SummaryMethods are described for working with Nosema apis and Nosema ceranae in the field and in the laboratory. For fieldwork, different sampling methods are described to determine colony level infections at a given point in time, but also for following the temporal infection dynamics.Suggestions are made for how to standardise field trials for evaluating treatments and disease impact. The laboratory methods described include different means for determining colony level and individual bee infection levels and methods for species determination, including light microscopy, electron microscopy, and molecular methods (PCR). Suggestions are made for how to standardise cage trials, and different inoculation methods for infecting bees are described, including control methods for spore viability. A cell culture system for in vitro rearing of Nosema spp. is described. Finally, how to conduct different types of experiments are described, including infectious dose, dose effects, course of infection and longevity tests. Métodos estándar para la investigación sobre Nosema ResumenSe describen procedimientos para trabajar con Nosema apis y Nosema ceranae en el campo y en el laboratorio. Para el trabajo de campo, se describen diferentes métodos de muestreo para determinar infecciones al nivel de colonia en un momento determinado, y también para el seguimiento de la dinámica temporal de infección. Se hacen sugerencias para la forma de estandarizar los ensayos de campo para evaluar los tratamientos y el impacto de la enfermedad. Los métodos de laboratorio descritos incluyen diferentes formas de determinar los niveles de infección al nivel de colonia y de abeja individual, y los métodos para la determinación de las especies, incluyendo microscopía óptica, microscopía electrónica y métodos moleculares (PCR). Se hacen sugerencias para estandarizar los ensayos con cajas, y se describen diferentes métodos de inoculación para infectar abejas, incluyendo métodos de control para la viabilidad de las esporas. Se describe un sistema de cultivo celular para la cría in vitro de Nosema spp. Finalmente, se describe cómo llevar a cabo diferentes tipos de experimentos, incluyendo la dosis infecciosa, efectos de la dosis, curso de la infección y las pruebas de longevidad.

-Worker and queen honey bees were fed individually with Nosema apis spores in sucrose solution and then returned to cages containing several hundred of their worker bee nestmates. After 3 to 7 days, the workers and queens that had been fed spores were sacrificed. Worker and queen ventriculi were removed and examined for spores by light microscopy, and DNA was extracted. The DNA was subjected to amplification with polymerase chain reaction, using primer sequences specific to N. apis DNA. The PCR analysis was more sensitive than examination for spores by light microscopy, in detecting N. apis infection. Worker bees and queen bees were infected at similar rates by the inoculation procedure.Apis mellifera / Nosema apis / PCR / queen / worker

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 is a widely prevalent microsporidian parasite in the western honey bee. There is considerable uncertainty regarding infection dynamics of this important pathogen in honey bee colonies. Understanding the infection dynamics at the colony level may aid in development of a reliable sampling protocol for N. ceranae diagnosis, and provide insights into efficient treatment strategies. The primary objective of this study was to characterize the prevalence (proportion of the sampled bees found infected) and intensity (number of spores per bee) of N. ceranae infection in bees from various age cohorts in a colony. We examined N. ceranae infection in both overwintered colonies that were naturally infected with N. ceranae and in quadruple cohort nucleus colonies that were established and artificially inoculated with N. ceranae. We also examined and quantified effects of N. ceranae infection on hypopharyngeal gland protein content and gut pH. There was no correlation between the prevalence and intensity of N. ceranae infection in composite samples (pooled bee samples used for analysis). Our results indicated that the prevalence and intensity of N. ceranae infection is significantly influenced by honey bee age. The N. ceranae infection prevalence values from composite samples of background bees (unmarked bees collected from four different locations in a colony) were not significantly different from those pertaining to marked-bee age cohorts specific to each sampling date. The foraging-aged bees had a higher prevalence of N. ceranae infection when compared to nurse-aged bees. N. ceranae did not have a significant effect on hypopharyngeal gland protein content. Further, there was no significant difference in mean gut pH of N. ceranae infected bees and non-infected bees. This study provides comprehensive insights into N. ceranae infection dynamics at the colony level, and also demonstrates the effects of N. ceranae infection on hypopharyngeal gland protein content and midgut pH.

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