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. 2023 May 26;62:e15. doi: 10.6620/ZS.2023.62-15

A New Polyclad Flatworm, Idiostylochus tortuosus gen. nov., sp. nov. (Platyhelminthes, Polycladida) from France. Can this Foreign Flatworm be Responsible for the Deterioration of Oyster and Mussel Farms?

Adrian Gutiérrez 1, Isabelle Auby 2, Benoit Gouillieux 3, Guillemine Daffe 3, Cecile Massé 4, Elvire Antajan 2, Carolina Noreña 1,*
PMCID: PMC10390326  PMID: 37533559

Abstract

A new species of polyclad flatworm, Idiostylochus tortuosus gen. nov., sp. nov. (Polycladida, Idioplanidae), from Arcachon Bay (France) is described. This description is based on a morphological analysis and a molecular analysis using partial sequences of the 28S and cytochrome Oxidase I (COI) genes. After the molecular analysis Idiostylochus gen. nov. appears to be the second genus of the Family Idioplanidae and closely related to the family Latocestidae as well as the genera Leptostylochus and Mirostylochus. The molecular data revealed that the new species may belong to an Indonesian or Indo-Pacific family, closely related to genera with origins in South Pacific Ocean waters. This species was found feeding on the oysters and mussels of the Arcachon farms.

Keywords: Acotylea, Idioplanidae, Non-native species, Magallana gigas, Oyster culture

BACKGROUND

France is the main producer and consumer of oysters in Europe. French oyster farms produce around 80,000 tons of Japanese oysters (Magallana gigas (Thunberg, 1793), formerly Crassostrea gigas) per year, representing 78% of European annual production (FAO 2021). With an annual production, in normal conditions, of around 10,000 tons of M. gigas (Vieira et al. 2020), Arcachon Bay is not only one of the most important areas for French oyster farming, it is also one of the first areas to have implemented this type of culture (Bouchet et al. 1997; Buestel et al. 2009). Over the years, three different species of oysters have been farmed in Arcachon Bay: the European flat oyster (Ostrea edulis Linnaeus, 1758), the Portuguese cupped oyster (Crassostrea angulata (Lamarck, 1819)), and the above-mentioned Japanese oyster. The appearance of different epizootics caused the collapse of the European and Portuguese species. At present, French oyster farmers focus their cultures almost exclusively on M. gigas. This species was introduced to Arcachon in the 1970s to gradually replace the declining oyster cultures of C. angulata (Buestel et al. 2009). Arcachon Bay benefited in May 1971 from an initial input of 52.5 tonnes of broodstock from British Columbia in Canada (very large oysters about 10 years old), reinforced by 60 tonnes in 1972 and 25 tonnes in 1973, making a total of 137.5 tonnes of mother oysters that were placed in several reserves along the Basin. At the same time, large quantities of spat from Japan were introduced (Grizel and Héral 1991). Magallana gigas is characterized by its large size, rapid growth and high tolerance to environmental changes compared to other oyster species (FAO 2005–2021).

Since the introduction of Magallana gigas from Japan, Canada and North America in the 1970s (Grizel and Héral 1991), several non-indigenous species have been observed (e.g., Bachelet et al. 2009; Lavesque et al. 2013; Gouillieux and Massé 2019), introduced directly from the Pacific or during regular transports of batches between French oyster farming areas. The description of a new species in the Arcachon Bay, which ultimately turned out to be a non-indigenous species, occurred recently with the annelid polychaete Marphysa victori Lavesque, Daffe, Bonifácio & Hutchings, 2017 (Annelida) (Lavesque et al. 2020).

In 2020, the presence of large amounts of unknown polyclads (Platyhelminthes, Polycladida) feeding on individuals of M. gigas and Mytilus sp. were recorded in Arcachon Bay. The occurrence of this polyclad may be related to the increasing oyster mortality recorded since 2019 in oyster farms (Vieira et al. 2020) and mussel beds in Arcachon Bay (local marine fisheries committee, pers. com.)

Most polyclads are predators of other small invertebrates, such as crustaceans, ascidians, cnidarians, gastropods, or bivalves (Barton et al. 2020; Jennings 1957; Newman and Cannon 2003; Lee 2006; Teng et al. 2022). At present, the suborder Acotylea (Polycladida) is divided into three superfamilies: Discoceloidea Dittmann, Cuadrado, Aguado, Noreña and Egger, 2019; Leptoplanoidea Faubel, 1984; and Stylochoidea Poche, 1926.

Within Acotylea, known predators of bivalves belong mainly to the superfamily Stylochoidea. They prey on mussels (Galleni et al. 1980), scallops (Heasman et al. 1998), giant clams (Newman et al. 1993) and oysters (Danglade 1919; Pearse and Wharton 1938; Littlewood and Marsbe 1990; Newman et al. 1993). Although the mechanism used to open the valves of the prey varies among species, this process usually starts with the polyclad gliding over the posterior end of the valves, followed by the secretion of a considerable amount of mucus. This secretion could serve to immobilize the prey (Hyman 1951), as well as to avoid desiccation and potential attacks by other predators (Gammoudi et al. 2017). Once in position, the polyclad inserts its pharynx into the bivalve, damages the adductor muscle to prevent the prey from closing its valves, and digests it (Gammoudi et al. 2017).

