*3.7. The Validation of DEGs by qRT*−*PCR*

In order to further validate the reliability of the DEGs identified by RNA−Seq, we randomly selected 10 DEGs from two comparisons: HC\_O vs. CG\_O and HC\_E vs. CG\_E. The qRT−PCR results were consistent with those of RNA−seq, indicating that the RNA−Seq data was accurate (Figure 7).

**Figure 6.** The heatmap of DEGs related to ovarian development in the HC\_E vs. CG\_E group.

**Figure 7.** The results were verified by qRT−PCR. (**a**) Relative fold change of DEGs between qRT−PCR and RNA−seq results in HC\_O vs. CG\_O group. (**b**) Relative fold change of DEGs between qRT-PCR and RNA−seq results in HC\_E vs. CG\_E group. Relative expression levels from the RNA−seq results were calculated as log2FC values. Fc011350 ecdysteroid regulated; Fc021284 legumain; Fc026977 lysosome−associated membrane glycoprotein 1; Fc008809 baculoviral IAP repeat-containing; Fc020229 N−acetylated−alpha−linked acidic dipeptidase; Fc010755 high−affinity choline transporter 1; Fc007027 Wnt 4; Fc019299 tachykinin−like peptides receptor; Fc005820 reelin 3; Fc012767 facilitated trehalose transporter; Fc026446 neuroligin 2; Fc015896 insulin receptor−related; Fc012259 serpin 1; Fc005350 UDP−glucosyltransferase 2; Fc008378 crustacyanin−C1; Fc001201 legumain; Fc002855 cathepsin L; Fc016036 multidrug resistance−associated protein; Fc004370 macrophage mannose receptor 1.

#### **4. Discussion**

Ovarian maturation is an elaborate and complex molecular process, and a large number of genes need to be regulated to ensure the normal development of the oocytes. In this study, we found that 8 mmol/L carbonate alkalinity stress retarded *E. carinicauda* ovarian development, even in non−developing females [21]. The ovaries and eyestalks are important organs in crustacean ovarian development. Eyestalk ablation is commonly used to induce ovarian maturation in shrimp farming [29]. Although the mechanism by which eyestalk ablation leads to ovarian maturation remains inconclusive, some genes expressed in the eyestalk regulate ovarian development. The histological study investigates the influence of high carbonate alkalinity on ovary and oocyte development. We used highthroughput sequencing of ovaries and eyestalks for the first time to understand the ovarian development mechanisms in *E. carinicauda* when in high carbonate alkalinity osmotic stress.

KEGG pathway enrichment can identify major biochemical metabolic and signal transduction pathways involved in genes. In the HC\_O group, the insect hormone biosynthesis pathway was significantly enriched, with methyl farnesoate epoxidase, which can catalyze methyl farnesoate into juvenile hormone (JH) III, differentially up−regulated. Methyl farnesoate is a non−epoxidized form of insect juvenile hormone III, which is secreted by the mandibular organs of crustaceans and has a significant stimulating effect on Vg synthesis in various Decapoda species [30–32]. Although JH III plays a very important role in regulating life processes such as growth, molting, and reproduction, it is considered to be an important endogenous hormone in insects and other arthropods [33,34]. Up-regulated methylfarnesoate epoxidase may lead to a decrease in methylfarnesoate synthesis. However, in *Macrobrachium rosenbergii* [35] and *Sagmariasus verreauxi* [36], metabolic enzymes convert methyl farnesoate into JH, which supports the view that JH is also an active hormone in crustaceans. Furthermore, methyl farnesoate epoxidase can directly convert farnesoic acid into JH III acid to form JH III. Therefore, the up−regulation of methyl farnesoate epoxidase promotes the synthesis of JH, thus affecting ovarian development in crustaceans.

Lysosomes were the most significant pathways, with a larger number of DEGs varying between the HC\_O and CG\_O groups as well as between the HC\_E and CG\_E groups. Lysosomes are important for intracellular trafficking, metabolic signaling, lipid metabolism, and immune responses [37]. Lysosomes are involved in the preparation of free cholesterol for steroidogenesis and degradation of steroidogenesis regulators, as well as follicle rupture during ovulation in the ovaries of vertebrates [38]. As the central digestive organ of cells, various macromolecules are sent to lysosomes for degradation. Vitellogenin is an important precursor of egg yolk in nearly all oviparous animals [39]. Lysosomes play an important role in the degradation of vitellogenin, which is internalized by endocytosis [40]. Lysosomes are related to the hydrolysis of vitellogenin and energy demand during *Macrobrachium nipponense* ovarian maturation [41]. Lysosomal enzymes, especially cathepsin B and L, are associated with ovarian development in crustaceans [42,43]. We also found that cathepsin B and L in the ovaries of *E. carinicauda* were significantly up−regulated in response to carbonate alkalinity stress, which was consistent with the above results. In addition, we found that the lysosomal pathway was significantly up−regulated in the ovaries and significantly down−regulated in the eyestalks. Therefore, we speculate that this change in lysosomes is closely related to the ovarian development of *E. carinicauda* under carbonate alkalinity stress. However, the specific mechanism is unknown.

