Polydorid species (Annelida: Spionidae) associated with commercially important oyster shells and their shell infestation along the coast of Normandy, in the English Channel, France

Polydorid species (Annelida, Spionidae), which inhabit the shells of the commercially important oyster Crassostrea gigas, were investigated along the coast of Normandy, France. Eight species, including five new records for Normandy (Polydora onagawaensis, Polydora websteri, Boccardia pseudonatrix, Boccardia proboscidea, and Boccardiella hamata) and two first records in European waters (P. onagawaensis and B. pseudonatrix), were identified based on morphological, molecular biological, and ecological characteristics. Polydora onagawaensis, which belongs to the Polydora ciliata/websteri complex, was discovered in the shells of wild and suspended cultured oysters, as well as in limestone substrates. In the phylogenetic analysis of mitochondrial COI gene sequences, specimens of P. onagawaensis collected from Normandy were grouped together with specimens from the USA into a single clade and were distinguished from the other three lineages that comprised Japanese and USA specimens. Polydora websteri inhabited shells of suspended cultured oysters. Polydora hoplura, Dipolydora giardi, and Dipolydora sp. were observed in shells from the sandy oyster culture grounds. Boccardiella hamata has been found in wild oyster shells from muddy oyster culture grounds. Boccardia pseudonatrix was observed in the shells of both the wild and cultured oysters. Adult and juvenile Boccardia proboscidea were observed in coralline algae, as well as in suspended cultured oysters. Mud tubes were observed to protrude from the outer surface of the shells, and abnormal black and calcareous deposits were secreted on the inner surface of the shells against polydorid penetration.


Introduction
The family Spionidae Grube, 1850, is found in a wide variety of marine environments worldwide. It is one of the most abundant polychaete groups in Annelida in terms of the number of species and biomass. Within the family Spionidae, the so-called polydorid species comprises nine genera: Polydora Bosc, 1802;Dipolydora Verrill, 1881;Pseudopolydora Czerniavsky, 1881;Boccardia Carazzi, 1893;Polydorella Augener, 1914;Tripolydora Woodwick, 1964;Boccardiella Blake & Kudenov, 1978;Carazziella Blake & Kudenov, 1978;and Amphipolydora Blake, 1983; each of them has the common morphological characteristics of specially modified major spines in the fifth chaetigerous segment (Blake 1996). They are widely distributed in coastal benthic environment. A variety of habitat types are observed depending on the species, that is, some species are known to create mud tubes in the bottom sediment, utilize sand or mud deposits in crevices of various substrates (e.g., rocks and mollusk shells), to inhabit the surface of various organisms (e.g., sponges, brachiopods, and mollusk shells), inside of various organisms (e.g., calcareous algae and sponges), and some excavate burrows in calcareous substrates, such as limestone, mollusk shells, and corals (Blake and Evans 1973;Simon and Sato-Okoshi 2015;Abe et al. 2019a;Abe and Sato-Okoshi 2020). Many have been reported to have symbiotic associations with other marine benthic invertebrates (Martin and Britayev 1998;, and new symbiotic relationships continue to be discovered Abe et al. 2022). Polydorids feed on suspended organic particles or deposited organic matter by exposing a pair of long palps from mud tubes (Dauer et al. 1981). In cases where symbiotic relationships have been observed, it is assumed that they compete with their hosts for food .
Some genera in polydorids have long been well known and reported, especially in fisheries (Takahashi 1937;Evans 1969;Kent 1979;Okoshi and Sato-Okoshi 1996;Mortensen et al. 2000;Lleonart et al. 2003;Simon et al. 2006;Sato-Okoshi et al. 2008). For species inhabiting the shells of commercially important mollusk species, whether they excavate the shells (boring) or just inhabit the shells as interstitial or epifaunal (non-boring), a significant infestation can negatively affect the host shells. They often reduce the commercial value of mollusks by damaging their shells, decreasing the growth rate and meat yield, and causing heavy mortality in some cases (Okoshi and Sato-Okoshi 1996;Sato-Okoshi et al. 2008;2013;Simon and Sato-Okoshi 2015). With the globalization of the aquaculture industry, commercially important mollusks are constantly transported between countries. Some mollusk-associated organisms, such as boring and non-boring polydorid species, are unintentionally introduced to newer, non-native regions. (Elton 1958;Carlton 1975;Bailey-Brock 2000;Sato-Okoshi et al. 2008;Simon and Sato-Okoshi 2015). Therefore, polydorids have established invasive populations in the waters where they have been introduced (Sato- . For species that cause damage to cultured shells, it is necessary to determine the species accurately and suggest preventive measures or control such infestations (Diggles et al. 2002;Simon et al. 2010), but this problem often remains unresolved. Moreover, these unintentionally introduced species are not only a source of economic concern but may also pose a threat ecologically as they can infest indigenous mollusk species in their invasive ranges if they disperse from aquaculture facilities (Elton 1958;Carlton 1975;Cohen and Carlton 1998;Bailey-Brock 2000). Ecological disturbances caused by invasive organisms associated with cultured mollusks, such as oysters, are currently a major cause of diversity losses worldwide (Mack et al. 2000;Miura 2007).
