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Fish Shellfish Immunol ; 93: 958-964, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31442589


The protozoan parasite Bonamia ostreae has been associated with the decline of flat oyster Ostrea edulis populations in some European countries. Control of shellfish diseases mostly relies on prevention measures including transfer restrictions and stock management measures such as breeding programmes. These prevention and mitigation measures require a better understanding of interactions between host and pathogens. Previous in vitro studies allowed identifying apoptosis as a mechanism activated by the flat oyster in response to B. ostreae. However, these experiments also suggested that the parasite is able to regulate apoptosis in order to survive and multiply within hemocytes. By simplifying the conditions of infection, in vitro studies allow identifying most distinct features of the response of the host. In order to appreciate the relative importance of apoptosis in this response at the oyster scale, in vivo trials were carried out by injecting with parasites oysters from two French locations, Quiberon Bay (Brittany) and Diana Lagoon (Corsica). Apoptosis was investigated on pools of hemolymph from oysters collected at early and later times after injection using previously developed tools. Apoptotic cellular activities including intracytoplasmic calcium concentration, mitochondrial membrane potential and phosphatidyl serine externalization were analysed using flow cytometry. Moreover, the expression of flat oyster genes involved in both extrinsic and intrinsic pathways was measured using real time quantitative PCR.

Sci Rep ; 8(1): 12494, 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30131502


Bonamia ostreae has been associated with the decline of flat oyster Ostrea edulis populations in some European countries. This obligatory intracellular parasite persists and multiplies into hemocytes. Previous in vitro experiments showed that apoptosis is activated in hemocytes between 1 h and 4 h of contact with the parasite. The flat oyster uses the apoptosis pathway to defend against B. ostreae. However, the parasite might be also able to modulate this response in order to survive in its host. In order to investigate this hypothesis the apoptotic response of the host was evaluated using flow cytometry, transmission electron microscopy and by measuring the response of genes involved in the apoptotic pathway after 4 h. In parallel, the parasite response was investigated by measuring the expression of B. ostreae genes involved in different biological functions including cell cycle and cell death. Obtained results allow describing molecular apoptotic pathways in O. edulis and confirm that apoptosis is early activated in hemocytes after a contact with B. ostreae. Interestingly, at cellular and molecular levels this process appeared downregulated after 44 h of contact. Concurrently, parasite gene expression appeared reduced suggesting that the parasite could inhibit its own metabolism to escape the immune response.

PLoS One ; 12(5): e0177448, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28542284


Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels.

Apoptose , Células Sanguíneas/citologia , Crassostrea/virologia , DNA Viral/metabolismo , Herpesviridae/fisiologia , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Animais , Células Sanguíneas/virologia , Crassostrea/genética , Fragmentação do DNA , Regulação da Expressão Gênica , Herpesviridae/genética , Herpesviridae/metabolismo , Fosfatidilserinas/metabolismo , Fatores de Tempo
Fish Shellfish Immunol ; 56: 322-329, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27431587


The in vitro model Ostrea edulis hemocyte - Bonamia ostreae is interesting to investigate host-parasite interactions at the cellular level. Indeed, this unicellular parasite infects the flat oyster Ostrea edulis and multiplies within hemocytes, the central effectors of oyster defenses. Apoptosis is a mechanism used by many organisms to eliminate infected cells. In order to study the potential involvement of this mechanism in the oyster response to B. ostreae, in vitro experiments were carried out by exposing hemocytes from the naturally susceptible oyster O. edulis and a resistant oyster species Crassostrea gigas to live and heat-inactivated parasites. Hemocyte apoptotic response was measured using a combination of flow cytometry and microscopy analyses. Whatever the host species was, the parasite was engulfed in hemocytes and induced an increase of apoptotic parameters including intracytoplasmic calcium concentration, mitochondrial membrane potential or phosphatidyl-serine externalization as well as ultrastructural modifications. However, the parasite appears more able to infect flat oyster than cupped oyster hemocytes and the apoptotic response was more important against live than dead parasites in the natural host than in C. gigas. Our results suggest that O. edulis specifically responds to B. ostreae by inducing apoptosis of hemocytes.

Apoptose , Haplosporídios/fisiologia , Interações Hospedeiro-Parasita , Ostrea/fisiologia , Ostrea/parasitologia , Animais , Citometria de Fluxo , Hemócitos/parasitologia , Hemócitos/fisiologia , Hemócitos/ultraestrutura , Microscopia Eletrônica de Transmissão
Fish Shellfish Immunol ; 46(2): 232-42, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26057459


Apoptosis is a fundamental feature in the development of many organisms and tissue systems. It is also a mechanism of host defense against environmental stress factors or pathogens by contributing to the elimination of infected cells. Hemocytes play a key role in defense mechanisms in invertebrates and previous studies have shown that physical or chemical stress can increase apoptosis in hemocytes in mollusks. However this phenomenon has rarely been investigated in bivalves especially in the flat oyster Ostrea edulis. The apoptotic response of hemocytes from flat oysters, O. edulis, was investigated after exposure to UV and dexamethasone, two agents known to induce apoptosis in vertebrates. Flow cytometry and microscopy were combined to demonstrate that apoptosis occurs in flat oyster hemocytes. Investigated parameters like intracytoplasmic calcium activity, mitochondrial membrane potential and phosphatidyl-serine externalization were significantly modulated in cells exposed to UV whereas dexamethasone only induced an increase of DNA fragmentation. Morphological changes were also observed on UV-treated cells using fluorescence microscopy and transmission electron microscopy. Our results confirm the apoptotic effect of UV on hemocytes of O. edulis and suggest that apoptosis is an important mechanism developed by the flat oyster against stress factors.

Apoptose/efeitos da radiação , Ostrea/efeitos da radiação , Raios Ultravioleta , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , Hemócitos/efeitos dos fármacos , Hemócitos/efeitos da radiação , Ostrea/efeitos dos fármacos , Ostrea/imunologia