In VitroGene Silencing of the Fish MicrosporidianHeterosporis sauridaby RNA Interference

Abstract

Heterosporis saurida, a microsporidian parasite of lizardfish, Saurida undosquamis, causes severe economic losses in marine aquaculture. Among the novel approaches being explored for treatment of parasitic infections in aquaculture is small interfering RNA molecules. The aim of the present study was to investigate the efficiency of using siRNA to knock down expression of specific genes of H. saurida in vitro. For this purpose, siRNAs specific for ATP/ADP antiporter 1 and methionine aminopeptidase II genes were designed and tested using a previously developed in vitro cultivation model. Silencing of H. saurida target genes was assessed and the efficacy of using siRNA for inhibition of gene expression was measured by quantitative real-time polymerase chain reaction (PCR). Silencing of ATP/ADP antiporter 1 or methionine aminopeptidase II by siRNA reduced H. saurida infection levels in EK-1 cells 40% and 60%, respectively, as measured by qRT-PCR and spore counts. Combined siRNA treatment of both ATP/ADP antiporter 1 and methionine aminopeptidase II siRNAs was more effective against H. saurida infection as seen by the 16S rRNA level and spore counts. Our study concluded that siRNA could be used to advance development of novel approaches to inhibit H. saurida and provide an alternative approach to combat microsporidia.

A secretory hexokinase plays an active role in the proliferation of Nosema bombycis

“…This was the first step toward the generation of successful transgenic microsporidia, but there were no follow-up reports regarding its application in protein function research. RNAi has been widely applied in other fungi including Nosema ceranae and Heterosporis saurida ( Paldi et al, 2010 ; Saleh et al, 2016 ), to study protein functions. N. bombycis has all of the genes required for RNA silencing and it functions in vivo , but the RNA interference efficiency was unstable in different experimental groups ( Pan et al, 2017 ; Wang et al, 2015 ).…”

“…The Sf9-III (Thermo Fisher, Santa Clara, CA, USA) cells were infected by using the previous method described above. After infection, two µg dsRNAs of NbHK or EGFP were transfected into Sf9-III respectively and then cultured in 6-well plates ( Saleh et al, 2016 ). The samples were collected at 1, 3 and 5 d. p. i.…”

Peer Review #4 of "A secretory hexokinase plays an active role in the proliferation of Nosema bombycis (v0.1)"

“…This was the first step toward the generation of successful Manuscript to be reviewed transgenic microsporidia, but there were no follow-up reports regarding its application in protein function research. RNAi has been widely applied in other fungi including Nosema ceranae and Heterosporis saurida (Paldi et al 2010;Saleh et al 2016), to study protein functions. N.…”

“…The Sf9-III (Thermo Fisher, CA, USA) cells were infected by using the previous method described above. After infection, 2 μg dsRNAs of NbHK or EGFP were transfected into Sf9-III respectively and then cultured in 6-wells plates (Saleh et al 2016). The samples were collected at 1, 3 and 5 d. p. i.…”

A new age in AquaMedicine: unconventional approach in studying aquatic diseases

“…RNAi treatment has shown promising results in the treatment of parasitic infections in fish. Saleh et al [ 128 ] demonstrated that in vitro RNAi knockdown of ATP/ADP antiporter and methionine aminopeptidase II of the Heterosporis saurida , parasite of the lizardfish ( Saurida undosquamis) , reduced targeted gene transcription and spore counts in cell culture assays. Potential for RNAi-based medicine has been demonstrated in vivo by treating the oligochaete host Tubifex tubifex for the cnidarian myxozoan parasite ( Myxobolus cerebralis ) which causes whirling diseases in salmonid fish [ 129 ].…”