In arthropods, neuropeptides are important signaling molecules to regulate metamorphosis, ecdysis, growth, and reproduction (Charmantier et al., 1997). In crustaceans, past studies have predominantly focus on the neurohormones derived from the eyestalk ganglia, where the X-organ and sinus gland complexes play central roles in the regulation of gonadal development. It has been long known that vitellogenesis inhibiting hormone (VIH) plays an inhibitory role in ovarian development (Tsutsui et al., 2013). Recently, many neuropeptides have been identified in the nervous system of crustaceans using high-throughput sequencing technology; however, their functions in reproduction remain largely unexplored. Therefore, it is intriguing to identify gonadotrophic neuropeptides in crustaceans, which can be applied to the artificial breeding technology of economically significant species.

SIFa is an amidated neuropeptide that characterized by a conserved Ser-Ile-Phe structure in its C-terminal, with 12 amino acid residues in length (Verleyen et al., 2009). There are three isoforms of SIFa that have been identified in arthropods, which are distinguished by a single amino acid residue at the end of the amino (N) terminus: Ala1-SIFa, Gly1-SIFa and Val1-SIFa. AYRKPPFNGSIFamide (Ala1-SIFa), Gly1-SIFa, and Val1-SIFa were first identified in the grey fly Neobellieria bullata (Janssen et al., 1996), the giant tiger prawn Penaeus monodon (Sithigorngul et al., 2002), and the American lobster Homarus americanus (Dickinson et al., 2008), respectively. To date, a number of SIFa and its paralogs have been identified in insects and crustaceans through the application of mass spectrometry and next generation sequencing, for example, the Jonah crab Cancer borealis (Huybrechts et al., 2003) the red swamp crayfish Procambarus clarkii (Yasuda et al., 2004) and the mud crab S. paramamosain (Bao et al., 2015).

The findings regarding physiological effect of SIFa are primarily mediated by insects. In Drosophila melanogaster, SIFa is involved in the regulating various physiological processes, such as courtship (Sellami and Veenstra, 2015), sleep (Huang et al., 2021) and feeding (Martelli et al., 2017). Similarly, the role of SIFa in regulating feeding behavior has been reported in Rhodnius prolixus (Ayub et al., 2020). In Rhyparobia maderae, injection of SIFa delayed locomotor activity rhythms in a circadian time-dependently (Arendt et al., 2015). To date, few studies have documented the biological functions of SIFa in crustacean species. SIFa has been reported to regulate heartbeat in C. borealis and Cancer irroratus (Dickinson et al., 2019), and aggression and dominant behavior in the freshwater prawn Macrobrachium rosenbergii (Vázquez-Acevedo et al., 2009).

The first SIFa receptor (SIFaR) was identified in D. melanogaster (Jørgensen et al., 2006). Subsequently, it was discovered in the genomes of the black-legged tick Ixodes scapularis (Šimo et al., 2013), silkworm Bombyx mori (Fan et al., 2010), red flour beetle Tribolium castaneum (Hauser et al., 2008) and bumblebee Bombus terrestris (Lismont et al., 2018). Two SIFa receptors exist in the American cockroach Periplaneta americana (Veenstra, 2021) and the brown planthopper Nilaparvata lugens (Tanaka et al., 2014). It was observed that activation of its receptor by the SIFa neuropeptide mobilizes Ca²⁺ accumulation in the cytoplasm, which regulates the expression of downstream genes and induces corresponding physiological effects (Jékely, 2013). SIFa/SIFaR is homologous to the vertebrate gonadotropin inhibitory hormone (GnIH) and its receptor (GnIHR), which is conserved from fish to humans and play a key role in inhibiting reproduction (Ubuka and Tsutsui, 2014). consequently, we hypothesized that SIFa/SIFaR are involved in the regulation of reproduction in crustaceans was proposed in view of this similarity.

