Preliminary Morphological Study and Food Uptake Experiment for Transmission of Gregarine Infection from Infected Bivalves, Anadara cornea (Reeve, 1844) to the Mud Crab Genus Scylla as an Intermediate Host.

Objective: Research about gregarine become important due to the problem reported by this parasite especially in commercial bivalve i.e. Oyster. Diagnose of these parasites are important to secure the aquaculture industry in the future. Due to the advanced technologies nowadays, this research regarding to these parasites are become relevant to be study. The objective of this study is to determine the occurrence of gregarine parasites in wild mud crab and the food uptake transmission of gregarine infection from infected bivalves, Anadara cornea (Reeve, 1844) to the mud crab genus Scylla. Result: Preliminary study show that high prevalence of infection was reported in the Hairy Cockle (Anadara cornea) from Setiu Lagoon, Malaysia. From the analysis, the infection intensity was high and each phagocyte (Pha) contain maximum of 15 oocyst (Oc). Each oocyst has a single cell wall, longitudinal shape and contained sporozoite (Sz). Parasitophorous Vacuole (Pv) cover by membrane wall. There is no transmission of parasites reported in this experiment. Malaysia Terengganu Animal Ethical Committee guilds lines. The examined of Scylla spp. and bivalves were part of this study. The sample of crab and bivalves are prepared with controlled the cleanliness of the samples as much as it can through the standard quarantine process.


Introductions
The apicomplexan gregarines generally are not noted for being highly detrimental to their hosts, the best-known effect being damage done to the epithelial cells to which the young trophozoites are attached in the intestine of the host [36]. This effect, whatever it may be, is directly related to the number of parasites present, since each parasite attacks a single host cell. This could suggest the possibility of a cumulative effect on the host. It also seems quite possible that such an effect may accompany the progressive mechanical obstruction of the blood vessels of the organs involved. It is also conceivable, as suggestion by Sprague and Orr [28] that toxic end products of metabolism might be liberated into the host tissues by the developing sporoblasts.

Parasitology Diagnosis
Parasitology diagnosis were prepared by referring to Tuntiwaranuruk et al. [37]. A piece of tissue (approximately 4 mm 2 ) from each sample was removed from the endo and ecto part of body on both sides of the bivalve. The tissue was immediately squashed between glass slides and observed under compound and light microscope using difference type of lens optics (Nikon Eclipse 80i) and captured for micrograph. In this study, sample for gill and digestive tract of the bivalve [38] are speci cally chosen as target area of infections according to previous report [3-4; 17; 20].

Transmission experiment
Thirty mud crab genus Scylla were used as a sample for three cycle of experiment. Data on mud crab size and weight were for this study. Total fteen crab were used for controlled and the other fteen crabs are using as a test sample. Tank that has been used for this study can be referring to Figure 4. All thirty crabs are following the moulting procedures to make sure the crab is preferably clear from the parasites. The forced-moulting techniques (leg cutting) were applied. The samples were divided into two tanks with complete aeration and sediment to build the real environment for the crab. The infected bivalves were fed into the crab in the experimental tank while the control tank was fed with thrash sh. After a month, the crab was dissected for diagnosis and genetic test were applied to con rm the transmission occurred or not.

DNA Analysis Genetic Sample Collection
Incubator must be set to 56 o C before running the extraction and all the work bench apparatus i.e. tip box, micropipette must be sterile with 70% ethanol to disinfect. A piece of gill and digestive tract of ten samples were isolated from the diagnosed hosts, washed three times in ltered distilled water or in 150 mM physiological NaCl solution and deposited into a 1.5-ml micro centrifuge tube for extraction using the Qaigen™ Complete analysis DNA Kit and RNA Puri cation Kit (Qaigen, US) [26-28]. Result were sent for sequencing and analysed using blasting software MEGA X.

