Introduction

Sex is the most universal biological phenomenon in nature, with significant differences in morphology, reproduction and behavior between the male and female sexes of almost all animals1. Gonadal differentiation is the gradual development of undifferentiated reproductive glands into sex-specific gonads during embryonic development, including formations of the testis and ovary2. Sex differences are formed as a result of gonadal differentiation, which directly influences anatomical, physiological, and behavioral differences between males and females. Crustaceans have a unique endocrine glandular tissue known as the androgenic gland (AG), which is widely believed to play a key role in the process of crustacean androgenization, including testis development, sperm maturation, and the formation of secondary sex characteristics3. A previous study suggested the existence of an endocrine axis of reproductive activity connecting the X organ-sinus gland (XO-SG), the AG, and the testis4, which may be subject to complex regulation by genes, neurodevelopment, hormones, and environmental factors, as well as other variables.

The oriental river prawn (Macrobrachium nipponense) is a species of freshwater prawn that belongs to the phylum Arthropods and the subclass Crustace5. This species has gained significant popularity in the aquaculture industry in China, Japan, South Korea, Vietnam, and Myanmar due to its short breeding cycle, delicious taste, low susceptibility to diseases, adaptability to various environments, and stable market price6. However, the ovaries of female M. nipponense periodically mature rapidly during the breeding season (June -October) when the water temperature exceeds 22 °C7,8. This leads to serious multigeneration recolonization in ponds, resulting in negative effects such as degradation of the germplasm, excessive density, and increased consumption of oxygen and feed. Previous studies have shown that in M. nipponense, the glands begin to develop at Post-larvae (PL10) developmental stages and sexual differentiation is complete at PL25 with the emergence of physiological males and females9,10. Therefore, research on sex control techniques for monosex culture could effectively solve this problem.

In 2000, 24-dehydrocholesterol reductase (DHCR24) was first identified in susceptible brain regions of Alzheimer’s disease (AD) patients11. In the post-lanosterol pathway of cholesterol synthesis, DHCR24 encodes an enzyme that catalyzes fibrillosterol, converting it to cholesterol12,13. DHCR24 is commonly regulated by steroids, dexamethasone, sex hormones, adrenocorticotropic hormone, thyroid hormones, neurotrophins, and exogenous organisms14,15. Initially, DHCR24 expression served as an indicator of selective AD because its down-regulation was closely associated with AD and other degenerative diseases, such as aging, diabetes related factors, hypoxia, oxidative stress, chronic inflammation, insufficiency of brain neurotrophic substances, and metabolic syndromes, etc16. Accumulating data showed that neurosteroid induced the expression of DHCR24, as well as the synthesis of cell cholesterol, in neurons and astrocytes. The distal promoter region of DHCR24 contains putative oestrogen receptor (ER) and androgen receptor (AR)-binding sites17. A study in mice found no sex difference in DHCR24 mRNA levels of the neonatal mouse cortex hippocampus, but its expression was increased by prenatal testosterone propionatetreatment in both sexes18.

Many studies reported the effects of feed additions of sex hormones such as testosterone, estradiol or their analogues on their phenotype19. For example, in Scylla paramamosain, appropriate addition of ARA to the feed promoted growth, improved ARA deposition and metabolism, increased sex steroid hormone and Vtg levels, and enhanced ovarian development in pre-developmental female mud crabs20. In insects, DHCR24 has two homologous genes which perform different functions, whileonly GbDHCR24-1 is the major enzyme promoting the conversion of bridging sterols to cholesterol in crickets21. However, the function of DHCR24 has not been studied in crustaceans. In evolutionary history, crustaceans diverged relatively early from other arthropods, and their lineage has distinctive features that differentiate them from vertebrates22,23.

In this study, we characterized the sequence features and phylogenetic relationships of the Mn-DHCR24 gene using bioinformatics methods. The expression of the Mn-DHCR24 gene in different tissues and developmental stages of embryos was determined by real-time PCR. The localization of Mn-DHCR24 mRNA in testis was detected using in situ hybridization. Moreover, we identified the role of the Mn-DHCR24 gene in the regulation of gonadal differentiation by knocking-down the Mn-DHCR24 gene using RNA interference (RNAi), and observing the changes of the sex ratio duing PL10 M. nipponense development. Furthermore, the content of 17α-methyltestosterone (MT) was measured by enzyme-linked immunosorbent assay (ELISA), and the development of gonads was observed in combination with tissue sections, confirming the role of the Mn-DHCR24 gene in gonadal development. Finally, we determined the regulatory role of the Mn-DHCR24 gene in relation to sex and hormone-related genes. However, this was a preliminary study conducted under experimental conditions. In practical farming, the complex polygenic nature of traits may involve factors such as temperature, water quality, diet and different developmental stages. In the future, our studies will integrate environmental factors and different developmental stages of M. nipponense to further reveal the role of Mn-DHCR24.

