Amyloodinium ocellatum (Dinoflagellate protozoa) Infestation in Dicentracus labrax (Sea bass) and trials of treatment

INTRODUCTION

Amyloodinium ocellatum, the causative agent of amyloodiniosis (marine velvet, or velvet disease), is one of the main problems in aquaculture, which was previously reported in the aquaculture of the Mediterranean region (Soares et al. 2012). These parasites can attach to the gills, the primary site of infestation for Amyloodiniosis. infestation may also involve the skin, fins, and eyes. It can penetrate deeply into the epithelium using a rhizoid root-like structure, leading to severe damage to tissue at the site of attachment. In addition, their rapid reproduction, causes high morbidity and mortalities, especially in cultured fish with high density. Therefore, A. ocellatum can cause serious economic losses in aquaria and the mariculture systems (Byadgi et al. 2020).  Concerning their life cycle, Amyloodinium ocellatum is an extracellular obligatory parasite that involves three developmental stages, one of which is the free-living phase; A.ocellatum requires its host fish to survive and complete its life cycle (Ranjbar and Khoramian, 2019). Sea bass is a marine fish of great economic importance particularly in Mediterranean aquaculture (Salama and Yousef, 2020). Dicentracus labrax seabass represents a major fisheries and aquaculture species in the Mediterranean, the European Atlantic coasts, and North Africa. Most fish living within its ecological range are susceptible to infestation with amyloodiniosis

Copper sulfate has been used extensively to treat infestations brought on by protozoa because it is one of the most effective chemicals against several parasitic infestations and diseases (Zhou et al. 2023). The cupric ion interferes in linking ionic regulatory proteins by obstructing their regulatory function (Hassan et al. 2017). Besides hydrogen peroxide plays an important role in cell protection against oxidative damage in unicellular eukaryotes which induces the expression of the detoxifying enzymes (Heo et al. 2020), which serves as a sensor and warning, enabling the cell to defend itself from oxidative stress. H2O2 is a significant Cellular Reactive Oxygen species that plays critical regulatory and signaling roles. It is highly damaging and reactive toward proteins, DNA, and lipids (Seixas et al. 2022).

The present study aimed to determine the pathogenesis and trials for the treatment of Amyloodinosis in naturally infested Mediterranean aquaculture associated with morbidity and mortalities in seabass Dicentrarchus labrax

MATERIAL AND METHODS

Sample collection and clinical examination:

A total of 300 European D. labrax fingerlings were collected randomly from a fishing farm at Port Said governorate, Egypt, in the Summer of 2022. The fish samples were transported alive in oxygenated plastic bags with marine water and supplied with an aerator according to El-Deen et al. (2013), they were acclimatized in fiberglass aquaria for conductions of treatment trial of A. ocellatum partially filled with marine water. Clinical investigations and Post-Mortem (PM) examinations were performed according to Noga, (2010). The infestation rate of collected fish with Amyloodiniosis was 195 (65 %), we have used 180 divided into three groups in the experiment. The fourth group was taken from the rest collected fish as a control.

Parasitological examination:

Macroscopic examination.

 Skin surfaces, fins, and gills were examined grossly for any abnormalities of parasites, and lesions.

Light microscopical examination

 Direct wet mount techniques (skin scrapping) and gill biopsy or gill clip (at which a gill filament is removed) are examined microscopically

Electron microscopical examination

 Samples were preserved in glutaraldehyde, and then they were mounted on SEM stubs and were coated with gold using SI5OA sputter coater and examined by scanning electron microscopy (JSMIT100 In Touch Scope™ Scanning Electron Microscope, JOEL, Damansara, Selangor, Malaysia) according to Abouelhassan, (2022).

Experimental design

A total of 240 from the collected 300 D. labrax fingerlings, 17 ± 3 g. weight, and (13 ± 1.50) cm length, 180 fish from 195 infected one divided into three equal groups of 60 (G1, G2, G3) and, 60 (G4) from the healthy one, distributed equally in fiberglass tanks size (1000L) Table 1.