The aim of this study is to describe and determine the systematic position of a new species of Polycladida found in Arcachon Bay. These animals are known as predators of bivalve mollusks, and the molecular evidence suggests that they are a non-native species. These preliminary results will guide future studies focused on the habitat, ecology and impact of this species in France.

MATERIALS AND METHODS

Sampling

The specimens were collected by hand in the Arcachon Bay (French Atlantic Coast) (Fig. 1) in October 2020. The live specimens were anesthetized with a solution of 7% MgCl2 and photographed. A small sample of tissue from the lateral margin was preserved in absolute ethanol for DNA extraction. The rest of the specimens were fixed individually in frozen 10% formalin buffered with filtered seawater. After 24 h, the worms were transferred to 30% ethanol for an hour, then to 50% ethanol for an hour and finally stored in 70% ethanol.

Fig. 1.

Fig. 1.

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Bay of Archachon. Sampling sites. The green dots show the localities inside the bay, the red stars mark out the localities outside the bay.

Histological processing

The fixed specimens were dehydrated in progressive ethanol solutions, embedded in Paraplast, and sectioned sagittally at 10 μm. The sections were stained with the Azan trichrome stain. To identify the species, reconstructions of the internal anatomy of the reproductive system were performed using a Zeiss Axio Scope A1 microscope.

DNA extraction, amplification and sequencing

DNA from four individuals (Table 1) was extracted using the phenol-chloroform protocol (Chen et al. 2010), using the tissue samples fixed in absolute ethanol. Once the DNA was extracted, its purity and concentration were calculated with a NanoDrop spectrophotometer (Thermo Fisher Scientific).

Two partial sequences, one of approximately 1,000 bp from the 28S gene and another of 700 bp from the COI gene, were amplified by PCR. For the 28S sequences, a forward primer (5'-AGCCCAGCACCGAATCCT-3') and a reverse primer (5'-GCAAACCAAGTAGGGTGTCGC-3') were used (Cuadrado et al. 2021). The reaction was carried out in a final volume of 25 μl with 1 μl of DNA, 12.5 μl of DreamTaq DNA polymerase and 1 μl of each primer. The amplification protocol used was an initial denaturation step at 95°C (4 min), followed by 35 cycles of denaturation at 95°C (1 min), annealing at 59°C (1 min) and extension at 72°C (1 min), with a final extension at 72°C (10 min).

For the COI sequences, the primers Acotylea_ COI_F (5'-ACTTTATTCTACTAATCATAAGGATATA GG-3') and Acotylea_COI_R (5'-CTTTCCTCTATAAA ATGTTACTATTTGAGA-3') were used (Oya and Kajihara 2020). The reaction was carried out with the same volumes as those used for the 28S gene. The amplification protocol was as follows: initial denaturation step at 94°C (5 min), 35 cycles of denaturation at 94°C (30 s), annealing at 50°C (30 s) and extension at 72°C (1 min), with a final extension at 72°C (7 min).

The PCR products were purified using ExoSAP (Bell 2008). The purified samples were sent to Secugen S.L. (www.secugen.es) for sequencing. Finally, the sequences obtained from the forward and reverse primers were combined and edited with Sequencher 4.1.4 (Gene Codes Corporation, Ann Arbor, MI, USA; http://www.genecodes.com).

Table 1.

List of species included in the molecular analysis with their respective locality, GenBank accession number or museum catalogue number (MNCN) and reference

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Sequence alignment and molecular analyses

The different trees were obtained applying the Maximum Likelihood (ML) and Bayesian Inference (BI) methods, using the sequences obtained in this work and those available in GenBank (NCBI) of representative species of the main families of the suborder Acotylea (Table 1). Two Cotylea were used as outgroups: Pericelis flavomarginata Tsuyuki, Oya, Jimi and Kajihara, 2020 and Cestoplana nopperabo Oya and Kajihara, 2018 (Table 1). Sequence alignment was performed with MAFFT (Katoh et al. 2018) with the default options. Ambiguous regions were removed using Gblocks ver. 0.91b (Talavera and Castresana 2007) with the least restrictive options. The alignments were checked manually with BioEdit (Hall 1999).

For this study, a dataset of 706 bp and 58 sequences for the 28S gene analysis and a dataset of 640 bp and 40 sequences for the COI gene analysis were used (the complete sequences of the new species can be found in GenBank, Table 1). The substitution model used for all the analyses was GTR+I+G, which was determined with ModelFinder (Kalyaanamoorthy et al. 2017) using the Akaike Information Criterion (AIC) (Akaike 1974).

ML analyses were performed with IQ-TREE (Trifinopoulos et al. 2016). Nodal support was calculated with a bootstrap standard test with 1,000 replicates. BI analyses were performed with MrBayes 3.2.3 (Ronquist et al. 2012). Two simultaneous analyses of 10,000,000 generations were run with four chains (one cold, three heated) and a tree sampling frequency of 1,000. The convergence of the chains was determined using the value of the standard deviation of the frequencies (< 0.05). The first 25% of the trees were discarded as burn-in. The trees resulting from both methods were visualized and edited with iTOL ver. 6.3 (Letunic and Bork 2021).

