Amyloodinium Research Articles

A quantitative real-time PCR assay for rapid detection and quantification of Amyloodinium ocellatum parasites in seawater samples

Amyloodinium ocellatum is one of the most important ectoparasites of marine fish, causing amyloodiniosis and mass mortality in aquaculture. To date, no diagnostic method has been developed to detect and quantify parasite abundance in seawater. In this study, a quantitative real-time PCR (qPCR) assay using the ITS-F3 and ITS-R3 primer pair based on the internal transcribed spacers of nuclear ribosomal DNA (ITS rDNA) of A. ocellatum was successfully established for the detection and quantification of dinospores in seawater. The dinospores were collected by suction filtration of seawater using a vacuum pump, and examination by light microscopy and scanning electron microscopy (SEM) showed that the dinospores were adsorbed on the surface of the cellulose acetate membrane. The linear relationship between seawater parasite abundance and Ct values followed the model y = −3.0607 x + 29.919 with a coefficient of determination (R2) of 0.985. The results of the assay showed high reproducibility and no cross-reactivity with other aquatic pathogens. The assay successfully detected dinospores (100 % success rate) at concentrations of 8 and 10 cell per 300 mL of seawater. The protective fluid buffer effectively protected the dinospores DNA from degradation for over 96 h. The method was used to investigate the life span of dinospores in seawater and the incubation patterns of tomonts in the tank, the results showed that a small fraction of dinospores can survive for more than 7 days in seawater and the process of dinospores incubation from the tomonts persisted up to 84 h. This study provides a valuable quantitative diagnostic tool to assist in the early monitoring and understanding of disease progression in amyloodinosis.

Establishment of a gill cell line from yellowfin seabream (Acanthopagrus latus) for studying Amyloodinium ocellatum infection of fish.

Amyloodinium ocellatum is among the most devastating protozoan parasites, causing huge economic losses in the mariculture industry. However, the pathogenesis of amyloodiniosis remains unknown, hindering the development of targeted anti-parasitic drugs. The A. ocellatum invitro model is an indispensable tool for investigating the pathogenic mechanism of amyloodiniosis at the cellular and molecular levels. The present work developed a new cell line, ALG, from the gill of yellowfin seabream (Acanthopagrus latus). The cell line was routinely cultured at 28°C in Dulbecco's modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS). ALG cells were adherent and exhibited an epithelioid morphology; the cells were stably passed over 30 generations and successfully cryopreserved. The cell line derived from A. latus was identified based on partial sequence amplification and sequencing of cytochrome B (Cyt b). The ALG was seeded onto transwell inserts and found to be a platform for invitro infection of A. ocellatum, with a 37.23 ± 5.75% infection rate. Furthermore, scanning electron microscopy (SEM) revealed that A. ocellatum parasitizes cell monolayers via rhizoids. A. ocellatum infection increased the expression of apoptosis and inflammation-related genes, including caspase 3 (Casp 3), interleukin 1 (IL-1), interleukin 10 (IL-10), tumour necrosis factor-alpha (TNF-α), invivo or invitro. These results demonstrated that the invitro gill cell monolayer successfully recapitulated invivo A. latus host responses to A. ocellatum infection. The ALG cell line holds great promise as a valuable tool for investigating parasite-host interactions invitro.

Phytoplankton population dynamics along the Casablanca - El Jadida Atlantic coast

