and based on the shape of the pterostigma and stigmal vein and by the presence in the forewing of three basal cells completely enclosed by pigmented veins. However, the presence of filiform antennae, a character present in very few females of dryinids and rare in Deinodryinus , makes this attribution somewhat suspect. Accordingly, our assignment of this species to Deinodryinus remains speculative and we hope for the eventual discovery of more completely preserved material so as to clarify the generic status of this ancient taxon.From a morphological standpoint,


The mid-point of the ligand is located on an inversion center, and shows a trans conformation. The ligands link the ZnII ions, forming a chain structure along [10-1].In the title compound, [ZnCl DOI: 10.1107/S1600536812014043/hy2529Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown.Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the N-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the M. sexta-N. attenuata interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated ≥ 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction.We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N. attenuata after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.The analysis of the early changes in the transcriptome of  Nicotiana attenuata is an annual native to Southwestern USA that germinates from seed banks in response to factors in wood smoke after fires . Statistical analysis of differentially expressed tags was calculated according to -dihydro-JA and [13C6]-JA-Ile. Homogenates were centrifuged for 10 min at 4°C, the organic phase collected and plant material re-extracted with 0.5 mL ethylacetate. Organic phases were combined and the samples evaporated to dryness. The dry residue was reconstituted in 0.2 mL of 70% (v/v) methanol/water for analysis on a LC-(ESI)-MS/MS system as previously described . The accession numbers for ESTs corresponding to the extended Unitag sequences are Tag-129: [GenBank: GT184388]; Tag-837: [GenBank: GT184389]; Tag-895: [GenBank: GT184390]; Tag-995: [GenBank: GT184391]; Tag-1439: [GenBank: GT184392]; Tag-1844: [GenBank: GT184393]; Tag-2067: [GenBank: GT184394]; Tag-2452: [GenBank: GT184395]; Tag-2815: [GenBank: GT184396]; Tag-2978: [GenBank: GT184397]; Tag-2990: [GenBank: GT184398]; Tag-4898: [GenBank: GT184399]; Tag-5283: [GenBank: GT184400]; Tag-5869: [GenBank: GT184401]; Tag-6032: [GenBank: GT184402]; Tag-6199: [GenBank: GT184403]; Tag-6205: [GenBank: GT184404]; Tag-6642: [GenBank: GT184405]; Tag-6938: [GenBank: GT184406]; Tag-7036: [GenBank: GT184407]; Tag-7795: [GenBank: GT184408]; Tag-9719: [GenBank: GT184409]; Tag-10039: [GenBank: GT184410]; Tag-11166: [GenBank: GT184411]; Tag-11559: [GenBank: GT184412]; Tag-12314: [GenBank: GT184413].The SuperSAGE data was deposited in the Gene Expression Omnibus (GEO) public domain under the accession [GenBank: PG and GB carried out the experiments, analyzed the data and drafted the manuscript. RJ and BR carried out experiments and analyzed the data. SS analyzed the data. ITB participated in the design and coordination of the study and helped to draft the manuscript. GB conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Complete list of UniTag sequences, copy numbers, and annotations to GenBank nucleotide databases.Click here for fileComplete list of differentially expressed UniTags and GO categorization.Click here for fileSupplementary Figures. Figure S1. Analysis of mRNA accumulation corresponding to 12 UniTags by qPCR. Figure S2. M.sexta larval performance on VIGS silenced plants.Click here for fileList of differentially expressed UniTags previously identified by differential expression techniques.Click here for fileSupplementary Tables. Table S1. Primers for elongation of cDNAs correspondig to UniTags. Table S2. Primers for qPCR. Table S3. Primers for VIGS analysis.Click here for file


