Pacific oysters, C. gigas (shell length 12–16 cm) were collected from a local farm in Dalian, Liaoning, China, and cultured in aerated seawater (SW) at 15 ± 2°C for 7 days. Different tissues (hemolymph, adductor muscle, gills, mantle, gonad, labial palps, and hepatopancreas) were collected from nine untreated oysters, and every three individuals were pooled together as one sample. There were three replicates for each tissue. The collected samples were added with TRIzol reagent (Invitrogen) for RNA extraction to examine the relative mRNA expressions of mitophagy-related genes. Hemolymph was collected from the posterior adductor muscle sinus of each oyster by using 1 ml of acid citrate-dextrose anticoagulant agent (20.8 g L⁻¹ glucose, 3.4 g L⁻¹ EDTA·2Na, 22.5 g L⁻¹ NaCl, and 8.0 g L⁻¹ disodium citrate) at a ratio of 1:1 and then centrifuged at 800 × g at 4°C for 4 min to harvest the hemocytes. The collected hemocytes were treated with 20 µM CCCP (Abcam) at 18°C with a 20 µM DMSO group as control. The samples were then analyzed by using Western blot or immunocytochemical assay.

The hemocytes were also collected after the oysters received an injection of 100 µl Vibrio splendidus at 2 × 10⁸ CFU ml⁻¹ dissolved in SW. The SW was used as the control. Nine oysters were randomly sampled from each group at 0, 3, 6, 12, 24, and 48 h after V. splendidus stimulation. The hemocytes from three oysters were pooled together as one sample, and there were three samples for each time point. The collected samples were then analyzed using Western blot.

Searching of mitophagy-related genes in C. gigas transcriptome data was conducted by using the tBLAST program acquired from the NCBI database (https://www.ncbi.nlm.nih.gov/) with known proteins (from mammals, yeast, insects, etc.) as queries. The primers for these genes (Supplementary Table S3) were designed in accordance with the sequence information acquired from the NCBI database (https://www.ncbi.nlm.nih.gov/). The obtained PCR products were inserted into a pMD 19-T vector (TaKaRa) and sequenced by Sangon Biotech (Sangong, China).

A translation tool (http://web.expasy.org/translate/) was used to predict the amino acid sequence of mitophagy-related genes. The simple modular architecture research tool (SMART) (http://smart.embl-heidelberg.de/) was used to predict the conserved domain. ClustalW multiple alignment program (http://www.ebi.ac.uk/clustalw/) and DNAMAN were used to create the multiple sequence alignment. The neighbor-joining (NJ) phylogenetic tree was constructed using the MEGA6.0 package.

The mRNA expressions of mitophagy-related genes in the different tissues were examined by quantitative reverse transcription PCR (qRT-PCR) with the elongation factor (CgEF; NP_001292242.2) fragment amplified with primers CgEF-RT-F and CgEF-RT-R (Supplementary Table S3) as an internal reference. The reaction was carried out in a total volume of 10 μl, including 5 µl of SYBR Green Master Mix (Applied Biosystems), 0.2 µl of each primer, 0.2 µl of ROX, 2 µl of the 20 times-diluted cDNA, and 2.4 µl of DEPC-treated water. qRT-PCR was programmed at 95°C for 10 min, followed by 40 cycles at 95°C for 10 s and 60°C for 45 s. The relative mRNA expression levels were calculated by the comparative Ct method (2^−∆∆Ct method) (Livak and Schmittgen, 2001). Significant differences were accepted at p < 0.05.

The hemocyte proteins were extracted from oysters after CCCP treatment or V. splendidus stimulation, separated by 15% SDS-polyacrylamide gel electrophoresis, and then transferred onto the nitrocellulose membrane by using a mini transfer tank for electrophoresis. The membranes were blocked with 5% nonfat milk in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.2% Tween 20) for 10 h at 4°C and then incubated with 1/1,000 diluted anti-LC3B antibody (ab51520, Abcam) or β-tubulin (Beyotime, China) in TBST, with 5% nonfat milk at 4°C for 10 h. HRP-conjugated Rabbit anti-mouse IgG (Shangon Biotech) diluted at 1/10,000 with TBS (10 mM Tris-HCl, pH 7.5, and 150 mM NaCl) was incubated with the membranes at room temperature for 2 h after the membranes were washed to remove the free nonspecifically bound antiserum. The membranes were finally immersed in the reaction system (1:1 mixing BeyoECL Moon A and B) in the dark for 1 min and imaged with Amersham Imager 600 (GE Healthcare, United States). The relative expression level of CgLC3Ⅱ protein was analyzed by ImageJ with β-tubulin protein as the control (Sun, et al., 2020).