Our taxonomic study from March 2014 to November 2015 along the atlantic coast between Casablanca and El Jadida revealed that phytoplanktonic structure is mainly represented by Diatoms (Bacillariophyceae), Dinoflagellates (Dinophyceae), Silicoflagellates (Dictyophyceae) and Euglenophyceae with a clear dominance of Diatoms and Dinoflagellates. A total of 101 taxa of planktonic algae have been identified revealing a relatively diversified taxocenosis. In terms of respective diversity, the Diatoms are represented by 62 taxa (61.4%), the Dinoflagellates by 36 taxa (35.6%) whereas the other two groups of Silicoflagellates and Euglenophyceae are only represented by 3 species (3.0%). Regarding potentially toxic species, more than fifteen taxa have been identified, most of which were Dinoflagellates and Diatoms (Pseudo-nitzschia australis and Pseudo-nitzschia cuspidata). The total phytoplankton densities exhibited great spatial and temporal variations as shown by analyses of diversity (H') and equitability (E) indices at the different coast sites investigated throughout the 2014-2015 years. Massive proliferation of some toxic species (e.g. Pseudo-nitzschia australis and Pseudo-nitzschia cuspidata, Lingulodinium polyedrum, Karenia mikimotoi) was also noticed. In addition, our study revealed the presence of opportunistic species (e.g. Eutreptiella, Thalassiosira, Prorocentrum scutellum) and of the new ectoparasite Dinoflagellate Amyloodinium ocellatum for the first time in Morocco. The detection of such diversity of toxic species, sometimes with alarming concentrations, should prompt the competent authorities to broaden the spectrum and frequency of biomonitoring to uncontrolled seafood harvesting sites.

Amyloodiniosis in aquaculture: A review

AbstractFish ectoparasites are one of the pathogens groups that pose great concern to the aquaculture industry. The dinoflagellate Amyloodinium ocellatum is responsible for amyloodiniosis, a parasitological disease with a strong economic impact in temperate and warm water aquaculture, mainly in earthen pond semi‐intensive systems. Amyloodiniosis represents one of the most important bottlenecks for aquaculture and, with the predictable expansion of the area of influence of this parasite to higher latitudes due to global warming it might also be a threat to other aquaculture species that are not yet parasitized by A. ocellatum. This review made a compilation of the existing knowledge about this parasite and the disease associated with it. It was noticed that, except from the life cycle characterisation, detection methods, histopathological analysis, and treatments, there are still a lot of areas that need a further investment in research. Areas like parasite–host interactions, epidemiological models, taxonomy, host physiological responses to parasitism, and genome sequencing, amongst others, can contribute to a better understanding of this disease. These proposed approaches and routes of investigation will enhance and contribute to a more standardised knowledge, creating the opportunity for a better understanding of amyloodiniosis impacts on fish and contributing for the development of new tools against A. ocellatum, that may reduce fish mortality in aquaculture production due to amyloodiniosis outbreaks.

Expert knowledge–based system for risk assessment of the occurrence of Amyloodinium ocellatum in semi-intensive fish farms

The implementation of a system to assess the risk of Amyloodinium ocellatum occurrence in rearing ponds in fish farms located in southern Spain is a fundamental aspect to ensure the economic viability of these facilities. For this purpose, a computer program (called Amy) for Windows PCs and an application for mobile devices (AmyAPP), based on the Android operating system, were developed integrating transformation functions and weightings associated with environmental parameters and fish behavioural factors from which it is possible to estimate the level of risk of occurrence of A. ocellatum. The weights for each of the environmental parameters and behavioural factors were estimated from the responses of a panel of experts (the fish farmers) using a Delphi methodology. The results indicate that, under operational validation, Amy/AmyAPP responses were statistically sensitive to the occurrence of A. ocellatum outbreaks in sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) rearing ponds.

Galvanized material is a promising approach to control Amyloodinium ocellatum infection in fishes

