The Pacific oysters C. gigas (about 2-year old, averaging 150 mm in shell length) were collected from a local farm in Dalian, Liaoning Province, China, and maintained in the aerated seawater at 20°C for 7 days before processing. The eggs and sperms were scraped from different adult oysters and mixed together for fertilization. After fertilization, the developing embryos were continuously observed by microscope until the second polar body was formed about 90 min post fertilization. Then, the embryos were transferred into an aquaculture tank containing 100 L aerated seawater of 20°C (Zhang et al., 2019).

Before acidification treatment on oyster larvae, trochophore were collected at 12 h post fertilization (hpf) according to previous description (Kin et al., 2009), and randomly divided into three groups. The remaining larvae were then transferred into three tanks containing seawater at a pH level of 8.10 ± 0.05 (control group), 7.80 ± 0.05 (moderate acidification treatment group) and 7.40 ± 0.05 (severe acidification treatment group), respectively. After acidification treatment, the larvae were collected at 15 hpf (early D-shape larvae) and 19 hpf (D-shape larvae), respectively. Samples for RNA extraction were added with 1 mL of Trizol reagent (Invitrogen, United States) in each tube. Larvae for whole-mount immunofluorescence assay were first relaxed by gradual addition of 7.5% MgCl₂, followed by fixation in fresh 4% paraformaldehyde (PFA) in 0.01 M phosphate buffer saline (PBS) (0.2 g KCl, 8.0 g NaCl, 2.9 g Na₂HPO₄⋅12H₂O, 0.2 g KH₂PO₄, pH 7.40) at 4°C for 3 h and washed three times (15 min for each time) with pre-cold PBS. After fixation, the samples were dehydrated and stored in pure methanol at −20°C for the subsequent whole-mount immunofluorescence assay.

As for the acidification treatment on adult oysters, 27 oysters were equally divided into three groups. Oyster without any treatment were designated as the control group (pH 8.10 ± 0.05), while the other bathed in acidified seawater at a pH level of 7.80 ± 0.05 and 7.40 ± 0.05 were employed as the moderate acidification treatment group and severe acidification treatment group (Wang et al., 2016). The mantles of three groups were collected at 7th day post treatment and every mantle sample collected from three oysters as one replicate. In addition, there are three replicates were conducted for each assay group. Samples for RNA extraction were added with 1 mL of Trizol reagent in each tube (Invitrogen, United States) and frozen immediately in liquid nitrogen. Tissues for sectioning and immunofluorescence assay were firstly fixed with Bouin’s solution (formalin 10%, picric acid saturated solution in distilled water, and glacial acetic acid, 15:5:1) for 24 h and then dehydrated with gradient ethanol.

The pH value of the acidification treatment group was controlled using an acidometer (AiKB, Qingdao, China). Total alkalinity was determined by end-point titration of 25 mmol L^–1 HCl on 50 mL samples. The pH value, total alkalinity and partial press of CO₂ (pCO₂) were measured in the experiment. The total alkalinity in the pH 8.1, pH 7.8 and pH 7.4 groups was 2849.1 ± 14.5, 2416.3 ± 125.1, and 2110.6 ± 75.3 μeq kg^–1, respectively. And the partial pressure of CO₂ was about 658.1 ± 11.0, 1217.3 ± 11.6, and 2268.4 ± 50.1 ppm in pH 8.1, pH 7.8 and pH 7.4 treatment groups, respectively (Zhang et al., 2019). All the animal-involving experiments of this study were approved by the Ethics Committee of Dalian Ocean University.

Total RNA was isolated from oyster tissues using Trizol reagent according to the standard protocol (Invitrogen). RNA concentration was measured on a NanoDrop reader (Saveen & Werner ApS, Denmark), and the integrity and purity of RNA was examined by running in 1.0% agarose gel. The total RNA was then treated with DNaseI (promega) to remove trace DNA contamination. The synthesis of the first-strand cDNA was carried out with Promega M-MLV RT with oligo (dT)-adaptor priming according to the manufactory’s protocol. The synthesis reaction was performed at 42°C for 1 h, terminated by heating at 95°C for 5 min. The cDNA mix was diluted to 1:40 and stored at −80°C for subsequent SYBR Green Fluorescent Quantificational real-time PCR.

