Identification and Characterization of the DMRT11E Gene in the Oriental River Prawn Macrobrachium nipponense

The doublesex and mab-3 related transcription factor (DMRT) gene family involvement in sex development is widely conserved from invertebrates to humans. In this study, we identified a DM (Doublesex/Mab-3)-domain gene in Macrobrachium nipponense, which we named MniDMRT11E because it has many similarities to and phylogenetically close relationships with the arthropod DMRT11E. Amino acid alignments and structural prediction uncovered conservation and putative active sites of the DM domain. Real-time PCR analysis showed that the MniDMRT11E was highly expressed in the ovary and testis in both males and females. Cellular localization analysis showed that DMRT11E was mainly located in the oocytes of the ovary and the spermatocyte of the testis. During embryogenesis, the expression level of MniDMRT11E was higher at the cleavage stage than at other stages. During the different stages of ovarian development, MniDMRT11E expression gradually increased from OI to OIII and decreased to the lowest level at the end of OIV. The results indicated that MniDMRT11E probably played important roles in embryonic development and sex maturity in M. nipponense. MniDMRT11E dsRNA injection also significantly reduced vitellogenin (VG) expression and significantly increased insulin-like androgenic gland factor (IAG) expression, indicating a close relationship in gonad development.


Introduction
The oriental river prawn, Macrobrachium nipponense, is an economically important freshwater prawn and is widely farmed in China, with an annual production of almost 240,739 tons in 2017 [1]. Male oriental river prawns grow faster than females. The average size of the male commodity is 2-2.5 times that of females. All male cultures will help to increase yields and economic value. The development of an all-male culture in M. nipponense is based on sexual control technology. Therefore, it is important to identity sex-determining genes and their regulatory mechanism.
Various sex determination methods in animals are the focus of current research. Sex-determining methods in insects that are close to crustaceans have recently been studied, especially in model species such as Drosophila [2,3]. The sex determination mechanism of crustaceans is relatively complex and impacted by environmental factors. At present, there are limited studies, and most species-related studies are still lacking. Insulin-like androgenic gland factor (IAG) is a unique gene in crustaceans   3D-structures of MniDMRT11E predicted by I-TASSER. Green rectangle (site I) and red rectangle (site II) represent the residues of two intertwined Zn 2+ -binding sites.

Tissue-Specific Expression Patterns of MniDMRT11E
The expression levels were analyzed in adult prawn tissues by qPCR, and the results showed that MniDMRT11E mRNA was distributed in all tissues ( Figure 4). MniDMRT11E was highest in the testis (p < 0.05), followed by the ovary (p < 0.05). MniDMRT11E was expressed more highly in the male hepatopancreas than in the female. However, the level of expression in the muscles was the opposite. In MniDMRT11E, the two intertwined Zn 2+ -binding sites (sites I C61/C64/H76/C80 and sites II H67/C85/C87/C90) are necessary for DNA binding ( Figure 2). The DM domain also contains a conserved nuclear localization signal (NLS) (Figure 2a). DM domains are more conserved in DMRT11Es (81.03-100%) and are similar to vertebrate DMRT2s (91.4%).

Tissue-Specific Expression Patterns of MniDMRT11E
The expression levels were analyzed in adult prawn tissues by qPCR, and the results showed that MniDMRT11E mRNA was distributed in all tissues ( Figure 4). MniDMRT11E was highest in the testis (p < 0.05), followed by the ovary (p < 0.05). MniDMRT11E was expressed more highly in the male hepatopancreas than in the female. However, the level of expression in the muscles was the opposite.

Expression of the MniDMRT11E Gene During Embryo Stages
The MniDMRT11E expression pattern was analyzed by qPCR in different developmental stages (from the cleavage stage to the first-day larvae after hatching) ( Figure 5). A relatively high level of MniDMRT11E expression was observed in the cleavage stage of embryos (p < 0.05). The expression at other developmental times was not significant (p > 0.05).

Expression of the MniDMRT11E Gene During Embryo Stages
The MniDMRT11E expression pattern was analyzed by qPCR in different developmental stages (from the cleavage stage to the first-day larvae after hatching) ( Figure 5). A relatively high level of MniDMRT11E expression was observed in the cleavage stage of embryos (p < 0.05). The expression at other developmental times was not significant (p > 0.05).

