Research Article |
Corresponding author: M. Leopoldina Aguirre-Macedo ( leopoldina.aguirre@cinvestav.mx ) Academic editor: David Gibson
© 2020 Andrés Martínez-Aquino, Jhonny Geovanny García-Teh, Fadia Sara Ceccarelli, Rogelio Aguilar-Aguilar, Victor Manuel Vidal-Martinez, M. Leopoldina Aguirre-Macedo.
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Citation:
Martínez-Aquino A, García-Teh JG, Ceccarelli FS, Aguilar-Aguilar R, Vidal-Martinez VM, Leopoldina Aguirre-Macedo M (2020) New morphological and molecular data for Xystretrum solidum (Gorgoderidae, Gorgoderinae) from Sphoeroides testudineus (Tetraodontiformes, Tetraodontidae) in Mexican waters. ZooKeys 925: 141-161. https://doi.org/10.3897/zookeys.925.49503
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Adults of trematodes in the genus Xystretrum Linton, 1910 (Gorgoderidae, Gorgoderinae) are parasites found exclusively in the urinary bladders of tetraodontiform fishes. However, limited and unclear morphological data were used to describe the type species, X. solidum Linton, 1910. Here, we present the first detailed morphological information for a member of Xystretrum. Morphological characters were described using light and scanning electron microscopy (SEM) of Xystretrum specimens from Sphoeroides testudineus (Linnaeus) (Tetraodontiformes, Tetraodontidae), collected at six localities off the northern Yucatan Peninsula coast of the Gulf of Mexico. We also compared sequence fragments of the 28S (region D1–D3) ribosomal DNA and mitochondrial Cytochrome c oxidase subunit 1 (COI) gene with those available for other gorgoderine taxa. We assigned these Xystretrum specimens to X. solidum, despite the incompleteness of published descriptions. The data provide a foundation for future work to validate the identities of X. solidum, X. papillosum Linton, 1910 and X. pulchrum (Travassos, 1920) with new collections from the type localities and hosts. Comparisons of 28S and COI regions described here also provide an opportunity to evaluate the monophyletic status of Xystretrum.
COI, molecular phylogenetics and systematics, parasites of marine fishes, scanning electron micrographs, 28S
The most reliable list of species of Xystretrum available is the public resource database of the World Register of Marine Species (
Despite the scarce taxonomic information from the western Atlantic,
Several S. testudineus from the coasts and lagoons of the Northern Yucatan Peninsula, Mexico were examined for parasites between 1995 and 2013. Gorgoderids were recovered from the urinary bladder of these hosts and preliminarily identified as Phyllodistomum sp. (
As
Trematode specimens in this study were collected from the urinary bladder of Sphoeroides testudineus. Hosts were collected between 1998 and 2016 (collection permit PPF/DGOPA-070/16 issued by Comisión Nacional de Acuacultura y Pesca, Mexico) at six localities in and off the northern Yucatan Peninsula, Mexico: Celestún tropical lagoon (20°45'N, 90°22'W, June 2005, August 2012, January 2016), Chelem lagoon (21°15'N, 89°45'W, August 2005, March 2007), Ría Lagartos lagoon (21°22'N, 87°30'W, July 2005), Chuburna port (coastal area) (21°15'N, 89°48'W, March 2005), Progreso port (coastal area) (21°16'N, 89°39'W, August 2006), and Chicxulub port (coastal area) (21°17'N, 89°36'W, June 2009) (Fig.
Unflattened trematode specimens were stained with Mayer’s paracarmine and mounted on permanent slides using Canada balsam. Specimens were measured, and drawings were made with the aid of a drawing tube attached to an Olympus BX50 microscope; measurements are presented in micrometres (μm) as ranges followed by the means in parentheses. For the SEM study, specimens were dehydrated through a graded series of ethyl alcohols and critical point dried with carbon dioxide. Specimens were mounted on metal stubs with silver paste, then coated with gold and examined in a Philips XL30 ESEM for variable pressure SEM at 10 kV. Trematodes were identified following/contrasting the taxonomic criteria of
Localities sampled (from east to west) for Sphoeroides testudineus, the host species of Xystretrum solidum, from the Yucatan Peninsula, Yucatan, Mexico. LM = Total number of measured individuals of X. solidum used for morphometric studies on light microscope slides. SEM = Total number of individuals of X. solidum used for scanning electron micrograph studies. CHCM = Voucher number from the Colección Helmintológica del CINVESTAV (CHCM) for specimens studied in this work.