An engrailed (CGI_10012208) was identified from oyster which was designated as Cgengrailed-1 (Huang et al., 2015). The open reading frame (ORF) of Cgengrailed-1 was cloned from cDNA library using specific primers (P1 and P2, Table 1). The PCR procedure of Cgengrailed-1 was as follows: 5 min at 95°C; 30 cycles at 94°C for 30 s, 56°C for 1 min, 72°C for 40 s, and 72°C for 10 min. The PCR product was gel-purified, cloned into PMD 19-T simple vector (Takara), and confirmed by DNA sequencing.

The homology searches of the cDNA sequence and protein sequence of Cgengrailed-1 was conducted with BLAST algorithm at the National Center for Biotechnology Information¹. The deduced amino acid sequence was analyzed with the Expert Protein Analysis System². The protein domain was predicted with the simple modular architecture research tool³ (SMART) version 5.1. Multiple sequence alignment of Cgengrailed-1 with other engraileds was created by the ClustalW multiple alignment program⁴ and multiple sequence alignment show program⁵.

The expression levels of Cgengrailed-1 in different development stages and different tissues were measured by an ABI PRISM 7500 Sequence Detection System with a total volume of 25.0 μL, containing 12.5 μL of SYBR Green Mix (Takara), 0.5 μL of each primer (10 μmol L^–1) (P5 and P6, Table 1), 2.0 μL of cDNA, and 9.5 μL of DEPC-water. The oyster elongation factor (EF) was used as internal control (P7 and P8, Table 1). Dissociation curve analysis of amplification products was performed to confirm that only one PCR product was amplified and detected. The comparative average cycle threshold method was used to analyze the mRNA expression level of the immune-related genes according to previous research (Zhang et al., 2008). All data were given in terms of relative mRNA expression using the 2^{–Δ Δ Ct} method (Yu et al., 2007).

The Cgengrailed-1 was induced and expressed through the prokaryotic expression system. The ORF fragment of Cgengrailed-1 was gained from the PCR reaction with pairs of primers (P3 and P4, Table 1). The PCR products and the pET-30a expression vector were linearized with double enzyme digestion, and then the PCR products and linearized expression vector were recombined to a complete vector through T4 ligase system. The DNA sequence combined to pET-30a expression vector was confirmed by DNA sequencing. The valid recombinant plasmid was extracted and transformed into Escherichia coli transetta DE3. Positive transformants were cultured in LB medium at 37°C, 120 rpm incubator. When the OD₆₀₀ value of bacteria solution reached 0.4–0.6, the bacteria were incubated for additional 8 h with the induction of Isopropyl β-D-thiogalactoside (IPTG) at the final concentration of 1 mM. The recombinant protein of Cgengrailed-1 labeled with 6× His-tag at the C-terminal was purified by Ni⁺ affinity chromatography, and desalted by extensive dialysis. The purified objective protein (rCgengrailed-1) was concentrated and stored at −80°C (Zong et al., 2019).

Six-week-old mice were immunized with rCgengrailed-1 to acquire polyclonal antibodies following the description of previous study (Cheng et al., 2006). Briefly, 100 μL rCgengrailed-1 (0.3 mg mL^–1) was emulsified with 100 μL complete Freund’s adjuvant (Sigma, United States) to immunize each mouse by subcutaneous implantation. The second and third injections were performed on the 14th and 21th day with incomplete Freund’s adjuvant (Sigma, United States). The fourth injection was performed with 100 μL rCgengrailed-1 on the 28th day. The anti-rCgengrailed-1 serum was obtained on the 36th day and stored at −80°C before use.

The specificity of polyclonal antibody against rCgengrailed-1 and natural Cgengrailed-1 (from mantles) was verified by western blot assay. Briefly, rCgengrailed-1 and natural Cgengrailed-1 was separated by 12% SDS-PAGE, and electrophoretically transferred onto a nitrocellulose membrane. The membrane was washed three times with TBS containing 0.1% Tween 20 (TBST), blocked by 5% skimmed milk (100 mL TBST, 5 g skimmed milk) at 4°C overnight, and then incubated with 1:500 diluted polyclonal antibodies against rCgengrailed-1 at 37°C for 3 h. After three times of washing with TBST, the membrane was incubated with 1:3,000 diluted secondary antibody Goat-anti-mouse IgG conjugated with HRP (ABclonal) at 37°C for 2 h. After the final three times of washing with TBST, the protein bands were developed by using Super ECL Detection Reagent (Sigma-Aldrich) for one minute, capture image and take picture by Amersham Imager 600 system (GE Healthcare, United States).