The similarity of morphological characteristics in polydorids has led to confusion in species identification (Sato-Okoshi 1999;Sato-Okoshi and Takatsuka 2001;Radashevsky and Pankova 2006;2013;Read 2010;Sato-Okoshi and Abe 2012;2013). Several species complexes within the polydorid group are morphologically indistinguishable, whereas others show high intraspecific morphological variations. Some species previously thought to be the same include more than one species (Abe et al. 2016;Kondoh et al. 2017;Simon et al. 2019), and cases have been reported in which two species thought to be different were the same (Sato- Malan et al. 2020). Thus, the identification of these species, based on morphology, is often difficult and complex. Accurate species identification is necessary to elucidate the means by which they are transported and to assess the severity of their impact as exotic invasive species.
The Japanese or Pacific oyster Crassostrea gigas (Thunberg, 1793) was introduced in European countries owing to the decline in native populations of the European flat oyster Ostrea edulis Linnaeus, 1758 (Robert et al. 1991) and the Portuguese oyster Crassostrea angulata (Lamarck, 1819) (Grizel and Héral 1991). In France, oyster production is thriving, and oysters have been cultured for many years. Crassostrea gigas oysters have been exported from Japan since 1880. After the disease of C. angulata (1969)(1970)(1971), large numbers of juvenile (spat) and adult C. gigas were introduced into France at the beginning of 1970, coming directly from Japan and also from British Colombia and Canada (Grizel and Héral 1991). Many polydorid species probably accompanied such voluntary introduction and have since been reported in cultured shells (Ruellet 2004;Royer et al. 2006). Polydorids are not the only annelids to be transported with cultured mollusks. Recently, Marphysa victori (Eunicidae) was reported to have accompanied oysters to France (Abe et al. 2019b;Lavesque et al. 2020).
Successive inventories have reported polydorids in the English Channel (Fauvel 1927;Dauvin et al. 2003;Ruellet 2004;Gully and Cochu 2020;Le Mao et al. 2020). In recent years, it has become necessary to distinguish the species of morphologically similar polydorids, especially the so-called Polydora ciliata/websteri complex (Blake 1996); however, this has not yet been sufficiently re-examined in France. Therefore, species identification using molecular biological methods as well as morphology is required in this region. In this study, we attempted to identify the polydorid species inhabiting commercially important oyster shells along the coast of Normandy using both morphology and molecular biology. Shell infestation by polydorids was investigated not only in oyster shells but also in other calcareous substrates from the Normandy intertidal zone in March 2018. Here, we report the species and their habitat conditions of polydorids 50 years after the mass introduction of Pacific oysters into France, which began in the 1970s.

Sampling of calcareous substrates and polydorid species
Sampling was conducted along the coast of Normandy in the English Channel from March 19 to 21, 2018 ( Fig. 1). Crassostrea gigas oysters were collected from four oyster culture sites: Blainville-sur-Mer (sandy oyster culture ground dotted with rocks together) from the western coast of Cotentin, Saint-Vaast-La-Hougue (muddy oyster culture ground), Quineville (suspended culture) from the eastern coast of Cotentin, and Asnelles (suspended culture) along the Calvados coast. In Normandy, which is currently the most important center of oyster production in France, there has been a rapid increase in the number of oyster farms just after the introduction of the Japanese oyster C. gigas, in two main places of the Cotentin Peninsula: Blainville-sur-Mer on the western coast and Saint-Vaast-La-Hougue on the eastern coast. Blainville-sur-Mer has an extended intertidal zone of up to 5 km at low tide during spring tide. Here, the oyster farms were located between 1 and 3 km from the seashore, and the tables were placed on heterogeneous sediment from sand to gravel. Saint-Vaast-La-Hougue is another important hot spot for oyster production in Normandy, with a moderate intertidal zone of up to 2 km, on muddy and sandy sediments, and the tables occupy the entire intertidal zone. Twenty cultured oysters that fell out of the net bags and 20 wild oysters that were attached to rocks in Blainville-sur-Mer were randomly collected during the March spring tide. Twenty wild oysters from muddy oyster beds in Saint-Vaast-La-Hougue were randomly collected at sites during the March spring tide. Twenty cultured oysters each, from Quineville and Asnelles, were obtained randomly from harvested ones cultured in cages suspended in the intertidal zone. Additional samples were 20 harvested scallops Pecten maximus collected by dredging near Asnelles. Calcareous algae and clumps of limestone and concrete blocks covered with barnacles and mussels were also sampled from intertidal zones in Barfleur Cape, northeast Cotentin, and Luc-sur-Mer (Calvados), respectively, to identify polydorid species that inhabit wild calcareous substrates.