Expression of the MniDMRT11E Gene During Embryo Stages
The MniDMRT11E expression pattern was analyzed by qPCR in different developmental stages (from the cleavage stage to the first-day larvae after hatching) ( Figure 5). A relatively high level of MniDMRT11E expression was observed in the cleavage stage of embryos (p < 0.05). The expression at other developmental times was not significant (p > 0.05).   Figure 6 shows the expression patterns of MniDMRT11E throughout the reproductive cycle, assessed by qPCR. The results confirmed a regular expression throughout the maturation of the ovary. The MniDMRT11E transcript level was low at the beginning of the reproductive cycle (stage I, undeveloped stage) and increased to a maximum at the nearly-ripe stage (stage III). Beyond this stage, the level fell to the lowest level at the ripe stage (stage IV). This implies that MniDMRT11E may play a role in reproductive development.

Expression of the MniDMRT11E Gene in Different Developmental Stages of the Ovaries
Bars with different letters were considered significant at p < 0.05. Values are means ± standard error of the mean (SE) for quadruplicate samples. Figure 6 shows the expression patterns of MniDMRT11E throughout the reproductive cycle, assessed by qPCR. The results confirmed a regular expression throughout the maturation of the ovary. The MniDMRT11E transcript level was low at the beginning of the reproductive cycle (stage I, undeveloped stage) and increased to a maximum at the nearly-ripe stage (stage III). Beyond this stage, the level fell to the lowest level at the ripe stage (stage IV). This implies that MniDMRT11E may play a role in reproductive development.

Localization of the MniDMRT11E Gene in the Gonad
According to previous results [26] and ovary color observation, the ovarian cycle of prawns was divided into five stages: transparent (undeveloped, oogonium proliferation, Stage I), yellow (developing, primary vitellogenesis, Stage II), light green (nearly-ripe, secondary vitellogenesis, Stage III), dark green (ripe, vitellogenesis termination, Stage IV), and gray (spent, Stage V) ( Figure 7A). The cellular localization of MniDMRT11E was examined in different development stages of ovaries by in situ hybridization. ISH revealed a MniDMRT11E signal in the same locations in all the oocytes types ( Figure 7B). The signal was visualized in all of the oocyte types, including yolk granules, the nucleus, and the cytoplasmic membrane ( Figure 7B). The signal appearance in the cytoplasm was closed to the nucleus. The male reproductive system consists of paired testes, vas deferens, terminal ampulla, and male gonopore. In mature testes, the MniDMRT11E mRNA was visualized in spermatogonia during spermatogenesis (Figure 8). In vas deferens, the MniDMRT11E mRNA was visualized in the eosinophilic matrix (Figure 8), and in the androgenic gland, the MniDMRT11E mRNA was visualized in the nucleus and cell membrane of three glandular cells (Figure 8).

Localization of the MniDMRT11E Gene in the Gonad
According to previous results [26] and ovary color observation, the ovarian cycle of prawns was divided into five stages: transparent (undeveloped, oogonium proliferation, Stage I), yellow (developing, primary vitellogenesis, Stage II), light green (nearly-ripe, secondary vitellogenesis, Stage III), dark green (ripe, vitellogenesis termination, Stage IV), and gray (spent, Stage V) ( Figure 7A). The cellular localization of MniDMRT11E was examined in different development stages of ovaries by in situ hybridization. ISH revealed a MniDMRT11E signal in the same locations in all the oocytes types ( Figure 7B). The signal was visualized in all of the oocyte types, including yolk granules, the nucleus, and the cytoplasmic membrane ( Figure 7B). The signal appearance in the cytoplasm was closed to the nucleus. The male reproductive system consists of paired testes, vas deferens, terminal ampulla, and male gonopore. In mature testes, the MniDMRT11E mRNA was visualized in spermatogonia during spermatogenesis (Figure 8). In vas deferens, the MniDMRT11E mRNA was visualized in the eosinophilic matrix (Figure 8), and in the androgenic gland, the MniDMRT11E mRNA was visualized in the nucleus and cell membrane of three glandular cells (Figure 8).

