Science de la vie, de la terre et agronomie REV. CAMES - VOL.05 NUM.02. 2017 ** ISSN 2424-7235 1 REV. CAMES - VOL.05 NUM.02. 2017 ** ISSN 2424-7235 Science de la vie, de la terre et agronomie CAMES Historique Plusieurs réunions de spécialistes chargés de définir le rôle et les fonctions de l’Enseignement Supérieur ont conduit à la constitution d’une "Commission consultative d’expert pour la réforme de l’Enseignement en Afrique et à Madagascar". Une résolution de la Conférence des Ministres de l’Éducation nationale tenue à Paris en 1966 donnait mandat à la commission d’entreprendre une recherche approfondie sur les structures et les enseignements des Universités Africaines et malgaches, dans un large esprit de coopération interafricaine. Les conclusions de la réflexion menée par la Commission leur ayant été soumises à la Conférence de Niamey, tenue les 22 et 23 janvier 1968, les Chefs d’Etats de l’OCAM décidèrent la création du "Conseil Africain et Malgache pour l’Enseignement Supérieur", regroupant à ce jour seize (16) Etats francophones d’Afrique et de l’Océan Indien. La convention portant statut et organisation du CAMES fut signée par les seize (16)Chefs d’Etat ou de Gouvernement, le 26 Avril 1972 à Lomé. Tous les textes juridiques ont été actualisés en 1998-1999 et le Conseil des Ministres du CAMES, a lors de la 17ème Session tenue à Antananarivo en Avril 2000, adopté l’ensemble des textes juridiques actualisés du CAMES, qu'on peut retrouver sur le site web http://www.lecames.org/spip.php?article1 Missions • Promouvoir et favoriser la compréhension et la solidarité entre les Etats membres ; • Instaurer une coopération culturelle et scientifique permanente entre les Etats membres ; • Rassembler et diffuser tous documents universitaires ou de recherche : thèses, statistiques, informations sur les examens, annuaires, annales, palmarès, information sur les offres et demandes d’emploi de toutes origines • Préparer les projets de conventions entre les États concernés dans les domaines de l’Enseignement Supérieur, de la Recherche et contribuer à l’application de ces conventions ; • Concevoir et promouvoir la concertation en vue de coordonner les systèmes d’enseignement supérieur et de la recherche afin d’harmoniser les programmes et les niveaux de recrutement dans les différents établissements d’enseignement supérieur et de recherche, favoriser la coopération entre les différentes institutions, ainsi que des échanges d’informations. Organisation Le Conseil des Ministres Le Conseil des Ministres est l’instance suprême du CAMES. Il regroupe tous les Ministres ayant en charge l’Enseignement Supérieur et/ou la Recherche Scientifique des pays membres. Il se réunit une fois l’an en session ordinaire et peut être convoqué en session extraordinaire. L’actuel Président du Conseil des Ministres est le Ministre de l’Enseignement Supérieur et de la Recherche de Côte d’Ivoire. Le Comité des Experts Le Comité des Experts prépare la session ministérielle. Il est composé de deux représentants par pays membre ou institution membre. Il se réunit une fois l’an en session ordinaire et peut être convoqué en session extraordinaire. Le Comité Consultatif Général (CCG) Il supervise et contrôle l’application de l’Accord portant création et organisation des Comités Consultatifs Interafricains. Ses membres sont des Recteurs ou Présidents d’Universités et des Directeurs des Centres Nationaux de Recherche. Les organismes signataires de l’Accord y sont représentés par leurs Directeurs. 2 Science de la vie, de la terre et agronomie REV. CAMES - VOL.05 NUM.02. 2017 ** ISSN 2424-7235 SOMMAIRE A proteome study of Myxobolus episquamalis parasite of flathead mullet (Mugil 06 cephalus Linné 1758) of Senegalese coasts Problématique de la qualité microbiologique des eaux de boisson en milieu scolaire 13 dans les zones rurales : Cas de la Région du Centre-Nord au Burkina Faso. Les minéralisations de manganèse du Nord Téra (Liptako, Ouest Niger) : origine 18 et conditions de mise en place First report of a tomato leaf curl virus (genus Begomovirus) on Corchorus olitorius 29 in Togo (SHORT NOTE) Successional dynamics of tree species during forest recovery in the southeast of 30 Côte d’Ivoire 3 REV. CAMES - VOL.05 NUM.02. 2017 ** ISSN 2424-7235 Science de la vie, de la terre et agronomie INSTRUCTIONS AUX AUTEURS Politique éditoriale Langue de publication La revue publie des articles rédigés en français ou en La Revue CAMES publie des contributions originales anglais. Cependant, le titre, le résumé et les motsclés (en français et en anglais) dans tous les domaines doivent être donnés dans les deux langues. de la science et de la technologie et est subdivisée en 9 séries : Ainsi, tout article soumis en français devra donc comporter, obligatoirement, «un titre, un abstract et • Sciences des structures et de la matière. Elle couvre les des keywords», idem, dans le sens inverse, pour tout domaines suivants : mathématiques, physique, chimie et article en anglais (un titre, un résumé et des informatique, motsclés). • Sciences