RESULTS

SYSTEMATICS

Order Polycladida Lang, 1881

Suborder Acotylea Lang, 1884

Superfamily Stylochoidea Poche, 1926

Family Idioplanidae Dittmann, Cuadrado, Aguado, Noreña and Egger, 2019

Idiostylochus gen. nov.

urn:lsid:zoobank.org:act:B1874F30-7A5B-45C6-AD8A-34A37FC66E5A

Diagnosis: Idioplanidae with the pharynx in the middle of the body. Cerebral and marginal eyes present. Male copulatory apparatus with spermiducal bulbs and prostatic vesicle. Seminal vesicle absent. Female apparatus with a tubular Lang’s vesicle and cement glands. The vagina makes a posterior turn before reaching the penis papilla.

Type species: Idiostylochus tortuosus sp. nov.

Etymology: The name Idiostylochus derives from a combination of Idioplana and Stylochus, regarding the presence of a unique combination of characters found in part in these genera.

Idiostylochus tortuosus sp. nov.

(Fig. 2)

urn:lsid:zoobank.org:act:BCF1166D-6D0A-4B79-899A-59D3B262644C

Type material: Holotype: 1 specimen. Arcachon Bay, France, October 20, 2020. Sagittal sections stained with Azan trichrome. MNCN 4.01/4263 to MNCN 4.01/4291 (29 slides). GeneBank accesion numbers: ON796526 (28S), ON796530 (COI).

Paratype: 1 specimen. Arcachon Bay, France,

October 20, 2020. Sagittal sections stained with Azan trichrome. MNCN 4.01/4292 to MNCN 4.01/4335 (44 slides).

Additional material: tissues preserved in 100% Ethanol and sagittal sections stained with Azan trichrome; Arcachon Bay, France, October 20, 2020.

For GeneBank accession numbers see table 1.

Diagnosis: Male copulatory apparatus with conspicuous spermiducal bulbs and a small prostatic vesicle. Elongated male atrium covered by glandular tissue. Female apparatus with a vagina bulbosa and well-developed cement glands that surround the two sections of the vagina (externa e interna).

Etymology: The specific name derives from the Latin tortuous, due to the winding and complex course of the distal portion of the vasa deferentia and spermiducal bulbs.

Description: Body shape rounded-oval with slightly undulated margins (Fig. 2A). Holotype 1.8 cm long and 1.1 cm wide, paratype 2.2 cm long and 1.5 cm wide. Body consistency firm and fleshy, more delicate and thinner towards the margins. Tentacles lacking. Cerebral and marginal eyes present. Background pigmentation chocolate brown to caramel in the margins. Numerous dark spots scattered over the dorsal surface, more abundant along the main body axis (Fig. 2A). Ventral surface pale, with grey to beige tonalities. Epithelium, basal membrane and body musculature more developed on the dorsal than on the ventral side (Fig. 2B). Pharynx ruffled, well developed, extends throughout the mid-body region. Oral pore at the beginning of the posterior body-half.

Reproductive system: male and female reproductive organs are located directly after the pharynx, in the posterior half of the animal. Male copulatory apparatus consisting of a conical penis papilla (or peneal bulb) and a small pyriform prostatic vesicle, seminal vesicle absent (Fig. 2C, E). Vasa deferentia forms bulky spermiducal bulbs. The diameter of the bulbs decreases as they approach each other until they join forming the common vas deferens, which opens into the middle region of the ejaculatory duct. Both vasa deferentia and spermiducal bulbs follow a tortuous course. Free prostatic vesicle. Male atrium elongated and covered with glandular, spongy tissue.

Female gonopore posterior to the male gonopore (Fig. 2D, E, F). With vagina bulbosa. The vagina externa curves anteriorly to the male reproductive system, then upwardly and continues posteriorly into the vagina interna. The oviduct opens between the vagina externa and interna. Connected to the vagina interna is an elongated and tubular Lang’s vesicle (Fig. 2D, E). The general appearance of the female apparatus is compact with small visible folds, mainly in the vagina externa, and surrounded by abundant cement glands.

Fig. 2.

Fig. 2.

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Idiostylochus tortuosus gen. nov., sp. nov. A–B, Live specimen. A, dorsal view; B, ventral view. C–D, histological section through male (left) and female (right) copulatory organs. E, sagittal reconstruction of the reproductive system. F, sagittal reconstruction of whole animal (anterior end at the left). Scale bars = 500 μm.

Biology and occurrence

Idiostylochus tortuosus was found in oyster cultures of Magallana gigas. Individuals were collected living in the mantle cavity of diseased or dead oysters as well as swimming around the oyster farming devices. Some individuals have also been observed feeding on natural beds of Mytilus edulis Linnaeus, 1758 and Mytilus galloprovincialis Lamarck, 1819 near the oyster farms (Vieira and Nowaczyk pers. com.). Although the presence of Idiostylochus was known long ago by oyster farmers, in recent years, the frequency and number of polyclads specimens seem to have increased, and their presence has caused noticeable damage to oyster and mussel crops.

Taxonomical remarks

The superfamily Stylochoidea, where the new species Idiostylochus tortuosus was placed, presents a free prostatic vesicle (Faubel 1983). From the molecular point of view, the closest related genera (see -tree 28S, Fig. 4) are Idioplana Woodworth, 1898 (Idioplanidae), Leptostylochus Bock, 1925 (Stylochidae), and also, but less related, Mirostylochus Kato, 1937 (Stylochidae), Latocestus Plehn, 1896 and Eulatocestus Faubel, 1983 (Latocestidae). All these genera present a free prostatic vesicle and either developed spermiducal bulbs or an elongated seminal vesicle. The new species shares some characters with these genera, while others are clearly different. A comparative discussion follows.

