Annette Bruun Jensen

Miscellaneous standard methods for Apis mellifera research

Summary A variety of methods are used in honey bee research and differ depending on the level at which the research is conducted. On an individual level, the handling of individual honey bees, including the queen, larvae and pupae are required. There are different methods for the immobilising, killing and storing as well as determining individual weight of bees. The precise timing of developmental stages is also an important aspect of sampling individuals for experiments. In order to investigate and manipulate functional processes in honey bees, e.g. memory formation and retrieval and gene expression, microinjection is often used. A method that is used by both researchers and beekeepers is the marking of queens that serves not only to help to locate her during her life, but also enables the dating of queens. Creating multiple queen colonies allows the beekeeper to maintain spare queens, increase brood production or ask questions related to reproduction. On colony level, very useful techniques are the measurement of intra hive mortality using dead bee traps, weighing of full hives, collecting pollen and nectar, and digital monitoring of brood development via location recognition. At the population level, estimation of population density is essential to evaluate the health status and using beelines help to locate wild colonies. These methods, described in this paper, are especially valuable when investigating the effects of pesticide applications, environmental pollution and diseases on colony survival.

Varying degrees of Apis mellifera ligustica introgression in protected populations of the black honeybee, Apis mellifera mellifera, in northwest Europe.

The natural distribution of honeybee subspecies in Europe has been significantly affected by human activities during the last century. Non‐native subspecies of honeybees have been introduced and propagated, so that native black honeybee (Apis mellifera mellifera) populations lost their identity by gene‐flow or went extinct. After previous studies investigated the remaining gene‐pools of native honeybees in France and Spain, we here assess the genetic composition of eight northwest European populations of the black honeybee, using both mitochondrial (restriction fragment length polymorphisms of the intergenic transfer RNAleu‐COII region) and nuclear (11 microsatellite loci) markers. Both data sets show that A. m. mellifera populations still exist in Norway, Sweden, Denmark, England, Scotland and Ireland, but that they are threatened by gene flow from commercial honeybees. Both Bayesian admixture analysis of the microsatellite data and DraI‐RFLP (restriction fragment length polymorphism) analysis of the intergenic region indicated that gene‐flow had hardly occurred in some populations, whereas almost 10% introgression was observed in other populations. The most introgressed population was found on the Danish Island of Læsø, which is the last remaining native Danish population of A. m. mellifera and the only one of the eight investigated populations that is protected by law. We discuss how individual admixture analysis can be used to monitor the restoration of honeybee populations that suffer from unwanted hybridization with non‐native subspecies.

Standard methods for fungal brood disease research

Summary Chalkbrood and stonebrood are two fungal diseases associated with honey bee brood. Chalkbrood, caused by Ascosphaera apis, is a common and widespread disease that can result in severe reduction of emerging worker bees and thus overall colony productivity. Stonebrood is caused by Aspergillus spp. that are rarely observed, so the impact on colony health is not very well understood. A major concern with the presence of Aspergillus in honey bees is the production of airborne conidia, which can lead to allergic bronchopulmonary aspergillosis, pulmonary aspergilloma, or even invasive aspergillosis in lung tissues upon inhalation by humans. In the current chapter we describe the honey bee disease symptoms of these fungal pathogens. In addition, we provide research methodologies and protocols for isolating and culturing, in vivo and in vitro assays that are commonly used to study these host pathogen interactions. We give guidelines on the preferred methods used in current research and the application of molecular techniques. We have added photographs, drawings and illustrations to assist bee-extension personnel and bee scientists in the control of these two diseases.

Sick ants become unsociable.

Parasites represent a severe threat to social insects, which form high‐density colonies of related individuals, and selection should favour host traits that reduce infection risk. Here, using a carpenter ant (Camponotus aethiops) and a generalist insect pathogenic fungus (Metarhizium brunneum), we show that infected ants radically change their behaviour over time to reduce the risk of colony infection. Infected individuals (i) performed less social interactions than their uninfected counterparts, (ii) did not interact with brood anymore and (iii) spent most of their time outside the nest from day 3 post‐infection until death. Furthermore, infected ants displayed an increased aggressiveness towards non‐nestmates. Finally, infected ants did not alter their cuticular chemical profile, suggesting that infected individuals do not signal their physiological status to nestmates. Our results provide evidence for the evolution of unsociability following pathogen infection in a social animal and suggest an important role of inclusive fitness in driving such evolution.

