Permission to conduct the experiments was obtained from the the Freshwater Fisheries Research Center (2021JBFM02, 12-06-2021). All experiments were performed in accordance with Jiangsu Laboratory’s Animal Management Guidelines (014000319/2008-00079). All M. nipponense used in this study were collected from the Dapu M. nipponense Breeding Base (120°13′44″E, 31°28′ 22″N; Wuxi, China). All prawns were maintained for 3 days in aerated freshwater with dissolved oxygen content ≥ 6 mg/L prior to tissue collection. An ice bath was used to anesthetize the prawns when tissues were collected (38).

Testis were collected for the synthesis of templates and used for rapid amplification of cDNA ends (RACE) cloning. Different tissues collected for qPCR analysis included eyestalks, brain, heart, hepatopancreas, muscle, gonad, and gills from both male (body weight of 2.87-4.12 g) and female (body weight of 1.84-2.12 g) M. nipponense. Specimens at different developmental stages were collected from a full-sib population every 5 days during their maturation process. The body weights of the full-sib population were from 0.003-0.021 g. Testis and androgenic gland were sampled during both the reproductive season in July (water temperature of 30 ± 2°C, illumination time ≥ 16 h) (body weight of 3.12-4.47 g) and the non-reproductive season in January (water temperature of 13 ± 2°C, illumination time ≤ 12 h) (body weight of 2.15-3.97 g). For each tissue, sex, and time point, five samples were pooled together to form a biological replicate, and three biological replicates were used for qPCR analysis. All collected tissues were immediately preserved in liquid nitrogen until used for qPCR analysis.

Table 1 lists the specific primers used for Mn-Plk1 cloning. The primers were designed using the Primer-BLAST tool from NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) based on the open reading frame (ORF) of Mn-Plk1. The full-length cDNA of Mn-Plk1 was cloned using the RACE technique following the methods described in previous studies (39, 40). Briefly, the total RNA from the testis was extracted using RNAiso Plus Reagent (Takara, Shiga, Japan), and the extracted total RNA was then used to synthesize the templates for 3′cDNA and 5′cDNA cloning using the 3′-Full RACE Core Set Ver.2.0 kit and the 5′-Full RACE kit (Takara), respectively. After the assembling, two pairs of primer were designed based on the full-length cDNA sequence of Mn-Plk1, in order to verify the accuracy of Mn-Plk1 cDNA sequence ( Table 1 ).

The structural characteristics of Mn- Plk1 were analysed using the BLASTx and BLASTN search program (http://www.ncbi.nlm.nih.gov/BLAST/) and the ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The theoretical isoelectric point and the molecular weight of Mn-Plk1 protein were measured using the ComputepI/Mwtool software (http://ca.expasy.org/tools/pi_tool.html). Table 2 lists the accession numbers of amino acid sequences of different species, which were used to construct the phylogenetic tree of Plk1 using MEGA X, the maximum-likelihood method, and the bootstrap method with 1000 replications.

qPCR was used to determine the relative mRNA expression of Mn-Plk1 in the different tissues. Table 1 lists the primers used for qPCR analysis, which was performed following previously described methods (39, 40). Briefly, RNAiso Plus Reagent was used to extract the total RNA from each tissue following the manufacturer’s protocol. After removing genomic DNA by gDNA Eraser, the total RNA was used to synthesize the cDNA template using PrimeScript™ RT reagent Kit (Takara) based on the manufacturer’s protocol. The UltraSYBR Mixture (CWBIO, Beijing, China) with the SYBR Green RT-qPCR assay was used to determine the expression level of Mn-Plk1 in each tissue, and the Bio-Rad iCycler iQ5 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) was used to perform the qPCR analyses. The amplification protocols for qPCR analysis were 95°C for 10 min, followed by 37 cycles of 95°C for 10 s and 60°C for 1 min. All primers for qPCR analysis were listed in Table 3 . The eukaryotic translation initiation factor 5A is a stable reference gene for qPCR analysis in M. nipponense, and it was used in the present study (41). The relative mRNA expression levels of Mn-Plk1 were calculated using on the 2^−ΔΔCT comparative CT method (42).

The potential function of Plk1 in reproductive development of male M. nipponense was investigated by RNAi, which has been widely used in gene functional analysis in this species. Table 1 lists the specific RNAi primer with a T7 promoter site, which was designed using Snap Dragon tools (http://www.flyrnai.org/cgibin/RNAifind_primers.pl) based on the ORF of Mn-Plk1. The Mn-Plk1 dsRNA (dsPlk1) was synthesized using the Transcript Aid™ T7 High Yield Transcription kit (Fermentas, Waltham, MA, USA) following the manufacturer’s protocol, and the green fluorescent protein dsRNA (dsGFP) was also synthesized and used as the negative control (43).

Six hundred male M. nipponense were collected from the Dapu M. nipponense Breeding Base and randomly divided into the dsPlk1 group (RNAi) and dsGFP group (control) (N = 300). These prawns were collected at approximately 5 months after hatching and had a body weight of 3.14−4.32 g. The dsPlk1 and dsGFP were injected into the prawns through the cardio coelom with the injected dose of 4 μg/g according to previously described methods (44). dsPlk1 and dsGFP were injected once and then again with the same dose after 7 days. Androgenic gland samples (N=15) were collected from both the control group and the RNAi group on days 1, 7, and 14 after dsGFP or dsPlk1 injection. The Mn-Plk1 mRNA expression levels were measured by qPCR to confirm the silencing efficiency. The mRNA expression of Mn-IAG was also measured using the same cDNA templates to analyse the regulatory relationship between Plk1 and IAG in M. nipponense.

