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Add To Calendar 29/04/2016 14:10:0029/04/2016 14:30:00America/ChicagoAsian-Pacific Aquaculture 2016THE EFFECT OF GIH-dsRNA ENCAPSULATED IN CHITOSAN-NANOPARTICLES ON THE LEVEL OF GONAD INHIBITING HORMONE (GIH) GENE EXPRESSION IN BLACK TIGER SHRIMP (Penaeus monodon) Crystal 2The World Aquaculture Societyjohnc@was.orgfalseanrl65yqlzh3g1q0dme13067DD/MM/YYYY

THE EFFECT OF GIH-dsRNA ENCAPSULATED IN CHITOSAN-NANOPARTICLES ON THE LEVEL OF GONAD INHIBITING HORMONE (GIH) GENE EXPRESSION IN BLACK TIGER SHRIMP (Penaeus monodon)

*Danang Crysnanto1 , Gianni Yosephine 1, Adi Pancoro1
1Genetics and Molecular Biotechnology Research Group, School of Life Science and Technology, Institut Teknologi Bandung, Bandung, Indonesia
adi@sith.itb.ac.id

 

Spawning process in black tiger shrimp (P. monodon) is negatively regulated by GIH hormone produced by X organ in eyestalk. Cutting the eyestalk (ablation) is the conventional way to accelerate the spawning process. However, this method may causes physiological problems in shrimp. The novel way to speed up the spawning process is through molecular mechanism by harnessing RNA interference system[2]. RNAi acts by repressing the GIH expression through posttranscriptional regulation mediated by dsRNA. Based on the prior study [1], injection of naked dsRNA to induce RNAi mechanism is not effective as it might cause dsRNA degradation during delivery. Nanoparticle encapsulation is expected to be able to protect the dsRNA from degradation, so that the gene expression could be more reduced and to produce long-lasting inhibition effect.

The goal of this experiment was to compare the inhibition level of dsRNA-GIH non-encapsulated to dsRNA-GIH encapsulated on the GIH expression in Penaeus monodon. Female shrimps (40 gr average weight) were divided into 2 groups and injected with non-encapsulated (first group) dan encapsulated (second group) on the second walking leg (pereiopoda) at dosage: 3 ug/g body weight. In the both groups, the eyestalks were sampled in 3 x 24 hours and 7 x 24 hours after injection. RNA from eyestalk was extracted and then was converted into cDNA. GIH expression level was analyzed using Real-Time quantitative PCR (RT-qPCR) with β-actin as the reference gene and LIVAK method was selected for quantifying the reduction of gene expression.

The result shown that the expression level of GIH was lower in shrimp injected with encapsulated dsRNA compared to the ones injected with naked dsRNA in both periods of sampling. The relative level of expressions for the former were 47% and 20% in day 3 and 7, and the corresponding gene expression for the latter were 5.4% and 7.9 % respectively. This experiment also shown that the gene expression was inhibited even further in days 7. This continued inhibition was assumed due to the protection from nanoparticles-encapsulation.

[1] Pancoro, A; Sony, S; Fainmarinat, I; Anggi, S. (2011). Silencing of Gonad-Inhibiting Hormone (GIH) gene Expression by GIH Intron Hairpin-Double Stranded RNA and Its Impact on Early Ovary Development Penaeus broodstock. Penelitian Unggulan Strategis Nasional DIKTI.

[2] Treerattrakool, Supattra; Panyim, Sakol; Udomkit, Apinunt. (2011). Induction of Ovarian Maturation and Spawning in Penaeus monodon Broodstock by Double-Stranded RNA. Mar Biotechnol 13, 1-7.




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