ENDOPARASITES INFECTION IN BLOOD COCKLE (Anadara granosa) IN ACEH BESAR WATERS, INDONESIA

This study aims to investigate the type, prevalence, and intensity of endoparasites infecting the organs of blood cockles (Anadara granosa) in the waters of Aceh Besar, Indonesia. A purposive sampling method was conducted from March to August 2020 consisting of two sampling sites, namely the waters of Ujung Pancu and Krueng Raya. Endoparasite identification and analysis was carried out at the Parasite Laboratory of the Fish Quarantine Center, Quality Control and Safety of Fishery Products in Blang Bintang, Aceh Besar. The results showed that there were two types of endoparasites living in the blood cockles: namely, Perkinsus sp., which belong to the group Protozoa, and Spiroxys sp. of the Nematode class. The prevalence levels of Perkinsus sp. was 73.33% and were found in the waters of Ujung Pancu; while the lowest prevalence level was 20% in blood cockles that were infected with Spiroxys sp., which were found both in Ujung Pancu and Krueng Raya. The highest intensity level was infections of Perkinsus sp. with a value of 89 ind/species in the Krueng Raya site. The lowest intensity value was infections of Spiroxys with a value of 2 ind/species in Krueng Raya and 2.3 ind/species in Ujung Pancu ____________________________________________________________________________________________________________________


INTRODUCTION
Indonesia is one of the largest marine areas in the world which is famous for its abundant marine resources, in term of both fisheries and crustaceans (Muchlisin et al., 2017;Rizwan et al., 2017;Putra et al., 2018;Muhammadar et al., 2019;Safriani et al., 2019). The blood cockle (Anadara granosa) is a type of shellfish that has a high economic value and is cultivated as a source of protein and minerals (Yulinda et al., 2020). There are various types of shellfish that have been recognized by the community and are also used as food ingredients. In addition, blood cockles can also be used as decoration, fertilizer, and for absorption of heavy metals (Solang, 2019). Blood cockles are a type of cockle belonging to the Arcidae family that live in intertidal zones with sandy substrates and originate from mud flats in Southeast Asia, particularly Indonesia, Malaysia, and Thailand (Khalil et al., 2017). Substrates, season, temperature, feed and other chemical factors which vary in each region are very important parameters to support oyster growth (Lindawaty et al., 2016). Thus, blood cockles can also be used as a bio-indicator of water pollution, because the habitat of these animals is at the bottom of the waters andas stated before they accumulate heavy metals (Supriatno and Lelifajri, 2009;Yona et al., 2020). If a high cadmium concentrationis found in the cokles that exceeds the normal limit of this organism, then this can be used as an indicator of pollution in the environment (Hossen et al., 2014).
Several recent studies that have been carried out on multiple blood cockle species. Among them was a study with collected data on blood cockles found in coastal waters and aquaculture ponds in the Gulf of Thailand (Theerachat and Glinwong, 2020). Other studies included descriptions and challenges of blood cockle cultivation in Malaysia (Saffian et al., 2020), microplastic concentrations in blood cockles found in Pandeglang Banten Indonesia (Ukhrowi et al., 2021), the concentration of heavy metals in blood cockles found on the coast of East Java and potential risks to human health (Soegianto et al., 2020), the health status of blood cockles in cultivation ponds in Malacca Strait (Mohamat-Yusuff et al., 2020), the economic potential of blood cockles in Riau province (Yulinda et al., 2020), and lastly studies on the bio-ecological aspects and reproductive biology of blood cockles (Khalil et al., 2017;Sulistiyaningsih and Arbi, 2020). Parasites are a type of infectious disease that attack many fishery commodities both in nature and in cultivated environments. In its application, parasites attack aquatic organisms with the principle of utilizing their host (Abowei et al., 2011). Parasites that are found outside the fish body are called ectoparasites and parasites inside the fish's body are called endoparasites (Sarjito et al., 2013). Apart from infecting teleost fish, parasites also infect various aquatic organisms such as bivalves, crustaceans, and mollusks (Wood and Lafferty, 2015).
Currently, research on parasites in blood cockles is still very limited. However, parasitic infections in blood cockles in Central Java and the Malacca Strait have been reported (Uddin et al., 2011;Karnisa et al., 2019). Parasitic infection studies in other mollusks have also been reported such as the green-lipped mussel (Trottier et al., 2012;Trottier and Jeffs, 2015), and oysters (Moss et al., 2008;Arzul et al., 2011;Ben-Horin et al., 2018). At present, research on endoparasite infections in blood cockles in the Aceh Besar District has not yet been carried out. Research on endoparasites in blood cockles is very important as it helps to provide a more comprehensive literature on endoparasite infections. Therefore, this study aims to determine the type, prevalence, and intensity of endoparasite infections in blood cockles in Aceh Besar.

