First report of Perkinsus olseni infections in blood cockles Tegillarca granosa on the south coast of Korea.

The protozoan parasite Perkinsus olseni has become a focus of attention since it has been responsible for mass mortalities and economic losses in a wide range of bivalve hosts globally. The P. olseni host range along the south coast of Korea may extend beyond what was previously understood, and blood cockles in the Family Arcidae are also suggested to be potential hosts of P. olseni. In the present study, we applied histology and molecular techniques to identify Perkinsus sp. infections in the blood cockles Tegillarca granosa, which have been commercially exploited on the south coast of Korea for several decades. Histology and molecular techniques, including genus-specific immunofluorescence assay, species-specific fluorescence in situ hybridization, and phylogeny based on the ribosomal DNA internal transcribed spacer region revealed that T. granosa is infected by P. olseni, although the prevalence was low (0.5%). Histology revealed massive hemocyte infiltrations in the mantle, gill, and digestive gland connective tissues, indicating that the infection exerts negative impacts on the host cockles.

The impact of climate change and pollution on trematode-bivalve dynamics.

Coastal ecosystems and their marine populations are increasingly threatened by global environmental changes. Bivalves have emerged as crucial bioindicators within these ecosystems, offering valuable insights into biodiversity and overall ecosystem health. In particular, bivalves serve as hosts to trematode parasites, making them a focal point of study. Trematodes, with their life cycles intricately linked to external factors, provide excellent indicators of environmental changes and exhibit a unique ability to accumulate pollutants beyond ambient levels. Thus, they act as living sentinels, reflecting the ecological condition of their habitats. This paper presents a comprehensive review of recent research on the use of bivalve species as hosts for trematodes, examining the interactions between these organisms. The study also investigates the combined impact of trematode infections and other pollutants on bivalve molluscs. Trematode infections have multifaceted consequences for bivalve species, influencing various aspects of their physiology and behavior, including population-wide mortality. Furthermore, the coexistence of trematode infections and other sources of pollution compromises host resistance, disrupts parasite transmission, and reduces the abundance of intermediate hosts for complex-living parasites. The accumulation process of these parasites is influenced not only by external factors but also by host physiology. Consequently, the implications of climate change and environmental factors, such as temperature, salinity, and ocean acidification, are critical considerations. In summary, the intricate relationship between bivalves, trematode parasites, and their surrounding environment provides valuable insights into the health and sustainability of coastal ecosystems. A comprehensive understanding of these interactions, along with the influence of climate change and environmental parameters, is essential for effective management and conservation strategies aimed at preserving these delicate ecosystems and the diverse array of species that rely on them.

Identification of glycine betaine as a host-derived molecule required for the vegetative proliferation of the protozoan parasite Perkinsus olseni.

Perkinsus olseni is an industrially significant protozoan parasite of Manila clam, Ruditapes philippinarum. So far, various media, based on Dulbecco's Modified Eagle Medium and Ham's F-12 nutrient mixture with supplementation of fetal bovine serum (FBS), have been developed to proliferate the parasitizing trophozoite stage of P. olseni. The present study showed that P. olseni did not proliferate in FBS-deficient Perkinsus broth medium (PBMΔF), but proliferated well in PBMΔF supplemented with tissue extract of host Manila clams, indicating that FBS and Manila clam tissue contained molecule(s) required for P. olseni proliferation. Preliminary characterization suggested that the host-derived molecule(s) was a heat-stable molecule(s) with a molecular weight of less than 3 kDa, and finally a single molecule required for the proliferation was purified by high-performance liquid chromatography processes. High-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses identified this molecule as glycine betaine (=trimethylglycine), and the requirement of this molecule for P. olsseni proliferation was confirmed by an assay using chemically synthesized, standard glycine betaine. Although glycine betaine was required for the proliferation of all examined Perkinsus species, supplementation of glycine betaine precursors, such as choline and betaine aldehyde, enhanced the proliferation of 4 Perkinsus species (P. marinus, P. chesapeaki, P. mediterraneus and P. honshuensis), but not of 2 others (P. olseni and P. beihaiensis). Thus, it was concluded that the ability to biosynthesise glycine betaine from its precursors varied among Perkinsus species, and that P. olseni and P. beihaiensis lack the ability required to biosynthesize glycine betaine for proliferation.

Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

Feeding of mussel-associated leeches Hemiclepsis kasmiana on bitterling embryos: Novel interaction between parasites in a shared host.

We investigated an interaction between bitterlings and a parasitic leech Hemiclepsis kasmiana in freshwater mussel hosts. We found that leeches fed on bitterling eggs and embryos; this may exert a considerable negative effect on bitterling fitness. Host choices by females of three bitterling species may be differently affected by the presence of leeches within mussels; Tanakia limbata apparently avoided laying eggs in infested mussels while T. lanceolata and Acheilognathus rhombeus did not. Our novel findings suggest that relationships between the parasitic leech and the host mussel may be context dependent.

Pathology and infectious agents of unionid mussels: A primer for pathologists in disease surveillance and investigation of mortality events.

Freshwater mussels are one of the most imperiled groups of organisms in the world, and more than 30 species have gone extinct in the last century. While habitat alteration and destruction have contributed to the declines, the role of disease in mortality events is unclear. In an effort to involve veterinary pathologists in disease surveillance and the investigation of freshwater mussel mortality events, we provide information on the conservation status of unionids, sample collection and processing techniques, and unique and confounding anatomical and physiological differences. We review the published accounts of pathology and infectious agents described in freshwater mussels including neoplasms, viruses, bacteria, fungi, fungal-like agents, ciliated protists, Aspidogastrea, Digenea, Nematoda, Acari, Diptera, and Odonata. Of the identified infectious agents, a single viral disease, Hyriopsis cumingii plague disease, that occurs only in cultured mussels is known to cause high mortality. Parasites including ciliates, trematodes, nematodes, mites, and insects may decrease host fitness, but are not known to cause mortality. Many of the published reports identify infectious agents at the light or ultrastructural microscopy level with no lesion or molecular characterization. Although metagenomic analyses provide sequence information for infectious agents, studies often fail to link the agents to tissue changes at the light or ultrastructural level or confirm their role in disease. Pathologists can bridge this gap between identification of infectious agents and confirmation of disease, participate in disease surveillance to ensure successful propagation programs necessary to restore decimated populations, and investigate mussel mortality events to document pathology and identify causality.

Factors at multiple scales drive parasite community structure.

Understanding how ecological communities are assembled remains a key goal of ecosystem ecology. Because communities are hierarchical, factors acting at multiple scales can contribute to patterns of community structure. Parasites provide a natural system to explore this idea, as they exist as discrete communities within host individuals, which are themselves part of a community and metacommunity. We aimed to understand the relative contribution of multi-scale drivers in parasite community assembly and assess how patterns at one level may mask those occurring at another. Specifically, we wanted to disentangle patterns caused by passive sampling from those determined by ecological drivers, and how these vary with scale. We applied a Markov Random Fields model and assessed measures of ß-diversity and nestedness for 420 replicate parasite infracommunities (parasite assemblages in host individuals) across two freshwater mussel host species, three sites and two time periods, comparing our results to simulations from four different ecologically relevant null models. We showed that ß-diversity between sites (explaining 25% of variation in parasite distribution) and host species (41%) is greater than expected, and ß-diversity between individual hosts is smaller than expected, even after accounting for parasite prevalence and characteristics of host individuals. Furthermore, parasite communities were significantly less nested than expected once parasite prevalence and host characteristics were both accounted for, but more nested than expected otherwise, suggesting a degree of modularity at the within-host level that is masked if underlying host and parasite characteristics are not taken into account. The Markov Random Fields model provided evidence for possible competitive within-host parasite interactions, providing a mechanism for the observed infracommunity modularity. An integrative approach that examines factors at multiple scales is necessary to understand the composition of ecological communities. Furthermore, patterns at one level can alter the interpretation of ecologically important drivers at another if variation at higher scales is not accounted for.

Twenty-Five Years of PSP Toxicity in Galician (NW Spain) Bivalves: Spatial, Temporal, and Interspecific Variations.

