Single-cell atlas of prawn gonads identifies NLRP2 regulated mitochondrial dysfunction in estrogen induced sex reversal

Single-cell atlas of prawn gonads identifies NLRP2 regulated mitochondrial dysfunction in estrogen induced sex reversal | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Single-cell atlas of prawn gonads identifies NLRP2 regulated mitochondrial dysfunction in estrogen induced sex reversal Hongtuo Fu, pengfei cai, Zijian Gao, sufei Jiang, Wenyi Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8343381/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Sex reversal is widely used in biological breeding, but its cellular and molecular consequences in crustaceans are poorly understood. Here, we combine estrogen induced feminization, single cell RNA sequencing, histology and RNA interference to dissect gonadal development in the oriental river prawn ( Macrobrachium nipponense ). Feeding post-larval males with 17β-estradiol generated neo-females with ovary like gonads. Single cell profiling of testis, ovaries and neo-female gonads produced a gonadal atlas in which, using newly validated markers, we resolved major oogenic and spermatogenic cell types. Cell-type composition and pseudotime analyses showed that neo-female gonads are dominated by early germ cells, whereas mature oocytes and spermatozoa are strongly depleted; histology confirmed this developmental arrest. Stage resolved differential expression and KEGG enrichment consistently highlighted oxidative phosphorylation as the most perturbed pathway across key germ-cell stages. Among the affected genes, MnNLRP2 displayed primary spermatocyte specific and testis enriched expression and was conserved among decapods. RNA interference of MnNLRP2 in PL10 prawns shifted phenotypic sex ratios toward females and altered sex steroid levels, reducing MT in males while elevating E 2 in females; knockdown in adults caused spermatogenic arrest and down-regulation of oxidative phosphorylation components. These findings provide unprecedented insights into crustacean germ cell dynamics and the metabolic and transcriptional networks governing incomplete sex reversal, thereby offering critical resources for reproductive biology and aquaculture research. Biological sciences/Genetics/Animal breeding Biological sciences/Developmental biology/Germline development/Oogenesis sex reversal scRNA-seq OXPHOS mitochondrial dysfunction NLRP2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Sex reversal through endocrine manipulation represents a promising aquaculture strategy for mono-sex populations in economically important species, including crustaceans [1,2]. While 17β-estradiol (E 2 ) has been shown to induce neo-females (sex-reversed males) in decapods such as Macrobrachium nipponense , these individuals exhibit impaired growth and gonadal maturation compared to natural females [3]. The mechanisms underlying these developmental defects, particularly at the cellular and metabolic levels, remain poorly understood. Given the pivotal role of redox homeostasis and energy metabolism in gametogenesis, we hypothesized that E 2 induced sex reversal disrupts these processes, leading to functional sterility in neo-females [4,5]. Crustaceans lack a canonical sex-determining pathway, and the molecular basis of germ cell development during sexual differentiation is largely uncharted. Previous histological studies confirmed testis-ovary coexistence in neo-females but failed to resolve the heterogeneity of germ cells or identify metabolic drivers of defective maturation [3]. Single cell RNA Sequencing (scRNA-seq) is a high-throughput genomics technology that resolves gene expression heterogeneity among cells by resolving cellular diversity [6]. In vertebrates, this approach has uncovered both conserved and species-specific profiling of gene expression in innate and adaptive immunity [7]. Recent studies of teleost gonads exemplify its power for deconvoluting sexual plasticity: scRNA-seq in protogynous groupers ( Epinephelus coioides ) identified ovarian pre-follicle cells co-expressing female and male sex determination genes prior to sex reversal, revealing a transitional transcriptional ‘bivalent state’ [8]. However, the application of such high-resolution approaches to crustaceans remains in its infancy and there are few reports of scRNA-seq in lower crustaceans (e.g., crabs, shrimps), especially in sex-reversed species [9,10]. The application of single-cell technology to the study of sex differentiation and gonadal development in crustaceans still faces unique challenges. Firstly, marker scarcity: Unlike mammals where conserved germline markers (vasa, piwil1) and somatic identifiers ( sox9 , cyp19a1 ) exist, most crustacean cell types lack definitive markers [11,12]. Secondly, transcriptomic and functional differences between sex-reversed neo-individuals and natural individuals challenge traditional cell typing paradigms, such as Epinephelus coioides [13], Larimichthys crocea [14], Mauremys reevesii [15], Siniperca chuatsi [16], etc. Oxidative phosphorylation (OXPHOS) serves as the cornerstone of cellular energy metabolism during gametogenesis, where its fidelity directly dictates germ cell viability and reproductive competence [17]. In vertebrates, mitochondrial OXPHOS not only generates adenosine triphosphate (ATP) for meiotic divisions and motility but also fine-tunes redox homeostasis by modulating reactive oxygen species (ROS) flux. Dysregulation of this system—whether through suppressed complex I/III activity or electron transport chain (ETC) overloading—triggers pathogenic ROS accumulation, DNA damage, and apoptotic cascades that devastate oocyte quality and spermatogenic efficiency [18]. For instance, mammalian studies confirm that OXPHOS deficiency impairs oocyte maturation [19]. Crucially, steroid hormones like estrogen inherently modulate mitochondrial function, yet whether exogenous E 2 disrupts OXPHOS-mediated redox balance in crustacean germ cells, where anaerobic metabolism dominates remains unexplored. This gap is particularly salient in crustaceans, which lack conserved sex-determining genes and exhibit unique metabolic adaptations. The differences between crustaceans and vertebrates have raised questions about the role of OXPHOS in germ cell development. In this study, we construct a scRNA-seq c atlas of M. nipponense gonads, define a marker framework for eight major germ-cell types, and uncover pronounced alterations of oxidative phosphorylation–related pathways in estrogen-induced neo-females. Using lineage-resolved scRNA-seq, we show that E 2 reshapes germ-cell composition and transcriptional trajectories and consistently perturbs oxidative phosphorylation across both oogenic and spermatogenic lineages. We further identify MnNLRP2 as a previously uncharacterized, spermatocyte enriched regulator whose knockdown disrupts testicular development and the expression of multiple oxidative-phosphorylation components. Together, these findings support a model in which estrogen induced sex reversal in M. nipponense is accompanied by mitochondrial dysfunction and germ-cell maturation failure, providing a mechanistic entry point to understand the redox and metabolic control of sexual plasticity in crustaceans. 2. Materials and Methods 2.1. Experimental Animals and Dietary Preparation The diets employed in this study were obtained from a commercial diet for shrimp produced by the Freshwater Fisheries Research Centre of the Chinese Academy of Fisheries Sciences (Wuxi). The composition of the commercial diets was as follows: crude protein, fish meal, shrimp meal, squid meal, starch, soybean meal, ash, rapeseed meal, soybean protein concentrate, and crude fat. E 2 (CAS number. 50-28-2, purity: 95.88%) was procured from Beijing Solarbio Technology Co, Ltd. (Beijing, China). The method of adding the hormone to the diet was outlined as follows: The hormone was dissolved in 95% ethanol to prepare a solution with a concentration of 20 mg/ml. The stock solution was then applied in a uniform manner to the feed (1 ml ethanol per 10 g diet) and agitated with a glass rod for a minimum of three minutes. Subsequently, the objects were placed in a ventilated laboratory hood and left in the shade for 15 minutes. The treated diets were transferred to 15 ml test tubes and stored in a refrigerator at 0°C to facilitate the natural evaporation of the residual alcohol. Preparation of feeds with an E 2 concentration of 200 mg/g. Previous studies have demonstrated that this concentration can effectively induce sex reversal in M. nipponense [20]: To ensure methodological rigor and reproducibility, the experimental and statistical protocols for obtaining neo-females were implemented as follows: Prior to the experiment, 100 male and 100 female PL30 (PL: post-larvae developmental stage) were selected and maintained in recirculating aquaculture systems under controlled conditions (water temperature: 22–24°C; pH: 7.5–8.0; salinity: 0.0‰). Prawns were fed daily at 5% body weight for 60 days, with the experimental group (males) receiving E 2 treated feed and the control group (females) receiving ethanol treated feed without E 2 supplementation. Body weight and length (mean ± SEM) were recorded every 15 days throughout the trial period. At the end of the experiment, neo-females (sex-reversed males) were identified in the experimental group based on specific morphological/gonadal criteria, with their quantity systematically recorded. 2.2. Sampling and scRNA sequencing At the end of the culture experiment, two adult females, two adult males and two neo-females were randomly sampled from both the control and treatment groups, and their gonads were collected. Suspension was prepared following the 10x Genomics User Guide ( https://support10xgenomicscom/single-cell-gene-expression/index/doc/user-guide-chromium-single-cell-3-reagent-kits-user-guide-v31-chemistry-dual-index ). Cellular suspensions were then loaded on a 10X Genomics GemCode Single-cell instrument to generate single-cell GelBead-In-EMlusion (GEMs). Using Chromium Next GEM Single Cell3’ Reagent Kits (v3.1), full-length cDNAs with barcode and Unique Molecular Identifier (UMI) were generated. The cDNAs were then sequenced on an Illumina NovaSeq 6000 platform [21]. 2.3. Data quality control The cell by gene matrices for each sample were individually imported to Seurat (v3.1.1) [22] for the downstream analysis. Raw data were processed using Cell Ranger (v7.1.0) [23] by aligning to the M. nipponense genome assembly (ASM1510439v2) [24]. Cells with > 8000 UMIs or > 10% mitochondrial reads were filtered out. During this process, we also excluded cells with less than 500 or more than 4000 genes detected. In addition, Doublet Finder (v2.0.3) [25] was used to filter out the doublet GEMs. 2.4. Cell clusting The integrated expression matrix was scaled and subjected to dimensionality reduction. To assess the significance of principal components (PCs), we performed a resampling test inspired by the jackStraw procedure. Subsequently, Uniform Manifold Approximation and Projection (UMAP) was applied to these PCs for two-dimensional visualization [26], using the parameters ‘n.neighbors = 50’, ‘min.dist = 0.2’, and ‘n.components = 2’. Finally, clustering analysis was conducted with a resolution parameter of 0.5 to identify distinct groups within the data. 2.5. Cell types identification The initial characterization of cell types within each cluster was conducted using the SingleR package [27] as a preliminary reference. Subsequently, differential expression analysis was primarily executed using the Seurat Find Markers function. This allowed us to visualize cluster-specific marker genes and generate a heatmap featuring the marker genes for each cluster. Cell types were ascertained based on the marker gene expression profiles. Representative makers were verified through in situ hybridization and qRT-PCR. 2.6. In situ hybridization Gonad samples for in situ hybridization were fixed in 4% paraformaldehyde (prepared with DEPC-treated water). Anti-sense and sense probes for chromogenic in situ hybridization (CISH) were designed using Primer5 software based on the cDNA sequence of maker genes and synthesized by Shanghai Sangon Biotech Company [28]. The DIG-labeled anti-sense probe served as the experimental probe, while the sense probe was used as the negative control. CISH was performed on 4-µm-thick formalin-fixed, paraffin-embedded sections using the Zytofast PLUS CISH Implementation Kit (ZytoVision GmbH, Germany). Mouse anti-DIG antibody (ZytoVision GmbH, Germany) was applied and incubated. After three 1-min TBS washes, slides were incubated with anti-mouse-HRP polymer (30 min, RT). Signal was developed using 3,3’-diaminobenzidine (DAB; 50 µL/slide, 10 min, RT) prepared according to the Zytofast PLUS CISH protocol. Counterstaining was performed with hematoxylin, followed by dehydration through graded alcohols. Slides were air-dried, mounted with DPX, and examined under a light microscope. 2.7. Quantitative Real-Time PCR To examine the expression of marker genes in testis and ovaries, qRT-PCR was performed. Total RNA was extracted from 100 mg of gonadal tissue using 1 mL TRIzol reagent (TaKaRa, Japan), and first-strand cDNA was synthesized with the Reverse Transcriptase MMLV Kit (TaKaRa). Gene-specific primers were used for amplification in a Bio-Rad iCycler iQ5 real-time PCR system (Hercules, CA, USA). Eukaryotic translation initiation factor 5A ( EIF ) served as the reference gene due to its stable expression across various conditions [29]. The PCR protocol consisted of 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min, followed by a final elongation step at 72°C for 10 min. Each sample was run in quadruplicate, along with three negative controls: nuclease-free water, primer-free water, and template-free water. Fluorescence data were automatically recorded, and dissociation curves were analyzed post-amplification to confirm specificity. Relative mRNA expression levels were quantified using the 2 −ΔΔCT method [30]. 2.8. Different expression analysis Expression value of each gene in given cluster was compared against the rest of cells using Wilcoxon rank sum test [31]. Significant upregulated genes were identified using a number of criteria. First, genes had to be at least 1.28-fold overexpressed in the target cluster. Second, genes had to be expressed in more than 25% of the cells belonging to the target cluster. Third, p value is less than 0.05. Based on the hypergeometric distribution, and a threshold of p-value < 0.05, the func-tional annotation and classification of DEGs were conducted with the Gene Ontology (GO) database [32] and KEGG database [33] and via the analysis of DEGs enrichment metabolic pathways. 2.9. RNA Interference To knockdown NLRP2 expression, target-specific primers flanked by T7 promoter sequences were designed using the SnapDragon online tool ( http://www.flyrnai.org ). Double-stranded RNA (dsRNA) targeting NLRP2 was then synthesized in vitro using the TranscriptAid™ T7 High Yield Transcription Kit (Fermentas, USA) following the manufacturer’s instructions. For the RNA interference assay, both long-term (PL10 prawns) and short-term (adult male prawns) interference experiments were conducted. (1) A total of 100 healthy PL10 prawns were selected and injected with dsMnNLRP2 at a dosage of 8 µg/g. Injections were administered once every 7 days, and sex ratios as well as steroid hormone levels were measured on days 15, 22, and 30 post-initial injection. (2) A total of 60 healthy adult male prawns were used for the short-term RNAi experiment. Prawns were injected with dsMnNLRP2 following the same procedure and dissected 7 days post-injection to collect androgenic gland for total RNA extraction, in order to assess knockdown efficiency and the expression of OXPHOS-related genes. 2.10. Enzyme linked Immunosorbent Assay (ELISA) The concentrations of MT and E 2 after RNAi treatment were quantified using a double-antibody, one-step sandwich ELISA following the manufacturer’s instructions (Shrimp EH ELISA Kits, Lot: 20230724-YJ923014 and 20230724-YJ950014; Mlbio, Shanghai). Prawn tissues were homogenized, centrifuged, and the supernatant was collected for analysis. Samples were loaded onto ELISA plates, incubated, washed, and subsequently treated with enzyme-labeled detection antibodies and substrate solution. Absorbance (OD) was recorded, and hormone concentrations were calculated based on standard curves. 2.11 Statistical analysis All morphometric data were analyzed using one-way ANOVA followed by Tukey's post-hoc test in SPSS 26.0 (IBM). The assumption of homogeneity of variances was verified using Levene's test, and normality was confirmed by Shapiro-Wilk test. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Agricultural University (Approval No. XYZ-2023-0012). 3. Results 3.1. Produce and single-cell RNA sequencing of neo-female Sex is the most common biological phenomenon in nature, and there are significant differences in morphology, reproduction and behaviour between the male and female sexes of almost all animals. The M. nipponense , with its high male-female differentiation and rapid reproduction rate [5], represents an ideal material for studying the mechanism of sex differentiation in crustaceans. Previous studies have shown that in oriental river prawns, the glands begin to develop at PL10 and sexual differentiation is complete at PL25 with the emergence of physiological males and females (Fig. 1 A). To study the mechanisms of sex differentiation in crustaceans, the 10× Chromium system was used to perform single-cell sequencing using whole. Figure 1 C shows the comparison between neo-female and normal male and female prawn. The average weight of male is 1.92 ± 0.55 g, and female is 1.33 ± 0.20 g, while neo-female is 0.93 ± 0.11 g (Table S1), which is much lower than normal male and female prawn (P < 0.05). In length (eye to caudal segment), male is 4.55 ± 0.21 cm and female 3.58 ± 0.10 cm, while neo-female is only 2.51 ± 0.22 cm (P < 0.05). Entire gonads were dissected, dissociated to single cells, and processed through the 10× chromium system (Fig. 1 D). Using cellranger, quality control of sequencing quality was performed by removing reads with low sequencing quality and performing preliminary counts of the number of reads measured and sequencing quality for each sample, which resulted in the Q30 of each sample being above 95%. After quality filtering, a total of 32,206 cells were sequenced, of which 4,867 and 5,061 are in the male group, accounting for 31.25% of all the sequenced cells, 6,166 and 3,879 are in the female group, accounting for 30.83% of the sequenced cells, and 6,105 and 6,110 are in the neo-female group, accounting for 37.93% of the sequenced cells. Figure 1 D-a shows the visualisation of the results after cell filtration, overall, the sample saturation in the female, male and neo-female groups are at 98.1%, 89.8% and 51.5%, respectively, which ensured the accuracy of the subsequent analyses. Gonadal cells were clustered based on transcriptional similarity. Clustering identified 25 transcriptionally distinct cell clusters, covering the three types of samples isolated (Fig. 1 D-b). Among the different cell clusters, cluster 0 contained the largest number of cells, accounting for 18.57% of the total number of sequenced cells (Table S2). More than 40% of the cells were contained in the largest 3 cell clusters, clusters 0,1 and 2, while 10 (clusters 16–25) of the 25 cell clusters sequenced accounted for less than 1% of the total number of cells. We assessed the proportion of cells in each group (female, male and neo-female) that could be determined with respect to changes in cells during sex differentiation. In the female group, the cell content of clusters 0, 1, 2, and 7 was greater than 5% in all clusters, implying that these clusters consisted predominantly of female cells. And in the male group, the cell content of clusters 3, 4, 6, 10 and 11 are all greater than 5%, which means that these cell groups are mainly composed of male cells. The rest of the cell clusters all have less than 5% of cells. It is noteworthy that in the neo-female group, clusters 0, 1, 2, 3, 5, 6, 10, and 12 were all with cell contents higher than 5%, and clusters 0, 1, 2, 3, 6 and 10 overlapped with females and males, respectively, implying that neo-females are likely to have communal germinal cells and to be involved in the process of sex differentiation. In contrast, the percentage of cells in cluster 25 was less than 1% in all three groups. 