Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

Sebastian Gisder,Elke Genersch,Wolfgang Blenau

doi:10.1371/journal.pone.0117200

Abstract

Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

Highlights

Wild and managed insect pollinators are crucial for the maintenance of natural ecosystems as well as for the agricultural production of many crops and fruit [1,2]
Quantification of N. ceranae infection is a prerequisite. To this end we adapted a recently published PCR-ELISA protocol, which had originally been developed for detection and quantification of Leishmania parasites in host tissues [61], to N. ceranae
A prerequisite for establishing an RT-PCR-ELISA for the detection and quantification of N. ceranae was the selection of appropriate primers detecting genes expressed during infection

Summary

Introduction

Wild and managed insect pollinators are crucial for the maintenance of natural ecosystems as well as for the agricultural production of many crops and fruit [1,2]. In most regions of the world, the supply with insect pollinators does not meet the steadily increasing demands in agriculture and it is this gap that makes the health of managed Western honey bee (Apis mellifera) colonies, which provide 90% of the commercial pollination in agriculture, a worldwide issue [3,4,5]. Among the pathogens obviously threatening honey bees and wild bees alike is the microsporidian parasite Nosema ceranae (N. ceranae) [6,8,9]. Honey bees are frequently found infected by Nosema apis (N. apis) and N. ceranae [12,13,14,15]. Once ingested by worker bees, e.g., in the course of cleansing activities inside the hive [16,17], the spores germinate in the midgut lumen thereby extruding their infection apparatus, the polar tube [18]. While N. apis has long since been associated with the Western honey bee [14] and is well studied [12,26,27], N. ceranae is considered an emerging pathogen of the Western honey bee that has switched host from the Eastern honey bee (Apis cerana) only quite recently [13,28,29,30,31,32,33,34]

Methods

Results

Conclusion