Effective Gene Silencing in a Microsporidian Parasite Associated with Honeybee ( Apis mellifera ) Colony Declines

Abstract

Honeybee colonies are vulnerable to parasites and pathogens ranging from viruses to vertebrates. An increasingly prevalent disease of managed honeybees is caused by the microsporidian Nosema ceranae. Microsporidia are basal fungi and obligate parasites with much-reduced genomic and cellular components. A recent genome-sequencing effort for N. ceranae indicated the presence of machinery for RNA silencing in this species, suggesting that RNA interference (RNAi) might be exploited to regulate Nosema gene expression within bee hosts. Here we used controlled laboratory experiments to show that double-stranded RNA homologous to specific N. ceranae ADP/ATP transporter genes can specifically and differentially silence transcripts encoding these proteins. This inhibition also affects Nosema levels and host physiology. Gene silencing could be mediated solely by Nosema or in concert with known systemic RNAi mechanisms in their bee hosts. These results are novel for the microsporidia and provide a possible avenue for controlling a disease agent implicated in severe honeybee colony losses. Moreover, since microsporidia are pathogenic in several known veterinary and human diseases, this advance may have broader applications in the future for disease control.

RNA interference in honeybees: off-target effects caused by dsRNA

“…Since its discovery in Caenorhabditis elegans (Fire et al 1998), RNAi has become the predominant reverse genetic method in a variety of non-model organisms. Moreover, as honeybees are among the few recognized beneficial insects with a large economical and ecological impact, the use of RNAi is increasingly used as a tool for fighting pests and pathogens in apiculture (Maori et al 2009;Paldi et al 2010).…”

Effective Silencing of Dicer Decreases Spore Load of the Honey Bee Parasite Nosema ceranae

“…Dicer expression was indeed suppressed after siRNA treatment for the first two days post infection. It is necessary to point out that N. ceranae is an intra-cellular parasite and the honey bee RNA interference pathway could also be activated to suppress N. ceranae gene expression, as suggested from previous study [21]. It is unknown whether honey bees use the siRNA to target the parasite gene Dicer or the parasite used the siRNA to silence the expression level of its own Dicer.…”

“…So far, there is no functional analysis of Dicer in N. ceranae. It is also unclear whether N. ceranae possesses a functional RNA interference pathway, although prior work has indicated that growth of this parasite is inhibited by RNAi [21]. In order to determine the importance of N. ceranae Dicer for its reproduction, we designed small interfering RNA (siRNA) to target the parasite gene for Dicer.…”

Negligible uptake and transfer of diet-derived pollen microRNAs in adult honey bees

“…In agreement, oral administration of dsRNA appears to be effective at regulation of gene expression in honey bees. [69][70][71][72][73][74][75][76] In addition, high dose injection of dsRNAs into the hemocoel has been used for delivery and inhibition of target genes in multiple tissues in this species [77][78][79][80][81][82][83][84][85][86] independent of the site of injection, demonstrating that these molecules can be efficiently taken up by disparate tissues once in the open circulatory system. Our results, in conjunction with the previous studies using dsRNA in other insect species, suggest that uptake of longer dsRNAs may be more efficient than shorter miRNAs.…”