Turkish Journal of Zoology Turk J Zool (2021) 45: 25-32 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-2005-25

Identification and characterisation of hyperglycaemic hormone (CHH) from Mediterranean shore aestuarii

1 1 2 2 2, Marsilda QYLI , Valbona ALIKO , Riccardo SGARRA , Piero Giulio GIULIANINI , Chiara MANFRIN * 1 Department of Biology, Faculty of Natural Sciences, University of Tirana, Tirana, Albania 2 Department of Life Sciences, University of Trieste, Trieste, Italy

Received: 15.05.2020 Accepted/Published Online: 11.11.2020 Final Version: 18.01.2021

Abstract: Crustacean hyperglycaemic hormone (CHH) is a neuropeptide that was originally identified in the X-organ/sinus gland complex of the eyestalks (ESs) in . Several CHH isoforms and spliced variants were later identified in other tissues, and their functions have still not been completely unveiled. In this study, the identification and characterisation of the conventional CHH prepropeptide from the ESs of the littoral crab, Carcinus aestuarii, via rapid amplification of cDNA ends was reported. The identified CHH resulted in a coding sequence of 429 bp, an estimatedprotein of 142 aa with a signal peptide of 26 aa, followed by a CHH precursor- related peptide of 40 aa and a mature peptide of 72 aa. The amino acid sequence ofC.aestuarii CHH was also compared, by similarity, with CHHs from Brachyura infraorder, which showed the highest similarity (98.6%) to the CHH peptide from . None of CHH members were reported from this species and being proved by several studies that CHH is produced also during stress conditions, the identification of the full length of the CHH in C. aestuarii opens a new wayin the possibly of studying stress response in Mediterranean shore crab by monitoring of the neuropeptide expression.

Keywords: CHH-precursor-related peptides, neuropeptide, RNA extraction, PCR amplification, phylogenetic tree

1. Introduction glycogen synthesis (Keller and Orth, 1990), and it regulates Crustacean endocrinology began with the discovery by different physiological processes, such as haemolymph Koller (1925) that the colour of shrimp changed as a result glucose levels, lipid metabolism, hepatopancreatic enzyme of blood-borne factors arising in their eyestalk (ES) tissues secretion, and Y-organ ecdysteroid production, growth, (Koller, 1925, 1928; Webster et al., 2012). These findings and reproduction (Dircksen, 1990, 1998; Van Herp, 1998; were investigated in depth by Hanström (1931, 1937), who Webster, 1998; Spanings-Pierrot et al., 2000). Among described the anatomy of the neurosecretory structures, species, CHH was also found to be involved in behavioural paying particular attention to those belonging to the optic responses linked to stress response (Lee et al., 2014). ganglia in the ES (Webster et al., 2012). The sinus gland The first CHH to be fully elucidated was isolated (SG) was elucidated as a neurohemal structure, namely a from the SG of shore crab Carcinus maenas (Kegel et al., group of terminalis of a neurosecretory perikarya cluster, 1989).This was immediately followed by the publication found in the medulla terminalis (MT), and this particular of the amino acid sequence of a peptide from Homarus cluster was labelled the X-organ (XO) (Hanström, 1931, americanus (Chang et al., 1990), which appeared to be 1937; Webster et al., 2012). A subsequent study by similar to the CHH of C. maenas in terms of its primary Abramowitz et al. (1944) demonstrated the existence of a structure, withthe same number of amino acids, namely diabetogenic factor in crustaceans following an injection 72, as well as a sequence identity of 61% (Webster et al., experiment of ES extracts from Uca pugilator into 2012). Callinectus sapidus. This factor, later named the crustacean Additional studies have expanded the number of the hyperglycaemic hormone (CHH), induced hyperglycaemia members in the CHH family, including molt-inhibiting via mobilisation of glucose from stored glycogen into the hormone (MIH), mandibular organ-inhibiting hormone hepatopancreas and muscles (Santos and Keller, 1993; (MOH) and vitellogenesis/gonad-inhibiting hormone Keller et al., 1999; Webster et al., 2012). CHH was found (VIH/GIH), which share common features with CHH, to be involved in the glycogenolysis and inhibition of such as a typical length within the range of 70 to 80 aa * Correspondence: [email protected] 25