AbstractEupolybothruscavernicolus Komerički & Stoev sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae), found in a remote karst region in Knin, Croatia, is the first eukaryotic species for which, in addition to the traditional morphological description, we provide a fully sequenced transcriptome, a DNA barcode, detailed anatomical X-ray microtomography (micro-CT) scans, and a movie of the living specimen to document important traits of its ex-situ behaviour. By employing micro-CT scanning in a new species for the first time, we create a high-resolution morphological and anatomical dataset that allows virtual reconstructions of the specimen and subsequent interactive manipulation to test the recently introduced ‘cybertype’ notion. In addition, the transcriptome was recorded with a total of 67,785 scaffolds, having an average length of 812 bp and N50 of 1,448 bp (see GigaDB). Subsequent annotation of 22,866 scaffolds was conducted by tracing homologs against current available databases, including Nr, SwissProt and COG. This pilot project illustrates a workflow of producing, storing, publishing and disseminating large data sets associated with a description of a new taxon. All data have been deposited in publicly accessible repositories, such as GigaScience GigaDB, NCBI, BOLD, Morphbank and Morphosource, and the respective open licenses used ensure their accessibility and re-usability.We demonstrate how a classical taxonomic description of a new species can be enhanced by applying new generation molecular methods, and novel computing and imaging technologies. A cave-dwelling centipede,  Index of Organism Names), animal diversity on the planet continues to decline with unprecedented speed , and a video of a living specimen showing behavioural features. In this increasingly data-driven era, a further aim of this study is to set a new standard for handling, management and publishing of various data types. It is essential that data are easily accessible to researchers in every field of science, and able to be integrated from many sources, to tackle complex and novel scientific hypotheses. Rapid advances and increasing throughput of technologies such as phenotyping, genome-scale sequencing and meta-barcoding are now producing huge volumes of data, but there has been a lag in efforts to curate, present, harmonise and integrate these data to make them more accessible and re-usable for the community. Furthermore, by employing micro-CT scanning we test for the first time in a new taxon the recently introduced ‘cybertype’ notion , Eupolybothruslitoralis  and Eupolybothrustridentinus  species-groups, as recently revealed by application of DNA barcoding . Two other studies , the National Museum of Natural History, Sofia (NMNHS) and the Natural History Museum of Denmark (ZMUC). The specimens were preserved in ethanol (70 or 96%) or RNAlater . The morphological study of the new species was performed at NMNHS and CBSSS with a Zeiss microscope. For scanning electron microscopy (performed at ZMUC), parts of the specimens were cleaned by ultrasonification, transferred to 96% ethanol and then to acetone, air-dried, mounted on adhesive electrical tape attached to aluminium stubs, coated with platinum/palladium and studied in a JEOL JSM-6335F scanning electron microscope. Images were edited in Adobe Lightroom 4.3 and Adobe Photoshop CS 5. All morphological images have been deposited in Morphbank. Terminology for external anatomy follows The present study is based on eight specimens of DNA barcode sequencingDNA extraction and amplification. All specimen data are stored in the Barcode of Life Data System (BOLD) online database and are available also in the dataset DS-EUPCAV (http://dx.doi.org/10.5883/DS-EUPCAV), where they are linked to the respective Barcode Index Numbers clusters. This dataset contains sequences from ten species: Eupolybothruscavernicolus Komerički & Stoev sp. n., Eupolybothrusleostygis , Eupolybothrusobrovensis , Eupolybothrusgrossipes , Eupolybothrusgloriastygis , Eupolybothrusnudicornis, Eupolybothruslitoralis, Eupolybothruskahfi, Eupolybothrustranssylvanicus  and Eupolybothrustridentinus. In addition, all sequences were registered in GenBank , one sequence of Eupolybothrusfasciatus  was recovered from GenBank (accession number AY214420). Two sequences from two Lithobiusspecies were included as outgroups: Lithobiusaustriacus  (MYFAB442-11) and Lithobiuscrassipes L. Koch, 1962 (MYFAB443-11). The final dataset comprises 39 sequences. Molecular delimitation of species was achieved by the implementation of the Automatic Barcoding Gap Discovery (ABGD) procedure as described in DNA extraction was conducted in the the Canadian Centre for DNA Barcoding, Guelph on complete animals or part of the leg of the specimens preserved in 96% ethanol. Standard protocols of the Canadian Centre for DNA Barcoding were used for both For the ABGD method, we tested various model combinations to cross-check the obtained results: relative gap with (X) ranging from 0.05 to 1.5, minimal intraspecific distance (Pmin) of 0.001 and maximal intraspecific distance (Pmax) ranging