Histopathologic Lesions in Bivalve Mollusks Found in Portugal: Etiology and Risk Factors

: Bivalve mollusks are an important resource due to their socioeconomic value and to the historical and genetic value of some species. Two nationally important oyster species-Portuguese oyster ( Crassostrea angulata ) and Japanese oyster ( Crassostrea gigas ) from distinctive areas in Portugal were studied to evaluate their sanitary status. Oysters were sampled from four different sites in Portugal. Oysters collected from Japanese oyster populations were cultivated in a strong ocean-inﬂuenced environment and Portuguese oyster populations were cultivated in wild-beds. The histopathological examination of both oyster species revealed the presence of parasites in gills, mantle epithelium, digestive gland tubules and connective tissue, with a moderate prevalence. In both populations was observed hemocytosis in the connective tissue, edema and metaplasia in the digestive gland and tissues necrosis. In wild populations from Sado and Mira estuaries the prevalence of mud blisters and gill lesions were higher than from populations produced on 0.50 m tables from mudﬂats. Biosecurity measures and diagnostic techniques are fundamental to control pathogenic agents, including the identiﬁcation of pathogens at an early stage in their life cycles. This will prevent diseases and improve pathogen reduction on transport of animals from different countries and regions to new production areas to avoid the transmission of diseases.


Introduction
In Portugal, in the late 20th century, the two most important commercial oysters were the flat Oyster (Ostrea edulis) and the Portuguese Oyster (Crassostrea angulata) [1]. Initially, flat oyster commerce was more important, but in the 20th century it was replaced by the Portuguese oyster. The culture of bivalve mollusks is an activity with high expression in Portuguese aquaculture. It represents 55% of the whole production, being the main species, clams, oysters and mussels [2].
There was a significant change in bivalve mollusks production. Initially, it was exclusively chosen for semi-intensive production in large areas. Nevertheless, at this moment there is an intensification of production in less area, increasing bivalve mollusks density. This option results in lower growth rates, lower product quality, lower fertility rates and increasing diseases [3].
Several diseases can affect different bivalve mollusks species and their production. Epizootics caused by fungi, viruses and protozoa can affect the entire bivalve production, as in Portugal and France in the 1970s where massive mortality caused by an iridovirus affected the Portuguese oyster production. Since 2009 a herpes virus (OsHV-1) has been the cause of massive mortalities in Japanese oyster (Crassostrea gigas), first in France and all over the most important European production countries of this species, including Portugal [3]. Japanese oyster aquaculture is a major primary production sector in many countries, but the industry has been threatened by mortality events for the last five decades [14]. In Portuguese and Japanese oyster, the main pathogens are virus, namely iridovirus, responsible for gill disease that affected Portuguese oyster production in the 1970's, and herpes viruses, namely Ostreid-herpesvirus 1 µvar (OsHV-1) that is related with summer oyster disease and affects the Japanese and Portuguese oyster populations [15]. From the first detection in the Eastern oysters (Crassostrea virginica) [14] in 1972, herpes virus has been identified in at least 20 bivalve species and has caused massive mortalities [14]. Herpes virus infecting bivalve mollusks are virulent pathogens of both larval and seed oysters. Mortalities of juvenile oysters associated with detection of herpes viruses have been reported from France, New Zealand, Spain and the USA [16][17][18][19].
In flat oyster, there are important diseases, including bonamiosis that is caused by Bonamia ostrea and Marteilia refringens that causes marteliosis and viruses such as Ostreidherpesvirus 1 µvar [20,21].
Regarding mussels Mytilus sp., the main pathogens are protozoa, such as Marteilia refringens that is the causative agent of marteliosis and copepods, such as Mytilicola intestinalis which causes red worm disease [22,23].
