The COLOSS BEEBOOK Volume I, Standard methods for Apis mellifera research: Introduction

The COLOSS BEEBOOK is a practical manual compiling standard methods in all fields of research on the western honey bee, Apis mellifera. The COLOSS network was founded in 2008 as a consequence of the heavy and frequent losses of managed honey bee colonies experienced in many regions of the world (Neumann and Carreck, 2010). As many of the world’s honey bee research teams began to address the problem, it soon became obvious that a lack of standardized research methods was seriously hindering scientists’ ability to harmonize and compare the data on colony losses obtained internationally. In its second year of activity, during a COLOSS meeting held in Bern, Switzerland, the idea of a manual of standardized honey bee research methods emerged. The manual, to be called the COLOSS BEEBOOK, was inspired by publications with similar purposes for fruit fly research (Lindsley and Grell, 1968; Ashburner 1989; Roberts, 1998; Greenspan, 2004).

The COLOSS BEEBOOK Volume II, Standard methods for Apis mellifera pest and pathogen research: Introduction

The COLOSS BEEBOOK is a practical manual compiling standard methods in all fields of research on the western honey bee, Apis mellifera. The COLOSS network was founded in 2008 as a consequence of the heavy and frequent losses of managed honey bee colonies experienced in many regions of the world (Neumann and Carreck, 2010). As many of the world’s honey bee research teams began to address the problem, it soon became obvious that a lack of standardized research methods was seriously hindering scientists’ ability to harmonize and compare the data on colony losses obtained internationally. In its second year of activity, during a COLOSS meeting held in Bern, Switzerland, the idea of a manual of standardized honey bee research methods emerged. The manual, to be called the COLOSS BEEBOOK, was inspired by publications with similar purposes for fruit fly research (Lindsley and Grell, 1968; Ashburner, 1989; Roberts, 1998; Greenspan, 2004).

Standard methods for Nosema research

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

Standard methods for maintaining adult Apis mellifera in cages under in vitro laboratory conditions

Adult honey bees are maintained in vitro in laboratory cages for a variety of purposes. For example, researchers may wish to perform experiments on honey bees caged individually or in groups to study aspects of parasitology, toxicology, or physiology under highly controlled conditions, or they may cage whole frames to obtain newly emerged workers of known age cohorts. Regardless of purpose, researchers must manage a number of variables, ranging from selection of study subjects (e.g. honey bee subspecies) to experimental environment (e.g. temperature and relative humidity). Although decisions made by researchers may not necessarily jeopardize the scientific rigour of an experiment, they may profoundly affect results, and may make comparisons with similar, but independent, studies difficult. Focusing primarily on workers, we provide recommendations for maintaining adults under in vitro laboratory conditions, whilst acknowledging gaps in our understanding that require further attention. We specifically describe how to properly obtain honey bees, and how to choose appropriate cages, incubator conditions, and food to obtain biologically relevant and comparable experimental results. Additionally, we provide broad recommendations for experimental design and statistical analyses of data that arises from experiments using caged honey bees. The ultimate goal of this, and of all COLOSS BEEBOOK papers, is not to stifle science with restrictions, but rather to provide researchers with the appropriate tools to generate comparable data that will build upon our current understanding of honey bees.

A direct role for secretory phospholipase A2 and lysophosphatidylcholine in the mediation of LPS-induced gastric injury.