Twenty-five years of paralytic shellfish poisoning (PSP) toxicity in Galician bivalves have been studied. PSP was detected in 4785 out of 73,740 samples of the commercially important bivalve species analyzed from 1995 to 2020. Its general prevalence in the area was 6.5%. Only 1.6% of all samples tested were over the regulatory limit (incidence). The maximum level of PSP in the area, 40,800 µg STX 2HCl-eq kg-1, was recorded in raft mussels from Bueu (PON-II, Pontevedra) in December 2005. The highest maximum PSP values were found in mussels, which were mostly affected by Gymnodinium catenatum, but not those of prevalence and incidence which were recorded in clams, mostly affected by Alexandrium. Average levels in mussels were higher than in any other studied species. Spatially, in general, the prevalence, incidence, maximum, and average PSP toxicity during episodes tend to decrease from south to northeast, but some hot points with high levels can be identified. PCA analysis separates the southern rías, associated to G. catenatum blooms, from the middle and northern ones, associated to Alexandrium blooms. Along the year, two main peaks of the four variables are observed, the first one in late autumn-winter and the other in summer, the summer peak being much more important for the infaunal species than for raft mussels. In the seasonal pattern obtained by time series analysis of the average PSP toxicity, the autumn-winter peak was only maintained (and very reduced) in the southern rías, indicating that this peak is seasonally much less important than the summer peak. The observed seasonality is expected based on the timing of the blooms of the two PSP-producing phytoplankton groups present in the area. Over the 25 years of monitoring, large differences in PSP toxicity have been observed. Apart from some special years, an ascending trend in prevalence and incidence seems to be present from 2011 to 2020. No trend seems to exist during the same period for average or maximum toxicity.

Molecular and microscopic identification of Eomarteilia granula infection in Manila clam Ruditapes philippinarum off the south coast of Korea.

A report on the new species Eomarteilia (=Marteilia) granula infecting Manila clam Ruditapes philippinarum from Japan in 2014 suggests the possibility of E. granula infecting other Manila clam populations in the Northwest Pacific region, including Korea. In this study, we report the first infections by E. granula in Manila clams off the south coast of Korea. Histology revealed Marteilia-like plasmodia in the digestive tubule epithelia. Tissue imprints demonstrated that each parasite sporangium enclosed 4 spores and transmission electron microscopy (TEM) revealed ultrastructure of primary cells enclosing secondary cells, which contained spores. Mature spores consisted of 3 sporoplasms: outermost, intermediate, and innermost. The outermost sporoplasm showed a peripheral electron-dense monolayer characteristic of E. granula. The 18S rDNA amplified from the Marteilia-like parasite yielded 1784-bp PCR amplicon sequences which were 99.8% similar to that of E. granula previously reported (as M. granula) from Japan. In the molecular phylogenetic analysis, the novel Marteilia-like organism formed a well-supported clade with E. granula. Accordingly, we concluded that the novel Marteilia-like parasite that we found infecting some Korean Manila clams is Eomarteilia granula. Field surveys revealed that the infection was limited to clams of the south coast of Korea, with the prevalence ranging from 3.3 to 5.0%.

Evaluation of the Ruditapes decussatus immune response after differential injected doses of Perkinsus olseni.

Ruditapes decussatus is a highly appreciated clam species, with a high demand, especially in European countries. However, natural populations of this species have been affected by over-exploitation, abiotic factors and also by diseases mainly caused by bacteria, virus and protozoans. Within the latter group of pathogens, Perkinsus spp. has been attracting attention for causing relevant mortality rates on bivalves around the world. From all species of this genus, Perkinsus olseni is one of the most impactful for R. decussatus, as it may cause chronic infection with fatal consequences under adverse conditions. Therefore, in this study, naïve clams were injected with two doses of P. olseni in order to better understand the response of R. decussatus against this parasite. So, one of the main goals of this experiment was to evaluate the differential response of the clams at gene expression level after injection and also to monitor the possible mortalities related to parasite. Results indicated that the methods applied for injection and detection of parasite load are effective. Furthermore, altogether, R. decussatus gene expression studies indicate differences on expression of some of the addressed genes among distinct levels of infection over time, which require further studies to confirm if these genes could be used as molecular markers of P. olseni infection. According to the results obtained, Adiponectin-C1q seems to be a promising gene to be used as molecular marker of chronic infection in clams haemolymph.

Invasion of fish parasite Prosorhynchoides ozakii (Trematoda: Bucephalidae) into Lake Kasumigaura and surrounding rivers of eastern Japan.