de la santé : médecine humaine, médecine Page de titre vétérinaire, pharmacie, odonto-stomatologie, La première page doit comporter le titre de l'article, productions animales ; les noms des auteurs, leur institution d'affiliation et leur adresse complète. Elle devra comporter • Sciences de la vie, de la terre et agronomie ; également un titre courant ne dépassant pas une soixantaine de caractères ainsi que l'adresse postale • Sciences appliquées et de l’ingénieur ; Littérature, del'auteur, à qui les correspondances doivent être langues et linguistique ; adressées. • Sciences humaines : Philosophie, sociologie, Résumé anthropologie, psychologie, histoire et géographie ; Le résumé ne devrait pas dépasser 250 mots. Publié • Sciences économiques et de gestion ; seul, il doit permettre de comprendre l'essentiel des travaux décrits dans l'article. • Sciences juridiques et politiques ; Introduction • Pharmacopée et médecine traditionnelles africaines ; L'introduction doit fournir suffisamment Toutes les séries publient en moyenne deux d'informations de base, situant le contexte dans numéros par an. lequel l'étude été entreprise. Elle doit permettre au lecteur de juger de l'étude et d'évaluer les résultats Les contributions publiées par la Revue CAMES acquis. représentent l'opinion des auteurs et non celle du comité de rédaction ou du CAMES. Tous les auteurs Corps du sujet sont considérés comme responsables de la totalité Les différentes parties du corps du sujet doivent du contenu de leurs contributions. apparaître dans un ordre logique. Soumission et forme des manuscrits Conclusion La soumission d'un manuscrit à la Revue CAMES Elle ne doit pas faire double emploi avec le résumé et implique que les travaux qui y sont rapportés n'aient la discussion. Elle doit être un rappel des principaux jamais été publiés auparavant, ne soient pas soumis résultats obtenus et des conséquences les plus concomitamment pour publication dans un autre importantes que l'on peut en déduire. journal et qu'une fois acceptés, ne fussent plus publiés nulle part ailleurs sous la même langue ou La rédaction du texte dans une autre langue, sans le consentement du La rédaction doit être faite dans un style simple et CAMES. concis, avec des phrases courtes, en évitant les répétitions. Les manuscrits, dactylographiés en interligne double en recto sont soumis aux rédacteurs en chef Remerciements des séries. Les remerciements au personnel d'assistance ou à des supports financiers devront être adressés en Les manuscrits doivent comporter les adresses terme concis. postales et électroniques et le numéro de téléphone de l'auteur à qui doivent être adressées les Références correspondances.Les manuscrits soumis à la Revue Les noms des auteurs seront mentionnés dans le CAMES doivent impérativement respecter les texte avec l'année de publication, le tout entre indications cidessous: parenthèses. 4 Science de la vie, de la terre et agronomie REV. CAMES - VOL.05 NUM.02. 2017 ** ISSN 2424-7235 Les références doivent être listées par ordre selon l’ordre d’appel dans le texte. alphabétique, à la fin du manuscrit de la façon La numérotation des tableaux se fera en chiffres suivante: romains et celle des figures en chiffres arabes, dans • Journal : noms et initiales des prénoms de tous les l’ordre de leur apparition dans le texte. auteurs, année de publication, titre complet de l’article, nom complet du journal, numéro et volume, Photographies les numéros de première et dernière page. Les photographies en noir & blanc et couleur, sont acceptées. • Livres : noms et initiales des prénoms des auteurs et année de publication, titre complet du livre, Procédure de révision éditeur, maison et lieu de publication. Les manuscrits sont soumis à la révision des pairs. Chaque manuscrit est soumis au moins à deux • Proceedings : noms et initiales des prénoms des référés spécialisés. Les auteurs reçoivent les auteurs et année de. publication, titre complet de commentaires écrits des référées. Il leur est alors l’article et des proceedings, année et lieu du congrès notifié, par lamême occasion, l’acceptation ou le ou symposium, maison et lieu de publication, les rejet de leur contribution. numéros de la première et dernière page. NB : Le manuscrit accepté doit, après correction Tableaux et figures conformément aux recommandations des référées, Chaque tableau sera soumis sur une feuille séparée être retourné aux différents rédacteurs en chef des et numéroté de façon séquentielle. Les figures seront séries, en format WORD ou DOC. soumises sur des feuilles séparées et numérotées, REDACTEURS EN CHEF DES REVUES Les auteurs sont invités à envoyer directement leurs articles aux rédacteurs en chef des différentes séries: • Sciences des structures et de la matière: Pr ABDOULA YB Alassane: [email protected] (Niamey) • Sciences de la santé: Pr TOURE Meissa [email protected] (Dakar) • Sciences de la vie, de la terre et agronomie: Pr GLITHO Adolé I. [email protected] (Lomé) • Sciences appliquées et de l'ingénieur: Pr FALL Meissa [email protected] (Thiès) • Littérature, langues et linguistique: Pr AINAMON augustin [email protected] (Cotonou) • Sciences humaines: Pr KADANGA Kodjona [email protected] (Lomé) • Sciences économiques et de gestion: Pr ONDO Ossa Albert [email protected] (Gabon) • Sciences juridiques et politiques: Pr SOMA Abdoulaye tikansonsoma@yahooJr (Ouagadougou) • Pharmacopée et médecine traditionnelles africaines Pr OUAMBA Jean Maurille [email protected] (Brazzaville) Les auteurs dont les articles ont été déposés sur la platforme en ligne de la revue ou acceptés par les rédacteurs en chef, doivent procéder au règlement des frais d'insertion qui s'élèvent à 50 000 FCFA auprès de l'agence comptable du CAMES, par transfert rapide. 