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      First spermatological study in the Atractotrematidae (Digenea, Haploporoidea): the case of Atractotrema sigani, intestinal parasite of Siganus lineatus Translated title: Première étude spermatologique chez les Atractotrematidae (Digenea, Haploporoidea) : le cas d’ Atractotrema sigani, parasite intestinal de Siganus lineatus

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          Abstract

          The ultrastructural organization of the mature spermatozoon of the digenean Atractotrema sigani (from Siganus lineatus off New Caledonia) was investigated by transmission electron microscopy. The male gamete of A. sigani exhibits the general morphology described in digeneans with the presence of two axonemes of different lengths showing the 9 + “1” pattern of the Trepaxonemata, a nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation, spine-like bodies and granules of glycogen. However, the mature spermatozoon of A. sigani has some specific characters such as the morphology of its anterior region and the submembranous electron-dense material. Although similar structures have been reported in some digenean species, the presence of a submembranous electron-dense material describing a complete ring is reported here for the first time in the mature spermatozoon of A. sigani. In addition, sperm characteristics are compared between the Haploporoidea and their supposed close superfamilies, and possible phylogenetic implications of these findings for the Digenea are discussed.

          Translated abstract

          L’organisation ultrastructurale du spermatozoïde mûr du digène Atractotrema sigani (parasite de Siganus lineatus en Nouvelle-Calédonie) est décrite au microscope électronique à transmission. Le gamète mâle d’ A. sigani présente la morphologie générale des spermatozoïdes décrits chez les digènes avec la présence de deux axonèmes de type 9 + “1” des Trepaxonemata, un noyau, deux mitochondries, deux champs de microtubules corticaux parallèles, une ornementation externe, des corps épineux et des granules de glycogène. Cependant, le spermatozoïde mûr d’ A. sigani présente certains caractères spécifiques tels que la morphologie de son extrémité antérieure et le matériel sous-membranaire opaque aux électrons. Bien qu’une structure similaire à cette dernière fût décrite précédemment chez certaines espèces, la présence d’un matériel opaque aux électrons décrivant un anneau complet est observée pour la première fois dans le spermatozoïde de A. sigani. De plus, les caractères spermatiques sont comparés entre les Haploporoidea et les superfamilles qui sont censées leur être proches et les possibles implications phylogénétiques de ces résultats pour les Digenea sont discutées.

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          Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda).

          Complete small subunit ribosomal RNA gene (ssrDNA) and partial (D1-D3) large subunit ribosomal RNA gene (lsrDNA) sequences were used to estimate the phylogeny of the Digenea via maximum parsimony and Bayesian inference. Here we contribute 80 new ssrDNA and 124 new lsrDNA sequences. Fully complementary data sets of the two genes were assembled from newly generated and previously published sequences and comprised 163 digenean taxa representing 77 nominal families and seven aspidogastrean outgroup taxa representing three families. Analyses were conducted on the genes independently as well as combined and separate analyses including only the higher plagiorchiidan taxa were performed using a reduced-taxon alignment including additional characters that could not be otherwise unambiguously aligned. The combined data analyses yielded the most strongly supported results and differences between the two methods of analysis were primarily in their degree of resolution. The Bayesian analysis including all taxa and characters, and incorporating a model of nucleotide substitution (general-time-reversible with among-site rate heterogeneity), was considered the best estimate of the phylogeny and was used to evaluate their classification and evolution. In broad terms, the Digenea forms a dichotomy that is split between a lineage leading to the Brachylaimoidea, Diplostomoidea and Schistosomatoidea (collectively the Diplostomida nomen novum (nom. nov.)) and the remainder of the Digenea (the Plagiorchiida), in which the Bivesiculata nom. nov. and Transversotremata nom. nov. form the two most basal lineages, followed by the Hemiurata. The remainder of the Plagiorchiida forms a large number of independent lineages leading to the crown clade Xiphidiata nom. nov. that comprises the Allocreadioidea, Gorgoderoidea, Microphalloidea and Plagiorchioidea, which are united by the presence of a penetrating stylet in their cercariae. Although a majority of families and to a lesser degree, superfamilies are supported as currently defined, the traditional divisions of the Echinostomida, Plagiorchiida and Strigeida were found to comprise non-natural assemblages. Therefore, the membership of established higher taxa are emended, new taxa erected and a revised, phylogenetically based classification proposed and discussed in light of ontogeny, morphology and taxonomic history.
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            A quick and simple method, usable in the field, for collecting parasites in suitable condition for both morphological and molecular studies.