Polydorid species that bore into or inhabit the shells, limestone, and coralline algae were extracted by fracturing calcareous substrates with cutting pliers. Five to thirty individuals of each species of extracted worms were examined to determine their morphological characteristics, state of sexual maturity, and presence and conditions of oocytes and egg capsules under a stereomicroscope (SZX16, Olympus, Tokyo, Japan) and a biological microscope (BX53F, Olympus, Tokyo, Japan), in live individuals and specimens fixed in 10% Fig. 1 Map of sampling sites along the coast of Normandy, northern France, in the English Channel formalin in seawater. For molecular analysis, one-eight worms per species were directly preserved in 99.5% ethanol.
Shell infestation was investigated by observing outer and inner shell surfaces and taking photographs using a digital camera (Tough TG-5, Olympus, Tokyo, Japan). Abnormal shell formation, along with the presence of black organic or calcareous deposits, indicates worm infestation. Worms, if present, were extracted from such shells and their specieslevel identification was determined. The number of shells with the abnormal formation due to polydorid invasion was examined for each species and valued as the occurrence rate of abnormal shell formation.
Specimens were deposited at the French National Natural History Museum, Paris, France, and the National Museum of Nature and Science, Tsukuba, Japan.

SEM observation
Six specimens of Polydora onagawaensis Teramoto et al., 2013 were used for scanning electron microscopy (SEM) (Hitachi SU8000, Hitachi, Japan). Each fixed specimen in 99.5% ethanol was freeze-dried in t-butyl alcohol, coated with platinum palladium, and viewed using an SEM equipped with a digital camera.
Gene sequences obtained in the present study were aligned using the MAFFT online service ver. 7 using the L-INS-i algorithm (Katoh et al. 2017) to reconstruct the molecular phylogeny with the sequences of other Polydora, Dipolydora, Boccardiella, and Boccardia species available in the DDBJ/ENA/GenBank databases (Table 1). For Dipolydora bidentata (Zachs, 1933 Williams et al. (2017) phylogenetic analyses of the 18S, 28S, and 16S rRNA gene regions, the gene sequences of Pseudopolydora paucibranchiata (Okuda, 1937), obtained from DDBJ/ENA/Gen-Bank, were used as outgroup taxa, according to Abe and Sato-Okoshi (2021). Phylogenetic analysis of the COI gene was performed to investigate the geographical genetic variation in P. onagawaensis, and the P. websteri sequence obtained in the present study was used as the outgroup. Ambiguously aligned regions were eliminated using Gblocks ver. 0.91b (Castresana 2000) with less stringent settings. The final lengths of the alignments were 1769 (18S), 766 (28S), 471 (16S), and 656 (COI) bp for the multiple sequence alignment. Two phylogenetic trees were constructed based on the concatenated sequences of 18S, 28S, and 16S, and the sequences of the COI gene region by maximum likelihood (ML) analyses performed using IQ-TREE (Nguyen et al. 2015) implemented in PhyloSuite v.1.2.2 (Zhang et al. 2020) under an edge-linked partition model. The TIM2e + I + G4, TNe + R2, GTR + F + I + G4, and TPM + F + G4 models were selected for the 18S, 28S, 16S rRNA, and COI gene regions, respectively, as the best substitution model by ModelFinder (Kalyaanamoorthy et al. 2017) as implemented in IQ-TREE under the Bayesian information criterion. The robustness of the ML trees was evaluated using the Shimodaira-Hasegawa-like approximate likelihood-ratio test (SH-aLRT) with 5000 replicates (Guindon et al. 2010), approximate Bayes (aBayes) test (Anisimova et al. 2011), and ultrafast bootstraps (UFBoot) with 5000 replicates (Hoang et al. 2018). SH-aLRT ≥ 80%, aBayes ≥ 0.95, and UFBoot ≥ 95% were defined as robust statistical supports.