3.2. Identification of eight major germ cell types in M. nipponense We next aimed to identify crustacean germ cell specific marker genes based on the 25 clusters we had already found. We screened out some low-quality gene clusters and further mapped female and male germ cell types by comparing cell type-specific marker genes in dot plots, selecting stage-specific enriched genes (Fig. 2 A, B). To distinguish the major germ cell types in the 25 different clusters, we performed differential gene expression analyses. A columned graph showing the highest differentially expressed genes was generated (Fig. 2 C, E). Based on the quantitative results, genes with large expression differences were selected, and designed probes based on the gene sequences, which were analysed by multicolour observation through microscope after reacting with nucleic acids in the sperm and the ovary (Fig. 2 D, F). Primer sequences and probe sequences of all genes are given in Table S3. 3.2.1. Oogonium cells (cluster 7) Cluster 7 represents oogonium, which the source of a renewing stem germ cell population in the ovary. It is enriched for genes like GABARAP . We performed qPCR analyses of stage I ovary (O1) and stage II ovary (O2) determined that the relative expression of GABARAP in the ovary was 2439.58 ± 584.48 and 4470.63 ± 219.66, whereas it was not expressed in spermatogonia. In situ hybridization localized GABARAP to oogonia in the oocyte interstitium, confirming it as a key marker for this cluster. 3.2.2. Primary oocyte cells (cluster 10) Primary oocytes are oocytes that are about to undergo meiosis after mitotic proliferation of the oogonium. In this study, PPAF2 expression in O1 and O2 was 3.54 ± 0.22 and 4.05 ± 0.03, respectively. To determine the location of PPAF2 within the ovary, we designed probes specific for this gene to hybridise with O1 and found that PPAF2 signals were detected in primary oocyte cells. 3.2.3. Secondary oocyte cells (cluster 8) As the primary oocyte develops, it accumulates yolk, mRNA and enzymes in the cytoplasm and grows into secondary oocyte. Our study demonstrated that Gsp-1 expression was 14.76-fold and 29.96-fold higher in O1 and O2 than in the testis, respectively, and was severely up-regulated in the crustacean ovary, proving that it is involved in crustacean ovarian development. In order to locate its position in different germ cells, our observation of its hybridisation signals revealed that Gsp-1 signals strongly in the Secondary oocyte cells. 3.2.4. Ovum cells (cluster 1) To date, the A1i3 gene has not been studied functionally in any species, but in the present study, qPCR results showed that the relative expression of A1i3 was 10.26 ± 0.84 in O2, which is 10-fold higher than that in O1. This implies that A1i3 is involved in the process of more mature female germ cell differentiation, and this speculation was confirmed by in situ hybridisation, where strong A1i3 signals were detected in mature oocytes, which were not observed at any other period. These results provide strong evidence that cluster 1 is the ovum cells. 3.2.5. Spermatogonia cells (cluster 11 and 17) The primordial germ cells (PGCs) arise by preformation or epigenesis [34]. The results of qPCR showed that Pch-2 was expressed 3982.77 ± 245.71 in the spermatheca, while only 2.11 ± 0.28 and 1.00 ± 0.10 in O1 and O2, which indicated that this gene was specifically expressed in the testis. This result was also demonstrated by in situ hybridisation, where Phc-2 signals were observed to be concentrated in spermatogonia cells in the sections. 3.2.6. Primary spermatocyte cells (cluster 13) Primary spermatocytes are the male germ cells before meiosis I. We compared the relative expression in testis and O1, O2 by qPCR and found that the expression of PF13 gene in testis was 14.77 ± 0.40, which was significantly different compared to 1.21 ± 0.30 and 1.01 ± 0.20 in O1 and O2. Strong hybridisation signals in primary spermatocyte cells also proved the accuracy of the results. 3.2.7. Secondary spermatocyte cells (cluster 20) The secondary spermatocyte is smaller in size than the primary spermatocyte, exists for a short period of time, and quickly enters a second maturational division. Our experiments demonstrated that MLC2 was expressed up to 61.36 ± 2.72 in the spermatheca, compared to only 1.72 ± 0.13 in O1, 35.67-fold higher than in the ovary. In situ hybridisation also detected a strong signal for MLC2 in sperm cells. Our study not only suggests that myosin may play an important role in crustacean spermatogenesis, but also demonstrates that the MLC2 gene can be used as a marker for crustacean secondary spermatocyte cells. 3.2.8. Sperm cells (cluster 6) Sperm, the terminal stage of spermatogenesis, marks the morphological transformation of secondary spermatocyte cells into mature sperm [35]. Interestingly, the gene VWDE has not been reported in any reference to its role in males, yet our experiments demonstrated that the expression in the spermatophore of the M. nipponense was as high as 8063.78 ± 1080.47, which is much higher than O1 and O2. Furthermore, after comparing the results of in situ hybridisation for several other genes, the signal for VWDE was strongest in sperm cells. 3.3. Construction of a single-cell transcriptome atlas of crustacean gonadal cells To study the progression of cell lines throughout development, we generated uniform manifold approximation and projection (UMAP) plot of samples based on previously distinguished marker genes. Cell clustering analysis grouped cells into nine distinct populations using UMAP in Fig. 3 C. Female germ cells are roughly categorised on the left side of the Fig. 3 C, male germ cells are grouped in the right bottom corner, and in the middle section are cells of undifferentiated types that may consist of blood cells, immune cells, support cells, follicle cells, stem cells, and so on. Based on literature search, qPCR and in situ hybridisation localisation, the germ cells of M. nipponense were classified into nine clusters, which were assigned as follows: Fig. 3 A corresponding to female germ cells. Figure 3 A-a (cluster 7) is the UMAP map of oogonium, and the marker gene is GABARAP (γ-aminobutyric acid A receptor–associated protein). Fig. A-b (cluster 10) is the primary oocyte, and the marker gene is PPAF2 . Figure 3 A-c (cluster 8) is the secondary oocyte. marker gene is Gsp-1 (grain softness proteins-1). Fig. A-d (cluster 1) is ovum, marker gene is A1i3 (alpha-1-inhibitor-3). Figure 3 B corresponds to male germ cells. Figure 3 B-a (clusters 11 and 17) is a UMAP map of spermatogonia, marker gene is Phc-2 . Figure 3 B-b (cluster 13) is primary spermatocyte, and the marker gene is PF13 . Figure 3 B-c (cluster 20) is a secondary spermatocyte, and the marker gene is Mlc2 (myosin regulatory light chain). Figure 3 B-d (cluster 6) corresponds to sperm, and the marker gene is c . The other 17 cell clusters are classified as Unknown cells. The visualised dot plot of the expression of the newly identified marker genes is shown in Fig. 3 D. Moreover, the selected genes could clearly be classified the cells into 25 classes as shown in Fig. 3 E. Next, we counted the proportion of these germ cells in male, female, and female (Fig. 3 F), and the details are shown in Table S4. Unknown cells accounted for the majority of the cells as they contained many unlabelled genes, 4751 (47.30%) in the female group, 7078 (71.20%) in the male group and 7521 (61.57%) in the neo-female group. A total of 10,045 cells were detected in the female group, of which 285 oogonium (2.84%), 127 primary oocyte (1.26%), 177 secondary oocyte (1.76%), and 4,292 ovum (42.73%). A total of 9928 cells were detected in male group, of which 296 spermaton (2.98%), 203 primary spermatocyte (2.04%), 107 secondary spermatocyte (1.08%), and 1292 sperm (13.01%). A total of 12215 cells were detected in neo-female group, of which 5 oogonium (0.04%), 667 primary oocyte (5.46%), 722 secondary oocyte (5.91%), 1,626 ovum (13.31%), 378 spermaton (3.09%), 5 primary spermatocyte (0.04%), 5 secondary spermatocyte (0.04%), and 155 sperm (1.27%). 3.4. Pseudotime reconstruction of male gonadal cells in neo-females Figure 4 shows the pseudotime analysis of the spermatogenic lineage in males and neo-females. The reconstructed trajectories show that spermatogonia, primary spermatocytes, secondary spermatocytes and sperm are ordered along a continuous developmental path (Fig. 4A). In the testes of control males (Fig. 4A-a), the cells were distributed across the entire trajectory, from the root to the terminal branch. This indicates complete progression from undifferentiated spermatogonia to fully mature sperm. By contrast, neo-female germ cells (Fig. 4A-b) accumulated primarily in the proximal segments of the trajectory, with only a few cells occupying the distal terminal region corresponding to late spermatids and spermatozoa. Consistent with this, a quantitative comparison of cell numbers at each stage revealed that the gonads of neo-females contained a higher proportion of spermatogonia, but a significantly lower proportion of other male gonadal cells (Fig. 4B). Further emphasising this phenomenon, kernel density estimation of pseudotime distributions showed that male cells exhibited a major density peak at late pseudotime, whereas neo-female cells showed a dominant peak at earlier pseudotime values (Fig. 4C). This confirms that the majority of spermatogenic cells in neo-females are arrested at early developmental stages. The gene expression dynamics along pseudotime were then examined (Fig. 4D, E). The heatmap in Fig. 4D shows three major gene modules that are sequentially activated from early to late pseudotime, corresponding to early, developing and mature spermatogenic cells. Expression of the early module is highest at proximal pseudotime, whereas the late module displays a marked increase toward terminal pseudotime in control males. In neo-female cells, the overall signal of this late module is noticeably reduced. Representative genes, including Mhc , wupA , TpnC41c , acta1 , NLRP2 and dnajc3 , exhibit a gradual increase in expression along pseudotime in males, with maximal levels at late spermatogenic stages (Fig. 4E). By contrast, in neo-female germ cells most data points are concentrated at early pseudotime values, and the high-expression domain at late pseudotime is largely absent. Figure 4 Pseudotime trajectory analysis reveals spermatogenic arrest in neo-female testis. (A) Reconstructed developmental trajectories of spermatogenic cells from males (a) and neo-females (b). Each dot represents a single cell and is coloured according to pseudotime, with earlier to later states shown from blue to green. Black circles indicate inferred branching points along the trajectory. (B) Stacked bar plots showing the numbers of cells from males and neo-females in each pseudotime state for the indicated spermatogenic stages. (C) Kernel density plots of pseudotime distributions for male and neo-female male gonadal cells. (D) Heatmap of dynamically regulated genes ordered by pseudotime. (E) Expression patterns of representative spermatogenesis-related genes along pseudotime. Dot plots show single-cell expression of (a) Mhc , (b) wupA , (c) TpnC41c , (d) acta1 , (e) NLRP2 and (f) dnajc3 overlaid on fitted pseudotime trends, highlighting the impaired up-regulation of late spermatogenic markers in neo-female testis. 3.5. Pseudotime reconstruction of female gonadal cells in neo-females Pseudotime analysis was then applied to female gonadal cells from females and neo-females (Fig. 5 ). The reconstructed trajectories arranged oogonia, primary oocytes, secondary oocytes and ova along a continuous developmental path (Fig. 5 A). In ovaries of control females, cells were distributed along the full trajectory from the root to the terminal branch, indicating the presence of oogenic cells at all stages. In contrast, cells from neo-females were mainly located in the proximal and middle segments of the trajectory, with only a small fraction occupying the distal region corresponding to late oocytes. Quantitative comparison of cell numbers in each pseudotime state confirmed this pattern (Fig. 5 B). In the case of oogonium, primary oocytes and secondary oocytes, neo-females exhibited a higher proportion of cells in early pseudotime states relative to females. By contrast, in ovum, cells from females predominated in later pseudotime states. Furthermore, kernel density plots of pseudotime distributions revealed a leftward shift for neo-female cells in comparison with female cells, with the primary density peak of neo-females situated at earlier pseudotime values (Fig. 5 C). Subsequently, the dynamics of gene expression along pseudotime were examined (Fig. 5 D, E). The heatmap (Fig. 5 D) demonstrates several gene modules that are sequentially activated from early to late pseudotime, corresponding to early, developing and mature oogenic cells. In females, early modules are enriched at low pseudotime, whereas late modules show increased expression towards terminal pseudotime. In neo-female, the intensity of the late module is reduced and concentrated in a narrower pseudotime window. Representative genes, including tdc-1 , pnt , pfn-3 , tsr , GRN and Gpdh1 , display characteristic changes in expression along pseudotime in females, with distinct expression domains at specific stages (Fig. 5 E). In neo-female oogenic cells, the majority of data points for these genes are concentrated at earlier pseudotime values, and the high-expression regions at late pseudotime observed in females are diminished. Collectively, these results indicate that the oogenic lineage in neo-females is biased towards early developmental states, with reduced representation of late-stage oocytes. 3.6. Histological observation and differentially expressed genes in neo-female To corroborate the results of single-cell sequencing, we performed serial, multisegmented histological observation of 1-month-old neo-female prawn gonads. Figure 6 A shows well the coexistence of testis-ovary in neo-female. On the left side is a part of the ovarian germ cells, in which a large number of primary oocytes are observed in the 100 µm bar, and it is characterised by gradual disappearance of the nucleus and larger egg diameter (Fig. 6 C-b). There are also a small number of oogonium (Fig. 6 C-a) and secondary oocyte (Fig. 6 C-c), the former with a distinct nucleus and a small diameter, the latter with a disappearing nucleus and a large accumulation of yolk granules in the cytoplasm. On the right side are the germ cells of testis, and as far as we can see there are only a few small amounts of spermation (Fig. 6 C-d), which are the most infantile spermatogonial cells and are finely granular. The development of mature oocytes was not observed in the entire neo-female gonadal tissue. Figure 6 B is a schematic representation of the tissue cross-sectioned for a better demonstration of testis-ovary coexistence. The results of the histological sections are consistent with the results of the scRNA-seq, thus we obtained an important result that some of the most primitive germ cells of males are still retained in neo-females. However, the differences between these four germ cells in neo-female gonads compared to normal males and females have not been clarified, so we compared up and down regulated differentially expressed genes (DEGs) of female vs neo-female and male vs neo-female, respectively (Fig. 6 D) to determine which genes were significant. The results showed that Pabpc1 , BCO1 , RpS27 , Gpdh1 , FLNC , GRN and other genes play important regulatory roles in the growth, maturation and differentiation of female germ cells. And Sam-S , Cd63 , RpL39 and Pabpc1 are likely to be the key genes responsible for the non-full sex reversal in neo-female. It is noteworthy that in these four comparison groups, there are some genes repeated multiple times, which means that they play and their important role in the gonadal development of M. nipponense . Furthermore, in addition to the differences between the male, female and neo-female groups, we screened for the presence of DEGs in the four germ cells in common by Wayne diagrams and found the top 5 genes most enriched in neo-females (Fig. 6 E, F). They were Syb , NPC1b , Zcchc24 , NLRP2 and Smg9 . The t-SNE plots for these five genes are shown in Fig. 6 -G, which illustrating the expression patterns of these candidates within each cell type demonstrated a high level of overlap and clear boundaries, emphasizing their conserved roles in crustacean oogenesis. 3.7. Consistent dysregulation of oxidative phosphorylation in neo-female germ cells Figure 7 presents the comparative pathway enrichment analysis that identifies oxidative phosphorylation as the key biological process altered in neo-female gonads. