Gustavus, a positive regulator in arthropod reproduction, features a conserved SPRY and a C-terminal SOCS box domain and belongs to the SPSB protein family. The SPSB family, encompassing SPSB1 to SPSB4, plays pivotal roles in higher animals, including immune response, apoptosis, growth, and stress responses. In Neocaridina denticulata sinensis, alternative splicing yielded two NdGustavus isoforms, NdGusX1 and NdGusX2, with distinct expression patterns-high in ovaries and muscles, respectively, and across all ovarian germ cells. These isoforms showed similar expression dynamics during embryogenesis and significant upregulation post-copper ion exposure (P < 0.05). The in situ hybridization result elucidated that NdGusX1 and NdGusX2 were expressed across the germ cell spectrum in the ovary, with NdGusX1 showing enhanced expression in oogonia and primary oocytes. In addition, RNA interference revealed functional complementation in ovaries and potential functional differentiation in muscles. Knockdown of NdGusX1 and NdGusX2 potentially disrupted endogenous vitellogenin synthesis, regulating vitellogenesis and reducing mature oocyte volume, affecting follicular cavity occupation. This study provides a theoretical framework for understanding the biological functions of the SPSB family in crustacean ovarian maturation.

NPC intracellular cholesterol transporter 1 (NPC1) plays an important role in sterol metabolism and transport processes and has been studied in many vertebrates and some insects, but rarely in crustaceans. In this study, we characterized NPC1 from Macrobrachium nipponense (Mn-NPC1) and evaluated its functions. Its total cDNA length was 4283 bp, encoding for 1344 amino acids. It contained three conserved domains typical of the NPC family (NPC1_N, SSD, and PTC). In contrast to its role in insects, Mn-NPC1 was mainly expressed in the adult female hepatopancreas, with moderate expression in the ovary and heart. No expression was found in the embryo (stages CS-ZS) and only weak expression in the larval stages from hatching to the post-larval stage (L1-PL15). Mn-NPC1 expression was positively correlated with ovarian maturation. In situ hybridization showed that it was mainly located in the cytoplasmic membrane and nucleus of oocytes. A 25-day RNA interference experiment was employed to illustrate the Mn-NPC1 function in ovary maturation. Experimental knockdown of Mn-NPC1 using dsRNA resulted in a marked reduction in the gonadosomatic index and ecdysone content of M. nipponense females. The experimental group showed a significant delay in ovarian maturation and a reduction in the frequency of molting. These results expand our understanding of NPC1 in crustaceans and of the regulatory mechanism of ovarian maturation in M. nipponense.

CYP302A1 is a member of the Halloween genes in the cytochrome P450 supergene family, which play an important regulatory role in the synthesis of 20-hydroxyecdysone (20E) in crustaceans and insects. In this study, we found that the Mn-CYP302A1sequence included typical CYP450 conserved domains. Phylogenic showed that it is closely related to crustaceans and insects. q-PCR analysis indicated that Mn-CYP302A1 was highly expressed in the ovaries and peaked before ovarian maturation. Mn-CYP302A1 expression was higher at the post-larval stage of day 15 than at other stages of embryogenesis. In situ hybridization indicated that Mn-CYP302A1 was mainly distributed in the nucleus, yolk granules, cell membrane and cytoplasm To further establish the function of CYP302A1, a 21-day RNA interference experiment was conducted. On day 16, the Gonad Somatic Index of the control group and the experimental group showed significant differences, with GSI of 11.72% in the control group and 3.21% in the experimental group. The cumulative proportion of the second entry into stage O-Ⅲ was 100% in the control group, while it was 41.67% in the experimental group on day 21. The ecdysone content was 8.91nmol/L in the control group and 6.11nmol/L in the experimental group on day 9. A significant difference in the molting proportion between the control group and the experimental group was also observed (49% in the control group and 34% in the experimental group) on day 16. Statistical results showed that the average molting cycle of the control group was 14.5 days, while that of the experimental group was 16.5 days. However, the morphological structure of ovarian tissue did not abnormal change. Therefore, the results of this study suggest that Mn-CYP302A1 can promote ovarian maturation and molting in female M. nipponense.