This work is licensed under a Creative Commons Attribution 4.0 International License. QYLI et al. / Turk J Zool and 6 conserved cysteine residues that form 3 disulphide to contribute to CHH superfamily characterisation in the bonds (Kegel et al., 1991; Böcking et al., 2002). They were genus Carcinus. In this study, the characterisation of the determined to be involved in metabolic control, moulting, CHH prepropeptide in specimens of C. aestuarii, using gonad maturation, ionic and osmotic regulation, and rapid amplification of cDNA ends(RACE), along with its methyl farnesoate synthesis in mandibular glands (Webster position within the phylogenetic tree of the infraorder et al., 2012). Brachyura presented for the first time. Until recently, the isolation and characterisation of CHH peptides has reached around 400 CHHs and CHH- 2. Materials and methods like peptides in crustaceans (Soyez et al., 1997; Lacombe et 2.1. and tissue preparation al., 1999; Dircksen et al., 2001, Böcking et al., 2002; Chan Adult male specimens (n = 20, carapax width 6.45 ± et al., 2003; Chen et al., 2005; Fanjul-Moles et al., 2006; 0.064 cm, mean ± SD) of Mediterranean green , C. Montagné et al., 2008; Chung et al., 2009; Christie et al., aestuarii, were caught by local fishermen in April 2019, 2010; Yu et al., 2020). within the Marano Lagoon (Friuli-Venezia Giulia, Italy, The existence of CHHs localised in different tissues, 45°44′06″N, 13°09′46.8″E) and transported immediately such as the pericardial organ, in addition to the presence to a laboratory for tissue preparation. After measurement, of many different isoforms, including truncated ones, the ESs were dissected from crabs anaesthetised on ice. has proven the existence of paralog genes and the Briefly, the medullae (externa, interna, and terminalis) were mechanisms ofalternative splicing,ashas been reported immediately placed in TRI Reagent (Sigma-Aldrich Corp., in Macrobrachium rosenbergii, Pachygrapsus marmoratus, St. Louis, MO, USA) for subsequent RNA extraction. No ibericum, Scylla olivacea, and Procambarus clarkii specific permits were required for these experiments, since (Soyez et al., 1998; Dircksen et al., 2001; Chan et al., 2003; there was no involvement of any endangered or protected Chung and Webster, 2003; Chen al., 2004; Chen et al., species. 2005; Toullec et al., 2006; Tsai et al., 2008; Chang et al., 2.2. Characterisation of the CHH transcript 2010; Wu et al., 2012; Fu et al., 2016; Duangprom et al., 2.2.1. RNA extraction and 5′, 3′ rapid amplification of 2017). cDNA ends Studies based on their precursor and primary structure The RNA from the medullae (n = 20) was extracted have reported a classification of the CHH family into 2 using the Tri Reagent (Sigma-Aldrich Corp.) according subfamilies, comprising type-I (CHH sensu stricto and to the manufacturer’s instructions and quantified using ion transport peptides) and type-II peptides (VIH/GIH, Nanodrop 2000 (Thermo Fisher Scientific Inc., Waltham, MOIH and MIH) (Webster et al., 2012). The differences MA, USA). The synthesis of both the ′5 and 3′ RACE between them are mainly due to the absence of the CHH cDNA was performed using the SMART cDNA synthesis precursor-related peptide (CPRP) and glycine at position kit (BD Biosciences, Franklin Lakes, NJ, USA), with 12 in the CHH type-II members (Böcking et al., 2002; specific CHH primers drawn with Primer3Plus v.4.0.0 Giulianini and Edomi, 2006, Webster et al., 2012). (Untergasser, 2012) and OligoCalc v.3.27 (Kibbe, 2007) C. aestuarii (Nardo, 1847) is a littoral endemic crab to exclude possible dimers and hairpin formations. CHH of the Mediterranean Sea and a species of economic from C. maenas was used as the template (GenBank: importance at seafood markets in the area. Even though X17596) and the resulting primers (shown in Table 1) were it bears some similarities to C. maenas, molecular studies further compared with additional CHH XO-type (namely, have found that substantial differences between the 2 AF286078, AF286079, AF286080, and AF286091) together taxa were sufficient to classify them as 2 separate species with CHH-XO, as characterised in the transcriptomic (Roman and Palumbi, 2004). C. aestuarii adaptability study by Oliphant et al. (2018), to verify correspondence with involvement of the CHH hormonehas been studied with the sequences. in relation to different environmental stressors (copper, Polymerase chain reactions (PCRs) were performed hypoxia, chloroform, adrenaline, and temperature) in a final volume of 20 µL with 1X PerfeCTa SYBR Green through biochemical, physiological, and immunological SuperMix (Quanta BioSciences, Inc., Beverly, MA, modulations (Matozzo and Marin, 2010; Matozzo et USA), 0.3 µM of each primer and 1 µL of cDNA. PCR al., 2011; Aliko et al., 2015; Qyli and Aliko, 2017, 2020; amplification was run on a Bio-Rad Touch real-rime PCR Qyli et al., 2017). The higher adaptive tolerance of C. detection system (Bio-Rad Laboratories, Inc., Hercules, aestuarii led to the inspiration to further characterize CA, USA) as follows: 95 °C 2′, 5 cycles 95 °C 20″, 72 °C the CHH hormone, expecting any possible structural 40″, 5 cycles 95 °C 20″, 70 °C 40″, 30 cycles: 95 °C 20″, 59 diversity when compared with C. maenas, in addition to °C 30″, 72 °C 20″ and a final melting curve analysis from65 attaining further data on CHH, since none are available °C to 95 °C, with an increment of 0.5 °C every 5″. The PCR for this species. Furthermore, the findings were expected products were checked on 1.5% TAE electrophoretic gel