Leptostylochus (Stylochidae) (Fig. 3A) is characterized by an elongated slender body shape; tentacular, cerebral, marginal and often frontal eyes; male copulatory apparatus with an unarmed penis papilla and without seminal vesicle, but with spermiducal bulbs that join into a common vas deferens before entering the medial region of the ejaculatory duct; female reproductive system with a developed Lang’s vesicle (Kato 1934; Faubel 1983; Beveridge 2017). Idiostylochus gen. nov. resembles Leptostylochus because of the presence of large spermiducal bulbs. Furthermore, the female system shows common features, like the well-developed shell glands around the vagina externa. In contrast, the Lang’s vesicle is conspicuous in Leptostylochus and reduced in Idiostylochus, which appears as a small tubular duct.

Latocestus and Eulatocestus (Latocestidae) share a similar morphology. Both genera have spermiducal bulbs, unarmed penis papilla and a simple female apparatus with Lang’s vesicle (Plehn 1896; Faubel 1983) (Fig. 3B). The main difference between Latocestus and Eulatocestus is the lining of the prostatic vesicle: irregular or fingered in Latocestus and a web of glandular follicles in Eulatocestus. Both genera are distinguishable from Idiostylochus by the morphology of the female apparatus. The presence of a well-developed Lang’s vesicle and a simple, non-bulbous vagina with much less abundant cement glands differentiates both genera from Idiostylochus.

Idioplana (Idioplanidae) is characterized by a male copulatory organ with an unarmed penis papilla, prostatic and seminal vesicle; female apparatus extended over the male apparatus; and an anchor-shaped Lang’s vesicle (Woodworth 1898; Meixner 1907; Faubel 1983; Rodríguez et al. 2021) (Fig. 3C).

Fig. 3.

Fig. 3.

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Comparison of the copulatory apparatuses of different genera of Stylochoidea. A, Leptostylochus Bock, 1925; B, Latocestus Plehn, 1896; C, Idioplana Woodworth 1898.

The analysis of the morphological data reveals external anatomical similarities, such as the oval body shape, the reddish-brown dorsal and whitish ventral coloration and the arrangement of the cerebral eyes, but both genera are clearly differentiated by the presence of conspicuous tentacles in Idioplana, absent in Idiostylochus, and the differences between the reproductive systems. In Idiostylochus the seminal vesicle is absent, the female canal is shorter, since the vagina interna do not extend anteriorly over the male copulatory organ, and the Lang’s vesicle is tubular. Within the male apparatus, there are clear differences. In Idioplana a seminal vesicle is present, while in Idiostylochus is absent. In Idiostylochus the function of the seminal vesicle is carried out by the spermiducal bulbs, since the latter replace the former in its absence.

Although the molecular analysis clusters Mirostylochus (Stylochidae) near Idiostylochus, the two genera are morphologically distinct. Mirostylochus is characterized by tentacular, cerebral and marginal eyes; and a female apparatus with ductus vaginalis, from which the vagina interna opens to the exterior behind the female gonopore. Lang’s vesicle is absent (Kato 1937; Tokinova 2003). Idiostylochus gen. nov. and Mirostylochus differ in the morphology of the female apparatus, as Idiostylochus possesses a tubular Lang’s vesicle and lacks ductus vaginalis.

Molecular approach

The molecular studies are based on two datasets of the genes 28S (nuclear) and COI (mitochondrial). The methods applied for the analyses were Maximum Likelihood (ML) and Bayesian Inference (BI).

Within the tree generated during the analysis of 28S (Fig. 4) we can distinguish three well-supported branches, the clusters of the superfamilies Discoceloidea, Leptoplanoidea and Stylochoidea respectively. The evidence provided by the 28S gene allows us to cluster the genera that are phylogenetically more related at the superfamily level within Acotylea. In our analysis, which is focused on determining the systematic position of the new species, Idiostylochus tortuosus, we found that the species is closely related to Idioplana atlantica and I. austaliensis, both of which belong to the Family Idioplanidae. On the other hand, the genera Mirostylochus and Leptostylochus do not cluster within the Family Stylochidae but with Latocestus and Eulatocestus, the selected representatives of the family Latocestidae in this study.

Fig. 4.

Fig. 4.

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Bayesian /Maximum likelihood tree based on partial sequences of the 28S gene (total length: 706 bp). Numbers in the nodes correspond to the posterior probability values of the BI analysis (> 0.70) and bootstrap support values of the ML analysis (> 50), respectively.

Other genera used in the 28S gene analysis are grouped in the same families as in previous molecular and morphological analyses carried out by previous authors (Dittmann et al. 2019; Litvaitis et al. 2019; Oya and Kajihara 2020).

In the COI analysis (Fig. 5) we have only taken into account values > 70 (ML), although we show values between < 70 and > 60 as they reflect a certain relationship among species or genera. Therefore, our analyses show strongly supported species within a genus, while low values at the family level are meaningless.

Fig. 5.

Fig. 5.

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Maximum likelihood /Bayesian Inference tree based on partial sequences of the COI gene (total length: 640 bp). Numbers in the nodes correspond to the bootstrap support values of the ML analysis (> 60) and the posterior probability values of the BI analysis (> 0.50), respectively.

DISCUSSION

Currently, the presence of Idiostylochus tortuosus sp. nov. has been confirmed in the oyster farms of Arcachon Bay (Atlantic basin, France) and is also likely to be in the Etang de Thau (Mediterranean basin, France) associated with oyster mortalities (Vieira et al. 2020).