Identification of polydorid species
Based on morphology and gene sequence results, a total of eight species were identified: three Polydora, P. onagawaensis, P. hoplura, and P. websteri; two Dipolydora, D. giardi, and Dipolydora sp.; two Boccardia, B. proboscidea, and B. pseudonatrix Day, 1961; and one Boccardiella, B. hamata. The sampling site, host species or associated substrate, culture or wild condition, habitat type, and condition of shell infestation and other calcareous substrates by each species are summarized in Table 3. Molecular phylogenetic analysis of concatenated sequences of nuclear 18S and 28S and mitochondrial 16S rRNA gene sequences of polydorid species obtained in the present study and from the DDBJ/EMBL/GenBank database supported the morphological identification results. The species identified in the present study were genetically close to the same species previously reported from other localities (Fig. 2).
Molecular phylogenetic analyses based on the mitochondrial COI gene sequences obtained in the present and previous studies showed that the individuals identified as P. onagawaensis were divided into four lineages (Fig. 3). One lineage consisted of individuals from Japan and China, two lineages consisted of individuals from the USA and Japan, and the remaining lineage consisted of individuals from the USA and France. The Japanese and USA specimens each contained three lineages, while the French specimens were of a single lineage.

Morphological characteristics of the specimens collected from Normandy
Polydora onagawaensis Teramoto et al., 2013 (Fig. 4) Adult morphology. Thirty individuals of Polydora onagawaensis were examined. After fixation, the maximum body length (95 chaetigers) was 15 mm, and the maximum width was 680 µm (at the 5th chaetiger). The body coloration of the live specimen was light tan. Body pigmentation in live and preserved specimens was weak; weak black pigmentation was present on and along the caruncle. Occasionally, intense black pigmentation was present on peristomium around the base of both palps (Fig. 4A-D). Palp pigmentation was either entirely absent or a faint black (Fig. 4D). Black pigment was occasionally present on the dorsal and lateral sides of the posterior chaetigers (Fig. 4E). The pygidium had no pigmentation or black pigmentation along the edge or on the side of the disc-like pygidium (Fig. 4E, F). The peristomium is wide and short. The prostomium was weakly incised on the anterior margin. The eyes were absent or up to four black eyes and were arranged in a trapezoid, with the anterior pair more widely spaced than the posterior pair (Fig. 4C). The caruncle extended posteriorly to the end of chaetigers 1 to 4, unrelated to body size, without a median antenna (Fig. 4G).
Chaetiger 1 had noto-and neuropodia; notochaetae were absent, and short capillary neurochaetae were present. Parapodial lobes and postchaetal lamellae were well-developed on anterior chaetigers, except chaetiger 5, gradually reducing on posterior chaetigers. Neuro-and notochaetae on chaetiger 2-4 and 6 were winged capillaries. The number of capillaries per fascicle and wings in the notopodia of succeeding chaetigers diminished gradually. Hooded hooks on neuropodia began from chaetiger 7 and were not accompanied by capillaries. Hooks were bidentate, with the main fang at a right angle to the shaft and an acute angle to the apical tooth. The shaft was slightly curved, with a constriction in the upper part. The size and number of hooks gradually diminished from anterior to posterior. Approximately six hooks were present in a series.
The modified chaetiger 5 was longer than adjacent chaetigers 4 and 6 ( Fig. 4G) and possessed curved horizontal rows of up to six major spines alternating with pennoned companion chaetae. Both dorsal and ventral tufts of several short and winged capillaries were present. The major spines were falcate with lateral flanges or sheaths, and unworn spines were tooth-like (Fig. 4H).
Pygidium was white and disc-shaped, with a dorsal gap (Fig. 4E, F). Gizzard-like structures were absent in the digestive tract. Glandular pouches began with chaetiger 7.
Juvenile morphology. Twenty chaetigers juveniles were examined (Fig. 4I, J). The prostomium was rounded anteriorly. Conspicuous black pigmentation occurred on peristomium around the base of the palps. Dorsal pigmentation consisted of two rows of melanophores from chaetiger 2 with those of the first six band-shaped pairs, which were then replaced by ramified melanophores in posterior chaetigers. Lateral pigments were found in chaetigers 1-3. Dorsolateral pigments at the base of the parapodia started from chaetiger 7. A pair of black pigments appeared on the pygidium. The ventral pigment was absent. Modified chaetae developed in chaetiger 5.
Reproduction. Only a few large individuals had oocytes in their coeloms. Egg capsules were absent; however, numerous juveniles with approximately 20 chaetigers, representing characteristic larval pigmentation on the dorsal side, were observed in early March (Fig. 4I).