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment was performed using the DEGs obtained from four critical germ cell stages—oogonium, primary oocyte, secondary oocyte, and spermaton (Fig. 7 A). Across all stages, oxidative phosphorylation (OXPHOS) ranked among the significantly enriched pathways, consistently appearing at the top of the enrichment lists for both female and male gonadal cells. Other pathways such as ribosome, spliceosome, and protein processing in the endoplasmic reticulum were also enriched but did not show the same stage-wide consistency. This indicates that mitochondrial energy metabolism is a common regulatory node affected in neo-female germ cells. Therefore, we organized the gene expression of OXPHOS in female germ cells (oogonium, primary oocyte and secondary oocyte) and male germ cell (spermation) separately and plotted the mechanism (Fig. 7 ). Critically, OXPHOS imbalance was the universal signature. A total of 12 genes were screened, covering the entire process of OXPHOS to produce ATP, including Ndufs1, Ndufb3, Ndufs5, SDHA, SDHC, QCR8, Cyt1, Cyc, COX7A, Nurf-38, ATPsynC and AAEL. Most of the genes were down-regulated in comparison with female germ cells and up-regulated in comparison with male germ cells. It is noteworthy that four genes, Ndufs1, Ndufs3, SDHA, and Cyt1, were expressed in the same pattern in both comparison groups. In female germ cells, dysregulation leads to downregulation of the majority of gene expression. This directly results in impaired OXPHOS function, significantly reducing ATP production, decreasing the efficiency of the mitochondrial ETC, and accelerating the accumulation of ROS. In male germ cells, excessive activation causes abnormal activation of downstream components of OXPHOS. This mitochondrial respiratory dysfunction leads to excessive consumption of the ETC, triggering electron transport overload. The abnormally steep proton gradient exacerbates the explosive production of ROS, causing redox imbalance. 3.8. NLRP2 is a germ cell specific gene of testis development and oxidative phosphorylation To verify the effect of the differential genes on M. nipponense gonadal development, we screened the distribution of these five genes in different germ cells and found that the expression of NLRP2 is germ cell specific and restricted to primary spermatocytes (Fig. 8 A). The UMAP plot of the dataset also confirms this result, as we observed that the NLRP2 is enriched for expression in primary spermatocytes. These results suggest that NLRP2 is a germ cell specific marker associated with a defined stage of spermatogenesis. Multiple sequence alignment of the MnNLRP2 protein from M. nipponense with representative crustacean MnNLRP2 homologs (Fig. 8 B-a) showed that the full-length protein (649 aa) exhibits a high degree of similarity within the conserved regions, with the canonical NACHT and LRR domain clearly identifiable. Tissue-level expression profiling further supported this conclusion (Fig. 8 B-b, c). In males, MnNLRP2 transcripts were detected in all examined tissues but were strongly enriched in the testis, where the relative expression reached 18.51 ± 1.03, significantly higher than in other tissues (p < 0.05). The hepatopancreas and androgenic gland also showed relatively high expression (16.02 ± 1.18 and 10.32 ± 0.40, respectively), whereas levels in eye stalk, cerebral ganglia, heart and gill were much lower. In females, MnNLRP2 expression was extremely low in the eye (undetectable) and ovary (1.00 ± 0.04), both significantly lower than in other female tissues (p < 0.05). In contrast to males, the highest expression in females was observed in muscle, reaching 383.72 ± 28.87. These patterns indicate pronounced tissue specificity and sexual dimorphism, with strong enrichment in male gonads. The phylogenetic tree constructed based on these amino acid sequences further resolved the evolutionary relationships of MnNLRP2 with other arthropod MnNLRP2 proteins (Fig. 8 B-d). M. nipponense clustered tightly with Macrobrachium rosenbergii and Palaemon carinicauda , with all major nodes supported by a bootstrap value of 100, highlighting the strong evolutionary conservation of this gene within the family Palaemonidae . Functional interference experiments were then performed. Injection of dsMnNLRP2 into PL10 prawns led to time-dependent changes in sex-related traits (Fig. 8 C). At 30 days post-injection (Fig. 8 C-a), the number of males in the control group was 30.33 ± 2.08, whereas the dsMnNLRP2 group contained only 14.33 ± 5.13 males. Female numbers showed a similar decline, decreasing from 31.00 ± 2.65 in controls to 17.33 ± 5.51 in the RNAi group (p < 0.05). Steroid hormone measurements showed a consistent endocrine shift following MnNLRP2 silencing (Fig. 8 C-b,c). In the control group, the 17α-Methyltestosterone (MT) concentrations in male prawns at days 15, 22, and 30 were significantly lower than those in the dsMnNLRP2 treated males (p < 0.05). Similarly, the E 2 levels of control females were markedly lower than those of the treated group (p < 0.05). These findings indicate that MnNLRP2 modulates endocrine status and influences sex differentiation during early developmental stages. In adult males,the qPCR analysis shows that 1.69 ± 0.06 and 1.00 ± 0.06 in the experimental group, compared to 22.57 ± 0.52 and 2.63 ± 0.09 in the control group, resulted in interference efficiencies of 92.21% and 61.98% (p < 0.05). The expression level of MnNLRP2 in the RNAi group is significantly lower than that in the control group throughout the whole experiment, this result indicates the efficiency of the interference experiment (Fig. 8 D-a). Interestingly, after 7 days of interference, the histological sections of the experimental and control groups are highly different (Fig. 8 D-b,c). A large number of sperm are observed in the testis of the control group, while a large number of spermatogonia were present in the experimental group despite the existence of sperm, suggesting that MnNLRP2 interfering effectively inhibited the spermatogenesis process, and that the testis of the control group more mature. To further validate the role of MnNLRP2 in OXPHOS in male M. nipponense , we identified gene expression during 7 days after RNAi (Fig. 8 D-d). Compared with the control group, the expression of most genes was significantly downregulated ( Ndufb3 , SDHC , QCR8 , Cyc , Nurf-38 , ATPSynO , and AAEL ), indicating that NLRP2 plays a protective role in ATP production in mitochondria. However, the gene affecting complex III ( Cyt1 ) exhibited an expression pattern opposite to that of other components. Collectively, these data indicate that MnNLRP2 is a conserved, gonad-enriched gene that participates in the regulation of steroidogenesis, testicular development and oxidative phosphorylation in M. nipponense . Primer sequences of all genes are given in Table S5. 4. Discussion Sex reversal achieved through sex steroid manipulation is a widely practiced technique in fish aquaculture. Previous studies reported successful induction of crustacean sex reversal by in vitro administration of E 2 [3]. Similar experiments have been studied in species such as Penaeus merguiensis [36], crayfish [37] and shrimp [38], and it seems possible to obtain all-female populations through exogenous E 2 . Our study obtained neo-females by feeding E 2 , which transformed from males to females and their body size and weight were smaller compared to normal females, implying that sex reversal caused by E 2 may be damaging to male health. Inspired by these advances and results, we utilised scRNA-seq to construct single-cell atlases of the gonads (testis and ovary) of females, males and neo-females to investigate the mechanisms of gonadal development, sex differentiation and sex reversal in crustaceans (Fig. 1 ). We integrated single-cell transcriptomics, in situ hybridisation and functional expression analyses, filling a gap in the molecular classification of crustacean germ cells. In this study (Fig. 2 ), eight different types of germ cell populations were identified for the first time, including oogonium ( GABARAP ), primary oocyte ( PPAF2 ), secondary oocyte ( Gsp-1 ), oocyte ( A1i3 ), spermation ( Phc-2 ), primary spermatocyte ( PF13 ), secondary spermatocyte ( Mlc2 ) and sperm ( VWDE ). At the cellular level, subclustering and quantitative comparison of cell-type proportions revealed that both the oogenic and spermatogenic lineages in neo-female gonads are dominated by early-stage cells, whereas late oocytes and spermatozoa are markedly reduced (Fig. 3 ). This is not the first reported case of sex reversal in a species. Histological observations of bluegill Lepomis macrochirus fed different concentrations of E 2 revealed that 13.3% and 5.0% of the intersex fish were determined to come from the 50 and 100 mg kg- 1 E 2 treatment groups, respectively, with 6.9% and 4.1% of the gonadal area containing spermatocytes [39]. Sex-reversed type XX Oryzias latipes showed isogenic spermatocysts with active spermatogenesis [40]. This pattern is mirrored in the histological sections, where neo-female gonads display abundant oogonia/early oocytes or spermatogonia with relatively few fully developed gametes (Fig. 6 A–C). This study also revealed the diversity of germ cell development (Fig. 4). For example, ovum contain three subtypes that may correspond to three different stages of yolk synthesis, nuclear maturation, and preovulatory activation [41]. The formation of fertile spermatozoa is the result of spectacular stages of cell differentiation that begin in the male gonad and finish in the female tract [42]. Trajectory analyses provide dynamic support for this conclusion. In males, spermatogenic cells span the entire pseudotime trajectory from spermatogonia to mature sperm (Fig. 4A), and in females, oogenic cells similarly extend from oogonia to ova (Fig. 5 A). In neo-females, however, the majority of cells cluster at proximal or intermediate pseudotime, with only a small fraction occupying terminal positions. Density plots show a clear leftward shift in pseudotime distributions for both spermatogenic and oogenic cells in neo-females (Fig. 4C, Fig. 5 C), indicating that germ-cell populations are biased toward earlier developmental states. These observations suggested that estrogen induced sex reversal in M. nipponense is accompanied not by a complete reprogramming of germ-cell fate, but rather by a failure of germ cells to progress to fully mature stages. Estrogen exposure disrupted the intrinsic maturation trajectory of germ cells, leading to an ovary-like phenotype with arrested gametogenesis. This study revealed that excessive E₂ induced redox metabolic disorders in germ cells during female sex reversal, despite inducing males to become neo-females (Fig. 6 ). This is consistent with the previous findings of organ damage and developmental abnormalities due to oestrogen overdose [43,44]. Differential expression analyses across corresponding stages further show that the transcriptional landscape of neo-female germ cells is extensively remodeled. Volcano plots for oogonia, primary and secondary oocytes and spermaton highlight numerous DEGs in the comparisons “female vs neo-female” and “male vs neo-female” (Fig. 6 D). We next examined the expression patterns of representative genes at single-cell resolution (Fig. 6 F,G). Synaptic vesicle-associated gene Syb and lipid transport–related NPC1b were preferentially expressed in selected oogenic clusters in control females, with clear, stage-specific enrichment along the oocyte lineage. In neo-females, the distribution and intensity of these signals were altered, consistent with their identification as DEGs. Zcchc24 and Smg9, which showed distinct cluster enrichment patterns in the integrated UMAP, also exhibited significant expression differences between controls and neo-females, indicating that mRNA processing and RNA surveillance pathways may be modulated in neo-female germ cells. Notably, NLRP2 displayed restricted expression in specific spermatogenic clusters, matching its primary localization to early/mid spermatogenic stages observed in the pseudotime analysis and providing a basis for subsequent functional studies. Among all functional categories enriched in the DEGs, oxidative phosphorylation emerged as the most consistent pathway across key germ-cell stages (Fig. 7 A). In oogonia, primary oocytes, secondary oocytes and spermaton, genes encoding components of the electron transport chain and ATP synthase were significantly altered in neo-females. Other pathways such as ribosome and spliceosome were also enriched, but oxidative phosphorylation was unique in spanning both oogenic and spermatogenic lineages and multiple developmental stages. OXPHOS is a central metabolic pathway for ATP production in mitochondria via the ETC and is essential for maintaining germ cell function [45]. In oogonium and primary oocytes, E₂ triggers aberrant metabolic activation, including accelerated ribosome synthesis and AMPK signaling pathway upregulation, indicating cellular energy stress and premature oocyte initiation under conditions of ATP/AMP imbalance [46,47]. This finding is consistent with the characterisation of the early stages of oocyte development, when oogonia are in transcriptional quiescence but reserve ribosome required for translation to support protein synthesis in subsequent developmental stages [48,49]. However, E₂ simultaneously impairs maturation in secondary oocytes, aberrant splicing function may result in a lack of transcription product diversity [50]. Combined with the disruption of the secondary oocyte pathway observed in neo-females, it is speculated that E 2 may interfere with the quality of RNA processing during the later stages of oocyte development, which may be one of the central mechanisms by which germ cell maturation is impaired in sex-reversed individuals. Abnormal carbon metabolism has been shown to inhibit spermatogenesis and disrupt DNA epitope modification [51,52], and the dysregulation of the same pathway found in this study in spermatons of neo-females implies that E 2 may interfere with normal spermaton differentiation by impairing the methylation cycle [53,54]. In persistent spermatogonia within neo-female gonads, E₂ causes significant dysregulation of the carbon metabolism pathway, directly linked to reactive oxygen species ROS overproduction and impaired DNA methylation cycles, thereby inhibiting normal spermatogenesis and potentially compromising genetic stability. These results are consistent with our previous findings that excessive estrogen quantities can cause harm to the animal, such as damage to organs, abnormal development, cancer risk, etc [55]. Thus, our results suggested that E₂ exerts a dual pathology: promoting precocious metabolic activity and energy stress in early-stage oocytes while disrupting redox balance, metabolic regulation, and epigenetic stability, ultimately impairing germ cell maturation and quality in sex-reversed gonads." To elucidate how OXPHOS dysfunction drives germ cell differentiation disorders during sexual reversal, we systematically screened and integrated the expression profiles of 12 core genes covering the entire ATP synthesis pathway (Fig. 7 B). In female reproduction, oocyte maturation, fertilisation and embryo development require stable mitochondrial function, and abnormal OXPHOS triggers the accumulation of ROS, leading to oxidative stress, which affects oocyte quality [56]. It has been shown that OXPHOS leads to abnormal meiosis and reduced fertilisation rate in mouse oocytes by inhibiting MPF (M-phase promoting factor) activity. Furthermore, OXPHOS damage leads to abnormal meiosis, decreased fertilisation rate and reduced embryo quality [57]. Our results indicate that dysregulation of female germ cells reduces ATP production and decreases ETC efficiency, accelerating ROS accumulation and ultimately disrupting normal oocyte maturation. Our results showed that OXPHOS is significantly inhibited in female germ cells in neo-female, resulting in dysfunctional oocyte reproduction. In male reproduction, sperm motility is highly dependent on ATP produced by mitochondria, and OXPHOS is the main pathway of sperm energy metabolism [58]. For example, human sperm rely primarily on mitochondrial OXPHOS to generate ATP in oxygen-rich environments, with glycolysis serving only as a secondary pathway [59]. Mitochondrial OXPHOS dysfunction directly affects sperm viability and morphology, and animal models have shown that increased mitochondrial ROS after testicular TD activate the caspase-9-dependent apoptotic pathway and induce germ cell apoptosis [60,61]. Our results confirmed this conclusion that OXPHOS was significantly activated in spermation in neo-females, leading to ETC overload, a sharp increase in ROS content, and ultimately a breakdown in redox homeostasis. These may be one of the main reasons for the lack of potential for spermation to continue to develop into sperm, suggesting that male reproductive function was greatly suppressed after E 2 feeding. On the other hand, we observed that the weight of neo-female was smaller than female. Therefore, we speculate that the reason for the incomplete reversal of male crustaceans by E 2 may be that the steroid hormone disrupts the normal synthesis of ATP by OXPHOS, which in turn causes DNA damage and apoptosis. The energy used by males to promote maturation of the testis, although suppressed, still has a tendency to develop spermation, which results in the neo-male still retaining a portion of spermation, and a defective maturation of the female germ cells and weight growth. Addressing this problem may require improved types and doses of hormones to be fed, or feeding at an earlier period of M. nipponense development to achieve full feminisation of the population. The results revealed a significant bidirectional imbalance pattern, which is the core mechanism of redox and energy metabolism disorders. NLRP2 (NOD-like receptor family pyrin domain-containing 2) emerged from the integrated analysis as a particularly interesting candidate. At the single-cell level, NLRP2 expression is highly restricted to primary spermatocytes in the spermatogenic lineage (Fig. 8 A), indicating a narrow developmental window of activity. NLRP2 is a member of the NLR family, which is mainly involved in the regulation of inflammatory responses, apoptosis and epigenetic modifications [62]. Sequence alignment shows that MnNLRP2 conserves the canonical NACHT and LRR domains characteristic of NLR proteins, while displaying species-specific variation in terminal regions, and phylogenetic analysis places it firmly within the decapod NLRP2 clade together with other shrimp, crab and crayfish species (Fig. 8 B-a,d). This supports that MnNLRP2 may participate in conserved cellular processes. The critical role in the reproductive system and embryo development has been progressively discovered in recent years, and it is decisive for female reproduction through the maintenance of oocyte quality, early embryo development and maternal genetic stability [63–65]. Its defects can lead to embryonic arrest, decline in fertility with age and abnormal development of the offspring [66,67]. In a mouse study, they knocked down MnNLRP2 transcription specifically in mouse germinal