Through the results obtained with the COI mitochondrial gene (Fig. 5) and 28S nuclear gene analysis (Fig. 4), three goals of this study have been achieved: the establishment of Idiostylochus tortuosus as a new species, the evidence of the close relation of Idiostylochus with species from the Pacific Ocean and the new combination of genera in some known families.

Therefore, together with the morphological evidence, Idiostylochus can be included with Idioplana in the Family Idioplanidae, diagnosed now as: Stylochoidea with or without tentacles; male copulatory organ with seminal vesicle or spermiducal bulbs. Unarmed penis; vasa deferentia or spermiducal bulbs unite before entering into the copulatory organ. Female copulatory organ developed with vagina bulbosa, abundant cement and shell glands and long vagina interna.

After COI analysis, Idiostylochus are closely related to Leptostylochus. However, in the 28S analysis, both genera appear separated into two different families. Idiostylochus was included in the family Idioplanidae, and Leptostylochus was included in the family Latocestidae, along with Mirostylochus and Latocestus (0.96) (Dittmann et al. 2019; Litvaitis et al. 2019; Oya and Kajihara 2020; Rodríguez et al. 2021).

Morphologically, Leptostylochus and Mirostylochus, clustered with Latocestus (Fig. 4), as they presented the external features of Latocestidae (absence of tentacles, cuneiform body, eyes scattered between frontal margin and brain). Within the anatomical characters, the free prostatic vesicle and the seminal receptacle developed as a false seminal vesicle or spermiducal bulb, but not as true seminal vesicle, are characteristic of the Latocestidae and not of the Stylochidae, the former family of Leptostylochus and Mirostylochus (Bock 1925; Kato 1937).

CONCLUSIONS

Idiostylochus tortuosus as well as other genera of polyclad flatworms like Stylochus, are predators of mollusks of economic interest and can be a serious threat to aquaculture, potentially expanding to where these species are being exploited (Danglade 1919; Galleni et al. 1980; Sluys et al. 2005; Gammoudi et al. 2017) (Fig 6). For this reason, it is recommended that control measures be implemented as soon as possible to reduce the impact on bivalve exploitation and prevent its possible expansion along the European Atlantic Coast. It is necessary to test the efficacy of nonspecific control methods such as hyper-and hyposaline baths (Espinosa 1981; O’Connor and Newman 2001), calcium hypochlorite solutions (Yang 1974), and the removal of fouling cover (Littlewood and Marsbe 1990). These control methods have already been used to combat other species of polyclad flatworms and could be effective in the control of this new species.

Fig. 6.

Fig. 6.

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Idiostylochus tortuosus photographed A–B, inside oysters (Bay d'Arcachon) and C, inside mussels (Biarritz).

Acknowledgments

This work and the new genus and the new species name were registered with ZooBank under urn:lsid:zoobank.org:pub:949287D5-3B25-4A4A-8182-D4424B9E352F. We would like to thank Gladys Fonteyraud from the regional shellfish farming committee (CRC Arcachon) for the sampling of specimens, Johann Vieira from the center for aquaculture, fisheries and environment of Nouvelle-Aquitaine (CAPENA) for the observations and Jeanne Bentejac for their work on this species during their license traineeship. The authors thank Annie Machordom and Iván Acevedo for access to the facilities of the Molecular Biology Lab (Museo Nacional de Ciencias Naturales, Madrid, Spain) and specially to Lourdes Alcaraz for her help and support during the investigations in this laboratory. We also thank the staff of the Histology Lab (Museo Nacional de Ciencias Naturales, Madrid, Spain). Last but not least, the authors are deeply thankful to Marta Novo of the Master’s Degree in Zoology of the Complutense University of Madrid and Almudena Puente for their help and support during all phases of the project. The authors declare that no funds or grants were received during the preparation of this manuscript. Only the infrastructure support of Ifremer and the National Museum of Natural Science (CSIC).

List of abbreviations

fp

female pore.

mp

male pore.

op

oral pore.

ovd

oviduct.

ph

pharynx.

spb

spermiducal bulbs.

shg

shell glands.

vd

vasa deferentia.

ve

vagina externa.

vi

vagina interna.

Lv

Lang’s vesicle.

pv

prostatic vesicle.

sv

seminal vesicle.

Footnotes

Authors’ contributions: All authors contributed to the study conception and design. Material preparation and data collection were performed by I. Auby, B. Gouillieux, G. Daffe, C. Massé, E. Antajan. Material processing and analysis were performed by A. Gutiérrez and C. Noreña. The first draft of the manuscript was written by A. Gutierrez and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Competing interests: The authors have no relevant financial or non-financial interests to disclose.

Availability of data and materials: The data and information used in this manuscript is available as follows: COI and 28S sequences have been submitted to NCBI-GenBank as reflected in table 1. Histologically processed individuals can be consulted in the collections of the National Museum of Natural Sciences (MNCN-CSIC) through the corresponding catalog number, as it appears in the “Material studied” in the description of the species. Other electronic information has not been generated for this manuscript.

Consent of publication: Not applicable.

Ethic approval consent to participate: Not applicable.

