Identification tools of <i>Diplostomum spathaceum</i> Rudolphi, 1819 (Diplostomida: Diplostomidae), a trematode parasite of herring gull (<i>Larus argentatus</i>)

Alshehri, E.; Abdel-Gaber, R.; Al-Quraishy, S.

doi:10.1590/1678-4162-13232

ABSTRACT

Specimens of the genus Diplostomum von Nordmann, 1832 (Trematoda, Diplostomidae) were collected from the intestine of the herring gull, Larus argentatus (Laridae), from El-Manzala Lake (Port Said City, Egypt). This parasite species was morphometrically, morphologically, and molecularly studied using the internal transcribed spacer (ITS1-5.8S-ITS2) gene region. The presence of a trematode species of Diplostomum spathaceum Rudolphi, 1819 (Diplostomidae) was observed in 70 % of the examined gull species. This species has generic features of the genus Diplostomum. Distinct criteria that discriminated this species from congeners were the division for body parts, the egg-shaped forebody with a smaller length than the hind body, the ventral sucker being smaller in size than the oral one, the position of the ventral sucker being near to the holdfast, vitellaria was compact and rarely extend anteriorly to the holdfast organ, and smaller egg size. Partial ITS1-5.8S-ITS2 sequences from diplosomite recovered in this study showed that they grouped with members of the genus Diplostomum and formed a monophyletic group supporting the morphological description. Findings obtained from molecular analysis are consistent with data from morphological classification where the parasite recorded was morphologically similar to Diplostomum spathaceum with a first record in Egyptian gulls.

Keywords:
gulls; diplostomidae; morphology; phylogeny

RESUMO

Espécimes do gênero Diplostomum von Nordmann, 1832 (Trematoda, Diplostomidae) foram coletados do intestino da gaivota de arenque, Larus argentatus (Laridae), coletada no lago El-Manzala (cidade de Port Said, Egito). Essa espécie de parasita foi estudada morfometricamente e morfologicamente, bem como molecularmente, usando a região do gene espaçador transcrito interno (ITS1-5.8S-ITS2). A presença de uma espécie de trematoda de Diplostomum spathaceum Rudolphi, 1819 (Diplostomidae) foi observada em 70% das espécies de gaivotas examinadas. Essa espécie tem características genéricas do gênero Diplostomum. Os critérios distintos que discriminaram essa espécie das congêneres foram a divisão das partes do corpo, o corpo dianteiro em forma de ovo com comprimento menor do que o corpo traseiro, a ventosa ventral de tamanho menor do que a oral, a posição da ventosa ventral próxima ao suporte, a viterlária compacta e raramente estendida anteriormente ao órgão do suporte e o tamanho menor do ovo. As sequências parciais ITS1-5.8S-ITS2 de diplostomídeos recuperadas neste estudo mostraram que eles se agruparam com membros do gênero Diplostomum e formaram um grupo monofilético que apoia a descrição morfológica. Os resultados obtidos com a análise molecular são consistentes com os dados da classificação morfológica, em que o parasita registrado era morfologicamente semelhante ao Diplostomum spathaceum, com um primeiro registro em gaivotas egípcias.

Palavras-chave:
gaivotas; diplostomídeos; morfologia; filogenia

INTRODUCTION

Gulls, particularly Larus species, are abundant and adaptable birds of the family Laridae with a worldwide cosmopolitan distribution (Burger and Gochfeld, 1996BURGER, J.; GOCHFELD, M. Family Laridae (Gulls). In: DEL HOYO, J.; ELLIOTT, A.;. SARGATAL, J. Handbook of the birds of the world: hoatzin to auks. Barcelona: Lynx, 1996. v.3, p.572-623.). These birds are susceptible to infection with a wide variety of intestinal flukes (Lee et al., 2020LEE, Y.I.I.; SEO, M.; CHAI, J.Y. Intestinal flukes recovered from a herring gull, Larus argentatus, in the Republic of Korea. Korean J. Parasitol., v.58, p.81-86, 2020.). Despite the importance of these aquatic birds as a source of protein, they also transport parasitic infections to fish and domestic birds, and even to humans (Yaseen and Abdullah, 2018YASEEN, A.M.; ABDULLAH, B.H. First record of three species of trematode in Caspin Gull (Larus cachinnans Pallas, 1811) in Faw township, southern of Basrah, Iraq. Basrah J. Sci., v.36, p.53-67, 2018.). DiplostomidaePoirier, 1886POIRIER, J. Sur les diplostomidae. Arch. Zool. Exp. Gen., v.2, p.327-346, 1886. is a family of trematodes in the order Diplostomida, with 42 genera split among four subfamilies (Heneberg et al., 2020HENEBERG, P.; SITKO, J.; TĚŠÍNSKÝ, M. Paraphyly of conodiplostomum Dubois, 1937. Parasitol. Int., v.76, p.102033, 2020.). Diplostomes have a complex life cycle, which includes development of larval stages in 2 intermediate hosts (cercaria in lymnaeid snails and metacercaria in a wide range of freshwater fish) and adult flukes in the digestive tract of various fish-eating bird species, such as gulls (Karvonen, 2012KARVONEN, A. Diplostomum spathaceum and related species. In: WOO, P.T.K.; BUCHMANN, K. (Eds.). Fish parasites: pathobiology and protection. Cambridge: CAB International, 2012. p.260-269.).