Amyloodinium ocellatum is a worldwide distributed dinoflagellate that infects marine and brackish water fish, causing mass mortality and economic loss. This study evaluated the efficacy of the seawater immersed with galvanized materials (IGM seawater) in controlling A. ocellatum reinfection in susceptible host Trachinotus ovatus. Furthermore, the toxicity of IGM seawater to the dinospores infectivity, tomonts development, and trophonts parasitism was determined in Acanthopagrus latus. The expression of zinc transporters in A. latus gills and livers was also detected after A. latus exposure to IGM seawater. The results revealed that parasite abundances on infected T. ovatus were significantly decreased in the seawater immersed with 0.1 m2/m3 of galvanized iron mesh for 2 days ([0.1–2 d] group) or galvanized iron plates with a 56.25% tank coverage for 1 day ([56.25% - 1 d] group). Besides, the survival rates of the infected T. ovatus in the [0.1–2 d] and [56.25% - 1 d] group were 100% on the 14th-day post-infection, while 0% in the control group. The infectivity of dinospores was significantly inhibited in the seawater immersed with 0.4 m2/m3 of galvanized iron mesh for 2 days ([0.4–2 d] group) by 89.50%. In addition, the reduction of trophonts in the [0.4–2 d] group was 46.24% compared to the control group. However, the hatching rate of tomonts in the seawater immersed with 0.4 m2/m3 of galvanized iron mesh for either 1, 2, or 3 days was 100% at 48th hr post-treatment. At the same time, the zinc ion concentrations in the IGM seawater group were continuously increased. On the contrary, the mRNA levels of zinc transporters ZIP7 and ZIP11 in the liver of A. latus exposure to IGM seawater were significantly down-regulated. Therefore, A. latus acclimatizes to a high‑zinc environment by reducing zinc ions influx to maintain intracellular zinc homeostasis. These results imply that using galvanized material is a promising approach to controlling A. ocellatum infection in fish.

An experimental animal model of yellowfin seabream (Acanthopagrus latus) for Amyloodinium ocellatum infection

Amyloodinium ocellatum is a worldwide distributed protozoan that infects almost all marine fish species, causing fatal amyloodiniosis. However, highly effective antiparasitic agents for controlling amyloodiniosis are lacking. This study was conducted to establish a laboratory model of A. ocellatum infection, facilitating the development of alternative treatment options and immunostimulants for A. ocellatum using yellowfin seabream (Acanthopagrus latus) as the experimental host. The life cycle and preservation strategies of A. ocellatum, the stability of dinospore infection in A. latus, and the tolerance of A. latus to A. ocellatum infection were observed and evaluated. Under suitable conditions (temperature: 27 ± 1 °C; salinity: 30 ± 1‰), A. ocellatum completes its life cycle on A. latus in 4–5 d and peak trophont detachment from A. latus occurs at 44–52 h post-infection. The peak period when tomonts release dinospores occurs 51–60 h after incubation at 28 °C. Dinospores have high vitality and infectivity within 6 h of release from tomonts. The infection model developed in this study is stable, with a 39.85–44.68% dinospore infection rate. Additionally, gills were the main A. ocellatum infection site, accounting for 70.49–72.68% of the total trophonts in infected A. latus. The maximal tolerance dose (MTD) and lethal concentration 50 (LC50) of A. ocellatum to A. latus were 14,686 and 28,901 dinospores per fish, respectively. Tomonts can be preserved for at least 3 months at 12 °C. This study established an A. ocellatum infection model under laboratory conditions, which is potentially helpful in exploring alternative treatments and investigating the pathogenic mechanisms of amyloodiniosis.

Investigating the etiologies behind emergent mass mortalities of farmed Liza carinata juveniles from coastal farms at Damietta, Egypt

This study aimed to identify the mortality present in private fish farm Amyloodinium ocellatum and Cryptocaryon irritans were isolated from this outbreak affecting Liza carinata fingerlings at an earthen-based aquaculture facility in Damietta, Egypt. A total of 140 moribunds, L. carinata, were collected from the fish ponds during the mortality events. Physico-chemical analysis of water was analyzed. The skin, fins, gills, and eyes of each fish specimen were scraped gently onto slides in areas over 2 cm area. All smears were examined separately under the light microscope. Molecular identification of the parasites using analysis of ITS rDNA regions flanking both 18S and 28S rDNA genes of Amyloodinium protozoa and C. irritans. Identities of the detected parasites were confirmed by gene sequence and phylogenetic analysis. The majority of the examined fish (90%) were infected, 66.42% had a mixed infection, and 23.57% had a single infection either with A. ocellatum (10.71%) or C. irritans (12.85%).The mean intensity of A. ocellatum was 16.5 ± 2.03 in the skin and 13.18 ± 1.90 in the gills of infected fish, while that of C. irritans was 4.75 ± 1.05 in gills and 7.43 ± 1.45 in the skin, respectively. To control the emergent mortalities, affected ponds were treated using copper sulfate pentahydrate, hydrogen peroxides solutions, and amprolium hydrochloride powder in feed. Fish across the treated ponds were gradually improved with low morbidity and mortalityrates during the treatment period. The clinical disease was almost diminished at the end of the second week of treatment. Coinciding with the clinical improvement of the treated juveniles, microscopical examination of skin/gill scraps exhibited a marked decline in the number of protozoan parasites at the end of the second week of treatment.