In 2019 to 2021, the golden mussel Limnoperna fortunei and several freshwater fishes were sampled from 22 sites of the Tone River system including Lake Kasumigaura, Honshu, Japan, to examine the invasion of bucephalid trematodes. The parasite species identification was performed by morphological observation and DNA barcoding based on the sequences of nuclear 28S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1). A total of 1719 mussels were collected from 10 sites, and trematode-infected mussels were detected from 8 sites with prevalences between 0.3 and 42.9%. The sporocysts and cercariae were identified as Prosorhynchoides ozakii, a newly introduced species in the river system. A total of 700 fish individuals belonging to 24 species were collected from 15 sites. Two species of catfishes (Silurus asotus and Ictalurus punctatus) harbored mature or immature adults of Pr. ozakii in the intestine with prevalences between 8.3 and 20% including both host species. The metacercariae of Pr. ozakii were found from the fins and epidermis of 13 fish species from 10 sites (prevalence 4.8-100%). Fishes were heavily infected with metacercariae in fins, which were surrounded by the infiltration of hemocytes and rodlet cells. A population genetic analysis of Pr. ozakii did not show an obvious bottleneck, suggesting the possibility that the parasite was intentionally and repeatedly introduced into the river system.

Histopathological survey of parasites harboured by the clam Tawera elliptica (Lamarck, 1818) from Chiloé Archipelago, southeastern Pacific.

Tawera elliptica (commonly known as "Almeja Juliana", is a venerid clam that inhabits sandy bottoms and is distributed from Valparaíso on the Pacific coast up to the Mar del Plata area along the Atlantic coast. Harvests of this clam have declined substantially over the last decade. Therefore, an analysis of common parasites and pathological conditions of this clam was undertaken along with histopathology. Monthly samples were prepared for routine histology for examination under light and electron microscopy. T. elliptica has a sex ratio of 1:1 and the relationship between the shell length and the wet tissue weight is not significantly different between females and males. The maximum values for de condition index and meat yield were found during the austral winter. The following parasites (and their overall prevalence) were detected: intracellular microcolonies of bacteria in digestive gland (22.9%), intestinal epithelium (9.3%) and gills (3.17%), an unidentified cyst in gills (59,3%), a Steinhausia-like intraoocytic microsporidian (5.2%), Gregarine spores (41.3%), ciliated protozoa (16.7%), two metazoa, a Paravortex like flatworm (4.3%), and a digenean trematode (8%). The monthly mean intensity of the most relevant parasites was between 2.3 and 35.6 for digestive gland intracellular microcolonies of bacteria (IMC), 0-5.1 for intestinal epithelium IMC, 0-2 for branchial IMC and 0 - 48 for intraoocytic microsporidium. The prevalence and the infection intensity were low-to very low, and no World Organisation for Animal Health OIE listed parasite was detected. It is concluded that this is a healthy clam, and no disease risks for the cultivation are visualized at present. However, IMC at high prevalence and intensities of infection could be potentially impactful, and the intraoocytic microsporidian could jeopardize reproduction if present in high intensities of infection.

Morphological, histopathological and molecular assessments of Prosorhynchoides sp. (Digenea: Bucephalidae) in Perna perna (Bivalvia: Mytilidae) mussels sampled off the coast of Rio de Janeiro, southeastern Brazil.

Mussel production is expanding worldwide, and in Brazil the main species currently produced is the mussel Perna perna. Bucephalid trematodes have been recorded in P. perna but their larval identification is problematic. In this context, the aims of this paper were to evaluate the prevalence of bucephalids in P. perna, perform taxonomic and phylogenetic trematode studies, and analyze potential histopathological alterations in the infected host. Mussels obtained by fishers from Guanabara Bay, Rio de Janeiro, Brazil were weighed and measured, and internal organ tissues and parasites were collected. Of the 69 analyzed mussels, 24.6 % (17/69) were parasitized by bucephalid larvae. Sporocysts were located mainly in host mantle. Mussels presented sporocysts and cercaria within the connective tissue of mantle, all without associated inflammatory reactions. Parasite loads varied from less than 5 % to > 50 % of parasitized tissue. Histopathological examinations indicated that male or female gonads were not observed in 77 % (10/13) of parasitized mussels and in 4 % (2/56) identified as non-parasitized in the histology but previously classified as parasitized in the stereomicroscopic analysis. Thus, the absence of gonads may be associated with parasitism. Prosorhynchoides sp. is reported herein for the first time in mussels sampled on the coast of Rio de Janeiro, with genetic and histological data reported for the intermediate host, sporocysts and cercariae. New 28S rDNA, 18S rDNA and ITS1, 5.8S and ITS2 sequences are provided.