In this study, significant differential genes provide evidence of response to high carbonate alkalinity stress in the ovary and eyestalk, which are the target tissues that regulate ovarian development. As the largest membrane receptor family in eukaryotes, G protein coupled receptors are involved in regulating many key physiological and biochemical processes, including sexual maturation and reproduction [44,45]. There are many important factors controlling the development and maturation of shrimp ovaries, such as neurotransmitters, hormones, and their receptors [46,47], which mainly bind and activate G protein coupled receptors on the cell surface [48] and initiate multiple downstream cascades [49]. In this study, G protein−coupled receptor and Mth were significantly up−regulated in the HC\_O group, which indicated that the expression of G protein−coupled receptor and Mth may regulate the ovarian development of *E. carinicauda* under high carbonate alkalinity stress.

As a glycoprotein bound to the cell surface, lectin specifically recognizes carbohydrates [50] and plays an important role in the innate immunity of invertebrates [51,52]. Qin et al. found that pmcl1 plays an important role in the immune response to pathogen infection and ammonia nitrogen stress [53]. Tateno et al. suggests that lectins in fish oocytes may prevent polysperm fertilization, regulate carbohydrate metabolism, participate in the formation of the fertilization shell after binding with glycoproteins, determine the source of disease, and have antibacterial effects [50]. Therefore, we speculate that C−type lectins may

perform similar functions in *E. carinicauda*, but the specific role requires further research. Additionally, lectin and vitellin VMO1/2 are closely bound to ovomucin to form the basic skeleton of the outer membrane of vitellin [54]. The VMO−1 protein is essential for the formation of the outer membrane in chicken eggs [55], and the adhesion between yolk membranes may be related to the VMO−2 protein in quail (*Coturnix japonica*) eggs [56]. VMO−1 is an important protein in the development of oocytes [57], and its main function is to prevent mixing between yolk and protein in crayfish [58]. In this study, the expression of VMO−1 and C−type lectin was up−regulated in the HC\_O group, suggesting that VMO−1 and lectin affects ovarian development in *E. carinicauda* under osmotic stress.

The crustacean eyestalk is known to regulate reproduction, molting, and energy metabolism [59,60]. Removing the eyestalk can induce ovarian maturation and oviposition in many crustaceans [61,62]. Eyestalk−derived neuropeptides regulate vitellogenesis in crustaceans. Pigment−dispersing hormone (PDH) is involved in the regulation of ovarian maturation in crustaceans [63]. The PDH may participate in vitellogenesis according to their spatiotemporal expression patterns, which maintained a high level from the pre−vitellogenesis stage and decreased significantly in the mature stage in *Scylla paramamosain* [64,65]. Wei et al. provided evidence of the inductive effect of PDH on oocyte meiotic maturation in *E. sinensis* [66]. In this study, the PDH was up−regulated in the HC\_E group, suggesting that it may participate in the ovarian development of *E. carinicauda* under high carbonate alkalinity stress.

Retinol and its derivatives play key roles in the meiosis of mammalian fetal ovarian germ cells [67], follicular development [68], ovarian steroidogenesis [69], and oocyte maturation [70]. The retinol dehydrogenase (RDH) is a member of the short−chain dehydrogenase/reductase (SDR) superfamily, which includes three RDHs, RDH11, RDH12, and RDH13 in transcriptome sequences. The expression level of RDH13 in a vitellogenesis ovary of zebrafish was significantly higher than that in a non−vitellogenesis ovary [71]. In addition, knockout of RDH11 resulted in decreased transcription of vitellogenin and vitellogenin receptors in *Procambarus clarkii* [72], suggesting that RDH11 might play an important role in the synthesis and conveyance of vitellogenin in crustaceans. Our previous study revealed that RDH11 is critical for ovarian development in *E. carinicauda* [73]. In this study, RDH11, associated with ovarian development in *E. carinicauda*, was significantly expressed under high carbonate alkalinity stress.

#### **5. Conclusions**

In the present study, the transcriptome analysis of the ovary and eyestalk of *E. carinicauda* under high carbonate alkalinity stress, as well as the effects of high carbonate alkalinity on ovarian development-related genes and signaling pathways, are described for the first time. Eighteen and thirteen DEGs in ovary and eyestalk tissue were identified, respectively. The key genes were identified to be involved in the folate biosynthesis, insect hormone biosynthesis, lysosome, and retinol metabolism, which play an essential role in the response of the ovaries and eyestalks of *E. carinicauda* to high carbonate alkalinity stress. This study provided new insights into the ovarian development of *E. carinicauda* under high carbonate alkalinity stress, which could be useful for saline–alkaline water aquaculture and related studies on the reproduction of crustaceans.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/w14223690/s1, Table S1: Primers of RT−PCR designed for validation experiment of DEGs; Table S2: Number of high-throughput clean reads and mapped clean reads generated from *E. carinicauda* ovary and eyestalk mRNA library.

**Author Contributions:** X.Z., J.W. and J.L. (Jitao Li) conceived and designed the research; X.Z., J.W., C.W., W.L., Q.G. and Z.Q. performed the experiments and analyzed the data; J.L. (Jian Li) provided research ideas for the experiments. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Key R&D Program of China (2019YFD0900404-03), National Natural Science Foundation of China (32072974), China Agriculture Research System of MOF and MARA (CARS-48) and Central Public-Interest Scientific Institution Basal Research Fund, CAFS (2020TD46).

**Institutional Review Board Statement:** No human subjects were included in this study. The animal study was reviewed and approved by the Animal Ethics Committee of Yellow Sea Research Institute, CAFS (ID Number: YSFRI–2022033).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Center for Biotechnology Information (NCBI) with the accession number PRJNA881755 and PRJNA881756.

**Conflicts of Interest:** The authors declare no conflict of interest.