Transcriptome Analysis of Greenfin Horse-Faced Filefish (Thamnaconus septentrionalis) Gills in Response to Amyloodinium ocellatum (AO) Infection

The greenfin horse-faced filefish (Thamnaconus septentrionalis) is susceptible to recurrent Amyloodinium ocellatum (AO) infestation over the grow-out production cycle. This parasite breeds mainly on the gills, causing hypoxia in the fish body, and leading to many deaths. The host-parasite response drives a complex immune reaction, which is poorly understood. To generate a model for host-parasite interaction and the pathogenesis of AO in greenfin horse-faced filefish, an RNA-seq approach, differential gene expression, GO, and KEGG analyses were employed. Overall, 624 new genes and 2076 differentially expressed genes (DEGs) were detected, including 942 upregulated and 1134 downregulated genes in the gills. Compared with the control group, the expression of leptin a, GTPase IMAP family member 4, and NLR family CARD domain-containing protein 3 was significantly higher in the AO-infected group. Conversely, cell wall integrity and stress response component 1-like, and hepcidin-like were significantly downregulated in the gills of AO-infected fish. GO and KEGG enrichment analysis indicated that DEGs were significantly enriched in signaling pathways associated with viral protein interaction with cytokine and cytokine receptor and cytokine–cytokine receptor interaction. Collectively, this transcriptomic study provides novel molecular insights into the pathology caused by AO infestation and alternative theories for future research implementing strategies to control and manage AO.

Report of Amyloodinium ocellatum in farmed black scraper (Thamnaconus modestus) in China

Black scraper, Thamnaconus modestus is a filefish with high market value but the standing stock has been decreased in the past years due to the overexploiting and environmental fluctuations. Industrial culture of black scraper relieved the tense between supply and demand but at the same time brought some disease problems. In this study, we reported a mass death case of cultured Thamnaconus modestus caused by Amyloodinium ocellatum in China. The disease outbroke at temperature of 22–24 °C and the main clinical symptoms were anoxia, decreased feeding activity, reduced mobility, lost balance and subcutaneous hemorrhage. According to the observation, the trophonts of A. ocellatum were non-transparent, suborbicula and the main parasitized tissues were gill and fin, accompanying with the necrocytosis of gill lamella, and hyperplasia of fin cell. The amplification and sequencing of the ribosomal RNA gene showed the parasite was 97.41–99.92% similar with A. ocellatum. The case that amyloodiniosis only occurred in T. modestu but not in turbot cultured with the same condition, indicated the tolerance of former was lower than the latter and farmers should take more surveillance to avoid the mass fish mortality in temperate season. This is the first report about the occurrence of amyloodiniosis in the black scraper.

Skin transcriptomic analysis and immune-related gene expression of golden pompano (Trachinotus ovatus) after Amyloodinium ocellatum infection