Remarks. Polydora onagawaensis resembles P. websteri and P. hoplura in that all three possess similar chaetal morphology, arrangement, and body pigmentation variation. However, P. onagawaensis can be distinguished from P. websteri by its small maximum size, few consecutive branchial chaetigers, wide and short peristomium, pygidium disc-like morphology rather than flared, and more pigment variations rather than only black pigment along the edge of the palps. Moreover, although body pigmentation resembles each other, P. onagawaensis can be distinguished from P. hoplura by its smaller body size, both in length and width, smaller pygidium, absence of a median  (Table 1). The gene sequences obtained in this study are highlighted in bold. SH-aLRT/approximate Bayes support/ultrafast bootstrap support values of ≥ 80%/ ≥ 0.95/ ≥ 95% are shown beside the respective nodes in red. Nodes with red circles indicate triple high support values of SH-aLRT ≥ 80, approximate Bayes support ≥ 0.95, and ultrafast bootstrap support ≥ 95. Scale bar represents the number of substitutions per site. The sequence of Pseudopolydora paucibranchiata was used for outgroup rooting antenna, and absence of posterior hooks in notopodia. The morphology of specimens from Normandy can be slightly distinguished from those from Japan (Teramoto et al. 2013), where the species was originally described by occasional intense black pigmentation around the base of both palps.  Twenty individuals of P. websteri were examined in this study. The maximum recorded body length after fixation was 18 mm. Polydora websteri from Normandy showed similar morphological characteristics to previous descriptions worldwide (Read 2010;Rodewald et al. 2021). Palps had weak-to-clear black pigmentation along the margin (Fig. 5A). Branchiae began from chaetiger 7, and were fairly long throughout, continuing almost till the posterior end. The pygidium was flared outward (Fig. 5B).

Polydora hoplura Claparède, 1868
Twenty individuals of P. hoplura were examined in this study. The maximum recorded body length after fixation was 35 mm Morphological characteristics of P. hoplura from Normandy were similar to those previously described worldwide (Sato-Okoshi and Abe 2013; Walker 2013; Radashevsky et al. 2017;Sato-Okoshi et al. 2017). Black pigmentation on the palps, prostomium, and peristomium was feebly visible or entirely absent. A median antenna was present. Special recurved hooks were present on the posterior notopodia. (Mesnil, 1893) We examined 20 individuals of Dipolydora giardi in this study. The maximum recorded body length after fixation was 5 mm. The morphological characteristics of D. giardi from Normandy were similar to those previously described in Asia (Sato-Okoshi 1999; and Australia (Sato-Okoshi et al. 2008;Walker 2013). Pigmentation was entirely absent on the body. Notochaetae were present on chaetiger 1, branchiae began from chaetiger 9, and capillaries were accompanied by hooded hooks.
The following description of Dipolydora sp. is based on an incomplete specimen. Branchiae began at chaetiger 9. While detailed morphological characteristics were unavailable, genetic data suggested that this species was different from D. giardi. Day, 1961 (Fig. 5C, D) We examined five individuals of Boccardia pseudonatrix in this study. The maximum recorded body length after fixation was 18 mm. The morphological characteristics of B. pseudonatrix from Normandy matched its previous descriptions from South Africa (Simon et al. 2010) and Australia (Sato-Okoshi et al. 2008, as B. knoxi: see Sato-Okoshi et al. 2015Walker 2013). Palps were transparent with irregular colorless spots crossing transversely, giving the appearance of being crossed by white bars in the lateral view. Conspicuous black pigmentation was observed in the prostomium (Fig. 5C, D). A mid-dorsal ridge appeared from chaetiger 5 to the middle of chaetiger 8 (Fig. 5D). Hartman, 1940 Twenty individuals of Boccardia proboscidea were examined in this study. The maximum recorded body length after fixation was 18 mm. The morphological characteristics of B. proboscidea from Normandy matched well with previous descriptions from Japan (Sato-Okoshi 2000), Australia (Blake and Kudenov 1978;Sato-Okoshi et al. 2008;Walker 2013), South Africa (Simon et al. 2006), USA (Bailey-Brock 2000, and Canada (Sato- Okoshi and Okoshi 1997). Black pigmentation was present on both sides of the caruncle. Their body color in life was greenish. The pygidium was divided into lobes. (Webster, 1879) (Fig. 5E, F) We examined 20 individuals of Boccardiella hamata. The maximum recorded body length after fixation was 38 mm. The morphological characteristics of B. hamata from Normandy matched those of B. hamata from North America (Dean and Blake 1966) and Asia (Sato-Okoshi 2000;2013). Pigmentation was entirely absent on the body (Fig. 5E). Special recurved hooks were present in posterior notopodia (Fig. 5F). Pygidium had two broad ventral lappets, each with a short process (Fig. 5F).