from 0.02 to 0.11. Pmin and Pmax refer to the genetic distance area where the barcoding gap should be detected, whereas X defines the width of the gap. Distance calculation was based on the Kimura-2-parameter model and a transition/transversion ratio of 2.0. The method was performed in 100 steps. Statistical Parsimony networks for the delineation of species were reconstructed on the basis of 95% statistical confidence (i.e. connection probability) using the program TCS 1.21 . The NJ-Transcriptome sequencingEupolybothruscavernicolus Komerički & Stoev sp. n. was crushed and preserved in liquid RNAlater  immediately after being captured. To extract total RNA, TRIzol reagent  was used according to the manufacturer’s instructions. Messenger RNA (mRNA) was isolated from total RNA using a Dynabeads mRNA Purification Kit . The mRNA was fragmented and transcribed into first-strand cDNA using SuperScript™II Reverse Transcriptase  and N6 primer (IDT). RNase H  and DNA polymerase I  were subsequently applied to synthesize the second-strand of the cDNA. The double-stranded cDNA then underwent end-repair, a single ‘A’ base addition, adapter ligation, and size selection, indexed and PCR amplified to construct a library. The extracted cDNA was utilised for library construction with an insert size of 250 bp. Finally, the library was sequenced on the Illumina HiSeq2000 sequencing platform  at BGI-Shenzhen using a 150bp pair-end strategy to generate a total of 2.5 Gb raw reads. Illumina HCS1.5.15.1 + RTA1.13.48.0 were applied to generate a “bcl” file which was then downloaded to local computers. Secondly, the “bcl” file was converted to qseq format using BclConverter-1.9.0-11-03-08. Finally, we separated individual sample data from multiplexed machine runs based on the specific barcode primer sequences, and converted the file format to fastq.One entire adult male specimen of The micro-CT scanning of one adult female specimen was performed at Bruker microCT, Kontich, Belgium, using a SkyScan 1172 system with the following settings: 40kV, 0.43° rotation step, acquiring 839 projection images from 360° with a pixel size of 8µm. Prior to scanning, the sample was dehydrated in graded ethanol: 50%, 70%, 90%, 100%, for 2 hours in total, and then transferred to HMDS (hexamethyldisilasane) for 2 hours, and air dried. Reconstruction was done with the SkyScan software NRecon, using a modified Feldkamp algorithm, and adjusting for beam hardening and applying ring artefact correction resulting in 3865 cross sections in. bmp format, with image size 2000x2000 pixels. The video of 3D volume renderings was created with CTVox, using the flight recorder function, and saved as an AVI (Audio Video Interface) file. The obtained data were processed through a transfer function where the different voxels with different grey value were (or weren't) made opaque and where the color was assigned to a certain grey value. The image stack is stored in GigaDB  under a – TergiteT, – TergitesTT, Legs: – leftL, – rightR; Plectrotaxy table: – coxaCx, – trochanterTr, – prefemurPf, – femurF, – tibiaT, a, m, p stand for spines in respectively, anterior, medial and posterior position.Komerički & Stoev, 2013sp. n.urn:lsid:zoobank.org:act:6F9A6F3C-687A-436A-9497-70596584678Chttp://www.ebi.ac.uk/ena/data/view/ERP003841http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1859http://dx.doi.org/10.5524/100063KF715043KF715049KF715050KF715059http://dx.doi.org/10.5883/DS-EUPCAV999021821&tsn=truehttp://morphosource.org/index.php/Detail/SpecimenDetail/Show/specimen_id/514Occurrence: recordedBy: M. Lukić; individualCount: 1; sex: male; lifeStage: adult; Location: country: Croatia; stateProvince: Knin; locality: NP Krka, village Kistanje, Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m; verbatimLatitude: 44°00'01.1''N; verbatimLongitude: 16°00'58.5''E; Event: samplingProtocol: hand collected under clay sediment; eventDate: 9 February 2013; Record Level: institutionCode: CBSS; collectionCode: CHP536Occurrence: recordedBy: A. Komerički; individualCount: 1; sex: female; lifeStage: adult; Location: country: Croatia; stateProvince: Knin; locality: NP Krka, village Kistanje, Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m; verbatimLatitude: 44°00'01.1''N; verbatimLongitude: 16°00'58.5''E; Event: samplingProtocol: hand collected under lump of clay; eventDate: 18 October 2012; Record Level: institutionCode: CBSS; collectionCode: CHP517Occurrence: recordedBy: M. Lukić; individualCount: 1; sex: male; lifeStage: adult; Location: country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje, Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m; verbatimLatitude: 44°00'01.1''N; verbatimLongitude: 16°00'58.5''E; Event: samplingProtocol: hand collected; eventDate: 9 February 2013; Record Level: institutionCode: BGIOccurrence: recordedBy: H. Cvitanović & A. Komerički; individualCount: 1; sex: female; lifeStage: adult; Location: country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje, Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m; verbatimLatitude: 44°00'01.1''N; verbatimLongitude: 16°00'58.5''E; Event: samplingProtocol: hand collected; eventDate: 18 October 2012; Record Level: institutionCode: NMNHS; collectionCode: NMNHS-CHILOPODA-1/2013Occurrence: recordedBy: A. Kirin & A. Komerički; individualCount: 1; sex: female; lifeStage: adult; Location: country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje, Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m; verbatimLatitude: 44°00'01.1''N; verbatimLongitude: 16°00'58.5''E; Event: samplingProtocol: hand collected; eventDate: 4 May 2010; Record Level: institutionCode: ZMUC; collectionCode: zmuc00029439Occurrence: recordedBy: M. Lukić; individualCount: 1; sex: male; lifeStage: adult; Location: country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje, cave Miljacka IV (= Špilja kod mlina na Miljacki); verbatimElevation: 115 m; verbatimLatitude: 44°00'12.8''N; verbatimLongitude: 16°01'08.8''E; Event: samplingProtocol: hand collected; eventDate: 4 May 2010; Record Level: institutionCode: ZMUC; collectionCode: zmuc00029440Occurrence: recordedBy: A. Kirin; individualCount: 1; sex: female; lifeStage: subadult; Location: country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje, Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m; verbatimLatitude: 44°00'01.1''N; verbatimLongitude: 16°00'58.5''E; Event: samplingProtocol: hand collected; eventDate: 4 May 2010; Record Level: institutionCode: CBSS; collectionCode: CHP420Occurrence: recordedBy: A. Komerički; individualCount: 1; sex: damaged female; lifeStage: subadult; Location: country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje, cave Miljacka IV (= Špilja kod mlina na Miljacki); verbatimElevation: 115 m; verbatimLatitude: 44°00'12.8''N; verbatimLongitude: 16°01'08.8''E; Event: samplingProtocol: hand collected; eventDate: 4 May 2010; Record Level: institutionCode: CBSS; collectionCode: CHP416Occurrence: recordedBy: A. Komerički; individualCount: 1; sex: damaged male; lifeStage: adult; Location: country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje, Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m; verbatimLatitude: 44°00'01.1''N; verbatimLongitude: 16°00'58.5''E; Event: samplingProtocol: hand collected; eventDate: 21 October 2012; Record Level: institutionCode: CBSS; collectionCode: CHP552Body length:approx. 30 mm ; leg 15 – 22.6 mm long, or 75% length of body.Color: uniformly yellow-brownish to chestnut, margins of cephalic plate slightly darker than inner parts , as wide as T1 , oval, situated on subtriangular sclerotisation below the inferiormost row of seriate ocelli  or T9 (left) when folded backwards, basal 2 articles enlarged, less pilose; posterior 30 articles visibly longer than broad, ultimate article approx. 1.3 times longer than penultimate one . All tergites smooth, setae present only on their lateral margins.Legs: leg 15 longest; leg 14 approx. 25% longer than legs 1-12, leg 13 only slightly longer than legs 1-12; pretarsus of legs 1–14 with expanded fundus, larger posterior accessory claw (approx. 1/3rd of fundus) and a slightly thinner and shorter anterior accessory claw  with characteristic bi- and tripartite tip , as wide as anterior part of T1; surface smooth, with several pits scattered throughout the head and giving rise to trichoid setae. Cephalic median sulcus contributing to biconvex anterior margin, marginal ridge with a median thickening; posterior margin slightly concave; transverse suture situated at about 1/3 of anterior edge; posterior limbs of transverse suture visible, connecting basal antennal article with anterior part of ocellar area. Ocelli: 18 blackish, subequal in size, in 3-4 rows. Tömösváry’s organ: moderately large (as large as or slightly larger than a medium ocellus), oval, situated slightly above the cephalic edge below the inferiormost row of ocelli. Clypeus: with a cluster of about 25 trichoid setae situated on the apex. Antennae: approx. 