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.
Highlights
Worldwide, diseases of aquatic animals impose considerable constraints to the expansion and management of aquaculture, attempts to control diseases have become a main concern in many fish-producing areas
EK-1 cells infected with H. saurida spores incubated in serum-free L-15 medium were transfected with a solution containing 2 mM FAM-labeled negative control small interfering RNA (siRNA) for 4 h at 26°C
EK-1 cells infected with H. saurida spores incubated in serum-free L-15 medium were transfected with a solution containing 2 mM FAMlabeled negative control siRNA and the uptake into H. saurida in EK-1 was observed (Fig. 4)
Summary
Diseases of aquatic animals impose considerable constraints to the expansion and management of aquaculture, attempts to control diseases have become a main concern in many fish-producing areas. Microsporidia are obligate, protozoan, intracellular, parasites that infect a broad range of animals, including fish, and are increasingly recognized as economically and medically important parasites [1]. Microsporidian infections by members of genus Heterosporis may lead to major pathogenic effects to their hosts. Heterosporis spp. do not produce a xenoma, but infect tissues diffusely, and may become bordered by host connective tissue [2]. Heterosporis saurida isolated from lizardfish, Saurida undosquamis in the Arabian Gulf, causes significant pathogenic effects on the host. The molecular basis of microsporidian pathogenicity and virulence is largely unexplored, due, in part, to the scarceness of suitable systems to support studies of host– pathogen interactions and allow genetic manipulation [1,4]
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