MATERIALS AND METHODS
This research was conducted for 2 months from March to August 2020. Blood cockles (Anadara granosa) were obtained from field collection in waters surrounding the Aceh Besar Regency. Sampling was carried out in two locations: namely, Ujung Pancu and Krueng Raya. The identification and observation of blood cockle (Anadara granosa) endoparasites was carried out at the Parasite Laboratory of the Fish Quarantine Center, Quality Control and Safety of Fishery Products in Blang Bintang, Aceh Besar.

Sampling Method
The research method used was a survey method by looking directly at the conditions of the waters at the research location and sampling using a purposive sampling method. Sampling took place in Aceh Besar District, Aceh, Indonesia at two locations. The number of samples taken per location was 15 individuals (Mardiana, 2014). The samples that had been taken were then collected in a bucket for further parasite examination in the laboratory.
Sampling was carried out three times a week for four weeks. The sample was put into a bucket then transported to the parasite laboratory of the Fish Quarantine Center, Quality Control and Safety of Fishery Products in Blang Bintang, Aceh Besar District for identification and analysis. The water quality was first measured before sampling, which includes taking the temperature using a thermometer; the degree of acidity was measured using a pH meter, dissolved oxygen using a DO meter, and salinity using a refractometer.
The blood cockles observed were transported from the container using a gutter and placed in a tray. Then the shells were opened and the targeted organs were cut off to be observed on a microscope. Observation of endoparasites were made on the blood cockles' gills, mantles, and gonads. The organs would first be dissected and then placed on a glass object that had been lubricated with distilled water as much as 1-2 drops and then observed under a microscope. Parasites were recorded and identified using the parasite manual book (Kabata, 1985).

Data Analysis Prevalence
Measurement of attack level (prevalence), according to (Fernando, 1972)  Measuring the intensity of parasite attacks according to (Fernando, 1972) as follows : = Total number of parasites N = The number of samples observed

Identification and Clinical Symptoms Of Endoparasites in Blood Cockles
Two endoparasites that were identified in the blood cockles (Anadara granosa) from two sampling locations in Aceh Besar were Perkinsus sp., and Spiroxys sp. Some endoparasites like Perkinsus sp., that were found still in the cyst stage which is commonly called a Perkinsus sp. cyst. The various endoparasites found in blood cockles within the sampling area is presented in Figure 1.
Clinical symptoms found in blood cockles infected with common parasites showed signs such as gills that were drizzled and the color of the gills looked pale while those in uninfected blood cockles had bright gill characteristics (Nainggolan, 2016). According to (Karnisa et al., 2019) the clinical symptoms of Perkinsus sp. infection in blood cockles are an imperfectly shaped shell and the presence of excessive mucus. Other symptoms as reported by (Baker et al., 2007) are blisters on the inner surface of the skin, sores on the mantle, pale flesh color, and excessive mucus. However, infection in blood cockles does not always cause these clinical symptoms. Interestingly, in this study, it found that blood cockles that were infected with endoparasites did not show any clinical symptoms. Moreover, this is in accordance with (Sarjito et al., 2013) who expressed that an infection of parasitic worms does not show clear external clinical symptoms so that it cannot be detected quickly.