Evolutionary Interaction Networks of Insect Pathogenic Fungi

Lineages of insect pathogenic fungi are concentrated in three major clades: Hypocreales (several genera), Entomophthoromycota (orders Entomophthorales and Neozygitales), and Onygenales (genus Ascosphaera). Our review focuses on aspects of the evolutionary biology of these fungi that have remained underemphasized in previous reviews. To ensure integration with the better-known domains of insect pathology research, we followed a conceptual framework formulated by Tinbergen, asking complementary questions on mechanism, ontogeny, phylogeny, and adaptation. We aim to provide an introduction to the merits of evolutionary approaches for readers with a background in invertebrate pathology research and to make the insect pathogenic fungi more accessible as model systems for evolutionary biologists. We identify a number of questions in which fundamental research can offer novel insights into the evolutionary forces that have shaped host specialization and life-history traits such as spore number and size, somatic growth rate, toxin production, and interactions with host immune systems.

Evidence for emerging parasites and pathogens influencing outbreaks of stress-related diseases like chalkbrood.

In agriculture, honey bees play a critical role as commercial pollinators of crop monocultures which depend on insect pollination. Hence, the demise of honey bee colonies in Europe, USA, and Asia caused much concern and initiated many studies and research programmes aiming at elucidating the factors negatively affecting honey bee health and survival. Most of these studies look at individual factors related to colony losses. In contrast, we here present our data on the interaction of pathogens and parasites in honey bee colonies. We performed a longitudinal cohort study over 6 years by closely monitoring 220 honey bee colonies kept in 22 apiaries (ten randomly selected colonies per apiary). Observed winter colony losses varied between 4.8% and 22.4%; lost colonies were replaced to ensure a constant number of monitored colonies over the study period. Data on mite infestation levels, infection with viruses, Nosema apis and Nosema ceranae, and recorded outbreaks of chalkbrood were continuously collected. We now provide statistical evidence (i) that Varroa destructor infestation in summer is related to DWV infections in autumn, (ii) that V. destructor infestation in autumn is related to N. apis infection in the following spring, and most importantly (iii) that chalkbrood outbreaks in summer are related to N. ceranae infection in the preceding spring and to V. destructor infestation in the same season. These highly significant links between emerging parasites/pathogens and established pathogens need further experimental proof but they already illustrate the complexity of the host-pathogen-interactions in honey bee colonies.

Nutritional limitation and resistance to opportunistic Aspergillus parasites in honey bee larvae

Honey bees are threatened by land use changes which reduce the availability and diversity of pollen and nectar resources. There is concern that poor nutrition may be involved in recent population declines, either directly or due to indirect effects on immunocompetence. The larval stage is likely to be the most vulnerable to a poor diet, but the effects of larval nutrition on the disease susceptibility of bees are not well known. In this study we used laboratory-reared honey bee larvae to investigate the effects of diet quality on disease susceptibility to the opportunistic fungal parasites Aspergillus flavus, Aspergillus phoenicis and A. fumigatus. Larvae fed on a nutritionally poor diet were found to be significantly more susceptible to A. fumigatus. Larval resistance to A. fumigatus was enhanced by feeding with a diet supplemented with either dandelion or polyfloral pollens. This indicates that dandelion and polyfloral pollens contain elements that enhance resistance to this fungal disease, illustrating an interaction between nutrition and parasitism and emphasising the benefit of diverse floral resources in the environment to maintain honey bee health.

Differential susceptibility across honey bee colonies in larval chalkbrood resistance

