New Report on the Occurrence of Gregarine Parasites in Wild Species of Macrobrachium rosenbergii from Setiu Lagoon, Terengganu, Malaysia

Studies on the parasite of prawns are likely to be important due to the commercial value of the prawn. Investigation study about gregarine parasites of M. rosenbergii in Malaysia is still lacking. Objective of this study is to determine the occurrence of gregarine parasites in shrimp, M. rosenbergii from Setiu Lagoon, Terengganu. The study was done specifically to observe the prevalence and the mean intensity of the parasites which include the morphological prevalence and parasitological study. Prevalence of the parasites in this study is 42.4% with 95% Cumulative Index (CI) 25.6% to 59.3% and value of Odd Ratio (OR) is 0.74 (CI = 0.29 to 1.88). Two structures of the gregarine which is gamont and syzygy found in this study are closely related to morphology characteristic of gregarine in the previous report. Cite this as: Zakariah, M. I., Daud, H. H. M., Abdullah, M. I., Wahab, W., Abdullah, F., Yusoff, N. A. H. M., Draman, A. S., & Hassan, M. (2022). New Report on The Occurrence of Gregarine Parasites in Wild Species of Macrobrachium rosenbergii from Setiu Lagoon, Terengganu, Malaysia. Jurnal Ilmiah Perikanan dan Kelautan, 14(1):142–149. http://doi.org/10.20473/jipk. v14i1.31087 Copyright ©2022 Faculty of Fisheries and Marine Universitas Airlangga 142 JIPK (JURNAL ILMIAH PERIKANAN DAN KELAUTAN) Scientific Journal of Fisheries and Marine

A survey of OIE listed parasites in tropical oysters (C. iredalei) in 2000 and 2003 reported the prevalence of gregarine Nematopsis spp. oocyte ranging from 33% to 73% (Kua and Taha, 2004). Despite, the commercial value and gradual increase in the production of crustacean species in Malaysia, there has been no report or any specific study on problematic diseases regarding these gregarine parasites. Several reports on the diseases, particularly those caused by parasites, have been described by various shellfish in other countries (Sindermann, 1990).
The objective of this study is to determine the occurrence of gregarine parasites in shrimp, M. rosenbergii from Setiu Lagoon, Terengganu. Nevertheless, a crustacean health monitoring programme has not yet been initiated by focusing on the OIE listed parasites. Subsequently, there is a lack of study about gregarine parasites in Malaysia. Most of the study is focusing on the species of mollusc that were previously reported by Kua et al. (2013). Ubiquitously, the infections of these types of parasites are commonly reported from bivalve and shrimp (Prasadan and Janardan, 2001). They report that all gregarines are known to inhabit crabs and prawns (Tuntiwaranuruk et al., 2004).

Samples Collection and Diagnosis Study
Setiu lagoon is a renowned wetland in the northern part of Terengganu and is extremely unique as it contains many ecosystems such as lagoon, rivers, mangrove forest, seagrass bed, beach, and small islands. This complete ecosystem is semi-enclosed with limited and poor tidal flushing and has a total water surface area of about 880 ha (Yahya et al., 2016). Setiu Wetlands has been divided into nine inter-connected ecosystems that served high biodiversity of flora and fauna from Penarik village to Beting Lintang village. This area is in making headway to be gazette by the state government as a protected area. This study area is of primary oceanographic importance since it is one of the largest estuaries of the east coast of Peninsular Malaysia which two main river systems flow, the Setiu River and Chalok River (Figure 1). These areas are of a diverse ecosystem with functional natural resources; an enormous array of biological diversity with the combination of terrestrial, coastal and riverine fishing activities.
For the diagnosis, all samples of M. rosenbergii were collected within several hours and were immediately taken into the laboratory and rapidly processed. The samples were collected for the parasitology test.
An extensive survey of estuarine crabs in Kerala, India revealed the presence of three species of cephaline gregarines parasites belonging to the genus Nematopsis were found. The objective of this study is to determine the occurrence of the gregarine parasite in M. rosenbergii from Setiu Lagoon, Terengganu. Besides, there is no report of gregarine species that infected crab but there is a new report of gregarine infection in the prawn and bivalves in Malaysia. The findings were significant and contributed new knowledge to the research community, especially on gregarine parasites infection in the commercially important crustacean culture worldwide.
Moreover, some studies proved that gymnospores and oocysts of gregarines are usually concomitant with a pivotal, nonthreatening inflammatory response and there is no serious effect or damage to the host (Hofman et al., 2009). Gymnospores need marine arthropods such as crabs or shrimps to complete their life cycle (Prytherch, 1940;Sindermann, 1990). Hence, the multiplication of gregarines is limited to crustacean and mollusc, respectively. Report of the occurrence of gregarine parasites are published but mass mortality cases of crustaceans and bivalves caused by gregarine have not yet been reported in Malaysia. However, the previous report mention the mass mortality of cockles and clams in Portugal had been associated with this parasite (Yee and Hassan, 2020).
In Malaysia, a monitoring programme on the molluscan health focusing on the Office International des Epizooties (OIE) revealed that gregarine was the most frequently listed parasite from 1999 until 2009, which showed that gymnospores of gregarine parasites were parasites most frequently encountered in tropical oysters (Crassostrea iredalei) and blood cockles (Anadara granosa) respectively (OIE, 2016). Hence, the previous study observation did not find any significant health effects. JIPK. Volume 14 No 1. April 2022 / Optimization of Medium Composition for Streptomyces sp. PB2 Chitinase...

