Occurrence of a New Apicomplexan Intracellular Parasite in the Digestive Gland of Bulla striata (Gastropoda: Cephalaspidea) from the South Coast of Portugal

Abstract

A new intracellular parasite of the phylum Apicomplexa is described infecting the digestive gland of the gastropod Bulla striata from the south coast of Portugal. Only merogonial stages enclosed within parasitophorous vacuoles were observed by light and electron microscopy. The meront cytoplasm contained lipid droplets, rough endoplasmic reticulum cisternae, and several round or oval electron-dense microbodies closely associated with amylopectin granules, suggesting that these microbodies may represent glycosomes. Mitochondria or related organelles were not identified. A reticulum of branched tubules extending from the parasitophorous vacuole membrane was observed, likely increasing the surface available for metabolite exchange between parasite and host cell. Merozoites resulting from meront division were present within the parasitophorous vacuoles. They were elongated and slightly curved, measuring 7–8 µm in length and about 2 µm in width, and possessed an apical complex comprising numerous rod-shaped micronemes, rhoptries, and a conoid. Phylogenetic analyses based on a partial 18S rDNA sequence placed this parasite within the coccidian lineage, at the base of the ichthyocolid clade, a recently recognized group of apicomplexans previously known from fish blood cells. This finding expands the host range of ichthyocolids to gastropods and provides the first ultrastructural observations of this lineage, although only of merogonic stages.

1. Introduction

The phylum Apicomplexa comprises more than 6000 described species of unicellular parasites and symbionts in both vertebrate and invertebrate hosts [1,2]. Some species are extracellular parasites, such as most gregarines, which attach to host cells using specialized structures called the mucron or epimerite [3]. Others, including coccidians and some gregarines, are intracellular parasites that develop within a parasitophorous vacuole. Apicomplexans possess complex and diverse life cycles comprising both asexual and sexual stages, typically organized into three successive schizogonies: merogony, gamogony, and sporogony. However, merogony is absent in most gregarines, being present in species of the order Neogregarinorida. Some apicomplexans are monoxenous, completing their entire life cycle within a single host, whereas others are heteroxenous, requiring an intermediate host, where asexual merogonial stages occur, and a definitive host, in which gamogonial and sporogonial stages take place to complete the life cycle. Proliferation within hosts and transmission between hosts are mediated by specialized infectious forms [4,5].

To attach and penetrate host cells, both the merozoites generated by asexual multiplication and the sporozoites released from oocysts resulting from sexual reproduction possess a specialized set of structures at the cell apex, collectively known as the apical complex. This complex—comprising micronemes, rhoptries, the conoid, and the polar ring—is a defining feature of Apicomplexa [6]. Micronemes are rod-shaped organelles that release proteins required for attachment of the parasite to the membrane of the host cell [7]. Rhoptries are elongated organelles characterized by a long narrow neck and a bulbous posterior region that discharge proteins at the onset of host cell invasion. In intracellular apicomplexans, rhoptry proteins are essential for the formation of the moving junction, a ring-shaped interface between the parasite membrane and the host cell membrane. This junction moves along the membrane surface, creating an invagination of the host membrane that envelops the invading parasite, ending in the formation of the parasitophorous vacuole [8]. The conoid is a small cone-shaped extrusive structure formed by tightly coiled tubulin fibres, and the apical polar ring serves as a microtubule-organizing centre [9]. In intracellular apicomplexans, the egress of infective stages from the host cell requires proteins secreted by micronemes and microneme-like organelles that compromise the integrity of the parasitophorous vacuole and host cell membrane [10].

