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Moult-inhibiting fusion protein augments while polyclonal antisera attenuate moult stages and duration in Penaeus
S Vrinda1, C Jasmin2, K C Sivakumar3
1National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Kochi 16, Kerala, India.
Abstract:
Moulting in crustaceans is regulated by moult-inhibiting hormone (MIH) of the CHH family neuropeptides. The inhibitory functions of MIH have pivotal roles in growth and reproduction of Penaeus monodon. In this study, we report the expression of a thioredoxin-fused mature MIH I protein (mf-PmMIH I) of P. monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmMIH I). The mature MIH I gene of 231bp, that codes for 77 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The translation expression vector construct (mf-PmMIH I+pET32a+) upon induction produced 29.85kDa mature MIH I fusion protein (mf-PmMIH I). The purified fusion protein was used as exogenous MIH I and as antigen to raise polyclonal antisera. When fusion protein (mf-PmMIH I) was injected into D2 and D3 stages of juvenile shrimp, the moult cycle duration was extended significantly to 16.67±1.03 and 14.67±1.03days respectively compared to that of 11.67±1.03days in controls. Moult duration was further reduced to 8.33±0.82days when polyclonal antiserum (anti-mf-PmMIH I - 1:500 dilutions) was injected. Anti-mf-PmMIH I immunolocalized MIH I producing neurosecretory cells in the eyestalk of P. monodon. In short, the present manuscript reports an innovative means of moult regulation in P. monodon with thioredoxin fused MIH I and antisera developed.
Insights
Researchers developed a thioredoxin-fused moult-inhibiting hormone (MIH) protein for Penaeus monodon. This protein and its antiserum offer innovative methods for regulating crustacean molting, impacting growth and reproduction.
Area of Science:
- Endocrinology
- Molecular Biology
- Aquaculture
Background:
- Crustacean molting is regulated by moult-inhibiting hormone (MIH), a neuropeptide crucial for growth and reproduction in Penaeus monodon.
- Understanding MIH function is key to controlling molting processes in commercially important shrimp species.
Purpose of the Study:
- To express a thioredoxin-fused mature MIH I protein (mf-PmMIH I) from P. monodon in a bacterial system.
- To utilize the expressed fusion protein as an antigen for generating polyclonal antiserum (anti-mf-PmMIH I).
- To investigate the potential of mf-PmMIH I and its antiserum in regulating the molting cycle of P. monodon.
Main Methods:
- Cloning the mature MIH I gene (231bp) into an Escherichia coli thioredoxin gene fusion expression vector (pET32a+).
- Inducing expression to produce a 29.85kDa mf-PmMIH I fusion protein.
- Purifying the fusion protein and using it to raise polyclonal antisera.
- Injecting the fusion protein and antiserum into juvenile shrimp (D2 and D3 stages) to observe effects on molting duration.
- Immunolocalization of MIH I-producing cells using the developed antiserum.
Main Results:
- The expression system successfully produced the 29.85kDa mf-PmMIH I fusion protein.
- Injection of mf-PmMIH I significantly extended the molting cycle duration in juvenile shrimp.
- Injection of anti-mf-PmMIH I antiserum significantly reduced the molting cycle duration.
- The antiserum successfully localized MIH I-producing neurosecretory cells in the shrimp eyestalk.
Conclusions:
- A functional thioredoxin-fused MIH I protein and specific antiserum were successfully developed for Penaeus monodon.
- These tools provide an innovative approach to manipulate and regulate crustacean molting.
- The findings have implications for aquaculture, potentially improving growth and reproductive management in shrimp farming.