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Table 1. Primer details used for the characterisation of the CHH prepropeptides of C. aestuarii.

Melting GC content Primer Sequence (5′-3′) temperature (°C) (% value) Caes_CHH_Forward GAATTCGCAGAAGGAAGACG 59.96 50 Caes_CHH_Reverse GTAGAGTGATGGCGGGTGTT 60.00 55 and the products were purified using Mag-Bind Total Pure family signature: [LIVM]-x(3)-C-[KR]-x-[DENGRH]- NGS (Omega Bio-tek Inc., Norcross, GA, USA) and sent C-[FY]-x-[STN]-x(2)-F-x(2)-C where 3′ cysteines were to be sequenced in both forward and reverse directions involved in the disulphide bonds. at the Macrogen EZ-Seq sequencing service (Macrogen Within the deduced peptide, a casein kinase II Europe B.V., Amsterdam, Netherlands). TheC. estuarii phosphorylation site, with the typical consensus pattern CHH sequence (526 bp encoding a putative prepropeptide [ST]-x(2)-[DE] (position 45–48 aa), and a protein kinase of 142 aa) was obtained by merging the sequence outputs C phosphorylation site, characterised by the consensus using CLC Genomics Workbench v.12 (Qiagen GmbH, pattern[ST]-x-[RK] (position 72–74 aa), were identified. Hilden, Germany) and the consensus was deposited into The C-terminal of the CHH contained a vGRKK GenBank (accession number: MW246807). tetrapeptide, which was an amidation site where the glycine