Apicomplexan species that cause severe diseases in humans and other terrestrial vertebrates, such as Plasmodium spp., Toxoplasma gondii, Cryptosporidium spp., Eimeria spp., among others [4], have been studied far more extensively than those infecting aquatic animals. Nevertheless, apicomplexans are also important parasites of fish, molluscs, annelids, crustaceans, and other marine animals. While more than one hundred species of coccidians have been described from marine fishes, primarily teleosts [11], gregarines are particularly common and represent the most frequently reported apicomplexans in marine invertebrates [12,13]. In addition to their role as parasites, the phylum Apicomplexa also includes species that appear to be non-pathogenic or even beneficial to their marine hosts. For example, Nephromyces spp., which inhabit the renal sac of tunicates, function as symbionts that use uric acid as their primary nitrogen source [14,15]. More recently, the order Corallicolida was erected to encompass an emerging lineage of intracellular apicomplexan symbionts of corals, commonly referred to as corallicolids [16,17]. Among molluscs, apicomplexan parasites have been detected in bivalves, cephalopods, gastropods, and even in a chiton [18]. In bivalves, these parasites have been found in the kidney, heart, adductor muscle, gill, and other tissues, where heavy infections potentially lead to significant organ damage [19,20,21]. Such infections can contribute to mass mortality events, particularly when associated with other environmental stressors [22,23,24]. Octopus and cuttlefish are definitive hosts of Aggregata spp. (Marosporida), which have crustaceans as intermediate hosts for asexual merogonial development [25,26].

To date, relatively few apicomplexan parasites have been reported from marine gastropods. Gregarines have been found in the intestines of Littorina spp. [27] and in various tissues of Nerita spp. [28,29]. Coccidians have been identified in the kidneys of Haliotis spp. [30], in the digestive glands of some species of the family Strombidae from the Caribbean Sea [31,32], and in the digestive gland and intestine of the limpet Patella vulgata [18]. In addition, the common whelk, Buccinum undatum, is the definitive host of Merocystis kathae, a species currently classified within the recently established apicomplexan class Marosporida [33], which uses a bivalve as its intermediate host [22]. Apicomplexa-like parasites have also been found in other marine gastropods [34], and additional species occur in terrestrial and freshwater gastropods [35,36,37]. Although the impact of these parasites on their hosts remains poorly understood, some species have been acknowledged as potential threats. For instance, an Eimeriidae-like parasite infecting the digestive gland of the queen conch Aliger gigas (formerly Strombus gigas) has been associated with reproductive decline in this gastropod [38].

Gastropoda is the mollusc class with greater number of species, including the subclass Heterobranchia, which comprises numerous marine species [39]. This highly diverse subclass includes the order Cephalaspidea, which comprises bubble-shell snails of the genus Bulla. Most Bulla species inhabit tropical seas, but Bulla striata spans across both temperate and tropical latitudes. This herbivorous marine snail is found in sheltered, shallow-water habitats with soft substrate along the Mediterranean and eastern Atlantic shores, from southern Portugal to Angola, and on several Atlantic islands [40,41]. Despite possessing a protective shell and chemical defences [42], B. striata is known to be preyed upon by Philinopsis depicta, a cephalaspidean of the family Aglajidae [43,44]. In gastropods and other molluscs, the digestive gland is the largest organ of the digestive system, mainly composed of digestive cells and basophilic cells. Digestive cells, rich in lysosomes, are responsible for the intracellular digestion of nutrient particles captured by endocytosis following extracellular digestion. Basophilic cells, abundant in endoplasmic reticulum and other organelles, are responsible for secreting enzymes required for extracellular digestion [45,46]. In this paper, we report the ultrastructural aspects and molecular data of an intracellular apicomplexan parasite found in the digestive gland of B. striata specimens collected from the Southern coast of Portugal.

2. Materials and Methods

2.1. Specimen Collection

Specimens of Bulla striata Bruguière, 1792 (Gastropoda: Cephalaspidea), about 2–3 cm in shell length, were collected manually during low tide from Ria de Alvor (37°8′ N, 8°37′ W), an estuarine system located on the southern coast of Portugal. The animals were transported live to the laboratory in aerated water from the sampling site and arrived in perfect conditions. Upon arrival, the digestive gland of five animals was removed and processed for microscopy and molecular analyses. The specimens analysed in this study were collected in April 2005 (2 animals), June 2007 (1 animal) and September 2013 (2 animals).