Japanese clam, European clam and cockles have been mainly affected by protozoa, such as Perkinsus olseni, the causative agent of Perkinsiosis [24][25][26][27] and Haplosporidium tapetis [26,28]. Perkinsus olseni is responsible for the high mortality of cockleshell populations. Perkinsiosis is caused by the protozoan P. olseni. This disease is a very common disease in clams and has caused massive mortalities in clam populations, contributing to the decrease in their production and consequently affecting their socioeconomic value [29]. Typical lesions that occur include cell disorganization, autolysis and necrosis of cells, both within and near the lesion [24].
The high prevalence of this pathogen in clams can be attributed to several factors, such as degraded sediments, high animal densities per unit area, lack of physical barriers between different culture beds, and transfer of animals carrying the disease [1].
Biotoxins can affect the nervous, the gastrointestinal and the respiratory systems, being fatal in some severe cases. They originate in various microalgae and dinoflagellates that produce these toxic elements in their metabolism. Mollusk bivalves are filter-feeder animals, and they can retain and accumulate those microalgae and the biotoxins, and as such, they can be a source of contamination for humans [30,31].
The accumulation of biotoxins in aquatic organisms depends on their feeding activity, on their metabolism, and on their elimination rate, modifying the transfer of toxins in the trophic chain [32].
Contaminant metals can cause several problems in humans, and the contamination of bivalve mollusks with different concentrations of heavy metals can be dangerous, namely lead, cadmium and mercury. They can accumulate high concentrations from water, and from the sediments and they have a wide bioaccumulation range of heavy metals depending on the species [33][34][35].
The aim of the present work was to study the sanitary condition in oyster production in four different populations of oysters. The objective was to describe the general health status, studying anatomo and histopathological lesions and parasites, in the Portuguese oyster (Crassostrea angulata) and Japanese oyster (Crassostrea gigas) from different production areas in Portugal, in order to evaluate their health condition.

Materials and Methods
The map (Figure 1) shows the four different areas where oysters were collected.

Materials and Methods
The map ( Figure 1) shows the four different areas where oysters were collected. Oysters (9-11 cm, aged 24 months old) were sampled from four different sites of the Portuguese coast. Crassostrea gigas is an exotic species that was introduced in Portugal, and so, its presence is restricted to some production areas (1 and 4). In areas 2 and 4, Crassostrea angulata were collected from wild beds.
Portuguese oysters from Sado estuary (n = 30) and Mira estuary (n = 30) and Japanese oyster from Alvor lagoon (n = 30) and Aveiro lagoon (n = 30) were collected in May 2019. In May, in the four sites, oysters have already spawned and are more susceptible to environment stressors and increasing chances of histopathological lesions.
Individuals were randomized collected in production areas present in four mesotidal estuarine areas indicated in the map (Figure 1). The areas are located on the west Portuguese coast (1, 2 and 3) and in the south Portuguese coast (4). All the four production areas are under a strong oceanic influence because of the proximity to the coastal water and they have an influence of fresh water. For production, oysters are placed inside plastic bags that are put on tables with a distance of 0.50 m from the bottom in areas 1 and 4. In Sado and Mira estuaries, oysters were collected in intertidal mudflats where oyster beds are located. In Aveiro lagoon there is a lagoon with several channels, stretching for 45 Km. Sado stuary includes a section of river, marshlands and channels and the dynamic tide extends for 65 Km. Mira estuary is a narrow estuary of the "Ria" pattern that extends for 40 km. Alvor lagoon has a water body and a mesotidal shallow lagoon that extends for 15 Km.  Oysters (9-11 cm, aged 24 months old) were sampled from four different sites of the Portuguese coast. Crassostrea gigas is an exotic species that was introduced in Portugal, and so, its presence is restricted to some production areas (1 and 4). In areas 2 and 4, Crassostrea angulata were collected from wild beds.
Portuguese oysters from Sado estuary (n = 30) and Mira estuary (n = 30) and Japanese oyster from Alvor lagoon (n = 30) and Aveiro lagoon (n = 30) were collected in May 2019. In May, in the four sites, oysters have already spawned and are more susceptible to environment stressors and increasing chances of histopathological lesions.