Localities | LM | SEM | CHCM |
Celestún | 4 | 1 | 529 |
Chuburna | 3 | 4 | 530 |
Chelem | 1 | 1 | 531 |
Progreso | 6 | 2 | 532 |
Chicxulub | 1 | – | 533 |
Ría Lagartos | 2 | 3 | 534 |
Deoxyribonucleic acid (DNA) was extracted from individual adult trematodes; DNA extraction was performed using the DNeasy blood and tissue extraction kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. Partial sequences of the 28S (region D1–D3) ribosomal DNA were amplified by Polymerase Chain Reaction (PCR) (
To obtain the consensus sequences of specimens of Xystretrum, we assembled and edited the chromatograms of forward and reverse sequences using the Geneious Pro v.5.1.7 platform (
Phylogenetic tree obtained using Bayesian inference for the 28S rRNA dataset. The scale bar represents the number of nucleotide substitutions per site. GenBank accession numbers of the new sequences of Xystretrum solidum are shown in bold. Filled circles above/below branches and at the nodes represent Bayesian Posterior Probability ≥ 0.95.
Phylogenetic trees were reconstructed for each gene separately (28S and COI), to test the monophyly of X. solidum analyzed in this study. Phylogenetic tree reconstructions were carried out using Bayesian Inference (BI) in MrBayes v.3.2.3 (
Phylogenetic tree obtained using Bayesian inference for the COI dataset. The scale bar represents the number of nucleotide substitutions per site. GenBank accession numbers of the new sequences of Xystretrum solidum are shown in bold. Filled circles above/below branches and at the nodes represent Bayesian Posterior Probability ≥ 0.95.
Specimens analyzed were assigned to Xystretrum solidum (Figs
Scanning electron microscopy (SEM) images of Xystretrum solidum (from three specimens collected at Progreso Port, Yucatan, Mexico) A whole adult specimen (ventral view) with scattered rosette papillae on forebody B forebody, showing 6 pairs of robust papillae (white arrowhead) C oral sucker, showing 13 pairs of papillae: 5 on interior margin surrounding mouth (yellow arrowhead); one posterolateral to interior margin (dark blue arrowhead); three anterolateral to interior margin (light blue arrowhead); 2 on stylet scar (dark green arrowhead); one lateral to stylet scar (light green arrowhead); one on posterior external margin of oral sucker (black arrowhead); one inside of mouth (red arrowhead) (only right hand side papillae are indicated) D genital atrium detail E ventral sucker (side view), showing long papillae on inner margin (white arrowhead) F ventral sucker (ventral view), showing long papillae on inner margin (white arrowhead). Scale bars: 1000 µm (A); 200 µm (B); 500 µm (C, D); 100 µm (E, F). For more details of observed characters by SEM from other localities analyzed in this study, see Suppl. material
Host: Sphoeroides testudineus (Tetraodontidae).
Site: Urinary bladder.
Localities: Dry Tortugas, Florida, USA (Gulf of Mexico). New localities from the Northern Yucatan Peninsula, Mexico: Celestún tropical lagoon (20°45'N, 90°22'W), Chelem lagoon (21°15'N, 89°45'W), Ría Lagartos lagoon (21°22'N, 87°30'W), Chuburna port (coastal area) (21°15'N, 89°48'W), Progreso port (coastal area) (21°16'N, 89°39'W), Chicxulub port (coastal area) (21°17'N; 89°36'W).
GenBank accession numbers: 28S rDNA sequences: MT215582–MT215584; COI mtDNA sequences: MT218558–MT218560.
In total, 12 bi-directional 28S and COI sequences were obtained from three individual adults of X. solidum. The final lengths (in number of base-pairs) of the 28S ribosomal sequence fragment were 892 (for two sequences) and 899 (for one sequence), with zero genetic variation, either among the new sequences or in the published 28S sequences of X. solidum (GenBank accession numbers MK648284 and KF013188). The total alignment length following the Gblocks exclusion was 814 bp. Nucleotide sequence variation in the 28S alignment from gorgoderids (excluding the outgroup taxon) included 330 conserved sites, 483 variable sites, 410 parsimony-informative sites and 73 singleton sites. The COI dataset consisted of 309 bp with a genetic distance of 0.3% between the three mitochondrial sequences. Nucleotide sequence variation (excluding the outgroup taxa) for each partition from COI (first, second and third codon positions) was 70/92/12 conserved, 33/11/91 variable, 29/7/79 parsimony-informative and 4/4/12 single sites, respectively.
We inferred the phylogenetic relationships from the 28S and COI sequence matrices separately. The 28S gene dataset contained 46 taxa (150 sequences) and the COI contained 18 taxa (63 sequences). Figures
The morphologies of the trematodes examined in this study are consistent with those of the genus Xystretrum provided by
Based on the observation of material from this study, plus the holotype of X. papillosum (voucher 1321174), we found that X. solidum presents a fluted tegument on the hindbody and that along the dorsoventral margin there are short dense fringe papillae (only readily visible using SEM, see Suppl. material
It is necessary to collect new X. solidum specimens from the original host (i.e., B. capriscus) and the type locality (i.e., off Bermuda) to compare their morphological measurements with our samples. Also, it is necessary to explore the possible presence of X. papillosum from L. triqueter co-distributed with B. capriscus off Bermuda, as part of a taxonomic revision of the genus Xystretrum, taking into consideration the morphological data presented here. In parallel, future revisions should seek to distinguish or synonymize X. solidum, X. papillosum and X. pulchrum, while being sensitive to the potential presence of cryptic species.