This study is the first to identify the function of the Mn-DHCR24 gene in crustaceans, and provided a solid foundation for understanding the mechanism of gonadal differentiation and development in M. nipponense, as well as for the development of monosex culture technology.

Results

Molecular cloning and structural analysis of the DHCR24 gene

As shown in Fig. 1, the full-length Mn-DHCR24 cDNA sequence was 1515 base pairs (bp). The open reading frame was 1515 bp, and encoded 504 amino acids (GenBank accession number: PP983041) Based on the amino acid sequence of the Mn-DHCR24 gene product shown in Fig. 2, the estimated protein molecular weight was 58.90 kDa and the isoelectric point was 6.186. The amino acid sequence of the Mn-DHCR24 gene product contained a common conserved structural domain, oxidoreductase (PDB). The other amino acid sequences showed 82.07% amino acid sequence homology with Mn-DHCR24. The highest similarity with Penaeus japonicus was 83.53%.

BLAST (http://blast.ncbi.nim.nih.gov/Blast.cgi) and phylogenetic analysis of Mn-DHCR24 from M. nipponense confirmed that it was related to crustaceans and vertebrates. As shown in Fig. 3, different colors represent different families, such as Procambarus clarkii, Camarhynchus parvulus, Carassius gibelio, Daphnia magna, Eriocheir sinensis, Penaeus vannamel, and Rhincodon typus. Mn-DHCR24 formed clusters with various crustaceans, including M. nipponense, P. vannamel, E. sinensis, P. clarkia, and other crustaceans, and then clustered with fleas such as D. magna and Daphnia pulicaria.

Fig. 1
figure 1

The nucleotide and deduced amino acid sequences of Mn-DHCR24. The numbers on the left of the sequence indicate the positions of nucleotides and amino acids. The start codon, ATG, and the stop codon, TGA, are marked with red, and the stop codon TGA in the amino acid sequence is represented by an asterisk (*).

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Fig. 2
figure 2

The alignment of amino acid sequences of the Mn-DHCR24 gene product from different species. The red boxes mark the single conserved domain sites of Macrobrachium nipponense (PP983041), Artemia franciscana (XP_065566811.1), Cherax quadricarinatus (XP_053640235.1), Daphnia galeata (CAH0099193.1), Eriocheir sinensis (XP_050690880.1), Penaeus indicus (XP_063609519.1), Penaeus japonicus (XP_042859734.1), Penaeus vannamei (XP_027224655.1), and Portunus trituberculatus (XP_045128680.1).

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Fig. 3
figure 3

Phylogenetic tree of Mn-DHCR24 sequences. The diagram was generated by the neighbor-joining method using the MEGA 7.0 program, with 1000 bootstrapping replications.

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Expression of the Mn-DHCR24 gene in different tissues in males

Mn-DHCR24 was expressed in the eyestalk, cerebral ganglion, heart, hepatopancreas, gill, testis, and androgenic gland as shown in Fig. 4, but was only minimally expressed in muscle. The highest expression level was observed in the testis, significantly surpassing other tissues (P < 0.05). Heart and cerebral ganglion showed moderate expressions, while the eyestalk, hepatopancreas, gill, and androgenic gland exhibited lower levels of expression.

Fig. 4
figure 4

Expression pattern of the Mn-DHCR24 gene in different tissues. E: eyestalk, Cg: cerebral ganglion, H: heart, He: hepatopancreas, G: gill, M: muscle, T: testis, and Ag: androgenic gland. Data are presented as the mean ± SEM (n = 6). Different letters indicate significant differences (P < 0.05).