References

  1. Aguado MT, Grande C, Gerth M, Bleidorn C, Noreña C. 2016. Characterization of the complete mitochondrial genomes from Polycladida (Platyhelminthes) using next-generation sequencing. Gene 575(2):199–205. doi:10.1016/j.gene.2015.08.054. . [DOI] [PubMed]
  2. Akaike H. 1974. A new look at the statistical model identification. IEEE Trans Automat Contr 19(6):716–723. doi:10.1109/TAC. 1974.1100705.
  3. Bachelet G, Blanchet H, Cotte M, Dang C, de Montaudouin X, de Moura Queiros A, Gouillieux B. 2009. A round-the-world tour almost completed: first records of the invasive mussel Musculista senhousia in the North-east Atlantic (southern Bay of Biscay). Mar Biodivers Rec 2:1–4. doi:10.1017/S1755267209001080.
  4. Bahia J, Padula V, Schrödl M. 2017. Polycladida phylogeny and evolution: integrating evidence from 28S rDNA and morphology. ODE 17(1):653–678. doi:10.1007/s13127-017-0327-5.
  5. Barton JA, Bourne DG, Humphrey C, Hutson KS. 2020. Parasites and coral-associated invertebrates that impact coral health. Rev Aquac 12(4):2284–2303. doi:10.1111/raq.12434.
  6. Bell JR. 2008. A simple way to treat PCR products prior to sequencing using ExoSAP-IT®. BioTechniques 44(6):834. doi:10.2144/000112890. . [DOI] [PubMed]
  7. Beveridge I. 2017. A new species of acotylean polyclad, Leptostylochus victoriensis sp. nov. (Platyhelminthes: Polycladida) from Victoria. Proc R Soc Vic 129(2):31–38. doi:10.1071/RS17006.
  8. Bock S. 1925. Papers from Dr. Th. Mortensen’s Pacific Expedition 1914–1916. XXV. Planarians. Parts I-III. Vidensk Medr dansk naturh Foren 79:1–84.
  9. Bouchet JM, Deltreil JP, Manaud F, Maurer D, Trut G. 1997. Etude intégrée du bassin d’Arcachon. Tome 5. Pêche et Ostréiculture. IFREMER, Brest, France.
  10. Buestel D, Ropert M, Prou J, Goulletquer P. 2009. History, status, and future of oyster culture in France. J Shellfish Res 28(4):813–820. doi:10.2983/035.028.0410.
  11. Chen H, Rangasamy M, Tan SY, Wang H, Siegfried BD. 2010. Evaluation of five methods for total DNA extraction from western corn rootworm beetles. PLoS ONE 5:e11963. doi:10.1371/journal.pone.0011963. . [DOI] [PMC free article] [PubMed]
  12. Cuadrado D, Rodríguez J, Moro L, Grande C, Noreña C. 2021. Polycladida (Platyhelminthes, Rhabditophora) from Cape Verde and related regions of Macaronesia. Eur J Taxon 736:1–43. doi:10.5852/ejt.2021.736.1249.
  13. Danglade E. 1919. The flatworm as an enemy of Florida oysters. Report of the U.S. Commissioner of Fisheries for 1918 Appendix 5.
  14. Dittmann IL, Cuadrado D, Aguado MT, Noreña C, Egger B. 2019. Polyclad phylogeny persists to be problematic. ODE 19:585–608. doi:10.1007/s13127-019-00415-1.
  15. Espinosa J. 1981. Stylochus megalops (Platyhelminthes: Turbellaria), nuevo depredador del ostión en Cuba. Poeyana 228:1–5.
  16. FAO. 2021. Fisheries and aquaculture statistics. World aquaculture production 1950–2019 (FishstatJ). In: FAO Fisheries Division (online). Rome. Available at: www.fao.org/fishery/statistics/software/fishstatj/en. Accessed 12 Aug. 2021.
  17. FAO. 2005–2021. Cultured Aquatic Species Information Programme. Crassostrea gigas. In: FAO Fisheries and Aquaculture Division (online). Rome. Available at: http://www.fao.org/fishery/culturedspecies/Crassostrea_gigas/en. Accessed 12 Aug. 2021.
  18. Faubel A. 1983. The Polycladida, Turbellaria. Proposal and establishment of a new system. Part I. The Acotylea. Mitt Hamb Zool Mus Inst 80:17–121.
  19. Galleni L, Tongiorgi P, Ferrero E, Salghetti U. 1980. Stylochus mediterraneus (Turbellaria: Polycladida), predator on the mussel Mytilus galloprovincialis. Mar Biol 55:317–326. doi:10.1007/BF00393784.
  20. Gammoudi M, Ben Ahmed R, Bouriga N, Ben-Attia M, Harrath AH. 2017. Predation by the polyclad flatworm Imogine mediterranea on the cultivated mussel Mytilus galloprovincialis in Bizerta Lagoon (northern Tunisia). Aquac Res 48(4):1608–1617. doi:10.1111/are.12995.
  21. Gouillieux B, Massé C. 2019. First record of Monocorophium uenoi (Stephenson, 1932) (Crustacea: Amphipoda: Corophiidae) in the Bay of Biscay, French Atlantic coast. BioInvasions Rec 8(1):87–95. doi:10.3391/bir.2019.8.1.09.
  22. Grizel H, Héral M. 1991. Introduction into France of the Japanese oyster (Crassostrea gigas). ICES J Mar Sci 47(3):399–403. doi:10.1093/icesjms/47.3.399.
  23. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98. doi:10.14601/Phytopathol_Mediterr-14998u1.29.
