Zoosyst. Evol. 99 (1) 2023, 117-121 | DOI 10.3897/zse.99.91381 Pi piss ae a _., D> PENSOFT. Nites A NATURKUNDE BERLIN First molecular identification of the trematode Maritrema bonaerense Etchegoin & Martorelli, 1997 (Plagiorchiida, Microphallidae) from its intermediate hosts, the gastropod Heleobia australis (d’Orbigny, 1835) (Littorinimorpha, Cochliopidae) and the crab Neohelice granulata (Dana, 1851) (Decapoda, Varunidae) in Argentina Lorena Martinez', Carmen Gilardoni*, Cintia Medina?, Juan José Lauthier*, Florencia Cremonte?, Jorge Etchegoin! 1 Instituto de Investigaciones en Produccion Sanidad y Ambiente (IIPROSAM), CONICET-UNMeéfP, Centro de Asociacion Simple CIC-PBA, Juan B. Justo 2550 (7600), Mar del Plata, Argentina 2 Instituto de Biologia de Organismos Marinos (IBIOMAR-CONICET), Boulevard Brown 2915, (9120), Puerto Madryn, Chubut, Argentina 3 Instituto de Diversidad y Evolucion Austral (IDEAus-CONICET), Boulevard Brown 2915, (9120), Puerto Madryn, Chubut, Argentina 4 Instituto de Investigaciones en Microbiologia y Parasitologia Médica (IMPaM), Facultad de Medicina, Universidad de Buenos Aires (UBA- CONICET), Paraguay 2155, Piso 12, (1121) Ciudad Autonoma de Buenos Aires, Buenos Aires, Argentina https://zoobank. org/EC975AED-6AE7-4848-9A 6F-63C0C84C916D Corresponding author: Carmen Gilardoni (gilardonicarmen@cenpat-conicet.gob.ar) Academic editor: Pavel Stoev # Received 8 August 2022 Accepted 8 November 2022 @ Published 26 January 2023 Abstract The genus Maritrema Nicoll, 1907 (Platyhelminthes, Trematoda, Plagiorchiida, Microphallidae) comprises cosmopolitan species that predominantly parasitize birds. Although approximately 65 species have been described worldwide, including 6 for Argentina, molecular data referring to Maritrema species are still scarce worldwide, especially in South America. Unfortunately, this lack of ref- erences for nucleotide sequences is an obstacle to understanding the taxonomy and life cycles of trematodes, and impedes advancing our studies on the phylogeny and geographical distribution of these parasites. For that reason, we performed the molecular study of developmental stages of Maritrema bonaerense: cercariae (collected from the snail first intermediate host Heleobia australis, inhab- iting Mar Chiquita lagoon) and metacercariae (collected from the crab second intermediate host Neohelice granulata, inhabiting Mar Chiquita lagoon and San Antonio Oeste, Argentina). The accordance between the ITS2 sequence of MZ. bonaerense cercaria from the snail H. australis and the sequences of metacercariae from the crab N. granulata was 100%, supporting previous findings of the life cycle of MZ. bonaerense based on morphological data. All Maritrema species are included in a monophyletic and well-supported clade. Maritrema bonaerense grouped more closely with Maritrema gratiosum. These findings contribute to the knowledge of dige- neans in coastal marine ecosystems. Key Words digeneans, ITS2 sequence, life cycle, South America Introduction cosmopolitan species that predominantly parasitize birds in brackish, marine and to a lesser extent, freshwater eco- The genus Maritrema Nicoll, 1907 (Platyhelminthes, systems (Deblock 2008; Capasso et al. 2019). Their life Trematoda, Plagiorchiida, Microphallidae) comprises cycles also involve gastropods and crustaceans as first Copyright Martinez, L. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 118 Martinez, L. et al.: First molecular identification of Maritrema bonaerense in Argentina and second intermediate hosts, respectively (Yamaguti 1975). To date, approximately 65 species of this ge- nus have been described worldwide (Presswell et al. 2014), including 6 species from Argentina: Maritrema bonaerense Etchegoin & Martorelli, 1997; MM. orensense Cremonte & Martorelli, 1998; M. madrynense Diaz & Cremonte, 2010; M. formicae Diaz, Gilardoni & Cremo- nte, 2012; M. patagonica Rauque, Flores & Brugni, 2013, and M. pichi Capasso D’ Amico & Diaz, 2019. As with other digeneans, molecular data referring to Maritrema species are still scarce in South America. For example, the only DNA sequences available in Argentina are from M. madrynense (Bagnato et al. 2015). Unfortu- nately, this lack of references for nucleotide sequences for South America is an obstacle to understanding the taxono- my and life cycles of trematodes (Lopez-Hernandez et al. 2019). Likewise, an increase in genetic data would be a sig- nificant step forward in our studies on the phylogeny and geographical distribution of these parasites in the region. The life cycle and developmental stages of M. bonaerense were originally described by Etchegoin and Martorelli (1997) from Mar