2.2.2. Phylogenetic network was the expected amide group donor for the terminal Val72 CHHs from infraorder Brachyura were downloaded of the hormone. from the National Center for Biotechnology Information The deduced molecular weights of the features of the repository, translated, and aligned using CLC Genomics peptide were: for the entire prepro-peptide, 16129.63 Da; Workbench v.12 (Qiagen GmbH). ModelTest-NG for the propeptide, 13347.22 Da; and for the MP, 9018.24 (Darriba et al. 2019) was used to test the best fitting Da. molecular evolution model, and based on the output, a From the alignment of the translated CHH peptide tree has been built using MrBayes 3.2.7a (Ronquist et from C. eastuarii with CHHs (XO-type) from C. maenas al. 2012). A Monte Carlo Markov chain with 1 million (Figure 2) it appeared to have a high level of conservation, generations (with 2 independent analyses run in parallel a part from very few amino acid differences. None of the with 64 chains) was generated and sampled every 1000 CHHs belonging to C. maenas shared 100% similarity, in generations. Convergence of the models was assessed using fact, CHH from C. aestuarii presented a Pro , while for Tracer_v.1.7.1 (Rambaut et al. 2018) and FigTree v.1.4.2. 30 variant 2 and the CHH isolated by Oliphant et al. (2018) software1 was used to graphically design the phylogenetic from C. maenas, the majority of the CHHs had a Gln . tree of the amino acid CHHs from Brachyura. 30 However, variant 2 and the XO-CHH (Oliphant et al., 2018) from C. maenas also had a Pro , within the SP, 3. Results 20 where other CHHs, including the one from C. aestuarii, 3.1. Nucleotide characterisation and deduced peptide had an Ala . sequences 20 Sanger sequencing provided the nucleotide sequences of 3.2. Phylogenetic tree the CHH prepropeptide of C. aestuarii (Figure 1), which Translated peptide sequences from CHHs from Brachyura resulted in an open reading frame sequence of 429 nt. The were aligned using CLC Genomics Workbench v.12 deduced protein consisted of 142 aa with a signal peptide (Qiagen) with the default settings. The peptide sequences (SP) of 26 aa, followed by a CPRP of 40 aa and a mature used are provided in Supplemental Material S1. The peptide (MP) of 72 aa. selected Brachyura CHHs were tested for the best fitting Using SP-5.0 (Henrik, 2017), the existence of a cleavage molecular evolution model, which resulted in JTT+I+G4, site was determined between positions 26 and 27 (AHA- where JTT is the general matrix of Jones et al. (1992), RS), with a probability of 0.8427 and a theoretical pI/Mw +I includes the empirical estimation of the proportion of 8.15/16129.63 g/mol. of invariant sites, and +G4 denotes the estimation of the The consensus pattern found in the newly- gamma distribution parameter with 4 rate classes. Figure characterised CHH sequence confirmed the presence of 3 shows the phylogenetic network among the complete the typical CHH/MIH/GIH neurohormones preprohormone CHHs from Brachyura. 1 Rambaut A (2007). FigTree [online]. Website http://tree.bio.ed.ac.uk/software/figtree/ [accessed 15th April 2020].

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Figure 1. A) Nucleotide and open reading frame of the CHH prepropeptide of C. aestuarii. B) Protein sequence. signal peptide (SP) and CHH precursor related peptide (CPRP), followed by the mature peptide (MP).

Figure 2. Multiple alignment of the CHH from C. aestuarii along with the CHHs (XO-type) from C. maenas.

4. Discussion that acclimation to different conditions is crucial to the C. aestuarii (Nardo, 1847) is a littoral endemic crab of survival of an organism, providing increasing evidence of the Mediterranean Sea; however, thus far, it has been the important role of neuropeptides in stress regulation found to be a global invader, just its relative, C. maenas. (Zhang et al., 2015). However, the fact that the precise Due to its important role in native coastal ecosystems, mechanism of how this process occurs remains elusive was as a result of its value as a food item for several high- the main drive to further characterise the C. aestuarii CHH, market-value species, and its high adaptability and expecting any possible structural diversity of it related resilience to live in coastal, estuarine, and lagoon eutrophic to the impact of environmental stressors. Furthermore, waters, C. aestuarii was chosen as the model organism the CHH peptide sequence may be used in the future to for this study. Significant environmental fluctuations, evaluate and compare CHH expression in other tissues or such as salinity levels, hypoxia, and temperature, as well in comparative studies. asthe presence of xenobiotics in water bodies, affect the The full-length sequence of the CHH prepropeptide was physiological wellbeing of aquatic organisms. Previous obtained from the MT of C. aestuarii. Once compared with studies on C. aestuarii revealed CHH-mediated responses CHHs from Brachyura, the highest similarity was found to environmental stress (Qyli and Aliko, 2017; Qyli to be between C. aestuarii and C. maenas, with a 98.6% et al., 2020). Other studies have corroborated the fact match. This high degree of similarity is not rare among

