Introduction
The eyestalk ganglia, also referred to as the X-organ-sinus gland (XO-SG) complex is composed of medulla terminalis X-organs (XO), which is involved in the synthesis of neuropeptides, aggregation of XO terminals, sinus gland (SG), as well as in neuropeptide storage and release (reviews, Fingerman, 1997; Webster et al., 2012). Crustacean hyperglycemic hormone superfamily neuropeptides (CHHs) are classic XO-SG complex neuropeptides that are involved in reproduction (reviews, Chung et al., 2010; Jayasankar et al., 2020), molting (reviews, Chung et al., 2010; Webster, 2015a; Chen et al., 2020; Mykles and Chang, 2020; Mykles, 2021), metabolism (reviews, Chung et al., 2010; Chen et al., 2020), immunity (reviews, Chen et al., 2020; Tong et al., 2021), stress (reviews, Chen et al., 2020; Tong et al., 2021), and osmotic regulation (reviews, Chung et al., 2010; Webster, 2015b; Chen et al., 2020).
In 1989, a 72aa peptide was isolated from XO-SG complex of the shore crab, Carcinus maenas. This peptide regulated hemolymph glucose levels and was therefore named “crustacean hyperglycemic hormone (CHH)” which serves as a hyperglycemic factor (Kegel et al., 1989). Subsequently, the moult-inhibiting hormone (MIH) with 78aa was also isolated from the SG of C. maenas and it showed a conserved six cysteine motif with CHH. MIH, produced and secreted by the XO-SG complex, inhibits the synthesis of ecdysteroids during intermoult (Webster, 1991). In the same year, the vitellogenesis-inhibiting hormone (VIH) with 77aa was isolated from the SG of the American lobster, Homarus americanus (Soyez et al., 1991) and characterized. VIH, which is now also referred to as the gonad-inhibiting hormone (GIH), suppresses the onset of vitellogenesis. Later, two neuropeptides, mandibular organ-inhibiting hormone 1 (MOIH-1) and MOIH-2, consisting of 78 residues were isolated from SG extracts of the crab, Cancer pagurus. MOIHs inhibit methyl farnesoate synthesis by the mandibular organ (Wainwright et al., 1996). These are classic crustacean hyperglycemic hormone family peptides, derived from the decapod eyestalk and are 72-78aa in length with conserved six aligned cysteines that form three disulphide bridges (Ohira, 2021). Based on their amino acid sequences, CHHs are divided into two subgroups: type I CHHs and type II CHHs. Type I CHHs have a CHH precursor-related peptide (CHH-PRP) between the signal and mature peptides, while type II CHHs lack CHH-PRP and have an additional glycine in position 5 after the first cysteine residue (review, Chen et al., 2020; Ohira, 2021).
Not only are CHH-family peptides present in crustaceans, they have also been found in several insects. The first non-crustacean member of CHHs was isolated from the desert locust, Schistocerca gregaria, and since it is involved in ion transport, this neuropeptide was named ion transport peptide (ITP) (Audsley and Phillips, 1990; Audsley et al., 1992; Meredith et al., 1996). ITP contains a dibasic cleavage site and a CHH-PRP, which led to it being classified as type I CHH (Montagné et al., 2010). However, phylogenetic analyses revealed that all ITP orthologs are clustered into a clade, which formed a newly named group, type III CHH (Montagné et al., 2010; Toullec et al., 2017). Transcriptomic analyses identified new decapod CHH peptides, whose BLAST hit matched ITPs of insects, therefore, they were named ITP-like peptides (Toullec et al., 2017). These peptides have six conserved cysteine residues, but neither do they contain CHH-PRP nor a specific glycine. For this reason, they were also named CHH-MIH-like (Veenstra, 2016). Based on their phylogeny, ITP-like peptide orthologs constitute a new set, type IV CHH (review, Chen et al., 2020). Hence, as more CHHs sequences were discovered from RNA-seq, reclassification of the CHH family peptides became necessary.
To reveal the varied and complicated functions of CHH peptides, determination of their receptors is crucial (Chung et al., 2010). The G protein-coupled receptors (GPCRs) (BNGR-A24 and BNGR-A34) have been revealed to be silkworm, Bombyx mori ITP and ITP-L receptors (Nagai et al., 2014). Subsequently, several GPCRs belonging to the BNGR-A34 orthologs have been identified as candidate receptors for CHH or MIH in decapods, including the crayfish, Procambarus clarkii (Veenstra, 2015, Rump et al., 2021), the Eastern rock lobster, Sagmariasus verreauxi (Buckley et al., 2016), the blackback land crab, Gecarcinus lateralis (Tran et al., 2019), the rock lobster, Jasus edwardsii (Christie and Yu, 2019), the spiny lobster, Panulirus argus, the blue crab, Callinectes sapidus, H. americanus (Rump et al., 2021), and the swimming crab, Portunus trituberculatus (Tu et al., 2021). Compared to the vast array of CHHs, their candidate GPCRs should be evaluated more.