2.2. Microscopy Procedures

Pieces of the digestive glands were fixed for about 2–3 h at 4 °C in 2.5% glutaraldehyde and 4% formaldehyde (obtained from hydrolysis of paraformaldehyde), buffered with 0.4 M sodium cacodylate pH 7.4 (final buffer concentration 0.28 M). After washing in buffer, samples were postfixed for 2–3 h at room temperature in 2% osmium tetroxide buffered with sodium cacodylate, dehydrated in an ascending ethanol series, embedded in epoxy resin, and polymerized at 60 °C for 2–3 days. Semithin sections (2 µm) for light microscopy were stained with methylene blue and azure. The PAS reaction for polysaccharide detection and the tetrazonium coupling reaction for proteins were also applied to semithin sections with the epoxy resin. For PAS reaction, after oxidation with 1% periodic acid for 10 min, semithin sections were washed with water and stained in Schiff reagent for about 15 min [47]. For the tetrazonium coupling reaction, the osmium tetroxide fixative was removed from the tissue by treatment of the semithin sections with a 0.6% solution of H₂O₂ for 10 min to improve staining. After washing in water, sections were treated for 10 min with a freshly prepared 0.2% solution of fast blue salt B in veronal-acetate buffer pH 9.2, washed in water and treated for 15 min with a saturated solution of ß-naphthol in veronal-acetate buffer pH 9.2 [48,49]. Ultrathin sections (about 90 nm thick) were double stained with uranyl acetate and lead citrate, observed, and photographed using a JEOL 100CXII transmission electron microscope (JEOL Ltd., Tokyo, Japan), operated at 60 kV, equipped with a Gatan Orius SC200 digital camera (Gatan Inc., Pleasanton, CA, USA).

2.3. Molecular Procedures

Genomic DNA was extracted from fresh fragments of three infected digestive glands using the GenElute^TM Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, MO, USA), following the manufacturer’s instructions for tissue samples. The extracted DNA was eluted in 150 μL of TE buffer and stored at −20 °C until further use.

Amplification and sequencing of the parasite 18S rDNA sequence were performed using the primer pair EF (5′-GAAACTGCGAATGGCTCATT-3′)/ER (5′-CTTGCGCCTACTAGGCATTC-3′) [50]. PCR reactions were prepared using 10 pmol of each primer, 10 nmol of each dNTP, 2 mM MgCl₂, 5 μL 10× Taq polymerase buffer, 2.5 U of Taq DNA polymerase (NZYtech, Lisbon, Portugal), and approximately 50–100 ng of genomic DNA, in a final reaction volume of 50 µL. PCRs were performed on a Hybaid PxE Thermocycler (Thermo Electron Corporation, Milford, MA, USA) with an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 53 °C for 45 s, and extension at 72 °C for 90 s, with a final elongation step at 72 °C for 7 min.

PCR products (5 μL) were electrophoresed on a 1% agarose gel prepared in 1× Tris-acetate-EDTA (TAE) buffer and stained with GreenSafe Premium (NZYtech, Lisbon, Portugal). Positive amplicons were purified and sequenced by STAB VIDA (Caparica, Portugal).

2.4. Phylogenetic Analyses

The parasite 18S rDNA sequence was obtained by aligning and trimming the forward and reverse sequences using ClustalW implemented in MEGA 11 [51,52]. Closely related sequences were identified using the Basic Local Alignment Search Tool (BLASTn) (BLAST+ 2.17.0), and an initial dataset was compiled including all 18S rDNA sequences sharing more than 93.4% similarity with the parasite sequence obtained in this study. This preliminary dataset was aligned using MAFFT version 7 [53] available online, and genetic distances were estimated in MEGA 11 using the p-distance model with the pairwise deletion option.