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Figure 3. Bayesian phylogeny based on the multiple sequence alignment of the full-length of the prepro-CHHs from Brachyura. Posterior probability values below 1 are displayed, while those fully supported are blanks. Each sequence is reported as follows: GenBank accession number, abbreviation of the species followed by the variant name of the CHH, as reported by each researcher. Circled in red is the position of the CHHs from C. maenas and C. aestuarii. See Supplemental File S1 and the main text for details. Caes: Carcinus aestuarii, Cmae: Carcinus maenas, Csap: Callinectes sapidus, Soli: Scylla olivacea, Ptri: Portunus triturbeculatus, Spar: Scylla paramamosain, Cpro: Cancer productus, Cbai: Chionoecetes bairdi, Cjap: Chionoecetes japonicas, Bthe: Bythograea thermydron, Oqua: Ocypode quadrata, Mthu: Metopograpsus thukunar, Pmar: Pachygrapsus marmoratus, Ngra: Neohelice granulate, Ppus: Ptychognathus pusillus, Esin: Eriocheir sinensis, Dcel: Discoplax celeste, Gnat: Gecarcinus lateralis, Gnat: Gecarcoidea natalis. decapods and was also described in lobsters, where H. CHH and the elucidation of its physiological function americanus and H. gammarus shared 100% CHHs similarity provides an excellent model to understand stress-related (GenBank AAB32871 and ABA42180, respectively). Given neuromodulation. the C. aestuarii CHH similarity with that of C. maenas, the same gene organisation can be hypothesised (Dircksen 5. Conclusion et al 2001; Webster et al., 2012); however, this remains Refereeing on GenBank, in addition to the complete CHH an assumption with no direct observations on the DNA. characterisation of C. maenas, this was the second complete Further studies need to be conducted to better understand CHH sequence given for a Carcinus species. When analysing the physiological function of C. aestuarii CHH. This work the nucleotide sequence, the most striking similarity of the represents an initial glimpse into C. aestuarii neuropeptides. CHH genes in C. aestuarii was reported with C. maenas Considering that C. aestuarii is also becoming an invasive (Dircksen et al., 2001). species that is capable of adaptation to a wide-range Nevertheless, to have a clearer and more detailed view of environmental fluctuations, the characterisation of of the CHH superfamily in C. aestuarii, further studies

29 QYLI et al. / Turk J Zool are needed to elucidate its specific expression in different Conflict of interest tissues. The characterisation and identification of CHH in The authors declare no potential conflicts of interest with C. aestuarii represents a use fulstarting point to further respect to the research, authorship, and/or publication of elucidate the biochemical and physiological pathways of this paper. this hormone superfamily. In this study, the tested primers to study CHH gene expression were also provided. Acknowledgments Furthermore, the phylogenetic position of C. aestuarii The authors thank Cristiano Ghenda for providing all of CHH within the Brachyura infragroup confirmed the the specimens used in this study. utility of CHH as a molecular model for understanding intergroup relationships.

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Supplemental Information 1. List of peptide sequences in fasta format used for the phylogenetic tree (Figure 3).