The type-genus Diplostomum von Nordmann, 1832 (subfamily Diplostominae) is a diverse group of digenean parasites with a complex taxonomy (Niewiadomska, 2010NIEWIADOMSKA, K. [Trematoda, the general part; systematic part - aspidogastrea, digenea: strigeida], Fauna Słodkowodna Polski, 34 A, Polish Hydrobiological Society. [Polish]: University of Łódź, Łódź, 2010. 388p.; Georgieva et al., 2013GEORGIEVA, S,. SOLDÁNOVÁ, M.; PÉREZ-DEL-OLMO, A. et al. Molecular prospecting for European Diplostomum (Digenea: Diplostomidae) reveals cryptic diversity. Int. J. Parasitol., v.43, p.57-72, 2013.; Blasco-Costa et al., 2014; Faltýnková et al., 2016FALTÝNKOVÁ, A.; SURES, B.; KOSTADINOVA, A. Biodiversity of trematodes in their intermediate mollusc and fish hosts in the freshwater ecosystems of Europe. Syst. Parasitol., v.93, p.283-293, 2016.; Hoogendoorn et al., 2020HOOGENDOORN, C.; SMIT, N.J.; KUDLAI, O. Resolution of the identity of three species of Diplostomum (Digenea: Diplostomidae) parasitising freshwater fishes in South Africa, combining molecular and morphological evidence. Int. J. Parasitol. Parasit. Wildl. v.11, p.50-61, 2020.) due to it includes numerous cryptic species-level lineages (Georgieva et al., 2013; Faltýnková et al., 2014; Selbach et al., 2015SELBACH, C.; SOLDÁNOVÁ, M.; GEORGIEVA, S.; KOSTADINOVA, A. SURES, B. Integrative taxonomic approach to the cryptic diversity of Diplostomum spp. in lymnaeid snails from Europe with a focus on the ‘Diplostomum mergi’ species complex. Parasit. Vectors, v.8, p.300, 2015.; Soldánová et al., 2017SOLDÁNOVÁ, M., GEORGIEVA, S., ROHÁČOVÁ, J. et al. Molecular analyses reveal high species diversity of trematodes in a sub-Arctic lake. Int. J. Parasitol., v.47, p.327-345, 2017.; Gordy and Hanington, 2019GORDY, M.A.; HANINGTON, P.C. A fine‐scale phylogenetic assessment of digenean trematodes in central Alberta reveals we have yet to uncover their total diversity. Ecol. Evol., v.9, p.3153-3238, 2019.; Hoogendoorn et al., 2020). Previous studies focused on various topics related to ecology, host-parasite relationships, systematics, and taxonomy of Diplostomum species (Galazzo et al., 2002GALAZZO, D.E.; DAYANANDAN, S.; MARCOGLIESE, D.J.; MCLAUGHLIN, J.D. Molecular systematics of some North American species of Diplostomum (Digenea) based on rDNA-sequence data and comparisons with European congeners. Can. J. Zool., v.80, p.2207-2217, 2002.; Karvonen et al., 2006KARVONEN, A.; TERHO, P.; SEPPÄLÄ, O.; JOKELA, J.; VALTONEN, E.T. Ecological divergence of closely related Diplostomum (Trematoda) parasites. Parasitology, v.133, p.229-235, 2006.; Seppälä et al., 2008SEPPÄLÄ, O.; LILJEROOS, K.; KARVONEN, A.; JOKELA, J. Host condition as a constraint for parasite reproduction. Oikos, v.117, p.749-753, 2008.; Niewiadomska, 2010; Locke et al., 2015LOCKE, S.A.; AL-NASIRI, F.S.; CAFFARA, M. et al. Diversity, specificity and speciation in larval Diplostomidae (Platyhelminthes: Digenea) in the eyes of freshwater fish, as revealed by DNA barcodes. Int. J. Parasitol., v.45, p.841-855, 2015.; Kudlai et al., 2017KUDLAI, O.; OROS, M.; KOSTADINOVA, A.; GEORGIEVA, S. Exploring the diversity of Diplostomum (Digenea: Diplostomidae) in fishes from the river Danube using DNA mitochondrial barcodes. Parasit. Vectors, v.10, p.592, 2017.; Vivas Muñoz et al., 2021). Identification of Diplo-stomum species has been carried out based on the morphological data of the adult specimens since the larval stages often lack reliable distinguishing morphological characters (Höglund and Thulin, 1992HÖGLUND, J.; THULIN, J. Identification of Diplostomum spp. In the retina of perch Perca fluviatilis and the lens of roach Rutilus rutilus from the Baltic Sea - an experimental study. Syst. Parasitol., v.21, p.1-19, 1992.; Galazzo et al., 2002; Pérez-del-Olmo et al., 2014; Sitko and Rząd, 2014SITKO, J.; RZĄD, I. Diplostomum and Ornithodiplostomum scardinii (Diplostomidae, Digenea) species from naturally infected birds (Anatinae) in the Czech Republic and in Poland: morphological, morphometric and ecological features. Helminthologia, v.51 p.215-224, 2014.; Yaseen and Abdullah, 2018YASEEN, A.M.; ABDULLAH, B.H. First record of three species of trematode in Caspin Gull (Larus cachinnans Pallas, 1811) in Faw township, southern of Basrah, Iraq. Basrah J. Sci., v.36, p.53-67, 2018.; Lee et al., 2020LEE, Y.I.I.; SEO, M.; CHAI, J.Y. Intestinal flukes recovered from a herring gull, Larus argentatus, in the Republic of Korea. Korean J. Parasitol., v.58, p.81-86, 2020.; Faltýnková et al., 2022).

