Chieu HD et al. (2019), Aquaculture Breeding Enhancement: Maturation an...

ECB-ART-47030

Front Genet 2019 Feb 19;10:77. doi: 10.3389/fgene.2019.00077.

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Aquaculture Breeding Enhancement: Maturation and Spawning in Sea Cucumbers Using a Recombinant Relaxin-Like Gonad-Stimulating Peptide.

Chieu HD , Turner L , Smith MK , Wang T , Nocillado J , Palma P , Suwansa-Ard S , Elizur A , Cummins SF .

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Wild sea cucumber resources have been rapidly exhausted and therefore there is an urgent need to develop approaches that will help restocking. Currently, there is a lack of information regarding the genes involved in sea cucumber reproductive processes. The neurohormone relaxin-like gonad-stimulating peptide (RGP) has been identified as the active gonad-stimulating peptide in sea stars (Asteroidea), which could also be present in other echinoderm groups. In this study, a sea cucumber RGP was identified and confirmed by phylogenetic analysis. A recombinant Holothuria scabra RGP was produced in the yeast Pichia pastoris and confirmed by mass spectrometry. To assess bioactivity, four levels of purification were tested in an in vitro germinal vesicle breakdown (GVBD) bioassay. The most pure form induced 98.56 ± 1.19% GVBD in H. scabra and 89.57 ± 1.19% GVBD in Holothuria leucospilota. Cruder levels of purification still resulted in some GVBD. Upon single injection into female H. scabra, the recombinant RGP induced head waving behavior followed by spawning within 90-170 min. Spawned oocytes were fertilized successfully, larvae settled and developed into juveniles. Our results provide a key finding for the development of a break-through new artificial breeding approach in sea cucumber aquaculture.

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Genes referenced: acp7 LOC100887844 LOC100893907 LOC105439578 LOC105439585 LOC115919910

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	Figure 1. Holothuria scabra RGP protein encoded within the vector construct. A-Chain (green), B-chain (Blue) and histidine linker (red) regions are shown. Yellow highlight backgrounds indicate conserved cysteine residues.
	Figure 2. Multiple sequence alignment and phylogenetic tree analysis of echinoderm RGP/IGF mature peptides. (A) Alignment of echinoderm RGP/IGF mature peptides. The yellow bars indicate the putative A- and B-chain peptides which contain the highly conserved cysteine residues (asterisks) important for disulphide bridge formation. The sequence logo above the alignment shows the conservation of amino acid composition at each position. (B) Phylogenetic tree analysis of sea cucumber and other echinoderm RGP mature peptides based on maximum likelihood estimation (500 bootstraps). The echinoderm IGFs were used as the outgroup. The numbers above nodes indicate the branch support values. The scale bar indicates the estimated amino acid substitutions per site. For species abbreviations and sequences used in the amino acid alignment and phylogenetic tree, see Supplementary Data S1.
	Figure 3. Recombinant Holothuria scabra RGP and mass spectrometry (MS) identification. (A) RGP sequence showing MS peptide coverage (light blue lines). (B) A representative structure of recombinant RGP. Green: A-chain, dark blue: B-chain, red: histidine linker “GSGSHHHHHHGSGS”. (C) Upper, potential energy of RGP peptide as a function of time during molecular dynamics (MD). Lower, backbone RMSD (root mean square deviation) during the same MD, compared to the lowest-energy conformation (the representative structure).
	Figure 4. Holothuria scabra oocyte maturation bioassay with recombinant RGP. Ovarian segments were incubated for 3 h in (A) crude supernatant, CS; (B) crude concentrated RGP, CR; (C) SepPak-purified RGP, SR; inset shows higher magnification of mature oocytes from ovarian segment incubated in 96 μg/μL SR; (D) His and Amicon-purified RGP, HR; (E) yeast solution without RGP, YS; (F) filtered artificial sea water, FASW; and (G) radial nerve extract, RNE. Two insets (red boxes) indicate higher magnification of mature oocytes inside ovarian segment incubated in SR (96 μg/μL) and RNE after 3 h. DO indicates the distorted oocyte; GV, germinal vesicle; GVBD, germinal vesicle breakdown. All scale bars indicate 200 μm.
	Figure 5. Summary of Holothuria scabra spawning bioassay with recombinant RGP. Negative controls included filtered artificial seawater (FASW) and yeast solution without RGP. Test solutions included crude RGP supernatant (CS) and His and Amicon-purified RGP (HR). The first injection was a 2mL volume and the second was a 3mL volume.
	Figure 6. Holothuria scabra spawning bioassay with recombinant RGP. (A) Total gonad from an individual showing tubules. (B) Immature oocytes (170 ± 6.4 μm in diameter) with germinal vesicle before spawning. (C) Injection of sample into the body wall. (D) Head waving observed prior to spawning. Inset, higher magnification of sea cucumber head (red box), showing the location of gonopore (single arrow) and mouth (double arrow). (E) Sea cucumber spawning oocytes from gonopore. Inset, high magnification view of spawned oocytes (red box) indicating they were mature.
	Figure 7. Embryonic, larval development and juvenile growth of Holothuria scabra from RGP-matured eggs. (A) the matured eggs were fertilized, then (B–D) 2-cell, 4-cell and multi-cell division was occurred until 8 h post-fertilization. (E) early auricularia stage at 2 days post-fertilization (dpf). Buccal ciliated cavity (bcc), cloaca (clo), ossicle (oss). (F) Mid-auricularia stage at 5 dpf. Mouth (mou), oesophagus (oes). (G) Late-auricularia at 14 dpf. Hyaline sphere (hs), axohydrocoel (axo), left somatocoel (ls). (H) Doliolaria at 16-day; digestive tract (dt), ciliary band (cb), primary tentacles primordium (ptp). (I) Pentactula at 21 dpf. Tentacle (ten), podia (pod). (J) Juvenile at 36 dpf. Papillae (pap). Scale bars in (A–D) are 50 μm. Scale bars in (E–H) are 100 μm. Scale bars in (I,J) are 500 μm.

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