Individuals were randomized collected in production areas present in four mesotidal estuarine areas indicated in the map ( Figure 1). The areas are located on the west Portuguese coast (1, 2 and 3) and in the south Portuguese coast (4). All the four production areas are under a strong oceanic influence because of the proximity to the coastal water and they have an influence of fresh water. For production, oysters are placed inside plastic bags that are put on tables with a distance of 0.50 m from the bottom in areas 1 and 4. In Sado and Mira estuaries, oysters were collected in intertidal mudflats where oyster beds are located. In Aveiro lagoon there is a lagoon with several channels, stretching for 45 Km. Sado stuary includes a section of river, marshlands and channels and the dynamic tide extends for 65 Km. Mira estuary is a narrow estuary of the "Ria" pattern that extends for 40 km. Alvor lagoon has a water body and a mesotidal shallow lagoon that extends for 15 Km.
Oyster were collected during low tide at a distance from the mouth of 1 Km (1) After being collected, oysters were immediately transported on ice to the laboratory. To survey the presence of lesions, parasites and diseases anatomic and histopathological examinations were used as main diagnostic methods. Oysters were opened with a knife and the samples, including all organs and tissues, were collected with a tweezer and a scalpel, Tissue samples were prepared for histopathology processing, following the protocol used in Pathology laboratory of IPMA. Samples were fixed in Davidson's fixative for 48 h, dehydrated and embedded in paraffin. Sections with 5 µm thick were stained with Hematoxylin-Eosin and mounted on a microscopic glass slide.
Histological preparations were carefully examined under light microscopy (Motic BA-410), looking for the presence of lesions and parasites in oysters.
After being collected, oysters were immediately transported on ice to the laboratory. To survey the presence of lesions, parasites and diseases anatomic and histopathological examinations were used as main diagnostic methods. Oysters were opened with a knife and the samples, including all organs and tissues, were collected with a tweezer and a scalpel, Tissue samples were prepared for histopathology processing, following the protocol used in Pathology laboratory of IPMA. Samples were fixed in Davidson's fixative for 48 h, dehydrated and embedded in paraffin. Sections with 5 μm thick were stained with Hematoxylin-Eosin and mounted on a microscopic glass slide.
Histological preparations were carefully examined under light microscopy (Motic BA-410), looking for the presence of lesions and parasites in oysters.

Results
In most of the samples no lesions were observed in organs (    Oyster were collected during low tide at a distance from the mouth of 1 Km (1) After being collected, oysters were immediately transported on ice to the laboratory. To survey the presence of lesions, parasites and diseases anatomic and histopathological examinations were used as main diagnostic methods. Oysters were opened with a knife and the samples, including all organs and tissues, were collected with a tweezer and a scalpel, Tissue samples were prepared for histopathology processing, following the protocol used in Pathology laboratory of IPMA. Samples were fixed in Davidson's fixative for 48 h, dehydrated and embedded in paraffin. Sections with 5 μm thick were stained with Hematoxylin-Eosin and mounted on a microscopic glass slide.
Histological preparations were carefully examined under light microscopy (Motic BA-410), looking for the presence of lesions and parasites in oysters.

Results
In most of the samples no lesions were observed in organs (Figure 2A     In samples of C. gigas from Aveiro lagoon, it was observed only 3% of oysters with edema, 3% with shell disease and 10% with M. ostrea. Finally, in samples from Aveiro lagoon, a prevalence of 13% Trichodina sp. infections, 13% Ancistrocoma sp. infections and 10% Mytilicola sp. infections. Concerning tissue lesions and morphological changes, 26% of wild oysters showed necrosis, 45% showed hemocytosis, indicating an inflammatory process, mainly in connective tissue. It was also observed a prevalence of 3% of ceroidosis in the connective tissue, edema in 15 % and 18% showed metaplasia in the digestive gland. Regarding farmed oysters, the following lesions and prevalence were found: necrosis (6%) in different tissues, hemocytosis (27%), ceroidosis (5%), edema (18%) and metaplasia (5%).