Therapeutic Doses Efficacy against Amyloodiniosis:

Each treatment was performed in triplicates. Fish were equally distributed into the four groups of D. labrax, the first was the infested non-treated group (G1), the second infested group was immersed with copper sulfate (aqua clear: it is a mixture of copper sulfate, acetic acid, citric acid, and formic acid) at 0.25 ml/m3 (G2). The third infested was immersed with H2O2 50% at 1.5 ml/m3 (G3) and the fourth one was apparently healthy non-treated group (G4). The experiment was inspected daily for 14 days, fish were fed on a well-balanced diet and changed 10 to 20% of water daily. The clinical signs and mortality were recorded.

Ethics:

This study was conducted following legal ethical guidelines of the Medical Ethical Committee of the National Research Center, Dokki, Egypt. (Ethical approval no. ARC AHRI 2306).

RESULTS

Clinical and post-mortem findings

Naturally infested fish samples with various clinical symptoms like darkening of the skin color, off food, abnormal swimming behavior as a jerky movement, and moving upside down with severe respiratory distress, depression, anorexia, anoxia, gasping of mouth and sudden collapse, the gills were very pale with excess mucous. Although the protozoan cannot be seen with the naked eye. The total infestation rate was 65%

Parasitological findings

Microscopically, Smears were prepared from the skins, fins, and gills of the infested fish before the treatment, showing Amyloodinium ocellatum trophonts which are spherical, oval, and or elliptical in shape and attached to the gills or the skin surface (Fig. 1A).

The SEM, showed the Amyloodinium trophonts were round or spherical with different sizes from 40 to 60 μm (Fig. 1C).  They were found in groups each group consisted of 3 to 6 trophonts or appeared separately attached firmly to the gill filaments, and the skin surface and between lamellae (Fig. 1D). The base of trophonts was narrow which is inserted in the tissues. This insertion of Amyloodinium trophonts led to changes in the gill and skin surface structures, for instance, the filaments appeared enlarged and swollen, with fusion of some lamellae (Fig. 1B )

Treatment trials

The effects of the treatment were evaluated by measuring both the infestation and mortality rates, besides the detection of the parasite number from the samples of each group, which showed a decrease of both infestation and mortality rates compared with the infected nontreated group, in the copper sulfate (aqua clear) treated group (G2), the recovery rate was 95%. Meanwhile, the hydrogen peroxide group (G3) was 71.66 % (Table 1 and Fig. 2&3). The treated fish regained its viability, appetite, and normal movement besides the disappearance of skin darkness, with normal gills appearance

Amyloodinium ocellatum (Dinoflagellate protozoa) Infestation in Dicentracus labrax (Sea bass) and trials of treatment

Dawlat A. Hassanin *, Eman M. Abouelhassan **, Walaa El-Shaer ***

Affiliations:  ‎

‎* Central Laboratory of Fisheries Resources, Al-Raswa branch, Port Saied, Egypt.

** Department of Parasitology, Faculty of Veterinary Medicine, ‎Suez Canal University, Egypt.

*** Agriculture Research Center  (ACR), Animal Health Research Institute, Ismailia branch, Unit of Fish Diseases, Egypt.

ABSTRACT

Amyloodiniosis (marine velvet, velvet disease) is one of the main problems in aquaculture, in this study, A total 300 of European Dicentracus labrax fingerlings, were collected randomly from a fishing farm at Port Said governorate, Egypt, in the summer of 2022. Samples were taken for the parasitological examination (light and electron microscopical examination) which revealed the presence of Amyloodinium ocellatum, for the experimental design. The total Prevalence of the 300 examined Dicentrarchus labrax fish, was 195 (65.00%), a total of 240 D. labrax fingerlings were divided into four equal groups of 60 each and distributed equally in fiberglass tanks, and both Copper sulfate and hydrogen peroxide were used in the treatments for 14 days, giving good recovery with decreasing the mortalities rates.  The identification is confirmed by scanning electron microscope.