vesicle oocytes, showed that MnNLRP2 is a member of the mammalian maternal effect genes and required for early embryonic development in the mouse [68]. Tissue distribution analysis showed that MnNLRP2 was strongly enriched in male gonads, with high expression in the testis and androgenic gland, low levels in most somatic tissues, and only weak expression in female ovaries, suggesting a close association with male reproduction. Functional knockdown experiments in PL10 prawns further supported this role. Repeated injection of dsMnNLRP2 during early post-larval development progressively altered the phenotypic sex ratio, leading to a significant decline in the proportion and number of males and a corresponding increase in females by 30 days post-injection, consistent with a partial male-to-female sex reversal (Fig. 8 C-a). After five rounds of interference, hormone measurements revealed that MT levels in phenotypic males of the RNAi group were significantly lower than those of controls, whereas E 2 concentrations in phenotypic females were significantly elevated compared with the corresponding control females (Fig. 8 C-b,c). These results indicated that MnNLRP2 knockdown disrupts normal androgen estrogen balance in a sex-specific manner and promotes feminization at early developmental stages. In adult males, sustained MnNLRP2 silencing led to pronounced histological changes in the testis. Testes from control prawns contained well-organized lobules filled with abundant mature spermatozoa, whereas testes from MnNLRP2 knockdown prawns were dominated by spermatogonia and early spermatocytes, with markedly fewer sperm (Fig. 8 D-b,c). This early-stage accumulation closely mirrors the spermatogenic arrest observed in neo-female gonads, supporting the conclusion that MnNLRP2 is required for proper progression of spermatogenesis in M. nipponense. Additionally, we found that MnNLRP2 inhibits sperm maturation and affects gene expression during OXPHOS (Fig. 8 D-d). MnNLRP2 functions as an inducible inflammatory mediator that regulates NF-κB activation [69]. MnNLRP2 inflammasome can be activated by IFN, ATP and LPS leading to inflammatory responses and involved in regulating NLRP3 inflammasome [70,71]. Given the crosstalk between NF-κB signaling and oxidative stress responses (e.g., ROS generation and antioxidant defense), MnNLRP2 likely modulates cellular redox balance through this inflammatory axis. Furthermore, NLRP family members (e.g., NLRP4 ) can interact with Fas-associated factor 1 (FAF1), a negative regulator of inflammatory signals [64,72]. If MnNLRP2 similarly modulates FAF1 or analogous partners, it may suppress pro-oxidant pathways (e.g., caspase activation or mitochondrial ROS production), thereby protecting cells from oxidative stress. Therefore, we speculated that MnNLRP2 may combat oxidative stress through multiple mechanisms, including tuning NF-κB-mediated inflammation to prevent ROS overproduction or scaffolding SCMC complexes to stabilize stress-defense factors. In conclusion, we establish a single-nucleus transcriptomic atlas of M. nipponense gonads and provide a framework of validated markers for staging crustacean germ cells. Using this atlas, we show that estrogen-induced neo-females are characterized by a strong shift in cell composition and developmental trajectories toward early oogenic and spermatogenic stages, with a marked depletion of mature gametes. Stage-resolved transcriptomics and enrichment analyses consistently implicate oxidative phosphorylation as the pathway most disturbed in neo-female germ cells. We further identify MnNLRP2 as a primary spermatocyte specific, testis enriched gene. Functional knockdown of MnNLRP2 alters sex ratios and sex steroid levels in PL10 prawns and causes spermatogenic arrest and dysregulation of oxidative-phosphorylation genes in adults. Together, our findings reveal that estrogen induced sex reversal in M. nipponense involves germ-cell arrest linked to mitochondrial dysfunction and highlight MnNLRP2 as a key regulator of male gonadal development and endocrine balance. Declarations Funding This research was supported by grants from National Key R&D Program of China (2023YFD2401000); Central Public-interest Scientific Institution Basal Research Fund CAFS (2023TD39); the earmarked fund for CARS-48-07; the seed industry revitalization project of Jiangsu province (JBGS [2021]118). Thanks to the Jiangsu Province Platform for the Conservation and Utilization of Agricultural Germplasm. Author Contributions P.C. designed the experiments, fed the prawns, analysed the data and wrote the manuscript; Z.G. wrote the materials and methods; H.F. supervised the experiments; S.J. (Sufei Jiang) and Y.X. provided the experimental prawns and the steroid hormones; W.Z., S.J. (Shubo Jin) and H.Q. carried out the qPCR analyses and made some further modifications. All authors have read and agreed to the published version of the manuscript. References G.A. Boxshall, Crustacean classification: on-going controversies and unresolved problems, Zootaxa 1668 (2007) 313–325. 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Supplementary Files Table.S1.xlsx Table.S1 Table.S2.xlsx Table.S2 Table.S3.xlsx Table.S3 Table.S4.xlsx Table.S4 Table.S5.xlsx Table.S5 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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(A) \u003cem\u003eM. nipponense \u003c/em\u003elife cycle. L1: The first day larvae; PL1: The first day post-larvae; PL25: The 25 day post-larvae. Corresponding to the period of nauplius, metamorphosis and gonadal development, respectively. (B) The addition of 17β-Estradiol to feed. Dose: 200mg/g (C) Comparison of neo-female with normal male and female prawn. (D) Schematic illustration of the experimental workflow in this study. (a) Sequencing data quality control and expression quantification. The horizontal coordinates represent the grouping of the samples, and the vertical coordinates are the logarithmic result of the gene number. (b) Each dot represents one cell colored according to assignment by clustering analysis. 10X Genomics Cell Ranger software (version3.1.0) was used to convert raw BCL files to FASTQ files, alignment and counts quantification. For visualization of clusters, t-distributed Stochastic Neighbor Embedding (t-SNE) were generated using the same PCs.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/f255458b4296c82bd77f528b.jpg"},{"id":99275456,"identity":"90f5e576-2590-4934-8b0c-21697e27d6f5","added_by":"auto","created_at":"2025-12-31 07:06:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4153992,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of eight major germ cell types in \u003cem\u003eM. nipponense\u003c/em\u003e by in situ hybridisation and qpcr. (A) Dot plot showing the relative expression of selected genes in clusters of germ cells in the ovary. (B) Dot plot showing the relative expression of selected genes in clusters of germ cells in the sperm. (C) Relative expression of four male marker genes in testis, stage I ovary (O1) and stage II ovary (O2). (D) In situ hybridisation of four female marker genes in the ovary of the female \u003cem\u003eM. nipponense\u003c/em\u003e. Strong signals (magenta) were observed in oogonia, primary oocytes, secondary oocytes and oocytes, respectively, and no signals were observed when negative RNA probes were used. (E) Relative expression of four marker genes in testis, stage I ovary (O1) and stage II ovary (O2). (E) In situ hybridisation of four marker genes in the testis of the male \u003cem\u003eM. nipponense\u003c/em\u003e. Strong signals (magenta) were observed in spermatogonia, primary spermatocytes, secondary spermatocytes and sperm, respectively, and no signals were observed when negative RNA probes were used. N: Nucleus; Oo: Oogonium; Po: Primary oocyte; So: Secondary oocyte; O: Oocyte; Sp: Spermatogonia; Ps: Primary spermatocyte; Ss: Secondary spermatocyte; S: Sperm. Scale bar = 20 µm and 10 µm.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/16b961e62fe505e9870aea5f.jpg"},{"id":99320291,"identity":"4c87e96f-bd9d-4518-926e-913d5720d966","added_by":"auto","created_at":"2025-12-31 16:38:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3301862,"visible":true,"origin":"","legend":"\u003cp\u003eSingle-cell transcriptome analysis identified germ cells of \u003cem\u003eM. nipponense\u003c/em\u003egonads. (A) Gene expression plots of cell- specific marker genes identify the major cell type that each cluster corresponds to. Cells expressing the indicated gene are colored blue, and the relative intensity indicates relative expression levels (intensity scale for each plot is on the right). (a): GABARAP; (b): PPAF2; (c): GSP-1; (d): Ali3. (B) Gene expression UMAP plots of select genes, same as in (A). (a): Phc-2; (b): PF13; (c): VWDE; (d): MLC2. (C) UMAP plot of eight germ cell clusters. Different cell types are shown in distinct colors. Each germ cell type is coloured differently. (D) Dot plot showing the relative expression of select genes in the germ cell subclusters. (E) Heatmap containing the highest differentially expressed genes in each cluster, 25 clusters in total. (F) Circle plots of cellular differences between females, males and neo-females.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/26e084d993142e228ebc6895.jpg"},{"id":99318944,"identity":"548346c2-280d-4ab6-aa08-8809c7dedbe0","added_by":"auto","created_at":"2025-12-31 16:35:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2286428,"visible":true,"origin":"","legend":"\u003cp\u003ePseudotime trajectory analysis reveals spermatogenic arrest in neo-female testis. (A) Reconstructed developmental trajectories of spermatogenic cells from males (a) and neo-females (b). Each dot represents a single cell and is coloured according to pseudotime, with earlier to later states shown from blue to green. Black circles indicate inferred branching points along the trajectory. (B) Stacked bar plots showing the numbers of cells from males and neo-females in each pseudotime state for the indicated spermatogenic stages. (C) Kernel density plots of pseudotime distributions for male and neo-female male gonadal cells. (D) Heatmap of dynamically regulated genes ordered by pseudotime. \u0026nbsp;(E) Expression patterns of representative spermatogenesis-related genes along pseudotime. Dot plots show single-cell expression of (a)\u003cem\u003e Mhc\u003c/em\u003e, (b)\u003cem\u003e wupA\u003c/em\u003e, (c) \u003cem\u003eTpnC41c\u003c/em\u003e, (d) \u003cem\u003eacta1\u003c/em\u003e, (e) \u003cem\u003eNLRP2\u003c/em\u003e and (f) \u003cem\u003ednajc3\u003c/em\u003e overlaid on fitted pseudotime trends, highlighting the impaired up-regulation of late spermatogenic markers in neo-female testis.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/8e28f41dab4e18fe6abee7db.jpg"},{"id":99320373,"identity":"ed12b247-7bb7-47d5-ba46-e970cfec1204","added_by":"auto","created_at":"2025-12-31 16:38:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2417907,"visible":true,"origin":"","legend":"\u003cp\u003ePseudotime trajectory analysis reveals oogenic arrest in neo-female ovary. (A) Reconstructed developmental trajectories of oogenic cells from females (a) and neo-females (b). (B) Stacked bar plots showing the numbers of cells from females and neo-females in each pseudotime state for the female gonadal stages (C) Kernel density plots of pseudotime distributions for female and neo-female ovarian germ cells. (D) Heatmap of dynamically regulated genes ordered by pseudotime. (E) Expression patterns of representative oogenesis-related genes along pseudotime. Dot plots show single-cell expression of (a) \u003cem\u003etdc-1\u003c/em\u003e, (b) \u003cem\u003epnt\u003c/em\u003e, (c) \u003cem\u003epfn-3\u003c/em\u003e, (d) \u003cem\u003etsr\u003c/em\u003e, (e) \u003cem\u003eGRN\u003c/em\u003eand (f) \u003cem\u003eGpdh1 \u003c/em\u003eoverlaid on fitted pseudotime trends, illustrating altered transcriptional dynamics of late oogenic markers in neo-female ovary.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/663792302a2b23f144915fcf.jpg"},{"id":99275455,"identity":"5c2bc145-2784-4313-aa9a-1f431bd6ef2c","added_by":"auto","created_at":"2025-12-31 07:06:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6157385,"visible":true,"origin":"","legend":"\u003cp\u003eHistological observation and differentially expressed genes in the gonads of neo-female (A) Histological and morphology of neo-female gonads by HE staining. a, c: scale bar = 100 µm; b: scale bar= 50 µm. (B) Schematic diagram of a transverse section of neo-female gonadal tissue. Oo: Oogonium, Po: Primary oocyte, So: Secondary oocyte, Sp: spermation, He: Hepatocyte. (C) HE staining sections of co-existence of testis-ovary contain germ cells. a: oogonium, b: primary oocyte, c: secondary oocyte, d: spermation. scale bar = 10 µm. (D) Volcano diagrams of four germ cell DEGs in female vs neo-female and male vs neo-female. a: oogonium, b: primary oocyte, c: secondary oocyte, d: spermation. (E) Analysis of DEGs by Venn diagram showing the number of DEGs in oogonium, primary oocyte, secondary oocyte and spermation. (F) Dot plots showing the relative expression of DEGs in the male, female and neo-female groups. (G) t-SNE plots of DEGs in male, female and neo-female groups of germ cells.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/00da5cf0ab687dd93228bc8c.jpg"},{"id":99275463,"identity":"4146c76f-bc0a-482a-99c6-90ae223751be","added_by":"auto","created_at":"2025-12-31 07:06:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3635036,"visible":true,"origin":"","legend":"\u003cp\u003eOxidative phosphorylation is consistently enriched among DEGs in neo-female germ cells. (A). KEGG pathway enrichment analysis of DEGs from four key germ cell stages. (B). A model of the OXPHOS pathway during gonadal development in the neo-female prawn. Red oval represents upward adjustment; green rectangle represents downward adjustment. Oo: oogonium, Po: primary oocyte, So: secondary oocyte, Sp: Spermatogonia, He: Hepatocyte, Sc: Sertoli cell, Fc: Follicle cell.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/f0cec150df3cf2ce8f74b746.jpg"},{"id":99318915,"identity":"1f2534a9-c708-4da4-bf61-1e46861d1643","added_by":"auto","created_at":"2025-12-31 16:35:44","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6581981,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification and functional characterization of \u003cem\u003eMnNLRP2\u003c/em\u003e in gonadal development. (A) Expression pattern of \u003cem\u003eMnNLRP2\u003c/em\u003e in single-nucleus data. (a) Dot plot of the distribution of the\u003cem\u003e NLRP2\u003c/em\u003e gene in eight germ cell species. (a) UMAP plots of \u003cem\u003eMnNLRP2\u003c/em\u003e gene in primary spermatocyte. (B) Molecular characterisation and tissue distribution of \u003cem\u003eMnNLRP2\u003c/em\u003e. (a) Multiple sequence alignment of \u003cem\u003eMnNLRP2\u003c/em\u003e with \u003cem\u003eNLRP2\u003c/em\u003e proteins from other species. (b,c) Expression pattern of the \u003cem\u003eMnNLRP2 \u003c/em\u003egene in different tissues. O: ovary, E: eyestalk, Cg: cerebral ganglion, H: heart, He: hepatopancreas, G: gill, M: muscle, T: testis, and Ag: androgenic gland. (d) Phylogenetic tree of \u003cem\u003eMnNLRP2\u003c/em\u003e proteins from \u003cem\u003eM. nipponense\u003c/em\u003e and other crustaceans. (C) Effects of\u003cem\u003e MnNLRP2\u003c/em\u003e knockdown in PL10 prawns. (a) Changes in the mean numbers of male and female individuals in control and \u003cem\u003edsMnNLRP2\u003c/em\u003e groups at 15, 22 and 30 days post-dsRNA injection. (b) MT content and (c) E\u003csub\u003e2\u003c/sub\u003e content in control and\u003cem\u003e dsMnNLRP2 \u003c/em\u003egroups at the indicated time points. (D) Effects of \u003cem\u003eMnNLRP2\u003c/em\u003e knockdown in adult male prawns. (a) Expression pattern of the \u003cem\u003eMn-NLRP2\u003c/em\u003e gene 7 days after RNAi. (b,c) Histological sections of testis from the control and\u003cem\u003e dsMnNLRP2\u003c/em\u003e groups after 7 days. Sp: Spermatogonia; Ps: Primary spermatocyte; Ss: Secondary spermatocyte; S: Sperm. scale bar = 200 µm. (d) A model of the OXPHOS pathway after RNAi in adult male prawns. Data are presented as the mean ± SEM (n = 9). Different letters indicate significant differences (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/a46b76267209580195b772f0.jpg"},{"id":99801719,"identity":"d144b34c-62bb-4c1a-96c5-e9cde995a2c5","added_by":"auto","created_at":"2026-01-08 14:07:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33242700,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/9b9d5d25-ce01-4e86-af9c-60470ac8da1f.pdf"},{"id":99319400,"identity":"3f70a69d-298c-4039-8180-53cdbbbee318","added_by":"auto","created_at":"2025-12-31 16:37:09","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10423,"visible":true,"origin":"","legend":"Table.S1","description":"","filename":"Table.S1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/405068643f04ac2e269b7b2b.xlsx"},{"id":99319661,"identity":"2f7d3a76-ab05-4d5f-b76e-323f3c4e994a","added_by":"auto","created_at":"2025-12-31 16:37:39","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11290,"visible":true,"origin":"","legend":"Table.S2","description":"","filename":"Table.S2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/018f45a9fc1725f413ec4cf8.xlsx"},{"id":99275452,"identity":"3a59ee88-e5d2-4f5f-9a28-ecfa12d9ada5","added_by":"auto","created_at":"2025-12-31 07:06:10","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10182,"visible":true,"origin":"","legend":"Table.S3","description":"","filename":"Table.S3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/082cf3b510dc445594e21d03.xlsx"},{"id":99275465,"identity":"20ba1a08-62ae-4d2c-bbaf-7c0583e1b5e0","added_by":"auto","created_at":"2025-12-31 07:06:11","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9577,"visible":true,"origin":"","legend":"Table.S4","description":"","filename":"Table.S4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/09e3b3938c9fd5ee587e2057.xlsx"},{"id":99321301,"identity":"aa3104ce-de9e-45e9-b33d-99e5e064d2af","added_by":"auto","created_at":"2025-12-31 16:39:19","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10235,"visible":true,"origin":"","legend":"Table.S5","description":"","filename":"Table.S5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8343381/v1/ea3b6289cd2f3592a0524fc2.