For phylogenetic inference, the preliminary dataset used for distance estimation was expanded to include representative sequences of the Eimeriorina and Adeleorina, as well as the piroplasmids Babesia microti (AB190459), Theileria parva (HQ895985) and Theileria annulata (JQ743630), which were used as outgroup, even if these were not retrieved in the BLASTn search. This approach was employed to ensure that all major lineages were adequately represented while avoiding an excessively large dataset. Multiple sequence alignments of the final dataset were performed using the E-INS-i refinement strategy in MAFFT version 7, followed by manual editing in MEGA 11. Phylogenetic relationships were inferred using maximum likelihood (ML) and Bayesian inference (BI) analyses. ML analyses were conducted in MEGA 11 using the general time-reversible substitution model with estimates of invariant sites and gamma-distributed rate variation among sites (GTR + I + G), with nodal support assessed by 1000 bootstrap replicates. BI analyses were performed in MrBayes v.3.2.6 [54], also using the GTR + I + G model. Posterior probabilities were estimated using the Markov chain Monte Carlo approach with four chains run simultaneously for 2 million generations, sampling every 500 generations, and discarding the first 25% of trees as burn-in.

3. Results

3.1. Light and Electron Microscopy Observations

Light microscopic examination of semithin sections of the digestive gland of Bulla striata disclosed a parasitic infection of the digestive gland in all five specimens analysed. The parasites, which exhibited apicomplexan features, were located within parasitophorous vacuoles, occupying a substantial portion of several digestive gland cells. Only merogonial stages were recognized, including developing and mature meronts, and merozoites. Infected cells contained either meronts or merozoites, but not both stages, and one or two meronts could be seen in each infected cell (Figure 1A). The meronts contained numerous granules strongly stained by the PAS reaction for polysaccharide detection (Figure 1B). Microbodies strongly stained by the tetrazonium coupling reaction for protein detection were also present in meronts, mostly at the periphery of these cells (Figure 1C).

The earliest merogonic stages recognized by transmission electron microscopy (TEM) corresponded to young meronts, enclosed by the membrane of the parasitophorous vacuole (Figure 1D). At this stage, a single nucleus was observed, presenting a prominent spherical nucleolus and low amounts of heterochromatin scattered in the electron-dense nucleus. The meront cytoplasm was packed with numerous round and oval electron-lucent amylopectin granules, some lipid droplets, several round or oval electron-dense microbodies—mostly located peripherally—and rough endoplasmic reticulum cisternae (Figure 1D). In ultrathin sections, most electron-dense microbodies had a diameter around 0.4–0.7 µm and were closely associated with the amylopectin granules that were highly diverse in size, mostly with sections ranging in diameter from about 1.0 to 0.2 µm. In some ultrathin sections, certain amylopectin granules were partially encircled by a microbody with a C-shaped section (Figure 2A,B), and several of these granules were surrounded by a rough endoplasmic reticulum cisterna (Figure 1D inset). Mitochondria or mitochondria-related organelles were not identified in the merogonial stages of the parasite.

More advanced meronts contained more than one nucleus, each closely surrounded by rough endoplasmic reticulum cisternae. Amylopectin granules, lipid droplets, and electron-dense microbodies were also present in the parasite cytoplasm at this stage (Figure 2C). A reticulum of branched tubules with a smooth membrane was observed at the periphery of the parasitophorous vacuoles (Figure 2C). The membrane of these tubules closely resembled the parasitophorous vacuole membrane, and in some ultrathin sections it was possible to see the connections of this reticulum with the parasitophorous vacuole (Figure 2D). Merozoites resulting from meront division were observed within the parasitophorous vacuoles (Figure 3A). The elongated and slightly curved merozoites had a length of 7–8 µm and were about 2 µm wide. At this stage the parasite was uninucleate, and contained rough endoplasmic reticulum cisternae, a Golgi stack, and amylopectin granules in the cytoplasm. The apical complex comprised numerous rod-shaped micronemes, rhoptries and conoid (Figure 3B–D).

