*3.3. Tissue Expression Analysis of PmMKK4*

Using real-time quantitative PCR technology, the expression differences of the *PmMKK4* gene in different tissues of *P. monodon* were explored. The results showed that the *PmMKK4* gene was expressed in all the tested tissues. Among them, the expression level was the highest in muscle tissue, and the lowest expression level occurred in the eye stalk nerve. The expression level in muscle was 41.25 times of that in the eye stalk nerve. Secondly, the expression was also higher in the thoracic nerve, intestine, epidermis, heart, gill, and lymphoid tissues, which was higher than that in other tested tissues (Figure 4).

**Figure 4.** mRNA relative expression of *PmMKK4* in different tissues. Abbreviations: M: muscle, T: thoracic nerve, I: intestines, Ep: epidermis, H: heart, G: gill, L: lymph, B: brain nerve, S: stomach, A: abdominal nerve, Hep: hepatopancreas, O: ootheca, Te: testis, Eye: eyestalk ganglion. Different letters between different tissues mean significant difference (*p <* 0.05).

#### *3.4. Expression Analysis of PmMKK4 under Bacterial Stimulation*

In gill tissues, the expression of *PmMKK4* showed different changes after injection of PBS, *S. aureus*, *Vibrio harveyi*, and *V. anguillarum* (Figure 5A). Taking the PBS group as the control, the *S. aureus* infection group was significantly up-regulated at 6 h and 24 h and reached the highest value at 24 h, which was 2.42 times that of the PBS group at this time, and there was a significant difference (*p* < 0.05). Significant down-regulation occurred at 48h, and there was no significant difference between the rest of the time and the PBS group. The group infected with *Vibrio harveyi* showed down-regulation from 12 h and returned to normal level at 72 h. In the group infected with *V. anguillarum*, the expression level remained down-regulated from 6 h, which was significantly different from that in the control group (*p* < 0.05).

**Figure 5.** Expression of *PmMKK4* in *P. monodon* after bacterial stimulation. Expression of *PmMKK4* in gill (**A**) and hepatopancreas (**B**) after stimulation by *S. aureus*, *V. harvey* and *V. anguillus*. Vertical bars represent the mean ± S.E (n = 3). Significant differences are indicated by \* (*p* < 0.05) and \*\* (*p* < 0.01).

In the hepatopancreas tissue, each experimental group showed different trends (Figure 5). Taking the PBS group as the control, after infection with *S. aureus*, except for 12 h, it maintained an upward trend, and there was a very significant difference at 3 h and 48 h. Compared with the control group, the *V. harveyi* infection group fluctuated; it was up-regulated at 3 h and 48 h and down-regulated at 12 h, 24 h, and 72 h (*p* < 0.05). There was no significant change within 9 h after infection with *V. anguillarum*, and it was down-regulated from 12 h.

#### *3.5. Expression Analysis of PmMKK4 under Low Salt Stress*

The overall expression level of *PmMKK4* after low-salt stress showed that gill tissue was more sensitive to low-salt stress than hepatopancreas tissue. In the gill tissue (Figure 6A), the overall change trend of salinity, which dropped sharply to 17 groups, was a very significant up-regulation and then turned to a significant up-regulation, which remained up-regulated except for 72 h. *PmMKK4* was up-regulated within 96 h after salinity suddenly dropped to the three groups, and the difference was extremely significant at 12 h, 24 h, and 72 h. In the hepatopancreas tissue (Figure 6B), the salinity 7 group was significantly up-regulated at 3 h, 12 h, and 72 h, and there was no significant difference between the expression levels at other time points and the control group. The salinity 3 groups were significantly up-regulated at 12 h and 96 h, and there was no significant difference at other time points.

**Figure 6.** Relative expression levels of *PmMKK4* in hepatopancreas (**A**) and gill (**B**) under acute low-salinity stress. Quantitative RT-PCR was performed to determine the expression of *PmMKK4* in gill (**A**) and hepatopancreas (**B**) of *P. monodon* at different time intervals (n = 3 for each group) after the salinity drops sharply to 17 and 3, ranging from 0 to 96 h. Vertical bars represent the mean ± S.E. (n = 3). Significant differences are indicated by \* (*p* < 0.05) and \*\* (*p* < 0.01).

