Biodiversity and adapta:ons of CYP enzymes in the Amazon Loricariidae fishes Colaborators STAFF Dr. John Stegeman – WHOI, USA PI_BR: Dr. Thiago E. Parente – FIOCRUZ, Rio de Janeiro, Brazil Dr. Francisco Paumgarien – FIOCRUZ, Brazil MSc. Daniel A. Moreira Dr. Paulo Buckup – MN, UFRJ, Brazil PI_US: Dr. Mark E. Hahn – WHOI, Woods Hole, MA, USA Paula C.C. de Andrade (undergrad) Dra. Carolina Furtado – INCA, Brazil Maithê G.P. Magalhães (undergrad) Dr. Adalberto Val – INPA, Brazil parente@ensp.fiocruz.br & [email protected] Dr. Brian Haas – Broad Ins8tute, USA Hypancistrus zebra Tangen:al project #1 Siluriformes 73 Figure R1: Annotaon of the liver transcriptome Abstract A B Mitogenomes assembled from transcriptome 1 Danio from Pterygoplichthys anisitsi. A - Top BLASTx hit Loricariidae is the most diverse family of the fish order Siluriformes . Currently, 4445 Molecular function 20% there are >700 valid species and this number is s8ll growing due to discovery of Homo species distribu8on. B - Func8onal classificaon We sequenced the transcriptome of three fish from the genus Ancistrus (Loricariidae, Results Rattus 9739 2676 Biological processes Siluriformes) using as start material total RNA isolated from the liver. The transcriptome data new species and the resolu8on of cryp8c ones1,2,3. Loricariids inhabit a wide 51% according to Gene Ontology (GO, first level) terms. Cellular components were used to assemble the mitogenome of each fish with 92%, 95% and 99% of the full 29% C - distribu8on of GOSlim2 terms over the spectrum of fresh water environments, have diverse ecological habitats and show C Mus Pongo length of their closest related species with a sequenced mitogenome. Taken the sequences 6827 848 4 assembled transcripts. of the three fish together, all the 13 protein-coding genes, two ribosomal RNAs, 22 tRNAs high endemism rate . This astonishing taxonomic and ecological diversity must rely 100000 and the D-loop known in the mitogenomes of vertebrates were sequenced. The use of on gene8c, molecular and biochemical adaptaons. In fact, our group has reported transcriptomic data also allowed the clear observaon of the punctuaon paern of mtRNA peculiar adaptaons in loricariids, which might increase their suscep8bility to the edi8ng, to analyze the transcrip8onal profile of mtRNA, and to detect heteroplasmic sites. 10000 5,6,7 . Ancistrus"sp."#1 Ancistrus"sp."#2a Ancistrus"sp."#2b toxic effects of pollutants . Here, the molecular biodiversity of Loricariidae fish is RNA"Integrity"Number"7"RIN 8.2 7.4 >7.00 Table TP1: Summary of the Library"insert"size"(bp) 230)*)800 268)*792 285)*)370 explored using Next-Generaon Sequencing (NGS) technologies. In total, 101 Reads"after"QC 43502597 53961751 60170745 Transcripts 1000 transcriptome and species has been collected; 34 of these will have their liver transcriptome total 67098 63847 67883 with"BLASTX"hit 35710 31886 33953 mitogenome data for the for"mitogenome 13 12 7 sequenced. For one species, Hypancistrus zebra, the transcriptome of brain, heart, mtRNA"reads"(%) 2.6 1.8 0.8 three fish (Ancistrus spp.). Mitogenome"coverage"(%) 99.2 92.5 94.7 kidney, gill, intes8ne and gonad will also be sequenced. The transcriptome of three 100 Heteroplasmic"sites 44 46 41 species has been sequenced; Pterygoplichthys anisitsi, Ancistrus spp. and Corydoras A$F$ V$ L$ I$Q$M$ W$A$N$C$Y$ S$D$ K$ G$ R$ H$S$L$ E$ T$P$ naereri. The results for P. anisitsi are shown bellow. Briefly, 34204 genes with 12S$rRNA$ 16S$rRNA$ nd1$ nd2$ cox1$ cox2$ atp8$ atp6$ cox3$ nd3$ nd4l$ nd4$ nd5$ nd6$ cob$ homologs in zebrafish were sequenced; 189 of these contained the complete coding sequence of proteins involved in mechanisms of defense against toxins. Great expansions were detected in subfamilies of Cytochromes P450 and cell death cilium plastid growth cytosol binding nucleus vacuole cell wall behavior ribosome transport cell cycle nucleolus lysosome cytoplasm cell death endosome catabolism cell growth intracellular peroxisome metabolism lipid particle lipid binding cytoskeleton nucleoplasm DNA bindingRNA binding actin binding biosynthesis ion transportreproduction chromosome cell envelope motor activity development viral life cycle mitochondrion kinase activity protein binding oxygen binding B$ cell recognition Golgi apparatus catalytic activity receptor activity morphogenesis receptor binding cell proliferation nuclease activity lipid metabolismprotein transport hydrolase activity peptidase activity DNA metabolism cell homeostasis chromatin binding Sulfotransferases. cell-cell signaling nucleotide binding transporter activity antioxidant activity References: 4: Covain & Fisch-Muller, 2007; Zootaxa 1462: 1-40 cell differentiation plasma membrane extracellular space nuclear membrane transferase activity extracellular region