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Expression of the Mn-DHCR24 gene during different developmental stages

Figure 5 shows the expression patterns of Mn-DHCR24 during different developmental stages. The expression of Mn-DHCR24 at all stages of embryonic development, and gradually increased during embryonic development, reaching a maximum of 124.83 (P < 0.05) at the critical period of metamorphosis (L15), followed by a rapid decline. Among the later stages, the highest expression was 126.20 observed at PL25 (P < 0.05), followed by 46.39 (PL5) and 46.36 (PL10). The expression was relatively low during the before-larvae, with a maximum of only 8.94 (ZS).

Fig. 5
figure 5

The expression levels of Mn-DHCR24 during embryonic development stages. CS = cleavage stage; BS = blastula stage; GS = gastrula stage; NS = nauplius stage; ZS = zoea stage; L1 = the first day after hatching; PL1 = the first day post-larvae after larvae, and so on. Data are shown as the mean ± SEM (n = 6). Different letters indicate significant differences (P < 0.05).

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Localization of the Mn-DHCR24 gene in testis

In situ hybridization was widely used in molecular biology studies to determine the location of expression of specific mRNAs in tissues. In this study, we used this technique to locate the expression of Mn-DHCR24 mRNA in the testis (Fig. 6). Strong signals were observed in both spermatogonium and spermatocyte cells. However, no signals were observed in sperm cells, and no signal was observed when using negative RNA probe.

Fig. 6
figure 6

In situ hybridization analysis in testis of M. nipponense. SG: spermatogonium; SC: spermatocyte; S: sperm, andCT: collecting tissues. Scale bars = 20 μm.

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Effects on PL10 M. nipponense of Mn-DCHR knockdown by RNAi

Sex ratio

One hundred PL10 M. nipponense were injected with 8 µg/g using dsMn-DHCR24. There were three replicates in each group. The sex ratio was counted after the third injection (day 15). As shown in Table 1, the number of prawns in both experimental and control groups decreased over time, but in similar ratios. The sex ratios for the three periods in the RNAi group were 1.01, 1.07, and 1.04, respectively, which were not significantly different from those in the control group (P > 0.05).

Table 1 The sex ratio on days 15, 22, and 30 after the injection of PL10 M. Nipponense.
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Steroid hormone content

The gonads of M. nipponense began to develop at PL10, and sexual differentiation was completed at PL25 with the emergence of physiological males and females. Therefore, results were only counted for 22 and 30 days in Fig. 7A, B. Figure 7A shows the MT content in male and Fig. 7B shows the 17β-estradiol (E2) content in females. The MT content of male M. nipponense decreased over time and was always lower than the control group (P < 0.05). The E2 content of female M. nipponense was similar with the control group at 22 days, however, at 30 days, the content was significantly lower (P < 0.05).

Fig. 7
figure 7

Sex hormone content in males and females after five injections of PL10 M. nipponense. (A). Content of 17α-methyltestosterone (MT) in males; (B). Content of 17β-estradiol(E2) in females. Data are presented as the mean ± SEM (n = 6); * Significance at P < 0.05.

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Effects on PL30 male M. nipponense using Mn-DCHR24 knockdown by RNAi

Histological observations of gonads

At 1 day after injection, the testes of male prawns in the control and Mn-DHCR24 groups were at the spermatid phase, with primary spermatocytes and secondary spermatids in the testis (Fig. 8A), while a few sperm were observed in Mn-DHCR24 group (Fig. 8D). At 15 days after injection, a large number of sperm appeared in the testis of the experimental group (Fig. 8E), but not in the control group (Fig. 8B). At 30 days after injection, there were still small numbers of primary and secondary spermatocytes, as well as some sperm in the control group (Fig. 8C). However, the testis of the Mn-DHCR24 group was more mature andfilled with sperm (Fig. 8F), with almost no spermatocytes.

Fig. 8
figure 8

Histological sections of testis from the control and dsMn-DHCR24 groups after five injections. (A)(D). Histological sections of testis on day 1; (B)(E). Histological sections of testis on day 15; (C)(F). Histological sections of testis on day 30. SC: spermatocyte; SC1: primary spermatocyte; and SC2: second spermatocyte; S: sperm. Scale bars: 200 μm and 20 μm.

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Steroid hormone content

After the Mn-DHCR24 gen was silenced, the MT content of the experimental groups were all significantly higher than the control group, and reached a maximum of 632.58 ± 36.07 pb/g on day 15, when compared to only 338.73 ± 34.77 pb/g in the control group (P<0.05). The MT content gradually increased with the time of interference, reaching a maximum on day 15, after which there was a decreasing trend, finally reaching 589.28 ± 26.36 pb/g on day 30 (Fig. 9).