Amyloodiniosis is a severe disease of marine and brackish water fish caused by Amyloodinium ocellatum. Golden pompano (Trachinotus ovatus) is often repeatedly infected by A. ocellatum, leading to extensive mortality. However, little is known about the immune response mechanisms of the T. ovatus following reinfection with A. ocellatum. In this study, an extensive analysis at the transcriptome level of T. ovatus skin was carried out at 24 h post-infection by A. ocellatum. During the transcriptomic analysis, 1367 differentially expressed genes (DEGs) in the skin of T. ovatus under A. ocellatum infection and control conditions were obtained. In Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotated analyses, the DEGs were significantly enriched in the immune-related pathways. To better understand the immune-related gene expression dynamics, a quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to assess the primary and secondary infection groups of T. ovatus at different stages (3 h, 12 h, 24 h, 48 h and, 72 h post-infection) of infection with A.ocellatum. The results showed that innate immunity-related genes [interleukin (IL-8), chemokine ligand 3 (CCL3), toll-like receptor 7 (TLR7), and G-type lysosome (LZM g)] and adaptive immunity-related gene [major histocompatibility complex (MHC) alpha antigen I and MHC alpha antigen II] expression levels in the primary and secondary infection groups were significantly increased compared to the control group. The expression of MHC I and MHC II was more rapidly upregulated in the secondary infection group compared with the primary infection group after A.ocellatum infection. However, no significant differences of A.ocellatum load were observed in primary and secondary infection groups. In addition, the serum of the primary infection group had significantly higher concentrations of triglyceride (TG), higher alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) activities than the control group. This study contributes to understanding the defense mechanisms in fish skin against ectoparasite infection.

Cytotoxic and Hemolytic Activities of Extracts of the Fish Parasite Dinoflagellate Amyloodinium ocellatum.

The dinoflagellate Amyloodinium ocellatum is the etiological agent of a parasitic disease named amyloodiniosis. Mortalities of diseased fish are usually attributed to anoxia, osmoregulatory impairment, or opportunistic bacterial infections. Nevertheless, the phylogenetic proximity of A. ocellatum to a group of toxin-producing dinoflagellates from Pfiesteria, Parvodinium and Paulsenella genera suggests that it may produce toxin-like compounds, adding a new dimension to the possible cause of mortalities in A. ocellatum outbreaks. To address this question, extracts prepared from different life stages of the parasite were tested in vitro for cytotoxic effects using two cell lines derived from branchial arches (ABSa15) and the caudal fin (CFSa1) of the gilthead seabream (Sparus aurata), and for hemolytic effects using erythrocytes purified from the blood of gilthead seabream juveniles. Cytotoxicity and a strong hemolytic effect, similar to those observed for Karlodinium toxins, were observed for the less polar extracts of the parasitic stage (trophont). A similar trend was observed for the less polar extracts of the infective stage (dinospores), although cell viability was only affected in the ABSa15 line. These results suggest that A. ocellatum produces tissue-specific toxic compounds that may have a role in the attachment of the dinospores’ and trophonts’ feeding process.

Whole transcriptome analysis provides new insight on immune response mechanism of golden pompano (Trachinotus ovatus) to Amyloodinium ocellatum infestation

Ectoparasitic dinoflagellate Amyloodinium ocellatum (AO) is one of the most pathogenic protozoan parasites, causing amyloodiniosis in temperate and tropical marine fish. Golden pompano is susceptible to AO and increasing outbreaks of AO have posed a major threat to the economic sustainability of intensive aquaculture industry. In this study, natural parasite infection was simulated, and whole transcriptome analysis was performed with infected golden pompano gills to explore the host immune response to AO infestation. Overall, 4388 differentially expressed (DE) genes, 395 DE long non-coding RNAs (lncRNAs), 10 DE circular RNAs (circRNAs) and 96 DE microRNAs (miRNAs) were identified. DE genes were involved in pathways of ECM-receptor interaction and cytokine-cytokine receptor interaction, including many crucial extracellular matrix (ECM) components (integrin, laminin and collagen), cytokines (interleukin and lymphotoxin) and C-C motif chemokines. DE lncRNAs were significantly enriched in the regulation of immune cells, such as monocytes, neutrophils and lymphocytes. DE circRNAs were notably correlated with NOD-like receptor signaling pathway. DE miRNAs were prominently associated with immune-related pathways of cytokine-cytokine receptor interaction, intestinal immune network for IgA production, herpes simplex infection and TGF-beta signaling pathway. Furthermore, a core competing endogenous RNA (ceRNA) network was constructed, consisting of 28 DE mRNAs, 21 DE miRNAs, 8 DE circRNAs and 58 DE lncRNAs. The key roles of cytokine-cytokine receptor interaction, focal adhesion, MAPK signaling, NOD-like receptor signaling and mTOR signaling pathway were highlighted in the ceRNA network. In total, the present study uncovered the molecular changes of coding and non-coding RNAs involved in host immune response induced by AO infestation. The results could contribute significantly to a comprehensive understanding of host immune response to AO infestation and provide new clues for developing efficient amyloodiniosis control measures and germplasm improvement in artificial breeding of golden pompano.