Molecular characterization of larval and adult diplostomes utilizing data from rRNA markers has proven to be useful in supplementing their morphology-based identification (Galazzo et al., 2002GALAZZO, D.E.; DAYANANDAN, S.; MARCOGLIESE, D.J.; MCLAUGHLIN, J.D. Molecular systematics of some North American species of Diplostomum (Digenea) based on rDNA-sequence data and comparisons with European congeners. Can. J. Zool., v.80, p.2207-2217, 2002.; Cavaleiro et al., 2012CAVALEIRO, F.I.; PINA, S.; RUSSELL-PINTO, F. et al. Morphology, ultrastructure, genetics, and morphometrics of Diplostomum sp. (Digenea: Diplostomidae) metacercariae infecting the European flounder, Platichthys flesus (L.) (Teleostei: Pleuronectidae), off the northwest coast of Portugal. Parasitol. Res., v.110, p.81-93, 2012.; Ndeda et al., 2013NDEDA, V.M.; OWITI, D.O.; AKETCH, B.O.; ONYANGO, D.M. Genetic relatedness of Diplostomum species (Digenea: Diplostomidae) infesting Nile Tilapia (Oreochromis niloticus L.) in Western Kenya. Open J. Appl. Sci., v.3, p.441-448, 2013.; Blasco-Costa et al., 2014; Faltýnková et al., 2014FALTÝNKOVÁ, A.; GEORGIEVA, S.; KOSTADINOVA, A. et al. Diplostomum von Nordmann, 1832 (Digenea: Diplostomidae) in the sub-Arctic: descriptions of the larval stages of six species discovered recently in Iceland. Syst. Parasitol., v.89, v.195-213, 2014.; Pérez-del-Olmo et al., 2014; Brabec et al., 2015BRABEC, J.; KOSTADINOVA, A.; SCHOLZ, T.; LITTLEWOOD, T.J. Complete mitochondrial genomes and nuclear ribosomal RNA operons of two species of Diplostomum (Platyhelminthes: Trematoda): a molecular resource for taxonomy and molecular epidemiology of important fish pathogens. Parasit. Vectors, v.8, p.336, 2015.; Aksenova et al., 2016AKSENOVA, O.V.; BESPALAYA, Y.V.; BOLOTOV, I.N.; KONDAKOV, A.V. First molecular identification of Diplostomum spathaceum (Rudolphi, 1819) (Trematoda: Digenea) from an intermediate host Radix auricularia (L., 1758) (Gastropoda: Lymnaeidae) in Russia. Biharean Biologist, v.10, p.90-92, 2016.; Achatz et al., 2022ACHATZ, T.J., MARTENS, J.R., KOSTADINOVA, A. et al. Molecular phylogeny of Diplostomum, Tylodelphys, Austrodiplostomum and Paralaria (Digenea: Diplostomidae) necessitates systematic changes and reveals a history of evolutionary host switching events. Int. J. Parasitol., v.52, p.47-63, 2022.; Khoshnaw and Abdullah, 2023KHOSHNAW, L.S.H.; ABDULLAH, S.M.A. Ultramorphology and molecular studies of Diplostomum paracaudum (Trematoda) collected in greater Zab River from Kurdistan region, Iraq. Appl. Ecol. Environ. Res., v.21, p.5163-5173, 2023.). However, most genetic sequences originate from the larval stages of diplostomes not accurately identified morphologically to species-level which prevents the resolution of the systematics of Diplostomum (Hoogendoorn et al., 2020HOOGENDOORN, C.; SMIT, N.J.; KUDLAI, O. Resolution of the identity of three species of Diplostomum (Digenea: Diplostomidae) parasitising freshwater fishes in South Africa, combining molecular and morphological evidence. Int. J. Parasitol. Parasit. Wildl. v.11, p.50-61, 2020.). The barcode region of the mitochondrial (mt) cytochrome c oxidase I (COI) gene has also been largely utilized as a tool elucidating life cycles and recognition of cryptic species diversity within Diplostomum (Blasco-Costa et al., 2014; Brabec et al., 2015; Kudlai et al., 2017KUDLAI, O.; OROS, M.; KOSTADINOVA, A.; GEORGIEVA, S. Exploring the diversity of Diplostomum (Digenea: Diplostomidae) in fishes from the river Danube using DNA mitochondrial barcodes. Parasit. Vectors, v.10, p.592, 2017.; Locke et al., 2020LOCKE, S.A.; DRAGO, F.B.; NÚÑEZ, V.; SOUZA, G.T.R.E.; TAKEMOTO, R.M. Phylogenetic position of Diplostomum spp. from new world herons based on complete mitogenomes, rDNA operons, and DNA barcodes, including a new species with partially elucidated life cycle. Parasitol. Res., v.119, p.2129-2137, 2020.; Achatz et al., 2022; Barata et al., 2022BARATA, S.D.; DORUCU, M.; GURSES, M. Identification and Molecular Investigation of Diplostomum in Capoeta umbla Caught from Freshwater Sources, Turkey. Genet. Aquat. Organ., v.6, p.GA454, 2022., 2023; Faltýnková et al., 2022; Sokolov et al., 2023SOKOLOV, S.G.; IESHKO, E.P.; LEBEDEVA, D.I. Resurrection of Diplostomum numericum Niewiadomska, 1988 (Digenea, Diplostomatoidea: Diplostomidae) based on novel molecular data from the Type-Host. Diversity, v.15, p.840, 2023.).

Although many reports have been published on helminths of various gull species from many parts of the world, few studies are available in Egypt (Abdel-Aal et al., 2001; Abdel-Aal and El-Sayed, 2003; Ghattas, 2004GHATTAS, M.W. Investigation on enteric helminth parasites of some aquatic birds at Port Said Governorate. J. Egypt. Med. Assoc., v.64, p.151-175, 2004.; Tadros et al., 2013TADROS, S.W.; ABO-ESA, J.F.K.; EL-GAYAR, A.K. Some studies on digenetic trematodes of herring gull (Larus argentatus) at Port Said governorate. Assiut Vet. Med. J., v.59, p.161-169, 2013.). The present study was carried out to shed more light on the Diplostomum species infecting herring gull (Larus argentatus) using integrative taxonomy with morphological description of the adult species associated with molecular analysis.