  24. Heasman MP, O’Connor WA, O’Connor SJ, Walker WW. 1998. Enhancement and farming of scallops in NSW using hatchery produced seedstock. NSW Dep Prim Ind Fish Final Rep Ser No 8.
  25. Hyman LH. 1951. The invertebrates. Volume 2, Platyhelminthes and Rhynchocoela: the acoelomate bilateria. McGraw-Hill, New York, United States.
  26. Jennings JB. 1957. Studies on feeding, digestion, and food storage in free-living flatworms (Platyhelminthes: Turbellaria). Biol Bull 112(1):63–80. doi:10.2307/1538879.
  27. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. 2017. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat Methods 14:587–589. doi:10.1038/nmeth.4285. . [DOI] [PMC free article] [PubMed]
  28. Kato K. 1934. Leptostylochus gracilis, a new polyclad turbellarian. ProcImp Acad 10:374–377. doi:10.2183/pjab1912.10.374.
  29. Kato K. 1937. The fauna of Akkeshi Bay, V. Polycladida. Annot Zool Jpn 16:124–133.
  30. Katoh K, Rozewicki J, Yamada KD. 2018. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 20(4):1160–1166. doi:10.1093/bib/bbx108. . [DOI] [PMC free article] [PubMed]
  31. Kenny NJ, Noreña C, Damborenea C, Grande C. 2019. Probing recalcitrant problems in polyclad evolution and systematics with novel mitochondrial genome resources. Genomics 111(3):343–355. doi:10.1016/j.ygeno.2018.02.009. [DOI] [PubMed]
  32. Lang A. 1884. Die Polycladen (Seeplanarien) des Golfes von Neapel und der angrenzenden meeres-abschnitte. Fauna und Flora des Golfes von Neapel. Engelmann W, Leipzig, Germany.
  33. Laumer E, Giribet G. 2014. Inclusive taxon sampling suggests a single, stepwise origin of ectolecithality in Platyhelminthes. Biol JLinn Soc 111(3):570–588. doi:10.1111/bij.12236.
  34. Lavesque N, Hutchings P, Abe H, Daffe G, Gunton LM, Glasby CJ. 2020. Confirmation of the exotic status of Marphysa victori Lavesque, Daffe, Bonifácio & Hutchings, 2017 (Annelida) in French waters and synonymy of Marphysa bulla Liu, Hutchings & Kupriyanova, 2018. Aquat Invasions 15(3):355–366. doi:10.3391/ai.2020.15.3.01.
  35. Lavesque N, Sorbe JC, Bachelet G, Gouillieux B, de Montaudouin X, Bonifacio P, Blanchet H, Dubois S. 2013. Recent discovery of Paranthura japonica Richardson, 1909 (Crustacea: Isopoda: Paranthuridae) in European marine waters (Arcachon Bay, Bay of Biscay). BioInvasions Rec 2(3):215–219. doi:10.3391/bir.2013. 2.3.07.
  36. Lee KM. 2006. Taxonomy and ecology of predatory marine flatworms (Platyhelminthes: Polycladida) in Botany Bay, New South Wales, Australia. PhD dissertation, University of New South Wales, Australia.
  37. Letunic I, Bork P. 2021. Interactive tree of life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res 49(1):293–296. doi:10.1093/nar/gkab301. . [DOI] [PMC free article] [PubMed]
  38. Littlewood DTJ, Marsbe LA. 1990. Predation on cultivated oysters, Crassostrea rhizophorae (Guilding), by the polyclad turbellarian flatworm, Stylochus (Stylochus) frontalis Verrill. Aquaculture 88:145–150. doi:10.1016/0044-8486(90)90289-Y.
  39. Litvaitis MK, Bolaños DM, Quiroga SY. 2019. Systematic congruence in Polycladida (Platyhelminthes, Rhabditophora): Are DNA and morphology telling the same story? Zool J Linn Soc 186(4):865–891. doi:10.1093/zoolinnean/zlz007.
  40. Lockyer AE, Olson PD, Littlewood DTJ. 2003. Utility of complete large and small subunit rRNA genes in resolving the phylogeny of the Neodermata (Platyhelminthes): Implications and a review of the cercomer theory. Biol J Linn Soc 78(2):155–171. doi:10.1046/j.1095-8312.2003.00141.x.
  41. Mallatt J, Winchell CJ. 2002. Testing the new animal phylogeny: First use of combined large-subunit and small-subunit rRNA gene sequences to classify the protostomes. Mol Biol Evol 19(3):289–301. doi:10.1093/oxfordjournals.molbev.a004082. . [DOI] [PubMed]
  42. Meixner A. 1907. Polycladen von der Somaliküste, nebst einer Revision der Stylochinen. Z wiss Zool 88:385–498.
  43. Newman LJ, Cannon LRG. 2003. Marine flatworms: the world of polyclads. CSIRO Publishing, Melbourne, Australia.
  44. Newman LJ, Cannon LRG, Govan H. 1993. Stylochus (Imogene) matatasi n. sp. (Platyhelminthes, Polycladida): pest of cultured giant clams and pearl oysters from Solomon Islands. Hydrobiologia 257(3):185–189. doi:10.1007/BF00765011.
  45. O’Connor WA, Newman LJ. 2001. Halotolerance of the oyster predator, Imogine mcgrathi, a stylochid flatworm from Port Stephens, New South Wales, Australia. Hydrobiologia 459(1):157–163. doi:10.1023/A:1012525015850.