The whole-mount immunofluorescence assay was conducted as previously described (Thisse and Thisse, 2008; Liu et al., 2015) with minor modification. In brief, the larvae stored in pure methanol were rehydrated by PBS and washed three times with PBST (PBS with 0.1% Tween20). The shells of D-shape larvae were decalcified in 25 mmol^–1 EDTA in PBS for 30 min, rinsed in PBST for 3 × 15 min, and blocked overnight in the blocking buffer (10% normal goat serum, 0.25% fetal bovine serum albumin, 1% TritonX-100 and 0.03% sodium azide in PBS). Then, the larvae were incubated with the primary antibodies (1:500 dilutions in blocking buffer) at 4°C for 3–4 days. These specimens were then washed for 3 × 15 min in PBST and incubated with goat anti-rat IgG conjugated to Alexa Fluor 488 (1:2,000 dilutions in blocking buffer; Invitrogen, Carlsbad, CA, United States) for 1 day at room temperature. After incubation in secondary antibodies, all the specimens were washed in PBST for 3 × 20 min and then mounted in 50% glycerol in PBS. The specimens incubated with pre-immune serum added with secondary antibodies were selected as negative control (Liu et al., 2015). All specimens were examined as whole-mount using the Zeiss Laser-Scanning Confocal Microscopy System LSM 710 (Zeiss, Jena, Germany).

The oyster mantles from control group and acidification treatment group were fixed in Bouin’s solution for 24 h and decolorized with 75% (v/v) ethanol. Then, the fixed samples were embedded in paraffin, and sectioned at 6 μm thickness. The slides were treated to remove the paraffin and rehydrated. Soak the slides in 0.01 M citrate buffer solution, conduct antigen renaturation in autoclave at 120° C for 6 min, and then cool it to room temperature naturally. After cooling down at room temperature, the slides were prepared for immunohistochemistry staining. The slides were first blocked in BSA, and Cgengrailed-1 polyclonal antibody was used as primary antibody (diluted at 1:500 in blocking buffer). Alexa Fluor 488 labeled goat-anti-mouse (diluted at 1:1,000, Abclonal, College Park, MD, United States) was used as secondary antibody. Finally, the slides were incubated with 4′, 6-diamidine-2-phenylindole dihydrochloride (DAPI, 10 mg mL^–1, diluted in PBS by 1:2,000) (Roche, Nutley, NJ, United States) for 5 min. After three times of washing, 50% glycerol was added on the slides before observation under Axio Imager A2 microscope (Carl Zeiss, Germany).

All the data were shown as mean ± S.D. The two-samples Student’s tested were performed for the comparisons between groups. Multiple group comparisons were executed by one-way ANOVA and followed by a Turkey multiple group comparison tests using Statistical Package for Social Sciences (SPSS) 16.0 Software. The difference was considered as significant at p < 0.05.

The ORF of a previously reported engrailed gene (Cgengrailed-1, CGI_10012208) was identified from the Pacific oyster C. gigas, which was of 690 bp encoding a polypeptide of 229 amino acids (Figure 1A). The putative molecular weight of Cgengrailed-1 was 25.70 kDa and its theoretical isoelectric point (pI) was 9.62. SMART analysis showed that there was a HOX domain (from Glu141 to Asn203) and a low expression region in Cgengrailed-1 (Figure 1B).

Results of multiple sequences alignment revealed that the deduced amino acid sequence of Cgengrailed-1 shared 48.50% identity with that from Mizuhopecten yessoensi, 41.30% identity with that from Xenopus laevis, 40.50% identity with that from Branchiostoma belcheri, 37.90% identity with that from Mus musculus, 27.50% identity with that from Homo sapiens, and 19.60% identity with that from Drosophila melanogaster (Figure 1C). A total of 14 engraileds from various vertebrate and invertebrate species were employed in phylogenetic analysis. There were two major distinct branches in the phylogenetic tree, the vertebrate and invertebrate branches. Cgengrailed-1 was firstly gathered with that from C. virginica and then clustered with those from M. yessoensis and other molluscs, and finally clustered with those from vertebrates (Figure 1D).