Shell infestation by polydorids
The conditions of shell infestation by each polydorid species at each sampling site are shown in Table 3. Shell infestation was determined by the occurrence rate of abnormal shell formation, and the degree of abnormal shell formation observed on the inner surface of the shell. The presence of abnormal black organic deposits or calcareous deposits is a direct response of the host mollusks to cover and defend the polydorid excavation and repair after their penetration. The degree "heavy infestation" was used only when multiple abnormal shell formations were observed per shell. We used the term "infestation" only when a host oyster formed an abnormal shell in response to an invasion, irrespective of the number of worms inhabiting the shell.
Sandy areas, including some rocks, were spread across Blainville-sur-Mer (Fig. 6A). Cultured Japanese oyster C. gigas that fell out of the net bags (Fig. 6B), along with wild Japanese oyster (Fig. 6C) and European flat oyster O. edulis (Fig. 6D) were collected and observed. They exhibited a large shell width (Fig. 6C-E) with calcareous layers (Fig. 6F). Shell chambers (French word: chambrage) are constructed with thin paper shells filled with jelly-like materials. Chalky deposits (Okoshi et al. 1987) or mud were observed in both shell valves. Small-sized D. giardi and large-sized P. hoplura were observed to inhabit the shells. Mud blisters, which were the responses against large-sized P. hoplura, were also confirmed. The number of individuals per shell fluctuated among the examined oysters, ranging from several to several tens of worms per shell. Although high densities of D. giardi (approximately 4 inds. /cm 3 in aggregation) were detected in the shells of cultured oysters that fell from the net bags, the damage to the shells was Wild oyster shells from oyster beds in the muddy ground at Saint-Vaast-La-Hougue ( Fig. 7A-C) were inhabited by P. onagawaensis, B. hamata, and B. pseudonatrix. Mud tubes were observed in, on, and within the shell crevices (Fig. 7D, E). The burrows created inside the shell are visible on the side of the inner surface of the shell (Fig. 7F, G). The number of individuals per shell fluctuated, ranging from several to several tens of worms per shell in P. onagawaensis. The number of worms extracted per shell (valve) was especially high (up to 135 individuals) in suspended cultured oysters from Quineville (Fig. 8A, B). Numerous mud tubes of P. onagawaensis and P. websteri protruded from the edge and near the umbo of the outer surface of the shell (Fig. 8C). The palps were observed waving from their tubes (Fig. 8D). The outer surface of the shell was peeled off, and a large number of burrows were found inside (Fig. 8E). Further, mud blisters were observed near the attachment site of the adductor muscle on the inner surface of the shell (Fig. 8F). Few cracks were observed along the burrows (Fig. 8G, H). Black or brown organic deposits and calcareous shell material were secreted against worm penetration on the inner surface of the shells (Fig. 8B, F-H).  Brown deposits and calcareous shell materials were observed to secrete against polydorid infestation on the inner surface of the right valve. C Each arrow shows the mud tube protruding from along the edge of the valves in a triploid oyster. D Each arrow shows polydorid palp waving from its tube. E Outer surface of the shell revealing many polydorid burrows which were formed inside after the shell surface peeling off. F Mud blister near the attachment site of the adductor muscle. G Crack observed running across the polydorid burrow over the adductor muscle attachment and mud blister. H Enlarged view of a part of a crack in G From the cultured oyster shells of Asnelles (Fig. 9A-D), three species, P. onagawaensis, P. websteri, and B. proboscidea, were documented. Mud blisters were also observed on the inner surface of the shells (Fig. 9D). Harvested scallops that were dredged near Asnelles had Cliona (Porifera) infestation; however, no polydorid infestations were observed on the shells.
In the wild, numerous burrows of P. onagawaensis were observed on the surface of limestone (ca. 2 burrows/cm 2 ), which covered the intertidal sea bottom in Luc-sur-Mer (Fig. 10A-D). Juveniles presenting larval pigmentation (Fig. 4I, J) were found from the shallow burrows on the surface of the concrete block covered with barnacles and mussel shells, which were installed during another study (Fig. 10E) (Dauvin et al. 2021).
High densities of approximately 1 ind./cm 2 of B. proboscidea were observed to inhabit coralline algae that covered the entire surface of the intertidal rocks at Point de Barfleur (Fig. 11A-D). They created mud tubes in the crevices of coralline algae. Adults with egg capsules in their tubes were obtained (Fig. 11E, F).

Diversity of environment along the coast of Normandy and calcareous substrates as a habitat
Eight species of polydorids were discovered during the present survey of the western half of the Normandy coast along approximately 450 km of the English Channel. In this study, Fig. 9 Cultured oysters from Asnelles. A Cultured oysters displayed in the market. B Harvested cultured oysters. Scale bar: 2 cm. C Inner surface of the left and right valves, respectively, left valve with soft body. Each arrow shows the polydorid burrows. D Mud blister and polydorid burrows visible from the inner surface of the right valve shell. Scale bar: 1 cm we investigated two large oyster culture grounds and two suspended culture sites in different environments and the surrounding natural calcareous substrates.
