Eva Forsgren

Symbionts as major modulators of insect health: Lactic Acid Bacteria and honeybees

Lactic acid bacteria (LAB) are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (>80 mya) history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis.

Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae

We evaluated the antagonistic effects of newly identified lactic acid bacteria (LAB) in the genera Lactobacillus and Bifidobacterium, originating from the honey stomach, on the honey bee pathogen, Paenibacillus larvae. We used inhibition assays on agar plates and honey bee larval bioassays to investigate the effects of honey bee LAB on P. larvae growth in vitro and on AFB infection in vivo. The individual LAB phylotypes showed different inhibition properties against P. larvae growth on agar plates, whereas a combination of all eleven LAB phylotypes resulted in a total inhibition (no visible growth) of P. larvae. Adding the LAB mixture to the larval food significantly reduced the number of AFB infected larvae in exposure bioassays. The results demonstrate that honey bee specific LAB possess beneficial properties for honey bee health. Possible benefits to honey bee health by enhancing growth of LAB or by applying LAB to honey bee colonies should be further investigated.ZusammenfassungDie Amerikanische Faulbrut (AFB) ist eine Krankheit, die junge Honigbienenlarven befällt. Sie ist eine der schädlichsten Bienenkrankheiten und hat große ökonomische Bedeutung für die Imkerei weltweit. Der Erreger der AFB ist das sporenbildende Bakterium Paenibacillus larvae, das den Mitteldarm junger Larven durch kontaminiertes Futter befällt. Die Besiedelung des larvalen Mitteldarms stellt einen der Schlüsselfaktoren für die Pathogenese von P. larvae dar und bestimmte Zusammensetzungen der Mikroflora des Darmes könnten das Wachstum des Krankheitserregers unterdrücken. Kürzlich wurden eine neue Flora von Milchsäurebakterien (LAB) der Gattungen Lactobacillus und Bifidobacterium aus dem Honigmagen der Bienen beschrieben. LAB sind zwar bekannt für die Produktion von antimikrobiellen Substanzen, jedoch gibt es Variationen bezüglich der nutzbringenden Eigenschaften zwischen Arten und Gattungen. In dieser Untersuchung wurde der antagonistische Effekt der Honigbienen-LAB auf P. larvae beurteilt. Wir verwendeten Hemmtests auf Agarplatten und Biotests mit Honigbienenlarven, um diese Effekte zu untersuchen. Die individuellen LAB-Phylotypen zeigten unterschiedliche Hemmeigenschaften gegenüber auf Agarplatten wachsenden P. larvae, während eine Kombination aller 11 LAB-Phylotypen sogar eine totale Hemmung (kein sichtbares Wachstum mehr) von P. larvae bewirkte. Eine Zugabe des LAB-Mix zum Larvenfutter reduzierte signifikant die Anzahl an AFB-infizierten Larven im Biotest.Die Ergebnisse zeigen, dass die für Honigbienen spezifischen LAB nutzbringende Eigenschaften für die Bienengesundheit besitzen. Der mögliche Nutzen einer Applikation von LAB in Bienenvölkern sollte untersucht werden.

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.

European foulbrood in honey bees

European foulbrood (EFB) is a severe bacterial brood disease caused by the Gram-positive bacterium Melissocccus plutonius. The disease has a worldwide distribution and is an increasing problem in some areas. Although the causative agent of EFB was described almost a century ago, many basic aspects of its pathogenesis are still unknown. This review presents both historical results and recent molecular data to synthesize present knowledge of this enigmatic honey bee disease.

Acaricide Treatment Affects Viral Dynamics in Varroa destructor-Infested Honey Bee Colonies via both Host Physiology and Mite Control

ABSTRACT Honey bee (Apis mellifera) colonies are declining, and a number of stressors have been identified that affect, alone or in combination, the health of honey bees. The ectoparasitic mite Varroa destructor, honey bee viruses that are often closely associated with the mite, and pesticides used to control the mite population form a complex system of stressors that may affect honey bee health in different ways. During an acaricide treatment using Apistan (plastic strips coated with tau-fluvalinate), we analyzed the infection dynamics of deformed wing virus (DWV), sacbrood virus (SBV), and black queen cell virus (BQCV) in adult bees, mite-infested pupae, their associated Varroa mites, and uninfested pupae, comparing these to similar samples from untreated control colonies. Titers of DWV increased initially with the onset of the acaricide application and then slightly decreased progressively coinciding with the removal of the Varroa mite infestation. This initial increase in DWV titers suggests a physiological effect of tau-fluvalinate on the hosts susceptibility to viral infection. DWV titers in adult bees and uninfested pupae remained higher in treated colonies than in untreated colonies. The titers of SBV and BQCV did not show any direct relationship with mite infestation and showed a variety of possible effects of the acaricide treatment. The results indicate that other factors besides Varroa mite infestation may be important to the development and maintenance of damaging DWV titers in colonies. Possible biochemical explanations for the observed synergistic effects between tau-fluvalinate and virus infections are discussed.