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Single-cell atlas of prawn gonads identifies NLRP2 regulated mitochondrial dysfunction in estrogen induced sex reversal","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSex reversal through endocrine manipulation represents a promising aquaculture strategy for mono-sex populations in economically important species, including crustaceans [1,2]. While 17β-estradiol (E\u003csub\u003e2\u003c/sub\u003e) has been shown to induce neo-females (sex-reversed males) in decapods such as \u003cem\u003eMacrobrachium nipponense\u003c/em\u003e, these individuals exhibit impaired growth and gonadal maturation compared to natural females [3]. The mechanisms underlying these developmental defects, particularly at the cellular and metabolic levels, remain poorly understood. Given the pivotal role of redox homeostasis and energy metabolism in gametogenesis, we hypothesized that E\u003csub\u003e2\u003c/sub\u003e induced sex reversal disrupts these processes, leading to functional sterility in neo-females [4,5].\u003c/p\u003e \u003cp\u003eCrustaceans lack a canonical sex-determining pathway, and the molecular basis of germ cell development during sexual differentiation is largely uncharted. Previous histological studies confirmed testis-ovary coexistence in neo-females but failed to resolve the heterogeneity of germ cells or identify metabolic drivers of defective maturation [3]. Single cell RNA Sequencing (scRNA-seq) is a high-throughput genomics technology that resolves gene expression heterogeneity among cells by resolving cellular diversity [6]. In vertebrates, this approach has uncovered both conserved and species-specific profiling of gene expression in innate and adaptive immunity [7]. Recent studies of teleost gonads exemplify its power for deconvoluting sexual plasticity: scRNA-seq in protogynous groupers (\u003cem\u003eEpinephelus coioides\u003c/em\u003e) identified ovarian pre-follicle cells co-expressing female and male sex determination genes prior to sex reversal, revealing a transitional transcriptional \u0026lsquo;bivalent state\u0026rsquo; [8]. However, the application of such high-resolution approaches to crustaceans remains in its infancy and there are few reports of scRNA-seq in lower crustaceans (e.g., crabs, shrimps), especially in sex-reversed species [9,10]. The application of single-cell technology to the study of sex differentiation and gonadal development in crustaceans still faces unique challenges. Firstly, marker scarcity: Unlike mammals where conserved germline markers (vasa, piwil1) and somatic identifiers (\u003cem\u003esox9\u003c/em\u003e, \u003cem\u003ecyp19a1\u003c/em\u003e) exist, most crustacean cell types lack definitive markers [11,12]. Secondly, transcriptomic and functional differences between sex-reversed neo-individuals and natural individuals challenge traditional cell typing paradigms, such as \u003cem\u003eEpinephelus coioides\u003c/em\u003e [13], \u003cem\u003eLarimichthys crocea\u003c/em\u003e [14], \u003cem\u003eMauremys reevesii\u003c/em\u003e [15], \u003cem\u003eSiniperca chuatsi\u003c/em\u003e [16], etc.\u003c/p\u003e \u003cp\u003eOxidative phosphorylation (OXPHOS) serves as the cornerstone of cellular energy metabolism during gametogenesis, where its fidelity directly dictates germ cell viability and reproductive competence [17]. In vertebrates, mitochondrial OXPHOS not only generates adenosine triphosphate (ATP) for meiotic divisions and motility but also fine-tunes redox homeostasis by modulating reactive oxygen species (ROS) flux. Dysregulation of this system\u0026mdash;whether through suppressed complex I/III activity or electron transport chain (ETC) overloading\u0026mdash;triggers pathogenic ROS accumulation, DNA damage, and apoptotic cascades that devastate oocyte quality and spermatogenic efficiency [18]. For instance, mammalian studies confirm that OXPHOS deficiency impairs oocyte maturation [19]. Crucially, steroid hormones like estrogen inherently modulate mitochondrial function, yet whether exogenous E\u003csub\u003e2\u003c/sub\u003e disrupts OXPHOS-mediated redox balance in crustacean germ cells, where anaerobic metabolism dominates remains unexplored. This gap is particularly salient in crustaceans, which lack conserved sex-determining genes and exhibit unique metabolic adaptations. The differences between crustaceans and vertebrates have raised questions about the role of OXPHOS in germ cell development.\u003c/p\u003e \u003cp\u003eIn this study, we construct a scRNA-seq c atlas of \u003cem\u003eM. nipponense\u003c/em\u003e gonads, define a marker framework for eight major germ-cell types, and uncover pronounced alterations of oxidative phosphorylation\u0026ndash;related pathways in estrogen-induced neo-females. Using lineage-resolved scRNA-seq, we show that E\u003csub\u003e2\u003c/sub\u003e reshapes germ-cell composition and transcriptional trajectories and consistently perturbs oxidative phosphorylation across both oogenic and spermatogenic lineages. We further identify \u003cem\u003eMnNLRP2\u003c/em\u003e as a previously uncharacterized, spermatocyte enriched regulator whose knockdown disrupts testicular development and the expression of multiple oxidative-phosphorylation components. Together, these findings support a model in which estrogen induced sex reversal in \u003cem\u003eM. nipponense\u003c/em\u003e is accompanied by mitochondrial dysfunction and germ-cell maturation failure, providing a mechanistic entry point to understand the redox and metabolic control of sexual plasticity in crustaceans.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Experimental Animals and Dietary Preparation\u003c/h2\u003e \u003cp\u003eThe diets employed in this study were obtained from a commercial diet for shrimp produced by the Freshwater Fisheries Research Centre of the Chinese Academy of Fisheries Sciences (Wuxi). The composition of the commercial diets was as follows: crude protein, fish meal, shrimp meal, squid meal, starch, soybean meal, ash, rapeseed meal, soybean protein concentrate, and crude fat. E\u003csub\u003e2\u003c/sub\u003e (CAS number. 50-28-2, purity: 95.88%) was procured from Beijing Solarbio Technology Co, Ltd. (Beijing, China). The method of adding the hormone to the diet was outlined as follows: The hormone was dissolved in 95% ethanol to prepare a solution with a concentration of 20 mg/ml. The stock solution was then applied in a uniform manner to the feed (1 ml ethanol per 10 g diet) and agitated with a glass rod for a minimum of three minutes. Subsequently, the objects were placed in a ventilated laboratory hood and left in the shade for 15 minutes. The treated diets were transferred to 15 ml test tubes and stored in a refrigerator at 0\u0026deg;C to facilitate the natural evaporation of the residual alcohol. Preparation of feeds with an E\u003csub\u003e2\u003c/sub\u003e concentration of 200 mg/g. Previous studies have demonstrated that this concentration can effectively induce sex reversal in \u003cem\u003eM. nipponense\u003c/em\u003e [20]:\u003c/p\u003e \u003cp\u003eTo ensure methodological rigor and reproducibility, the experimental and statistical protocols for obtaining neo-females were implemented as follows: Prior to the experiment, 100 male and 100 female PL30 (PL: post-larvae developmental stage) were selected and maintained in recirculating aquaculture systems under controlled conditions (water temperature: 22\u0026ndash;24\u0026deg;C; pH: 7.5\u0026ndash;8.0; salinity: 0.0\u0026permil;). Prawns were fed daily at 5% body weight for 60 days, with the experimental group (males) receiving E\u003csub\u003e2\u003c/sub\u003e treated feed and the control group (females) receiving ethanol treated feed without E\u003csub\u003e2\u003c/sub\u003e supplementation. Body weight and length (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM) were recorded every 15 days throughout the trial period. At the end of the experiment, neo-females (sex-reversed males) were identified in the experimental group based on specific morphological/gonadal criteria, with their quantity systematically recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Sampling and scRNA sequencing\u003c/h2\u003e \u003cp\u003eAt the end of the culture experiment, two adult females, two adult males and two neo-females were randomly sampled from both the control and treatment groups, and their gonads were collected. Suspension was prepared following the 10x Genomics User Guide (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://support10xgenomicscom/single-cell-gene-expression/index/doc/user-guide-chromium-single-cell-3-reagent-kits-user-guide-v31-chemistry-dual-index\u003c/span\u003e\u003cspan address=\"https://support10xgenomicscom/single-cell-gene-expression/index/doc/user-guide-chromium-single-cell-3-reagent-kits-user-guide-v31-chemistry-dual-index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Cellular suspensions were then loaded on a 10X Genomics GemCode Single-cell instrument to generate single-cell GelBead-In-EMlusion (GEMs). Using Chromium Next GEM Single Cell3\u0026rsquo; Reagent Kits (v3.1), full-length cDNAs with barcode and Unique Molecular Identifier (UMI) were generated. The cDNAs were then sequenced on an Illumina NovaSeq 6000 platform [21].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Data quality control\u003c/h2\u003e \u003cp\u003eThe cell by gene matrices for each sample were individually imported to Seurat (v3.1.1) [22] for the downstream analysis. Raw data were processed using Cell Ranger (v7.1.0) [23] by aligning to the \u003cem\u003eM. nipponense\u003c/em\u003e genome assembly (ASM1510439v2) [24]. Cells with \u0026gt;\u0026thinsp;8000 UMIs or \u0026gt;\u0026thinsp;10% mitochondrial reads were filtered out. During this process, we also excluded cells with less than 500 or more than 4000 genes detected. In addition, Doublet Finder (v2.0.3) [25] was used to filter out the doublet GEMs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cell clusting\u003c/h2\u003e \u003cp\u003eThe integrated expression matrix was scaled and subjected to dimensionality reduction. To assess the significance of principal components (PCs), we performed a resampling test inspired by the jackStraw procedure. Subsequently, Uniform Manifold Approximation and Projection (UMAP) was applied to these PCs for two-dimensional visualization [26], using the parameters \u0026lsquo;n.neighbors\u0026thinsp;=\u0026thinsp;50\u0026rsquo;, \u0026lsquo;min.dist\u0026thinsp;=\u0026thinsp;0.2\u0026rsquo;, and \u0026lsquo;n.components\u0026thinsp;=\u0026thinsp;2\u0026rsquo;. Finally, clustering analysis was conducted with a resolution parameter of 0.5 to identify distinct groups within the data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cell types identification\u003c/h2\u003e \u003cp\u003eThe initial characterization of cell types within each cluster was conducted using the SingleR package [27] as a preliminary reference. Subsequently, differential expression analysis was primarily executed using the Seurat Find Markers function. This allowed us to visualize cluster-specific marker genes and generate a heatmap featuring the marker genes for each cluster. Cell types were ascertained based on the marker gene expression profiles. Representative makers were verified through in situ hybridization and qRT-PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. In situ hybridization\u003c/h2\u003e \u003cp\u003eGonad samples for in situ hybridization were fixed in 4% paraformaldehyde (prepared with DEPC-treated water). Anti-sense and sense probes for chromogenic in situ hybridization (CISH) were designed using Primer5 software based on the cDNA sequence of maker genes and synthesized by Shanghai Sangon Biotech Company [28]. The DIG-labeled anti-sense probe served as the experimental probe, while the sense probe was used as the negative control. CISH was performed on 4-\u0026micro;m-thick formalin-fixed, paraffin-embedded sections using the Zytofast PLUS CISH Implementation Kit (ZytoVision GmbH, Germany). Mouse anti-DIG antibody (ZytoVision GmbH, Germany) was applied and incubated. After three 1-min TBS washes, slides were incubated with anti-mouse-HRP polymer (30 min, RT). Signal was developed using 3,3\u0026rsquo;-diaminobenzidine (DAB; 50 \u0026micro;L/slide, 10 min, RT) prepared according to the Zytofast PLUS CISH protocol. Counterstaining was performed with hematoxylin, followed by dehydration through graded alcohols. Slides were air-dried, mounted with DPX, and examined under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Quantitative Real-Time PCR\u003c/h2\u003e \u003cp\u003eTo examine the expression of marker genes in testis and ovaries, qRT-PCR was performed. Total RNA was extracted from 100 mg of gonadal tissue using 1 mL TRIzol reagent (TaKaRa, Japan), and first-strand cDNA was synthesized with the Reverse Transcriptase MMLV Kit (TaKaRa). Gene-specific primers were used for amplification in a Bio-Rad iCycler iQ5 real-time PCR system (Hercules, CA, USA). Eukaryotic translation initiation factor 5A (\u003cem\u003eEIF\u003c/em\u003e) served as the reference gene due to its stable expression across various conditions [29]. The PCR protocol consisted of 35 cycles of denaturation at 94\u0026deg;C for 30 s, annealing at 50\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 1 min, followed by a final elongation step at 72\u0026deg;C for 10 min. Each sample was run in quadruplicate, along with three negative controls: nuclease-free water, primer-free water, and template-free water. Fluorescence data were automatically recorded, and dissociation curves were analyzed post-amplification to confirm specificity. Relative mRNA expression levels were quantified using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method [30].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Different expression analysis\u003c/h2\u003e \u003cp\u003eExpression value of each gene in given cluster was compared against the rest of cells using Wilcoxon rank sum test [31]. Significant upregulated genes were identified using a number of criteria. First, genes had to be at least 1.28-fold overexpressed in the target cluster. Second, genes had to be expressed in more than 25% of the cells belonging to the target cluster. Third, p value is less than 0.05. Based on the hypergeometric distribution, and a threshold of p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, the func-tional annotation and classification of DEGs were conducted with the Gene Ontology (GO) database [32] and KEGG database [33] and via the analysis of DEGs enrichment metabolic pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. RNA Interference\u003c/h2\u003e \u003cp\u003eTo knockdown \u003cem\u003eNLRP2\u003c/em\u003e expression, target-specific primers flanked by T7 promoter sequences were designed using the SnapDragon online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.flyrnai.org\u003c/span\u003e\u003cspan address=\"http://www.flyrnai.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Double-stranded RNA (dsRNA) targeting \u003cem\u003eNLRP2\u003c/em\u003e was then synthesized in vitro using the TranscriptAid\u0026trade; T7 High Yield Transcription Kit (Fermentas, USA) following the manufacturer\u0026rsquo;s instructions. For the RNA interference assay, both long-term (PL10 prawns) and short-term (adult male prawns) interference experiments were conducted.\u003c/p\u003e \u003cp\u003e(1) A total of 100 healthy PL10 prawns were selected and injected with \u003cem\u003edsMnNLRP2\u003c/em\u003e at a dosage of 8 \u0026micro;g/g. Injections were administered once every 7 days, and sex ratios as well as steroid hormone levels were measured on days 15, 22, and 30 post-initial injection.\u003c/p\u003e \u003cp\u003e(2) A total of 60 healthy adult male prawns were used for the short-term RNAi experiment. Prawns were injected with \u003cem\u003edsMnNLRP2\u003c/em\u003e following the same procedure and dissected 7 days post-injection to collect androgenic gland for total RNA extraction, in order to assess knockdown efficiency and the expression of OXPHOS-related genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. \u003cem\u003eEnzyme linked Immunosorbent Assay (ELISA)\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe concentrations of MT and E\u003csub\u003e2\u003c/sub\u003e after RNAi treatment were quantified using a double-antibody, one-step sandwich ELISA following the manufacturer\u0026rsquo;s instructions (Shrimp EH ELISA Kits, Lot: 20230724-YJ923014 and 20230724-YJ950014; Mlbio, Shanghai). Prawn tissues were homogenized, centrifuged, and the supernatant was collected for analysis. Samples were loaded onto ELISA plates, incubated, washed, and subsequently treated with enzyme-labeled detection antibodies and substrate solution. Absorbance (OD) was recorded, and hormone concentrations were calculated based on standard curves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll morphometric data were analyzed using one-way ANOVA followed by Tukey's post-hoc test in SPSS 26.0 (IBM). The assumption of homogeneity of variances was verified using Levene's test, and normality was confirmed by Shapiro-Wilk test. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Agricultural University (Approval No. XYZ-2023-0012).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Produce and single-cell RNA sequencing of neo-female\u003c/h2\u003e \u003cp\u003eSex is the most common biological phenomenon in nature, and there are significant differences in morphology, reproduction and behaviour between the male and female sexes of almost all animals. The \u003cem\u003eM. nipponense\u003c/em\u003e, with its high male-female differentiation and rapid reproduction rate [5], represents an ideal material for studying the mechanism of sex differentiation in crustaceans. Previous studies have shown that in oriental river prawns, the glands begin to develop at PL10 and sexual differentiation is complete at PL25 with the emergence of physiological males and females (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To study the mechanisms of sex differentiation in crustaceans, the 10\u0026times; Chromium system was used to perform single-cell sequencing using whole.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC shows the comparison between neo-female and normal male and female prawn. The average weight of male is 1.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 g, and female is 1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 g, while neo-female is 0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 g (Table S1), which is much lower than normal male and female prawn (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In length (eye to caudal segment), male is 4.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 cm and female 3.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 cm, while neo-female is only 2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 cm (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Entire gonads were dissected, dissociated to single cells, and processed through the 10\u0026times; chromium system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Using cellranger, quality control of sequencing quality was performed by removing reads with low sequencing quality and performing preliminary counts of the number of reads measured and sequencing quality for each sample, which resulted in the Q30 of each sample being above 95%.