Memórias do Instituto Oswaldo Cruz AnalyzingFor ambiguities Review in trypanosomatids Only taxonomy by barcoding Journal: Memórias do Instituto Oswaldo Cruz Manuscript ID MIOC-2019-0504.R1 Manuscript Type: Original Article Date Submitted by the n/a Author: Complete List of Authors: Boucinha, Carolina; Instituto Oswaldo Cruz Caetano, Amanda; Instituto Oswaldo Cruz Santos, Helena; Instituto Oswaldo Cruz Healers, Raphael; University Catholique of Louvain, de Duve Institute Vikkula, Miikka; University Catholique of Louvain, de Duve Institute Branquinha, Marta; Instituto de Microbiologia Paulo de Góes - Universidade Federal do Rio de Janeiro, Microbiologia Geral Santos, André; Universidade Federal do Rio de Janeiro, Grellier, Philippe; Muséum National d'Histoire Naturelle, RDDM Morelli, Karina; Universidade do Estado do Rio de Janeiro, Ecologia d'Avila-Levy, Claudia; IOC, barcode, barcoding, Kinetoplastea, taxonomy, Trypanosomatida, Keyword: Trypanosomatidae Theme: Taxonomy, Phylogeny, Parasitology https://mc04.manuscriptcentral.com/mioc-scielo Page 1 of 41 Memórias do Instituto Oswaldo Cruz 1 2 3 4 5 6 7 8 Analyzing ambiguities in trypanosomatids taxonomy by barcoding Commented [CL1]: Running title deleted, as requested. 9 10 11 Carolina Boucinha1, Amanda C. Rodrigues1, Helena L. C. Santos1, Raphael Healers2, Miikka 12 Vikkula2, Marta Helena Branquinha3, André Luis Souza dos Santos3, Philippe Grellier4, 13 1,5 1,2* 14 Karina Alessandra Morelli , Claudia Masini d'Avila-Levy 15 16 1 Coleção de Protistas, Laboratório de Estudos Integrados em Protozoologia, Instituto 17 Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. 2 de Duve Institute, University 18 19 of Louvain, Brussels, Belgium. 3 Instituto de Microbiologia Paulo de Góes, UFRJ, Rio de 20 Janeiro, Brazil. 4 Unité Molécules de Communication et Adaptation des Microorganisme 21 For Review Only (UMR 7245 CNRS MCAM), Muséum National d'Histoire Naturelle, Paris, France. 5 Instituto 22 23 de Biologia Roberto Alcântara Gomes, Departamento de Ecologia, UERJ, Rio de Janeiro, 24 Brazil. 25 26 27 * Corresponding author: https://orcid.org/0000-0001-8042-4695 (d'Avila-Levy CM):: 28 Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil 4365, Manguinhos, 21040-360, 29 Rio de Janeiro, Brazil. Tel.: +5521 2562-1014; Fax: +5521 2590-3495; email: 30 [email protected] 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 https://mc04.manuscriptcentral.com/mioc-scielo Memórias do Instituto Oswaldo Cruz Page 2 of 41 1 2 3 4 5 6 7 8 Abstract 9 10 BACKGROUND: Biodiversity screens and phylogenetic studies are dependent on 11 reliable DNA sequences in public databases. Biological collections possess vouchered 12 specimens with a traceable history. Therefore, DNA sequencing of samples available at 13 14 institutional collections can greatly contribute to taxonomy, and studies on evolution and 15 biodiversity. METHODS: We sequenced part of the glycosomal glyceraldehyde phosphate 16 dehydrogenase (gGAPDH) and the SSU rRNA (V7/V8) genes from 102 trypanosomatid 17 cultures, which are available on request at www.colprot.fiocruz.br. OBJECTIVE: The main 18 19 objective of this work was to use phylogenetic inferences, using the obtained DNA sequences 20 and those from representatives of all Trypanosomatidae genera, to generate phylogenetic trees 21 For Review Only that can simplify new isolates screenings. FINDINGS: A DNA sequence is provided for the 22 23 first time for several isolates, the phylogenic analysis allowed the classification or 24 reclassification of several specimens, identification of candidates for new genera and species, 25 as well as the taxonomic validation of several deposits. MAIN CONCLUSIONS: This survey 26 27 aimed at presenting a list of validated species and their associated DNA sequences combined 28 with a short historical overview of each isolate, which can support taxonomic and biodiversity 29 research and promote culture collections. 30 31 32 Keywords: barcode, barcoding, Kinetoplastea, taxonomy, Trypanosomatida, 33 Trypanosomatidae, voucher 34 35 36 Sponsorships: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), 37 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos 38 Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) 39 40 41 42 43 44 45 46 47 Introduction 48 49 The class Kinetoplastea is a noted group of protists that has one or two flagella, 50 emerging from a flagellar pocket and that may exist in nature either as free-living species or 51 as parasites of vertebrates, invertebrates or plants. The class is defined by the presence of a 52 53 54 55 56 57 58 59 60 https://mc04.manuscriptcentral.com/mioc-scielo Page 3 of 41 Memórias do Instituto Oswaldo Cruz 1 2 3 4 5 6 7 8 characteristic structure, the kinetoplast, a highly condensed DNA from a single mitochondrion 9 (1) 10 that ramifies throughout the cell body. 11 The family Trypanosomatidae encompasses members, which are the causative agents 12 of severe human diseases that are mainly transmitted by an insect vector, such as Chagas 13 14 disease (caused by