Samples of wild C. angulata from Sado estuary show the following results (Tables 1 and 2): 20% presented mud blisters, mainly caused by Polydora sp.; 33% with gills lesions; 10% had shell disease associated with the presence of the fungus Ostracoblabe implexa; and 17% showed Myicola ostrea copepods.
Portuguese oysters sampled in Sado estuary, it was found a prevalence of 30% infections with Trichodina sp., characterized by a shape similar to a disc, a circlet of eosinophilic denticles, a ciliary fringe and a horseshoe shaped nucleous, in gills and mantle epithelium. A prevalence of 20% infections with Ancistrocoma sp., characterized by a Spindle-shaped ciliates with large, granular and polymorphic nuclei, in the digestive gland tubules and connective tissue. Copepods (Mytilicola sp.), characterized by an elongated cylindrical shape and a redish colour were observed within the intestine of oysters with the prevalence of 17% [36].
In samples from Mira estuary, it was found a prevalence of 13% infections with Trichodina sp., 30% infections with Ancistrocoma sp. and 3% infections with Mytilicola sp.
Finally, in samples of C. gigas from Alvor lagoon 10% presented mud blisters.
In farmed oysters sampled from Alvor lagoon, it was observed a prevalence of 23% Trichodina sp. infections and a prevalence of 27% Ancistrocoma sp. Copepod (Mytilicola sp.) were observed within the intestine of oysters at a prevalence of 3%. In samples of C. gigas from Aveiro lagoon, it was observed only 3% of oysters with edema, 3% with shell disease and 10% with M. ostrea.
Samples of wild C. angulata from Sado estuary show the following results (Tables 1 and 2): 20% presented mud blisters, mainly caused by Polydora sp.; 33% with gills lesions; 10% had shell disease associated with the presence of the fungus Ostracoblabe implexa; and 17% showed Myicola ostrea copepods.  Portuguese oysters sampled in Sado estuary, it was found a prevalence of 30% infections with Trichodina sp., characterized by a shape similar to a disc, a circlet of eosinophilic denticles, a ciliary fringe and a horseshoe shaped nucleous, in gills and mantle epithelium. A prevalence of 20% infections with Ancistrocoma sp., characterized by a Spindle-shaped ciliates with large, granular and polymorphic nuclei, in the digestive gland tubules and connective tissue. Copepods (Mytilicola sp.), characterized by an elongated cylindrical shape and a redish colour were observed within the intestine of oysters with the prevalence of 17% [36].
In samples from Mira estuary, it was found a prevalence of 13% infections with Trichodina sp., 30% infections with Ancistrocoma sp. and 3% infections with Mytilicola sp.
Finally, in samples of C. gigas from Alvor lagoon 10% presented mud blisters.
In farmed oysters sampled from Alvor lagoon, it was observed a prevalence of 23% Trichodina sp. infections and a prevalence of 27% Ancistrocoma sp. Copepod (Mytilicola sp.) were observed within the intestine of oysters at a prevalence of 3%.

Discussion
Cultivation and harvesting of bivalve mollusks are two important activities, and it is fundamental to preserve the natural ecosystems that support those activities and lead to increasing natural production and improving its quality. Bivalve mollusks production depends on the external factors [37], including water parameters, such as temperature variations, food change, biotoxins and pathogens (including parasites, bacteria, viruses) or anthropogenic, such as chemical contaminants (heavy metals, pesticides), organic contaminants (fecal contamination) and overexploitation of natural banks [1,38]. It is wildly known that the marine environment provides stress to oysters during their life cycle, namely changes in environmental parameters, changes in the availability of food and the display of various toxic pollutants [39][40][41]. Oysters are very resistant to changes of temperature and salinity that are present in the ecosystems where they live, namely estuaries and lagoons. As other euryhaline organisms, oysters can live in water where large variations of salinity occur. For short periods of time salinity values could range between 2% and 38% [1,42]. Similarly, oysters are resistant to temperature changes between 8 • C and 30 • C [1]. There are several factors that influence the growth of mollusk bivalves such as temperature, oxygen and the presence of different species of phytoplankton. Once they are filter-feeders, the quality and the quantity of filtered food define the outcome of their growth [43].