The Cholesterol 7-desaturase gene plays an important role in insect ecdysone synthesis, but its role in ovarian development has not been reported. In this study, characteristics and the phylogenetic relationship of Cholesterol 7-desaturase were identified by bioinformatics. qPCR showed that the Mn-CH7D gene was highly expressed in the ovary, which was much higher than that in other tissues, and the expression level of Mn-CH7D reached the highest level at the third stage of the ovarian development stage (O-III). During embryonic development, the Mn-CH7D gene expression was highest in the zoea stage. The function of the Mn-CH7D gene was explored by RNA interference. The experimental group was injected with Mn-CH7D dsRNA through the pericardial cavity of M. nipponense, while the control group was injected with the same volume of dsGFP. Statistical analysis of gonadal development and GSI calculation showed that the silencing of Mn-CH7D resulted in the suppression of gonadal development. In addition, the molting frequency of the experimental group was significantly lower than that of the control group during the second molting cycle after silencing Mn-CH7D. On the seventh day after silencing, ecdysone content in the experimental group was significantly reduced. These results demonstrated that the Mn-CH7D gene played a dual role in ovarian maturation and molting of M. nipponense.

Optimistic and pessimistic cognitive biases have been described in many animals and are related to the perceived valence of the environment. We, therefore, hypothesize that such cognitive bias can be adaptive depending on environmental conditions. In reward-rich environments, an optimistic bias would be favoured, whereas in harsh environments, a pessimistic one would thrive. Here, we empirically investigated the potential adaptive value of such bias using zebrafish as a model. We first phenotyped female zebrafish in an optimistic/pessimistic axis using a previously validated judgement bias assay. Optimistic and pessimistic females were then exposed to an unpredictable chronic stress protocol for 17 days, after which fish were euthanized and the sectional area of the different ovarian structures was quantified in both undisturbed and stressed groups. Our results show that zebrafish ovarian development responded to chronic stress, and that judgement bias impacted the relative area of the vitellogenic developmental stage, with pessimists showing higher vitellogenic areas as compared with optimists. These results suggest that pessimism maximizes reproductive investment, through increased vitellogenesis, indicating a relationship between cognitive bias and life-history organismal decisions.

The Japanese eel (Anguilla japonica) experiences dramatic internal and external environmental changes during its transoceanic reproductive migrations. Here, we assess immune function changes in the primary and secondary immune organs (head kidney and spleen) of A. japonica during artificial ovarian maturation at the previtellogenic (PV), midvitellogenic (MV), and ovulating (OV) stages by transcriptome analyses. Stress responses were also assessed by determining the serum concentrations of lysozyme, alkaline phosphatase, acid phosphatase, total antioxidant capacity, and superoxide dismutase. Our results showed that together with increased serum 17β-estrogen and testosterone, lysozyme activity and antioxidant capacity were suppressed during artificial ovarian maturation. Comparisons across these developmental stages identified 60 (head kidney) and 36 (spleen) differentially expressed genes associated with the immune system. Genes related to the key activation markers of innate immune function, such as CXCL10, CXCL11, CCL20, HSP90B, MMP9, and MMP13, were upregulated and significantly enriched in the interleukin-17 signaling pathway. Adaptive immune function-related genes (IGM and MHC1) were upregulated in the head kidney from PV to MV, and their levels increased thereafter in the spleen. Moreover, a correlation between Pax5 expression and IGM expression in the spleen of MV (IGM+/Pax5+) and OV (IGM++/Pax5-) stage suggests that adaptive immune function was enhanced during ovarian maturation. To our knowledge, the present study is the first to describe transcriptome profiling of immune organs during ovarian maturation in teleost. Our findings suggest that the interleukin-17 pathway and IgM may play important roles in spawning.

Pyrokinins (PKs) are neuropeptides that have been found to regulate a variety of physiological activities including reproduction in various insect and crustacean species. However, the reproductive roles of PKs in the giant freshwater prawn, Macrobrachium rosenbergii, have not yet been investigated. In this study, we identified the MroPK gene from next-generation sequence resources, which encodes a MroPK precursor that shares a high degree of conservation with the C-terminal sequence of FxPRLamide in other arthropods. MroPK is expressed within most tissues, except the hepatopancreas, stomach and gill. Within developing ovarian tissue, MroPK expression was found to be significantly higher during the early stages (stages 1-2) compared with the late stages (stages 3-4), and could be localized to the oogonia, previtellogenic and early vitellogenic oocytes. A role for PK in M. rosenbergii reproduction was supported following experimental administration of MroPK to ovarian explant cultures, which led to an increase in the production of progesterone and estradiol and upregulation of expression of steroidogenesis-related genes (3β-HSD and 17β-HSD) and vitellogenin (Vg). Together, these results support a role for MroPK in regulating ovarian maturation via steroidogenesis.