5 REV. CAMES - VOL.05 NUM.02. 2017 ** ISSN 2424-7235 Science de la vie, de la terre et agronomie A proteome study of Myxobolus episquamalis parasite of flathead mullet (Mugil cephalus Linné 1758) of Senegalese coasts Malick FALL 1,*, Pascal COSETTE 2, Julie HARDOUIN 2, BhenSikina TOGUEBAYE 1, Emmanuel CORNILLOT3 Abstract Myxozoa are endoparasites characterized by a simple cytoplasmic organization and multicellular spores containing polar capsules with extrusible polar filaments. Myxozoans have a complex life-cycle, typically alternating between teleost fish and invertebrate hosts, in which are formed myxospores and actinospores respectively. Both spores contain polar capsules, which are strikingly similar in their morphogenesis and mature structure to the nematocysts found in the phylum Cnidaria (Canning and Okamura 2004). Similarity with cnidarians was also supported by several phylogenetic molecular studies. In this context, we have performed the first large scale proteomic study of a Myxozoa, Myxobolus episquamalis which is a parasite of flathead mullet in Senegal. Comparisons with different databases combining different softwares confirm importance of cytoskeleton and central metabolism in the invasion process. The present study identified for the first time neuropeptides and analgesic toxins encoded by the M. episquamalis. It also highlighted long peptides presenting strong homologies with proteins of unknown function, a large fraction of them being identified from Nematostella vectensis proteome. Homologies with other lower Bilateria raise again the question of the position of Myxozoa at the transition between Cnidarian and Bilaterian. Keywords: Myxozoa, M. episquamalis, Proteomics, Phmmer, Neuropeptide. 1Département de Biologie Animale Faculté des Sciences et Techniques, du Cancer Montpellier (ICM) & Université de Montpellier, France. Université Cheikh Anta DIOP de Dakar. Corresponding author : Malick FALL, 2UMR CNRS 6270, Plateforme Protéomique PISSARO (IRIB), Département de Biologie Animale - Faculté des Sciences et Techniques Université de Rouen, 76821 Mont-Saint-Aignan, France. Université Cheikh Anta DIOP - Dakar, Sénégal 3Institut de Biologie Computationnelle (IBC), Institut de Recherche en Mail: [email protected] Cancérologie de Montpellier (IRCM - INSERM U1194), Institut régional Phone:00221776451810/ 00221338252529. Introduction phylogenetic analysis of the SSU-rDNA allowed placing Myxozoa within the Metazoan (Smothers et al, 1994; Myxozoans are parasites of poikilothermic vertebrates Siddall et al, 1995). More recent phylogenomic analyses and invertebrates, especially fishes and count amongst placed Myxozoa in Cnidaria (Holland et al, 2011; the most known enigmatic organisms. Markiw and Wolf Jimenez-Guri et al, 2007; Nesnidal et al, 2013, Feng et (1983, 1984) were the first discovering a tubificid annelids al, 2014). Polar capsules differ from typical nematocysts as alternate hosts for Myxobolus cerebralis. Later, it of cnidarians in lacking chemo- and/or mechano-sensory has been established that, there may be an obligatory or structures and neural connections that modulate discharge facultative invertebrate host in most, if not all of the life (Jiménez-Guri et al, 2007). cycle of myxozoans. Next this two-stage life cycle was described in several marine species (Køie et al, 2004; Myxosporean are fish parasites. They are present in both 2007; 2008; 2013; Rangel et al, 2009; Karlsbakk and freshwater and marine environments and have a significant Køie, 2012). Vertebrates are considered as intermediate incidence on wild and cultured stocks (Timi and Mackenzie, hosts since the sexual process (gametogony) has been 2015). Few strong epizootic diseases have been described observed during the actinospore phase in invertebrates and associated with Myxobolus pfeifferi, responsible for (annelid oligochaete was described as definitive host for the boil disease of barbels or Myxobolus exiguus which some species). A possible direct fish-to-fish transmission caused epidemic in flathead mullet of the northern sector of was demonstrated for some marine species (Diamant the Black Sea along the Crimean coast (Schulman, 1957). 