            Many methods have been proposed for collecting and fixing parasites, but most were written before the molecular age, and were intended to be practised by experienced parasitologists in well-equipped laboratories. We describe here a very simple method, illustrated by photographs, for collecting helminths from the digestive tract of vertebrates. It only requires a few plastic vials, some ethanol and a means to heat water. Basically, the method consists of: (a) the extraction of all organs from the abdominal cavity; (b) opening the digestive system longitudinally; (c) agitate gut and contents in a saline solution (i.e. ca. 9% NaCl or 1/4 sea water in tap water); (d) decant in saline as many times as needed to clean contents; (e) immediately fix parasites in near-boiling saline; (f) discard saline and keep specimens in 95% ethanol. Additional information is given for collecting parasites from fish gills with a similar process. The method will collect most helminths (digeneans, larval cestodes, nematodes, acanthocephalans) from the digestive tract, and monogeneans and isopod and copepod crustaceans from fish gills. The specimens will be suitable for both morphological study and DNA sequencing. The method is simple, fast, inexpensive and can be used by untrained personnel, even in the field without electricity and without a binocular microscope. It can also be used by trained parasitologists who need to expedite treatment of abundant samples.
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              Spermatozoa as phylogenetic characters for the Eucestoda.

              Spermatozoon ultrastructure and spermiogenesis are significant characters for phylogenetic inference. Sperm ultrastructure is reviewed from the literature in 56 species of Eucestoda. Data are available for 11 of the 12 orders of Eucestoda (Lecanicephalidea excepted), but in some orders data are scarce and often limited to a single species. Spermiogenesis and sperm ultrastructure in the Eucestoda is compared to that of other parasitic Platyhelminthes, with emphasis on structures of phylogenetic interest. Not only the descriptions of sperm structure, but those of the process of spermiogenesis, are necessary to define characters. Synapomorphies based on sperm ultrastructure for the Eucestoda include the absence of a mitochondrion in mature sperm and the presence of a crested body. A proposed synapomorphy for the Cyclophyllidea + Tetrabothriidea is the twisting of the peripheral microtubules; the absence of intercentriolar body and the absence of striated roots in the spermatid may constitute additional synapomorphies for this assemblage. Absence of flagellar rotation during spermiogenesis is synapomorphic for the Cyclophyllidea, and absence of proximodistal fusion could be synapomorphic for a part of the Cyclophyllidea. Several other characters could be useful for understanding phylogeny within the Eucestoda. The polarity of these characters could in several cases be determined, but diagnoses for taxa or relationships based on synapomorphies cannot be specified unequivocally due to putative convergence. Such characters and their putative polarity include: (1) the number of axonemes in mature spermatozoon (plesiomorphic=2; apomorphic=1); (2) size and number of crested bodies (p=1; a=several); and (3) angle of twisted microtubules; shape of nucleus (p=compact cord; a=crescent and annulus). Additional apomorphic attributes include (1) presence of a periaxonemal sheath (a putative synapomorphy for the Cyclophyllidea + Tetrabothriidea if reversals are postulated in certain cyclophyllideans); (2) presence of proteinaceous transverse walls; (3) presence of dense granules; and (4) shape of apical cones and posterior structures. Studies of sperm structure in the poorly known orders and additional comparative studies in the Cyclophyllidea are expected to provide new information for elucidation of phylogenetic relationships.
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                Author and article information

                Journal
                Parasite
                Parasite
                parasite
                Parasite
                EDP Sciences
                1252-607X
                1776-1042
                2015
                16 October 2015
                : 22
                : ( publisher-idID: parasite/2015/01 )
                : 26
                Affiliations
                [1 ] CNRS-Università di Corsica, UMR 6134-SPE, SERME Service d’Étude et de Recherche en Microscopie Électronique Corte 20250 Corsica France
                [2 ] Laboratory of Evolutionary Biology, Ecology and Management of Ecosystems, Cheikh Anta Diop University of Dakar BP 5055 Dakar Senegal
                [3 ] ISYEB, Institut de Systématique, Évolution, Biodiversité (UMR7205 CNRS, EPHE, MNHN, UPMC), Muséum National d’Histoire Naturelle, Sorbonne Universités CP 51 55 rue Buffon 75231 Paris cedex 05 France
                [4 ] Department of Life Sciences, Natural History Museum Cromwell Road SW7 5BD London UK
                [5 ] Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona Av. Joan XXIII, sn 08028 Barcelona Spain
                [6 ] Institut de Recerca de la Biodiversitat, Facultat de Biologia, Universitat de Barcelona Av. Diagonal 645 08028 Barcelona Spain
                Author notes
                [* ]Corresponding author: quilichini@ 123456univ-corse.fr
                Article
                parasite150063 10.1051/parasite/2015026
                10.1051/parasite/2015026
                4609001
                26475645
                82a97f37-ab55-458b-a928-47254df34f4f
                © A.J.S. Bakhoum et al., published by EDP Sciences, 2015

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 14 August 2015
                : 25 September 2015
                Page count
                Figures: 4, Tables: 0, Equations: 0, References: 49, Pages: 10
                Categories
                Research Article

                cell biology,platyhelminthes,digenea,ultrastructure,spermatozoon,phylogeny

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