The mud crab (S. paramamosain) is an important marine culture crustacean. It takes a long time for female crabs to develop into gonadally mature crabs, which results in high culture costs in aquaculture and constrains the development of mud crab culture industry. In this study, we aimed to investigate the putative role of Sp-SIFa and its receptors in the ovarian development of mud crabs. First, cDNA sequences of Sp-SIFa and its possible receptors were obtained by transcriptome sequencing and molecular cloning. Subsequently, their expression profiles were determined by reverse transcription polymerase chain reaction (RT-PCR) and Real-time Quantitative polymerase chain reaction (qPCR). Finally, in vitro and in vivo experiments were performed to explore the putative effects of Sp-SIFa on ovarian development.

SIFa is extensively present in the central nervous systems of arthropods, and its conservation across various species suggests its potential significance (Verleyen et al., 2009). Leveraging advancements in high-throughput sequencing technology, SIFa has been widely recognized in several crustaceans (Sithigorngul et al., 2002; Dickinson et al., 2008; Huybrechts et al., 2003; Yasuda et al., 2004; Ma et al., 2009); however, its physiological functions remain largely unexplored. This study characterized SIFa and its two putative receptors and described their expression profiles in the mud crab S. paramamosain, illustrating a potential stimulatory role in ovarian development.

In the mud crab S. paramamosain, sequences encoding an Sp-SIFa precursor and its two potential SIFa receptors were identified. The Sp-SIFa precursor exhibited a characteristic similarity with the documented insect SIFa, comprising a signal peptide, mature peptide, and precursor-related peptide (Ayub et al., 2020). There was an amidated signal at the C-terminus of the mature peptide ( Figure 1 ), suggesting it was an amidated hormone. Sequence alignment revealed that the crustacean SIFa precursor exhibited high sequence similarity to that in insects, suggesting that SIFa is highly conserved among arthropods ( Figure 2 ). Previous research has demonstrated that SIFaR is structurally related to the GnIHR; however, there is low similarity between their ligands SIFa and GnIH (Ubuka and Tsutsui, 2014; Elphick and Mirabeau, 2014). It has been proposed that GnIH/GnIHR and SIFa/SIFaR may be distinct from the ancestral Famide peptide system, which is a family of peptides that have independently co-evolved in vertebrates and insects, including FMRFamide, neuropeptide F, short neuropeptide F, sulfakin, myosuppressin and SIFa (Ubuka and Tsutsui, 2014). G protein-coupled receptors (GPCRs) are the largest family of receptors characterized by seven α-helical transmembrane structural domains, which are involved in regulating a wide variety of physiological processes (Horn et al., 1998; Pierce et al., 2002). The GPCRs are classified into six major classes, and SIFaR belongs to class A, Rhodopsin-like receptors (Šimo et al., 2013). Sequence alignment revealed that SIFa receptors from crustaceans and insects exhibited relatively high conservation in the seven transmembrane structural domains ( Figure 3 ). In S. paramamosain, the two SIFaRs (Sp-SIFaR1 and Sp-SIFaR2) exhibited low sequence identity (less than 32%), implying that they may be involved in a distinct physiological process.