The hemocytes were extracted from the oysters at 3 h after CCCP treatment or at 24 h after V. splendidus stimulation. The hemocytes from the dimethyl sulfoxide (DMSO) or SW group were used as control, respectively. The mitochondrial membrane potential of hemocytes was measured using a mitochondrial membrane potential assay kit with JC-1 (Beyotime, China). The treated hemocytes were deposited on clean slides in the wet chamber and mounted on buffered glycerin (50%) for observation using a fluorescence microscope (ZEISS, Germany). ImageJ was used to calculate the fluorescence signals of JC-1 monomers and JC-1 aggregates.

The hemocytes were collected and resuspended in an L15 medium. The suspension was deposited on clean slides (a drop on each) in the wet chamber. After the monolayer was sedimented on the side, the hemocytes were fixed with 4% paraformaldehyde (PFA) for 10 min. After being washed with L15 medium for 5 min, the hemocytes were blocked by incubating in 3% BSA at 37°C for 30 min. The supernatant was then removed, and the dishes were incubated with 500 µl of anti-LC3 (diluted 1:200 in 3% BSA) at 4°C for 2 h. After washing three times with L15 medium, the hemocytes were incubated with Alexa Fluor 488-labeled goat anti-Rabbit secondary antibody (diluted 1:200 in 3% BSA) at 37°C for 1 h. After washing another three times with an L15 medium, the hemocytes were incubated with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Solarbio Life Sciences, China, diluted 1:1,000 in 3% BSA) to stain the nucleus. The slides were mounted on buffered glycerin (50%) and observed using a fluorescence microscope (ZEISS, Germany) after the final three times of washing with the L15 medium. ImageJ was used to calculate the fluorescence signals of CgLC3 in the hemocytes.

An immunocytochemical assay was performed to observe the colocalization of mitochondria with lysosomes in hemocytes 3 h after the treatment with 20 μM CCCP. DMSO was used as the control. A measure of 1 ml of hemocyte resuspension (about 1 × 10⁶ cells in L15 medium) was incubated with MitoTracker Green (Beyotime) and LysoTracker Red (Beyotime) dyes (diluted 1:15,000 in L15 medium) at 37°C in the dark for 30 min, respectively. The hemocytes were washed thrice with L15 medium and fixed with 1 ml of a mixture containing an L15 medium and 4% paraformaldehyde (1:1 in volume) for 10 min. After being washed thrice with L15 medium, the hemocyte resuspension was deposited on poly-lysine microscope slides for adhesion at room temperature for 30 min. Fluorescence was observed under an inverted fluorescence microscope (ZEISS, Germany).

A measure of 1 mL of the hemocyte resuspension (about 1 × 10⁶ cells in L15 medium) was incubated with LysoTracker Red or mitochondria at 37°C in the dark for 30 min. After being washed thrice with L15 medium, the hemocytes were fixed with 1 ml of a mixture containing an L15 medium and 4% paraformaldehyde (1:1 in volume) for 15 min. The hemocytes were resuspended in an L15 medium and deposited on the poly-L-lysine–coated slides. After 1 h, the slides were incubated successively with 500 µl of anti-LC3B antibody (ab51520, Abcam) (diluted 1:200 in 3% BSA) at 4°C for 2 h and Alexa Fluor 488-labeled goat anti-Rabbit as a secondary antibody (diluted 1:200 in 3% BSA) at 37°C for 1 h. The slides were washed with L15 medium three times and mounted on buffered glycerin (50%) for observation under a fluorescence microscope (ZEISS, Germany).

The autophagy assay kit (Abcam) was used to analyze the autophagy levels of hemocytes at 24 h after the oysters received an injection of V. splendidus. SW was used as the negative control and untreated oysters as the blank control. The hemocytes about 1 × 10⁶ cells mL⁻¹ were resuspended in 250 μl 1 × assay buffer and then incubated with 250 μl of diluted CYTO-ID^® Green stain solution at room temperature in the dark for 30 min. The treated hemocytes were then washed with 1 × assay buffer three times. The flow cytometry assay was conducted using Amnis ImageStream MkII to count the fluorescence signals of autophagy through IDEAS analysis software. A total of 5,000 hemocytes were analyzed for each sample.

The oyster hemolymph was collected from the sinus of five oysters 3 h after CCCP treatment and centrifuged at 1,500 rpm, 4°C for 4 min. The hemocytes were resuspended in an L15 medium. Then, 1 ml of hemocyte suspension (1 × 10⁶ cells) was centrifuged at 1,500 rpm, 4°C for 8 min. The samples were fixed in 3% glutaraldehyde solution (Sigma-Aldrich, G5882) at 4°C for 24 h and then analyzed by JEM-1400 TEM.

All the data were represented as mean ± standard deviation, and they were analyzed by one-way ANOVA and multiple comparisons using SPSS 22.0. The statistically significant differences were designated at p < 0.05 and were extremely significant at p < 0.01 (N = 3).