Fig. 9
figure 9

Content of 17α-methyltestosterone (MT) in the testis after five injections of male M. nipponense. Data are presented as the mean ± SEM (n = 6); * Significance at P < 0.05.

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RNA interference efficiency

The real-time PCR analysis showed that the expression of Mn-DCHR24 in the control group increased on days 1, 8 and 15, and gradually decreased on days 22 and 30 (Fig. 10). The expression of Mn-DCHR24 in the RNAi group was always lower than that in the control group during the whole experiment (P < 0.05), and the interference efficiencies were 46.70%, 82.51%, 73.70%, 66.02% and 76.23% on day 1, 8, 15, 22 and 30, respectively.

Fig. 10
figure 10

Expression levels of Mn-DHCR24 in testis after injection with dsMn-DHCR24. Data are shown as the mean ± SEM (n = 6); * Significance at P < 0.05.

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Male related and hormone related genes expression

Figure 11 shows the expressions of male related and hormone related genes in male M. nipponense at different stages after 30 days of different treatments (Control, RNAi, and E2). As shown in Fig. 11A, B, hormone-related genes Mn-CHH and Mn-GIH had similar expression patterns, the expression of the RNAi group were significantly lower than the control group, while the E2 group was higher (P < 0.05). It is noteworthy that E2 affected the expressions of Mn-IAG and Mn-SG only on day 1, and was not significantly different from the control group thereafter (P > 0.05), as shown in Fig. 11C, D. The expression levels of Mn-IAG and Mn-SG after dsMn-DHCR24 treatment decreased and then increased. The expressions of both Mn-LIPA and Mn-DHCR24 after dsMn-DHCR24 treatment gradually decreased, whereas E2 treatment resulted in negative feedback of each other, as shown in Fig. 11E, F.

Fig. 11
figure 11

Expression levels of sex-related genes in testis after treatment with control, dsMn-DHCR24, and 17β-estradiol. (A) Mn-CHH; (B) Mn-GIH; (C) Mn-IAG; (D) Mn-SG; (E) Mn-LIPA; and (F) Mn-DHCR24. Data are shown as the mean ± SEM (n = 6); * Significance at P < 0.05.

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Discussion

Sterol compounds including cholesterol are very important for almostall animals because of their use as components of cellular membranes and precursors of steroid hormones. In the DHCR24 silencing neuronal cell model, it was found that cellular desmosterol level is significantly elevated, and cellular cholesterol content is greatly decreased, suggesting the remarkable effects of DHCR24 expression on cholesterol homeostasis24. In the cholesterol biosynthesis of vertebrates, two pathways, which are known as the Bloch pathway and the Kandutsch-Russell pathway, are involved25. In both pathways, an enzyme called 24-dehydrocholesterol reductase (DHCR24) reduces ∆24 in sterols. Two homologs of DHCR24 (DHCR24-1 and DHCR24-2) have been reported in phytophagous insects26, and it was found that DHCR24-1 contributed to conversion of phytosterols to cholesterol, mainly in the midgut of the phytophagous lepidopteran larvae. However, there is only one homolog in other animals, such as humans and mice27. Another study showed that DHCR24 regulates the rate of cholesterol synthesis and cholesterol homeostasis through the Bloch pathway and/or the K-R pathway, and that knockout results in a significant reduction of plasma membrane and intracellular cholesterol28. In the present, only one DHCR24 homolog was found in M. nipponense, We successfully cloned the full-length Mn-DHCR24 cDNA from M. nipponense (Fig. 1), which exhibited one typical conserved domain of oxidoreductase (PDB). An alignment analysis showed that the amino acid similarities between Mn-DHCR24 and other crustacean DHCR24 were greater than 50% (Fig. 2). Phylogenetic analyses showed that crustaceans and vertebrates were clearly delineated and clustered together (Fig. 3), indicating that Mn-DHCR24diverged from vertebrates during crustacean evolution, and was more conserved within the same class, which may have led to changes in function.