MATERIAL AND METHODS

A total of 20 birds of herring gull, Larus argentatus (Laridae), were hunted from February through September 2019 from the shore of El-Manzala Lake (Gamil Outlet in Port Said City, Egypt), and then moved to the laboratory for examination. Each bird was euthanized, within 8-24hr of capture, by receiving an intraperitoneal injection of sodium pentobarbital (Blink Health, NY, US). Birds were dissected, and the digestive tract was isolated and divided into main parts (Esophagus, proventriculus + gizzard, intestine, intestinal caeca, rectum, and cloaca) then each part was placed in 0.9% saline solution and examined under a dissecting microscope (Nikon SMZ18, NIS ELEMENTS software). The worms were collected and counted. The prevalence and intensity of infection were analyzed statistically following Bush et al. (1997BUSH, A.O.; LAFFERTY, K.D.; LOTZ, J.M.; SHOSTAK, A.W. Parasitology meets ecology on its own terms: Margolis, revisited. J. Parasitol., v.83, p.575-583, 1997.) guidelines. Worms were fixed with 10% formalin, stained with Semichon’s aceto-carmine, dehydrated in the ethyl alcohol series, cleared with xylene, and mounted in Canada balsam. Photomicrographs were taken with a Leica DM 2500 microscope (NIS ELEMENTS software, ver. 3.8). All measurements in the description and table were made with an Olympus ocular micrometer then given in micrometers and expressed as range followed by mean in parentheses.

gDNA was extracted from the ethanol-preserved worms by using procedure of the manufacturer’s Qiagen DNeasy^TM tissue kit. PCR reaction was performed to amplify the gDNA target using specific primers for the ITS1-5.8S-ITS2 gene: the forward primer D1 (5`-AGG AAT TCC TGG TAA GTG CAA G-3`) and the reverse one D2 (5`-CGT TAC TGA GGG AAT CCT GGT-3`), designed by Galazzo et al. (2002GALAZZO, D.E.; DAYANANDAN, S.; MARCOGLIESE, D.J.; MCLAUGHLIN, J.D. Molecular systematics of some North American species of Diplostomum (Digenea) based on rDNA-sequence data and comparisons with European congeners. Can. J. Zool., v.80, p.2207-2217, 2002.). PCR reactions of 25µL were included in 2 mM MgCl₂, 1.25 U rTaq polymerase buffer, 2.5µL 10× rTaq DNA buffer, 0.2 mM each of dNTPs, 2.5µM of each primer, 1 µl of DNA sample and completed to 25μL with dist. H₂O. The thermal cycle was performed in a thermocycler (BioRad) with the following conditions: 94°C for 2 min, then 30 cycles of 1 min at 94°C, 1 min at 56°C, and 2 min at 72°C and finally post-PCR extension for 5 min at 72°C. PCR products were examined using 1.5% w/v agarose gel (Sigma-Aldrich, Missouri, USA) in 1× Tris-acetate-EDTA (TAE) and stained with SYBR Safe DNA gel dye (Thermo Fischer Scientific, Ottawa, Canada) against the GeneRuler 100bp Plus ready-to-use DNA ladder (Fermentas, Lithuania). BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) used for sequencing PCR amplicons with 310 Automated DNA Sequencer (Applied Biosystems, USA). ITS1-5.8S-ITS2 sequences were deposited in GenBank^TM and compared with those data available in the NCBI database. Phylogenetic analysis was performed in MEGA X (Kumar et al., 2018KUMAR, S.; STECHER, G.; LI, M.; KNYAZ, C.; TAMURA, K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol., v.35, p.1547-1549, 2018.). Phylogenetic tree was constructed using maximum parsimony with 1000 replicates of bootstrapping.

RESULTS

Trematode parasites were naturally infected fourteen out of twenty (70%) specimens of the examined herring gull species, Larus argentatus. Adult worms were observed in the intestinal region of infected gull and identified as Diplostomum spathaceumRudolphi, 1819RUDOLPHI, K.A. Entozoorum synopsis cui accedunt mantissa duplex et indices locupletissimi/auctore, Carolo Asmund Rudolphi. 1819. Available in: https://www.biodiversitylibrary.org/page/9698017. Accessed in: 1, April, 2019.
https://www.biodiversitylibrary.org/page... and described in Figure (1) with measurements for different body parts in Table 1.

Figure 1
Photomicrographs of the adult Diplostomum spathaceum infecting Larus argentatus. (A) Whole mount preparation. (B-J) High magnifications for different body parts showing: (B-E) Forebody region. (F-J) Hindbody region. Note: EG, Eggs; EP, Excretory pore; FB, Forebody; GP, Genital pore; HB, Hindbody; HFO, Holdfast organ; OS, Oral sucker; OV, Ovary; PH, Pharynx; PrP, Pre-Pharynx; PS, Pseudosucker; SV, Seminal vesicle; TE, Testes; VS, Ventral sucker; VF, Vitelline follicles.

The body was bipartite with a total length of 1890-2021 (1984). The forebody was oval, dorso-ventrally flattened, 725-1062 (832) long with 40-45 (42)% of the total body length, with maximum width 483-682 (509) at the level of the holdfast organ. The hind body was elongated-oval, narrower anteriorly, measured 1131-1297 (1204) long, with a maximum width of 325-496 (437) at the level of the anterior testis. The oral sucker was ventro-subterminal, subspherical, measured 68-98 (82) long and 65-96 (80) wide.

The ventral sucker was sub-globular, situated just anteriorly to the mid-forebody, 60-91 (73) long and 75-96 (81) wide. Pseudosuckers were two in number, present on each side of the oral sucker, measured 102-143 (131) long and 39-58 (51) wide. Holdfast organ was large, sub-globular, close to the ventral sucker and measured 154-243 (236) long and 223-297 (257) wide. Prepharynx was short. The pharynx was oval, measured 51-84 (63) long and 40-54 (47) wide. Esophagus was indistinct; the caeca was narrow and directed posteriorly to the level of the holdfast organ.

The testes were two in number, located in the posterior half of the hind body; the anterior testis measured 167-198 (186) long and 150-218 (176) wide; posterior testis was 187-298 (283) long and 235-401 (354) wide. Seminal vesicle was voluminous.

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Table 1
Comparative measurements for the present Diplostomum spathaceum with those described previously

Genital pore was dorso-subterminal. Ovary was sub-globular, pre-testicular, measured 79-91 (89) long and 80-99 (90) wide. Vitellarium follicular was in shape and numerous, arranged in lateral bands surrounding the holdfast organ and posterior margin of the forebody, and well-distributed on both sides of the hind body reaching to the posterior extremity of the body. Eggs were few, measured 84-91 (87) long and 56-68 (61) wide.