Chiquita lagoon (Buenos Aires province, Argentina). Later, Alda et al. (2013) re- described the adult and metacercaria, and experimentally confirmed the life cycle of this species which includes the cochliopid snail Heleobia australis as first intermediate host and the crabs Cyrtograpsus angulatus and Neohelice granulata as second intermediate hosts, and the birds Chroicocephalus maculipennis, Larus atlanticus and 40°S 42°S 44° 52°S 54°S — fa Cs a ae San : 70° a6" e2" L. dominicanus as definitive hosts. Both mentioned stud- ies were conducted using only morphological analyses. For that reason, and taking into account the scarcity of genetic data related to the species of Maritrema, we per- formed the molecular study of cercariae and metacercari- ae of M. bonaerense, collected from the snail H. australis inhabiting Mar Chiquita lagoon and from the crab N. granulata inhabiting Mar Chiquita lagoon and San Antonio Oeste (Rio Negro province, Argentina). It is important to mention here that although adult stag- es from the definitive hosts could not be obtained because Mar Chiquita is a Man and Biosphere Reserve (UNESCO) within which the birds are protected, M. bonaerense is the only species of Maritrema that parasitizes H. australis and N. granulata in this location (Etchegoin 2001; Parietti et al. 2013). Therefore, there was no possibility of misidenti- fications of developmental stages collected for this study. Materials and methods The specimens of H. australis were collected in Mar Chiquita lagoon, Buenos Aires province, Argentina (37°45'08"S, 57°26'18"W). In the laboratory, molluscs were isolated individually in 45 ml plastic cups and main- tained under a 12-12 light-dark photoperiod for 48 h to stimulate shedding of cercariae. Crabs (N. granulata) col- lected in Mar Chiquita lagoon and in San Antonio Oeste (40°43'36"S, 64°54'49"W) (Fig. 1) were transported to Mar Chiquita Lagoon SOUTH AMERICA R= 0 200 400 km 58° 54° 50° Figure 1. Map of sampling sites from Argentina: Mar Chiquita Lagoon (Buenos Aires province) where the snail Heleobia australis and the crab Neohelice granulata were collected and San Antonio Oeste (Rio Negro province) where N. granulata were collected. Invertebrate drafts extracted from Alda et al. (2013). zse.pensoft.net Zoosyst. Evol. 99 (1) 2023, 117-121 Table 1. Molecular data of Maritrema species considered in this study. 119 Species Life stage Host Habitat type Country ITS2 p-distance Reference Maritrema bonaerense cercaria Heleobia australis brackish Argentina ON833442 this study Maritrema bonaerense metacercaria Neohelice granulata brackish Argentina ON833466 0.00 this study Maritrema bonaerense metacercaria Neohelice granulata marine Argentina ON833467 0.00 this study Maritrema gratiosum metacercaria Semibalanus balanoides marine lreland HM584171 0.04 Galaktionov et al. (2012) Maritrema subdolum cercaria Peringia ulvae brackish Russia HM584172 0.08 Galaktionov et al. (2012) Maritrema eroliae cercaria Clypeomorus bifasciata marine Kuwait HQ650132 0.11 AlKandari et al. (2011) Maritrema novaezealandense _—cercaria © Zeacumantus subcarinatus marine New Zealand KJ540203 0.10 Born-Torrijos et al. (2014) Maritrema madrynense adult Larus dominicanus marine Argentina KF575167 0.10 Diaz and Cremonte (2010) Maritrema brevisacciferum — metacercaria Caridina indistincta freshwater Australia KT355824 0.09 Kudlai et al. (2015) Maritrema oocysta cercaria Hydrobia ulvae marine lreland HM584170 0.10 Galaktionov et al. (2012) Microphallus similis metacercaria Carcinus maenas marine Russia HM584180 0.14 Galaktionov et al. (2012) the laboratory and maintained in aerated water. Posteri- orly, infected snails and crabs were necropsied, and the developmental stages (sporocyst, cercariae and metacer- cariae) were stored in 96% ethanol for molecular studies. Cercariae and metacercariae of M. bonaerense were iden- tified according to Etchegoin and Martorelli (1997) and Alda et al. (2013). The molecular characterization of the developmental stages of M. bonaerense was made using rRNA ITS2 sequences. The DNA extraction, PCR amplification, and sequencing were performed using the protocol de- scribed in Gilardoni et al. (2020). Newly generated ITS2 sequences were deposited in GenBank and aligned us- ing MAFFT (Katoh et al. 2019) together with available Maritrema spp. and with Microphallus similis as out- group (Table 1). Maximum likelihood (ML) and Bayes- ian inference (BI) analyses were conducted in MEGA X (Kumar et al. 2018) and MrBayes version 3.2.7a (Ronquist et al. 2012) respectively. Genetic divergences amongst taxa were calculated as uncorrected p-distances using MEGA X. For both, ML and BI, to determine the