22 mm long, composed of 67 articles, reaching the middle of T10 when folded backwards, basal 2 articles enlarged, less setose; posterior 30 articles visibly longer than broad, ultimate article approx. 1.3 times longer than penultimate one. Forcipules: coxosternite subpentagonal, shoulders almost absent, lateral margins straight; anterior margin set off as a rim by furrow; coxosternal teeth 7+7, median diastema well-developed, V-shaped, subparallel and narrow, porodont arising from a pit below the dental rim, situated lateral to the lateralmost tooth; base of porodont thinner then adjacent tooth, coxosternite sparsely setose anteriorly; setae moderately large, irregularly dispersed. Medial side of forcipular trochanteroprefemur, femur and tibia and proximal part of forcipular tarsungulum setose. Distal part of forcipular tarsungulum about 3 times longer than proximal part.Tergites: T1 wider than long, subtrapeziform wider anteriorly, posterior margin slightly concave; TT3 and 5 more elongated than T1, posterior margin slightly concave medially, posterior angles rounded; T2 almost entirely covered by T1, only posteriormost part surpassing the margin of T1; posterior margin of TT4 and 6 straight, posterior angles abruptly rounded; T7 rectangular, posterior margin straight, posterior angles abruptly rounded; T8 approx. 1.4 times longer than T7, posterior margin of T8 slightly concave medially, angles abruptly rounded; TT9, 11, 13 with a well-developed posterior triangular projections; TT10 and 12 subequal in size, approx. 1.2 times longer than T8, posterior margin slightly emarginated; posterior margin of T14 slightly emarginated, surface smooth, posterior-most part covered with just a few trichoid setae  absent.Legs: leg 15 longest, leg 14 latter approx. 25% longer than legs 1-12, leg 13 only slightly longer than legs 1-12; pretarsus of legs 1–14 with a more expanded fundus, larger posterior accessory claw (approx. 1/3rd of fundus) and a slightly thinner and shorter anterior accessory claw  , comprised of 67-71 articles; 11-15 ocelli; 6+6-8+8 coxosternal teeth; tergites 9, 11, 13 with posterior triangular projections; intermediate tergite subpentagonal, posterior margin deeply emarginated, middle part of posterior third of tergite densely covered with setae; laterally, on both sides of the central setose area, there are two specific seta-free regions; pretarsus 15 without accessory spines; leg 15 long (approx. 70-75% body length), prefemur of male leg 15 with a large, apically rounded proximal knob protruding mediad, latter slightly bent dorsad and bearing a cluster of long setae on tip; distal part of prefemur with a well-defined circular protuberance covered with setae; posterior margin of male first genital sternite deeply emarginated, nearly as deep as half of the sternite’s length.The species can be readily distinguished from all other congeners by the following set of molecular and morphological characters: interspecific genetic distance in COI from the closest neighbour, Eupolybothruscavernicolus Komerički & Stoev sp. n. is so far known only from the caves Miljacka II and Miljacka IV (= Špilja kod mlina na Miljacki), situated near the village of Kistanje, Krka National Park, Knin District, Croatia ; RH = 100%; Tsediment = 12.5-13.2°C; Twater = 12.6-13.2°C; in Miljacka IV (measured on 4 May 2010): Tair = 13.1-13.6°C (Kestrel); RH = 100%; Tsediment = 12.5°C; Twater = 12.5°C. In Miljacka II, the specimens were collected in the aphotic zone, approx. 50 m from the cave entrance, in a passage where water never occurs in a periodic flow. In Miljacka IV, they were found closer to the entrance, under stones.tia Fig. . The twotia Fig. . Most ofAssociated fauna:Gastropoda: Oxychiluscellarius , Hauffeniajadertina Kuščer, 1933, Hadziellasketi Bole, 1961; Araneae: Episinuscavernicola , Nesticuseremita Simon, 1879, Tegenariadomestica , Metellinamerianae , Histopona sp.; Pseudoscorpiones: Chthoniustetrachelatus , Chthoniuslitoralis Hadži, 1933, Neobisiumcarsicum Hadži, 1933, Pselaphocherneslitoralis Beier, 1956; Opiliones: Nelimatroglodytes Roewer, 1910; Acari: Parasitus sp.; Isopoda: Monolistrapretneri Sket, 1964, Sphaeromidesvireimediodalmatina Sket, 1964, Alpioniscusbalthasari , Cyphopleonkratochvili ; Amphipoda: Niphargus sp.; Decapoda: Troglocaris sp.; Chilopoda: Eupolybothrustridentinus, Harpolithobius sp., Lithobius sp., Cryptops sp.; Diplopoda: Brachydesmussubterraneus Heller, 1858; Collembola: Troglopedetespallidus Absolon, 1907, Heteromurusnitidus , Pseudosinellaheteromurina , Lepidocyrtus sp.; Diplura: Plusiocampa (Stygiocampa) dalmatica Conde, 1959, Japygidae gen. spp.; Coleoptera: Laemostenuscavicolamülleri , Athetaspelaea ; Orthoptera: Dolichopodaaraneiformis , Troglophilusovuliformis Karny, 1907, Gryllomorphadalmatina Ocskay, 1832; Psocoptera: Psyllipsocus sp.; Lepidoptera: Apopestesspectrum ; Amphibia: Proteusanguinus Laurenti, 1768; Chiroptera: a colony of bats, Myotiscapaccinii   .Cavernicolus means “living in caves or caverns”, to emphasise that the species inhabits caves.http://dx.doi.org/10.5883/DS-EUPCAVhttp://plazi.org:8080/GgSRS/html?CCEB9C62C87766E980DD858BC13468C8KF715047KF715051KF715053KF715060KF715055KF715056KF715057KF715058999021822&tsn=trueLithobius (Polybothrus) leostygis Verhoeff, 1899 - Occurrence: recordedBy: J. Bedek; individualCount: 1; sex: male; lifeStage: adult; Location: country: Croatia; stateProvince: Dubrovnik-Neretva; locality: Jama u Zabirađu, Zabirađe, Osojnik; Event: samplingProtocol: hand collected; eventDate: 30 March 2008; Record Level: institutionCode: ZMUC; collectionCode: zmuc00029441Eupolybothrusleostygis is still insufficiently known, we provide here scanning electron microscope images  – Eupolybothrustridentinus (GER2) to 24.5 % for Eupolybothrusgrossipes – Eupolybothruscavernicolus Komerički & Stoev sp. n. (Table Eupolybothrusnudicornis (7.2 %) and minimal for individuals of Eupolybothrusobrovensis and Eupolybothrusgloriastygis and the species Eupolybothruscavernicolus Komerički & Stoev sp. n. (0.0 %)  Fig. .http://cegg.unige.ch/orthodb6) , Ixodesscapularis Say, 1821 and Strigamiamaritima . HaMstR (The raw data was first filtered by removing inadequate reads with: 1) adapter contamination; 2) ≥10 Ns; 3) ≥50 base pairs of low quality . The resulting 2 Gb of clean data were processed into subsequent assemblies using SOAPdenovo_trans  under de20rthodb6) , 2,188 o. HaMstR  was appl. HaMstR ).Eupolybothrus of Eupolybothruscavernicolus Komerički & Stoev sp. n. falls into subgenus Schizopolybothrus Verhoeff, 1934, characterized by the presence of triangular projections on tergites 9, 11, 13, a VCm spine on leg 15, one or more VCa spines and a single claw on the pretarsus of leg 15. The same author further distinguishes three species groups in the subgenus based on the morphology of male gonopods and presence/absence of modifications on leg 15:According to the division of the subgenera of Eupolybothruscaesar , from Bosnia-Herzegovina, Albania, mainland Greece (incl. Ionian Is.) and Macedonia (FYROM); Eupolybothrusspiniger , from Bosnia-Herzegovina; Eupolybothrusacherontis , from Bosnia-Herzegovina (Eupolybothrusacherontisacherontis) and Macedonia (FYROM) ); Eupolybothrusstygis , from Bosnia-Herzegovina; and Eupolybothrusleostygis, from Croatia and Bosnia-Herzegovina  from the Western Alps and Eupolybothrusexcellens  from the Ligurian Apennines.Group II, lacking any specific modifications on male legs while gonopods are also short, encompassing Eupolybothruszeus  from Central Greece and Eupolybothrussissii  from Euboea Island, Greece. Both species are currently considered junior synonyms of the widespread Carpathian-Balkan species Eupolybothrus (Mesobothrus) transsylvanicus  by the presence of a large proximal knob surmounted by a characteristic cluster of setae, and distal setose protuberance of male prefemur 15. In addition, the species presents a different arrangement of spiniform setae on the intermediate tergite.Jeekel’s division of the genus  is quitesee Fig. b. Two othrus cf. . ShowingThe new generation imaging technologies, such as magnetic resonance imaging (MRI) and micro-computed tomography (micro-CT) are opening new horizons in biology . Micro-CThe ‘cybertype’ notion is herewith tested for the first time with the newly described taxon Fig. , for whiAlipesgrandidieri Lucas, 1864 , exist in the literature  MINSEQE compliant metadata . To maxihttp://dicom.nema.org/) used by the medical community is not tailored for taxonomic purposes, thus its usefulness for this research field still has to be investigated (http://morphosource.org). The same set of descriptors has been used by GigaDB, where we also applied the ISA-TAB format in order to ensure re-usability and interoperability of the data  files:Eupolybothrus_cavernicolus_sequenced_vaucher_ paratype.csvEupolybothrus_cavernicolus_micro-CT_vaucher_ paratype.csvEupolybothrus_cavernicolus_all_types.csvISA-TAB metadata that ensure retrievability and interoperability.Eupolybothruscavernicolus Komerički & Stoev sp. n. The