Transcriptomic profiles of Florida pompano (Trachinotus carolinus) gill following infection by the ectoparasite Amyloodiniumocellatum

The dinoflagellate Amyloodinium ocellatum is an important pathogenic parasite infecting cultured marine and brackish water fishes worldwide. This includes cultured Florida pompano (Trachinotus carolinus), which is one of the most desirable marine food fish with high economic value in the USA. A. ocellatum infects fish gills and causes tissue damage, increased respiratory rate, reduced appetite, and mortality, especially in closed aquaculture systems. This study mimicked the natural infection of A. ocellatum in cultured pompano and conducted a transcriptomic comparison of gene expression in the gills of control and A. ocellatum infected fish to explore the molecular mechanisms of infection. RNA-seq data revealed 604 differentially expressed genes in the infected fish gills. The immunoglobulin genes (including IgM/T) augmentation and IL1 inflammation suppression were detected after infection. Genes involved in reactive oxygen species mediating parasite killing were also highly induced. However, excessive oxidants have been linked to oxidative tissue damage and apoptosis. Correspondingly, widespread down-regulation of collagen genes and growth factor deprivation indicated impaired tissue repair, and meanwhile the key executor of apoptosis, caspase-3 was highly expressed (25.02-fold) in infected fish. The infection also influenced the respiratory gas sensing and transport genes and established hypoxic conditions in the gill tissue. Additionally, food intake and lipid metabolism were also affected. Our work provides the transcriptome sequencing of Florida pompano and provides key insights into the acute pathogenesis of A. ocellatum. This information can be utilized for designing optimal disease surveillance strategies, future selection for host resistance, and development of novel therapeutic measures.

News Insights into the Host-Parasite Interactions of Amyloodiniosis in European Sea Bass: A Multi-Modal Approach.

Amyloodiniosis is a disease resulting from infestation by the ectoparasitic dinoflagellate Amyloodinium ocellatum (AO) and is a threat for fish species such as European sea bass (ESB, Dicentrarchus labrax), which are farmed in lagoon and land-based rearing sites. During the summer, when temperatures are highest, mortality rates can reach 100%, with serious impacts for the aquaculture industry. As no effective licensed therapies currently exist, this study was undertaken to improve knowledge of the biology of AO and of the host-parasite relationship between the protozoan and ESB, in order to formulate better prophylactic/therapeutic treatments targeting AO. To achieve this, a multi-modal study was performed involving a broad range of analytical modalities, including conventional histology (HIS), immunohistochemistry (IHC) and confocal laser scanning microscopy (CLSM). Gills and the oro-pharyngeal cavity were the primary sites of amyloodiniosis, with hyperplasia and cell degeneration more evident in severe infestations (HIS). Plasmacells and macrophages were localised by IHC and correlated with the parasite burden in a time-course experimental challenge. CLSM allowed reconstruction of the 3D morphology of infecting trophonts and suggested a protein composition for its anchoring and feeding structures. These findings provide a potential starting point for the development of new prophylactic/therapeutic controls.

Immune response of silver pomfret (Pampus argenteus) to Amyloodinium ocellatum infection.

