*3.3. Expression Analysis of PmNHE mRNA in Different Tissues*

In order to study the expression of the *PmNHE* gene in different tissues, ten tissues including the muscle, hepatopancreas, gill, epidermis, intestine, heart, hemolymph, stomach, ovary, and testis were mainly detected (Figure 4). The order of expression from high to low is intestine > muscle > hemolymph > heart > hepatopancreas > stomach > epidermis > gill > testis > ovary, and the intestine and muscle had the highest levels of *PmNHE* mRNA expression.

#### *3.4. Expression Changes of PmNHE under Acute Ammonia Nitrogen Stress*

The gills and intestines of *P. monodon* were subjected to two concentrations of ammonia nitrogen, and RT-qPCR was used to detect changes in *PmNHE* expression. The expression levels in the intestine and gills varied in both the safe and half-lethal concentrations of ammonia nitrogen when compared to the control group (Figure 5).

In the gill tissue, the fluctuation range of *PmNHE* in the safe concentration was small, and with the increase in stress time, the expression of the *PmNHE* gene was not significantly different from that of the control group. Under the stress of half-lethal ammonia nitrogen concentration, the expression of *PmNHE* at 3, 12, 48, 72, and 96 h was up-regulated compared with the control group (*p* < 0.01). In the intestine, *PmNHE* showed a trend of first decreasing and then increasing in the safe concentration, which was higher than that of the control group at 6 h and lower than that of the control group at 24 h. The expression of *PmNHE* under the half-lethal concentration stress was inhibited.

**Figure 3.** Multiple alignment of amino acid sequences of NHE gene from *Peenaeus monodon* and other species. Sequence ID: *Litopenaeus vannamei*, QCR98514.1; *Limulus Polyphemus*, XP\_013784312.1; *Portunus trituberculatus*, ANV19765.1.

**Figure 4.** Relative expression of *PmNHE* in different tissues of *Penaeus monodon*. The values are x ± SD (*n* = 3). Bars with different lowercase letters indicate significant differences (*p* < 0.05).

**Figure 5.** The mRNA expression levels of *PmNHE* in the gills and intestine at different time intervals of different-concentration ammonia nitrogen stress treatments. (**a**) Gill and (**b**) intestine under ammonia nitrogen stress. Data are shown as means ± SD (standard deviation) of three separate individuals. Significant differences are indicated with asterisks, \* *p* < 0.05, \*\* *p* < 0.01.

#### *3.5. Mortality in High-Concentration Ammonia Nitrogen Environment after PmNHE Silencing*

Under high-concentration ammonia nitrogen, the expression of *PmNHE* was significantly up-regulated in gills and down-regulated in the intestine. It is suggested that *PmNHE* may be involved in the mechanism of response to high-concentration ammonia nitrogen stress. In order to explore the role of *PmNHE* in the process of high-concentration ammonia nitrogen stress, in this study, RNA interference experiments were performed by injecting ds*PmNHE* synthesized in vitro, and 24 h after ds*PmNHE* injection, acute highconcentration ammonia nitrogen stress was applied to *P. monodon*. The results show that

with phosphate-buffered saline (PBS) and dsGFP as controls, the expression of *PmNHE* was lower than that of the control group at 24 h after injection (Figure 6), and there was always a difference between the ds*PmNHE* injection group and the two control groups during the subsequent acute ammonia nitrogen stress experiment (*p* < 0.01). It showed that the whole experimental process had good interference efficiency.

**Figure 6.** Silencing efficiency of *PmNHE* under ammonia-nitrogen stress. Data are shown as means ± SD (standard deviation) of three separate individuals. Significant differences are indicated with asterisks, \* *p* < 0.05, \*\* *p* < 0.01.

The relative expression of *PmNHE* in the ds*PmNHE* and GFP groups at different time points under high-concentration ammonia-nitrogen stress was determined.

We compared the survival curves of the *PmNHE* silenced group and the control group under high levels of ammonia nitrogen stress to explore the relationship between the *PmNHE* gene and high-concentration ammonia nitrogen stress (Figure 7). The shrimp in the GFP group all died within 69 h, with an average survival time of 40.66 h, the shrimp in the PBS group all died within 69 h, with an average survival time of 42.32 h, the average survival time of the shrimp in the *PmNHE*-dsRNA group was 37.96 h. From the trend of the survival curve, it can be seen that the survival rate of shrimp in the silenced group was higher than that in the control group in the first 24 h under high-concentration ammonia nitrogen stress; after 36 h of stress, the death rate of shrimp in the *PmNHE*-silenced group began to increase, which was different from that of the two control groups.

