Ingemar Fries

Seed coating with a neonicotinoid insecticide negatively affects wild bees

Understanding the effects of neonicotinoid insecticides on bees is vital because of reported declines in bee diversity and distribution and the crucial role bees have as pollinators in ecosystems and agriculture. Neonicotinoids are suspected to pose an unacceptable risk to bees, partly because of their systemic uptake in plants, and the European Union has therefore introduced a moratorium on three neonicotinoids as seed coatings in flowering crops that attract bees. The moratorium has been criticized for being based on weak evidence, particularly because effects have mostly been measured on bees that have been artificially fed neonicotinoids. Thus, the key question is how neonicotinoids influence bees, and wild bees in particular, in real-world agricultural landscapes. Here we show that a commonly used insecticide seed coating in a flowering crop can have serious consequences for wild bees. In a study with replicated and matched landscapes, we found that seed coating with Elado, an insecticide containing a combination of the neonicotinoid clothianidin and the non-systemic pyrethroid β-cyfluthrin, applied to oilseed rape seeds, reduced wild bee density, solitary bee nesting, and bumblebee colony growth and reproduction under field conditions. Hence, such insecticidal use can pose a substantial risk to wild bees in agricultural landscapes, and the contribution of pesticides to the global decline of wild bees may have been underestimated. The lack of a significant response in honeybee colonies suggests that reported pesticide effects on honeybees cannot always be extrapolated to wild bees.

Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae)

Summary Based on light microscopic and ultrastructural characteristics as well as on the nucleotide sequence of the small subunit ribosomal RNA coding region, the microsporidium Nosema ceranae n. sp., a parasite of the Asian honey bee Apis cerana is described. Merogonial stages and sporonts are diplokaryotic. Merozoites are mostly formed by cytoplasmic fission in quadrinucleate meronts and the number of merogonial cycles may vary. The sporogony is disporoblastic. The living mature spore is ovocylindrical, straight to slightly curved and measures 4.7 × 2.7 μm whereas fixed and stained spores measure 3.6 × 1.7 μm. The polar filament is isofilar with a diameter of 96–102 nm and is arranged in 20–23 coils in the posterior and mid-part of the spore. In the anterior part of the polaroplast there are closely packed approximately 11 nm thick lamellae. The lamellae of the posterior polaroplast are thicker and less regular. In the posterior part of the mature spore a well fixed posterior body interpreted as a posterosome was often observed. Phylogenetic analysis, based on the sequence of the small subunit ribosomal RNA, places Nosema ceranae in the Nosema clade, as defined by Nosema bombycis , the type species of the Nosema genus.

Nosema ceranae in European honey bees (Apis mellifera).

Nosema ceranae is a microsporidian parasite described from the Asian honey bee, Apis cerana. The parasite is cross-infective with the European honey bee, Apis mellifera. It is not known when or where N. ceranae first infected European bees, but N. ceranae has probably been infecting European bees for at least two decades. N. ceranae appears to be replacing Nosema apis, at least in some populations of European honey bees. This replacement is an enigma because the spores of the new parasite are less durable than those of N. apis. Virulence data at both the individual bee and at the colony level are conflicting possibly because the impact of this parasite differs in different environments. The recent advancements in N. ceranae genetics, with a draft assembly of the N. ceranae genome available, are discussed and the need for increased research on the impacts of this parasite on European honey bees is emphasized.

Nosema ceranae has infected Apis mellifera in Europe since at least 1998 and may be more virulent than Nosema apis

Nosema ceranae, a microsporidian formerly regarded as confined to its Asiatic host Apis cerana, has recently been shown to parasitise Apis mellifera and to have spread throughout most of the world in the past few years. Using a temporal sequence of N = 28 Nosema isolates from Finland from 1986–2006, we now find (i) that N. ceranae has been present in Europe since at least 1998 and (ii) that it has increased in frequency across this time period relative to Nosema apis, possibly leading to higher mean spore loads per bee. We then present results of a single laboratory infection experiment in which we directly compare the virulence of N. apis with N. ceranae. Though lacking replication, our results suggest (iii) that both parasites build up to equal numbers per bee by day 14 post infection but that (iv) N. ceranae induces significantly higher mortality relative to N. apis.ZusammenfassungNosema ceranae, ein Microsporidien-Parasit der früher auf seinen asiatischen Wirt Apis cerana beschränkt war, ist kürzlich in Völkern von Apis mellifera nachgewiesen worden und hat sich in den vergangenen Jahren nahezu weltweit verbreitet. Wir untersuchten Nosema-Proben aus Finnland (N = 28), die in den Jahren 1986–2006 aus Bienen isoliert wurden (Abb. 1). Wir ermittelten die durchschnittliche Anzahl an Sporen pro Biene und bestimmten über PCR und Restriction Fragment Length Polymorphism (RFLP) des 16S rRNA-Gens auf der Parasiten-DNA die Nosema-Art. Wir konnten nachweisen, dass N. ceranae bereits seit mindestens 1998 in Finnland vorkommt, und dass seitdem der Anteil von N. ceranae im Vergleich zu N. apis zugenommen hat (Abb. 2). Dies hat möglicherweise zu einer insgesamt höheren Sporenbelastung pro Biene geführt (Abb. 3). Neuere Infektionen (2006) bestehen ausschließlich aus N. ceranae oder einer Mischinfektion von N. apis und N. ceranae.Wir führten daraufhin ein einziges Laborexperiment durch, in dem wir die Virulenz von N. apis direkt mit der von N. ceranae verglichen. Dazu wurden Käfige mit jeweils 50 Bienen, die mit 105 Sporen von N. apis bzw. von N. ceranae gefüttert wurden, gefüllt. Die negative Kontrolle erhielt Zuckerwasser. Während der nächsten 15 Tage wurden die Anzahl der Sporen im Mitteldarm sowie die Mortalität der Bienen erfasst. Obwohl unsere Ergebnisse wegen der fehlenden Wiederholung nur vorläufigen Charakter haben, scheint bei beiden Parasiten die Anzahl der Sporen pro Biene während der ersten 14 Tage in ähnlichem Umfang zuzunehmen (Durchschnitt 27±2× 106 Sporen, Abb. 4). Allerdings verursachte N. ceranae eine signifikant höhere Bienenmortalität im Vergleich zu N. apis (Abb. 5). Wir diskutieren unsere Ergebnisse in Zusammenhang mit der beobachteten Zunahme an Völkerzusammenbrüchen in Südeuropa und wir beschreiben Experimente, die notwendig sind um den kausalen Zusammenhang zwischen N. ceranae-Infektionen und Völkerzusammenbrüchen nachzuweisen.