To date, 14 species of the genus Xystretrum are considered valid (
In the phylogenetic tree obtained from the 28S dataset, all members of the genus Xystretrum included in the analysis formed a well-supported clade, but without nodal support with their sister clade. A similar result has been reported in previous phylogenetic analyses carried out for similar taxa using the same gene (e.g.,
The COI phylogenetic topology shows a clade with X. solidum from this study, and the clade formed by the freshwater taxa P. centropomi + Phyllodistomum sp. (from
As indicated by
Our thanks go to the staff of the laboratory of the Patología Acuática: Gregory Arjona-Torres, Francisco Puc-Itzá, Clara Vivas and Nadia Herrera Castillo from CINVESTAV-IPN, Unidad Mérida, Mexico. We are indebted to Dr. José Q. García-Maldonado for lending us space for sequencing and to MSc. Abril Gamboa-Muñoz for technical assistance in the molecular lab. We are grateful to Anna Philips and Freya Goetz, Museum Technician and Curator of the Clitellata and Parasitic Worms at the Smithsonian Institution National Museum of Natural History, USA, for the loan of the X. papillosum holotype. Dr. Cristina Damborenea (Curator of Colección Helminthológica of Museo de La Plata, Argentina [MLP-He]), Dr. Fabiana B. Drago (MLP-He), Dr. Melissa Q. Cárdenas (Laboratório de Helmintos Parasitos de Peixes, Instituto Oswaldo Cruz, Brasil), and Dr. Carlos Mendoza-Palmero (Institute of Parasitology, Czech Republic) kindly helped with the literature on Xystretrum spp. Our thanks also go to Dr. John Mclaughlin and the editor Dr. David Gibson for reviewing the manuscript and greatly improving the style and grammar. Special thanks are due to Beatriz Heredia-Cervera and Ana Ruth Cristobal Ramos, at the CLSM and SEM unit at CINVESTAV-IPN-Unidad Mérida, Mexico. AM-A thanks Dr. Juan Tapia, Director of the Facultad de Ciencias, UABC, for invaluable support during the writing of the present work. JGG-T thanks CONACyT for a scholarship to accomplish his MSc degree within the Posgrado en Biología Marina, CINVESTAV–IPN, Mérida, Yucatán, Mexico. Financial support for AM-A at the Laboratory of Aquatic Pathology, CINVESTAV–IPN came from Grant No. 201441 SENER Hidrocarburos–CONACYT.
Table S1
Explanation note: Comparative data of relevant taxonomic characters of Xystretrum solidum from this study, in contrast to the 14 congeneric Xystretrum spp.
Figure S1
Explanation note: Scanning electron microscopy (SEM) images of Xystretrum solidum (from five specimens collected in the Northern Yucatan Peninsula, Mexico). (A) Oral sucker, showing 13 pairs of papillae surrounding margin of oral sucker (locality: Chelem). (B) Stylet scar, showing 2 pairs of surrounding papillae and one lateral (dark and light green arrowhead, respectively) (locality: Ría Lagartos). (C) Forebody, showing 6 pairs of robust papillae (white arrowhead) (locality: Chuburna). (D) Oral sucker, showing 13 pairs of papillae surrounding anterolateral margin (locality: Chuburna). (E) Oral sucker, showing single pair in upper region inside oral sucker (red arrowhead) (locality: Chuburna). (F) Genital pore detail (locality: Chuburna). (G) Ventral sucker, showing long papillae on inner margin (white arrowhead) (locality: Chuburna). (H) Details of hindbody corrugation and demarcation of folds (locality: Chuburna). (I) Details of “pseudopapilles” on smooth lateral margins of hindbody (white arrowhead) (locality: Chuburna). (J) Oral sucker, showing 13 pairs of papillae surrounding margin of oral sucker (locality: Celestún). Colors of arrowheads in A, D and J as follows: Yellow arrowhead = 5 pairs of papillae on interior margin, surrounding mouth; Dark blue arrowhead = one pair of papillae posterolateral to interior margin; Light blue arrowhead = three pairs of papillae anterolateral to interior margin; Dark green arrowhead = 2 pairs of papillae on stylet scar; Light green arrowhead = one pair of papillae lateral to stylet scar; Black arrowhead = one pair of papillae on posterior external margin of oral sucker. Scale bars: 100 µm (A, J); 50 µm (B, D, E, F, G); 200 µm (C, I); 20 µm (H); 200 µm (I).