generation of large molecular and morphological data pools that originate from type specimens increases the applicability of taxonomic data in other scientific disciplines such as comparative morphology, evolutionary biology, medicine, ecology. The new holistic approach raises important questions and shows up new directions for developments of biodiversity data management about the lack of mechanisms for cross-linking molecular and morphological data and global metadata standards for micro-CT and transcriptomic data, as well as absence of reliable data repositories for micro-CT image libraries.In combination with the Darwin Core files describing the specimen data, we thus fully annotate and document the ‘cybertype’ of Eupolybothrus (Schizopolybothrus) species with their original descriptions that were extracted from the original publications through applying optical character recognition (OCR) and additionally tagged by using Golden Gate software .Also, as a pilot project, we annotate all currently valid software . All speTo create reliable links between the published sequence IDs and BOLD, an online dataset DS-EUPCAV was generated in the BOLD system, through which the respective Barcode Index Numbers (BINs) of the specimens barcoded for this study may be tracked  or even distributed . Further problems to be addressed are the lack of appropriate mechanisms for cross-linking molecular and morphological data, as well as the absence of global metadata standards and reliable data repositories for micro-CT image libraries. The metadata descriptors for micro-CT files used by the Morphosource and GigaDB repositories are a good starting point for that, as is the use of ISA-TAB to integrate everything together. Whatever the answers to these questions, there is one mandatory requirement for data that we can already identify: discoverability and accessibility.With complex taxon descriptions such as the present one, we are entering new dimensions of data volumes that have to be managed properly to realise their true value. The deposition of large data pools in appropriate repositories is not yet straightforward, and such initiatives have started to emerge only recently. It is our task to ensure from the beginning that they do not develop into isolated data worlds but that they support community standards, describing the datasets in a way that they can be retrieved and cross-linked. Currently, even in modern taxon descriptions, different pieces of data are only linked through a central locus: the published article. In a future, data-centric world of taxonomy, articles published through next generation journal workflows will become an even more important node in a linked network of data elements describing the taxon. These data elements have to be defined and made accessible through persistent identifiers – not unlike the traditional practice for physical specimens that are accessible through their museum accession number. In combination with rich metadata standards, taxonomy will thus open itself up to the semantic Web with its possibilities for intelligent, complex queries.In this study, we have taken a first step towards that direction. All data have been deposited in publicly accessible repositories, such as GigaDB, NCBI, BOLD, Morphbank and Morphosource, and the respective open licenses used ensure their accessibility and re-usability. GigaDB in this example provides direct links between the genomic and micro-CT data, through a Darwin Core CSV dataset describing the type specimens, as well as capturing all of the metadata in the interoperable ISA-TAB format. Molecular data and images are annotated with rich metadata to ensure discoverability and reuse. Techniques such as micro-CT are, however, still in their infancy, and no standardised metadata schemas exist yet – a gap that needs urgently to be addressed by the community if we are to avoid a proliferation of isolated datasets.Taxonomy is at a turning point in its history. New technologies allow for creation of new types of information at high speed and in gigantic volumes, but without clear rules for communication standards, we will not be able to exploit their full potential. We need to focus our efforts on linking these bits and pieces together, by documenting them, by standardising them and by making them retrievable. If such an infrastructure is in place, unforeseen analytical powers can be unleashed upon these data, creating a revolution in our abilities to understand and model the biosphere.