Discovery of the parasite Marteilia cocosarum sp. nov. In common cockle (Cerastoderma edule) fisheries in Wales, UK and its comparison with Marteilia cochillia.

Diseases of bivalve molluscs caused by paramyxid parasites of the genus Marteilia have been linked to mass mortalities and the collapse of commercially important shellfish populations. Until recently, no Marteilia spp. have been detected in common cockle (Cerastoderma edule) populations in the British Isles. Molecular screening of cockles from ten sites on the Welsh coast indicates that a Marteilia parasite is widespread in Welsh C. edule populations, including major fisheries. Phylogenetic analysis of ribosomal DNA (rDNA) gene sequences from this parasite indicates that it is a closely related but different species to Marteilia cochillia, a parasite linked to mass mortality of C. edule fisheries in Spain, and that both are related to Marteilia octospora, for which we provide new rDNA sequence data. Preliminary light and transmission electron microscope (TEM) observations support this conclusion, indicating that the parasite from Wales is located primarily within areas of inflammation in the gills and the connective tissue of the digestive gland, whereas M. cochillia is found mainly within the epithelium of the digestive gland. The impact of infection by the new species, here described as Marteilia cocosarum n. sp., upon Welsh fisheries is currently unknown.

Gonadal cycle, reproductive indices and detection of parasitism in the clam Ameghinomya antiqua in natural beds of importance for fisheries.

The clam Ameghynomia antiqua is a highly important resource for fisheries due to its high catches volume. It is the bivalve mollusc with the highest fisheries landings from natural beds on the Pacific coast of southern South America; however, studies of the reproductive conditions of this species are scarce and date back many years. The object of the present work was to evaluate the reproductive characteristics of the species, analysing its gametogenic and gonadal cycle, and reproductive indices, in fishery locations that present the natural beds with the highest fisheries catches, as well as parasite loads in the species. The gonads of the individuals were sampled monthly over a year and classified into one of three states called: "in development", "ripe" and "spawned". Synchrony between the sexes was observed in the indicators of the Gonadosomatic Index and Condition Index in each of the locations, although no synchrony was observed between locations. In the gametogenic cycle, the "ripe" state was observed in females in spring-summer, followed by rapid recovery to new development of the gonads; in males the "ripe" state was observed throughout the year. It was observed that males entered the "spawned" state one month ahead of females. The presence of digenean parasites in the state of metacercariae was detected in the gonads and mantle. No significant differences were found in the prevalence or intensity of infection when analysed by sex and month. The metacercariae were identified, by sequencing of three DNA regions, as belonging to the clade shared by species of the genus Parvatrema and close to the Gymnophalloides; both these genera belong to the family Gymnophallidae of the superclass Digenea. Infection was observed to reduce the gonadal tissue, in some cases causing castration. This is the first record of the presence of these parasites of A. antiqua, with genetic identification at genus level. These results are relevant for act proper management of this resource, which is important for fishing.

Molecular detection of a novel perkinsid associated with the deep-sea clam Phreagena okutanii.

Based on environmental DNA surveys, it is widely held that phylogenetically diverse protists exist in chemosynthetic ecosystems. However, knowledge regarding the protists associated with the endemic animals inhabiting these environments is still very limited. In the present study, utilizing polymerase chain reaction (PCR) techniques, we detected fragments of the small subunit ribosomal RNA (SSU rRNA) gene and the internal transcribed spacer (ITS) region of the ribosomal RNA genes from a particular protist in the gills of the vesicomyid clam Phreagena okutanii (formerly described as Calyptogena okutanii), a representative animal in chemosynthetic ecosystems. Based on the phylogeny of the SSU rRNA gene, the organism in question belongs to the genus Perkinsus, which is exclusively composed of protistan parasites infecting mollusks. Intriguingly, based on the ITS phylogeny, this protist was not related to any known Perkinsus species and was deeply branched within the radiation of this genus, thus represents an undescribed species. In addition, the protist detected by PCR was localized to the intercellular spaces in the gills of the host clam with fluorescence in situ hybridization. Although the ecological significance of this novel deep-sea perkinsid remains unclear, our present findings may provide important insights into the diversity of the genus Perkinsus.

New insights about Haplosporidium pinnae and the pen shell Pinna nobilis mass mortality events.