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.

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.

Strain- and Genotype-Specific Differences in Virulence of Paenibacillus larvae subsp. larvae, a Bacterial Pathogen Causing American Foulbrood Disease in Honeybees

ABSTRACT Virulence variations of Paenibacillus larvae subsp. larvae, the causative agent of American foulbrood disease of honeybees, were investigated by analysis of 16 field isolates of this pathogen, belonging to three previously characterized genotypes, as well as the type strain (ATCC 9545) of P. larvae subsp. larvae, with exposure bioassays. We demonstrated that the strain-specific 50% lethal concentrations varied within an order of magnitude and that differences in amount of time for the pathogen to kill 100% of the infected hosts (LT100) correlated with genotype. One genotype killed rather quickly, with a mean LT100 of 7.8 ± 1.7 days postinfection, while the other genotypes acted more slowly, with mean LT100s of 11.2 ± 0.8 and 11.6 ± 0.6 days postinfection.

Natural infections of Nosema ceranae in European honey bees

The classification of the large and heterogeneous group of microorganisms called protozoans has been recently updated as increased knowledge of the biology and phylogenetics is acquired. The Committee on Systematics and Evolution of the Society of Protozoologists established seven phyla in the elevated subkingdom Protozoa under the Kingdom Protista more than two decades ago (Levin, et al., 1980). Later, it was suggested that the protists should be reorganized into the broader category protoctists that would also include fungi and algae besides the small eukaryotes consisting of a single or a few cells (Margulis et al., 1990). However, with the rapidly increasing phylogenetic data accumulating, this higher order taxonomy is no longer accurate. Based on molecular phylogenies, microsporidia are actually included into the cluster Fungi, rank Opisthokonta which comprises the animals, the fungi and others eukaryotes (Sina et al., 2005). Microsporidia are, thus, to be regarded as highly specialized parasitic fungi. Only two microsporidian parasites are described so far from honey bees (Nosema apis Zander 1909 and Nosema ceranae Fries et al. 1996). Nosema apis was detected in the European honey bee (Apis mellifera L 1758) and is one of the first microsporidia to be described (Zander, 1909). Although the parasite and its life cycle have been described by many authors (see Gray et al., 1969), vegetative stages are difficult to recognize and identify by light microscopy. These early descriptions have later been complemented with ultrastructural features of the parasite (Liu, 1984; Fries, 1989) and also with a molecular characterization (Gatehouse & Malone, 1998). Nosema ceranae, isolated from the Asian honey bee (Apis cerana Fabricius 1793) in China is a more recent description (Fries et al., 1996). There are good reasons to assume that other microsporidia species are also present in honey bees and await full descriptions (i.e. Buys, 1977; Clark 1980). Prior to the description of N. ceranae, observations of microsporidian infections in A. cerana had already been made (Sing, 1975). Yakobson (1992) observed microsporidia infections in both A. cerana and A. mellifera in apiaries with both honey bee species mixed and suggested that cross infection experiments using N. apis spores could perhaps elucidate the question of host specificity in N. apis. Many species of microsporidia cannot be distinguished using light microscopy and only with difficulty using electron microscopy (Larsson, 1986; Rice, 2001) and it cannot be excluded that some earlier observations of microsporidia infections in A. cerana, and possibly also in A. mellifera, may in fact have been observations of N. ceranae. Reports in the past of damage to A. cerana colonies attributed to N. apis infections (Lian, 1980) may actually be reports of N. ceranae, since differences between N. ceranae and N. apis may have gone unnoticed when investigated under the light microscope (Fries et al., 1996). Cross infections between the two host species have demonstrated that N. apis is in fact infective for A. cerana, but also that this parasite develops less well in the Asian host compared to the European host (Fries & Feng, 1995). It has also been stated that N. ceranae is infective for A. mellifera and multiplies more readily in A. mellifera than N. apis does in A. cerana (Fries, 1997), although detailed data were never published. At the annual meeting of Society for Invertebrate Pathology in Anchorage, Alaska, 2005 it was reported that N. ceranae had been found in natural infections in A. mellifera in Taiwan (Huang et al., 2005). The apiary where the infection was detected had harboured both A. mellifera and A. cerana. Thus, it was apparent that N. ceranae could cross the host species barrier, although no data on bee pathological repercussions due to N. ceranae in Apis mellifera were mentioned by the authors. Almost at the same time and following progressively increased incidences of problems with nosema disease in Spain (Martin et al., 2005), the laboratory of Centro Apícola Regional, involved in Journal of Apicultural Research 45(3): 230–233 (2006)