3.2. Molecular and Phylogenetic Analyses

A partial 18S rDNA sequence of 1453 bp was assembled and deposited in GenBank under accession number PX990216. BLASTn searches revealed that the parasite sequence showed highest similarity to ichthyocolids (94.7–98.3%) and corallicolids (93.4–97.2%). Distance estimation analysis based on the preliminary dataset indicated the highest sequence identity with ichthyocolids (96.9–98.5%), particularly with sequences obtained from the apicomplexan parasite infecting Stegastes spp. (MH401637-42, KT806397-98), which shared 98.4–98.5% identity, and from the apicomplexan infecting Ophioblennius macclurei (OR832857, KY940307, KT806396), which shared 97.8–97.9% identity.

ML and BI analyses yielded largely congruent tree topologies, recovering consistent branching patterns among the main coccidian lineages. Sequences of the suborder Adeleorina formed a clade sister to a small clade comprising Nephromyces sp. ex Molgula occidentalis (SOZB01005269) and Cardiosporidium cionae (EU052685), although this sister-group relationship was not strongly supported. Together, these two clades occupied a basal position relative to the remaining coccidian lineages. Sequences of the suborder Eimeriorina formed a clade sister to the assemblage comprising ichthyocolids and corallicolids, which themselves resolved as two distinct sister clades. The 18S rDNA of the parasite in study clustered at the base of the ichthyocolid clade, which includes all ichthyocolid sequences currently available (Figure 4). Although the ichthyocolid clade was well supported in both analyses, its internal relationships were poorly resolved.

4. Discussion

4.1. Microscopic Identification and Ultrastructural Features

Electron microscopy has played a significant role in advancing our understanding of apicomplexans [55]. In this case, a parasite species was incidentally discovered during an ultrastructural study of the digestive gland of B. striata and other cephalaspideans [46]. The parasite, which specifically infected the digestive cells, displayed ultrastructural features typical of the phylum Apicomplexa, namely the presence of an apical complex at the infective life cycle stages, which in this case were limited to merozoites.

Amylopectin reserve granules found in apicomplexan parasites are considered a legacy from their microalgae ancestors. These granules are composed of a branched glucose polymer that serves as an energy reserve, supplying glucose for ATP production [56]. In Toxoplasma gondii, amylopectin granules are present in encysted bradyzoites, responsible for transmission from the intermediate host to the feline definitive host, and in sporozoites within sporulated oocysts, which mediate transmission from the definitive to an intermediate host. Conversely, these granules are absent in merozoites involved in asexual proliferation within feline intestinal cells, and in tachyzoites responsible for rapid asexual replication in intermediate hosts. This pattern suggests that, in T. gondii, the amylopectin granules serve as critical energy reserves for encysted forms that may endure nutrient deprivation. Bradyzoites, although encysted, retain metabolic activity and can divide, playing an important role in chronic infection within intermediate hosts. They can also remain viable for days or even a few weeks in the tissues of dead hosts, which may subsequently be consumed by a feline. Similarly, sporozoites must survive in sporulated oocysts in the environment until ingested by an intermediate host [57,58]. In contrast, in the apicomplexan parasite infecting B. striata digestive gland, non-encysted merozoites were found to contain a substantial amount of amylopectin granules. This observation suggests a potentially different biological strategy or a specific metabolism of this parasite species. Encysted stages of the parasite were not observed in the digestive gland of B. striata. However, amylopectin reserves might also be important for survival of hypothetical encysted stages that could be released to the environment as part of the life cycle.

Based on their size and distribution in the cytoplasm, the electron-dense microbodies observed by TEM in the apicomplexan parasite infecting the digestive gland of B. striata likely correspond to the structures that stained intensely in semithin sections using the tetrazonium coupling reaction for protein detection under light microscopy. Their close spatial association with amylopectin granules—particularly the presence of microbodies with a C-shaped section partially encircling these granules—strongly suggests a metabolic relationship. Thus, it is conceivable that these microbodies may contain glycolytic enzymes. If this is the case, they could be glycosomes: organelles that contain enzymes of the glycolytic pathway, typically observed in TEM as round or oval-shaped microbodies with a protein-dense matrix, surrounded by a single membrane [59,60]. Despite the ultrastructural similarities between glycosomes and the microbodies observed in the parasite of B. striata, this hypothesis requires confirmation through enzymatic or molecular characterization. Glycosomes are well known in kinetoplastids and diplonemids [61,62], but have never been reported in Apicomplexa.