Chalkbrood susceptibility of in vitro reared honey bee larvae was investigated. Larvae were grafted from 3–4 colonies headed by pure mated queens of Apis mellifera carnica, A. m. ligustica and A. m. mellifera, respectively. Three day old larvae were fed with different dosages of Ascophaera apis spores and a clear dose-response relationship was shown. Over the whole experiment LD50 estimates ranged from 55 to 905 spores. The response differed significantly (up to a factor ten) between colonies of the same subspecies. The mean time to death decreased with increased dose, with more larvae dying faster after eating more fungal spores. The A. m. ligustica larvae used in this study were less susceptible to A. apis than A. m. mellifera and A. m. carnica larvae. However due to the limited number of colonies included and the high variation shown we cannot predict that any A. m. ligustica colony is better adapted to cope with A. apis than colonies of A. m. carnica and A. m. mellifera.ZusammenfassungLarven aus verschiedenen Honigbienenvölkern wurden mit Kalkbrut-Sporen (Ascosphaera apis) infiziert. Die Larven stammten von Königinnen, die sich an isolierten Paarungsplätzen (z.B. Inseln) natürlich gepaart hatten. Wir verwendeten Königinnen von Apis mellifera carnica, A. m. ligustica und A. m. mellifera. Dadurch konnten wir sowohl zwischen den drei Gruppen als auch innerhalb der Gruppen die Unterschiede in der Anfälligkeit gegen eine Kalkbrutinfektion testen. Dreitägige Larven wurden dazu mit unterschiedlichen Dosen von A. apis-Sporen gefüttert, während die Kontrollgruppe nicht kontaminiertes Futter erhielt. Alle Larven wurden täglich unter dem Mikroskop beobachtet und nach den Kriterien „lebend“, „tot“ oder „durch Pilzbefall getötet“ (mit Pilzhyphen am Körper) eingeteilt. Es gab einen klaren Zusammenhang zwischen der gefütterten Sporen-menge und der Anzahl infizierter Larven. Es waren zwischen minimal 55 und maximal 905 Sporen notwendig, um 50 % der behandelten Larven zu töten (Tab. I). Die Unterschiede in der Anfälligkeit betragen also mehr als den Faktor 10 und erwartungsgemäß nahm die durchschnittliche Überlebensdauer mit zunehmender Sporendosis ab (Abb. 1). Bei der höchsten verabreichten Sporendosis von 10000 Sporen gab es keine Unterschiede in der Überlebensdauer zwischen bzw. innerhalb der Gruppen. Wurden die Larven aber nur mit 1.000 Sporen gefüttert, traten zwischen den Gruppen signifikante Unterschiede auf. Insgesamt weisen die LD50—Werte und die durchschnittliche Überlebensdauer darauf hin, dass die in dieser Untersuchung verwendeten A. m. ligustica-Larven weniger anfällig gegen A. apis-Infektionen waren als Larven von A. m. mellifera und A. m. carnica. Allerdings werden aufgrund der begrenzten Anzahl an Testvölkern und der hohen Variation innerhalb der Unterarten weitere Daten benötigt, um zuverlässige Vorhersagen zur spezifischen Anfälligkeit bzw. Toleranz verschiedener Apis-Unterarten gegenüber einer Kalkbrutinfektion zu machen. Die großen Unterschiede in der Kalkbrut-Anfälligkeit zwischen den verschiedenen Völkern zeigen aber, dass es durchaus ein Potential für die Zucht auf Kalkbrut-Resistenz gibt.

Genetic variation within the insect-pathogenic genus Entomophthora , focusing on the E. muscae complex, using PCR–RFLP of the ITS II and the LSU rDNA

The ITS II and the first part of the LSU rDNA were amplified from 26 isolates within the genus Entomophthora. The specificity of the primers allowed the use of both in vivo and in vitro material. Size polymorphism and long amplification of the ITS II regions, ranging from 1200 to 2000 bp, were observed. The PCR-products were cut with eight different restriction endonucleases and analysed by UPGMA, one analysis from each of the two regions. Conidial morphology was of predictive value for the overall taxonomy of the genus Entomophthora, as the genus clustered together in the analysis of the LSU rDNA. In both analyses the E. muscae complex clustered into three different clades, which support the validity of E. schizophorae and E. syrphi as separate species. Considerable variation was detected in the E. muscae clade, but it could not be grouped by host, geographic origin or conidial morphology, though the E. muscae s. sir. isolates in both analyses grouped together. One isolate with E. muscae-like conidia found on Hymenoptera clustered out within the E. muscae clade, widening the host range for E. muscae significantly.

‘Entomophagy’: an evolving terminology in need of review

There is growing interest in insects as human food in academia, food and agricultural industries, public institutions and the public at large. Yet many of the words and concepts used to describe these organisms and the human practices surrounding them are still rudimentary, compared to the diversity of the organisms themselves and the existing complexity and rapid evolution of the practices they aim to describe. The goals of this paper are to: (1) show how the roots of the term ‘entomophagy’ and its uses have evolved over time; (2) illustrate some of the term’s problems that necessitate its review; and (3) offer recommendations for use of the term in future research and other practice. Our paper offers a brief historical review of insect eating as described by certain Western cultural sources, explores some of the taxonomic ambiguities and challenges surrounding the category ‘insects’, and ultimately argues for more precise and contextual terminology in this both richly traditional and rapidly developing ...