DNA amplification of the partial ITS1-5.8S-ITS2 gene region from D. spathaceum resulted in a fragment of ~847 bp. One sequence was obtained from the organism detected in the present study, which was morphologically identified as D. spathaceum, and deposited in GenBank under the accession number PP177554. In GenBank, there are 13 sequences belonging to the members of the Genus Diplostomum, which belong to the adult stages of 3 species infecting Larus species. D. spathaceum was represented by 5 sequences, D. pseudospathaceum was represented by 4 sequences whereas Diplostomum sp. was represented by 4 sequences. Sequence identity ranged between 98.47-98.58% in D. spathaceum, to 97.76% in D. pseudospathaceum, and 97.40-97.99% in Diplostomum sp., respectively. Phylogenetic analysis revealed that sequences obtained in the present study clustered with the clade which included Diplostomum members including D. spathaceum, D. pseudospathaceum, and Diplostomum sp. with strong bootstrap support (Figure 2). Members of Diplostomum were distinct from those in the diplostomidid genera of Austrodiplostomum, Tylodelphys, and Neodiplostomum (Figure 2). Gigantobilharzia huronensis (EF071986.1) was used in analysis as an outgroup.

DISCUSSION

The Diplostomum species (family Diplostomidae Poirier, 1886POIRIER, J. Sur les diplostomidae. Arch. Zool. Exp. Gen., v.2, p.327-346, 1886.) are trematode parasites that spend part of their life cycle in the intestine of fish-eating avian hosts, with D. spathaceum being found in gulls and terns (Höglund and Thulin, 1992HÖGLUND, J.; THULIN, J. Identification of Diplostomum spp. In the retina of perch Perca fluviatilis and the lens of roach Rutilus rutilus from the Baltic Sea - an experimental study. Syst. Parasitol., v.21, p.1-19, 1992.; Marcogliese et al., 2001; Blasco-Costa and Locke, 2017).

In this study, the recovered parasite species was consistent with the diagnosis of the genus Diplostomum by Niewiadomska and Laskowski (2002NIEWIADOMSKA, K.; LASKOWSKI, Z. Systematic relationships among six species of Diplostomum Nordmann, 1832 (Digenea) based on morphological and molecular data. Acta Parasitol., v.47, p.20-28, 2002.) in distinctly bipartite body, a trilobate anterior extremity with pseudosuckers, vitelline follicles that distributed in the prosoma and opisthosoma, tandem testes with the anterior one being asymmetrical, absence of a genital cone, a non-protrusible copulatory bursa, and ovary being pre-testicular. Herein, the intestinal region of fourteen herring gulls (70%) was infected with a diplostomidid species within the Diplostomum genus.

Figure 2
Molecular Phylogenetic analysis using the Maximum Likelihood method based on the Jukes-Cantor model. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

This prevalence is similar to those mentioned previously in Yaseen and Abdullah (2018YASEEN, A.M.; ABDULLAH, B.H. First record of three species of trematode in Caspin Gull (Larus cachinnans Pallas, 1811) in Faw township, southern of Basrah, Iraq. Basrah J. Sci., v.36, p.53-67, 2018.) for D. pseudospathaceum in L. cachinnans from Faw township (Southern Basrah, Iraq) (prevalence (p) = 70%). Moreover, this prevalence is lower than the previous data of D. spathaceum reported by Yaseen and Abdullah (2018) in L. cachinnans from Faw township (Southern Basrah, Iraq) (p = 100%). This prevalence is higher than the previous data of diplostomidid infection reported by Ehrhardt et al. (1966EHRHARDT, W.R.; HARKEMA, R.; MILLER, G.C. Trematodes from two species of gulls from North Carolina. J. Elisha Mitchell Sci. Soc., v.82, p.208-212, 1966.) in L. atricilla (p=59%), Threlfall (1967THRELFALL, W. Diseases and Pathological conditions of the herring gull (Larus argentatus Pontopp.) excluding helminth infestations. Bull. Wildl. Dis. Assoc., v.3, p.62-67, 1967.) in L. argentatus from UK (p = 0.15%), Al-Allousi (1985) in L. ridibundus from Baghdad (Iraq) (p = 26.5%), Mhaisen et al. (1990MHAISEN, F.T.; KHAMEES, N.R.; AL-SAYAB, A.A. Flat worms (Platyhelminthes) of two species of gull (Larus ichthyaetus and L. canus) from Basrah. Zool. Middle East, v.4, p.113-116, 1990.) in L. ichinyaetus and L. canus from Basrah (Iraq) (p = 47%), Tameemi (2013TAMEEMI, I.A.A. Helminthes parasitized on some aquatic birds and the importance of insects in the life cycle of some of them in Basrah province. 2013. 241f. Thesis (Doctor) - College of Science, University of Basrah, IRQ.) in L. genei from Basrah (Iraq) (p = 13.3%), and Pérez-del-Olmo et al. (2014) in L. argentatus and L. ridibundus in fish and birds from Spain (p = 60-65%).

