Monitoring Autophagy at Cellular and Molecular Level in Crassostrea gigas During an Experimental Ostreid Herpesvirus 1 (OsHV-1) Infection

Abstract

Mortality outbreaks of young Pacific oysters, Crassostrea gigas, have seriously affected the oyster-farming economy in several countries around the world. Although the causes of these mortality outbreaks appear complex, a viral agent has been identified as the main factor: a herpesvirus called ostreid herpesvirus 1 (OsHV-1). Autophagy is an important degradation pathway involved in the response to several pathologies including viral diseases. In C. gigas, recent studies indicate that this pathway is conserved and functional in at least haemocytes and the mantle. Furthermore, an experimental infection in combination with compounds known to inhibit or induce autophagy in mammals revealed that autophagy is involved in the response to OsHV-1 infection. In light of these results, the aim of this study was to determine the role of autophagy in the response of the Pacific oyster to infection by virus OsHV-1. For this purpose, an experimental infection in combination with a modulator of autophagy was performed on Pacific oysters known to have intermediate susceptibility to OsHV-1 infection. In haemolymph and the mantle, the autophagy response was monitored by flow cytometry, western blotting, and real-time PCR. At the same time, viral infection was evaluated by quantifying viral DNA and RNA amounts by real-time PCR. Although the results showed activation of autophagy in haemolymph and the mantle 14 hours post infection (after viral replication was initiated), they were also indicative of different regulatory mechanisms of autophagy in the two tissues, thus supporting an important function of autophagy in the response to virus OsHV-1.

Keywords: Pacific oyster (Crassostrea gigas); autophagy; herpesvirus; innate immunity; invertebrate.

Figure 1

Kaplan-Meier survival curves of the pacific oyster Crassostrea gigas injected with the virus OsHV-1, exposed to NH₄Cl or injected with OsHV-1 and exposed to NH₄Cl. The control condition corresponds to pacific oysters injected with artificial sea water. In each condition the mortality was monitored on n=30 oysters. a: significant difference with the artificial seawater condition (p < 0.05). b: significant difference with the virus condition (p < 0.05). c: significant difference with the virus+NH₄Cl condition (p < 0.05).

Figure 2

Detection of OsHV-1 DNA and RNA in haemolymph and mantle of Crassostrea gigas. (A) Viral DNA amounts detected by real time PCR in Pacific oysters of the four different tested conditions (artificial seawater, NH₄Cl, virus and virus+NH₄Cl) after injection by OsHV-1 (i) in haemolymph (n=2 pools of 15 animals) and (ii) in the mantle. (n=6 animals) The line represents the trend curve of viral DNA amount in the virus condition (red line) and the virus+NH₄Cl condition (purple line). No significant difference was observed between the virus and virus+NH₄Cl conditions in the mantle and haemolymph. (B) Relative genes expressions of ORF 80, ORF 87 and ORF 99 estimated by RT-PCR at different time of exposure for the virus and virus+NH₄Cl condition in haemolymph (n= 2 pools of 15 animals) and mantle (n=3 animals). The line represents the trend curve of the expression of each gene in the virus condition (red line) and the virus+NH₄Cl condition (purple line). No significant difference was observed between the virus and virus+NH₄Cl condition in haemolymph and the mantle of the Pacific oyster. VGE, viral gene expression.

Figure 3

Modulation of the autophagy in Crassostrea gigas exposed to OsHV-1. (A) Monitoring of autophagy in haemocytes of C. gigas by flow cytometry. Scatterplot of the difference of the percentage of haemocytes presented autophagosomes between the seawater condition (ASW) and the virus condition from 6 to 30 hpi (n=2 pools of 15 animals). The line represents the trend curve of the difference of percentage of cells between the two conditions. *Significant increase of the difference of the percentage of cells presented autophagosomes between the virus and seawater condition (p < 0.05). (B) Relative gene expression of key autophagy genes in haemolymph and the mantle of the Pacific oysters, C. gigas, in ASW condition (blue) and virus condition (red) at each sampling time point (T0, 6, 10, 14, 18, 24 and 30 hpi) detected by real time PCR (haemolymph: n=2 pools of 15 animals; mantle: n=3 animals). The line represents the trend curve of the expression of each gene in the virus condition (red) and the ASW condition (blue). Significant difference between the virus and ASW condition at early (6-14 hpi) or late time points (18-30 hpi) of the experimental infection (p < 0.05). Significant difference between the virus and ASW conditions of the same time point (p < 0.05). RGE, relative gene expression. (C) Detection of the autophagy protein SQSTM1 in the mantle of C. gigas during the kinetic of infection by OsHV-1 in the virus condition at each sampling time point (for each time point n=1 pool of 3 animals). Significant differences between the virus and ASW condition at early (6-14 hpi) or late time points (18-30 hpi) of the experimental infection (p < 0.05).