Results
In the preliminary study, the gregarine has been found infected the gill and intestine tissue of Anadara cornea. The illustrated of the parasites are showed in Fig. 1. From the analysis, the infection intensity was high and each phagocyte (Pha) contain maximum of 15 oocyst (Oc). Each oocyst has a single cell wall, longitudinal shape and contained sporozoite (Sz). Parasitophorous Vacuole (Pv) cover by membrane wall. Histopathology study (Fig. 2) is showing Phagocyte (Pha) in digestive tract and oocysts (Oc) in parasitophorous vacuole (PV); sprozoites (Sz) are clearly visible (Magni cation: 400X). Oocysts are commonly found in the parasitized phagocytes. Further analysis will be correlated with the tissue changes by using lesion scoring obtained from previous study. Previous study of oocyst also reported by Azevedo and Matos [4] and Clopton [11]. For Scylla spp. Preliminary study, thirty mud crab genus Scylla were collected from Setiu Wetland, Terengganu and observed for preliminary study and one of the crab's sample show positive ndings. Average of carapace length (7.4 ± 0.7 cm), carapace width (5.3 ± 0.4 cm) and body weight (71.1 ± 13.5gram) of mud crab genus Scylla recorded for this study. But when the transmission study has been applied, no positive results are showed. The preliminary nding of syzygy-like of gregarine parasites (Fig. 3) was assumedly accidentally found cases. Table 1 show the summarized results of transmission experiment between two species of host. For the genetic study, there is no positive result that showing the transmission of the parasites between intermediate and de nitive host. Table 1 show the test replication with the diagnosis of every mud crab. Sample for DNA study are randomly collected from different part of mud crab possibilities infected organ. Sample that has been collected is standardized taken from gut, intestine, gill and hepatopancreas which is suggested from previous study. Overall result did not show any positive infections.
Further study is needed to double con rm whether the transmission is really happened. The sample of crab and bivalves are prepared with controlled the cleanliness of the samples as much as it can through the standard quarantine process. The crabs for experiment were through the moulting process and the next step is fed with pellet which is assumedly free from gregarine parasites. The crabs also were bath in the 10% formalin for 24 hours to make sure that another suspected parasite is eliminated. The infected bivalve is selected by the diagnosis of the sample. All the infected of bivalves were collected and fed to the crab within 2-3 weeks. The number of bivalves which is fed to the crab is not speci ed because this experiment is only wanted to explore the transmission is happen or not. Details for the transmission should be applied if future experiments of lifecycle of this parasite are done.

Discussions
Effect of the gregarine infections in aquatic organisms nowadays are reported due to the importance of this parasite [5][6][7]. Most of the infections reported from the commercial species culture are related to the requirement of World Organization for Animal Health (OIE) [30; 35]. This information is important because of these parasites are that can give serious impact to the sustainability of aquaculture in the future. Movement of the parasites is very important to ful l the complete lifecycle [34]. Some study explained this parasite able to shift and can adjust the direction along the surface through gliding movement [13; 40] without use of any organs such as cilia, agella, etc. Previous research stated that the motility is happen using complex combination including myosin with the involvedness of actin cytoskeleton to perform the gliding motion [40]. Some study stated that the epicytes of all the gregarines is differentiated into a system of regular longitudinal folds [9]. Some gregarine species folds heave supports the parasites to glide along [39]. Most of the study shows that abiotic and biotic factors give an effect to the changes of terrestrial and aquatic habit in natural system of the parasites [12;16]. Normally host can interact precisely and implicit the situation of transmission process. This can also detrimentally to regulate the natural parasite transmission process [9;15;41]. Natural habitat should be maintained the ecosystem because if the structure, biodiversity, host-demographics, evolution are disturbed, the sustainability of the ecosystem will be destroyed [8][9]. Signi cant climate change commonly affects the water quality such as temperature, salinity [16], dissolved oxygen and pH. This factor is very important to relate the distribution of this parasites among the local host in the speci c area of sampling study [21;22]. Some information relates to this mechanism of interaction between environmental stressors [2] and the subsequent effect on parasites transmission are precisely complicated [9; 19]. The factor that increasing the prevalent parasites intensity [31], growth, reproduction and infection are thoroughly de ne by the uctuation of temperature and yet can contribute to the high mortality if it is not controlled properly.

Conclusion
Nowadays, the seafood consumption demand by human is increase throughout year. The common cultured species did not support the demand and now a lot of new species are introduced for species culture including crustacean species. This study purposely shows the morphology of the gregarine and some information added to explore the further study of this parasites. Histopathology of gregarine infection also show the infection of the parasites. Previous review of this parasites has been potential to explore this scope of study to the next level of research. Transmission experiments show negative result. Further tests need to be done to con rm the transmission.

Limitations
This work was carried out as a preliminary study at a single site. In this study, sample that were used declare a seafood, we investigated the experiment and get the sample from the live market by local sherman. Parasite that were collected for morphology study are too tiny and live specimen were used for identi cation. No latest journal is available.

Funding
This study did not receive any speci c grants from funding agencies in the public, commercial, or not-for-pro t sectors.

Availability of data and materials
All data generated or analysed during this study are included in this published article. The original datasets are available upon request to the corresponding author.
Ethics approval and consent to participate Both species used in the present study, mud crab, Scylla spp. and Blood cockle, Anadara spp. are common commercial shery caught species and require no approval to utilise them. Both species also are been handled accordingly following Universiti Malaysia Terengganu Animal Ethical Committee guilds lines. The examined of Scylla spp. and bivalves were part of this study. The sample of crab and bivalves are prepared with controlled the cleanliness of the samples as much as it can through the standard quarantine process.

Consent for publication
Not applicable.

Competing interests
The authors declared no con icts of interest    Gregarine parasite transmission experiment. Whole experiment takes about 2 months starting from the moulting process to in-vitro test.