The existence of varied habitats, such as the different environments at oyster farms, the diverse natural environments of mud, sand, and rocky areas, as well as the existence of artificial environments such as oyster farming facilities and structures, could be the reason for the coexistence of many of the eight species found in Normandy area. We also observed different morphological characteristics of shells between wild and cultured oysters. For example, all oysters cultivated in mesh bags were "single oysters" and were not stuck to each other. In contrast, the wild oysters that live on the muddy bottom in Saint-Vaast-La-Hougue were attached to each other (Fig. 7B). Crevices between individual oysters and mud accumulates created a conducive habitat for polydorids. Moreover, at Saint-Vaast-La-Hougue, oyster shells that live on the muddy bottom grow longitudinally upward and are thin to avoid burial under the mud (Fig. 7B, C). Since oysters with thin shells are less suitable as long-term habitats for large numbers of polydorid individuals, the abundance of mud provides a suitable alternate for species that build mud tubes to live in (Fig. 7D, E).
Oyster shells cultured in mesh bags on gravel and sandy ground farms and a wild single oyster grown on rocks or coarse sand tended to be thicker in Blainville-sur-Mer (Fig. 6B-E) compared to wild oyster shells in Saint-Vaast-La-Hougue. The cross-section of the shell was thicker (Fig. 6F), and a large number of shell chambers (Okoshi et al. 1987) were also observed. Owing to the thickness of the shell and the tendency for mud to collect in the shell chamber, it is inhabitable by more polydorid individuals. Triploid-cultured oysters were characterized by a cup-shaped valve on the left (Fig. 8C). In this case, the surface area of the shell increases, becoming a substrate on which more individuals can live. Thus, it is speculated that these microhabitats can be created, and multiple species can coexist within them.
Interestingly, the species composition differed between the west and east across the Cotentin Peninsula. While from Blainville-sur-Mer, which is a gravel and sandy oyster culture ground located in the west, P. hoplura and D. giardi, both species were already known to be distributed in this locality, were observed to inhabit the oyster shells. In contrast, the two species were not reported from the east except Quineville. However, P. onagawaensis and P. websteri, both species previously unknown from this locality, abundantly inhabited muddy oyster beds in Saint-Vaast-La-Hougue and suspended culture oysters from Quineville and Asnelles, respectively, located in the east. None of these two species was observed on the west side of the peninsula during the present survey. Differences in species composition between the west and the east may be related to differences in habitat environment conditions, such as coastal current, sediment type, and sea water temperature, which was higher in the eastern part of the Cotentin (Dauvin 2019). At the same time, it is crucial to clarify the origin and distribution route of oysters brought to each farm since cultured oysters are transported and moved manually.
In this study, a newly recorded species, P. onagawaensis, was found to be distributed on the east side of the Cotentin Peninsula from wild and cultured oyster shells and limestone substrates. Notably, P. websteri was extracted only from suspended cultured oysters, and not from the wild. Further, our previous survey showed that P. websteri did not inhabit the shells of cultured oysters from Arcachon Bay, located along the Bay of Biscay, south of Normandy, in 2017 (Sato- Okoshi et al. in prep). Polydora websteri was recently reported to be abundant in the shells of cultured oysters and naturalized oyster reefs in the Wadden Sea, Netherlands, and Germany (Waser et al. 2020). These results suggest the possibility of new transportation of the species to the east through anthropogenic factors. Further studies on the distribution of P. onagawaensis and P. websteri along the western European coast, based on accurate species identification, are required.
Specimens of a newly recorded species, P. onagawaensis, reported for the first time in Normandy, were compared to the type locality in northeastern Japan and USA (Silverbrand et al. 2021). Polydora onagawaensis in France was grouped with a USA specimen in a single clade and was differentiated from three other lineages containing specimens from Japan and the USA (Fig. 3). Moreover, the morphological characteristics of French P. onagawaensis differed slightly from those of Japanese P. onagawaensis, as described in the remarks section. In the future, the P. onagawaensis complex should be studied in more detail by examining its genetic information, morphology, and ecology from additional habitat areas.
Boccardia proboscidea was originally described on the Pacific coast of North America (Hartman 1940), but the number of reports of this species from new locations globally continues to increase (Sato-Okoshi 2000;Sato-Okoshi et al. 2008;Simon and Sato-Okoshi 2015;Jaubet et al. 2018;Spilmont et al. 2018;Radashevsky et al. 2019). Boccardia proboscidea was found in coralline algae and the shells of cultured oysters during the present survey. Moreover, it was recently reported along the French coast of the English Channel, on the Opal Coast in northern France in 2014 (Spilmont et al. 2018), and in 2018 in North Brittany (Gully and Cochu 2020).