nucleotide substitution model that gave the best fit to our data set, the program MEGAX which held the JModel test anal- ysis was employed, with model selection based on the Akaike information criterion (AIC). Results indicat- ed that the general time reversible model with an es- timate of gamma distributed among-site rate variation (GTR+G) was the most appropriate. For the ML tree, the percentage of trees in which the associated taxa clustered together is shown next to the branches. Ini- tial trees for the heuristic search were obtained auto- matically by applying Neighbor-Join and BioNJ algo- rithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log like- lihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis in- volved 35 nucleotide sequences. There are a total of 771 positions in the final dataset. For the BI tree, GTR was selected as the substitution model (command Iset nst=6). We ran four independent chains of 100 million generations each, sampling every 5000 generations, with the first 1000 trees discarded as “burn-in”. Chain convergence was confirmed using Tracer v.1.6 (Ram- baut et al. 2018). Finally, a 50% majority rule consensus tree was constructed. Results and discussion The PCR amplification of the ITS2 rRNA from cercaria from Mar Chiquita Lagoon and metacercariae from Mar Chiquita Lagoon and San Antonio Oeste gave products of 540 bp, 557 bp and 543 bp respectively. The accordance between the ITS2 sequence of M. bonaerense cercaria from H. australis and the sequences of metacercariae from N. granulata was 100% (Fig. 2). This result sup- ports previous findings of the life cycle of MZ. bonaerense based on morphological data (Etchegoin and Martorelli 1997, Alda et al. 2013). The genus Maritrema constitutes a monophyletic and well-supported clade. Among all the species of Maritrema compared in this work, M. bonaerense seems to be more closely related to M. gratiosum Nicoll, 1907. Both spe- cies constitute a well-supported clade separated from the other Maritrema spp. The genetic divergence (p-dis- tance) revealed M. bonaerense presents 0.04 variation with M. gratiosum, 0.08—0.11 with the other Maritrema spp. and 0.14 with Microphallus similis (outgroup). The molecular data support the morphological taxonomy of the genus Maritrema, which 1s distinguished by the vitel- lartum in symmetrical ribbons reaching close to margin of hindbody, surrounding uterine coils and testes, horse- shoe-shaped with posteriorly directed opening or com- plete ring (Deblock 2008). Despite the high number of Maritrema species morphologically described, molecular data are very scarce: rRNA 18S (6 spp sequenced), 28S (10 spp.), ITS1 (5 spp.), ITS2 (7 spp.), mitochondrial DNA cox! (1 sp.). To date, most available sequences belong to species infecting marine or brackish hosts (Table 1). How- ever, Maritrema species are present in freshwater habitat as M. brevisacciferum (Kudlai et al. 2015). Our findings contribute to the development of molecular database that may be used in future studies about these common and widespread parasites infecting birds worldwide. zse.pensoft.net 120 Martinez, L. et al.: First molecular identification of Maritrema bonaerense in Argentina HM5841 78 Microphallus similis HM584170 Maritrema oocysta 0.99/100 | KT355824 Maritrema brevisacciferum | 1/100 1/100 KF575167 Maritrema madrynense 1/80 1/100 KJ540203 Maritrema novaezealandense 0.66/52 HQ65013? Maritrema eroliae HM584172 Maritrema subdolum 0.82/75 Maritrema bonaerense met-Ng-SAO ON833467 Maritrema bonaerense met-Ng-MCh 1/100)" 09N833466 Maritrema bonaerense cerc-Ha-MCh we ON833442 HM584171 Maritrema gratiosum 0.02 Figure 2. Phylogram for Maritrema species (Microphallus similis as outgroup), inferred by ML/BI of sequence data for ITS2 of the rRNA genes. The newly generated sequences are indicated in bold. Values on the branches correspond to posterior probabil- ities > 0.85 followed by bootstrap support > 60. Values below these thresholds were not reported. Abbreviations: cerc-cercaria, met-metacercaria, Ng-Neohelice granulta, Ha-Heleobia australis, MCh-Mar Chiquita Lagoon, SAO-San Antonio Oeste. Drafts of life stages extracted from Etchegoin and Martorelli (1997). Acknowledgements This study was supported by ANPCyT (PICT 2017-1819 to Etchegoin JA, PICT 2019-00837 to Gilardoni C, PICT 2020-2120 to Cremonte F) and from Universidad Nacio- nal de Mar del Plata (Grant number 15/E935 EXA997/20 to Etchegoin JA). References Al-Kandari WY, Al-Bustan SA, Alnaqeeb M (2011) Ribosomal DNA sequence characterization of Maritrema cf. eroliae Yamaguti, 1939 (Digenea: Microphallidae) and its life cycle. 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