1997; Redondo et al 2002; Yasuda et al 2002). In these In addition, there are other serious pathogenic concerns later cases, the proliferative stages are responsible for the with important commercial impact. Thus, in cultured transmission of the disease, contradictorily to spores. salmonids, many cases of losses were due to M. cerebralis but also to Tetracapsuloides bryosalmonae, responsible for There are two classes of Myxozoans, the clades the mysterious disease called PKD, observed in USA and Myxosporea and Malacosporea. Their cell organization is Europe. Besides, carp Eurasia infection caused by other typically eukaryotic with nucleus, endoplasmic reticulum, parasites (such as Sphaerospora arenicola, Sphaerospora mitochondria and Golgi cisternae (Canning and Okamura, molnari, Myxobolus eucephalicus, Thelohanellus nikolskii, 2004).Their spores are made of three cell groups: valve Thelohanellus kitauei or Chloromyxum cristatum) may cells, polar capsules and a sporoplasm generally with also cause serious damages in fish farms. Myxobolus two nuclei, or two sporoplasms, each containing one episquamalis Egusa, Maeno and Sorimachi, 1990 was nucleus. Their polar capsules, used to attach to a new host, found for the first time in flathead mullet (Mugil cephalus are similar to the stinging organelles (nematocysts) of Linneaus 1758) in Senegal. It was previously described cnidarians. Structure similarities between the myxozoan in M. cephalus from Israel, Portugal, Japan and Tunisia. polar capsules and nematocysts of cnidarians cnidocysts The infected specimens of flathead gray mullet (Mugil led to consider myxozoan to be aberrant cnidarians (Weill, cephalus) present whitish myxosporidian cystic masses on 1938), a hypothesis further supported by ultrastructural their bodies, measuring approximately 5-7 mm. The cystic studies (Lom and de Puytorac, 1965; Lom, 1969). First 6 Science de la vie, de la terre et agronomie REV. CAMES - VOL.05 NUM.02. 2017 ** ISSN 2424-7235 masses are distributed over the entire body and cover 10 to using the Bradford protein assay (Bio-Rad) with BSA used 70% of the body. They are located on the distal part of the as standard. For the first dimension (isoelectric focusing, scales and also affected the fins causing malformations. IEF), 50 µg of proteins (final volume 350 µL) were loaded The cyst mass is ovoid, containing numerous spores. The onto immobilized pH gradients (ImmobilineDryStrip pH spore is pyriform in front view and each valve presents 7 3-10, 18 cm; GE Healthcare). IEF was performed using to 9 markings along the suture wall. The polar capsules an IEF-CELL (Bio-Rad) as follows: active rehydration for are pyriform and extending half of the spore length and 1h at 350 V, 1h at 750V, 1h at 1500V and final focusing formed 5 or 6 coils inside of the polar capsule. Finally, the for 28h at 3500 V. The strips containing focused proteins sporoplasm is binucleate, occupying the posterior part of were then equilibrated for 10 min in a buffer containing 6 the spore. M urea, 30% glycerol, 2% SDS,50 mMTris-HCl (pH 6.8) with 5 mM dithiothreitol, and then for an additional 10 Up to now, very few molecular investigations were min in the same solution except that DTT was replaced by dedicated to M. episquamalis. Thus we performed a 25 mM iodoacetamide. Thereafter, the second-dimension proteomic study to identify M. episquamalis cyst gel separation was ensured by sodium dodecyl sulfate expressed proteins. To achieve this goal. Thus, in this polyacrylamide gel (SDS-PAGE) electrophoresis using a study, we performed a proteomics study to address 12.5 % polyacrylamide resolving gel (width 16 cm, length identifications of proteins expressed in M. episquamalis 20 cm, thickness 0.75 cm). SDS-PAGE was carried out cysts. To reach this objective, peptide sequencing was using Protean II Xi vertical systems (Bio-Rad) according combined to computational methods to identify major to the protocol described by the manufacturer. Migrations functions associated to this stage of development of the were performed at 10 mA per gel in the stacking gel and 20 parasite. mA in the running gel. Finally, proteins were revealed using Materials and methods silver staining and the gels were scanned using a dedicated imager (ProXpress, Perkin-Elmer). Protein patterns were Biological material analyzed using the ProgenesisSameSpotssoftware (v3.0; Mugil cephalus Linnaeus1758 is a eurythermal and Nonlinear Dynamics). euryhaline fish having a wide geographical range. It hosts Analysis by Mass Spectrometry many parasites including Myxobolus episquamalis which belongs to the phylum Myxozoa and parasitizes apical Spots were excised from the polyacrylamide gel and sliced part of the scales where it induces the formation of cysts into small pieces. Excised fragments were then washed (Fig.1). The scales of parasitized fishes were removed and several times and dried in a SpeedVac centrifuge for 15 ground with cysts in a mortar with PBS. The homogenate minutes. Trypsin digestion of protein spots was performed was centrifuged for ten minutes at 2000 tr/mn. The pellet overnight at 37°C by using a dedicated automate were distributed into 1.5ml tubes and stored at 4°C. (MultiPROBE