Amyloodinium ocellatum (AO) infection in silver pomfret (Pampus argenteus) causes extensive mortality. Insufficient information exists on the molecular immune response of silver pomfret to AO infestation, so herein we simulated the process of silver pomfret being infected by AO. Translucent trophosomes were observed on the gills of AO-infected fish. Transcriptome profiling was performed to investigate the effects of AO infection on the gill, kidney complex and spleen. Overall, 404,412,298 clean reads were obtained, assembling into 96,341 unigenes, which were annotated against public databases. In total, 2730 differentially expressed genes were detected, and few energy- and immune-related genes were further assessed using RT-qPCR. Moreover, activities of three immune-related (SOD, AKP and ACP) and three energy-related (PKM, LDH and GCK) enzymes were determined. AO infection activated the immune system and increased interleukin-1 beta and immunoglobulin M heavy chain levels. Besides, the PPAR signalling pathway was highly enriched, which played a role in improving immunity and maintaining homeostasis. AO infection also caused dyspnoea, leading to extensive lactic acid accumulation, potentially contributing towards a strong immune response in the host. Our data improved our understanding regarding the immune response mechanisms through which fish coped with parasitic infections and may help prevent high fish mortality in aquaculture.

Innate immune-gene expression during experimental amyloodiniosis in European seabass (Dicentrarchus labrax)

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the causative agent of amyloodiniosis in European seabass (ESB, Dicentrarchus labrax). There is a lack of information about basic molecular immune response mechanisms of ESB during AO infestation. Therefore, to compare gene expression between experimental AO-infested ESB tissues and uninfested ESB tissues (gills and head kidney) RNA-seq was adopted. The RNA-seq revealed multiple differentially expressed genes (DEG), namely 679 upregulated genes and 360 downregulated genes in the gills, and 206 upregulated genes and 170 downregulated genes in head kidney. In gills, genes related to the immune system (perforin, CC1) and protein binding were upregulated. Several genes involved in IFN related pathways were upregulated in the head kidney. Subsequently, to validate the DEG from amyloodiniosis, 26 ESB (mean weight 14 g) per tank in triplicate were bath challenged for 2 h with AO (3.5 × 106/tank; 70 dinospores/mL) under controlled conditions (26−28 °C and 34‰ salinity). As a control group (non-infested), 26 ESB per tank in triplicate were also used. Changes in the expression of innate immune genes in gills and head kidney at 2, 3, 5, 7 and 23 dpi were analysed using real-time PCR. The results indicated that the expression of cytokines (CC1, IL-8) and antimicrobial peptide (Hep) were strongly stimulated and reached a peak at 5 dpi in the early infestation stage, followed by a gradual reduction in the recovery stage (23 dpi). Noticeably, the immunoglobulin (IgM) expression was higher at 23 dpi compared to 7 dpi. Furthermore, in-situ hybridization showed positive signals of CC1 mRNA in AO infested gills compared to the control group. Altogether, chemokines were involved in the immune process under AO infestation and this evidence allows a better understanding of the immune response in European seabass during amyloodiniosis.

Transcriptome Analysis of Amyloodinium ocellatum Tomonts Revealed Basic Information on the Major Potential Virulence Factors.

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the etiological agent of amyloodiniosis in European seabass (Dicentrarchus labrax) (ESB). There is a lack of information about basic molecular data on AO biology and its interaction with the host. Therefore, de novo transcriptome sequencing of AO tomonts was performed. AO trophonts were detached from infested ESB gills, and quickly becoming early tomonts were purified by Percoll® density gradient. Tomont total RNA was processed and quality was assessed immediately. cDNA libraries were constructed using TruSeq® Stranded mRNA kit and sequenced using Illumina sequencer. CLC assembly was used to generate the Transcriptome assembly of AO tomonts. Out of 48,188 contigs, 56.12% belong to dinophyceae wherein Symbiodinium microadriaticum had 94.61% similarity among dinophyceae. Functional annotations of contigs indicated that 12,677 had associated GO term, 9005 with KEGG term. The contigs belonging to dinophyceae resulted in the detection of several peptidases. A BLAST search for known virulent factors from the virulence database resulted in hits to Rab proteins, AP120, Ribosomal phosphoprotein, Heat-shock protein70, Casein kinases, Plasmepsin IV, and Brucipain. Hsp70 and casein kinase II alpha were characterized in-silico. Altogether, these results provide a reference database in understanding AO molecular biology, aiding to the development of novel diagnostics and future vaccines.