In the kinetoplastid parasite, Trypanosoma brucei, mitochondrial volume and metabolic activity have been reported to increase under low-glucose conditions and decrease when glucose is abundant. In periods of high glucose availability, the relative volume occupied by glycosomes increases in this parasite, favouring glycolysis in detriment of mitochondrial aerobic respiration [63,64]. A similar inverse relationship between mitochondrial and glycosomal volume in response to glucose availability has been reported in another kinetoplastid. When glucose was the main energy source for Herpetomonas roitmani promastigotes, mitochondria occupied a smaller proportion of the cell volume, while glycosomes were very numerous [65,66]. Considering this, it can be that the apicomplexan parasite infecting the digestive gland of B. striata has a metabolic strategy similar to H. roitmani, with glycolysis likely being its major pathway for energy production. This is plausible given that the parasite resides within digestive cells actively involved in nutrient absorption and intracellular digestion, where glucose availability is expected to be high [45]. The abundance of amylopectin granules accumulated in the meronts further support the idea that a good supply of glucose is available to the parasite in the digestive cells of B. striata.

Mitochondria with typical ultrastructural morphology are present in Plasmodium spp. [67] and Toxoplasma gondii [68], whereas Cryptosporidium spp. possess a highly reduced mitochondrial-related organelle (mitosome), which has lost the mitochondrial genome and several metabolic pathways typical of mitochondria [69]. In general, loss of mitochondrial functions is strongly associated with structural reduction, often making these modified organelles difficult to recognize in TEM observations. Interestingly, although gregarines possess mitochondria with standard ultrastructure [70], most appear to lack mitochondrial ATP production [71]. Mitochondria or related organelles were not recognised in the parasite of the digestive gland of B. striata, which, like several other apicomplexans, may rely primarily on glycolysis for ATP production.

The smooth membrane reticulum, functioning as a tubular extension of the parasitophorous vacuole, substantially increases the membrane surface area available for metabolite exchanges between the host cell and the parasite located within the parasitophorous vacuole [72]. This reticulum that extends through the cytoplasm of the infected digestive cells of B. striata resembles the tubovesicular network described in Plasmodium-infected cells [73,74]. In the parasitophorous vacuoles containing Toxoplasma a nanotubular network is present but, in this case, located inside the parasitophorous vacuole [75].

4.2. Phylogenetic Placement

Molecular analyses revealed that the parasite infecting the digestive gland of B. striata shows highest similarity to ichthyocolid apicomplexans. Congruently, ML and BI phylogenetic inferences positioned the parasite within the broader coccidian assemblage, specifically at the base of the ichthyocolid clade. Under current phylogenetic frameworks, Coccidia encompasses not only the more classically recognized coccidian lineages, but also more recently resolved groups such as corallicolids and ichthyocolids [16,17,76].

Ichthyocolids were initially classified as Haemogregarina-like and Haemohormidium-like parasites of tropical fishes, based on morphological observations of life cycle stages in erythrocytes [77,78]. However, phylogenetic analyses have consistently recovered them within a distinct lineage of Coccidia [79,80,81], closely related to corallicolids [76,82]. Recently, Bonacolta et al. [76] termed these fish blood parasites ichthyocolids and characterized the group using analyses of the entire rRNA operon and complete mitochondrion and apicoplast genomes of the parasite infecting Ophioblennius macclurei erythrocytes, confirming that they are distinct from true Haemogregarina (suborder Adeleorina) and Haemohormidium (order Piroplasmida). Our phylogenetic analyses agree with these previous findings, consistently recovering a sister-group relationship between ichthyocolids and corallicolids, together forming an assemblage sister to the Eimeriorina.

