*2.3. Sample Collection and cDNA Synthesis*

After 1 week of seawater culturing, healthy male and female *P. monodon* were randomly selected. Hepatopancreas, gills, intestines, stomach, lymph, heart, muscle, epidermis, eye stalk nerve, cranial nerve, thoracic nerve, ventral nerve, and ovary (testis) tissue were dissected for collection. The same tissue samples from 3 shrimps were mixed into one tube. Furthermore, during summer, the mating season of *P. monodon*, samples of shrimp larvae at different stages of development were collected. Those samples including zygote, nauplius, zoea, mysis, and postlarval stages. Zygote stage samples were collected immediately after egg laying. When 80% of the population had reached the objective stage according to their morphologies, as observed using an optical microscope, samples of the larvae of different stages were collected. The samples mentioned above were organized into three parallel groups, stored in RNAlater® RNA stabilization solution (Invitrogen, Carlsbad, CA, USA) at 4 ◦C overnight, and then stored at −80 ◦C.

Following the manufacturer's instructions of Trizol reagent (Invitrogen, USA), the total RNA of all collected samples was extracted. The ratios of ultraviolet absorbance at 260/280 nm were measured using a NanoDrop2000 device (NanoDrop Technologies, Waltham, MA, USA); 1.5% agarose gel electrophoresis was used to ensure the integrity. A template of cDNA was synthesized from the RNA using the PrimeScript II 1st strand cDNA synthesis kit (Takara, Tokyo, Japan). For the sake of real-time quantitative PCR (qRT-PCR), cDNA was synthesized in accordance with the manufacturer's instructions for the PrimeScript TM RT reagent kit with a gDNA eraser (Perfect Real-time, Takara, Japan) and diluted to 50 ng/μL for use as the template.

#### *2.4. Bacterial Infection Challenge*

In total, 200 healthy *P. monodon* with an average weight of 15–18 g were selected for immune challenge experiments. Three pathogenic bacteria provided by the Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization were used. According to a previous study [11], the culture schedule and injection concentration for each strain were determined. Four experimental groups were included: PBS (shrimp specimen injected with sterile phosphate-buffered saline as control), *Staphylococcus aureus*, *Vibrio harveyi*, and *Vibrio anguillarum* groups. Each group of 50 shrimp specimens was injected into the second abdominal segment with 100 μL of sterile phosphate-buffered saline (PBS, pH 7.4) or 100 μL (1.0 × 108 cfu/mL) of *S. aureus*, *Vibrio harveyi*, and *V. anguillarum*, respectively. Healthy and intact shrimp were randomly selected at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after injection for the dissection of hepatopancreas and gill tissues. Hepatopancreas and gill tissues were stored overnight in RNAlater solution at 4 ◦C and then kept at −80 ◦C.

#### *2.5. Low-Salinity Stress*

The experiment site of the low-salt stress test was in the South China Sea Fisheries Research Institute (Shenzhen City, Guangdong Province, China). A total of 360 shrimps (7–10 cm) was selected for these experiments. The salinity concentration was adjusted to the target salinity by mixing the cultured seawater with freshwater using a salinity meter (AZ8371, Hengxin, Taiwan). According to the pre-test results, the 96 h half-lethal salinity was 3 psu [12]. Therefore, the stress salinity was set to 3 psu. Another experimental group with salinity of 17 psu was set up, and conventional aquaculture seawater (about 25 psu) was used as the control group [13]. Three parallel groups (*n* = 40/group) were created. The incubation temperature and pH were maintained at 25–28 ◦C and 7.0 ± 0.5, respectively, and 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after exposure to different salinity stresses, shrimp specimens with optimal activity during the intermolt were selected for dissection to collect gills and hepatopancreas tissues, and they were preserved in RNAlater solution. The tissues were then maintained at −80 ◦C after overnight storage at 4 ◦C.

