Abstract:The total saline-alkaline land area in China is approximately 99.13 million hectares, distributed throughout northern China, coastal areas, and areas along the Huanghe River. About 45.87 million hectares of saline-alkaline water areas are spread around these lands, most of which are athalassic waters characterized by a high pH value above 8.8, associated with high-carbonate alkalinity and various types of ions imbalances. The saline-alkaline land and water cannot be directly used for agriculture, and most of them are arid. The development of aquaculture in saline-alkaline land is not only beneficial to expanding the aquaculture area but also can restore the saline-alkaline soil, which is of great significance to food security and ecological restoration. Saline-alkaline aquaculture is one of the main inland aquaculture models developed in the past ten years. With the maturity of aquaculture technologies, the saline-alkaline aquaculture area has expanded year by year, which has brought earnings to local farmers. China has abundant saline-alkaline water resources. The high pH and high-carbonate alkalinity of these waters restrict the survival, growth, and reproduction of aquatic animals. Litopenaeus vannamei is highly resistant to stress and has a certain tolerance to saline-alkaline water. Under short-term saline-alkaline stress, the expression of the carbonic anhydrase (CA), Na+/K+-ATPase, and other ion-regulated genes of L. vannamei were induced, and the acid-base and osmolality balance were determined by strengthening ion regulation. At present, relatively few studies on gene regulation of L. vannamei under long-term stress have been performed. Although L. vannamei farming has been successful in saline-alkaline water, the survival rate is unstable, and there are few reports on the selective breeding of L. vannamei tolerant to salinity and alkalinity. Through independent innovation, a family-based "multi-trait compound breeding technology for aquatic animals" has been established in China. These techniques have laid a good foundation for developing improved L. vannamei strains. To effectively utilize saline-alkaline water resources, it is urgent to conduct L. vannamei salt-alkali-tolerant breeding and promote the healthy development of the saline-alkaline aquaculture industry. L. vannamei has strong environmental adaptability and relatively high tolerance to saline-alkaline water. It is one of the main species of saline-alkaline aquaculture. However, its survival rate in high pH and high-alkaline environments is not stable. To explore the response mechanism to long-term high-alkaline stress, L. vannamei was exposed to low-alkaline water as the control group (LSW, carbonate alkalinity of 3 mmol/L, salinity of 6, pH of 8.1) and to high-alkaline stress (AW, carbonate alkalinity of 10 mmol/L, salinity of 6, pH of 8.8) for 42 days. The intestine and gill of L. vannamei raised for 42 days were used as the experimental materials. Transcriptome sequencing was performed using the Illumina platform. After splicing analysis and gene annotation, the differentially expressed genes and regulatory pathways regulated under high-alkaline stress were screened and analyzed, with further verification by qRT-PCR. The results showed 243 differentially expressed genes in both tissues, of which 98 were up-regulated and 145 were down-regulated. The differentially expressed genes in the intestine were enriched for glucose metabolism, carbohydrate digestion and absorption, bile secretion, ABC transmembrane transport, and tight junction related pathways. The differentially expressed genes in gills were enriched for glutathione metabolism, bicarbonate transport, arginine synthesis, sugar metabolism, and ion transport related pathways. The ten most significant differentially expressed genes were further studied and verified by qRT-PCR. Carbonic anhydrase (CA1, CA10), ecdysone-inducible protein (Eip74EF), and β-galactosyltransferase (UGT8) genes in gills were down-regulated. However, the expression of Na+/K+-ATPase-α (NKA-α), Na+/K+ transporting ATPase interacting (NKAIN), ammonia transporter (Rhbg), and malate dehydrogenase (MDH) were up-regulated under high-alkaline stress. The transcriptome expression pattern and qRT-PCR results were consistent. We speculated that these genes may be involved in the shrimp stress response to high-alkaline stress. L. vannamei showed a relatively strong high-alkaline tolerance, which may be compensated by down-regulating the expression of CA to prevent alkalosis, up-regulating Rhbg to prevent ammonia accumulation and NKA-related genes to maintain the osmotic balance. The ecdysone function was probably affected as the Eip74EF gene was down-regulated. This study provides basic data for further analyzing the physiological response mechanisms of L. vannamei under long-term highly alkaline stress.