Effects of MniDMRT11E Knockdown on Gonad by RNAi
Considering that MniDMRT11E exhibits a dimorphic expression pattern, we used RNAi to investigate the role of MniDMRT11E function in male/female phenotypic development/maintenance of M. nipponense. Intravenous injection of RNAi-mediated knockout with dsRNA was apparently successful, and on the seventh day after injection, MniDMRT11E expression was down-regulated by 86% (p < 0.01, Figure 9A) compared with control levels. After MniDMRT11E RNAi, we observed that the VG (female) transcript decreased significantly by 60 % in the ovary and 94% in the hepatopancreas (p < 0.01, Figure 9B). However, we observed a nearly two-fold increase in the IAG transcript (p < 0.01, Figure 9C). These results indicated that MniDMRT11E RNAi reduced VG accumulation and increased IAG accumulation.

Effects of MniDMRT11E Knockdown on Gonad by RNAi
Considering that MniDMRT11E exhibits a dimorphic expression pattern, we used RNAi to investigate the role of MniDMRT11E function in male/female phenotypic development/maintenance of M. nipponense. Intravenous injection of RNAi-mediated knockout with dsRNA was apparently successful, and on the seventh day after injection, MniDMRT11E expression was down-regulated by 86% (p < 0.01, Figure 9A) compared with control levels. After MniDMRT11E RNAi, we observed that the VG (female) transcript decreased significantly by 60 % in the ovary and 94% in the hepatopancreas (p < 0.01, Figure 9B). However, we observed a nearly two-fold increase in the IAG transcript (p < 0.01, Figure 9C). These results indicated that MniDMRT11E RNAi reduced VG accumulation and increased IAG accumulation.

Discussion
DMRT or dmrt-like genes have been cloned in several invertebrates, but due to a lack of genomic information, it is difficult to annotate and identify DMRT genes in different invertebrate groups [27,28]. In this study, we identified a DMRT gene corresponding to MniDMRT11E in M. nipponense and performed a series of in silico analyses, such as amino acid translation, domain/motif identification and comparison, and phylogenetic analysis, to characterize the MniDMRT11E gene [22]. This is the first gene with a DM-domain to be identified in M. nipponense and it was shown that the DM domain was well-conserved, as in other species. All DMRT amino-acid sequences contain a single conserved DNA-binding motif known as the DM domain. The DM motif was a cysteine-rich DNA-binding domain comprising interwoven CCHC and HCCC Zn 2+ binding sites and a putative nNLS consisting of KGHKK/R (Figure 2) [29]. Phylogenetic analysis indicated that the MniDMRT11E protein clustered with the DMRT11E sequences of other species.
The DMRT11E gene is usually expressed in a sex-specific manner in many species. In Drosophila melanogaster, three DM genes were identified: DMRT11E, DMRT93B, and DMRT99B. Quantitative gene expression analysis in gonads showed that DMRT11E is expressed higher in the ovary than in the testis [29]. In Daphnia magna, the DMRT11E was also highly expressed in the ovary [29]. However, in Macrobrachium rosenbergii, DMRT11E transcription was prominent in the testis, while much lower in the ovary [10]. In our study, DMRT11E transcription was prominent in both the testis and ovary; moderate in hepatopancreas (female) and muscle (male); and much lower in the eyestalk, brain, and gill ( Figure 4) (p<0.05). However, the levels were much higher in the testis than in the ovary. The expression profile of the MniDMRT11E is quite similar to that of Macrobrachium rosenbergii MroDMRT11E in the testis [10]. The cellular localization of the MniDMRT11E transcripts has also been examined in spermatogonia in the testis and oocyte in the ovary. Together, these findings suggest that MniDMRT11E is an important gene in gonad maturity.
During embryo stages, we founded that the abundance of MniDMRT11E mRNA in the cleavage stage is much higher than in other stages ( Figure 5). Due to the elaborations in arthropods, the clues to the exact role of DMRT11E can be indicated by the function of their vertebrate homologs (DMRT2s; Figure 5). In zebrafish, two genes, DMRT2a and DMRT2b, are present. In adult tissues, the zebrafish DMRT2a and DMRT2b mRNA is expressed highly in muscle tissues. In different stages of embryos, DMRT2b transcripts appear in all stages of embryos, and the level of DMRT2b transcripts increases during late development. DMRT2a transcripts appeared at the blastula stage and reached a peak at the bud stage immediately before segmentation. DMRT2a is necessary for symmetric somite formation, while DMRT2b regulates somite differentiation impacting on slow muscle development [30]. In mice, DMRT2 is expressed in the dermomyotome of developing vertebrate somites [31]. This could explain why MniDMRT11E is highly expressed in muscle (male) (Figure 4).
Ovogenesis refers to the process in which primordial oogonium cells develop into oocytes cells and develop into mature eggs. Oogonium cells first mitotically propagate in the ovary, and then enter meiosis to become oocytes. In the current study, different expression patterns were also detected in the ovary. The expression level of MniDMRT11E was lower at the beginning of the reproductive cycle (stage I), and then increased and peaked at the oil globules stage (stage III), after which a decrease in levels was observed. In addition, the cellular localization of MniDMRT11E s mRNA was visualized in all the oocyte, including yolk granules, the nucleus, and the cytoplasmic membrane ( Figure 5). This means it is important for oocyte development.
Among the known invertebrate model species, Caenorhabditis elegans and Drosophila have sex-determining genes called mab-3 and doublesex, respectively, which have one common DNA-binding motif in the gate (DM) domain. The DM domain has regulatory relationships with certain genes, such as VG and IAG [10,29,[32][33][34][35][36]. Although the foregoing results suggest a relationship between MniDMRT11E and gonad maturity, the precise function of MniDMRT11E during gonad maturity remains unclear. RNAi has been helpful in studying more and more crustaceans and revealing the function of novel crustacean genes [21,[37][38][39]. Although the above results indicated a link between MniDMRT11E and gonadal maturation, the exact function of MniDMRT11E during gonadal maturation remains unclear. Therefore, we further investigated its function in M. nipponense by injecting MniDMRT11E dsRNA. MniDMRT11E mRNA expression was significantly inhibited seven days after the dsRNA injection in vivo compared to the control group, confirming that RNAi using dsRNA was an effective and valuable tool for studying specific gene functions by gene silencing.

