**3. Results**

#### *3.1. Histopathology of Ovary*

Normal reproduction characteristics were observed in the histological sections of the ovary. The *E. carinicauda* ovary was in Phase I. The oogonia were oval and proliferative, and the nuclei were round. Most cells had one nucleolus, while a few had two nucleoli. The nucleolus stained the deepest color. The cells of the oogonia were closely arranged. A single layer of follicle cells was closely arranged around the oogonia (Figure 1a). After 60 days of high carbonate alkalinity stress, most of the cells in the ovarian cavity were previtellogenic follicles, and the nuclei were large and original, accounting for about half of the cell volume (Figure 1b).

**Figure 1.** Histological sections of the ovarian status of *E. carinicauda*: (**a**) CG\_O group; (**b**) HC\_O group. Bar: 50 μm. N: nucleus; Nu: nucleolus; Fc: follicular cell.

#### *3.2. Summary of the RNA–Sequencing Data*

Six mRNA libraries (*n* = 6) were constructed from the ovary and eyestalk, respectively, to identify the effects of high carbonate alkalinity on the underlying molecular signaling pathways in *E. carinicauda* ovarian development. The raw data was submitted to NCBI with accession numbers PRJNA881755 and PRJNA881756. A total of 273,545,646 clean reads were obtained from ovaries, with the Q30 (%) varying from 92.63–94.20%. Of these reads, 70.91–72.74% mapped to the reference genome of *E. carinicauda*. A total of 264,648,418 clean

reads were obtained from eyestalks, with the Q30 (%) varying from 91.72–92.67%. Of these reads, 79.84–83.02% mapped to the reference genome of *E. carinicauda* (Table S2).

#### *3.3. Differential Gene Expression (DEGs) Analysis*

A total of 1102 significant DEGs were identified between the HC\_O and CG\_O groups. Compared to the control group, 758 up−regulated genes and 344 down−regulated genes were expressed in the ovarian tissue of the HC group (Figure 2a). Compared with the CG\_E group, 468 DEG (139 up−regulated and 329 down−regulated) were identified in the HC\_E group (Figure 2b). These results suggest that carbonate alkalinity has a significant effect on transcription in the ovary and eyestalk.

**Figure 2.** The number of differentially expressed genes between different groups. (**a**) HC\_O vs. CG\_O group; (**b**) HC\_E vs. CG\_E group. Significantly up−regulated and down−regulated genes are indicated in red and blue, respectively, and those not significantly different are in gray.

#### *3.4. Gene Ontology (GO)Analysis of Significant DEGs*

The DEGs of the comparison of HC\_O and CG\_O were classified into biological process (BP), cellular component (CC), and molecular function (MF). Compared with the CG\_O group, up−regulated genes in the HC\_O group had enriched steroid metabolic process, sterol homeostasis, molting cycle, follicle cell microvillus organization, steroid biosynthetic process, sterol transport, and estrogen secretion. Conversely, the down−regulated genes in the HC\_O group had an enriched positive regulation of ovulation, regulation of ovulation, sterol transport, ovulation, and sterol homeostasis (Table 1).

The DEGs of the comparison of HC\_E and CG\_E were also classified into biological process (BP), cellular component (CC), and molecular function (MF). Compared with the CG\_E group, the up−regulated genes in the HC\_E group were enriched in the positive regulation of sterol transport, regulation of sterol transport, embryonic morphogenesis, embryonic organ morphogenesis, embryo development, entry into reproductive diapause, and sterol metabolic process. The down−regulated genes in the HC\_E group were enriched in sterol transport, regulation of metaphase/anaphase transition of meiosis I, ecdysteroid metabolic process, sterol homeostasis, maternal placenta development, metaphase/anaphase transition of meiosis I, and the molting cycle (Table 2).


**Table 1.** The GO term of related to ovarian development in the HC\_O vs. CG\_O group.

**Table 2.** The GO term of related to ovarian development in the HC\_E vs. CG\_E group.


#### *3.5. Kyoto Encyclopedia of Genes and Genomes (KEGG) Analysis*

Comparing the DEGs to the KEGG database of pathway enrichment, the potential functions of the significant DEGs were analyzed in order to further understand the ovarian development of *E. carinicauda* under high carbonate alkalinity stress.

For high carbonate alkalinity stress treatment, 24 KEGG pathways (19 up−regulated and 5 down−regulated pathways) were significantly enriched in the HC\_O group (Figure 3). Among these KEGG pathways, some were associated with ovarian development, such as ECM–receptor interaction, folate biosynthesis, FoxO signaling pathway, insect hormone biosynthesis, lysosome, metabolic pathways and neuroactive ligand–receptor interaction.

In the comparison of HC\_E and CG\_E group, 17 KEGG pathways (5 up−regulated and 12 down−regulated pathways) were significantly enriched (Figure 4). Concurrently, the ECM-receptor interaction, folate biosynthesis, lysosome, metabolic pathways, and retinol metabolism were associated with ovarian development.

**Figure 3.** The KEGG significant enrichment pathway in HC\_O vs. CG\_O group. (**A**) Up−regulation pathways in HC\_O vs. CG\_O group; (**B**) down−regulation pathways in HC\_O vs. CG\_O group.

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#### *3.6. DEGs Involved in Ovarian Development*

In the ovary, differential genes involved in ovarian development were selected high carbonate alkalinity stress, such as the G protein−coupled receptor, vitelline membrane outer layer protein 1 (VMO−1), retinol dehydrogenase 11, ecdysone−induced protein 78, ecdysteroid regulated−like protein, voltage−dependent calcium channel gamma−7, methyl farnesoate epoxidase, and estradiol 17−beta−dehydrogenase. A clustering heatmap of these results is provided (Figure 5).

In the eyestalk, the participation of differential genes in ovarian development was observed, and genes were selected which showed high carbonate alkalinity stress. Some DEGs related to ovarian development were screened, such as the vitelline membrane outer layer protein 1, pigment−dispersing hormone 1, insulin−like growth factor binding protein, insulin receptor−related protein, NF−kappa B inhibitor alpha, neuronal acetylcholine receptor, neuroligin−2, and DNA replication complex GINS. A clustering heatmap of these results is provided (Figure 6).
