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 bee's 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.

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.

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.

SummarySummaryMethods 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.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.本文描述了实验室及野外实验中关于孢子虫Nosema apis 和 Nosema ceranae 的研究方法。对于野外实验，本文列举了多种取样方法，用于研究特定时间段内，蜂群群体感染水平，以及随后开展长期感染规律的研究。同时也对如何标准化评估蜂场治疗效果、感病程度及选用哪些指标用于标准化评估提出了建议。实验室方法包括，确定蜂群感染水平及个体蜜蜂感染水平的方法以及测定孢子虫种类的方法，如光学显微镜法，电子显微镜法以及分子方法（PCR）。对于如何标准化蜂笼实验的各项指标提出了建议，并描述了感染蜜蜂的不同接种方法，包括孢子生存能力的对比法。描述了孢子虫的一种体外细胞培养体系。最后描述了如何进行不同类型的实验，包括感染剂量、剂量效能、感染过程以及寿命试验。Keywords: Nosema apisNosema ceranaefield methodslaboratory methodssampling methodsinfection dynamicsinfection levelmicroscopyspecies identificationstandardised cage trialsinoculation methodsspore viabilitycell cultureinfectious dosedose effectscourse of infectionlongevity testshoney beeBEEBOOKCOLOSS

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.

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

Nosema ceranae, a honey bee pathogen now known worldwide, may be detected quickly by either light microscopy or by antibody tests. While these tests are less sensitive than polymerase chain reaction, they may be more practical for routine diagnosis. Phase contrast light microscopy allows one to distinguish between primary, environmental, and germinated spores. Fluorescent stains may demonstrate the maturity of spores and the integrity of the spore membranes. Polar filaments from the spores are also seen by proper microscopic techniques. Antibodies are also helpful in identifying Nosema spores and can be specific to the Nosema ceranae species. The antibodies attach to protein in the wall of the spore. This test is able to detect an infestation as few as 1,000 spores, a tiny fraction of the spores present in a highly infected bee. We hope to see this test commercialized so that it is available to beekeepers.

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 1x10(3) and higher spore numbers per assay. Considering that a single infected bee can produce over 50 x 10(6) spores, this level of sensitivity will allow detection of a very low level of Nosema infection in bee colonies.

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.

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.

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.

The proportion of Varroa jacobsoni Oudemans that were alive and mobile when they fell from honey bees, Apis mellifera L., in hives was measured during a 20-wk period to determine the potential use of systems that prevent these mites from returning to the bees. Traps designed to discriminate between the live, fallen mites and those that are dead or immobile were used on hive bottom boards. A large fraction of the fallen mites was alive when acaricide was not in use and also when fluvalinate or coumaphos treatments were in the hives. The live proportion of mitefall increased during very hot weather. The proportion of mitefall that was alive was higher at the rear and sides of the hive compared with that falling from center frames near the hive entrance. More sclerotized than callow mites were alive when they fell. A screen-covered trap that covers the entire hive bottom board requires a sticky barrier to retain all live mites. This trap or another method that prevents fallen, viable mites from returning to the hive is recommended as a part of an integrated control program. It also may slow the development of acaricide resistance in V. jacobsoni and allow the substitution of less hazardous chemicals for the acaricides currently in use.

Honey bees are found associated with forests globally. Flowers of forest trees provide subsistence for honey bees and the trees physically provide shelter for a swarm or bee hive. Forest management and beekeeping have each had a long history both in the United States and globally, but have seldom been integrated or studied in a systematic fashion. Purposeful plantings of trees, as in agroforestry systems, could be designed to favor bee forage or hive protection.

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 host finding behavior of other arthropods. Understanding this behavior in tracheal mites would be useful for developing survey or control tactics of this serious pest.

Two formulations of bicyclohexylammonium fumagillin, Fumagilin B and Fumidil B, were equally effective treatments for Nosema apis Zander infecting honey bees (Apis mellifera L.). Caged worker bees consumed either formulation in sucrose syrup as readily as syrup without fumagillin. Either formulation suppressed infection when fed with Nosema spores to caged workers for 7 d or when fed to workers for 3 d after they had consumed spores without fumagillin for 4 d. When consumed in sucrose syrup by bee colonies, neither fumagillin formulation significantly affected queen survival. More uncapped brood was reared by the colonies consuming fumagillin than those consuming syrup without fumagillin. Capped brood and total brood rearing was similar for colonies consuming fumagillin or syrup without fumagillin. Fumagilin B is more soluble in sucrose solution than Fumidil B, but both are easily prepared at the U.S. Food and Drug Administration approved level of 26 mg/liter in sucrose solution.

Journal Article Electronic System for Monitoring Queen Cell Provisioning in Honey Bee (Hymenoptera: Apidae) Colonies Get access Thomas C. Webster Thomas C. Webster Community Research Service, Kentucky State University, Frankfort, Kentucky 40601. Search for other works by this author on: Oxford Academic Google Scholar Annals of the Entomological Society of America, Volume 82, Issue 1, 1 January 1989, Pages 121–125, https://doi.org/10.1093/aesa/82.1.121 Published: 01 January 1989 Article history Received: 09 November 1987 Accepted: 25 July 1988 Published: 01 January 1989

Journal Article Short-Term and Long-Term Effects of Methamidophos on Brood Rearing in Honey Bee (Hymenoptera: Apidae) Colonies Get access Thomas C. Webster, Thomas C. Webster 1 Department of Entomology, University of California, Davis, California 95616 1Current address: Community Research Service, Kentucky State University, Frankfort, Ky. 40601. Search for other works by this author on: Oxford Academic PubMed Google Scholar Ying-Shin Peng Ying-Shin Peng Department of Entomology, University of California, Davis, California 95616 Search for other works by this author on: Oxford Academic PubMed Google Scholar Journal of Economic Entomology, Volume 82, Issue 1, 1 February 1989, Pages 69–74, https://doi.org/10.1093/jee/82.1.69 Published: 01 February 1989 Article history Received: 04 January 1988 Accepted: 01 September 1988 Published: 01 February 1989

AbstractA food‐producing role for cephalic exocrine glands has arisen independently in both taxa of highly eusocial bees, Apis and Meliponini. With several exceptions, there is little evidence that food is produced by glands of solitary bees or by most bees at lower levels of sociality. We suggest that this association with sociality is due to four adaptive features of these glands: (1) food from the glands allows feces from queens and larvae to have a small volume, (2) the queen's fecundity can be increased, (3) nutrient recovery via cannibalism can be facilitated, and (4) rearing of emergency replacement queens is accelerated. Acceleration of the rearing of other castes and of queens in the normal process of colony fission is not clearly an advantage ascribed to these glands. Trophic eggs produced by meliponine colony workers are analogous to the secretions from food‐producing glands in Meliponini and Apis workers.