Based on a comprehensive screening of publicly available fish microbiome datasets using the apicoplast 16S rRNA gene, Bonacolta et al. [76] suggested that ichthyocolids are geographically and taxonomically widespread among marine teleosts, with potential implications for commercial fisheries and oceanic food webs. Low-abundance amplicon sequence variants (ASVs) with 95% similarity to the ichthyocolid infecting O. macclurei were also recovered from environmental samples, mollusks, flatworms, and even echinoderms, and were hypothesized to originate from sparse ichthyocolid cysts that, similarly to other apicomplexans, likely persist in the environment [1,13]. Our finding of an ichthyocolid infecting B. striata on the Portuguese South coast not only corroborates the widespread geographic distribution of these parasites, which has so far been mostly documented in coral-reef environments, but also highlights gastropods as potential invertebrate hosts for ichthyocolids. Notably, the B. striata ichthyocolid occupies a sister position to all previously characterized ichthyocolids, representing a basal branch within the clade and expanding our understanding of host range and evolutionary relationships within this group. In addition, this study reveals for the first time the ultrastructure of an ichthyocolid.

4.3. Life-Cycle Considerations

Most blood-borne apicomplexans require two hosts to complete development. Asexual proliferation, leading to gamont formation in peripheral blood, occurs in a vertebrate intermediate host, whereas sexual development occurs in a haematophagous invertebrate definitive host. Transmission of infective sporozoites from invertebrate hosts occurs either via inoculation, as in haemosporidia (Plasmodium spp.), piroplasms (Babesia spp.), and some haemogregarines (Haemogregarina spp.), or via ingestion of the infected invertebrate, as in most haemogregarines (Hepatozoon spp.). Haemococcidia such as Lankesterella and Schellackia complete their development entirely within vertebrates, with invertebrates acting only as paratenic or mechanical hosts [83].

To date, no direct data exist regarding ichthyocolid transmission, although gnathiid isopod larvae have been suggested as potential vectors based on the recovery of parasite 18S rDNA from blood-fed larvae [84]. In the present study, the absence of gamogonial and sporogonial stages in the digestive gland of B. striata supports the hypothesis of a heteroxenous life cycle, as the presence of multiple life-cycle stages in a single host is generally associated with monoxenous parasites [30,32].

In many Apicomplexa, the definitive and intermediate hosts have a trophic relationship [4]. However, B. striata is an herbivorous species that primarily feeds on green algae, seagrass (Zostera), cyanobacteria and diatoms [41], and do not have known natural fish or crustacean predators mainly due to chemical deterrence. In addition to their strong chemical defences, these gastropods also rely on the protection provided by a strong shell and nocturnal habits [42,85]. Moreover, apicomplexan stages were not observed in previous studies of the digestive system of P. depicta, a well-documented predator of B. striata [44,46]. Therefore, although B. striata may constitute a viable reservoir host for a potential fish-infecting apicomplexan, a conceivable transmission route remains unclear, warranting future studies. Such studies should screen B. striata for additional developmental stages and examine fish for parasite development.

Apicomplexa can act as pathogenic agents to their intermediate hosts, as merogonic stages alone may cause significant pathology. For example, in Plasmodium spp. infections, humans—the intermediate host—can suffer severe pathology from the merogonic stages, whereas the definitive host, Anopheles mosquitoes, remains largely unaffected [86]. Similarly, Merocystis kathae, an apicomplexan that infects the renal tissues of its definitive host—the common whelk B. undatum—without apparent pathology, has been implicated in the collapse of populations of its intermediate host, the Iceland scallop Chlamys islandica [22]. In this study, although infection intensity was not quantitatively assessed, even the specimens with a higher parasite load did not show signs of significant tissue damage or organ impairment. However, the absence of observed pathology in this study does not exclude the possibility of gamogonial and sporogonial stages exhibiting pathogenic potential to B. striata, if the life cycle of the parasite is actually monoxenous. If the life cycle is heteroxenous, the possibility of gamogonial and sporogonial stages exhibiting pathogenic potential to a definitive host cannot be excluded. Therefore, additional research is needed to understand the life cycle of this newly discovered ichthyocolid parasite and its interaction with the host.