>Carcinus aestuarii_CHH* MYSKTIPAMLAIITVAYLCALPHAHARSTPGYGRMDRILAALKTSPMEPSAALAVEHGTTHPLEKRQIYDTSCKGVYDRALFNDLE HVCDDCYNLYRTSYVASACRSNCYSNLVFRQCMDDLLMMDEFDQYARKVQMVGRKK >DQ507375_Gecarcinus lateralis_CHH_D MTSRMTSAMALVVVAVCASLYSLPHAHARSADGFGRMERLLSSLRGSAESSGALGELRGAGEASAAHPLEKRQIYDRSCKGVYDRS LFNKLEHVCDDCYNLYRTSFVYSSCRENCYSNLVFRQCMEDLLLMNVFDEYAKAVQVVGRKKK >DQ492296_Gecarcinus lateralis_CHH_A MTSRMTSAMALVVVAVCASLYSLPHAHARSADGFGRMERLLSSLRGSAESSGALGELRGAGEASAAHPLEKRQIYDRSCKGVYDRS LFNKLEHVCDDCYNLYRTSFVYSSCRENCYSNLVFRQCMEDLLLMDVFDEYAKAVQVVGRKKK >EF095546_Gecarcoidea natalis_CHH MTSRMTSAMALVVVAVCVSLYSLPHAHARSADGFGRMERLLNSLRGSAESSAALGELRGAGEASAAHPLEKRQIYDRSCKGVYDR SLFNKLEHVCDDCYNLYRTSFVYSSCRENCYSNLVFRQCMEDLLLMDVFDEYAKAVQVVGRKK >JF894384_Discoplax celeste_CHH MLTSRMTSTMALVAVAVFASMHALPHAHARSADGFGRMERLLTSLRGSAESPAALGEASAAHPLEKRQIYDRSCKGVYDRSLFSKL EHVCDDCYNLYRTSYVSSACRENCYSNLVFRQCMDDLLLMDVFDEYAKAVQMVGRKKK >DQ507376_Gecarcinus lateralis_CHH_HT MTSRMTSAMALVVVAVCASLYSLPHAHARSADGFGRMERLLSSLRGSAESSGALGELRGAGEASAAHPLEKRQIYDRSCKGVYDRS LFNKLEHVCDDCYNLYRTSFVYSSCRRDCFDNEMFNYCVKELQLPSPGEYILIRDALRG >JX485644_Eriocheir sinensis_CHH MVAYRMTSTVALVVVVALGASILILPHAHARSAEGFGRMERLLGQLRGGADSSAAVGDLRVAVEGPAGHPLDKRQAYDRSCKGIY DRSLFSKLEHVCDDCFNLYRSSHVASGCRENCYSNLVFRQCMDDLLLMDMFDEYAKAIRVVGRKK >JN048802_Ptychognathus pusillus_CHH MVAYRMTSTVALVVVVALGASILLLPHAHARSAEGFGRMERLLSQLRGGADSPAALGELSIAAEGPAGHPLEKRQVYDRSCKGIYD RSLFSKLEHVCDDCFNLYRSSHVASGCRANCYSNLVFRQCMDDLLLMDMFDEYAKAIRVVGRKK >KP192910_Neohelice granulata_CHH MVTYRMTSTVALVVVVALGASILILPHAHARSAEGFGRMERLLSQIRGGSDSSAALGEMRVAGEGPAGHPLEKRQIYDRSCKGIYD RSLFSKLEHVCDDCYNLYHTSHVASGCRENCYSNLVFRQCMDDLLLMDMFDEYAKAIQVIGRKKK >AY094983_Pachygrapsus marmoratus_CHH MVTCRMASTVALMLVTIFATLAVLPHASARSADGFGRMERLLASLRGSADQPSALGELRAAEEGSSVPHPLEKRQIYDRSCKGVYD RSLFGKLQHVCDDCYNLYRTHHVASACRENCYSNLVFRQCMDDLLLMDVFDEYAKAVQMVGRKK >AY180334_Pachygrapsus marmoratus_CHH_B MVTCRMASTVALMLVTIFATLAVLPHASARSADGFGRMERLLASLRGSADQPSALGELRAAEEGSSVPHPLEKRQIYDRSCKGVYD RSLFGKLEHVCDDCYNLYRTHHVASACRENCYSNLVFRQCMDDLLLMDVFDEYAKAVQMVGRKK >AY180333_Pachygrapsus marmoratus_CHH_A MVTCRMASTVAFMLVTIFASLAVLPHASARSADGFGRMERLLASLRGSADQPSALGELRAAEEGSSVPHPLEKRQIYDRSCKGVYD RSLFGKLEHVCDDCCNLYRTHHVASACRENCYSNLVFRQCMDDLLLMDVFDEYAKAVQMVGRKK >KM052164_Ocypode quadrata_CHH MTSRMTSVAVMVACLCVCGSVLPIATARSADGFSRMERLLTSLRGPAESSGESAHPLEKRQIYDHSCKGVYDRSLFSKLEHVCDDC YNLYRTSYVASACRSNCYSNLVFRQCIDDLLLMDVFDEYAKAIQVVGRKK >KY284578_Metopograpsus thukuhar_CHH_2 MTSRMTSTLALMLLALHVTLPGLPHASARSTDGFGRMERLLTSLRGTTDQATALGDLRAPGEGAAAHPLEKRQIYDRSCKGVYD RSLFNKLEHVCDDCYNLYRTHRVASACREGCYENFVFNECVEDLMLPRELLDIRDAVRG >AF241264_Bythograea thermydron_CHH MLTSRTISSLVTVMVVVCIAVWVLPIAHARSAEGYGRMERLLASIRGGADSMGHLGELTGAGEGAGHPLEKRQIYDRSCKGLYDR RLFSDLDHVCDDCYNLYRNSRVANACRENCYSNLVFRQCMEDLLLMDQFDKYARAVQTVGKK >EF592483_Cancer productus_CHH_I MLTSRTLPTIILGVLCIYLSTIPNAHARSAQGMGKMEHLLASYRGALESNTPTGDLPGGLVHPVEKRQIYDSSCKGVYDRGLFSDLE HVCDDCYNLYRNSYVASACRSNCYSNVVFRQCMEELLLMEEFDKYARAVQIVGKKK >EF592486_Cancer productus_CHH_III MLTSRTLPTIILGVLCIYLSTIPNAHARSAQGMGKMEHLLASYRGALESNTPIGDLPGGLVHPVEKRQIYDSSCKGVYDRGLFSDLE HVCDDCYNLYRNSYVASACRSNCYSNVVFRQCMEELLLMEEFDKYARAVQIVGKKK >EF592485_Cancer productus_CHH_IIb MLTSRTLPTLILGVLCIYLSTLPNAHARSAQGMGKMERLLASYRGAVEPNTPLGDLPGGLVHPVEKRQIYDTSCKGVYDRGLFSDL EHVCDDCYNLYRNSYVASACRSNCYSNVVFRQCMEELLLMEEFDKYARAVQIVGKKK