To the best of our knowledge, The function of Mn-DHCR24 has not been well defined or analyzed in crustaceans, so this is the first real-time PCR analysis of Mn-DHCR24 expression. We performed real-time PCR analysis of Mn-DHCR24 in various tissues and at various developmental stages of M. nipponense. In Bombyx mori, it is expressed highest in the intestine and fat body, and also in the brain and gonads26. However, tissue-specific analyses showed that Mn-DHCR24 was observed in all tissues of M. nipponense, with the highest expression in the testis, followed by the heart and cerebral ganglion, and the least in muscle (Fig. 4). Cholesterol synthesis induces many complex direct or indirect physiological changes. In a mouse study, DHCR24 was involved in cell invasion and migration by modulating cholesterol biosynthesis and lipid raft formation. We suggested that Mn-DHCR24 may play a potential role in sex differentiation and gonadal development by controlling steroid synthesis, thus affecting the endocrine axis of male reproductive activity, which connect the XO-SG, AG, and testes in crustaceans. Ecdysteroids are synthesized from dietary cholesterol to inactive precursors by the Y-organ (YO) in crustaceans and the prothoracic gland (PG) in insects29. A study that compared the effects of different lipids on ovarian development in Macrobrachium Rosenbergii detected sex hormones specific to mammals, such as E2 and progesterone, in the ovaries of female prawns, and found that they promote the synthesis of serum steroid hormones and lipid metabolism, and affect ovarian development and overall health30. The results of different developmental periods showed the highest expression at L15 (day 15 after hatching) and PL25, which are critical periods before metamorphosis and gonadal differentiation31, indicating that Mn-DHCR24 was a crucial participant in specific physiological activities such as metamorphosis and gonadal differentiation in M. nipponense (Fig. 5). In the present study, strong signaling was detected in the testis, suggesting that Mn-DHCR24 was involved in steroid synthesis in this organ, which in turn promoted sperm maturation and testis development in M. nipponense (Fig. 6). This is the first report of the role of DHCR24 in sex differentiation and gonadal development in crustaceans.

The gonads of M. nipponense began to develop at PL10 and sexual differentiation was complete by PL25, with distinctive secondary sexual characteristics enabling the identification of males and females. We selected PL10 and PL30 (male) and used RNAi technology to determine whether Mn-DHCR24 was involved in sex differentiation and gonadal development, respectively, in M. nipponens. At 30 days after dsMn-DHCR24 injection, the sex ratio of PL10 M. nipponense in the experimental group remained constant at 1:1 (PL10), which did not significantly change, when compared to the control group (Table 1). We collected both male and female prawns from the experimental group according to the previous method used to differentiate the sex of M. nipponense, which was measured using an ELISA. Importantly, both MT production by male and E2 production by female M. nipponense were reduced in the experimental group (Fig. 7). dsMn-DHCR24 effectively promoted sperm development and maturation in RNAi experiments on PL30 male M. nipponense (Fig. 8). This result was confirmed by the results of an ELISA assay in the testis, which showed a higher MT content in the testis of male M. nipponense in the experimental group, when compared to the control group (Fig. 9). The results shown in Fig. 10 indicated that dsRNA effectively inhibited the expression of Mn-DHCR24.

Of the two main sex determination systems, genetic sex determination and environmental sex determination, the former is controlled by genetic factors (e.g., sex chromosomes), while the latter is driven by environmental cues (e.g., temperature and photoperiod)32. Crustaceansare lowly aquatic animals whose sex determination system is usually controlled by factors such as multiple genes or the environment33,34. Sex determination and sexual differentiation processes in crustaceans generally occur in a cell-autonomous manner, contrasting with the cell non-autonomous regulation found in most mammals and other vertebrate classes35. Malacostraca crustaceans, including decapods, exhibit a cell non-autonomous mode of sexual development that differs from the gonad-dependent endocrine regulation observed in vertebrates36. Our study showed that Mn-DHCR24 was not the master gene controlling sex differentiation in M. nipponense, but it was involved in the synthesis of MT and E2.

