Thomas C. Webster

Standard methods for Nosema research

Summary Methods 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.

Nosema apis infection in honey bee (Apis mellifera) queens

Summary Queen honey bees were fed Nosema apis spores in sucrose solution, returned to their hives, and examined later for N. apis infection by a polymerase chain reaction test. Eggs, larvae and pupae from the hives were also examined for infection on three observation dates during a 39 day period following the inoculation of the queens. Six of seven surviving queens developed N. apis infections in their ventriculi, but none had detectable N. apis in their ovaries. No eggs, larvae or pupae taken from the hives of Nosema inoculated queens contained detectable N. apis. These results suggest that N. apis is not transmitted vertically, unlike many other Microsporidia in other invertebrate hosts. In an effort to determine if the stress of shipping increased the susceptibility of Nosema exposed bees, another set of mated, queen honey bees was fed N. apis spores in sucrose, and the queens were then either mailed in small shipping cages, from Kentucky to Indiana and back, or kept caged for the same period in the laboratory. These queens were then dissected and their ovaries and ventriculi examined for N. apis. Of the mailed queens, all developed infection, whilst 86% of the queens not mailed became infected, but this difference was not statistically significant. N. apis was not detected in any of the ovaries of mailed queens nor of those queens not mailed.

Live Varroa jacobsoni (Mesostigmata: Varroidae) fallen from honey bee (Hymenoptera: Apidae) colonies.

Abstract The proportion of Varroa jacobsoni Oudemans that werealive and mobile when they fell from honey bees, Apismellifera L., in hives was measured during a 20-wk period todetermine the potential use of systems that prevent these mites fromreturning to the bees. Traps designed to discriminate between the live,fallen mites and those that are dead or immobile were used on hivebottom boards. A large fraction of the fallen mites was alive whenacaricide was not in use and also when fluvalinate or coumaphostreatments were in the hives. The live proportion of mitefall increasedduring very hot weather. The proportion of mitefall that was alive washigher at the rear and sides of the hive compared with that fallingfrom center frames near the hive entrance. More sclerotized than callowmites were alive when they fell. A screen-covered trap that covers theentire hive bottom board requires a sticky barrier to retain all livemites. This trap or another method that prevents fallen, viable mitesfrom returning to the hive is recommended as a part of an integratedcontrol program. It also may slow the development of acaricideresistance in V. jacobsoni and allow the substitution ofless hazardous chemicals for the acaricides currently in use.

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 RNAi-mediated 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. IMPORTANCE Given 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.

Visible and near-infrared spectroscopy detects queen honey bee insemination

The abdomens of honey bee queens and semen from drone bees were analyzed by visible and near-infrared spectroscopy. Mated honey bee queens could be distinguished from virgin queens by their absorption spectra with 100% accuracy. Spectra of semen showed that classifications of queens were likely influenced by the presence or absence of semen in the queen spermathecae. However, physiological or morphological changes that occur in the queens after mating probably influenced the classifications also.ZusammenfassungIm Allgemeinen können die inneren Organe der Honigbienen nicht untersucht werden, ohne die Bienen zuvor zu töten. Eine schnelle und nicht invasive Methode zum Nachweis von Sperma in der Spermatheka der Bienenkönigin wäre aber äußerst nützlich. Die VIS-NIR-Spektroskopie (parallele Spektroskopie im sichtbaren Wellenlängenbereich und im Nahen Infrarot) wurde bereits erfolgreich bei anderen Untersuchungen von Insektengeweben eingesetzt; hier wurde sie dazu benutzt, unbegattete Königinnen, begattete Königinnen und das zugehörige Drohnensperma zu identifizieren. Wir untersuchten 52 begattete und 52 unbegattete Königinnen, indem wir den Strahl des Spektrometers auf den Abschnitt des Königinnenabdomens richteten, in dem sich die Spermatheka befindet. Dabei konnten begattete von unbegatteten Königinnen zu 100% unterschieden werden (Abb. 1 und Abb. 2). Die Unterschiede in den Spektren könnten durch das Sperma in der Spermatheka der begatteten Königinnen hervorgerufen worden sein (Abb. 3). Allerdings scheinen auch physiologische und morphologische Veränderungen, die bei der Königin nach der Paarung auftreten, eine wichtige Rolle bei den Unterschieden zwischen den Spektren zu spielen.

Evaluation of Nosema ceranae spore-specific polyclonal antibodies

Summary A new genomic antibody (Ab) has been developed against a spore-wall protein SWP-32 of the honey bee intracellular pathogen, Nosema ceranae. In dot blots and Western blots this Ab specifically recognized N. ceranae spore antigens and did not cross-react with N. apis spore lysates, unless blots were overdeveloped. The detection sensitivity depends on both the concentration of the anti-SWP-32 Ab and the concentration of Nosema spores in the lysates. To avoid non-specific staining, we suggest using this new Ab at 1:5000 dilution for detection of 1×103 and higher spore numbers per assay. Considering that a single infected bee can produce over 50 × 106 spores, this level of sensitivity will allow detection of a very low level of Nosema infection in bee colonies.

A serological method for detection of Nosema ceranae

We developed a new method for detection of the intracellular parasite, Nosema ceranae, one of the most economically devastating pathogens of the honeybee.

Preliminary studies of honey queen bee conditions using Cyranose 320 nose technology

Over the last ten years, the bee keeping industry has been struggling to understand and stop the sudden widespread loss or collapse of honey bee colonies, known collectively as Colony Collapse Disorder (CCD), in the U.S. and around the world. While honey bee colonies experience many stressors that could cause a colony to collapse, we are focusing on the quality, health and reproductive ability of honey bee queens. The purpose of this line of research is to identify relationships between the pheromone signatures of honey bee queens and the quality of honey bee queens. The ultimate goal of this research is to find a reliable, non-invasive tool that does not harm the queen, but still allows beekeepers to make informed decisions about purchasing honey bee queens and deciding when to replace a queen bee before a colony collapses. In this portion of the research, we use an electronic nose (e-nose) device, which is a device that digitizes smells. The scope of this paper is to determine whether an e-nose device is viable for our research, and if so, to determine the best way to configure the settings to improve data collection. Also, to gather data on queen bee pheromones production was considered since that is an indicator of a queen bees reproductive ability. We were able to use the e-nose device to digitize pheromone signatures from 20 queen bees. Using Microsoft excel and R programming language, we were able to see patterns that will be useful in configuring the e-nose device for future research. We also noticed an early indication that the e-nose can distinguish between a healthy bee and a sick bee.

Attractiveness of CO2 and Synthetic honey bee (Apis Mellifera L.) (Hymenoptera: Apidae) cuticular hydrocarbons to the honey bee tracheal mite, Acarapis woodi (Rennie) (Acari: Tarsonemidae)

Abstract Despite the devastation caused by the honey bee tracheal mite, Acarapis woodi, over its range, it is difficult to detect and little is known about what attracts the mite to its host. Based on previous studies of tracheal mites and of other blood sucking arthropods, we developed bioassay procedures to study the attractiveness of CO2 and of honey bee cuticular hydrocarbons to dispersing tracheal mites. The CO2 assay consisted of a three-choice test between streams of air, a CO2 /air mix, and a no-gas control. Air was chosen most frequently overall. Weighted scores were calculated based on the strength and frequency of response, and in this case the CO2 /air mixture was favored. Known hydrocarbon mixtures and hexane extracts of bees were applied to pipecleaners and inserted into small test hives. No tracheal mites were recovered from any of these, failing to support other studies that found these substances to be attractive in the lab. The results are discussed in relation to what is known about the...