Parasitology Study
According to Tuntiwaranuruk et al. (2004), tissue (4 mm 2 ) from each sample was cut out at a mid-ventral position from the inner and outer parts of the gut and intestine. The tissue was immediately squashed between glass slides and observed under a light microscope by differential interference lens optics (Nikon Microscopes Eclipse 80i) and captured for micrograph. In this study, samples for prawns are specifically chosen as the target area of infections according to the previous report.

Prevalence and Mean Intensity Study
Two descriptions of parasite distribution are used: prevalence and infection intensity (Ihwan et al., 2013). Prevalence describes the proportion of individuals in the population that are infected by a specific parasite or pathology and is calculated as:

Prevalence
= Number of prawn with gregarine

Number of prawn analyzed
Infection intensity is calculated as the average number of occurrences of the gregarine or pathology in infected bivalves. This is a measure of the intensity of infection in infected individuals.

A B
and previously infected hosts. Have been studying the case of gregarine infection in prawns and have developed the lifecycles of the parasites. The study of morphological characteristics is referring to Tuntiwaranuruk et al. (2004).

Micrograph
For micrographs, samples were viewed live under the light microscope (Nikon Eclipse 80i) at Advanced Microscope Laboratory, Institute of Tropical Aquaculture (AKUATROP), Universiti Malaysia Terengganu (UMT). Ten samples were selected for micrograph viewing. The measurement of length and width of the gregarine were recorded.

Drawing
The live samples were placed into the glass slide in the condition of a semi-dry slide. The selected samples were then viewed under light microscopes and directly drawn using a Leica Lucida camera tube. The captured sample was sketched on A4 paper and later transferred to tracing paper for detailed morphology drawing. Lucida camera tubes attached with Leica compound microscopes were used for drawing the parasites. The important characteristics were focused to confirm the internal and external parts of parasites which is stated for identification purposes.

Data Analysis
SPSS version 24 software were used as a tool for analysis. The selection of the test method formula is depended on the objectives of the study. The prevalence and odds ratio study were calculated using Microsoft Excel version 2010.    Table 1. One-Sample statistics analysis for gregarine parasites found on M. rosenbergii from Setiu lagoon gregarine parasites in the prawn was carried out with detailed morphological characteristic study for identification purposes. The morphological characteristic of the gregarine found in this study is closely related to reports, presumably because it originates from the same region. Research done by Shanavas et al. (1989) showed different characteristics and sizes of Nematopsis rosenbergii even though it was found in the same host as in the present study (Table 3).
Fajer-Avila et al. (2005) explained that the presence of gregarine is usually associated with the slow growth of the prawn. Nonetheless, the presence of this gregarine parasite can be seen in the wild and it can indicate the situation that related to present if no action was carried out on the prawn for broodstock purposes. The potential of heavy infection by this gregarine will increase the absorption of nutrients and yet can decrease the nutrients that are prioritized for the development of prawns. The infection also can result in a lesion due to the penetration of the epimerite structure which can damage the epithelium cell and can lead to secondary infection of bacteria and virus which eventually caused mortalities.

Conclusion
Investigation study about gregarine parasites of M. rosenbergii in Malaysia is still lacking. The prevalence of the parasites in this study is 42.4% with 95% Cumulative Index (CI) 25.6% to 59.3% and value of Odd Ratio (OR) is 0.74 (CI = 0.29 to 1.88). Two structures belonging to different stages of parasites were found in this study, which are gamont and syzygy structure stages. The morphological characteristic of the gregarine found in this study is closely related to the previous report. Further study using genetics applications is needed to confirm the species. Zakariah et al. / JIPK,

Results and Discussion
Gregarine gamont and syzygy were discovered in the gut and intestine of M. rosenbergii from Nyatoh River, Setiu Lagoon. Observation using light microscopy (Nikon Eclipse 80i) showed that gamont was presumably gliding freely in the lumen or penetrate in the epithelium wall cells. Two spherical nuclei were seen on the structure of the body of the parasites. Location of nuclei generally situated at one-third of the body from both ends. Every sample that was found is characterized with epimerite structure which is functional as an anchoring part in their host gut and intestine. Two structures belonging to different stages of parasites were found in this study, which are gamont and syzygy structure stages.
The total number of 33 samples (N=33) with mean weight (23.88±32.23) and length (124.84±33.29) of freshwater prawn was diagnosed for gregarine parasites ( Table 1). The prevalence of the parasites is 42.4% with 95% CI 25.6% to 59.3% and value of Odd Ratio (OR) 0.74 (CI 0.29 to 1.88). One sample T-test was applied for data analysis and the result showed p-value (p<0.05) to be significant especially for weight, length, sex, and infections ( Table 2). For the parasite measurement, the mean length is 244.74±16.53 and the mean length is 54.04±8.42, respectively. From this study, the size of gregarine parasites are closely related to the parasites that have been found by Prasadan and Janardan (2001) and Tuntiwaranuruk et al. (2008) with the measurement range size is 213.0-317.0 µm and 230.0-361.0µm, respectively. Detailed information on the measurement range size of the present study and previous report are varied (Table 3).
Studies of the parasite of prawn are likely to be important due to the commercial value of the prawn. Parasites may be considered as a pathogenic agents who affected the growth and reproductive performance of their host. The previous study had carried out a parasitological diagnosis of freshwater prawn, M. rosenbergii, in different geographical regions which relate to the defense mechanism and their economic importance to the aquaculture industry. Investigation study about gregarine parasites of M. rosenbergii in Malaysia is still lacking. Even though the study of this parasite has been described in other parts of the world region, it might not be identical and their pathogenicity could be different to those found in Malaysia.
Disease outbreaks in this prawn as a host will occur if the knowledge of this parasite was not critically explored. Thus, the investigation of the occurrence of