The ORF fragment of Cgengrailed-1 was inserted into the pET-30a vector to build the recombinant plasmid and transferred into the expression bacterial strain (E. coli Transetta, DE3). After IPTG induction, the purified recombinant protein of rCgengrailed-1 was ascertained by the 12% SDS-PAGE through electrophoresis (Figure 2A). There is one single band about 35 kDa detected by SDS-PAGE, which was in consistent with the predicted molecular weight of rCgengrailed-1 (Figure 2A, lane 3). There was no corresponding band in the cell lysate without induction (Figure 2A).

The purified rCgengrailed-1 was used to immune the female mice to prepare the polyclonal antibody. The polyclonal antibody specificity of the rCgengrailed-1 was verified by western blot. There was only a distinct band detected, whose molecular weight was in accordance with that of rCgengrailed-1 (Figure 2B). Meanwhile, no distinct band was observed in the negative control.

The expression level of Cgengrailed-1 mRNA in oyster tissues was detected by quantitative real-time PCR. The mRNA transcripts of Cgengrailed-1 were widely expressed in all the tested tissues including hepatopancreas, hemocytes, adductor muscle, gill, mantle and labial palp (Figure 2C). The highest expression level of Cgengrailed-1 transcripts was observed in labial palp, which was approximately 86.83-fold of that in hepatopancreas (p < 0.05). In addition, the expression level of Cgengrailed-1 transcripts in mantle and gill was 75.87-fold (p < 0.05) and 28.52-fold (p < 0.05) higher than that in hepatopancreas, respectively.

In the control (pH 8.1) group, the mRNA expression levels of Cgengrailed-1 decreased dramatically at early D-shape larvae and D-shape larvae stages, which were 0.64- and 0.15-fold compared to trochophore (p < 0.05). After the larvae received acidification treatment at trochophore stage, the mRNA expression levels of Cgengrailed-1 increased significantly in D-shape larvae stage, which was 3.11- (pH 7.8, moderate acidification treatment) and 4.39-fold (pH 7.4, severe acidification treatment) of that in control group (p < 0.05). No significant change of Cgengrailed-1 mRNA was observed in early D-shape larvae after acidification treatment, which was 1.12- (pH 7.8) and 1.23-fold (pH 7.4) of that in control group (p < 0.05, Figure 3).

The expression of Cgengrailed-1 in oyster larvae in response to the acidification treatment was also measured by whole-mount immunofluorescence assay. In the control group (pH 8.1), the positive signals (the green point) were observed on the margin of shell gland or periostracum in trochophore, early D-shape larvae and D-shape larvae, and positive signals gradually decreased following the developmental progresses. After moderate or severe acidification treatment, the positive signals were observed on the same location of oyster larvae. No signal was detected in the negative group, indicating the specificity of polyclonal antibody and the reliability of the present results. In trochophore, lots of small and dense positive signals were observed on the dorsal edge of the periostracum. Once the oyster larvae received acidification treatment from trochophore stage, the positive signals in both early D-shape larvae and D-shape larvae were stronger than that in the control group (Figure 4).

The expression level of Cgengrailed-1 mRNA in the mantle of adult oysters after different acidification treatment was determined by quantitative real-time PCR. After the adult oysters received moderate (pH 7.8) and severe acidification (pH 7.4) treatment for one week, the mRNA expression level of Cgengrailed-1 in mantle decreased significantly, which were 0.75- and 0.15-fold (p < 0.05) of that in the control (pH 8.1) group. The decreasing trend in pH 7.4 group was more obvious than that in pH 7.8 group (Figure 5).

The expression of Cgengrailed-1 in mantle responsive to experimental acidification treatment was further examined by immunofluorescence assay. No positive signal was observed in the negative and control (pH 8.1) groups. When adult oysters received acidification treatment, positive signals were detected in the middle fold of mantle, and the signal intensity in moderate acidification treatment group was significantly stronger than that in severe acidification treatment group (Figure 6).