Sexual differentiation in crustaceans involves a complex regulatory mechanism, and male sexual differentiation and secondary sexual characteristics were thought to be primarily influenced by insulin-like androgenic gland factor (IAG)37,38, which is synthesized and secreted by the AG39,40, an endocrine gland unique to males. Like the vertebrate hypothalamic/neurohypophyseal system, the crustacean XO–SG releases neurohormones from axons into the circulation to exert responses at distant target tissues41. To further investigate the regulatory relationships of Mn-DHCR24 on sex-related genes and hormone-related genes, male M. nipponense, it was fed with E2 at a concentration of 200 mg/kg. Crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) belong to the crustacean hyperglycemic hormone superfamily of neurohormones, which are stored and released by sinus glands (SG), and have been shown to play important roles in crustacean reproduction42, molting43 and metabolism44. In the present, RNAi results showed that Mn-DHCR24 and E2 positively regulated the expression of both CHH and GIH (Fig. 11A-B). This cross-bioactivity is reflected in other crustaceans. Crustacean female sex hormone may suppress IAG expression in male Callinectes sapidus and there may be putative regulatory interactions between IAG, and steroidogenic genes during testosterone synthesis45.

Sperm gelatinase (SG) is a key player in regulating various reproductive functions, including acrosome reaction46,47, and sperm-egg fusion48, and it also responds to sperm maturation and testis development. Previous studies have reported that exogenous hormone-induced changes in the gonads led to changes in their own synthesis and secretion of sex hormones20. The results in Fig. 11C-D showed that IAG and SG had similar expression patterns after treatment with dsMn-DHCR24 and E2. The long-term interference of GsiRNA was found to inhibit the development of male Procambarus clarkii external sexual characteristics during the juvenile stage, and delayed it during the adult stage49. Moreover, male Cancer borealis were shown to have an endocrine axis between the androgen glands and the eyestalk ganglia, and the expression of IAG became significantly higher during the breeding season50. Mn-LIPA has previously been shown to have a similar role in gonadal development in M. nipponense with Mn-DHCR2451, and Mn-LIPA expression was reduced after Mn-DHCR24 was inhibited. However, the expression pattern of Mn-DHCR24 after E2 feeding was in contrast to Mn-LIPA, indicating that exogenous hormone-induced genetic changes at the level of the “steroid biosynthesis” pathway are complex, but our study was unable to reveal the reasons. However, it is reasonable to speculate that excessive sex hormone supplementation causes weight gain, which involves a variety of biological mechanisms, including immunity, growth, and so on.

In conclusion, we identified, cloned, and analyzed a single DHCR24 homologue (Mn-DHCR24) in M. nipponense. Mn-DHCR24 had the highest expression in the testis and played a role in metamorphosis and gonadal differentiation. RNAi experiments showed that Mn-DHCR24 was not a master gene for sex differentiation but had an important role in reproductive regulation, and affected sex hormone synthesis, sperm development, and complex regulatory interactions in crustaceans.

Materials and methods

Experimental prawns

Larvae were fed with Artemia until their body weight reached PL30 male M. nipponense (1.4351 ± 0.1621 g), and assigned randomly in the control and experimental groups. The injection was performed every 7 days, samples of testis were collected for tissue observation, steroid hormone, q-PCR and sex ratio after each injection. The experimental design is shown in Fig. 12.

Fig. 12
figure 12

Experimental design.

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Nucleotide sequence analysis

Total RNA from the various tissues of M. nipponense was extracted using RNAiso Plus Reagent (TaKaRa, Dalian, China), according to the manufacturer’s protocol. The quality of RNA were assessed by measuring their absorbance at 260 and 280 nm using NanoDrop One. The cDNA synthesis was then performed using random hexamers (Invitrogen, USA) and SuperScript® III reverse transcriptase following the manufacturer’s protocol. The completed cDNA solution was stored at – 20 ºC for further analysis. The full-length Mn-DHCR24 cDNA sequence was obtained from the testis transcriptome library of female M. nipponense (accession SAMN27687877-SAMN27687891) under Bioproject PRJNA830321.

Tissue expression analysis by real-time PCR

The Bio-Rad iCycler iQ5 Real-Time PCR system (Bio-Rad, Hercules, CA, United States) was used to detect the expression of the Mn-DHCR24 gene in the above samples52. The amplification was carried out in a 96-well plate using a 20 µl reaction volume. Each reaction contained 1 µl of cDNA from each tissue, 10 µl of Sensi FASTTM SYBR® No-ROX kit, 0.4 µl of each primer (10 µM/µl), and 8.2 µl of sterile ddH2O. The following steps were followed in the thermal real-time PCR cycling profile according to the manufacturer’s protocol: firstly, one 2-minute enzyme activation cycle at 95 °C, followed by 40 cycles of 5-second denaturation at 95 °C and 40 cycles of 20-second annealing/extension at 60 °C. Sterile distilled water (ddH2O) was used as the negative control instead of the template. The specific primers are listed in Table 2. MnEIF was selected as the reference gene for real-time PCR53. The real-time PCR expression level was calculated by the 2−ΔΔCT method54, and MnEIF used as the reference gene.