Trypanosoma cruzi), sleeping sickness (caused by Trypanosoma brucei 15 sensu lato) and the various forms of cutaneous and visceral leishmaniasis (caused 16 by Leishmania spp.). (2) In addition, there are currently 18 formally described genera that are 17 generically known as insect trypanosomatids, because they are thought to have the life cycle 18 19 restricted to an insect host, namely: Angomonas, Blastocrithidia, Blechomonas, Borovskyia, 20 Crithidia, Herpetomonas, Jaenimonas, Kentomonas, Lafontella, Leptomonas, Lotmaria, 21 For Review Only Novymonas, Paratrypanomsoma, Rhynchoidomonas, Sergeia, Strigomonas, Wallacemonas 22 (3) (4-8) 23 and Zelonia. (reviewed by and new genera described in ) Finally, there is one genus, 24 Phytomonas, which alternates its life cycle between a plant and an insect. (9, 10) 25 In a historical perspective, trypanosomatids taxonomy and diversity have been 26 27 completely revolutionized by the so-called “molecular era”. For instance, from 1966 to 1990, 28 only one new genus has been described,(1, 11) after the 90’s, 14 new genera were described, 29 with a clear increase of genera description in the last years. (4-8, 12) This augment in the number 30 of described taxa is not a simple consequence of more field expeditions and sample collection, 31 32 it also reflects taxonomic revisions and proposals of new taxa to better reflect the phylogeny 33 of previously described trypanosomatid isolates, whose true diversity has been hidden by 34 traditional taxonomy approaches.(3, 8, 13) 35 36 It is clear nowadays that trypanosomatid taxonomy, previously strongly based on 37 morphotypes, host specificity or site/mode of host colonization, must also be supported by 38 phylogenetic inferences. To this end, glycosomal glyceraldehyde-3-phosphate dehydrogenase 39 40 (gGAPDH) and the V7/V8 variable region of the 18S small subunit ribosomal RNA (SSU) 41 have been widely used and have been advocated as barcodes for trypanosomatid taxonomy, 42 while the spliced leader (SL) RNA and the internal transcribed spacer (ITS) region of the 43 ribosomal RNA are suitable to analyze strain or isolate diversity.(3, 8, 14) The use of these 44 45 markers allows the identification of species through comparison with sequences available in 46 public databases. However, it is not uncommon that the public databases lack one or more of 47 the aforementioned molecular markers, or even worse, the associated data of the sequenced 48 49 organism is imprecise, incomplete, outdated or even incorrect. Therefore, culture collections 50 with vouchered species and associated information are of outmost importance to pave the road 51 for trypanosomatid taxonomy.(15) 52 53 54 55 56 57 58 59 60 https://mc04.manuscriptcentral.com/mioc-scielo Memórias do Instituto Oswaldo Cruz Page 4 of 41 1 2 3 4 5 6 7 8 The Fiocruz Protist Culture Collection (Fiocruz-COLPROT, www.colprot.fiocruz.br) 9 10 has more than 400 live specimens of protists from several families, most of them 11 representatives of the Kinetoplastea class, mainly members of the Trypanosomatidae family. 12 This collection is constantly enriched with deposits from various collaborators, as well as field 13 14 expeditions performed by its research team. To ensure reliable management of a culture 15 collection is extremely important because it greatly increases the chances of sample 16 availability and preservation for future generations.(15) 17 Here, we sequenced gGAPDH and SSU (region V7/V8) to provide an unequivocal 18 19 molecular signature to 102 trypanosomatid specimens that compose part of the COLPROT 20 catalogue. In addition, a detailed revision of the archived information regarding sample 21 For Review Only precedency, host, place and year of isolation and any relevant associated information was 22 23 detailed, revised and made public. Our data led to the reclassification of several specimens 24 based on recent taxonomic revisions and phylogenetic inferences, the identification of 25 candidates to new genus and species, as well as the confirmation of the molecular identity of 26 27 several deposits. Overall, this “molecular certification” offers to the scientific community a 28 broader view of the family Trypanosomatidae. 29 30 Materials and Methods 31 32 Chemicals 33 Media constituents, reagents used in electrophoresis, buffer components, agarose and 34 all other reagents are of analytical grade or superior. The following kits were purchased from 35 36 Promega: Wizard® Genomic DNA Purification kit and Wizard® SV Gel and PCR Clean-Up 37 System. GelRed™ fluorescent DNA stain was obtained from Biotium. The BigDye® 38 Terminator v3.1 Cycle Sequencing was obtained from Life Technologies. Fetal bovine serum 39 40 (FBS) was purchased from Cultilab and heat inactivated. 41 42 Trypanosomatid cultures 43 All organisms examined in this study are cryopreserved at Fiocruz Protist Culture 44 45 Collection (COLPROT) (http://colprot.fiocruz.br). The host, geographical origin, isolator, 46 depositor and year of isolation are summarized in Table S1.