The present diplostomidid species are completely matched with the adult stages of D. spathaceum described previously by Krause (1914KRAUSE, R. Beitrag zur kenntnis der hemistominen. Z. Wissenschaftl. Zool., v.112, p.93-238, 1914.), Dubois (1938DUBOIS, G. Monographie des Strigeida (Trematoda). Mem. Soc.Neuchatel. Sci. Nat., v.6, p.161-196, 1938., 1970), Cichowlas (1961CICHOWLAS, Z. The life-cycle of Diplostomum spathaceum (Rud. 1819) in brackish waters of the Baltic Sea. Acta Parasitol. Pol., v.9, p.33-46, 1961.), Sweeting (1976SWEETING, R.A. An experimental demonstration of the life cycle of a Diplostomum from Lampetra fluviatilis Linnaeus, 1758. Z. Parasitenkd., v.49, p.233-242, 1976.), Dick and Rosen (1981DICK, T.A., ROSEN, R. Identification of Diplostomum spp. From the eyes of lake whitefish, Coregonus clupeaformis (Mitchell), based on experimental infection of herring gull chicks, Larus argentatus Pontoppidan. Can. J. Zoo, v.59, p.1176-1179, 1981.), Niewiadomska (1984NIEWIADOMSKA, K. Present status of Diplostomum spathaceum (Rudolphi, 1819) and differentiation of Diplostomum pseudospathaceum nom. nov. (Trematoda: Diplostomidae). Syst. Parasitol., v.6, p.81-86, 1984.), Sonin (1986SONIN, M.D. A key of the trematoda of piscivorous birds within the Palaearctic area. Moscow: Nauka, 1986. p.127-145.), Brady (1989BRADY, A. Studies on the Taxonomy and biology of Diplostomum species (Digenea). 1989. These (Doctor) - Faculty of Natural Sciences,University of Stirling, Stirling, UK.), Pérez-del-Olmo et al. (2014), Yaseen and Abdullah (2018YASEEN, A.M.; ABDULLAH, B.H. First record of three species of trematode in Caspin Gull (Larus cachinnans Pallas, 1811) in Faw township, southern of Basrah, Iraq. Basrah J. Sci., v.36, p.53-67, 2018.), and Lee et al. (2020LEE, Y.I.I.; SEO, M.; CHAI, J.Y. Intestinal flukes recovered from a herring gull, Larus argentatus, in the Republic of Korea. Korean J. Parasitol., v.58, p.81-86, 2020.) with the correspondence in the morphology and dimensions of the body and internal organs, host type (ex Larus species), and infection-specific sites. However, there is a slight difference between the current D. spathaceum and those described previously by Krause (1914), Dubois (1970), Sweeting (1976), Dick and Rosen (1981), Lee et al. (2020) in the smaller dimensions of testes in our specimens. This is the first report on the presence of D. spathaceum in an avian species in Egypt.

The adults of the current D. spathaceum are most similar to D. pseudospathaceum infecting L. argentatus and L. ridibundus, which is consistent with Niewiadomska (1984NIEWIADOMSKA, K. Present status of Diplostomum spathaceum (Rudolphi, 1819) and differentiation of Diplostomum pseudospathaceum nom. nov. (Trematoda: Diplostomidae). Syst. Parasitol., v.6, p.81-86, 1984.). However, there are some differences between both parasite species, including: (I) the length of the pseudosuckers (smaller in D. pseudospathaceum), (II) the shape of the forebody (elongate or linguiform in D. pseudospathaceum vs. egg-shaped or oval in D. spathaceum), (III) the position of the ventral sucker to the holdfast organ (distant from it in D. pseudospathaceum vs. near to the holdfast or partly covered by it in D. spathaceum), (IV) the distribution of vitellaria in the forebody (strands of small follicles reaching the ventral sucker in D. pseudospathaceum vs. compact and rarely extend anteriorly to the holdfast organ), and (V) the shape of testes (horseshoe-shaped for posterior one in D. pseudospathaceum).

Moreover, the recovered D. spathaceum is be differentiated from other Diplostomum species mentioned previously in Höglund and Thulin (1992HÖGLUND, J.; THULIN, J. Identification of Diplostomum spp. In the retina of perch Perca fluviatilis and the lens of roach Rutilus rutilus from the Baltic Sea - an experimental study. Syst. Parasitol., v.21, p.1-19, 1992.), Galazzo et al. (2002GALAZZO, D.E.; DAYANANDAN, S.; MARCOGLIESE, D.J.; MCLAUGHLIN, J.D. Molecular systematics of some North American species of Diplostomum (Digenea) based on rDNA-sequence data and comparisons with European congeners. Can. J. Zool., v.80, p.2207-2217, 2002.), Pérez-del-Olmo et al. (2014), Sitko and Rząd (2014SITKO, J.; RZĄD, I. Diplostomum and Ornithodiplostomum scardinii (Diplostomidae, Digenea) species from naturally infected birds (Anatinae) in the Czech Republic and in Poland: morphological, morphometric and ecological features. Helminthologia, v.51 p.215-224, 2014.), Yassen and Abdullah (2018), Lee et al. (2020LEE, Y.I.I.; SEO, M.; CHAI, J.Y. Intestinal flukes recovered from a herring gull, Larus argentatus, in the Republic of Korea. Korean J. Parasitol., v.58, p.81-86, 2020.), and Faltýnková et al. (2022FALTÝNKOVÁ, A.; KUDLAI, O.; PANTOJA, C.; YAKOVLEVA, G.; LEBEDEVA, D. Another plea for 'best practice' in molecular approaches to trematode systematics: Diplostomum sp. Clade Q identified as Diplostomum baeri Dubois, 1937 in Europe. Parasitology, v.149, p.503-518, 2022.), by the following: (I) the extent of the body division (indistinct in D. parviventosum in Melanitta fusca and Mergus merganser, D. pungitiiShigin, 1965SHIGIN, A.A. Some results of the study of the systematics of metacercariae of Diplostomum - the pathogens causing diplostomosis of freshwater fishes of the USSR. Mater. Nauk. Konf., v.1, p.261-265, 1965. in Clangula hyemalis, Somateria mollissima, Bucephala clangula, and Aythya fuligula), (II) the dimensions of the body and internal organs (smaller in D. mergi infecting M. merganser, D. parviventosum, D. pungitii, D. pusillum from M. merganser, D. phoxini infecting M. merganser, D. baeri from Stercoraius parasiticus, S. longicaudus, L. delawarensis, D. huronense, and D. indistinctum), (III) the fore/hindbody ratio (fore- is longer than hind part in D. mergi, D. parviventosum, D. pungitii, and D. phoxini), (IV) the ventral/oral sucker ratio (ventral larger than oral sucker in D. mergi, D. parviventosum, D. pungitii, D. pusillum, D. phoxini, D. baeri, D. volvens, D. huronense, and D. indistinctum), (V) the size of pseudosuckers (larger in D. pungitii, D. baeri, and D. volvens from L. ridibundus), (VI) the distribution of vitellaria (three forward-oriented strands in D. phoxini), and (VIII) the size of eggs (larger in D. mergi, D. parviventosum, D. pungitii, D. pusillum, D. phoxini, D. baeri, D. volvens, D. huronense, and D. indistinctum).