II, Perkin Elmer) with an enzyme/protein ratio of 1/20 (w/w). The gel fragments were then incubated several times for 15 min in water/acetonitrile mixtures acidified with 0.1% TFA, to allow peptide extraction from the gel pieces. After lyophilization, the peptide extracts were suspended in 10 µL of 0.2% formic acid (FA). The resulting samples were analyzed with an LTQ Orbitrap mass spectrometer Velos (Thermo Scientific) equipped with a nano-ESI and coupled to a nano-chromatography system (Easy-nLC II, Thermo Scientific). After loading the Figure 1. Flathead mullet (Mugil cephalus) presenting numerous sample on an enrichment column, separation was achieved cysts on its scales. on a C18 column (NikkyoTechnos, Japan) using a linear These cysts contain mature spores of the myxosporean parasite gradient of 15% to 45% of B buffer in 30 min (buffer A: Myxobolus episquamalis. Microscopic cysts were measured between H O/0.1% FA; buffer B: CH CN/0.1% FA) 150 and 400 microns and form a cystic mass of around 6-7 mm length. 2 3 The mass spectrometer was operated in positive mode Arrow indicates cystic mass containing microscopic cysts. with a capillary voltage set at 1.5 kV and the source Protein extraction temperature set at 200 ° C. The samples were analyzed by HCD fragmentation using a method referenced "top20". The proteins of the spores of Myxobolus episquamalis were The initial MS scan was recorded in the Orbitrap analyzer extracted in 1ml of a lysis buffer containing 7M urea, 2M with a resolution of 60000 on a mass range m/z 400-1800 thiourea, 4% CHAPS, 2% DTT and10mM Tris. To allow (mono-charged ions were not selected for fragmentation this, spores were sonicated 5 times using a microtip probe. and dynamic exclusion was applied). This initial treatment was followed by 5 freezing-thawing cycles at -80°C and 37°C, respectively. The resulting Label Free material was centrifuged at 20800g for 30 min at 4°C. The For these analysis, the spore proteins were obtained in the pellets were stored at – 20°C. same manner as described above. Then, 50μg of the whole Two-dimensional gel electrophoresis (2-DE) protein extract were concentrated within the stacking gel of SDS-PAGE experiment. After a short period of migration For all experiments, two biological replicates were (< 1 h), the revealed protein band was cut and digested obtained and the corresponding two-dimensional gels with 2 µg of trypsin. After solvent evaporation, the whole made in duplicate. Protein concentrations were evaluated 7 REV. CAMES - VOL.05 NUM.02. 2017 ** ISSN 2424-7235 Science de la vie, de la terre et agronomie peptide pool was suspended in aqueous solution of 0.1% for proteins that were correctly identified with at least formic acid prior to analysis by mass spectrometry. MS two peptides. This combination of analysis enabled the settings were similar to those described previously except unambiguous identification of actin, collagen, histone, heat the gradient duration which was increased to 120 min. shock protein 70 (HSP70), glyceraldehyde-3 phosphate deshydrogenase (GA3PDH) and triosephosphate Protein identification isomerase. For protein identification, raw data files were processed These proteins have been conserved during evolution. either using Proteome Discoverer (V1.3, Thermo It was not possible to distinguish whether they were Scientific) or Peaks (V6.0, Bioinformatic Solutions, corresponding to M. episquamalis proteins or host Canada) for de novo sequencing of fragmentation data. contamination as no genome data were available for To achieve these identifications, parameters used included both organisms. The total protein extracts were analyzed 2 missed cleavages, variable modifications of cysteine using shotgun analysis for whole proteome analysis of M. (carbamidomethylation) and methionine (oxidation). The episquamalis proteins. A set of 3121 peptides were kept tolerance of the precursor ion mass was set to10.0 ppm as significant after a filtering stage based on the peptide and that of fragment ions to 0.05 Da. To validate protein score threshold application using Peaks. Peptides above identification, the protein had to be characterized by at the threshold did not yield much significant hit allowing a least 2 peptides with individual ion fragmentation score very low FDR (Fig. 3A). higher than 53 (default value of Mascot). Sequence comparison Mascot and Peaks analysis was used to identify protein homologues and there by infer functional annotation. Secondly HMMER was used to query sequences, using phmmer web interface at EBI (http://www.ebi.ac.uk/Tools/hmmer/). We choose to compare our peptides of length over 11 amino-acids (1497 sequences) with the Uniprot data base (http://www.uniprot.org/). Best homologues Figure 3. Validation of proteins identification. were kept for functional and taxonomic analysis. A. The total number of peptide sequence match (PSM) was plotted against the peptide probability score (true database in blue and decoy database Results in pink). This allowed to estimate a probality