#### *3.6. Expression Analysis after PmMKK4 Interference*

According to the quantitative results of whole tissue expression, the expression of *PmMKK4* was the highest in muscle, so after the RNA interference test, muscle tissue was selected for subsequent quantitative research. Quantitative detection of the *PmMKK4* gene was performed at 24 h, 48 h, and 72 h after RNA interference to verify the interference efficiency. Taking the non-injected group as the control, as shown in Figure 7, the expression of *PmMKK4* in the ds*MKK4* group was only 35.7% of that in the non-injected group at 24 h after injection. At 48 h, the expression level of the injected ds*MKK4* group was 54.1% of that of the non-injected group, and at 72 h, the expression level of the injected ds*MKK4* group was 16.9% of that of the non-injected group, indicating that the interference efficiency of dsPm*MKK4* was obvious, and the interference was effective during the experiment. The mortality rate of each group in the RNAi experiment at each time point after low salt stress is shown in Figure 8. The mortality rate of the dsPm*MKK4* injection group was always higher than that of the other three groups. At 18 h, the mortality rate started to exceed 20%, and the final mortality rate was 25.58%. The lowest mortality rate was in the PBS injection group, with a final mortality rate of 13.04%. The three experimental groups under low-salt stress had more death in the first 24 h, and less death after that.

**Figure 7.** *PmMKK4* mRNA expression profiles after silencing by RNA interference. Quantitative RT-PCR was performed to determine the expression of *PmMKK4* in gills of *P. monodon* at different time intervals (n = 3 for each group) after dsRNA injection. Vertical bars represent the mean ± S.E. (n = 3). Significant differences are indicated by \* (*p* < 0.05).

**Figure 8.** Mortality changes of *Penaeus monodon* in different gene silencing groups under acute low salinity stress.

Figure 9 shows the expression levels of *PmMKK7* and *PmJNK* in the muscles under acute low-salt stress after the injection of dsPm*MKK4*. Taking the PBS injection group as the control, the expression of the *MKK7* gene in *P. monodon* showed a significant upwardregulated trend as a whole, except for 3 h and 24 h, and the expression was significantly up-regulated at other time points. The expression level of *PmJNK* fluctuated throughout the experiment. After acute low-salt stress, the dsMKK4 group first increased at 3 h, then decreased at 6 h, then increased expression, down-regulated at 24 h, and then continued to increase until the end of the experiment.

**Figure 9.** Relative expression levels of *PmMKK7* (**A**) and *PmJNK* (**B**) after dsRNA-*MKK4* under acute low salinity stress. \* (*p* < 0.05), \*\* (*p* < 0.01). (Significant difference from the CG group).

#### **4. Discussion**

The *MKK4* gene of *P. monodon* was cloned in this study. The results of amino acid sequence analysis showed that *PmMKK4* has 22 phosphorylation sites. The phosphate groups can regulate different functions of the protein and may play a synergistic or reverse role in various reactions [14]. Structural prediction indicated that *PmMKK4* contains a conserved serine/threonine protein kinase (S–T–T–K–C) region, which is a potential double phosphorylation site. The *MKK4* gene has two downstream pathways: the JNK signaling pathway, and the p38 signaling pathway. These two branch pathways play important roles in immunity and anti-stress processes in organisms. Phylogenetic tree analysis showed that the *MKK4* gene of *P. monodon* was most closely related to *P. vannamei* and *P. chinensis*, clustered into a clade. The results of multiple sequence alignment showed that *PmMKK4* has a high similarity with *MKK4* genes of other species, among which the similarity with *P. vannamei* and *P. chinensis* was the highest (99.65%), indicating that *MKK4* genes were relatively similar among closely related species. It is speculated that its conserved

protein kinase domain may play an important role in many aspects such as physiology and biochemistry.