Table 2 Primers for cDNA clone, real-time PCR analysis, and RNAi that were involved in this study.
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In situ hybridization (ISH) and histological observations

The testis and of mature male M. nipponense were collected and fixed in 4% paraformaldehyde in PBS (pH 7.4) at 4 ◦C overnight. Following the method of Li et al.55, we used the Zytofast PLUS CISH imple mentation kit and embedded the samples in paraffin. Primers were designed using Primer5 software based on the Mn-DHCR24 cDNA sequence. The sequence 5′-CATTCTCTAACCTGCTT CTGGACTTCCTTCACTC-3′ was used as the ISH probe. The slides were examined under a light microscope.

Hematoxylin and eosin (HE) staining was used in order to study the histological changes in the testes and ovaries. On days 1, 15, and 30 after injection, testis samples from the control and Mn-DHCR24 groups were mounted on slides, which staining with Hematoxylin, eosin staining followed by cleaning with different concentrations of alcohol56. Observations took place using a stereo microscope (SZX16; Olympus Corporation, Tokyo, Japan). Comparative labeling was performed with various cell types based on cell morphology57.

Sex ratio statistics

In this study, prawns in both control and experimental groups (PL10) were randomly selected to ensure that the number of individuals in each group exceeded 90 for statistical analysis.

RNA interference of Mn-DHCR24

Primers for ds-MnDHCR24 were designed using the online software Snap Dragon. The T7 promoter sequence was added to the 5’ end of the RNAi primer sequence. T7: TAATACGACTCACTATAGGG. The target gene fragments were amplified using the RNAi primers. The integrity of dsRNA was verified by agarose gel electrophoresis. Afterwards, the concentration of dsRNA was measured using a BioPhotometer (Eppendorf, Hamburg, Germany). Finally, dsRNA was stored at – 80 °C for long-term storage.

Enzyme linked Immunosorbent Assay (ELISA)

The content of MT and E2 in prawn tissues was detected by ELISA after RNAi. The Sex hormone content in prawns was determined using double-antibody one-step sandwich ELISA following the instructions of the Shrimp EH ELISA Kit instruction (Lot number: 20230724-YJ923014 and 20230724-YJ950014; Mlbio Shanghai). Prawn tissues were taken and centrifuged and the supernatant was collected. Afterwards, samples were added to the ELISA plate, incubated and washed. Then enzyme-labelled detection antibody was added and the reaction was stopped by adding substrate solution. Finally, the absorbance (OD value) was measured, and the concentrations of MT and E2 in the samples were calculated according to the standard curve to analyse the effect of RNAi on their contents.

Dietary preparation

The diets used in this study came from the commercial prawn diet produced by Freshwater Fisheries Research Center, Chinese Academy of Fisheries Sciences, China (Wuxi, China). The method of incorporating hormones into feeds was as follows: firstly, the hormones were dissolved in 95% ethanol and diluted to form a stock solution at a concentration of 20 mg/ml; later, the prepared hormone stock solution was uniformly sprayed on the commercial diets at the rate of 1 ml of ethanol per 10 g of diet. Finally, the diet was blown dry at low temperature (0 °C) to allow the alcohol to evaporate naturally. The MT (CAS number: 58-18-4, purity: 97.65%) and E2 (CAS number: 50-28-2, purity: 95.88%) were purchased from Beijing Solarbio Technology Co, Ltd. (Beijing, China).

Data analysis

The statistical analyses were all conducted using IBM SPSS Statistics for Windows, version 23.0. (IBM Corporation, Armonk, NY, USA). We calculated the relative expression of genes using the 2–ΔΔCt method. The significant differences between groups were determined by one-way analysis of variance and two-tailed t-tests. In addition, to determine if the effect was linear and/or quadratic, a follow-up trend analysis using orthogonal polynomial contrasts was performed60. Real-time PCR data are expressed as mean ± standard error (SEM). Probability (P) values < 0.05 were considered statistically significant.