In insects, SIFa is mainly expressed in the nervous system (Ayub et al., 2020). In crustaceans, including H. americanus and P. clarkii, SIFa is predominantly expressed in the stomatogastric nervous system and the middle gut (Dickinson et al., 2008). Similarly, the Sp-SIFa transcript is confined to nervous tissues (eyestalk, cerebral, and thoracic ganglion) and the middle gut of S. paramamosain ( Figure 5A ), suggesting that SIFa functions as a brain-gut hormone in most crustaceans. SIFa is present in the nerve fibers of the olfactory lobes of certain insect species, suggesting a possible neuromodulatory role for SIFa in the olfactory system (Verleyen et al., 2004). In Drosophila, SIFa is expressed in only four medial neurons of the brain (Terhzaz et al., 2007), which can inhibit sexual behavior (Dickson, 2008), promote sleep (Park et al., 2014), and induce appetitive and feeding behaviors (Martelli et al., 2017). In R. prolixus, SIFa is released by the interneurons and dorsal vessel, where it also has a role in facilitating ingestion and inducing heart rate in a dose-dependent manner (Ayub et al., 2020). Similarly, SIFa functions as a cardioactive peptide in C. borealis and C. irroratus to promote cardiac output, however, this regulation differs from that of R. prolixus. SIFa appears to be synthesized in the middle gut and delivered to the heart to regulate C. borealis and C. irroratus (Dickinson et al., 2019). In this study, Sp-SIFa was highly expressed in the cerebral ganglion, implying that it may be involved in similar physiological activities, including reproduction, sleeping, and feeding, which was further supported by the wide expression of Sp-SIFaR1 ( Figure 5A ). Sp-SIFaR1 was extensively expressed in the eyestalk ganglion, cerebral ganglion, ovary, and hepatopancreas, whereas the Sp-SIFaR2 expression was restricted to the eyestalk ganglion and ovary ( Figure 5A ). This outcome is broadly comparable to the tissue distribution of SIFaR in insects (Sellami and Veenstra, 2015; Lismont et al., 2018; Veenstra, 2021). Moreover, the Sp-SIFa expression in the eyestalk and cerebral ganglia was significantly altered during vitellogenesis, implying its possible role in ovarian development ( Figure 5B ). The presence of two Sp-SIFa receptors in the ovary of S. paramamosain further supports their significance in regulating ovarian development. Sp-SIFaR1 levels was significantly elevated in the early and late vitellogenic stages ( Figure 5C ), while Sp-SIFaR2 was significantly increased at the late vitellogenic stage ( Figure 5D ). These results indicated that Sp-SIFaR1 and Sp-SIFaR2 may involve in different regulation during ovarian development, which warrants future study. Two receptors for SIFa in I. scapularis collaborate to regulate salivary secretion (Guerrib et al., 2023).

Vitellogenesis, the process by which Vg synthesized by the hepatopancreas and ovary is endocytosed into the oocyte mediated by the Vg receptor, is essential for ovarian development in crustaceans (Subramoniam, 2010). Although vitellogenesis in crustaceans has been extensively studied, the mechanisms underlying its regulation remain incompletely comprehended. Multifunctional neuropeptides, neurotransmitters, neuromodulations, and other factors also regulate ovarian development in crustaceans (Subramoniam, 2010). VIH, crustacean hyperglycaemic hormone (CHH), and molt-inhibiting hormone (MIH) are eyestalk peptides of the CHH family. VIH inhibits the Vg production in the hepatopancreas and the Vg uptake by oocytes during vitellogenesis (Chen et al., 2014; Qiao et al., 2015). In contrast, CHH and MIH may act as positive regulators of vitellogenesis and ovarian development (de Kleijn et al., 1998; Luo et al., 2015). 5-HT is a neurotransmitter that significantly contributes to ovarian development by modulating downstream endocrine factors, hormones, and reproduction-associated proteins (Jayasankar et al., 2020). The present study revealed the role of Sp-SIFa in the vitellogenesis of mud crabs by detecting the effects of Sp-SIFa on the expression of Sp-Vg and Sp-VgR. The in vitro results demonstrated that Sp-SIFa significantly induced Sp-Vg expression in the hepatopancreas and Sp-VgR expression in the ovary ( Figure 6 ). Short-term injection of SIFa into female crabs consolidated its stimulatory effects on vitellogenesis by promoting Sp-Vg and Sp-VgR expression ( Figure 7 ). A similar effect was observed with the addition of hEGF protein, which can induce Sp-VgR expression (Lu et al., 2020). In the long-term experiment, the Sp-Vg level did not change significantly ( Figure 8 ), which may have been caused by the sampling point. However, the level of Sp-VgR ( Figure 8 ), GSI, and oocyte diameter ( Figure 9 ) were significantly increased by the prolonged injection of Sp-SIFa, suggesting a stimulatory role in ovarian development.

In conclusion, this study isolated Sp-SIFa and its two putative receptors from mud crabs. It delineated their expression patterns in females, offering insight into their physiological activities. This study further confirmed the stimulatory role of Sp-SIFa in ovarian development by promoting Vg biosynthesis and oocyte uptake.