\u003c/p\u003e \u003cp\u003eAfter quality filtering, a total of 32,206 cells were sequenced, of which 4,867 and 5,061 are in the male group, accounting for 31.25% of all the sequenced cells, 6,166 and 3,879 are in the female group, accounting for 30.83% of the sequenced cells, and 6,105 and 6,110 are in the neo-female group, accounting for 37.93% of the sequenced cells. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-a shows the visualisation of the results after cell filtration, overall, the sample saturation in the female, male and neo-female groups are at 98.1%, 89.8% and 51.5%, respectively, which ensured the accuracy of the subsequent analyses.\u003c/p\u003e \u003cp\u003eGonadal cells were clustered based on transcriptional similarity. Clustering identified 25 transcriptionally distinct cell clusters, covering the three types of samples isolated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-b). Among the different cell clusters, cluster 0 contained the largest number of cells, accounting for 18.57% of the total number of sequenced cells (Table S2). More than 40% of the cells were contained in the largest 3 cell clusters, clusters 0,1 and 2, while 10 (clusters 16\u0026ndash;25) of the 25 cell clusters sequenced accounted for less than 1% of the total number of cells. We assessed the proportion of cells in each group (female, male and neo-female) that could be determined with respect to changes in cells during sex differentiation. In the female group, the cell content of clusters 0, 1, 2, and 7 was greater than 5% in all clusters, implying that these clusters consisted predominantly of female cells. And in the male group, the cell content of clusters 3, 4, 6, 10 and 11 are all greater than 5%, which means that these cell groups are mainly composed of male cells. The rest of the cell clusters all have less than 5% of cells. It is noteworthy that in the neo-female group, clusters 0, 1, 2, 3, 5, 6, 10, and 12 were all with cell contents higher than 5%, and clusters 0, 1, 2, 3, 6 and 10 overlapped with females and males, respectively, implying that neo-females are likely to have communal germinal cells and to be involved in the process of sex differentiation. In contrast, the percentage of cells in cluster 25 was less than 1% in all three groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Identification of eight major germ cell types in M. nipponense\u003c/h2\u003e \u003cp\u003eWe next aimed to identify crustacean germ cell specific marker genes based on the 25 clusters we had already found. We screened out some low-quality gene clusters and further mapped female and male germ cell types by comparing cell type-specific marker genes in dot plots, selecting stage-specific enriched genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). To distinguish the major germ cell types in the 25 different clusters, we performed differential gene expression analyses. A columned graph showing the highest differentially expressed genes was generated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, E). Based on the quantitative results, genes with large expression differences were selected, and designed probes based on the gene sequences, which were analysed by multicolour observation through microscope after reacting with nucleic acids in the sperm and the ovary (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, F). Primer sequences and probe sequences of all genes are given in Table S3.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Oogonium cells (cluster 7)\u003c/h2\u003e \u003cp\u003eCluster 7 represents oogonium, which the source of a renewing stem germ cell population in the ovary. It is enriched for genes like \u003cem\u003eGABARAP\u003c/em\u003e. We performed qPCR analyses of stage I ovary (O1) and stage II ovary (O2) determined that the relative expression of \u003cem\u003eGABARAP\u003c/em\u003e in the ovary was 2439.58\u0026thinsp;\u0026plusmn;\u0026thinsp;584.48 and 4470.63\u0026thinsp;\u0026plusmn;\u0026thinsp;219.66, whereas it was not expressed in spermatogonia. In situ hybridization localized \u003cem\u003eGABARAP\u003c/em\u003e to oogonia in the oocyte interstitium, confirming it as a key marker for this cluster.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Primary oocyte cells (cluster 10)\u003c/h2\u003e \u003cp\u003ePrimary oocytes are oocytes that are about to undergo meiosis after mitotic proliferation of the oogonium. In this study, \u003cem\u003ePPAF2\u003c/em\u003e expression in O1 and O2 was 3.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 and 4.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, respectively. To determine the location of PPAF2 within the ovary, we designed probes specific for this gene to hybridise with O1 and found that \u003cem\u003ePPAF2\u003c/em\u003e signals were detected in primary oocyte cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3. Secondary oocyte cells (cluster 8)\u003c/h2\u003e \u003cp\u003eAs the primary oocyte develops, it accumulates yolk, mRNA and enzymes in the cytoplasm and grows into secondary oocyte. Our study demonstrated that \u003cem\u003eGsp-1\u003c/em\u003e expression was 14.76-fold and 29.96-fold higher in O1 and O2 than in the testis, respectively, and was severely up-regulated in the crustacean ovary, proving that it is involved in crustacean ovarian development. In order to locate its position in different germ cells, our observation of its hybridisation signals revealed that \u003cem\u003eGsp-1\u003c/em\u003e signals strongly in the Secondary oocyte cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4. Ovum cells (cluster 1)\u003c/h2\u003e \u003cp\u003eTo date, the \u003cem\u003eA1i3\u003c/em\u003e gene has not been studied functionally in any species, but in the present study, qPCR results showed that the relative expression of \u003cem\u003eA1i3\u003c/em\u003e was 10.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84 in O2, which is 10-fold higher than that in O1. This implies that \u003cem\u003eA1i3\u003c/em\u003e is involved in the process of more mature female germ cell differentiation, and this speculation was confirmed by in situ hybridisation, where strong \u003cem\u003eA1i3\u003c/em\u003e signals were detected in mature oocytes, which were not observed at any other period. These results provide strong evidence that cluster 1 is the ovum cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.2.5. Spermatogonia cells (cluster 11 and 17)\u003c/h2\u003e \u003cp\u003eThe primordial germ cells (PGCs) arise by preformation or epigenesis [34]. The results of qPCR showed that \u003cem\u003ePch-2\u003c/em\u003e was expressed 3982.77\u0026thinsp;\u0026plusmn;\u0026thinsp;245.71 in the spermatheca, while only 2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 and 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 in O1 and O2, which indicated that this gene was specifically expressed in the testis. This result was also demonstrated by in situ hybridisation, where \u003cem\u003ePhc-2\u003c/em\u003e signals were observed to be concentrated in spermatogonia cells in the sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.2.6. Primary spermatocyte cells (cluster 13)\u003c/h2\u003e \u003cp\u003ePrimary spermatocytes are the male germ cells before meiosis I. We compared the relative expression in testis and O1, O2 by qPCR and found that the expression of \u003cem\u003ePF13\u003c/em\u003e gene in testis was 14.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40, which was significantly different compared to 1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 and 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 in O1 and O2. Strong hybridisation signals in primary spermatocyte cells also proved the accuracy of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.2.7. Secondary spermatocyte cells (cluster 20)\u003c/h2\u003e \u003cp\u003eThe secondary spermatocyte is smaller in size than the primary spermatocyte, exists for a short period of time, and quickly enters a second maturational division. Our experiments demonstrated that \u003cem\u003eMLC2\u003c/em\u003e was expressed up to 61.36\u0026thinsp;\u0026plusmn;\u0026thinsp;2.72 in the spermatheca, compared to only 1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 in O1, 35.67-fold higher than in the ovary. In situ hybridisation also detected a strong signal for \u003cem\u003eMLC2\u003c/em\u003e in sperm cells. Our study not only suggests that myosin may play an important role in crustacean spermatogenesis, but also demonstrates that the \u003cem\u003eMLC2\u003c/em\u003e gene can be used as a marker for crustacean secondary spermatocyte cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.2.8. Sperm cells (cluster 6)\u003c/h2\u003e \u003cp\u003eSperm, the terminal stage of spermatogenesis, marks the morphological transformation of secondary spermatocyte cells into mature sperm [35]. Interestingly, the gene \u003cem\u003eVWDE\u003c/em\u003e has not been reported in any reference to its role in males, yet our experiments demonstrated that the expression in the spermatophore of the \u003cem\u003eM. nipponense\u003c/em\u003e was as high as 8063.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1080.47, which is much higher than O1 and O2. Furthermore, after comparing the results of in situ hybridisation for several other genes, the signal for \u003cem\u003eVWDE\u003c/em\u003e was strongest in sperm cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Construction of a single-cell transcriptome atlas of crustacean gonadal cells\u003c/h2\u003e \u003cp\u003eTo study the progression of cell lines throughout development, we generated uniform manifold approximation and projection (UMAP) plot of samples based on previously distinguished marker genes. Cell clustering analysis grouped cells into nine distinct populations using UMAP in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. Female germ cells are roughly categorised on the left side of the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, male germ cells are grouped in the right bottom corner, and in the middle section are cells of undifferentiated types that may consist of blood cells, immune cells, support cells, follicle cells, stem cells, and so on. Based on literature search, qPCR and in situ hybridisation localisation, the germ cells of \u003cem\u003eM. nipponense\u003c/em\u003e were classified into nine clusters, which were assigned as follows: Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA corresponding to female germ cells. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-a (cluster 7) is the UMAP map of oogonium, and the marker gene is \u003cem\u003eGABARAP\u003c/em\u003e (γ-aminobutyric acid A receptor\u0026ndash;associated protein). Fig. A-b (cluster 10) is the primary oocyte, and the marker gene is \u003cem\u003ePPAF2\u003c/em\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-c (cluster 8) is the secondary oocyte. marker gene is \u003cem\u003eGsp-1\u003c/em\u003e (grain softness proteins-1). Fig. A-d (cluster 1) is ovum, marker gene is \u003cem\u003eA1i3\u003c/em\u003e (alpha-1-inhibitor-3). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB corresponds to male germ cells. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-a (clusters 11 and 17) is a UMAP map of spermatogonia, marker gene is \u003cem\u003ePhc-2\u003c/em\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-b (cluster 13) is primary spermatocyte, and the marker gene is \u003cem\u003ePF13\u003c/em\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-c (cluster 20) is a secondary spermatocyte, and the marker gene is \u003cem\u003eMlc2\u003c/em\u003e (myosin regulatory light chain). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-d (cluster 6) corresponds to sperm, and the marker gene is \u003cem\u003ec\u003c/em\u003e. The other 17 cell clusters are classified as Unknown cells. The visualised dot plot of the expression of the newly identified marker genes is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. Moreover, the selected genes could clearly be classified the cells into 25 classes as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE.\u003c/p\u003e \u003cp\u003eNext, we counted the proportion of these germ cells in male, female, and female (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), and the details are shown in Table S4. Unknown cells accounted for the majority of the cells as they contained many unlabelled genes, 4751 (47.30%) in the female group, 7078 (71.20%) in the male group and 7521 (61.57%) in the neo-female group. A total of 10,045 cells were detected in the female group, of which 285 oogonium (2.84%), 127 primary oocyte (1.26%), 177 secondary oocyte (1.76%), and 4,292 ovum (42.73%). A total of 9928 cells were detected in male group, of which 296 spermaton (2.98%), 203 primary spermatocyte (2.04%), 107 secondary spermatocyte (1.08%), and 1292 sperm (13.01%). A total of 12215 cells were detected in neo-female group, of which 5 oogonium (0.04%), 667 primary oocyte (5.46%), 722 secondary oocyte (5.91%), 1,626 ovum (13.31%), 378 spermaton (3.09%), 5 primary spermatocyte (0.04%), 5 secondary spermatocyte (0.04%), and 155 sperm (1.27%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Pseudotime reconstruction of male gonadal cells in neo-females\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;4 shows the pseudotime analysis of the spermatogenic lineage in males and neo-females. The reconstructed trajectories show that spermatogonia, primary spermatocytes, secondary spermatocytes and sperm are ordered along a continuous developmental path (Fig.\u0026nbsp;4A). In the testes of control males (Fig.\u0026nbsp;4A-a), the cells were distributed across the entire trajectory, from the root to the terminal branch. This indicates complete progression from undifferentiated spermatogonia to fully mature sperm. By contrast, neo-female germ cells (Fig.\u0026nbsp;4A-b) accumulated primarily in the proximal segments of the trajectory, with only a few cells occupying the distal terminal region corresponding to late spermatids and spermatozoa.\u003c/p\u003e \u003cp\u003eConsistent with this, a quantitative comparison of cell numbers at each stage revealed that the gonads of neo-females contained a higher proportion of spermatogonia, but a significantly lower proportion of other male gonadal cells (Fig.\u0026nbsp;4B). Further emphasising this phenomenon, kernel density estimation of pseudotime distributions showed that male cells exhibited a major density peak at late pseudotime, whereas neo-female cells showed a dominant peak at earlier pseudotime values (Fig.\u0026nbsp;4C). This confirms that the majority of spermatogenic cells in neo-females are arrested at early developmental stages.\u003c/p\u003e \u003cp\u003eThe gene expression dynamics along pseudotime were then examined (Fig.\u0026nbsp;4D, E). The heatmap in Fig.\u0026nbsp;4D shows three major gene modules that are sequentially activated from early to late pseudotime, corresponding to early, developing and mature spermatogenic cells. Expression of the early module is highest at proximal pseudotime, whereas the late module displays a marked increase toward terminal pseudotime in control males. In neo-female cells, the overall signal of this late module is noticeably reduced. Representative genes, including \u003cem\u003eMhc\u003c/em\u003e, \u003cem\u003ewupA\u003c/em\u003e, \u003cem\u003eTpnC41c\u003c/em\u003e, \u003cem\u003eacta1\u003c/em\u003e, \u003cem\u003eNLRP2\u003c/em\u003e and \u003cem\u003ednajc3\u003c/em\u003e, exhibit a gradual increase in expression along pseudotime in males, with maximal levels at late spermatogenic stages (Fig.\u0026nbsp;4E). By contrast, in neo-female germ cells most data points are concentrated at early pseudotime values, and the high-expression domain at late pseudotime is largely absent.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;4 Pseudotime trajectory analysis reveals spermatogenic arrest in neo-female testis. (A) Reconstructed developmental trajectories of spermatogenic cells from males (a) and neo-females (b). Each dot represents a single cell and is coloured according to pseudotime, with earlier to later states shown from blue to green. Black circles indicate inferred branching points along the trajectory. (B) Stacked bar plots showing the numbers of cells from males and neo-females in each pseudotime state for the indicated spermatogenic stages. (C) Kernel density plots of pseudotime distributions for male and neo-female male gonadal cells. (D) Heatmap of dynamically regulated genes ordered by pseudotime. (E) Expression patterns of representative spermatogenesis-related genes along pseudotime. Dot plots show single-cell expression of (a) \u003cem\u003eMhc\u003c/em\u003e, (b) \u003cem\u003ewupA\u003c/em\u003e, (c) \u003cem\u003eTpnC41c\u003c/em\u003e, (d) \u003cem\u003eacta1\u003c/em\u003e, (e) \u003cem\u003eNLRP2\u003c/em\u003e and (f) \u003cem\u003ednajc3\u003c/em\u003e overlaid on fitted pseudotime trends, highlighting the impaired up-regulation of late spermatogenic markers in neo-female testis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Pseudotime reconstruction of female gonadal cells in neo-females\u003c/h2\u003e \u003cp\u003ePseudotime analysis was then applied to female gonadal cells from females and neo-females (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The reconstructed trajectories arranged oogonia, primary oocytes, secondary oocytes and ova along a continuous developmental path (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In ovaries of control females, cells were distributed along the full trajectory from the root to the terminal branch, indicating the presence of oogenic cells at all stages. In contrast, cells from neo-females were mainly located in the proximal and middle segments of the trajectory, with only a small fraction occupying the distal region corresponding to late oocytes.