Meanwhile, MniDMRT11E
RNAi caused a significant negative regulation of VG gene expression in the female ovary and hepatopancreas. However, MniDMRT11E RNAi resulted in significantly positively regulation of the IAG transcripts in the abdominal ganglia in males. In M. nipponense, the main sites of VG synthesis are the ovary and hepatopancreas. VG RNAi inhibited maturation of the ovary [38]. The relationship between MniDMRT11E and MniVG also explains the high expression of MniDMRT11E in the hepatopancreas(female) (Figure 4). In M. rosenbergii, silencing of MrIAG led to the arrest of testicular spermatogenesis and of spermatophore development in the terminal ampullae of the sperm duct, accompanied by hypertrophy and hyperplasia of the AGs. In addition, the sex reversal of male M. rosenbergii occurred through the silencing of a single IAG-encoding [4,36]. This also explains why the MniDMRT11E mRNA was visualized in glandular cells of AG (Figure 8). Considering the important involvement of IAG in crustacean males [4][5][6][7][8]36] and the vitally important involvement of VG in females [32,38,40], we hypothesized that MniDMRT11E may participate in this pathway, either directly or indirect upstream of IAG/VG. This result provides strong evidence for an important role of MniDMRT11E in promoting ovary maturity and inhibiting testis maturity. It is noteworthy that MroDMRT11E has a positive regulatory effect on IAG in patients with M. rosenbergii, but in M. nipponense, MniDMRT11E is the opposite. Crustaceans are less developed animals than vertebrates. Even in related species, the functions of the same gene among different species are different. The results of RNAi suggest that further research is needed to elucidate the function of MniDMRT11E.
In this study, we have identified a DMRT gene from Macrobrachium nipponense. These results suggested that the MniDMRT11E gene did not have dimorphic gene expression, but it could promote gonadal development, as well as embryogenetic expression patterns. The MniDMRT11E RNAi displayed negative regulation of the VG gene in the ovary and hepatopancreas and positive regulation of the IAG gene in abdominal ganglia. This study advances our understanding of the biological functions of the MniDMRT11E gene in M. nipponense.

