Silencing the Honey Bee (Apis mellifera) Naked Cuticle Gene ( nkd ) Improves Host Immune Function and Reduces Nosema ceranae Infections

Abstract

Nosema ceranae is a new and emerging microsporidian parasite of European honey bees, Apis mellifera, that has been implicated in colony losses worldwide. RNA interference (RNAi), a posttranscriptional gene silencing mechanism, has emerged as a potent and specific strategy for controlling infections of parasites and pathogens in honey bees. While previous studies have focused on the silencing of parasite/pathogen virulence factors, we explore here the possibility of silencing a host factor as a mechanism for reducing parasite load. Specifically, we used an RNAi strategy to reduce the expression of a honey bee gene, naked cuticle (nkd), which is a negative regulator of host immune function. Our studies found that nkd mRNA levels in adult bees were upregulated by N. ceranae infection (and thus, the parasite may use this mechanism to suppress host immune function) and that ingestion of double-stranded RNA (dsRNA) specific to nkd efficiently silenced its expression. Furthermore, we found that RNAimediated knockdown of nkd transcripts in Nosema-infected bees resulted in upregulation of the expression of several immune genes (Abaecin, Apidaecin, Defensin-1, and PGRP-S2), reduction of Nosema spore loads, and extension of honey bee life span. The results of our studies clearly indicate that silencing the host nkd gene can activate honey bee immune responses, suppress the reproduction of N. ceranae, and improve the overall health of honey bees. This study represents a novel host-derived therapeutic for honey bee disease treatment that merits further exploration. IMPORTANCEGiven the critical role of honey bees in the pollination of agricultural crops, it is urgent to develop strategies to prevent the colony decline induced by the infection of parasites/pathogens. Targeting parasites and pathogens directly by RNAi has been proven to be useful for controlling infections in honey bees, but little is known about the disease impacts of RNAi silencing of host factors. Here, we demonstrate that knocking down the honey bee immune repressor-encoding nkd gene can suppress the reproduction of N. ceranae and improve the overall health of honey bees, which highlights the potential role of host-derived and RNAi-based therapeutics in controlling the infections in honey bees. The information obtained from this study will have positive implications for honey bee disease management practices. E uropean honey bees, Apis mellifera, play a critical role in the pollination of important crops. However, honey bee populations have suffered high losses in much of the world (1), coincident with an increase in agricultural demand for honey bee pollination (2). Specifically, honey bee colony losses in the United States have been exacerbated since the report of colony collapse disorder (CCD), a syndrome that comprises large-scale, unexplained losses of managed honey bees (3-9). High levels of parasites and pathogens have been linked to the decline of honey bee colonies (10,11).Nosema is a genus of obligate, intracellular microsporidian parasite...

Nosema ceranae parasitism impacts olfactory learning and memory and neurochemistry in honey bees (Apis mellifera)

“…Something similar may be occurring in honey bees with Nosema infection. Nosema ceranae has been found to suppress the immune system of the honey bee (Antunez et al, 2009;Holt et al, 2013;Li et al, 2016). It upregulates the naked cuticle gene, nkd, a negative regulator of host immune function (Li et al, 2016).…”

“…Nosema ceranae has been found to suppress the immune system of the honey bee (Antunez et al, 2009;Holt et al, 2013;Li et al, 2016). It upregulates the naked cuticle gene, nkd, a negative regulator of host immune function (Li et al, 2016). This has the effect of suppressing the host's immune response and, when nkd is knocked down, several immune genes are upregulated and N. ceranae spore loads are reduced (Li et al, 2016).…”

“…It upregulates the naked cuticle gene, nkd, a negative regulator of host immune function (Li et al, 2016). This has the effect of suppressing the host's immune response and, when nkd is knocked down, several immune genes are upregulated and N. ceranae spore loads are reduced (Li et al, 2016). An important distinction to be made, however, is that evasion of the immune system does not indicate parasitic manipulation necessarily: there may be an effect on the immune system that leads to behavioral manipulation over evolutionary time (Adamo, 2002).…”

Dicer regulates Nosema ceranae proliferation in honeybees

“…Different host genes of the apoptosis pathway were reported to be regulated to inhibit the apoptosis across different parasite species (del Aguila et al ., ; Higes et al ., ; He et al ., ; Doublet et al ., ; Martín‐Hernández et al ., ). Additional studies have tried to identify the important proteins for microsporidian infection, including parasite polar tube proteins, cell wall proteins, adenosine triphosphate transporters and host cuticle proteins (Xu and Weiss, ; Paldi et al ., ; Li et al ., ; ; Han et al ., ). Nevertheless, there have been few attempts to identify more broadly the pathways used by microsporidia to infect hosts, and the counter‐defences hosts might use to reduce the impacts of infection.…”

“…Correspondence: Qiang Huang, Institute of Bee Health, Vetsuisse Faculty, University of Bern, Schwarzenburgstrasse 161, Liebefeld,Switzerland; transporters and host cuticle proteins (Xu and Weiss, 2005;Paldi et al, 2010;Li et al, 2016Li et al, , 2012Han et al, 2017). Nevertheless, there have been few attempts to identify more broadly the pathways used by microsporidia to infect hosts, and the counter-defences hosts might use to reduce the impacts of infection.…”