\u003c/p\u003e \u003cp\u003eQuantitative comparison of cell numbers in each pseudotime state confirmed this pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In the case of oogonium, primary oocytes and secondary oocytes, neo-females exhibited a higher proportion of cells in early pseudotime states relative to females. By contrast, in ovum, cells from females predominated in later pseudotime states. Furthermore, kernel density plots of pseudotime distributions revealed a leftward shift for neo-female cells in comparison with female cells, with the primary density peak of neo-females situated at earlier pseudotime values (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eSubsequently, the dynamics of gene expression along pseudotime were examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). The heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) demonstrates several gene modules that are sequentially activated from early to late pseudotime, corresponding to early, developing and mature oogenic cells. In females, early modules are enriched at low pseudotime, whereas late modules show increased expression towards terminal pseudotime. In neo-female, the intensity of the late module is reduced and concentrated in a narrower pseudotime window. Representative genes, including \u003cem\u003etdc-1\u003c/em\u003e, \u003cem\u003epnt\u003c/em\u003e, \u003cem\u003epfn-3\u003c/em\u003e, \u003cem\u003etsr\u003c/em\u003e, \u003cem\u003eGRN\u003c/em\u003e and \u003cem\u003eGpdh1\u003c/em\u003e, display characteristic changes in expression along pseudotime in females, with distinct expression domains at specific stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In neo-female oogenic cells, the majority of data points for these genes are concentrated at earlier pseudotime values, and the high-expression regions at late pseudotime observed in females are diminished. Collectively, these results indicate that the oogenic lineage in neo-females is biased towards early developmental states, with reduced representation of late-stage oocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Histological observation and differentially expressed genes in neo-female\u003c/h2\u003e \u003cp\u003eTo corroborate the results of single-cell sequencing, we performed serial, multisegmented histological observation of 1-month-old neo-female prawn gonads. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA shows well the coexistence of testis-ovary in neo-female. On the left side is a part of the ovarian germ cells, in which a large number of primary oocytes are observed in the 100 \u0026micro;m bar, and it is characterised by gradual disappearance of the nucleus and larger egg diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-b). There are also a small number of oogonium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-a) and secondary oocyte (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-c), the former with a distinct nucleus and a small diameter, the latter with a disappearing nucleus and a large accumulation of yolk granules in the cytoplasm. On the right side are the germ cells of testis, and as far as we can see there are only a few small amounts of spermation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-d), which are the most infantile spermatogonial cells and are finely granular. The development of mature oocytes was not observed in the entire neo-female gonadal tissue. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB is a schematic representation of the tissue cross-sectioned for a better demonstration of testis-ovary coexistence. The results of the histological sections are consistent with the results of the scRNA-seq, thus we obtained an important result that some of the most primitive germ cells of males are still retained in neo-females.\u003c/p\u003e \u003cp\u003eHowever, the differences between these four germ cells in neo-female gonads compared to normal males and females have not been clarified, so we compared up and down regulated differentially expressed genes (DEGs) of female vs neo-female and male vs neo-female, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) to determine which genes were significant. The results showed that \u003cem\u003ePabpc1\u003c/em\u003e, \u003cem\u003eBCO1\u003c/em\u003e, \u003cem\u003eRpS27\u003c/em\u003e, \u003cem\u003eGpdh1\u003c/em\u003e, \u003cem\u003eFLNC\u003c/em\u003e, \u003cem\u003eGRN\u003c/em\u003e and other genes play important regulatory roles in the growth, maturation and differentiation of female germ cells. And \u003cem\u003eSam-S\u003c/em\u003e, \u003cem\u003eCd63\u003c/em\u003e, \u003cem\u003eRpL39\u003c/em\u003e and \u003cem\u003ePabpc1\u003c/em\u003e are likely to be the key genes responsible for the non-full sex reversal in neo-female. It is noteworthy that in these four comparison groups, there are some genes repeated multiple times, which means that they play and their important role in the gonadal development of \u003cem\u003eM. nipponense\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, in addition to the differences between the male, female and neo-female groups, we screened for the presence of DEGs in the four germ cells in common by Wayne diagrams and found the top 5 genes most enriched in neo-females (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). They were \u003cem\u003eSyb\u003c/em\u003e, \u003cem\u003eNPC1b\u003c/em\u003e, \u003cem\u003eZcchc24\u003c/em\u003e, \u003cem\u003eNLRP2\u003c/em\u003e and \u003cem\u003eSmg9\u003c/em\u003e. The t-SNE plots for these five genes are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e-G, which illustrating the expression patterns of these candidates within each cell type demonstrated a high level of overlap and clear boundaries, emphasizing their conserved roles in crustacean oogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Consistent dysregulation of oxidative phosphorylation in neo-female germ cells\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e presents the comparative pathway enrichment analysis that identifies oxidative phosphorylation as the key biological process altered in neo-female gonads. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment was performed using the DEGs obtained from four critical germ cell stages\u0026mdash;oogonium, primary oocyte, secondary oocyte, and spermaton (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Across all stages, oxidative phosphorylation (OXPHOS) ranked among the significantly enriched pathways, consistently appearing at the top of the enrichment lists for both female and male gonadal cells. Other pathways such as ribosome, spliceosome, and protein processing in the endoplasmic reticulum were also enriched but did not show the same stage-wide consistency. This indicates that mitochondrial energy metabolism is a common regulatory node affected in neo-female germ cells.\u003c/p\u003e \u003cp\u003eTherefore, we organized the gene expression of OXPHOS in female germ cells (oogonium, primary oocyte and secondary oocyte) and male germ cell (spermation) separately and plotted the mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Critically, OXPHOS imbalance was the universal signature. A total of 12 genes were screened, covering the entire process of OXPHOS to produce ATP, including Ndufs1, Ndufb3, Ndufs5, SDHA, SDHC, QCR8, Cyt1, Cyc, COX7A, Nurf-38, ATPsynC and AAEL. Most of the genes were down-regulated in comparison with female germ cells and up-regulated in comparison with male germ cells. It is noteworthy that four genes, Ndufs1, Ndufs3, SDHA, and Cyt1, were expressed in the same pattern in both comparison groups.\u003c/p\u003e \u003cp\u003eIn female germ cells, dysregulation leads to downregulation of the majority of gene expression. This directly results in impaired OXPHOS function, significantly reducing ATP production, decreasing the efficiency of the mitochondrial ETC, and accelerating the accumulation of ROS. In male germ cells, excessive activation causes abnormal activation of downstream components of OXPHOS. This mitochondrial respiratory dysfunction leads to excessive consumption of the ETC, triggering electron transport overload. The abnormally steep proton gradient exacerbates the explosive production of ROS, causing redox imbalance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.8. NLRP2 is a germ cell specific gene of testis development and oxidative phosphorylation\u003c/h2\u003e \u003cp\u003eTo verify the effect of the differential genes on \u003cem\u003eM. nipponense\u003c/em\u003e gonadal development, we screened the distribution of these five genes in different germ cells and found that the expression of \u003cem\u003eNLRP2\u003c/em\u003e is germ cell specific and restricted to primary spermatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The UMAP plot of the dataset also confirms this result, as we observed that the \u003cem\u003eNLRP2\u003c/em\u003e is enriched for expression in primary spermatocytes. These results suggest that \u003cem\u003eNLRP2\u003c/em\u003e is a germ cell specific marker associated with a defined stage of spermatogenesis. Multiple sequence alignment of the \u003cem\u003eMnNLRP2\u003c/em\u003e protein from \u003cem\u003eM. nipponense\u003c/em\u003e with representative crustacean \u003cem\u003eMnNLRP2\u003c/em\u003e homologs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-a) showed that the full-length protein (649 aa) exhibits a high degree of similarity within the conserved regions, with the canonical NACHT and LRR domain clearly identifiable. Tissue-level expression profiling further supported this conclusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-b, c). In males, \u003cem\u003eMnNLRP2\u003c/em\u003e transcripts were detected in all examined tissues but were strongly enriched in the testis, where the relative expression reached 18.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03, significantly higher than in other tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The hepatopancreas and androgenic gland also showed relatively high expression (16.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18 and 10.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40, respectively), whereas levels in eye stalk, cerebral ganglia, heart and gill were much lower. In females, \u003cem\u003eMnNLRP2\u003c/em\u003e expression was extremely low in the eye (undetectable) and ovary (1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04), both significantly lower than in other female tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast to males, the highest expression in females was observed in muscle, reaching 383.72\u0026thinsp;\u0026plusmn;\u0026thinsp;28.87. These patterns indicate pronounced tissue specificity and sexual dimorphism, with strong enrichment in male gonads. The phylogenetic tree constructed based on these amino acid sequences further resolved the evolutionary relationships of \u003cem\u003eMnNLRP2\u003c/em\u003e with other arthropod \u003cem\u003eMnNLRP2\u003c/em\u003e proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-d). M. nipponense clustered tightly with \u003cem\u003eMacrobrachium rosenbergii\u003c/em\u003e and \u003cem\u003ePalaemon carinicauda\u003c/em\u003e, with all major nodes supported by a bootstrap value of 100, highlighting the strong evolutionary conservation of this gene within the family \u003cem\u003ePalaemonidae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFunctional interference experiments were then performed. Injection of \u003cem\u003edsMnNLRP2\u003c/em\u003e into PL10 prawns led to time-dependent changes in sex-related traits (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). At 30 days post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-a), the number of males in the control group was 30.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08, whereas the \u003cem\u003edsMnNLRP2\u003c/em\u003e group contained only 14.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.13 males. Female numbers showed a similar decline, decreasing from 31.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65 in controls to 17.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51 in the RNAi group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Steroid hormone measurements showed a consistent endocrine shift following \u003cem\u003eMnNLRP2\u003c/em\u003e silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-b,c). In the control group, the 17α-Methyltestosterone (MT) concentrations in male prawns at days 15, 22, and 30 were significantly lower than those in the \u003cem\u003edsMnNLRP2\u003c/em\u003e treated males (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, the E\u003csub\u003e2\u003c/sub\u003e levels of control females were markedly lower than those of the treated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicate that \u003cem\u003eMnNLRP2\u003c/em\u003e modulates endocrine status and influences sex differentiation during early developmental stages.\u003c/p\u003e \u003cp\u003eIn adult males,the qPCR analysis shows that 1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 and 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 in the experimental group, compared to 22.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 and 2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 in the control group, resulted in interference efficiencies of 92.21% and 61.98% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The expression level of \u003cem\u003eMnNLRP2\u003c/em\u003e in the RNAi group is significantly lower than that in the control group throughout the whole experiment, this result indicates the efficiency of the interference experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-a). Interestingly, after 7 days of interference, the histological sections of the experimental and control groups are highly different (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-b,c). A large number of sperm are observed in the testis of the control group, while a large number of spermatogonia were present in the experimental group despite the existence of sperm, suggesting that \u003cem\u003eMnNLRP2\u003c/em\u003e interfering effectively inhibited the spermatogenesis process, and that the testis of the control group more mature. To further validate the role of \u003cem\u003eMnNLRP2\u003c/em\u003e in OXPHOS in male \u003cem\u003eM. nipponense\u003c/em\u003e, we identified gene expression during 7 days after RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-d). Compared with the control group, the expression of most genes was significantly downregulated (\u003cem\u003eNdufb3\u003c/em\u003e, \u003cem\u003eSDHC\u003c/em\u003e, \u003cem\u003eQCR8\u003c/em\u003e, \u003cem\u003eCyc\u003c/em\u003e, \u003cem\u003eNurf-38\u003c/em\u003e, \u003cem\u003eATPSynO\u003c/em\u003e, and \u003cem\u003eAAEL\u003c/em\u003e), indicating that \u003cem\u003eNLRP2\u003c/em\u003e plays a protective role in ATP production in mitochondria. However, the gene affecting complex III (\u003cem\u003eCyt1\u003c/em\u003e) exhibited an expression pattern opposite to that of other components. Collectively, these data indicate that \u003cem\u003eMnNLRP2\u003c/em\u003e is a conserved, gonad-enriched gene that participates in the regulation of steroidogenesis, testicular development and oxidative phosphorylation in \u003cem\u003eM. nipponense\u003c/em\u003e. Primer sequences of all genes are given in Table S5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSex reversal achieved through sex steroid manipulation is a widely practiced technique in fish aquaculture. Previous studies reported successful induction of crustacean sex reversal by in vitro administration of E\u003csub\u003e2\u003c/sub\u003e [3]. Similar experiments have been studied in species such as \u003cem\u003ePenaeus merguiensis\u003c/em\u003e [36], crayfish [37] and shrimp [38], and it seems possible to obtain all-female populations through exogenous E\u003csub\u003e2\u003c/sub\u003e. Our study obtained neo-females by feeding E\u003csub\u003e2\u003c/sub\u003e, which transformed from males to females and their body size and weight were smaller compared to normal females, implying that sex reversal caused by E\u003csub\u003e2\u003c/sub\u003e may be damaging to male health. Inspired by these advances and results, we utilised scRNA-seq to construct single-cell atlases of the gonads (testis and ovary) of females, males and neo-females to investigate the mechanisms of gonadal development, sex differentiation and sex reversal in crustaceans (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We integrated single-cell transcriptomics, in situ hybridisation and functional expression analyses, filling a gap in the molecular classification of crustacean germ cells. In this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), eight different types of germ cell populations were identified for the first time, including oogonium (\u003cem\u003eGABARAP\u003c/em\u003e), primary oocyte (\u003cem\u003ePPAF2\u003c/em\u003e), secondary oocyte (\u003cem\u003eGsp-1\u003c/em\u003e), oocyte (\u003cem\u003eA1i3\u003c/em\u003e), spermation (\u003cem\u003ePhc-2\u003c/em\u003e), primary spermatocyte (\u003cem\u003ePF13\u003c/em\u003e), secondary spermatocyte (\u003cem\u003eMlc2\u003c/em\u003e) and sperm (\u003cem\u003eVWDE\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eAt the cellular level, subclustering and quantitative comparison of cell-type proportions revealed that both the oogenic and spermatogenic lineages in neo-female gonads are dominated by early-stage cells, whereas late oocytes and spermatozoa are markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This is not the first reported case of sex reversal in a species. Histological observations of bluegill \u003cem\u003eLepomis macrochirus\u003c/em\u003e fed different concentrations of E\u003csub\u003e2\u003c/sub\u003e revealed that 13.3% and 5.0% of the intersex fish were determined to come from the 50 and 100 mg kg- 1 E\u003csub\u003e2\u003c/sub\u003e treatment groups, respectively, with 6.9% and 4.1% of the gonadal area containing spermatocytes [39]. Sex-reversed type XX \u003cem\u003eOryzias latipes\u003c/em\u003e showed isogenic spermatocysts with active spermatogenesis [40]. This pattern is mirrored in the histological sections, where neo-female gonads display abundant oogonia/early oocytes or spermatogonia with relatively few fully developed gametes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;C).\u003c/p\u003e \u003cp\u003eThis study also revealed the diversity of germ cell development (Fig.\u0026nbsp;4). For example, ovum contain three subtypes that may correspond to three different stages of yolk synthesis, nuclear maturation, and preovulatory activation [41]. The formation of fertile spermatozoa is the result of spectacular stages of cell differentiation that begin in the male gonad and finish in the female tract [42]. Trajectory analyses provide dynamic support for this conclusion. In males, spermatogenic cells span the entire pseudotime trajectory from spermatogonia to mature sperm (Fig.\u0026nbsp;4A), and in females, oogenic cells similarly extend from oogonia to ova (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In neo-females, however, the majority of cells cluster at proximal or intermediate pseudotime, with only a small fraction occupying terminal positions. Density plots show a clear leftward shift in pseudotime distributions for both spermatogenic and oogenic cells in neo-females (Fig.\u0026nbsp;4C, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), indicating that germ-cell populations are biased toward earlier developmental states. These observations suggested that estrogen induced sex reversal in \u003cem\u003eM. nipponense\u003c/em\u003e is accompanied not by a complete reprogramming of germ-cell fate, but rather by a failure of germ cells to progress to fully mature stages. Estrogen exposure disrupted the intrinsic maturation trajectory of germ cells, leading to an ovary-like phenotype with arrested gametogenesis.