This study was the first to evaluate multiple hormonal manipulations to hepatopancreas over the ovarian development stages of the mud crab, Scylla paramamosain. A total of 1258 metabolites in 75 hepatopancreas explants from five female crabs were induced by juvenile hormone III (JH III), methyl farnesoate (MF), farnesoic acid (FA) and methoprene (Met), as identified from combined metabolomics and lipidomics (LC-MS/MS). 101 significant metabolites and 47 significant pathways were selected and compared for their comprehensive effects to ovarian maturation. While MF played an extensive role in lipid accumulation, JH III and Met shared similar effects, especially in the commonly and significantly elevated triglycerides and lysophospholipids (fold change≥2 and ≤0.5, VIP≥1). The significant upregulation of β-oxidation and key regulators in lipid degradation by FA (P ≤ 0.05) resulted in less lipid accumulation from this treatment, with a shift toward lipid export and energy consumption, unlike the effects of MF, JH III and Met. It was possible that MF and FA played their own unique roles and acted in synergy to modulate lipid metabolism during crab ovarian maturation. Our study yielded insights into the MF-related lipid metabolism in crustacean hepatopancreas for the overall regulation of ovarian maturation, and harbored the potential use of juvenoids to induce reproductive maturity of this economic crab species.

Research on nutrition and feed development for the broodstock of the Pacific white shrimp, Litopenaeus vannamei, is rare, and a poor broodstock quality is a critical factor restricting the seed supply in shrimp farming. As an essential nutrient for the gonadal development of L. vannamei, one control diet (no phospholipid) and three typical phospholipids (soybean lecithin, egg yolk lecithin, and krill oil) were evaluated in a semipurified diet of 4% phospholipid for a 28-day trial (initial weight 34.7 ± 4.2 g). Dietary phospholipid supplementation significantly promoted the ovarian maturation of female L. vannamei. Compared with soybean lecithin and egg yolk lecithin, krill oil showed the best positive results. Shrimp fed with a diet krill oil has obtained a significantly higher gonadosomatic index, yolk particle deposition, lipid accumulation, and estrogen secretion than from other sources. Ovary lipidomic analysis showed that the krill oil enriched the lipid composition of the ovary. The \"glycerophospholipid metabolism\" and \"sphingolipid metabolism\" pathways were significantly varied via topological pathway analysis. Genes and hub genes, with significantly different expression levels, were significantly enriched in the \"fatty acid metabolism pathway,\" \"glycerophospholipid metabolism,\" and \"arachidonic acid metabolism\" pathways by transcriptomic analysis. Correlation analysis of the transcriptome and lipidomics showed that the differential gene \"hormone-sensitive lipase-like\" (HSL) was positively correlated with various lipids [triglycerides (TG), phosphatidic acid (PA), phosphatidylserine (P), phosphatidylethanolamine (PE), glucosylceramide (GlcCer), phosphatidylglycerol (PG), and phosphatidylinositol (PI)] but was negatively correlated with diacylglycerol (DG), lysophosphatidylethanolamine (LPE), and sphingomyelin (SM). In conclusion, the dietary phospholipids, especially krill oil as a phospholipid source, can promote the development of L. vannamei ovaries by increasing the accumulation of nutrients such as triglycerides and sterols, and the secretion of estrogen or related hormones, such as estradiol and methylfarneside, by affecting the metabolism of glycerol phospholipids and some key fatty acids.

CircRNAs are novel endogenous non-coding small RNAs involved in the regulation of multiple biological processes. However, little is known regarding circRNAs in ovarian development and maturation of fish. Our study, for the first time, provides the genome-wide overview of the types and relative abundances of circRNAs in tongue sole tissues during three ovarian developmental stages. We detected 6790 circRNAs in the brain, 5712 in the pituitary gland, 4937 in the ovary and 4160 in the liver. Some circRNAs exhibit tissue-specific expression, and qRT-PCR largely confirmed 6 differentially expressed (DE) circRNAs. Gene Ontology and KEGG pathway analyses of DE mRNAs were performed. Some DE circRNA parental genes were closely associated with biological processes in key signalling pathways and may play essential roles in ovarian development and maturation. We found that the selected circRNAs were involved in 10 pathways. RNase R digestion experiment and Sanger sequencing verified that the circRNA had a ring structure and was RNase R resistant. qRT-PCR results largely confirmed differential circRNA expression patterns from the RNA-seq data. These findings indicate that circRNAs are widespread in terms of present in production-related tissues of tongue sole with potentially important regulatory roles in ovarian development and maturation.