Infected Organ
In this study, we have examined endoparasite infections on organs in the blood cockle such as the gills, mantle, and gonads as presented in table 1. The most common sites for endoparasite infection are the gills, followed by the mantle and gonads. This finding was in accordance with a study by Elston et al. (2003) who reported afinding of parasite infection in Manila cockles (Venerupis philippinarum). They also found that Perkinsus sp. is usually found on the gills, mantle, and palp. A similar finding was observed in waters surrounding Jakarta and Central Java that also reported that the dominant parasitic infection in blood cockles there was Perkinsus sp. (Choi and Park, 2010;Karnisa et al., 2019). However different results were found in blood cockles infected with endoparasites in the Malacca Strait. They reported the organs that were frequently affected by endoparasites were the gonads, followed by the stomach epithelium, mantle, digestive glands, and foot muscle (Uddin et al., 2011).
Apart from Perkinsus, the endoparasite that was also found in this study was Spiroxys sp. Spiroxys sp. is in the nematode class of endoparasite that infects almost all species of aquatic biota. In several reports, Spiroxys sp. has also been found in several freshwater fish (Santos et al., 2009;Galaviz-Silva et al., 2013;Vieira-Menezes et al., 2017;Mhaisen et al., 2018;Šimková et al., 2019), farmed fish (Soler-Jiménez et al., 2017), turtles (Rakhshandehroo et al., 2020) and some marine fishes (Öktener, 2016). However, in this study, Spiroxys sp was also found in the blood cockles' gonads. The characteristics of Spiroxys sp., found in the blood cockles samples had a non-segmented body shape and had a pointed tip. Spiroxys sp. larvae are motile as they are capable of active movement. once   (Broom, 1985) DO ppm 3-4 4.9-6.4 (Atmaja et al., 2014) these larvae reach the adult phase, they can be commonly found in marine mammals, such as dolphins and whales, and live within their muscle tissue (Tamba et al., 2012). The identification of this parasite is in accordance with the research of (Uddin et al., 2011) who also found parasites in blood cockles. However, the parasites detected in the gonadaltissue og these blood cockles were Turbellarian (Bower et al., 1994) stated that parasites that reside in the gonads can cause interference with gametogenesis and result in failure to reproduce. Besides infecting gonads, some endoparasites also attack the gill and mantle area. Gills and mantles are the main target organs of Perkinsus sp. infection (Choi and Park, 2010). The gills on shellfish have a way of feeding by filtering (filter feeder) organic matter suspended in the waters using siphons. Another function of the gills of a shellfish can be as an organ for gas exchange or as a means of breathing. Shellfish can select food particles to be filtered and consumed, this is because each shellfish siphon has different sensors and behaviors towards food (Bachok et al., 2006). This was confirmed by (Allam et al., 2013) which stated that Perkinsus sp. cells live and swim freely in the waters, and are pumped into the pallial cavity and then captured within the gills. Perkinsus sp. develop and undergo incubation for three days inside the gills, then move on to infect other tissues. Parasitic cells enter the mantle cavity which will then develop and the remaining parasites that are not swallowed will be discharged as feces. The rest of these feces will be contagious towards healthy shellfish.

Intensity and Prevalence
The intensity and prevalence of blood cockle endoparasite in Aceh Besar have been presented in table 2. In general, the level of prevalence and intensity of each sampling site in Aceh Besar was different; the prevalence value of blood cockles in Ujung Pancu was higher than in Krueung Raya. This is assumed to be due to the existence of residential areas, stalls, and also fishing industry activities such as salted fish and octopuses that are very close to these waters. Therefore as a result of water pollution, this may stimulate parasite infection. However, differences in the prevalence and intensity levels of parasites from one area to another are often related to many different factors, including: spatial variations, population densities of the animals and the presence of alternative hosts (Uddin et al., 2011). Meanwhile, according to (Nainggolan, 2016), the difference in prevalence at each location is due to the factors that influence it: for example, the differences in water quality conditions at each shellfish fishing location, the distribution pattern of endoparasites, and also blood cockle immunity. The incubation period of Perkinsus sp. for blood cockle infections at each location has a different period as well.
Based on Table 2, the highest prevalence value of Perkinsus sp. found in Ujung Pancu was 73.33%, which is categorized as the usual amount which means that the level of infection is common (Nandlal and Pickering, 2004), moreover, the prevalence value of Perkinsus sp. in Krueng Raya was 66.7% which belongs to a very frequent category where the prevalence level can be monitored. However, the intensity value in Ujung Pancu was 85.54 ind/species and in Krueng Raya was 89 ind/species which means that these two values are included in the very heavy category.
The prevalence value of Spiroxys in the waters of Ujung Pancu and Krueung Raya showed the same value of (20%) which is included in the frequent category which means that the level of infection often occurs (Nandlal and Pickering, 2004). The intensity value in Ujung Pancu was 2.3 ind/species, which is included in the light category. The intensity value of Spiroxys sp attacking blood cockles in Krueung Raya is 2 ind/species which is also included in the light category. A high stocking density can also accelerate the spread of parasites. Apart from the stress factor due to a high density, poor nutrition, and poor water quality, allow for the rapid development of parasites (Anshary, 2010;Anshary et al., 2013).

CONCLUSION
Two identified endoparasites were found to be infecting blood cockles at Ujung Pancu and Krueng Raya, namely Spyroxys sp. and Perkinsus sp., (including cysts Perkinsus sp.). The highest prevalence value was obtained in Perkinsus sp. infections with a value of 73% included in the usual category, while the lowest prevalence rate was Spiroxys sp. with a value of 20% included in the frequent category. The highest intensity level was obtained in Perkinsus sp with a value of 94.1 ind/species, which was included in the very heavy category, while the lowest intensity level was found in Spiroxys sp. with a value of 2 ind/species which belongs in the light category.