**1. Introduction**

Saline–alkaline water, as the third kind of water, differs from seawater and freshwater and accounts for a large proportion of the world's water resources [1,2]. There are about 46 million hectares of saline–alkaline water areas, which are widely distributed in the northwest, northeast, and north of China, involving 19 provinces, cities, and autonomous regions [3–5]. Saline–alkaline water has a poor buffering capacity, which is characterized by high pH, high carbonate alkalinity, and various types of ion imbalances [1]. Excessive carbonate alkalinity in water substantially affects the development, survival, and reproduction of organisms. Several fishes can adapt to high alkalinity, such as *Leuciscus waleckii* [6], *Gymnocypris przewalskii* [7], and tilapia [8], which provide excellent breeding organisms for the development and utilization of carbonate alkalinity waters for aquaculture.

**Citation:** Zhang, X.; Wang, J.; Wang, C.; Li, W.; Ge, Q.; Qin, Z.; Li, J.; Li, J. Effects of Long-Term High Carbonate Alkalinity Stress on the Ovarian Development in *Exopalaemon carinicauda*. *Water* **2022**, *14*, 3690. https://doi.org/10.3390/w14223690

Academic Editor: Heiko L. Schoenfuss

Received: 17 October 2022 Accepted: 12 November 2022 Published: 15 November 2022

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The ridgetail white prawn *E. carinicauda* is one of the most important commercial shrimps [9], which is widely distributed in the coastal areas of the Yellow Sea and the Bohai Sea in China [10]. Due to its multiple advantages, such as fast growth, high reproductive performance, and good disease resistance [11], the breeding area of *E. carinicauda* in China has expanded in recent years, which may account for one third of the total output of mixed culture ponds [12,13]. Recently in China, *E. carinicauda* has been successfully bred and cultured in saline–alkaline ponds at Dongying city, Shandong province (with salinity of 5–8 and carbonate alkalinity of 1.4–8.0 mmol/L) and Cangzhou city, Hebei province (with salinity of 10–20 and carbonate alkalinity of 3.5–13.0 mmol/L), suggesting that *E. carinicauda* have a high tolerance to saline–alkaline stress [13,14]. *E. carinicauda* shows strong tolerance to salinity and high carbonate alkalinity, and can carry eggs during culture. Therefore, it is a potential species suitable for large-scale culture in saline–alkaline waters. However, as most studies focused on changes in the physiology and gill transcriptomics in *E. carinicauda*, the effects of long-term carbonate alkalinity stress on the reproductive mechanisms in *E. carinicauda* still remains unknown.

Carbonate alkalinity has been considered to be the main stress source affecting the survival, growth, and reproductive traits of aquatic animals in saline–alkaline water [15–17]. High carbonate alkalinity can alter normal metabolism, osmotic pressure regulation capacity, and antioxidant capacity [18]. Yao et al. [3] reported that when the carbonate alkalinity exceeded 15.7 mmol/L, the survival rate of medaka (*Oryzias latipes*) decreased, and morphological abnormalities such as embryo coagulation, embryonic development stagnation, and hatching failure were present. Previous studies have shown that the growth, development, and reproduction indexes in *Moina mongolica* Daday are optimal when the alkalinity is 2.05–4.58 mmol/L. However, when the alkalinity is 6.43–8.98 mmol/L, all indicators showed a downward trend [19]. Xu et al. [20] found that survival rate was not affected by water with a salinity < 3.2 and alkalinity < 14.32 mmol/L in a 72 h embryo tolerance experiment with *Barbus capito*. Water with salinity < 5.1 and alkalinity < 14.32 mmol/L did not affect the 96 h survival of larvae. Liu et al. [21] also found the growth and reproduction of *E. carinicauda* was not affected by low carbonate alkalinity. *E. carinicauda* is better adapted to environments with high carbonate alkalinity, as it adjusts immune enzyme activities. However, research on the influence of carbonate alkalinity in aquatic organisms mainly focuses on survival and growth. Few reports address carbonate alkalinity influences on the mechanisms involved in gonadal development. Therefore, we urgently need to understand the reproduction of aquatic organisms in saline–alkaline waters to aid aquaculture development, especially breeding in saline–alkaline water.

In recent years, transcriptome sequencing technology has been widely used to study the differential expression and molecular pathways of genes under specific environmental stresses [22,23]. For example, RNA–seq compares the transcriptomic responses of *Litopenaeus vannamei* under salinity stress [24]. Li et al. [25] used transcriptome sequencing to reveal the genes and pathways related to salt stress in *Eriocheir sinensis*. However, research on the effect of carbonate alkalinity on aquatic organisms focuses on osmoregulation, with relatively few studies involved in ovarian development. This is the first study involving the transcriptome of the *E. carinicauda* ovary and eyestalk being sequenced by RNA–seq technology. We analyzed the transcriptome data of the eyestalks and ovaries under high carbonate alkalinity stress in order to identify the genes and pathways involved in ovarian development. This study helps to clarify the ovarian development mechanisms in *E. carinicauda* when adapting to high carbonate alkalinity.

#### **2. Materials and Methods**

#### *2.1. Sample Collection*

Adult female shrimps (body length 2.67 ± 0.20 cm, body weight 0.12 ± 0.08 g) were collected from Haichen Aquaculture Co., Ltd., in Rizhao, Shandong province, China. The shrimps were domesticated in the environment (25 ◦C) for two weeks. The experiment was performed in 200 L PVC tanks. One hundred and eighty shrimps were divided into two groups randomly, including the high carbonate alkalinity group (carbonate alkalinity 8 ± 0.5 mmol/L, salinity 25 ppt, temperature 26 ± 0.5 ◦C, pH 8.1 ± 0.3, dissolved oxygen 7.5 ± 0.5 mg L−1) and the control group (carbonate alkalinity 2 ± 0.5 mmol/L, salinity 25 ppt, temperature 26 ± 0.5 ◦C, pH 8.8 ± 0.5, dissolved oxygen 7.5 ± 0.5 mg L−1). The experimental design included three replicates of 30 shrimps in each group. The shrimp were fed 3–5% of their body weight twice daily (8:00 and 18:00) during the experimental period. The water was aerated and 30% of the water was changed daily (with the new sea-water adjusted to maintain the original carbonate alkalinity).

After 60 days, thirty–six female shrimps (6 individuals × 3 three replicates × 2 groups) were randomly sampled for Illumina (San Diego, CA, USA) RNA–seq. The ovary and eyestalk samples were obtained and rapidly frozen in liquid nitrogen, then stored at −80 ◦C until RNA isolation, respectively. The ovary samples in the high carbonate alkalinity group are labeled HC\_O and the eyestalk samples are labeled HC\_E. The ovary samples in the control group are labeled CG\_O and the eyestalk samples are labeled CG\_E. Six female shrimps (2 individuals × 3 three replicates × 2 groups) were sacrificed for the tissue slices.