Recently, due to advances in sequencing technologies, several decapod genomes have been sequenced and assembled (Gutekunst et al., 2018; Zhang et al., 2019; Tan et al., 2020; Tang et al., 2020, 2021; Van Quyen et al., 2020; Cui et al., 2021; Uengwetwanit et al., 2021; Zhao et al., 2021), providing an important basis for comprehensive understanding and differentiation of decapod CHHs and their putative receptors, as well as for further exploration of CHHs genomic structures and characteristics. In this study, CHHs and their putative GPCRs were identified and characterized in three species of economically important crabs: the mud crab, Scylla paramamosain, P. trituberculatus, and the Chinese mitten crab, Eriocheir sinensis. Genomic mining, full-length transcriptome, reference transcriptomic assembly and phylogenetic analyses were performed to determine the genomic structures of CHHs and their putative receptors. Moreover, transcriptome data of the three crabs were analyzed to establish the expression levels of CHHs and their putative receptors in different tissues and under different treatments. Our findings provide a reference basis for the classification and functional studies of CHHs in decapods.
Discussion
Several CHHs and their putative GPCRs from crabs are described here. We focused on their genomic structures and expression patterns. The RNA-seq is a powerful technique for identifying mud crab neuropeptides and their GPCRs (Bao et al., 2015, 2018a). We combined genomic assembly, full-length transcriptome sequencing and RNA-seq analyses to identify crabs CHHs and their putative GPCRs.
In typical classification, CHHs were divided into type I CHH and type II CHH. Alternative splicing of type I CHH gene has been shown in almost all reported decapod species (review, Webster et al., 2012). The type I CHH gene is composed of four exons, one splice variant lacks the 3rd exon and transcript end in the 4th exon. The other one includes all exons, and is hereby named CHH-L. However, CHH-L transcript ends in the 3rd exon, therefore, it exhibits similar amino acid lengths with the first splice variant (review, Webster et al., 2012). These features match the findings from the CHH1 genes of P. trituberculatus and E. sinensis in this study. However, we did not identify CHH1 genomic sequences in the S. paramamosain genome, which might be attributed to incomplete genomic assembly (Zhao et al., 2021). There are three indications suggesting that the Sp-CHH1 gene has four exons and generates two splice variants. First, consistency of the CHH1 gene structure in several decapods. Second, in a previous study, two CHH isoforms (“Sp-CHH1” and “Sp-CHH2”) exhibited identical nucleotide sequences, except the insert (equivalent to the 3rd exon) in the “Sp-CHH2” isoform (Fu et al., 2016). Finally, full-length transcriptome analyses revealed that two transcripts that are identical to the above reported CHH isoforms exist in mud crab (Fu et al., 2016). Given another type I CHH gene was identified from crabs in this study, we named this typical type I CHH as “CHH1,” and the new-found type I CHH was referred to as “CHH2.” CHH1-v1 and CHH1-v2 were used to describe the C-terminal amidated CHH peptide and long-isoform CHH peptide, respectively. Generally, CHH1-v1 transcripts were mainly detected in the neurons of the X-organ, which are located in the eyestalk, while CHH1-v2 transcripts were found in the pericardial organ, thoracic ganglia, antennal glands, gills and heart (review, Webster et al., 2012). Tissues expression profiles of the CHH1 gene in S. paramamosain has also been reported (Fu et al., 2016). However, common areas from two splice variants were selected to design qRT-PCR primers, resulting in expression patterns of two splice variants that could not be distinguished (Fu et al., 2016). We used the StringTie software to re-analyze the expression patterns of two splice variants that originate from the Sp-CHH1 gene. Our results were consistent with those of previously reported species (review, Webster et al., 2012). The Sp-CHH1-v1 transcript was majorly expressed in the eyestalk while the Sp-CHH1-v2 transcript was mainly detected in various tissues. Just like S. paramamosain, two splice variants from P. trituberculatus and E. sinensis CHH1 gene have also shown differential expression patterns. CHH1-v1 was expressed in the eyestalk throughout decapods, suggesting that it might be functionally conserved. Non-eyestalk splice variant was expressed in a variety of tissues, their expression patterns in three crabs were different. The Sp-CHH1-v2 transcript was highly expressed in the cerebral ganglia, hepatopancreas under salinity stress and post-molt, and in ovarian follicular cells, while Pt-CHH1-v2 and Es-CHH1-v2 exhibited low expression levels in our study. These findings suggest that CHH1-v2 might have species specific functions. The expression levels of Sp-CHH1-v2 significantly increased when mud crabs were subjected to salinity changes, which might speculatively implicate the hepatopancreas in, as yet undefined, roles in osmo/iono regulation. Elevated expression levels of Sp-CHH1-v2 in the follicular cells were comparable to those of Sp-BMP7 (Shu et al., 2016) and Sp-sNPF (Bao et al., 2018b) in the ovary, which are involved in vitellogeneses and ovarian maturation (Shu et al., 2016; Bao et al., 2018b).