Since early autumn 2016, Mass Mortality Events (MME) have drastically impacted the population of the fan mussel Pinna nobilis in the Mediterranean Sea. Haplosporidium pinnae, a newly described Haplosporidian species, has been considered the causative agent of the mortality outbreaks in association to opportunistic bacterial pathogens. In the present study, we first reported a cytological description of H. pinnae in moribund specimens of P. nobilis which were collected in the Gulf of Taranto (Ionian Sea, Italy) during summer 2018. Different life-cycle stages of the parasite, including uni- and binucleate cells, small plasmodia, big multinucleate plasmodia and sporocysts with spores, were detected in all the examined animals and most of the parasite cells were present in gills, mantle and digestive gland, while the spores were found only in the latter organ. Histology and molecular biology were also performed, confirming the nature of the infectious agent, as already reported in the area. Additionally, molecular study revealed the presence of bacteria from the Mycobacterium ulcerans - M. marinum complex but no evident macroscopical or microscopical lesions, just as no bacteria referred to Mycobacterium were observed by histology. In conclusion, the present study aimed to provide further contributions to the understanding of the mortality of P. nobilis, pointing to the role of the cytological method of investigation both for diagnostic and epidemiological purposes, and discussing the current epidemic situation in the Adriatic sea.

Pathophysiological Responses of Pinna nobilis Individuals Enlightens the Etiology of Mass Mortality Situation in the Mediterranean Populations.

Due to the rapid decrease of Pinna nobilis populations during the previous decades, this bivalve species, endemic in the Mediterranean Sea, is characterized as 'critically endangered'. In addition to human pressures, various pathogen infections have resulted in extended reduction, even population extinction. While Haplosporidium pinnae is characterized as one of the major causative agents, mass mortalities have also been attributed to Mycobacterium sp. and Vibrio spp. Due to limited knowledge concerning the physiological response of infected P. nobilis specimens against various pathogens, this study's aim was to investigate to pathophysiological response of P. nobilis individuals, originating from mortality events in the Thermaikos Gulf and Lesvos and Limnos islands (Greece), and their correlation to different potential pathogens detected in the diseased animals. In isolated tissues, several cellular stress indicators of the heat shock and immune response, apoptosis and autophagy, were examined. Despite the complexity and limitations in the study of P. nobilis mortality events, the present investigation demonstrates the cumulative negative effect of co-infection additionally with H. pinnae in comparison to the non-presence of haplosporidian parasite. In addition, impacts of global climate change affecting physiological performance and immune responses result in more vulnerable populations in infectious diseases, a phenomenon which may intensify in the future.

The colorful mantle of the giant clam Tridacna squamosa expresses a homolog of electrogenic sodium: Bicarbonate cotransporter 2 that mediates the supply of inorganic carbon to photosynthesizing symbionts.

Giant clams live in symbiosis with phototrophic dinoflagellates, which reside extracellularly inside zooxanthellal tubules located mainly in the colourful and extensible outer mantle. As symbiotic dinoflagellates have no access to the ambient seawater, they need to obtain inorganic carbon (Ci) from the host for photosynthesis during illumination. The outer mantle has a host-mediated and light-dependent carbon-concentrating mechanism to augment the supply of Ci to the symbionts during illumination. Iridocytes can increase the secretion of H+ through vacuolar H+-ATPase to dehydrate HCO3- present in the hemolymph to CO2. CO2 can permeate the basolateral membrane of the epithelial cells of the zooxanthellal tubules, and rehydrated back to HCO3- in the cytoplasm catalysed by carbonic anhydrase 2. This study aimed to elucidate the molecular mechanism involved in the transport of HCO3- across the apical membrane of these epithelial cells into the luminal fluid surrounding the symbionts. We had obtained the complete cDNA coding sequence of a homolog of electrogenic Na+-HCO3- cotransporter 2 (NBCe2-like gene) from the outer mantle of the fluted giant clam, Tridacna squamosa. NBCe2-like gene comprised 3,399 bp, encoding a protein of 1,132 amino acids of 127.3 kDa. NBCe2-like protein had an apical localization in the epithelial cells of zooxanthellal tubules, denoting that it could transport HCO3- between the epithelial cells and the luminal fluid. Furthermore, illumination augmented the transcript level and protein abundance of NBCe2-like gene/NBCe2-like protein in the outer mantle, indicating that it could mediate the increased transport of HCO3- into the luminal fluid to support photosynthesis in the symbionts.