Experimental Animals and Sampling
Our study does not involve endangered or protected species. This study was approved by the Institutional Animal Care and Use Ethics Committee of the Freshwater Fisheries Research Center, the Chinese Academy of Fishery Sciences (Wuxi, China, FFRC125, 26 August 2016). Healthy adult prawns, M. nipponense, were collected from Tai Lake in Wuxi, China (120 • 13 44"E, 31 • 28 22"N) in June 2017. The weight of each male prawn was 2.8±0.5g and the weight of each female was 1.8±0.5g. All prawns were transferred to an aquarium, cultured in an inflated freshwater pool in an indoor facility, and fed parudina twice per day.

Tissue Expression Analysis by Quantitative Real-Time PCR
After one week of culture in the laboratory, the eyestalk, brain, heart, hepatopancreas, gill, muscle, ovary, and testis were dissected from mature prawns (n = 5). The development of embryos was divided into seven stages based on a study of Bai et al. [26] (from the unfertilized egg (UE) to the first day larvae after hatching (L1)). The samples were dissected separately, immediately frozen in liquid nitrogen, and stored at −80 • C until processed.
The procedures for RNA isolation and cDNA synthesis were as described previously [21]. The expression profiles of MniDMRT11E in different tissues were determined using qPCR assays (CWBIO, Beijing, China) [21,41]. The relative copy numbers of MniDMRT11E mRNA were calculated according to the 2 −∆∆CT comparative CT method [42]. Beta actin was constantly smooth expressed in different developmental stages of prawns. Bestkeeper analysis and similar methods were performed for expression levels of beta actin [43]. Differences in expression levels were considered significant at p < 0.05.

Expression Profiles of DMRT11E in Ovarian Cycle
The determination of ovarian stage was based on the color of the oocytes, according to the criteria stated in [28]: Stage I (transparent), Stage II (yellow), Stage III (light green), Stage IV (dark green), and Stage V (gray). Ovarian samples were treated in the same way as other tissue. Then, we used qPCR to detect the expression level.

In Situ Hybridization (ISH)
ISH was performed on 4µ-thick formalin fixed paraffin-embedded ovary and testis sections using the Zytofast PLUS CISH implementation kit, after they were embedded in paraffin, as described in [21]. The slides were examined under a light microscope. The anti-sense and sense probes of the chromogenic in-situ hybridization study were designed by Primer5 software based on the cDNA sequence of MniDMRT11E. Both anti-sense and sense probes were hybridized with the slide. The anti-sense probe (5 -GTCUUCGCGUAGGACUUCGGCGAGUAUUCUGGAGUG-3 ) was prepared for the experimental group, whereas the sense probe (5 -CACUCCAGAAUACUCGCC GAAGUCCUACGCGAAGAC-3 ) was prepared for the control group.

RNA Interference of DMRT11E
Deliberate dsDMRT11E synthesis and preservation were performed according to Li et al. [21]. The template for DMRT11E dsRNA synthesis was prepared by the amplification of testis cDNA with the primers DMRT-iF and DMRT-iR (Table 1). Eighty healthy mature female (stage I) prawns and 80 healthy mature male prawns were respectively assigned to two groups. The experimental group (n = 40) was injected with DMRT11E dsRNA (4 µg/g of body weight). DEPC water was injected at an equal dose based on gram body weight in the control group (n = 40). All tissues (androgenic gland, hepatopancreas, and gonad) from each group were randomly collected on the seventh day after the injection and dissected, frozen immediately in liquid nitrogen, and stored at −80 • C until analysis. VG and DMRT11E mRNA expression in female tissue and IAG and DMRT11E mRNA expression in male tissue were investigated to detect the interference efficiency by qPCR on the seventh day after the injection (n = 4).

Data Analysis
All data are expressed as means ± standard. Statistical differences were estimated by one-way ANOVA followed by LSD and Duncan's multiple range test in tissue distribution, embryo stages, and ovary cycle. A two-side t-test was used to compare expression levels in RNAi. All statistical analyses were performed using SPSS 20.0 (SPSS, Chicago, IL, USA).

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.