  46. Oya Y, Kajihara H. 2017. Description of a new Notocomplana species (Platyhelminthes: Acotylea), new combination and new records of Polycladida from the northeastern Sea of Japan, with a comparison of two different barcoding markers. Zootaxa 4282(3):526–542. doi:10.11646/zootaxa.4282.3.6.
  47. Oya Y, Kajihara H. 2018. A new bathyal species of Cestoplana (Polycladida: Cotylea) from the West Pacific Ocean. Mar Biodivers 49:905–911. doi:10.1007/s12526-018-0875-8.
  48. Oya Y, Kajihara H. 2020. Molecular Phylogenetic Analysis of Acotylea (Platyhelminthes: Polycladida). Zool Sci 37(3):271–279. doi:10.2108/zs190136. . [DOI] [PubMed]
  49. Oya Y, Kajihara H. 2021. Description and phylogenetic relationships of a new genus of Planoceridae (Polycladida, Acotylea) from Shimoda, Japan. J Mar Biolog AssocUK 101(1):81–88. doi:10.1017/S0025315421000060.
  50. Oya Y, Kimura T, Kajihara H. 2019. Description of a new species of Paraplehnia (Polycladida, Stylochoidea) from Japan, with inference on the phylogenetic position of plehniidae. ZooKeys 864:1–13. doi:10.3897/zookeys.864.33955. . [DOI] [PMC free article] [PubMed]
  51. Oya Y, Tsuyuki A, Kajihara H. 2020. A new species of Zygantroides (Platyhelminthes: Polycladida) from Amakusa, Japan. Species Divers 25(2):189–196. doi:10.12782/specdiv.25.189.
  52. Pearse AS, Wharton GW. 1938. The oyster ‘leech,’ Stylochus inimicus Palombi, associated with oysters on the coasts of Florida. Ecol Monogr 8(4):605–656. doi:10.2307/1943085.
  53. Plehn M. 1896. Neue Polycladen, gesammelt von Herrn Kapitän Chierchia bei der Erdumschiffung der Korvette Vettor Pisani, von Herrn Prof. Dr. Kükenthal im nördlichem Eismeer und von Herrn Prof Dr. Semon in Java. Jena Zeitschr 30:137–176.
  54. Rodríguez J, Hutchings PA, Williamson JE. 2021. Biodiversity of intertidal marine flatworms (Polycladida, Platyhelminthes) in southeastern Australia. Zootaxa 5024(1):1–63. doi:10.11646/ZOOTAXA.5024.1.1. . [DOI] [PubMed]
  55. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. Mrbayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3):539–542. doi:10.1093/sysbio/sys029. . [DOI] [PMC free article] [PubMed]
  56. Salvitti L, Wood SA, Taylor DI, McNabb P, Cary SC. 2015. First identification of tetrodotoxin (TTX) in the flatworm Stylochoplana sp.; a source of TTX for the sea slug Pleurobranchaea maculata. Toxicon 95:23–29. doi:10.1016/j.toxicon.2014.12.006. . [DOI] [PubMed]
  57. Sluys R, Faubel A, Rajagopal S, van der Velde G. 2005. A new and alien species of ‘oyster leech’ (Platyhelminthes, Polycladida, Stylochidae) from the brackish North Sea Canal, The Netherlands. Helgol Mar Res 59:310–314. doi:10.1007/s10152-005-0006-3.
  58. Talavera G, Castresana J. 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56(4):564–577. doi:10.1080/10635150701472164. . [DOI] [PubMed]
  59. Teng CJ, Su YJ, Yeh CY, Jie WB. 2022. Predation of oysters using an autonomic pharynx in the oyster leech Cryptostylochus sp. (Polycladida: Stylochidae). Zool Stud 61:7. doi:10.6620/ZS.2022.61-07. . [DOI] [PMC free article] [PubMed]
  60. Tokinova R. 2003. Two new species of the genus Mirostylochus (Turbellaria, Polycladida, Stylochidae) from the Russian far-eastern seas. Zool Zhurnal 82(8):1010–1016.
  61. Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ. 2016. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res 44(1):232–235. doi:10.1093/nar/gkw256. . [DOI] [PMC free article] [PubMed]
  62. Tsunashima T, Hagiya M, Yamada R, Koito T, Tsuyuki N, Izawa S, Kosoba K, Itoi S, Sugita H. 2017. A molecular framework for the taxonomy and systematics of Japanese marine turbellarian flatworms (Platyhelminthes, Polycladida). Aquat Biol 26:159–167. doi:10.3354/ab00682.
  63. Tsuyuki A, Oya Y, Kajihara H. 2020. Description of Pericelis flavomarginata sp. nov. (Polycladida: Cotylea) and its predatory behavior on a scaleworm. Zootaxa 4894(3):403–412. doi:10.11646/zootaxa.4894.3.6. . [DOI] [PubMed]
  64. Vieira J, Bourgés A, Béchade M, Barbier P. 2020. Observatoire ostréicole du Bassin d’ Arcachon. Rapport annuel 2020. CREAA, Gujan-Mestras, France.
  65. Woodworth W. 1898. Some planarians from the Great Barrier Reef of Australia. Bull Mus Comp Zool 32(4):63–67.
  66. Yang HC. 1974. On the extermination of polyclads: calcium hypochlorite treatment in the period of high water temperature. Korean J Fish Aquat Sc 7:121–125.

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