Through quantitative tissue analysis, it was determined that *PmMKK4* was expressed in all tested tissues, which is the same as the tissue distribution results of other species. In the detected tissues, the expression level of Pm*MKK4* in muscle was the highest, which was significantly higher than other tissues. Similarly, in the study of *P. chinensis*, the expression level of *MKK4* in muscle tissue was also significantly higher than that in other tissues [7], suggesting that muscle may be an important tissue for the function of the *MKK4* gene. In addition to muscle, *PmMKK4* was also highly expressed in tissues such as intestine, heart, gill, and lymph. Intestine and lymph were important immune tissues, and gills were important tissues for crustaceans to exchange ions with the environment and regulate osmotic pressure. Therefore, it was speculated that *PmMKK4* plays an important role in the immune and salinity stress responses of *P. monodon*.

In order to explore the role of the *PmMKK4* gene in the innate immunity of *P. monodon*, we carried out pathogen infection experiments. Although the expression of *PmMKK4* was the highest in muscle, considering that it had been widely accepted that gill and hepatopancreas were important immune organs in previous studies, while muscle and nerve were rarely considered immune-related tissues, we chose gill and hepatopancreas. Tissue responses to bacterial stimulation were studied. The experimental results showed that *PmMKK4* was up-regulated in hepatopancreas and gill tissues after infection with *S. aureus*, which is similar to the *MKK4* gene in *L. vannamei* in response to *S. aureus* [3]. After infection with *Vibrio harveyi*, *PmMKK4* was down-regulated for a period of time in gill tissue, while fluctuating between up- and down-regulation in hepatopancreas tissue. After *P. monodon* was infected with *Vibrio harveyi*, the expression levels of three related genes in the JNK pathway were significantly reduced, indicating that some effector molecules of the JNK signaling pathway may be involved in the process of immune regulation, which is consistent with the research results of Shi et al. [15]. After infection with *V. anguillarum*, gill tissue and hepatopancreas tissue showed a continuous downward trend. However, the *MKK4* gene was significantly up-regulated after *L. vannamei* infection with the Gram-negative bacteria *Vibrio parahaemolyticus* [3]. The physiological role of the post-*MKK4* gene is different, and therefore the response mode is different. In addition, when *Pinctada fucata* are infested by exogenous pathogens, the expression level of *MKK4* gene is significantly changed and phosphorylated, suggesting that it is involved in the self-protection mechanism of *Pinctada fucata* to defend against the occurrence of diseases [16,17].

Changes in salinity can cause changes in the osmotic pressure and the activities of various non-specific immune enzymes in the shrimp, affecting the immune defense ability of the shrimp. The experimental results of acute low-salt stress in this study showed that the *PmMKK4* gene in the gill tissue and hepatopancreas tissue of *P. monodon* was activated after stress, resulting in different degrees of up-regulation. Among them, the expression change of *PmMKK4* in gill tissue was more significant. It can be speculated that under low salt stress, the gill tissue undertakes more physiological and biochemical reactions related to the *MKK4* gene. In the process of ammonia nitrogen stress in *P. chinensis*, the expression of the *MKK4* gene in muscle, hepatopancreas, gill, and other tissues is significantly increased, suggesting that the *MKK4* gene is involved in the stress resistance process of *P. chinensis*. *MKK4* can activate downstream JNK signaling pathway. Studies have confirmed that the JNK branch pathway plays an important role in the salinity adaptation process of aquatic animals. Under salinity stress, the expression of *MAPK 8 (JNK1)* and *MAPK 9 (JNK 2)* was significantly up-regulated [18–20]. To further explore the role of the *PmMKK4* gene in low-salt stress, we performed double-stranded RNA injection experiments. In a low-salt environment, the mortality rate after knocking down the *PmMKK4* gene increased rapidly in a short period of time, while the mortality rates of the other two groups were lower. It is speculated that the low expression of the *MKK4* gene reduces the adaptability of *P. monodon* to the low-salt environment and causes more deaths. The *PmMKK4* gene may play an important role in adaptation to the low-salt environment. After the interference, we

further conducted a quantitative study on other genes in the JNK signaling pathway. The results showed that the expression of the *MKK7* gene in *P. monodon* showed a significant upward trend as a whole, while the expression of *PmJNK* fluctuated in the early stage and stably increased in the later stage. It is speculated that the *JNK* signaling pathway is further activated under low-salt stress, and the *PmMKK7* gene maintains high expression, while the *PmMKK4* gene is expressed less to ensure the stable functioning of the JNK signaling pathway.