Figure 4

Modulation of autophagy in Crassostrea gigas exposed to NH₄Cl. (A) Monitoring of autophagy in haemocytes of C. gigas by flow cytometry. Scatterplot of the difference of the percentage of haemocytes presented autophagosomes between the seawater condition (ASW) and the NH₄Cl condition from 6 to 30 hpi (n=2 pools of 15 animals). The line represents the trend curve of the difference of percentage of cells between the two conditions. *Significant increase of the difference of the percentage of cells presented autophagosomes between the NH₄Cl and ASW condition between 18 to 30hpi (p < 0.05). (B) Relative gene expression of key autophagy genes in haemolymph and the mantle of the Pacific oysters, C. gigas, in ASW condition (blue) and NH₄Cl condition (green) at each sampling time point (T0, 6, 10, 14, 18, 24 and 30 hpi) detected by real time PCR (haemolymph: n=2 pools of 15 animals; mantle: n=3 animals). The line represents the trend curve of the expression of each gene in the NH₄Cl condition (green) and the ASW condition (blue). Significant difference between the NH₄Cl and ASW conditions at early (6-14 hpi) or late time points (18-30 hpi) of the experimental infection (p < 0.05). Significant difference between the NH₄Cl and ASW conditions of the same time point (p < 0.05). RGE, relative gene expression. (C) Detection of the autophagy protein SQSTM1 in the mantle of C. gigas during the kinetic of exposition to NH₄Cl at each sampling time point (for each time point n=1 pool of 3 animals). Significant differences between the NH₄Cl and ASW condition at early (6-14 hpi) or late time points (18-30 hpi) of the experimental infection (p < 0.05).

Figure 5

Modulation of autophagy in Crassostrea gigas exposed to virus+NH₄Cl. (A) Monitoring of autophagy in haemocytes of C. gigas by flow cytometry. Scatterplot of the difference of the percentage of haemocytes with autophagosomes between the seawater condition (ASW) and the virus+NH₄Cl condition from 6 to 30 hpi (n=2 pools of 15 animals). The line represents the trend curve of the difference of percentage of cells between the two conditions. *Significant increase of the difference of the percentage of cells presented autophagosomes between the virus+NH₄Cl and ASW condition between 18 to 30hpi (p < 0.05). (B) Relative gene expression of key autophagy genes in haemolymph and the mantle of the Pacific oysters, C. gigas, in seawater condition (blue) and the virus+NH₄Cl condition (purple) at each sampling time point (T0, 6, 10, 14, 18, 24 and 30 hpi) detected by real time PCR (haemolymph: n=2 pools of 15 animals; mantle: n=3 animals). RGE, relative gene expression. The line represents the trend curve of the expression of each gene in the virus+NH₄Cl condition (purple) and the ASW condition (blue). Significant difference between the virus+NH₄Cl and ASW conditions at early (6-14 hpi) or late time points (18-30 hpi) of the experimental infection (p < 0.05). Significant difference between the virus+NH₄Cl and ASW conditions of the same time point (p < 0.05). (C) Detection of the autophagy protein SQSTM1 in the mantle of C, gigas during the kinetic of infection by OsHV-1 in the virus+NH₄Cl condition at each sampling time point (for each time point n=1 pool of 3 animals). Significant differences between the virus+NH₄Cl and ASW condition at early (6-14 hpi) or late time points (18-30 hpi) of the experimental infection (p < 0.05).

Figure 6

Modulation of autophagy in the mantle and haemolymph of the Crassostrea gigas, during the process of an infection by the virus OsHV-1. (A) in the NH₄Cl condition; (B) in the virus condition; (C) in the virus+NH₄Cl condition. The thickness of each color square represents an increase of the parameter considered.