Molecular confirmation of the recovered parasite was brought about by studying the sequence variation on the ITS1-5.8S-ITS2 gene region, which agreed with Faltýnková et al. (2022FALTÝNKOVÁ, A.; KUDLAI, O.; PANTOJA, C.; YAKOVLEVA, G.; LEBEDEVA, D. Another plea for 'best practice' in molecular approaches to trematode systematics: Diplostomum sp. Clade Q identified as Diplostomum baeri Dubois, 1937 in Europe. Parasitology, v.149, p.503-518, 2022.) showed that this nuclear genetic region is useful for the identification of the adult stages of Diplostomum species infecting fish-eating birds. In the present study, one sequence was obtained from the organism which clustered with the adult stages of the genus Diplostomum infecting Larus species. This is consistent with Galazzo et al. (2002GALAZZO, D.E.; DAYANANDAN, S.; MARCOGLIESE, D.J.; MCLAUGHLIN, J.D. Molecular systematics of some North American species of Diplostomum (Digenea) based on rDNA-sequence data and comparisons with European congeners. Can. J. Zool., v.80, p.2207-2217, 2002.), Pérez-del-Olmo et al. (2014), Brabec et al. (2015BRABEC, J.; KOSTADINOVA, A.; SCHOLZ, T.; LITTLEWOOD, T.J. Complete mitochondrial genomes and nuclear ribosomal RNA operons of two species of Diplostomum (Platyhelminthes: Trematoda): a molecular resource for taxonomy and molecular epidemiology of important fish pathogens. Parasit. Vectors, v.8, p.336, 2015.), and Lee et al. (2020LEE, Y.I.I.; SEO, M.; CHAI, J.Y. Intestinal flukes recovered from a herring gull, Larus argentatus, in the Republic of Korea. Korean J. Parasitol., v.58, p.81-86, 2020.) reported that the Larus species considered as the specific host type for the Diplostomum taxa. The branch that grouped members of the genus Diplostomum was distinct from the branch that grouped the related organisms with the genera Austrodiplostomum, Tylodelphys, and Neodiplostomum. In this study, all diplostomidid genera formed a monophyletic group. Our molecular findings confirmed the distinction of the organism which is identified as D. spathaceum from D. pseudospathaceum. This agreed with the previous data of Pérez-del-Olmo et al. (2014) studied D. spathaceum infecting Larus argentatus from Ebro Delta, Cunit (Tarragona, Spain) and confirmed its molecular identity which grouped, in one clade, with D. pseudospathaceum and D. mergi.

CONCLUSION

This study represents a first report for the morphological description of the Diplostomum species infecting herring gull Larus argentatus in Egypt, in association with the molecular data used for identification.

ACKNOWLEDGMENTS

This study was supported by the Researchers Supporting Project (RSP2024R25), King Saud University, Riyadh, Saudi Arabia.

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YASEEN, A.M.; ABDULLAH, B.H. First record of three species of trematode in Caspin Gull (Larus cachinnans Pallas, 1811) in Faw township, southern of Basrah, Iraq. Basrah J. Sci., v.36, p.53-67, 2018.

Publication Dates

Publication in this collection
24 June 2024
Date of issue
Jul-Aug 2024

History

Received
21 Jan 2024
Accepted
20 Feb 2024

This is an open-access article distributed under the terms of the Creative Commons Attribution License

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[61] YASEEN, A.M.; ABDULLAH, B.H. First record of three species of trematode in Caspin Gull (Larus cachinnans Pallas, 1811) in Faw township, southern of Basrah, Iraq. Basrah J. Sci., v.36, p.53-67, 2018.