Deformed wing virus associated with Tropilaelaps mercedesae infesting European honey bees (Apis mellifera)

Mites in the genus Tropilaelaps (Acari: Laelapidae) are ectoparasites of the brood of honey bees (Apis spp.). Different Tropilaelaps subspecies were originally described from Apis dorsata, but a host switch occurred to the Western honey bee, Apis mellifera, for which infestations can rapidly lead to colony death. Tropilaelaps is hence considered more dangerous to A. mellifera than the parasitic mite Varroa destructor. Honey bees are also infected by many different viruses, some of them associated with and vectored by V. destructor. In recent years, deformed wing virus (DWV) has become the most prevalent virus infection in honey bees associated with V. destructor. DWV is distributed world-wide, and found wherever the Varroa mite is found, although low levels of the virus can also be found in Varroa free colonies. The Varroa mite transmits viral particles when feeding on the haemolymph of pupae or adult bees. Both the Tropilaelaps mite and the Varroa mite feed on honey bee brood, but no observations of DWV in Tropilaelaps have so far been reported. In this study, quantitative real-time RT-PCR was used to show the presence of DWV in infested brood and Tropilaelapsmercedesae mites collected in China, and to demonstrate a close quantitative association between mite-infested pupae of A. mellifera and DWV infections. Phylogenetic analysis of the DWV sequences recovered from matching pupae and mites revealed considerable DWV sequence heterogeneity and polymorphism. These polymorphisms appeared to be associated with the individual brood cell, rather than with a particular host.

Increased Tolerance and Resistance to Virus Infections: A Possible Factor in the Survival of Varroa destructor -Resistant Honey Bees ( Apis mellifera )

The honey bee ectoparasitic mite, Varroa destructor, has a world-wide distribution and inflicts more damage than all other known apicultural diseases. However, Varroa-induced colony mortality is more accurately a result of secondary virus infections vectored by the mite. This means that honey bee resistance to Varroa may include resistance or tolerance to virus infections. The aim of this study was to see if this is the case for a unique population of mite-resistant (MR) European honey bees on the island of Gotland, Sweden. This population has survived uncontrolled mite infestation for over a decade, developing specific mite-related resistance traits to do so. Using RT-qPCR techniques, we monitored late season virus infections, Varroa mite infestation and honey bee colony population dynamics in the Gotland MR population and compared this to mite-susceptible (MS) colonies in a close by apiary. From summer to autumn the deformed wing virus (DWV) titres increased similarly between the MR and MS populations, while the black queen cell virus (BQCV) and sacbrood virus (SBV) titres decreased substantially in the MR population compared to the MS population by several orders of magnitude. The MR colonies all survived the following winter with high mite infestation, high DWV infection, small colony size and low proportions of autumn brood, while the MS colonies all perished. Possible explanations for these changes in virus titres and their relevance to Varroa resistance and colony winter survival are discussed.

Variability in germination and in temperature and storage resistance among Paenibacillus larvae genotypes

There are several methods for cultivation of Paenibacillus larvae, the causative agent of American foulbrood (AFB) in honey bees. Protocols for detection of sub-clinical levels of the bacterium from honey and bee samples include heat treatment of samples. The main objective of this study was to investigate if there is variability in temperature resistance among P. larvae genotypes, potentially leading to biased diagnose and disease monitoring. The variation in germination and proliferation ability among type collection (N=4) and field isolates (N=4) of P. larvae representing four different genotypes was investigated. Results demonstrate a significant variability between P. larvae genotypes in germination rate on solid media as well as in endospore resistance to heat treatment and storage. It is concluded that strains of different genotypes should be included in evaluation of standard laboratory protocols for cultivation of P. larvae to avoid bias in disease monitoring and quantification of the pathogen.

Distribution of Melissococcus plutonius in Honeybee Colonies with and without Symptoms of European Foulbrood

A sensitive hemi-nested polymerase chain reaction (PCR) was used for detection of Melissococcus plutonius, the causative agent of European foulbrood (EFB). Sampling was made in Switzerland, where EFB is a widespread disease and incidences have increased in recent years. Larvae from brood samples with and without clinical signs of disease (n = 92) and honey (n = 92) from the same colonies were investigated. Individual larvae (n = 60) and pupae (n = 30) from diseased brood in single colonies were also investigated to study the distribution of the bacterium within the brood between larvae. M. plutonius was detected in larvae in all apiaries where symptoms of EFB could be seen, but not in all colonies judged as cases of EFB in the field, when healthy-looking larvae from such colonies were tested. The occurrence of the bacterium within the brood was not limited to larvae with symptoms only, but was mainly found in diseased larvae. The bacterium was also found in pupae. Healthy-looking larvae—even from heavily diseased combs—failed, in a number of cases, to amplify product in the PCR. M. plutonius could only be detected in 35% of the brood nest honey from clinically diseased colonies.

Standard methods for European foulbrood research

Summary European foulbrood (EFB) is a severe bacterial honey bee brood disease caused by the Gram-positive bacterium Melissocccus plutonius. The disease is widely distributed worldwide, and is an increasing problem in some areas. Although the causative agent of EFB was described almost a century ago, many basic aspects of its pathogenesis are still unknown. Earlier studies were hampered by insensitive and unspecific methods such as culture based techniques. Recent advances in molecular technology are making it increasingly easy to detect and characterize microbes, and nucleic acid detection technologies are quickly displacing the traditional phenotypic assays in microbiology. This paper presents selected methodologies which focus on EFB and its causative agent M. plutonius.