**Figure 7.** Mortality under ammonia-nitrogen stress after silencing of *PmNHE*.

ds*PmNHE*, GFP, and PBS group shrimp were simultaneously stressed under a higher concentration of ammonia nitrogen; the number of dead shrimp in each group was observed and recorded every 3 h.

#### **4. Discussion**

Numerous studies have shown that NHEs play an important role in the transport of various ions in animals [18]. In this research, the full-length cDNA encoding NHE was cloned from *P. monodon* for the first time. The amino acid sequence analysis of *PmNHE* by the SMART program found that *PmNHE* had 12 transmembrane domains, and the sequence contained a sodium/hydrogen ion exchanger domain (78–490 aa). Exploring this domain revealed that NHE transporters contained 10–12 transmembrane domains at the amino terminus and a larger cytoplasmic domain at the carboxy terminus. The transmembrane region M3–M12 had the same characteristics as other members of this family. The M6 and M7 transmembrane regions were highly conserved and therefore considered to be involved in the transport of sodium and hydrogen ions [19]. Phylogenetic tree analysis showed that the evolution relationship between *PmNHE* of *P. monodon* and NHE of *Penaeus vannamei* was the closest. The similarity between *PmNHE* and the NHE of *Litopenaeus vannamei* reached 93.92%, the similarity with the protein of *Limulus americanus* was 42.71%, and the similarity with the protein of *Portunus trituberculatus* was 35.77%; these data indicated that NHE sequences were species specific.

Quantitative analysis of various tissues showed that *PmNHE* was expressed in a variety of tissues, and the expression of *PmNHE* was the most abundant in the intestines of *P. monodon*, and the expression level in the muscle was second only to that of the intestinal tissues. It has been shown that the expression of NHE was the highest in the intestine of *Penaeus vannamei*, which was similar to our results [20]. Osmoregulation and ion regulation in crustaceans were mainly performed by gill tissues with multiple functions and the intestines, which was the excretory and digestive organ [21,22]. In addition, muscle has also been demonstrated to have an ion regulation function [23]. Therefore, we selected the gill and intestine as the research objects to explore their expression patterns under ammonia nitrogen stress.

The quantitative results show that the expression of *PmNHE* in the gill tissue of *P. monodon* was significantly up-regulated under a high concentration of ammonia nitrogen stress, which was similar to the result of the significant up-regulation of the NHE-2 gene in the gill tissue of *Oncorhynchus mykiss* when the concentration of ammonia nitrogen increased [24]. Other researchers found that it could block the excretion of ammonia nitrogen from their gills by inhibiting the NHE activity of *Carcinus maenas*, *Cancer pagurus*, and *Astacus leptodactylus* by non-specific inhibitor amiloride [7,8]. Therefore, we speculated that *PmNHE* also played a role in excreting ammonia in the gills of *P. monodon*, which may be in line with the "Na+/NH4+ exchange complex" model of ammonia excretion in freshwater fish. This model suggested that ammonia transport was carried out through Rh glycoproteins, and the hydration of CO2 and H<sup>+</sup> by *Na+/NH4 +-exchange* (NHE-2) and *H+-ATPase* led to the acidification of the gill boundary layer, thereby promoting ammonia transport [25]. Furthermore, we also conducted a quantitative analysis of the intestines, and the results show that the expression of *PmNHE* in the intestines of *P. monodon* under high levels of ammonia nitrogen stress was significantly inhibited compared with the control group. Silva et al. quantitatively analyzed the foregut and hindgut of *Monopterus albus* treated with high ammonia nitrogen and showed that the expression of NHE3 was increased in the foregut after 6 h of ammonia exposure, about 28 times that of the control group [25]. The expression of the NHE1 gene in the foregut and hindgut changed little over time. The expression of the NHE1 and NHE3 genes in the foregut and hindgut following chronic exposure was not different. They speculated that NHE3 in the foregut of *Monopterus albus* may have a significant impact on the active excretion of NH4 <sup>+</sup> under the high-concentration ammonia nitrogen environment. The high ammonia nitrogen environment made *PmNHE* up-regulated in the gill tissue of *P. monodon* and inhibited in

the intestine. We speculated that it may be due to the differences between species; *PmNHE* in the gill tissue of *P. monodon* played an important role under ammonia nitrogen stress. To further explore the role of *PmNHE* in an environment with high levels of ammonia nitrogen, we compared the survival curves of the *PmNHE* silenced group and control group under high-concentration ammonia nitrogen stress. We found that after high levels of ammonia nitrogen stress, the death curves of shrimp in the GFP and PBS control groups were essentially consistent, and the average death time was similar. The average survival time of the shrimp in the *PmNHE*-dsRNA injection group was lower than that in the two control groups. This result indicates that *PmNHE* may have a significant impact on the high ammonia nitrogen environment.