Another type I CHH gene (CHH2) from several decapods has also been found (Veenstra, 2016). In S. paramamosain, full-length Sp-CHH2 cDNA was cloned, and it was shown to regulate hyperglycemic activity (Liu et al., 2019). In this study, the expression levels of hepatopancreatic Sp-CHH2 were found to be significantly elevated as a result of salinity stress, adding to the evidence that Sp-CHH2 plays a key role in metabolism (Liu et al., 2019). This expression profile is comparable to that of Sp-CHH1-v2. Furthermore, Sp-CHH2 mRNA was highly expressed in follicular cells, indicating that Sp-CHH1-v2 and Sp-CHH2 play synergetic roles in S. paramamosain. The functions of Pt-CHH2 and Es-CHH2 have not been verified, however, their expressions in various tissues indicate that they have multiple functions.
Even though type II CHH has three classifications (MIH, VIH/GIH and MOIH), most decapods have two type II CHH genes (review, Webster et al., 2012). For instance, C. pagurus has two MOIHs (Wainwright et al., 1996). In the spider crab, Libinia emarginata, three peptides were purified to show MOIH activity (Liu and Laufer, 1996), however, one of them belonged to type I CHH (Liu et al., 1997). These designations were based on their biological activities when they were identified for the first time. Two type II CHH genes were also been found in S. paramamosain, and were named MIH and VIH, respectively as their first identifications (Huang et al., 2015; Liu et al., 2018). However, only one MIH was found in P. trituberculatus and E. sinensis, respectively. Despite we searched the type II CHH in all accessible genome, Pacbio full-length transcriptome and RNA-seq. We postulate that there is only one type II CHH gene in P. trituberculatus and E. sinensis. This could be because one copy of type II CHHs might have been lost from the genome during evolution. Pt-MIH expression has been negatively correlated with the ecdysteroid (20-hydroxyecdysone, 20E) titer during molt stages (Wang et al., 2013). MIH has also been identified in E. sinensis, and the recombinant Es-MIH (rEs-MIH) inhibits ecdysteroid secretion by Y-organs in this species (Zhang et al., 2011). It is now timely to determine whether the single copy type II CHHs have inhibitory roles in gonad maturation or methyl farnesoate (MF) synthesis by mandibular organs. Alternatively, we postulate that the lost roles of type II CHHs may have been replaced by type I CHHs. Conversely, six type II CHH genes were found in the P. vannamei genomic assembly (Zhang et al., 2019). Expansion of type II CHH seems to be universal phenomenon in the Penaeidae, as we also found thirteen type II CHH genes in P. monodon. In C. pagurus, MIH and MOIH were clustered in the same chromosome (Lu et al., 2000). Due to the lack of assembled genome, this feature had not been found in other decapods until six tandem MIH genes were determined in the P. vannamei genome (Zhang et al., 2019). In this study, all identified type II CHHs (more than one copy) from every species were located in adjacent positions or showed tandem structures in the same chromosome. This characteristic of type II CHH is ubiquitous in decapods.
The ITP-like peptide exhibited a higher sequence similarity with ITP isoforms of insects rather than CHHs or MIHs (Toullec et al., 2017). In addition, 4 exon/3 intron genomic structure of the ITP-like peptide is identical to that of ITP. The 4-exon structure of the ITP gene can generate splice variants, just like CHH1 (reviews, Webster et al., 2012; Chen et al., 2020). Nevertheless, in this study none were found in the PacBio generated full-length transcriptomes. Whilst deeper level transcriptome sequencing might show alternative splice variants, it is also possible that such transcripts are expressed at extremely low levels, which could have no functional relevance. Specific expressions in eyestalk indicate that the ITP-like peptide has a conserved function in decapods. However, it should be determined whether the newly identified ITP-like peptide functions as an ion transport factor.