Comparative Therapeutic Effects of Natural Compounds Against Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae).

The fish parasites Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae) cause important losses in freshwater and marine aquaculture industry, respectively. The possible adverse effects of compounds used to control these parasites in aquaculture resulted in increased interest on the search for natural products with antiparasitic activity. In this work, eighteen plant-derived compounds (2′,4′-Dihydroxychalcone; 7-Hydroxyflavone; Artemisinin; Camphor (1R); Diallyl sulfide; Esculetin; Eucalyptol; Garlicin 80%; Harmalol hydrochloride dihydrate; Palmatine chloride; Piperine; Plumbagin; Resveratrol; Rosmarinic acid; Sclareolide; Tomatine, Umbelliferone, and Usnic Acid) have been tested in vitro. Sixteen of these were used to determine their effects on the gill cell line G1B (ATCC®CRL-2536™) and on the motility of viable dinospores of Amyloodinium ocellatum, and thirteen were screened for inhibitory activity against Saprolegnia spp. The cytotoxicity results on G1B cells determined that only two compounds (2′,4′-Dihydroxychalcone and Tomatine) exhibited dose-dependent toxic effects. The highest surveyed concentrations (0.1 and 0.01 mM) reduced cell viability by 80%. Upon lowering the compound concentration the percentage of dead cells was lower than 20%. The same two compounds revealed to be potential antiparasitics by reducing in a dose-dependent manner the motility of A. ocellatum dinospores up to 100%. With respect to Saprolegnia, a Minimum Inhibitory Concentration was found for Tomatine (0.1 mM), Piperine and Plumbagin (0.25 mM), while 2′,4′-Dihydroxychalcone considerably slowed down mycelial growth for 24 h at a concentration of 0.1 mM. Therefore, this research allowed to identify two compounds, Tomatine and 2′,4′-Dihydroxychalcone, effective against both parasites. These compounds could represent promising candidates for the treatment of amyloodiniosis and saprolegniosis in aquaculture. Nevertheless, further in vitro and in vivo tests are required in order to determine concentrations that are effective against the considered pathogens but at the same time safe for hosts, environment and consumers.

Studies on Prevailing Parasitic Fish Diseases in Pre-mature Cultured Sea bass, Dicentrarchus labrax, Sea bream and Mugil cephalus at Ismailia, Province with Special References to Control

This study concentrated on the assessment of the prevailing parasitic fish diseases in some marine fishes at Ismailia province and how to control the infestation using microalgae. This study was carried out on 1080 pre-mature fish (360 D. labrax (225±25 g) and 360 S aurata (150±25 g) and 360 M. cephalus (125±25 g) collected from similar ponds of studies to be examined at the end of treatment. In addition to that we followed non-treated fish (1080 premature). The infested fish showed dark colour and respiratory signs. Post mortem lesions were a presence of congestion or paleness and destruction of gill filaments. The total prevalence of infestation was the total prevalence of parasitic infection of non-treated fishes was 45.83 %. The highest percentage was in D. labrax 56.94 % followed by S. aurata 47.22%, the lowest percentage in M. cephalus 33.33. The total prevalence of parasitic infection in premature treated with 2 g algae was 28.79%, followed by 3 g algae was 23.60 %, while the lowest percentage with 5 g algae was 20.37 % respectively. The detected species of parasites were protozoal parasites, Amyloodinium ocellatum and Riboscyphidia in additions of marine monogenea, Lamellodiscus diplodicus isolated from D Labrex, Mugil Cephalus and S aurata. The present study concluded that, the use of microalgae instead of fish meal decreased parasitic infestation in marine fish. The histopathological alteration of natural infested examined fishes was also recorded.