score of 30 to propose a low Protein identification. FDR while maintening the highest level of protein identifications. B. The peptide score was also plotted against the difference of mass (in ppm). Two-dimensional gel electrophoresis revealed the presence This graph highlighted different distributions of Dm depending on the of a large panel of proteins distributed all over the gel (Fig. 2). database used for identification searches. On the pH gradient (range from 3 to 10), we noticed that high molecular weight proteins had some trouble getting out This was supported by the fact that average ∆mass of the staking gel. Added to this is the presence of spots of (difference between observed and calculated masses) was varying size and intensity between replicates. much lower for true positives than for hits obtained when searching against the decoy database (Fig. 3B). About 350 peptides at the 3121 full set of filtered peptides had homologies with788 proteins present in the database. Many peptides match with several proteins but in most cases they were orthologues. Reciprocally, 196 proteins of 788 homologues were identified with at least two peptides. Altogether, PEAKS analysis defines a non-redundant set of 254 proteins matching with the 3121 peptides that were initially selected. Functional analysis of Myxobolus episquamalis peptides. Functional analysis was performed using the 254 protein groups characterized after shot-gun mass spectrometry analysis and compared with a second analysis that was performed only on 1497 peptides that were longer than10 residues from the initial Figure 2. Two dimension electrophoresis gel of protein extracts set of 3121 peptides. These 1497 peptides were compared from Myxobolus episquamalis. with the Uniprot database using Phmmer online server. We Protein spots that were excised and analyzed by mass spectrometry are obtained 352 significant homologies. The level of proteins with highlighted. Unfortunately, probably due to lack of genomic sequence unknown function was of 37.5% and 78.5% after Peaks and available, many excised spots remain unannotated. Phmmer analysis respectively (Fig. 4A and 4B). The low percentage of homologues of known function with A set of 21 reliable spots were extracted from gel and Phmmer could result from the composition of the Uniprot submitted to trypsic digestion. Mass spectrometry analysis data base which contains a large proportion of peptides from and Mascot searches provide annotation for only few recently sequenced and automatically annotated genome. On proteins with strong background of contaminant (among the opposite, the Peaks database was composed of peptides which keratin and trypsin). Positive annotation was kept that were considered to be relevant for mass-spectrometry 8 Science de la vie, de la terre et agronomie REV. CAMES - VOL.05 NUM.02. 2017 ** ISSN 2424-7235 analysis. Most of them are selected among proteins from well Comparison to the Uniprot data base provided more information annotated genomes. about proteins from M. episquamalis, including cellular periphery and adhesion: a protein from the adhesive plaque matrix, a putative adhesin/hemaglutinin found in bacteria, a mucin protein with matches in worm eukaryotes. Homology with the transmembrane protease serine 9 could be related with maturation of the extracellular matrix but could also be considered as a host-interacting factor. Functions related to signaling were more heterogeneous. In addition, homology was found with creatine kinase from sea anemone (1 peptide) and calmodulin from oyster (3 peptides). Taxonomic analysis of Myxobolus episquamalis peptides The distribution of organisms presenting protein homologues provided interesting insight about the taxonomic position of Figure 4. Distribution of main functions associated with protein M. episquamalis. Analysis was performed on the full set of homologues of Myxobolus episquemalis peptides identified 788 homologues and synthetic constructs were removed. We by label-free mass spectrometry analysis. observed that 664 of the 769 remaining homologues were A. Frequency of proteins with known function after Peaks de novo originating from metazoan (Fig. 5A). Indeed Peaks database was sequencing. A total of 254 non redundant protein groups were built to promote peptide matching and composition is therefore characterized after Peaks analysis. strongly based for well characterized peptides. Nevertheless, two B. Frequency of proteins with known function after Phmmer analysis. matches with a virus polyprotein were intriguing. The Phmmer A total of 352 peptides displayed significant level of homology (e-value analysis was performed against Uniprot database which could < 0.01) against the Uniprot database using phmmer software. be considered as unbiased even though no myxozoan genome has been sequenced so far. C. Major functional categories of the 159 proteins homologues with known annotation identified by Peaks. Number of hits per function is By keeping only the best homologies (homologue with lower given