Biochemical characterization of receptor binding characteristics using 125-I labeled CHH, MIH and membrane preparations in C. maenas (Webster, 1993) and C. sapidus (Chung et al., 2010) has been used to identify receptor binding sites in various tissues. In C. sapidus, CHH1-v1 (ES-CHH) and CHH1-v2 (PO-CHH or CHH-L) have multiple binding sites from various tissues, including hepatopancreas, gills, abdominal muscles, scaphognathites, hindgut, midgut, and heart (Katayama and Chung, 2009). The Y-organs of juveniles and hepatopancreas of females were confirmed to be binding sites for C. sapidus MIH (Zmora et al., 2009). In C. maenas, CHH1-v1 and MIH have highly specific binding sites of Y-organs (Webster, 1993). In this study, only the transcriptome data for E. sinensis Y-organs were available in public databases, and all Es-CHHR-likes were not detected in this tissue. Therefore, more YO RNA-seq data should be generated to investigate the expression profiles of CHHR-likes in other crabs. In addition, C. maenas CHH1-v1 has many binding sites in the hepatopancreas, gills and hindgut (Kummer and Keller, 1993; Webster, 1993; Chung and Webster, 2006). Hepatopancreas membranes from crayfish, Orconectes limosus and C. maenas were shown to have a CHH1-v1 binding specificity. Membranes from the hepatopancreas of C. maenas had lower affinities for O. limosus CHH1-v1, while C. maenas CHH1-v1 did not bind O. limosus CHH1-v1 (Kummer and Keller, 1993). This species-specificity of CHH1-v1 is consistent with its hyperglycaemic activity in different crabs, and this was attributed to co-evolution of CHH1-v1/receptors (review, Chen et al., 2020). In this study, although hepatopancreatic CHHR-like GPCRs were clustered in a clade, they exhibited differences in terms of their amino acid sequences. These differences might have led to protein structural differences, leading to CHH species-specificity in the hepatopancreas. Differences in genomic structures of hepatopancreatic CHHR-like GPCRs were that copies of Sp-GPCR-A36 and Sp-GPCR-A37 overlapped on the chromosome, while hepatopancreatic GPCRs from P. trituberculatus and E. sinensis showed an isolated copy located in adjacent positions in the same chromosome, which might be evolutionary traces. Based on previous findings, three decapods (C. sapidus, C. maenas, and O. limosus) CHH1-v1 have common binding tissues, hepatopancreas, the major metabolic site (Kummer and Keller, 1993; Webster, 1993; Chung and Webster, 2006). The newly identified hepatopancreatic GPCRs from S. paramamosain, P. trituberculatus, and E. sinensis might be receptors for CHH1-v1, which regulates hyperglycemic activity. This postulate was supported by the significant increase in expression levels of Sp-GPCR-A36 in salinity stress conditions. Hepatopancreas is a major vitellogenin source and a metabolic site, and the female hepatopancreas for C. sapidus is a target tissue for MIH (Zmora et al., 2009), suggesting that hepatopancreatic CHHR-like GPCRs bind MIH/VIHs in this species. However, in C. maenas specific binding of MIH to hepatopancreas membrane preparations was not observed (Webster, 1993). Two copies of hepatopancreatic CHHR-like GPCRs were differentially expressed, indicating that they play different functions in hepatopancreas. However, it has not been determined whether one of them is a CHH1-v1 receptor and another a MIH/VIH receptor. Furthermore, Sp-GPCR-A34 and its ortholog, Pt-CHHR-like2, were specifically expressed in the hepatopancreas and in the ovary. Sp-GPCR-A34, which showed higher expressions in the hepatopancreas during the post-molt stage, exhibited the MIH receptor feature (reviews, Chung et al., 2010; Chen et al., 2020). Expressions of Sp-GPCR-A33, Sp-GPCR-A35 and their orthologs in various tissues suggests pleiotropic functions, and it should be determined whether they were activated by the multi-tissue expressed CHH1-v2/CHH2 or by the pleiotropic CHH1-v1. Previous review revealed that decapod putative CHHRs were clustered into two clades (CHHR1 and CHHR2) (review, Mykles, 2021). In this study, more decapod putative CHHR sequences were collected to construct phylogenetic tree to clarify their clustering. Of which, our hepatopancreatic CHHR-like clade was consistent with the CHHR2 clade, and four putative CHHRs (Sv_GPCR_A12, Gl_GPCRA12, Pc-GPCRA52, and Pc-GPCRA63) in the CHHR2 clade also expressed in hepatopancreas (Veenstra, 2015; Buckley et al., 2016; Tran et al., 2019). In the review, only four CHHRs in the CHHR1 clade (review, Mykles, 2021), our analysis expanded this clade, which three subgroups were separated from each other. Moreover, three putative CHHRs constitute a new CHHR set.
In summary, we show the genomic structures and expressions of CHHs and their putative receptors in S. paramamosain, P. trituberculatus, and E. sinensis. Our findings provide an important basis for subsequent studies on CHHs binding sites and on CHHs/GPCRs signaling pathways.