#### *2.6. qRT-PCR Analysis of PmMKK4 mRNA Expression*

In this study, qRT-PCR was used to detect *PmMKK4* mRNA expression in different tissues at different developmental stages following bacterial challenge and low-salinity stress exposure. Since the reaction component and cycle condition for EF1a are consistent with *PmMKK*, the reference gene was chosen to be 1α (EF1a) (Table 1). The solution in each hole (12.5 μL) was a mixture of 6.25 μL of 2 × TB GreenTM Premix ExTaq (Takara, Beijing, China), 0.5 μL each of *PmMKK4*-qF and *PmMKK4*-qR (50 μmol/L), 1 μL qRT-PCR diluted cDNA, and 4.25 μL double-distilled water. Green fluorescence measurement qRT-PCR was carried out in the quantitative real-time PCR system, Roche Light Cycler® 480II. The following four steps were conducted: degeneration for 30 s at 95 ◦C, a quantitative analysis stage with 40 cycles of 94 ◦C for 5 s and 60 ◦C for 30 s, dissolution curve analysis for 5 s at 95 ◦C and 60 ◦C and up to 95 ◦C for 1 min, and an according stage of 30 s at 50 ◦C. The relative CT method (2−ΔΔCT) was used to obtain the PCR data. One-way ANOVA was used to work on statistical analysis. SPSS statistics version 23.0 software (IBM, Armonk, New York, USA) was used to carry out the Tukey's multiple range test. The differences were considered to be significant at *p* < 0.05. Tested data were presented as mean ± SD (standard deviation).

### *2.7. Low-Salinity Stress Testing in P. monodon following RNA Interference (RNAi)-Mediated Knockdown of PmMKK4*

Primer Premier 5.0 was used to design the following primers: dsMKK-f, dsMKK-r, dsMKK-T7-f, and dsMKK-T7-r (Table 1). A DNA fragment containing the T7 promoter was amplified by Ex Taq using normal cDNA as a template. PCR conditions were as follows: 3 min at 94 ◦C, 35 cycles of 30 s at 94 ◦C, 30 s at 58 ◦C, 1 min at 72 ◦C, and a final cycle of 10 min at 72 ◦C. Excess bands were clipped after agarose gel electrophoresis, leaving clear and bright bands. DNA fragments were recovered according to the gel recovery kit instructions. dsRNA synthesis was performed according to the T7 RiboMAXTMExpress RNAi System kit instructions. Reaction system: RiboMAXTM Express T7 2X Buffer 10.0 μL, linear DNA template (total 1 μg) 1.0–8.0 μL, Nuclease-Free Water 0–7.0 μL (8 μL-DNA volume), Enzyme Mix-T7 Express 2.0 μL. A final volume of 20.0 μL was incubated at 37 ◦C for 30 min to obtain single-stranded RNA (ssRNA). Then, equal volumes of complementary ssRNA were mixed and incubated at 70 ◦C for 10 min, 65 ◦C for 10 min, and 25 ◦C for 10 min. Then, 2.0 μL of freshly diluted RNase solution (1 μL RNase solution: 199 μL Nuclease-Free Water) and 2.0 μL RQ1 RNase-Free DNase was added and incubated at 37 ◦C for 30 min. The obtained dsRNA was purified for later use. dsGFP-F/R primers and pD-GFP recombinant vectors were used to synthesize green fluorescent protein (GFP) double-stranded RNA in the same way.

The weight of *P. monodon* was 5.0 ± 1.0 g, and the dsRNA injection volume was 3–5 μg/g shrimp. Each shrimp was injected intramuscularly in the second abdominal segment. The injection experiments were divided into three groups: PBS group, dsGFP group, and dsMKK group. Before injection, samples of healthy shrimp in the intermolt phase were randomly collected, and their gill tissues were placed in RNAlater as a 0 h sample to check the RNAi efficiency. Twenty-four hours after injection, the three groups of shrimp specimens were transferred to plastic buckets adjusted to salinity of 3 psu. The dead shrimps of each group were collected and recorded every 3 h.

After 3 h, 6 h, 9 h, 12 h, 24 h, 48 h, 72 h, and 96 h exposure to salt stress, healthy shrimp specimens were collected from each treatment group, and their gill tissues were dissected and placed in RNAlater. Furthermore, 10 shrimp injected with dsGFP and dsMKK, respectively, were placed in two separate plastic buckets filled with seawater (about 25 psu). After 24 and 48 h, gill tissues were randomly collected and placed in RNAlater as samples to measure dsRNA interference efficiency. All RNA samples were mixed in RNAlater and stored at 4 ◦C overnight, then kept at −80 ◦C.