The mitophagy inducer CCCP was used to induce mitophagy in oyster hemocytes, and mitophagy was observed by TEM. The organelles were observed to be intact without swelling and deformation in the DMSO group under an electron microscope, and there were few vacuoles present in hemocytes. In the CCCP treatment group, the accumulation of vacuolar structures and autophagosomes was observed in the cytoplasm of hemocytes, which was 2.36-fold (p < 0.05) of that in the DMSO group (Figures 1A,B).

A total of 10 mitophagy process–related genes were identified from C. gigas, including CgNIX, CgFUNDC1, CgPHB2, CgCardiolipin, CgP62, CgOPTN, CgVDAC2, CgMFN2, CgPARL, and CgMPP. They all contained the conserved domains similar to their homologs in mammals and yeast, and their detailed sequence features are shown in Supplementary Table S1. Multiple sequence alignments revealed that the amino acid sequences of PHB2s, cardiolipins, P62s, OPTNs, VDAC2s, MFNs, and MPPs were relatively conserved between all the tested species (Supplementary Figures S1A–S7C). The 10 mitophagy process–related proteins were clustered with their corresponding molecules from C. virginica, and all fell in the invertebrate branch in the phylogenetic tree (Supplementary Figures S1B–S7D). CgOPTN contained three LC3-interacting regions (LIRs). CgNIX, CgFUNDC1, and CgCardiolipin contained two LIRs. One LIR was identified in CgPHB2 and CgP62 (Figure 2).

The mRNA transcripts of the 10 mitophagy process–related genes were found to be distributed in all the tested tissues, including the gills, mantle, hepatopancreas, adductor muscle, gonad, hemolymph, and labial palps, with some differences in the expression levels (Figures 3, 4; Supplementary Table S2). The 10 genes had relatively lower expression levels in gills and mantle, and their expression levels were relatively higher in adductor muscle, gonad, and hemocytes (Figures 3, 4; Supplementary Table S2). The detailed information about their mRNA expression levels is listed in Supplementary Table S2.

The mitochondrial membrane potential of hemocytes in the CCCP-treated oysters was determined by using the mitochondrial membrane potential assay kit with the mitochondrial probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). The positive signal of JC-1 monomers in the cell cytoplasm was observed in green, while JC-1 aggregates were stained in red. After CCCP treatment, the fluorescence intensity of JC-1 monomers (green signals) in hemocytes increased significantly, which was 4.60-fold of that in the DMSO group. The value of JC-1 aggregates (red signals) in hemocytes from the CCCP treatment group decreased significantly, which was 0.20-fold of that in the DMSO group (Figures 5A,B). Western blot assay with the CgLC3 antibody revealed that there was a CgLC3B-II band in CCCP-treated hemocytes, while no bands were detected in the DMSO treatment group (Figure 5C). The immunocytochemical assay with the anti-LC3 antibody was used to analyze the subcellular distribution of CgLC3 in the hemocytes in the CCCP treatment group. The positive signals of CgLC3B were observed in green, which were accumulated in hemocytes in the form of speckled dots in the CCCP treatment group, compared with that in the DMSO treatment group (Figures 5D,E).

The content of lysosomes was analyzed with LysoTracker Red in the hemocytes in the CCCP treatment group. The fluorescence intensity of LysoTracker Red–labeled lysosomes (red signals) in the hemocytes of the CCCP treatment group increased significantly, which was 3.91-fold of that in the DMSO group (Figures 6A,B). CgLC3 was marked by the CgLC3 antibody, and Alexa Fluor 488 conjugated antibody was observed in green. In the CCCP treatment group, the statistic value of CgLC3B (green signals) co-localized with the LysoTracker Red–labeled lysosomes (red signals) in hemocytes increased significantly (4.88-fold of that in the DMSO group) (Figures 6C,D). The mitochondria were stained by MitoTracker Green in green signals. The statistic value of mitochondria (green signals) co-localized with the LysoTracker Red–labeled lysosomes (red signals) in the hemocytes of the CCCP treatment group increased significantly, which was 5.73-fold of that in the DMSO group (Figures 6E,F).

The fluorescence intensity of JC-1 monomers (green signals) in hemocytes increased after the oysters were stimulated by V. splendidus, which was 1.29-fold of that in the SW group (Figures 7A,B). The band intensity of CgLC3B-II in hemocytes increased at 3 and 12 h and peaked at 24 h after V. splendidus stimulation (Figure 7C). CgLC3 was marked by the CgLC3 antibody and Alexa Fluor 488 conjugated antibody in green signals, and mitochondria were stained by using MitoTracker Red in red signals. The co-localization of CgLC3 (green signals) with mitochondria (red signals) was enhanced at 24 h after V. splendidus stimulation, compared with that of the SW group (Figures 7D,E). The autophagy assay kit was used to analyze the autophagy levels of the hemocytes at 24 h after V. splendidus stimulation. The fluorescence intensity of autophagy in hemocytes increased after the oysters were stimulated by V. splendidus, which was 1.60-fold of that in the SW group (Figures 7F,G).