Comparable parameters	Krause (1914KRAUSE, R. Beitrag zur kenntnis der hemistominen. Z. Wissenschaftl. Zool., v.112, p.93-238, 1914. )	Dubois (1938DUBOIS, G. Monographie des Strigeida (Trematoda). Mem. Soc.Neuchatel. Sci. Nat., v.6, p.161-196, 1938. )	Cichoelas (1961)	Dubois (1970DUBOIS, G. Synopsis des strigeidae et des diplostomatidae (Trematoda). Mém. Soc. Neuchâte. Sci. Natur., v.10, p.259-727, 1970. )			Sweeting (1976SWEETING, R.A. An experimental demonstration of the life cycle of a Diplostomum from Lampetra fluviatilis Linnaeus, 1758. Z. Parasitenkd., v.49, p.233-242, 1976. )	Dick and Rosen (1981DICK, T.A., ROSEN, R. Identification of Diplostomum spp. From the eyes of lake whitefish, Coregonus clupeaformis (Mitchell), based on experimental infection of herring gull chicks, Larus argentatus Pontoppidan. Can. J. Zoo, v.59, p.1176-1179, 1981. )
Host species	L. argentatus, L. fuscus, L. canus	L. argentatus	L. ridibundus	Larus spp.	Larus spp.	Larus spp., Sterna spp., Rissa tridactyla, Alca torda	L. argentatus	L. argentatus
Locality	BRD	-	Baltic	USA	USA	-	-	-
Body	1500-3370	1250-4450	-	3250	3400	1250-4450	737-2277	2730-3484
Forebody	600-1360 × 360-690	640-1800 × 270-960	731-1003 × 510-935	520-1620 × 450-920	820-1500 × 270-850	600-1800 × 270-960	737-862	1326-1651 × 585-884
Hind body	770-2100 × 230-710	520-3220 × 210-750	850-1105 × 306-442	520-1800 × 400-710	880-2280 × 250-810	520-3220 × 210-750	550-1416	1482-1872 × 355-637
Oral sucker	54-94 × 54-99	40-100 × 48-104	68-75	65-112 × 51-105	55-110 × 49-97	40-100 × 46-104	70-91	65-105 × 65-110
Pseudo sucker (right)	-	-	-	80-135	-	75-180	-	90-105
Pseudo sucker (left)	-	-	-	80-135	-	75-180	-	90-105
Ventral sucker	54-110 × 59-140	48-101 × 48-130	-	60-120 × 73-138	60-110 × 60-135	48-110 × 48-140	98-102	75-115 × 100-125
Holdfast organ	220-410 × 240-390	125-450 × 90-432	119-165	180-360 × 150-340	110-310 × 120-320	125-450 × 90-390	200-234	225-415
Pharynx	41-81 × 46-72	48-91 × 26-75	-	51-98 × 36-64	50-94 × 30-79	39-91 × 25-75	-	65-95 × 50-70
Testis (anterior)	170-340 × 280-520	95-460 × 130-560	-	200-435 × 330-630	170-420 × 220-490	95-460 × 130-560	494-273	325-400 × 285-500
Testis (posterior)	210-400 × 320-590	90-485 × 190-650	-	225-500 × 370-610	220-560 × 240-560	90-485 × 190-650	385-519	320-425 × 355-510
Ovary	77-130 × 100-170	50-205 × 70-235	-	69-170 × 123-225	80-190 × 90-200	50-205 × 70-235	136-197	90-170 × 105-195
Egg	-	-	-	-	-	84-115 × 52-76	-	-
Comparable parameters	Niewiadomska (1984NIEWIADOMSKA, K. Present status of Diplostomum spathaceum (Rudolphi, 1819) and differentiation of Diplostomum pseudospathaceum nom. nov. (Trematoda: Diplostomidae). Syst. Parasitol., v.6, p.81-86, 1984.)	Sonin (1986SONIN, M.D. A key of the trematoda of piscivorous birds within the Palaearctic area. Moscow: Nauka, 1986. p.127-145.)	Brady (1989BRADY, A. Studies on the Taxonomy and biology of Diplostomum species (Digenea). 1989. These (Doctor) - Faculty of Natural Sciences,University of Stirling, Stirling, UK.)	Pérez-del-Olmo et al. (2014)	Yaseen and Abdullah (2018YASEEN, A.M.; ABDULLAH, B.H. First record of three species of trematode in Caspin Gull (Larus cachinnans Pallas, 1811) in Faw township, southern of Basrah, Iraq. Basrah J. Sci., v.36, p.53-67, 2018.)	*Lee et al. (2020*LEE, Y.I.I.; SEO, M.; CHAI, J.Y. Intestinal flukes recovered from a herring gull, Larus argentatus, in the Republic of Korea. Korean J. Parasitol., v.58, p.81-86, 2020.)	Present study
Host species	L. fuscus, L. ridibundus	Larus spp.	L. argentus	L. argentatus, L. ridibundus	L. cachinnans	L. argentatus	L. argentatus
Locality	Poland	Eurasia	Scotland	Spain	Iraq	Korea	Egypt
Body	Up to 4000	3830	1313-2550 (1952)	1971-2189	2440-3800 (3100)	1750-2000	1890-2021 (1984)
Forebody	1110-1480 × 590-850	850-1630 × 400-1090	440-1000 (718) × 430-610 (529)	782-1155 × 504-726	900-1350 (106) × 600-800 (700)	680-850 × 650-700	725-1062 (832) × 483-682 (509)
Hind body	1560-2920 × 560-660	1090-2800 × 325-770	775-1550 (1222) × 300-470 (371)	1252-1368 × 387-575	1420-2500 (2090) × 580-650 (600)	1080-1100 × 500-520	1131-1297 (1204) × 325-496 (437)
Oral sucker	57-95 × 74-102	55-100 × 60-111	60-88 (75) × 63-90 (76)	71-93 × 70-92	60-110 (80) × 80-160 (950)	60-65 × 75-100	68-98 (82) × 65-96 (80)
Pseudo sucker (right)	102-153	70-130	-	109-155 × 44-62	90-120 (110) × 50-80 (60)	-	102-143 (131) × 39-58 (51)
Pseudo sucker (left)					110-140 (120) × 40-70 (50)
Ventral sucker	78-95 × 89-102	65-110 × 65-130	60-85 (74) × 73-100 (90)	65-95 × 80-99	80-150 (89) × 90-120 (110)	75-85 × 80-100	60-91 (73) × 75-96 (81)
Holdfast organ	238-374 × 259-399	150-460 × 150-440	-	150-236 × 202-288	200-300 (270) × 210-390 (250)	175-275 × 125-160	154-243 (236) × 223-297 (257)
Pharynx	59 - 74 × 51 - 74	50-85 × 45-70	53-73 (64) × 40-53 (46)	55-89 × 45-59	60-120 (80) × 50-90 (70)	50-52 × 55-65	51-84 (63) × 40-54 (47)
Testis (anterior)	185-540 × 421-629	190-430 × 220-500	-	171-203 × 154-224	170-420 (250) × 300-550 (470)	325-350 × 140-150	167-198 (186) × 150-218 (176)
Testis (posterior)	348-592 × 466-658	220-550 × 280-550	-	190-317 × 240-399	160-530 (390) × 350-620 (520)	300-325 × 175-225	187-298 (283) × 235-401 (354)
Ovary	138-222 × 163-236	90-180 × 70-270	90-130 (114) × 90-150 (122)	87 × 83	130-200 (150) × 160-200 (180)	100-135 × 75-100	79-91 (89) × 80-99 (90)
Egg	-	-	80-100 (90)	89-99 × 61-66	110-150 (130) × 80-100 (90)	108-125 × 65-85	84-91 (87) × 56-68 (61)

Brasil