The morphological differences between testis samples taken from the dsPlk1- and dsGFP-injected prawns were assessed by histological observations of tissues stained with HE. The histological observations were performed following previously described methods (45, 46). The slides were observed under an Olympus SZX16 microscope (Olympus Corporation, Tokyo, Japan). The cell types were labelled according to the previous studies (14, 15), and the numbers of each cell type were counted through the whole testis after the treatment of dsGFP and dsPlk1.

SPSS Statistics 23.0 (IBM, Armonk, NY, USA) was used to evaluate differences in Mn-Plk1 expression levels, followed the independent samples t-test and analysis of ANOVA variance (least significant difference and Duncan’s multiple range tests). Quantitative data were expressed as mean ± standard deviation. P < 0.05 and P < 0.01 were considered to be differentially expressed and significantly differentially expressed, respectively.

The full-length cDNA sequence of Mn-Plk1 was 2,360 base pairs (bp) long, with a 5′ and a 3′ untranslated region of 92 bp and 432 bp, respectively. The ORF of Mn-Plk1 was 1,836 bp long and encoded 611 amino acids ( Figure 1 ). The cDNA sequence of Mn-Plk1 was submitted to NCBI with the accession number OP379748. The theoretical isoelectric point of Mn-Plk1 was 4.90, and the molecular weight of the protein was 18.466 kDa.

The protein sequence of Mn-Plk1 shared over 80% identity with the Plk1 sequences from other crustacean species in NCBI based on the BLASTx analysis. The species included Procambarus clarkii (83.03%, XP_045623220.1), Penaeus monodon (82.42%, XP_037785985.1), Penaeus chinensis (82.42%, XP_047493190.1), Penaeus japonicus (82.06%, XP_042864001.1), and Penaeus vannamei (81.20%, XP_027211725.1) ( Figure 2 ). Protein sequence alignment of Plk1 from different species revealed that Mn-Plk1 contains the common structure of Polo members, including a highly conserved N-terminal kinase and C-terminal protein binding domain (PBD). The conserved N-terminal kinase is composed of a serine/threonine kinase domain, of which the sub-domains I to XI have been labelled ( Figure 2 ). The C-terminal PBD is composed of two Polo-boxes called Polo-box 1 and Polo-box 2 ( Figure 2 ).

The phylogenetic tree analysis revealed that the protein sequences of Plk1 from different species were mainly divided into two branches: crustacean species and mollusks species ( Figure 3 ). Mn-Plk1 was clustered into the crustacean branch and had the closest evolutionary distance with that of crayfish, and then it clustered with the Plk1s of marine shrimp, followed by that of crabs. The evolutionary distance of Mn-Plk1 with Plk1s of mollusks species was much more distant.

The qPCR analysis revealed that the highest expression of Mn-Plk1 occurred in the testis and ovary of male and female prawns, respectively, and it was significantly higher than the expression in the other tested tissues (P < 0.01). The expression levels in the testis and ovary were 505.56-fold and 840.39-fold higher than that of the female eyestalk, which had the lowest expression of all of the tested tissues. Mn-Plk1 expression levels were higher in the eyestalk, brain, heart, and hepatopancreas of male prawns than of female prawns, whereas the expression levels in the muscle, gonad, and gill showed the opposite pattern ( Figure 4A ).

Mn-Plk1 expression was widely detected during the whole developmental process of juvenile prawns, but peak expression occurred at the cleavage stage and was significantly higher than that at the other tested stages (P < 0.01). The expression levels during the larval and post-larval developmental stages (PL) generally remained stable and showed no significant differences (P > 0.05) ( Figure 4B ). The lowest expression of Mn-Plk1 was observed at PL25, and the value at cleavage stage was 117.57 folds higher than that of PL25.

The qPCR analysis also revealed that Mn-Plk1 expression was higher in the testis and androgenic gland from samples collected during the reproductive season than during the non-reproductive season. The values in the testis and androgenic gland from the reproductive season were 2.69-fold and 3.36-fold higher than those from the non-reproductive season, respectively, and the differences were statistically significant (P < 0.01) ( Figure 5 ).

In the RNAi experiment, qPCR analysis revealed that the Mn-Plk1 expression remained at a stable level in the androgenic gland after the injection of dsGFP (P > 0.05), whereas it was significantly decreased after the injection of dsPlk1. The decrease reached 94.8% and 88.9% at days 7 and 14, respectively, after the injection of dsPlk1 compared with the injection of dsGFP, and the values were significantly different (P < 0.01) ( Figure 6A ).

The expression of Mn-IAG in the same cDNA templates also remained at a stable level in the dsGFP-injected prawns (P > 0.05), but it decreased in the dsPlk-injected prawns. Decreases of > 55% and 60% were observed at days 7 and 14, respectively, in the dsPlk1 group compared to the dsGFP group (P < 0.01) ( Figure 6B ).

Histological observation revealed significant morphological differences between the testis from dsGFP- and dsPlk1-injected prawns. The cells in the testis were almost the same on different days after dsGFP injection, and sperm were the dominant cells, which were dramatically more than that of spermatogonia and spermatocyte ( Figures 7A, B ). However, the number of sperm decreased with time after dsPlk1 injection, while the percentages of spermatogonia and spermatocytes increased. Sperm (approximate 5%) were rarely observed on day 14 in dsPlk1-injected prawns, while over 80% cells were observed as spermatogonia ( Figures 7A, C ).