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>EF592484_Cancer productus_CHH_IIa MLTSRTLPTIILGVLCIYLSTLPNAHARSAQGMGKMERLLASYRGAVEPNTPLGDLPGGLVHPVEKRQIYDTSCKGVYDRGLFSDL EHVCDDCYNLYRNSYVASACRSNCYSNVVFRQCMEELLLMEEFDKYARAVQIVGKKK >X17596_Carcinus maenas_CHH MYSKTIPAMLAIITVAYLCALPHAHARSTQGYGRMDRILAALKTSPMEPSAALAVENGTTHPLEKRQIYDTSCKGVYDRALFNDL EHVCDDCYNLYRTSYVASACRSNCYSNLVFRQCMDDLLMMDEFDQYARKVQMVGRKK >KF792072_Chionoecetes japonicus_CHH MTTTTLITMLLLLVCVSMNALNLAQARSTQGYGHMDKLLQSLRGNGDPTTPIDNMAHSLEKRQVYDTSCKGMYDRGLFSDLEH VCDDCYNLYRNPHVATACRGNCYSNLVFRQCMEDLLLMEEFDKYARAIQTVGKKK >EU677192_Chionoecetes bairdi_CHH MTTTTLIAMLLLLVCVSMNALNLAQARSAQGYGHMDKLLQSLRGNGDPTTPIDNMAHSLEKRQVYDTSCKGLYDRGLFSDLEH VCDDCYNLYRNPHVATACRGNCYSNLVFRQCMEDLLLMEEFDKYARAIQTVGKKK >JQ812807_Scylla paramamosain_CHH MSTFTSVIQMAVLVACIAMATLPHTQGRSADGFGRMGRLLASLKADSLGPVQDYGVEGAAHPLEKRQTFDSSCKGVYDRAIFSEL EHVCNDCYNLYRTSRVASGCRSNCYSNVVIRQCMEDLLLMDNFEEIARKIQMVGKK >JQ421463_Scylla paramamosain_CHH_2 MSTFTSVIQMAVLVACIAMATLPHTQGRSADGFGRMGRLLASLKADSLGPVQDYGVEGAAHPLEKRQIFDSSCKGVYDRAIFSEL EHVCNDCYNLYRTSRVASGCRSNCYSNVVIRQCMEDLLLMDNFEEIARKIQMVGKK >JQ855711_Scylla paramamosain_CHH_2 MSTFTSVIQMAVLVACIAMATLPHTQGRSADGFGRMGRLLASLKADSLGPVQDYGVEGAAHPLEKRQTFDSSCKGVYDRAIFSEL EHVCNDCYNLYRTSRVASGCRANCFENHVFDDCVYDLLLHNPDEVLLMRDAIRG >KR078357_Scylla paramamosain_CHH_2 MSTFTSVIQMAVLVACIAMATLPHTQGRSADGFGRMGRLLASLKADSLGPVQDYGVEGAAHPLEKRQIFDSSCKGVYDRAIFSEL EHVCNDCYNLYRTSRVASGCRANCFENHVFDDCVYDLLLHNPDEVLLMRDAIRG >JQ421462_Scylla paramamosain_CHH MSTFTSVIQMAVLVACIAMATLPQTQGRSADGFGRMGRLLASLKGDSLGPVQDYGVEGAAHPLEKRQIFDSSCKGVYDRAIFSEL EHVCNDCYNLYRTSRVASGCRANCFENHVFDDCVYDLLLHNPDEVLLMRDAIRG >AY372181_Scylla olivacea_CHH MSALTSIMQMAVLVACITMATLPDTQARSAEGFGRMGRLLASLKADSLGPVQDFGVEGAAHPLEKRQIFDSSCKGVYDRAIFNEL EHVCNDCYNLYRTSHVASGCRSNCYSNVVIRQCMEDLLLMDNFEEIARKIQMVGKK >AY536012_Callinectes sapidus_CHH MQSIKTVCQITLLVTCMMATLSYTHARSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHPLEKRQIYDSSCKGVYDRAIFNELEH VCDDCYNLYRNSRVASGCRSNCYSNMVIRQCMEDLLIMDNFEEYARKIQVVGKK >DQ343306_Callinectes sapidus_CHH MQSIKTVCQITLLVTCMMATLSYTHARSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHPLEKRQIYDSSCKGVYDRAIFNELEH VCDDCYNLYRNSRVASGCRENCFDNMMFETCVQELFYPEDMLLVRDAIRG >KJ813806_Portunus trituberculatus_CHH_2 MQSIKSVCQVSLVAACIIFTLPWTQARSAEGFGRMGRLLASLKADSLTPMQGYGTETGHPLEKRQIYDSSCKGVYDRAIFSELEHV CDDCYNLYRTSRVASGCRENCFENDLFEECVFELMLPDEMFLIRDAIRG > EF095546_ Gecarcoidea natalis_CHH MALVVVAVCVSLYSLPHAHARSADGFGRMERLLNSLRGSAESSAALGELRGAGEASAAHPLEKRQIYDRSCKGVYDRSLFNKLEH VCDDCYNLYRTSFVYSSCRENCYSNLVFRQCMEDLLLMDVFDEYAKAVQVVGRKK

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