\u003c/p\u003e \u003cp\u003eThis study revealed that excessive E₂ induced redox metabolic disorders in germ cells during female sex reversal, despite inducing males to become neo-females (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This is consistent with the previous findings of organ damage and developmental abnormalities due to oestrogen overdose [43,44]. Differential expression analyses across corresponding stages further show that the transcriptional landscape of neo-female germ cells is extensively remodeled. Volcano plots for oogonia, primary and secondary oocytes and spermaton highlight numerous DEGs in the comparisons \u0026ldquo;female vs neo-female\u0026rdquo; and \u0026ldquo;male vs neo-female\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). We next examined the expression patterns of representative genes at single-cell resolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eF,G). Synaptic vesicle-associated gene Syb and lipid transport\u0026ndash;related NPC1b were preferentially expressed in selected oogenic clusters in control females, with clear, stage-specific enrichment along the oocyte lineage. In neo-females, the distribution and intensity of these signals were altered, consistent with their identification as DEGs. Zcchc24 and Smg9, which showed distinct cluster enrichment patterns in the integrated UMAP, also exhibited significant expression differences between controls and neo-females, indicating that mRNA processing and RNA surveillance pathways may be modulated in neo-female germ cells. Notably, NLRP2 displayed restricted expression in specific spermatogenic clusters, matching its primary localization to early/mid spermatogenic stages observed in the pseudotime analysis and providing a basis for subsequent functional studies.\u003c/p\u003e \u003cp\u003eAmong all functional categories enriched in the DEGs, oxidative phosphorylation emerged as the most consistent pathway across key germ-cell stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In oogonia, primary oocytes, secondary oocytes and spermaton, genes encoding components of the electron transport chain and ATP synthase were significantly altered in neo-females. Other pathways such as ribosome and spliceosome were also enriched, but oxidative phosphorylation was unique in spanning both oogenic and spermatogenic lineages and multiple developmental stages. OXPHOS is a central metabolic pathway for ATP production in mitochondria via the ETC and is essential for maintaining germ cell function [45]. In oogonium and primary oocytes, E₂ triggers aberrant metabolic activation, including accelerated ribosome synthesis and AMPK signaling pathway upregulation, indicating cellular energy stress and premature oocyte initiation under conditions of ATP/AMP imbalance [46,47]. This finding is consistent with the characterisation of the early stages of oocyte development, when oogonia are in transcriptional quiescence but reserve ribosome required for translation to support protein synthesis in subsequent developmental stages [48,49]. However, E₂ simultaneously impairs maturation in secondary oocytes, aberrant splicing function may result in a lack of transcription product diversity [50]. Combined with the disruption of the secondary oocyte pathway observed in neo-females, it is speculated that E\u003csub\u003e2\u003c/sub\u003e may interfere with the quality of RNA processing during the later stages of oocyte development, which may be one of the central mechanisms by which germ cell maturation is impaired in sex-reversed individuals. Abnormal carbon metabolism has been shown to inhibit spermatogenesis and disrupt DNA epitope modification [51,52], and the dysregulation of the same pathway found in this study in spermatons of neo-females implies that E\u003csub\u003e2\u003c/sub\u003e may interfere with normal spermaton differentiation by impairing the methylation cycle [53,54]. In persistent spermatogonia within neo-female gonads, E₂ causes significant dysregulation of the carbon metabolism pathway, directly linked to reactive oxygen species ROS overproduction and impaired DNA methylation cycles, thereby inhibiting normal spermatogenesis and potentially compromising genetic stability. These results are consistent with our previous findings that excessive estrogen quantities can cause harm to the animal, such as damage to organs, abnormal development, cancer risk, etc [55]. Thus, our results suggested that E₂ exerts a dual pathology: promoting precocious metabolic activity and energy stress in early-stage oocytes while disrupting redox balance, metabolic regulation, and epigenetic stability, ultimately impairing germ cell maturation and quality in sex-reversed gonads.\"\u003c/p\u003e \u003cp\u003eTo elucidate how OXPHOS dysfunction drives germ cell differentiation disorders during sexual reversal, we systematically screened and integrated the expression profiles of 12 core genes covering the entire ATP synthesis pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In female reproduction, oocyte maturation, fertilisation and embryo development require stable mitochondrial function, and abnormal OXPHOS triggers the accumulation of ROS, leading to oxidative stress, which affects oocyte quality [56]. It has been shown that OXPHOS leads to abnormal meiosis and reduced fertilisation rate in mouse oocytes by inhibiting MPF (M-phase promoting factor) activity. Furthermore, OXPHOS damage leads to abnormal meiosis, decreased fertilisation rate and reduced embryo quality [57]. Our results indicate that dysregulation of female germ cells reduces ATP production and decreases ETC efficiency, accelerating ROS accumulation and ultimately disrupting normal oocyte maturation. Our results showed that OXPHOS is significantly inhibited in female germ cells in neo-female, resulting in dysfunctional oocyte reproduction. In male reproduction, sperm motility is highly dependent on ATP produced by mitochondria, and OXPHOS is the main pathway of sperm energy metabolism [58]. For example, human sperm rely primarily on mitochondrial OXPHOS to generate ATP in oxygen-rich environments, with glycolysis serving only as a secondary pathway [59]. Mitochondrial OXPHOS dysfunction directly affects sperm viability and morphology, and animal models have shown that increased mitochondrial ROS after testicular TD activate the caspase-9-dependent apoptotic pathway and induce germ cell apoptosis [60,61]. Our results confirmed this conclusion that OXPHOS was significantly activated in spermation in neo-females, leading to ETC overload, a sharp increase in ROS content, and ultimately a breakdown in redox homeostasis. These may be one of the main reasons for the lack of potential for spermation to continue to develop into sperm, suggesting that male reproductive function was greatly suppressed after E\u003csub\u003e2\u003c/sub\u003e feeding. On the other hand, we observed that the weight of neo-female was smaller than female. Therefore, we speculate that the reason for the incomplete reversal of male crustaceans by E\u003csub\u003e2\u003c/sub\u003e may be that the steroid hormone disrupts the normal synthesis of ATP by OXPHOS, which in turn causes DNA damage and apoptosis. The energy used by males to promote maturation of the testis, although suppressed, still has a tendency to develop spermation, which results in the neo-male still retaining a portion of spermation, and a defective maturation of the female germ cells and weight growth. Addressing this problem may require improved types and doses of hormones to be fed, or feeding at an earlier period of \u003cem\u003eM. nipponense\u003c/em\u003e development to achieve full feminisation of the population. The results revealed a significant bidirectional imbalance pattern, which is the core mechanism of redox and energy metabolism disorders.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNLRP2\u003c/em\u003e (NOD-like receptor family pyrin domain-containing 2) emerged from the integrated analysis as a particularly interesting candidate. At the single-cell level, \u003cem\u003eNLRP2\u003c/em\u003e expression is highly restricted to primary spermatocytes in the spermatogenic lineage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), indicating a narrow developmental window of activity. \u003cem\u003eNLRP2\u003c/em\u003e is a member of the NLR family, which is mainly involved in the regulation of inflammatory responses, apoptosis and epigenetic modifications [62]. Sequence alignment shows that \u003cem\u003eMnNLRP2\u003c/em\u003e conserves the canonical NACHT and LRR domains characteristic of NLR proteins, while displaying species-specific variation in terminal regions, and phylogenetic analysis places it firmly within the decapod NLRP2 clade together with other shrimp, crab and crayfish species (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-a,d). This supports that \u003cem\u003eMnNLRP2\u003c/em\u003e may participate in conserved cellular processes.\u003c/p\u003e \u003cp\u003eThe critical role in the reproductive system and embryo development has been progressively discovered in recent years, and it is decisive for female reproduction through the maintenance of oocyte quality, early embryo development and maternal genetic stability [63\u0026ndash;65]. Its defects can lead to embryonic arrest, decline in fertility with age and abnormal development of the offspring [66,67]. In a mouse study, they knocked down \u003cem\u003eMnNLRP2\u003c/em\u003e transcription specifically in mouse germinal vesicle oocytes, showed that \u003cem\u003eMnNLRP2\u003c/em\u003e is a member of the mammalian maternal effect genes and required for early embryonic development in the mouse [68]. Tissue distribution analysis showed that \u003cem\u003eMnNLRP2\u003c/em\u003e was strongly enriched in male gonads, with high expression in the testis and androgenic gland, low levels in most somatic tissues, and only weak expression in female ovaries, suggesting a close association with male reproduction. Functional knockdown experiments in PL10 prawns further supported this role. Repeated injection of \u003cem\u003edsMnNLRP2\u003c/em\u003e during early post-larval development progressively altered the phenotypic sex ratio, leading to a significant decline in the proportion and number of males and a corresponding increase in females by 30 days post-injection, consistent with a partial male-to-female sex reversal (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-a). After five rounds of interference, hormone measurements revealed that MT levels in phenotypic males of the RNAi group were significantly lower than those of controls, whereas E\u003csub\u003e2\u003c/sub\u003e concentrations in phenotypic females were significantly elevated compared with the corresponding control females (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-b,c). These results indicated that \u003cem\u003eMnNLRP2\u003c/em\u003e knockdown disrupts normal androgen estrogen balance in a sex-specific manner and promotes feminization at early developmental stages.\u003c/p\u003e \u003cp\u003eIn adult males, sustained \u003cem\u003eMnNLRP2\u003c/em\u003e silencing led to pronounced histological changes in the testis. Testes from control prawns contained well-organized lobules filled with abundant mature spermatozoa, whereas testes from \u003cem\u003eMnNLRP2\u003c/em\u003e knockdown prawns were dominated by spermatogonia and early spermatocytes, with markedly fewer sperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-b,c). This early-stage accumulation closely mirrors the spermatogenic arrest observed in neo-female gonads, supporting the conclusion that \u003cem\u003eMnNLRP2\u003c/em\u003e is required for proper progression of spermatogenesis in \u003cem\u003eM. nipponense.\u003c/em\u003e Additionally, we found that \u003cem\u003eMnNLRP2\u003c/em\u003e inhibits sperm maturation and affects gene expression during OXPHOS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-d). \u003cem\u003eMnNLRP2\u003c/em\u003efunctions as an inducible inflammatory mediator that regulates NF-κB activation [69]. \u003cem\u003eMnNLRP2\u003c/em\u003e inflammasome can be activated by IFN, ATP and LPS leading to inflammatory responses and involved in regulating \u003cem\u003eNLRP3\u003c/em\u003e inflammasome [70,71]. Given the crosstalk between NF-κB signaling and oxidative stress responses (e.g., ROS generation and antioxidant defense), \u003cem\u003eMnNLRP2\u003c/em\u003e likely modulates cellular redox balance through this inflammatory axis. Furthermore, \u003cem\u003eNLRP\u003c/em\u003e family members (e.g., \u003cem\u003eNLRP4\u003c/em\u003e) can interact with Fas-associated factor 1 (FAF1), a negative regulator of inflammatory signals [64,72]. If \u003cem\u003eMnNLRP2\u003c/em\u003e similarly modulates FAF1 or analogous partners, it may suppress pro-oxidant pathways (e.g., caspase activation or mitochondrial ROS production), thereby protecting cells from oxidative stress. Therefore, we speculated that \u003cem\u003eMnNLRP2\u003c/em\u003e may combat oxidative stress through multiple mechanisms, including tuning NF-κB-mediated inflammation to prevent ROS overproduction or scaffolding SCMC complexes to stabilize stress-defense factors.\u003c/p\u003e \u003cp\u003eIn conclusion, we establish a single-nucleus transcriptomic atlas of \u003cem\u003eM. nipponense\u003c/em\u003e gonads and provide a framework of validated markers for staging crustacean germ cells. Using this atlas, we show that estrogen-induced neo-females are characterized by a strong shift in cell composition and developmental trajectories toward early oogenic and spermatogenic stages, with a marked depletion of mature gametes. Stage-resolved transcriptomics and enrichment analyses consistently implicate oxidative phosphorylation as the pathway most disturbed in neo-female germ cells. We further identify \u003cem\u003eMnNLRP2\u003c/em\u003e as a primary spermatocyte specific, testis enriched gene. Functional knockdown of \u003cem\u003eMnNLRP2\u003c/em\u003e alters sex ratios and sex steroid levels in PL10 prawns and causes spermatogenic arrest and dysregulation of oxidative-phosphorylation genes in adults. Together, our findings reveal that estrogen induced sex reversal in \u003cem\u003eM. nipponense\u003c/em\u003e involves germ-cell arrest linked to mitochondrial dysfunction and highlight \u003cem\u003eMnNLRP2\u003c/em\u003e as a key regulator of male gonadal development and endocrine balance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by grants from National Key R\u0026amp;D Program of China (2023YFD2401000); Central Public-interest Scientific Institution Basal Research Fund CAFS (2023TD39); the earmarked fund for CARS-48-07; the seed industry revitalization project of Jiangsu province (JBGS [2021]118). Thanks to the Jiangsu Province Platform for the Conservation and Utilization of Agricultural Germplasm.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eP.C. designed the experiments, fed the prawns, analysed the data and wrote the manuscript; Z.G. wrote the materials and methods; H.F. supervised the experiments; S.J. (Sufei Jiang) and Y.X. provided the experimental prawns and the steroid hormones; W.Z., S.J. (Shubo Jin) and H.Q. carried out the qPCR analyses and made some further modifications. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eG.A. Boxshall, Crustacean classification: on-going controversies and unresolved problems, Zootaxa 1668 (2007) 313\u0026ndash;325. DOI: 10.11646/zootaxa.1668.1.16\u003c/li\u003e\n\u003cli\u003eZ. Ye, T. Bishop, Y. Wang, R. Shahriari, M. Lynch, Evolution of sex determination in crustaceans, Mar. Life Sci. 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DOI: 10.1111/imm.13699\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"sex reversal, scRNA-seq, OXPHOS, mitochondrial dysfunction, NLRP2","lastPublishedDoi":"10.21203/rs.3.rs-8343381/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8343381/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSex reversal is widely used in biological breeding, but its cellular and molecular consequences in crustaceans are poorly understood. Here, we combine estrogen induced feminization, single cell RNA sequencing, histology and RNA interference to dissect gonadal development in the oriental river prawn (\u003cem\u003eMacrobrachium nipponense\u003c/em\u003e). Feeding post-larval males with 17β-estradiol generated neo-females with ovary like gonads. Single cell profiling of testis, ovaries and neo-female gonads produced a gonadal atlas in which, using newly validated markers, we resolved major oogenic and spermatogenic cell types. Cell-type composition and pseudotime analyses showed that neo-female gonads are dominated by early germ cells, whereas mature oocytes and spermatozoa are strongly depleted; histology confirmed this developmental arrest. Stage resolved differential expression and KEGG enrichment consistently highlighted oxidative phosphorylation as the most perturbed pathway across key germ-cell stages. Among the affected genes, \u003cem\u003eMnNLRP2\u003c/em\u003e displayed primary spermatocyte specific and testis enriched expression and was conserved among decapods. RNA interference of \u003cem\u003eMnNLRP2\u003c/em\u003e in PL10 prawns shifted phenotypic sex ratios toward females and altered sex steroid levels, reducing MT in males while elevating E\u003csub\u003e2\u003c/sub\u003e in females; knockdown in adults caused spermatogenic arrest and down-regulation of oxidative phosphorylation components. These findings provide unprecedented insights into crustacean germ cell dynamics and the metabolic and transcriptional networks governing incomplete sex reversal, thereby offering critical resources for reproductive biology and aquaculture research.\u003c/p\u003e","manuscriptTitle":"Single-cell atlas of prawn gonads identifies NLRP2 regulated mitochondrial dysfunction in estrogen induced sex reversal","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-31 07:06:04","doi":"10.21203/rs.3.rs-8343381/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e3eb5b32-0686-4604-9800-9342ea23b860","owner":[],"postedDate":"December 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60326538,"name":"Biological sciences/Genetics/Animal breeding"},{"id":60326539,"name":"Biological sciences/Developmental biology/Germline development/Oogenesis"}],"tags":[],"updatedAt":"2026-05-07T06:50:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-31 07:06:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8343381","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8343381","identity":"rs-8343381","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}