after the coma. E-value) for each peptide, only a small proportion of metazoans D. Major functional categories of the 75 proteins with known were retained during Peaks analysis (Fig. 5B). No function was annotation identified by phmmer analysis. Number of hits per function associated to bacterial homologues. Fungi proteins with known is given after the coma. function were related to signalization (2 peptides) and lipid metabolism (one peptide). Two peptides were similar to surface Functional annotation associated to proteins was classified into antigens of protists. Three peptides displayed homologies with four groups: cellular functions, biological functions, metabolism mimiviridae proteins which are known pathogens of protists and genome expression (Fig. 4B and 4C). Peaks analysis gave and lower metazoan. 159 protein homologues with known function and Phmmer 75 homologues with known function. They correspond to major functions found in eukaryotes. Only 17 peptides have been found in common between Peaks and Phmmer analysis. Analysis at functional level revealed high redundancy for some proteins such as Myosin, actin or glyceraldehyde-3-phosphate dehydrogenase. Redundancy was higher for Peaks analysis. Protein functions that were in common between both methods represented about one third from the analyses (Peaks: 61/159; Phmmer: 24/75). The most redundant protein functions in common with both sets of homologues were cytoskleton proteins (actin, myosin, nebulin and troponin) and enzymes of the glycolysis, (glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and phosphoglucomutase). Ubiquitin was also found in common in analysis as well as creatine kinase and calmodulin, two enzymes sufficiently present in muscles. More specific proteins and enzymes were identified after either the Peaks or Phmmer analysis. The Peaks analysis identified a large number of cytosketon enzymes and a large proportion Figure 5. Taxonomic distribution of protein homologues of Myxobolus of enzymes of the central metabolism (Fig. 4C). Keratin episquemalis peptides identified by mass spectrometry analysis. contamination was less observed than during 2DE analysis. The A.Frequency of proteins of metazoan origin after Peaks analysis and same analysis also identified hemoglobin and albumin. Possible comparison with other major kingdom or branches of living organisms. A contamination with host tissue cannot be excluded even if total of 769 natural protein homologues were characterized. homologues among lower Metazoan were also found for most B. Frequency of proteins of metazoan origin after Phmmer analysis and common proteins ‘e.g. actin, tubulin, calmodulin, elongation comparison with other major kingdom or branches of living organisms. factor EF1alpha or histone H2A). Phmmer analysis revealed A total of 352 peptides had significant level of homology (e-value < 0.01). less homology with muscle proteins as demonstrated by the composition of cytoskeleton function which included mainly C. Distribution of proteins identified by Peaks analysis among major actin and myosin. groups of metazoan. Number of hits per groups is given after the coma. 9 REV. CAMES - VOL.05 NUM.02. 2017 ** ISSN 2424-7235 Science de la vie, de la terre et agronomie D. Distribution of proteins identified by Phmmer analysis among major of Hox genes in Myxozoa genome (Anderson et al 1998). groups of metazoan. Number of hits per groups is given after the coma. The identification of peptides matching with neuropeptides and analgesic toxins offers new output on the interaction Phmmer analysis shed original light on M. episquamalis between Myxozoa and their host. It is the first time that peptide composition (Fig. 5D). Almost 30%o homology pathogenic factors are described in Myxozoa. A major search results were cnidarian proteins of Nematostella output of peptide annotations was the presence of a large vectensis (Starlet sea anemone). These results are in proportion of records within microorganism including agreement with the taxonomic position of Myxozoa among bacteria, archaea, protists, fungi and virus (112 proteins cnidarian. The second larger organism providing homology 352 Phmmer homologues) About one third of these results was the mollusk Lottia gigantean whose genome has organisms were marine microbes such as Cyanobacteria, been sequenced. More generally, Phmmer analysis provided Oceanospirillales, Algae, Perkinsus and Stramenopiles, homologues in a wide range of organism, including animals which revealed the composition of the microbiome which are often described at the transition between groups associated with the host scale and M. episquamalis such as Amphoxius, Lamprey or Coelacanth. Despite the cysts The presence of Mimivirus peptides in the protein availability of the tunicate Ciona intestinalis genome, we set may suggested that Myxozoans may be infected by observed little homology with this organism. specific viruses. Among identified proteins were actin, Discussion tropomyosin, myosin, tubulin, troponin, also proposed in