Reference 46 was cited incorrectly. The corrected reference is:Tsitsanou KE, Thireou T, Drakou CE, Koussis K, Keramioti MV, Leonidas DD, Eliopoulos E, Iatrou K, Zographos SE (2012)Anopheles gambiae odorant binding protein crystal complex with the synthetic repellent DEET: implications for structure-based design of novel mosquito repellents. Cellular and Molecular Life Sciences 69(2): 283-297. doi:10.1007/s00018-011-0745-z. PubMed: 21671117.An incorrect reference appears in the Materials and Methods section, in the second to last sentence of the first paragraph under '4: Hit Expansion and Molecular docking.' The corrected sentence should read: "Despite the fact that the Anopheles gambiae OBP1 structure  [46] was solved at a higher resolution, we selected the Aedes aegypti 3K1E structure for docking because of our interest in this particular mosquito species."


Acid-sensing ion channels (ASICs) have long been known to sense extracellular protons and contribute to sensory perception. Peripheral ASIC3 channels represent natural sensors of acidic and inflammatory pain. We recently reported the use of a synthetic compound, 2-guanidine-4-methylquinazoline (GMQ), to identify a novel nonproton sensing domain in the ASIC3 channel, and proposed that, based on its structural similarity with GMQ, the arginine metabolite agmatine (AGM) may be an endogenous nonproton ligand for ASIC3 channels.2+. AGM-induced ASIC3 channel activation was not through the chelation of extracellular Ca2+ as occurs with increased lactate, but rather through a direct interaction with the newly identified nonproton ligand sensing domain. Finally, AGM cooperated with the multiple inflammatory signals to cause pain-related behaviors in an ASIC3-dependent manner.Here, we present further evidence for the physiological correlation between AGM and ASIC3. Among arginine metabolites, only AGM and its analog arcaine (ARC) activated ASIC3 channels at neutral pH in a sustained manner similar to GMQ. In addition to the homomeric ASIC3 channels, AGM also activated heteromeric ASIC3 plus ASIC1b channels, extending its potential physiological relevance. Importantly, the process of activation by AGM was highly sensitive to mild acidosis, hyperosmolarity, arachidonic acid (AA), lactic acid and reduced extracellular Cain vivo conditions.Nonproton ligand sensing domain might represent a novel mechanism for activation or sensitization of ASIC3 channels underlying inflammatory pain-sensing under  Acid-sensing ion channels (ASICs) represent a new subgroup of the epithelial sodium channel/degenerin (ENaC/DEG) family of ion channels. To date, functional cloning studies revealed four genes that give rise to at least six ASIC informs  . ASIC3 i+ channels )n], where I is the normalized current at a given pH, Imax is the maximum normalized current, EC50 is the concentration of proton yielding a current that is half of the maximum, and n is the Hill coefficient.Results were expressed as means ± S.E.M. Unless otherwise noted, statistical comparisons were made with the Student's N-methyl-D-aspartate; NO: nitric oxide; PcTX1: psalmotoxin 1; TRPM: Transient Receptor Potential Melastatin; TRPV1: Transient Receptor Potential Vanilloid Type 1; WT: wild-type.5-HT: serotonin; AA: arachidonic acid; AGM: agmatine; ARC: arcaine; ASIC: acid-sensing ion channel; BDNF: brain-derived neurotrophic factor; CHO: Chinese Hamster Ovary; DRG: dorsal root ganglion; ENaC/DEG: epithelial sodium channel/degenerin; GFP: green fluorescent protein; GMQ: 2-guanidine-4-methylquinazoline; H-Osm: Hyperosmolarity; MTSEA: 2-aminoethyl-methanethiosulfonate; MTSET: 2-(trimethylammonium)ethyl methanethiosulfonate; NGF: nerve growth factor; NMDA: The authors declare that they have no competing interests.WGL, YY, and TLX designed the project. WGL, YY, and ZDZ performed cell culture, patch-clamp recording, behavior tests, and data analysis. ZDZ and HC did mutations. WGL and TLX wrote the manuscript. All authors read and approved the final manuscript.