The regularly consumption of bivalve mollusks by different populations and the growth of shellfish aquaculture production show the importance of these animals and their monitoring must be undertaken to avoid diseases and mortality in oyster populations. Hereby we are presenting the results of the sanitary monitoring of oyster production in four different production areas. In general, it was observed that low numbers of parasites were present in oysters being Trichodina sp. and Ancistrocoma sp. infections the most common ones present in the areas that were studied. The presence of Trichodina sp., Ancistrocoma sp., Mytilicola sp. and Myicola ostrea are commonly observed in oyster populations and in massive infestations could lead to host weakness [36,[44][45][46]. Lesions of the internal organs, such as metaplasia, may reflect physiological stress, contaminants or the presence of large parasitic loads [1,44]. In our study, hemocytosis, cerodosis and edema were mainly present in the interstitial connective tissue. These lesions and those observed in the epithelium of the diverticula of the digestive gland, including the hemocytic infiltration and necrosis were usually associated and could be related with the presence of the parasites. In wild populations from Sado and Mira estuaries the prevalence of mud blisters and gill lesions were higher than from populations produced on tables 0.50 m from mudflats.
The identification, characterization and registration of pathologic processes in oysters constitute a set of important measures for sanitary control. Based on the results of the sanitary control appropriate biosecurity tools could be developed and implemented leading to the control of diseases spread and transmission. Finally, knowledge and ability to manage risk of different pathogen will contribute to the sustainability of oyster production and protection of animal and human health.
Aquatic ecosystems can be modified due to human activities such as overharvesting and destruction of substrates [47]. In addition, transfers of oysters, between countries and regions can spread diseases and invasive species [47][48][49]. Concurrently with increase of the socio-economic importance of mollusk farming in Portugal all associated risk factors are affecting this production. It is essential to have stricter control at all stages of production of the different bivalve mollusk species. This is the only way to improve economic performance without jeopardizing animal welfare and at the same time to prevent diseases in oyster populations and their dissemination to different areas. In Portugal, IPMA (https://www.ipma.pt/en/) (accessed on 14 September 2021) undertakes regular analysis to the water, including in the areas where animal species that are produced to be consumed by populations. In the areas where animals were collected no contaminants were detected. Biotoxins are not harmful to bivalve mollusks but, when they are consumed by humans, they can cause several serious problems. As such, the control and monitoring of levels of contaminants are fundamental to prevent any risk to public health.
Diseases are important risk factors, specific or not, of different species of bivalve mollusks, which are caused, in many cases, by non-compliance with basic management rules, namely the level of animal load in production areas, the length of time the bivalve mollusks remain in these areas, and the introduction of seeds of unknown origin. Effective biosecurity measures and correct diagnostic techniques are essential to control pathogenic agents. The identification of pathogens should include those at an early stage in their life cycles, to control and to prevent their multiplication and proliferation. In the production areas from Aveiro and Alvor lagoons these measures are implemented by producers and by the authorities. Producers understand that it is essential to prevent mortalities in bivalve mollusk populations, namely avoiding overcrowding and preventing diseases. In Sado and Mira estuaries, wild populations show that oysters are more susceptible to lesions than oysters produced on tables under the supervision of producers.
Research on genetic resistant bivalve populations to different pathogenic agents and the use of probiotic bacteria are important strategies to prevent diseases caused, namely by different Vibrio species [50,51] that can cause mortality outbreaks, that did not occur in the areas that were studied. Furthermore, restoration programs can be implemented in areas where ecosystems were negatively affected [49]. The success of bivalve mollusk production can only be achieved, if appropriate measures are applied, namely biosecurity, correct and efficient diagnostic methods, effective compliance with legislation and the dissemination of knowledge among populations.