calcium ion binding ion channel activity response to stress signal transduction nucleic acid binding cell communicationprotein metabolism protein modification protein biosynthesis nuclear chromosome carbohydrate binding protein kinase activity 1: Covain & Fisch-Muller, 2007; Zootaxa 1462: 1-40 5: Parente et al., 2009; Comp. Bioch. Physiol. C 150: 252-260 endoplasmic reticulum secondary metabolism nutrient reservoir activity signal transducer activity enzyme regulator activity embryonic development cytoplasmic chromosome transcription factor activity structural molecule activity carbohydrate metabolism cytoskeletal protein binding translation regulator activity 2: Ferraris, 2007; Zootaxa 1418: 1-628 6: Parente et al., 2011; Gene 489: 111-118 response to abiotic stimulus response to biotic stimulus microtubule organizing center response to external stimulus 3: Taphorn et al., 2013; Zootaxa 3641(4): 343-370 7: Parente et al., 2014; Aquac Toxicology 154: 193-199 external encapsulating structure cell organization and biogenesis response to endogenous stimulus neurotransmitter transporter activity extracellular matrix (sensu Metazoa) phosphoprotein phosphatase activity cytoplasmic membrane-bound vesicle organelle organization and biogenesis cytoplasm organization and biogenesis cytoskeleton organization and biogenesis regulation of gene expression, epigenetic translation factor activity, nucleic acid binding mitochondrion organization and biogenesis generation of precursor metabolites and energy nucleoside, nucleotide and nucleic acid metabolism Table R1: Table 3.3.1: Number of sequenced components in the hepatic transcriptome of P. anisitsi with A complete coding sequence (CDS), >75% of the CDS, >50% of the CDS and the total number of C$ Material & Methods Figure R2: Mitochondrial contigs for each defensome gene family. genome of Pterygoplichthys Full length CDS >75% CDS >50% CDS Contigs spp.. A – Circular coverage coverage • (FIOCRUZ, INPA and MN UFRJ) representaon of P. AHR & ARNT 3 3 3 9 Fish sampling disjuncvus mtDNA (Gi: Aldo Keto Reductase 5 5 5 5 1kb$ Corydoras RJ Corydoras AM 339506171). Red circles Callichthydae ATP Binding Cassette (ABC) 13 19 25 91 indicate the tRNA n o t Figure TP1: The assembled mitogenomes of Ancistrus sp.#1 (A), Ancistrus sp.#2a (B) and Ancistrus sp.#2b (C). The number of aLRT Basic leucine zipper 3 3 5 8 1 suppor8ng reads along the sequence is shown in a logarithmic scale and below the schemac view of each mitogenome. Hoplosternum litorale AM Corydoras sp. Hoplosternum litorale RJ sequenced In P. anisitsi mtDNA Heteroplasmic sites are highlighted with a different color on the graphic of suppor8ng reads. Each feature of the mitogenomes is Delturinae Catalase 1 1 1 1 Hemipsilichthys RJ named on the top of the figure. The tRNAs are named using the one-leier code of the amino acid they transport. and red arrows the Otothryni Hysonotus RJ Cytochrome P450 34 35 39 82 Parotocinclus RJ Hypoptomatinae Schizolecis RJ approximate region of the Epoxide hidroxilase 2 2 2 2 Otocinclus RJ Hypoptomatini Hypoptopoma AM three smallest gaps of 10, 24 Kronichthys RJ Glucuronosyltransferase 8 8 10 15 Neoplecostominae Pareiorhapis garbie RJ and 31 nucleo8des. B – Linear Neoplecostomus RJ aLRT Rineloricaria sp. 1 Glutatione Peroxidase 6 6 8 11 representaon of the Glutatione-S-transferase 8 10 10 10 annotated mitochondrial IDENTIFICAÇÃO MOLECULAR DASTangen:al project #2 ESPÉCIES DE LORICARIIDAE (SILURIFORMES) DA Yet to be done Loricariinae Loricarichthys AM n-acetyl-transferases 10 11 11 14 BACIA DO RIO PARAÍBA DO SUL E DRENAGENS COSTEIRAS ADJACENTES Loricaria AM genome of P. anisitsi. Loricariidae Loricarichthys RJ Paulo A . Buckup1 , W. Bryan Jennings1 , Thiago E. M. Parente2 , Maithê G. P. Magalhães2 , Rineloricaria RJ Nuclear receptor 23 32 33 54 B Paula C. C. Andrade2 , Gustavo A. Ferraro1 Rineloricaria AM 1 2 • Sequence the transcriptome of 31 species Museu Nacional, UniversidadeBarcoding Federal do Rio de Janeiro. Fundação Osvaldoloricariids Cruz, Rio de Janeiro. Ancistrus sp. Sulfotransferases (SULT) 47 49 49 53 [email protected]. Dekeyseria AM Ancistrini Ancistrus RJ A bacia do Para íba do Sul e os rios das baixadas MNLM6149NmicPAG • Assemble the 31 transcritomes 0 BACIAS MNLM6150NmicPAG Ancistrus AM costeiras adjacentesà sua foz no Estado do Rio de 95 Superoxide desmutase 3 3 3 3 SUDESTE MNLM6148NmicPAG MNLM6151NmicPAC Panaque AM Janeiro abrigam 37 espécies de peixes loricariídeos NEOPLECOSTOMINAE PARAÍBA DO SUL 100 88 MNLM6152NmicPAC Neoplecostomus microps Bariancistrus AM usualmente consideradas válidas, além de LESTE MNLM6279NmicPAG Peckoltia AM Bariancistrus sp. DOCE MNLM5092NmicRON representantes de um gê nero ainda não descrito. O 100 55 MNLM6121NmicPAP • Analyze CYPs and AHR genes in the 31 species