others works (Hartigan et al 2016; Gema et al, 2012; Myxosporidians are major fish parasites causing outbreaks Uspenskaya and Raikova 2004). Indeed, the presence with serious economic impact. The objective of this work of F-actin is consistent with previous investigations that was to provide an insight on the inventory of proteins in have highlighted for the first time cellular components M.episquamalis by mass spectrometry. To reach this goal, and mechanisms involved in the motility of Myxozoans, we used two approaches, namely 2-DE and shotgun mass through a study dedicated to Ceratomyxa puntazzi (Gema spectrometry, coupled to sequence homology searches after et al, 2012). Their work based on a morphological study de novo sequencing. To get high quality results, samples were (ultra-structural and three-dimensional structure) revealed analyzed by a high resolution mass spectrometer associated the role of filamentous actin in the different developmental to a very good mass accuracy (~ 1ppm). One major problem stages of C. puntazzi. In addition, Monteiro et al (2002) was related to the presence of chitin in the spore of Myxozoa characterized the presence of muscle blocks in the as evidenced by the work of Julius et al (1993). The later Myxozoan worm Buddenbrokia. Uspenskaya and Raikova demonstrated resistance of spores to alkaline hydrolysis, (2004) demonstrated the role of F-actin and β-tubulin which strongly complicated the extraction of proteins from cytoskeletal proteins in the anchoring mechanism of spores of M.episquamalis. Evidence of contamination with myxosporidians during the infestation phase. fish proteins was provided by the distribution of protein homologues. The Peaks analysis was more responsive to this The presence of collagen was shown by 2-DE analysis but not contamination. Major homologues were found with the most confirmed after shotgun analysis. Jason et al, (2011) reported common protein in the animal cell such as actin or enzymes the presence of a gene encoding a mini-collagen called Tb- of the glycolysis. Presence of Myosin provides also evidence Ncol-1 in a Tetracapsuloides bryosalmonae. Interestingly, of contamination with muscle cell. Taxonomic analysis a similar protein had been identified in nematocysts of of Peaks homologues confirmed the host contamination cnidarians (Ulrike et al, 2002). More recently, transcriptomic as87.5% of the proteins were part of the phylum Metazoans. analysis revealed the presence of several genes encoding The contamination with animal proteins was also found nemato-galectins and mini-collagens in myxozoan and with Phmmer analysis where 68% of the homologues had cnidarian genomes (Shpirer et al 2014). homologies with Metazoan peptides. Nevertheless, both It has been demonstrated some phylogenetic relationship functional and taxonomic analysis confirmed that relevant between Cnidarians and Myxozoa, or nematocysts, homologies were found with M. episquamalis peptides. which are special cells of cnidarians containing a polar Even some of the homologies with animal proteins could be filament whose primary role is the defense and capture of the result of M. episquamalis peptides matches with nearly prey which could further confirm the mechanical role of identical residues of highly conserved proteins. The confusion myxosporidians polar filament in the spore attachment to of the analysis may result from the fact that the genome of the host cells. Noteworthy, a polyclonal antibody (pAbMPPF) flathead mullet and of a Myxozoa is not available. was generated against the polar filament of the myxozoan The combination of 2-DE and shotgun analyses identified Myxobolus pendula (Ringuette et al, 2011). In western blot proteins that were classified into five groups: cellular analysis, many bands located at 35, 40, 42, 55, 70, 100, and function, biological function, metabolism, genome 200 kDa were revealed in M. pendula while a major band expression and proteins of undetermined function, this located at 40 kDa and 3 minor bands at 48, 70, and 130 latter comprising the majority of proteins. One of the main kDa were found in M. bartai. The proteins corresponding reason for this relies to the fact that the closest organisms to pAbMPPF reactive spots possess glycine-rich that has been sequenced remain poorly annotated. Strong hydrophobic domains with sequence similarity to spinalin homologies with the Nematostella vectensis starlet sea (Ringuette et al, 2011). Spinalin is a 24-kDa glycine- and anemone and Lottia gigantean genome do not help histidine-rich protein in spines of Hydra nematocysts, with us to address a complete map of the pathways. Strong regions presenting homology to loricrins and keratins. homologies with functions found in bilateria raised Unfortunately, these proteins were not identified during again the origin of the Myxozoa and their position in the this analysis. The presence of the polar tube was also evolution as it was questioned in the past by the discovery described in microsporidia. Proteins from the polar tube of 10