RNA interference mechanism (RNAi) as a biotechnological tool for the health and reproduction of White Shrimp Litopenaeus vannamei
Author: Rubens Galdino Feijó (Currículo Lattes)
Supervisor: Dr Luis Fernando Fernandes Marins
Co-supervisor: Dr Rodrigo Maggioni
Abstract
The studies described in this thesis involve the application of methods for the development of RNAi technology aimed at gene therapy of L. vannamei shrimp infected with the Infectious Myonecrosis virus (IMNV) and the study of the reproduction of L. vannamei through post-silencing transcriptional gene encoding gonad inhibitory hormone (GIH). In Chapter I, methodologies for the detection and quantification by RT-qPCR and qPCR of the Infectious Myonecrosis virus (IMNV) and the White Spot Syndrome virus (WSSV), respectively, were developed and effective for the unprecedented detection of the occurrence of co -infection with both viruses in specimens of L. vannamei shrimp. In Chapter II it was demonstrated that the transcription pattern of the main key genes of the RNAi mechanism (Sid-1, Dicer-2 and Argonauta-2) of L.vannamei is unaffected by the infection caused by IMNV when injecting different concentrations of the viral inoculum and that the average number of 2.55 x 103 copies of IMNV detected in hemocytic tissue can be considered safe for L. vannamei cultivation systems under optimal conditions. Chapter III describes the production of dsRNA molecules related to different ORFs in the IMNV genome (dsRNA-IMNV ORF1a, dsRNA-IMNV ORF1b or dsRNA-IMNV ORF2) for the therapy of L. vannamei experimentally infected with 1.02 x 106 copies of IMNV. The treatments with dsRNA-IMNV (ORF1a) and dsRNA-IMNV (ORF1b) were effective in inhibiting the replication of IMNV resulting in survival of 90% and 80%, respectively, after 34 days of experimental challenge.The results obtained in this chapter suggest an export of the RNAi signal through the SID-1 transmembrane channel, this event being directly associated with the inhibition of IMNV replication and, consequently, with the survival of challenged shrimp. In Chapter IV, it was demonstrated that despite the knockdown of GIH mRNA levels detected in the eye peduncle of L. vannamei females injected with dsRNA-GIH, the effect on the expression of vitellogenin mRNA in the ovaries and in the diameter of the oocytes it was only verified on the 37th day after injection. The results presented in this thesis confirm the potential of using RNAi technology both for gene therapy of viral diseases and for the study of reproduction in L. vannamei shrimp.this event being directly associated with inhibition of IMNV replication and, consequently, with the survival of challenged shrimp. In Chapter IV, it was demonstrated that despite the knockdown of GIH mRNA levels detected in the eye peduncle of L. vannamei females injected with dsRNA-GIH, the effect on the expression of vitellogenin mRNA in the ovaries and in the diameter of the oocytes it was only verified on the 37th day after injection. The results presented in this thesis confirm the potential of using RNAi technology both for gene therapy of viral diseases and for the study of reproduction in L. vannamei shrimp.