Boccardiella hamata was also reported for the first time in the English channel. This species was originally described from the Atlantic coast of North America (Webster 1879) and is now commonly known from the Pacific coast, for example, Vancouver Island, Canada (Sato- Okoshi and Okoshi 1997), Japan (Sato-Okoshi 2000), China (Zhou et al. 2010), Korea (Sato-Okoshi et al. 2012, and recently near Belgium and the North Sea (Kerckhof and Faasse 2014). The species has been reported from muddy environments, particularly sandstones (Sato- Okoshi and Okoshi 1997) and rigid man-made substrates such as coastal defense structures on sandy coasts (Kerckhof and Faasse 2014). Boccardiella hamata was also recently found to inhabit sponges (Abe et al. 2019a).
Boccardia pseudonatrix, which has never been reported in European waters, was found in the shells of wild and cultured oysters. Boccardia pseudonatrix was originally described in South Africa (Day 1967;Simon et al. 2010), from which it may have been artificially transferred to rest of the world by aquaculture or ship ballast water. This species has recently been found in Australia (Sato- Okoshi et al. 2008Okoshi et al. (as B. knoxi), 2015Walker 2013) and Japan Abe et al. 2019a) and is likely to be found worldwide after accurate identification in the future.
It was unclear from this survey whether each of the eight species inhabiting this water area was introduced as non-indigenous or misidentified due to confusion in species identification. Since the life history of polydorids has been reported to range from less than a year to several years, it can be assumed that the species extracted from the shells of cultured and wild oysters from the oyster farms of Saint-Vaast-La-Hougue and Blainville-sur-Mer could include both the species that originally inhabited the water areas and those that were transported to the areas accompanying the host shells.
Notably, all species of polydorids were identified to the species level in the present study (except Dipolydora sp.), which are also found in Japan, especially along the Pacific coast of Tohoku District (Sato-Okoshi 1999;Abe and Sato-Okoshi 2021). Considering the historical background of cultured oysters in France and Japan, Japanese oysters were abundantly exported from the Pacific coast of Tohoku District of Japan to France (Koike 2015). Future discussions on the species of polydorids inhabiting the waters should consider the anthropogenic transfer of oysters, which could be their hosts and species genetic information.
Analyzing historical trends may be challenging; however, considering the future range expansion of these species, their larval developmental patterns should be studied. Of the eight species confirmed during the survey in March, only a few large individuals of P. onagawaensis were found to have oocytes. Further, only B. proboscidea produced egg capsules with developed larvae and many nurse eggs. Polydora onagawaensis, D. giardi, and B. hamata have shown simultaneous development and produce planktotrophic larvae with a relatively long planktonic larval phase before settling (Sato-Okoshi 1999;Teramoto et al. 2013). In contrast, P. websteri, P. hoplura, and B. proboscidea are known to produce adelphophagic larvae, that usually emerge soon before settlement or as a mixture of these and planktotrophic larvae (reviewed by Simon and Sato-Okoshi 2015); B. pseudonatrix is known to produce adelphophagic larvae (Sato- Okoshi et al. 2008, as B. knoxi;Simon et al. 2010). Further confirmation of the developmental pattern of larvae in this water may lead to the elucidation of distribution expansion.

Effects of polydorids on cultured oysters in Normandy
A high occurrence rate of abnormal shell formation was observed in wild and cultured oysters, with particularly high rates in wild oysters caused by P. hoplura and in suspended cultured oysters caused by P. websteri and P. onagawaensis. Host oysters secrete excess organic and calcareous shell materials to cover and defend against polydorid excavation and repair part of the shell after its penetration. Although we did not confirm oyster death or apparent soft body shrinkage due to the infestation of the oyster shells by polydorids, based on the levels of shell infestation observed, negative impacts on oysters were a serious concern. The decrease in the commercial value of the host oysters, unpredictable environmental conditions, and other unfavorable factors could easily lead to growth inhibition and mortality. For these high-risk polydorid species groups, biological and ecological control measures, such as avoiding the period when the planktonic larvae settle on the host shells and avoiding water areas where larvae aggregate, to prevent invasion, are required to minimize damage to the culture system. At the same time, considering the high density and wide distribution of these species reported today, we need to recognize that invasive alien species have already established their populations in non-indigenous waters and continue to expand their distribution. It is necessary to discuss and evaluate global impacts on ecosystems.