Endotoxemia from sepsis can injure the gastrointestinal tract through mechanisms that have not been fully elucidated. We have shown that LPS induces an increase in gastric permeability in parallel with the luminal appearance of secretory phospholipase A2 (sPLA2) and its product, lysophosphatidylcholine (lyso-PC). We proposed that sPLA2 acted on the gastric hydrophobic barrier, composed primarily of phosphatidylcholine (PC), to degrade it and produce lyso-PC, an agent that is damaging to the mucosa. In the present study, we have tested whether lyso-PC and/or sPLA2 have direct damaging effects on the hydrophobic barriers of synthetic and mucosal surfaces. Rats were administered LPS (5 mg/kg, i.p.), and gastric contents were collected 5 h later for analysis of sPLA2 and lyso-PC content. Using these measured concentrations, direct effects of sPLA2 and lyso-PC were determined on (a) surface hydrophobicity as detected with an artificial PC surface and with intact gastric mucosa (contact angle analysis) and (b) cell membrane disruption of gastric epithelial cells (AGS). Both lyso-PC and sPLA2 increased significantly in the collected gastric juice of LPS-treated rats. Using similar concentrations to the levels in gastric juice, the contact angle of PC-coated slides declined after incubation with either pancreatic sPLA2 or lyso-PC. Similarly, gastric contact angles seen in control rats were significantly decreased in sPLA2 and lyso-PC-treated rats. In addition, we observed dose-dependent injurious effects of both lyso-PC and sPLA2 in gastric AGS cells. An LPS-induced increase in sPLA2 activity in the gastric lumen and its product, lyso-PC, are capable of directly disrupting the gastric hydrophobic layer and may contribute to gastric barrier disruption and subsequent inflammation.

The composition and timing of flower odour emission by wild Petunia axillaris coincide with the antennal perception and nocturnal activity of the pollinator Manduca sexta

In the genus Petunia, distinct pollination syndromes may have evolved in association with bee-visitation (P. integrifolia spp.) or hawk moth-visitation (P. axillaris spp). We investigated the extent of congruence between floral fragrance and olfactory perception of the hawk moth Manduca sexta. Hawk moth pollinated P. axillaris releases high levels of several compounds compared to the bee-pollinated P. integrifolia that releases benzaldehyde almost exclusively. The three dominating compounds in P. axillaris were benzaldehyde, benzyl alcohol and methyl benzoate. In P. axillaris, benzenoids showed a circadian rhythm with an emission peak at night, which was absent from P. integrifolia. These characters were highly conserved among different P. axillaris subspecies and P. axillaris accessions, with some differences in fragrance composition. Electroantennogram (EAG) recordings using flower-blends of different wild Petunia species on female M. sexta antennae showed that P. axillaris odours elicited stronger responses than P. integrifolia odours. EAG responses were highest to the three dominating compounds in the P. axillaris flower odours. Further, EAG responses to odour-samples collected from P. axillaris flowers confirmed that odours collected at night evoked stronger responses from M. sexta than odours collected during the day. These results show that timing of odour emissions by P. axillaris is in tune with nocturnal hawk moth activity and that flower-volatile composition is adapted to the antennal perception of these pollinators.

The COLOSS BEEBOOK Volume II: Standard methods for Apis mellifera pest and pathogen research

n recent years, declines of honey bee populations have received massive media attention worldwide, yet attempts to understand the causes have been hampered by a lack of standardisation of laboratory techniques. Published as a response to this, the COLOSS BEEBOOK is a unique collaborative venture involving 234 bee scientists from 34 countries, who have produced the definitive guide to how to carry out research on honey bees. It is hoped that these volumes will become the standards to be adopted by bee scientists worldwide. Volume II includes approximately 600 separate protocols dealing with the study of the pests and diseases of the honey bee, Apis mellifera. These cover epidemiology and surveying techniques, virus diseases, bacterial diseases such as European and American foulbrood, fungal and microsporidian diseases such as Nosema, mites such as Acarapis, Varroa and Tropilaelaps, and other pests such as the small hive beetle.

The COLOSS BEEBOOK Volume I: Standard Methods for Apis mellifera Research

In recent years, declines of honey bee populations have received massive media attention worldwide, yet attempts to understand the causes have been hampered by a lack of standardisation of laboratory techniques. Published as a response to this, the COLOSS BEEBOOK is a unique collaborative venture involving 234 bee scientists from 34 countries, who have produced the definitive guide to how to carry out research on honey bees. It is hoped that these volumes will become the standards to be adopted by bee scientists worldwide. Volume I includes approximately 1,100 separate protocols dealing with the study of the honey bee, Apis mellifera. These cover anatomy, behavioural studies, chemical ecology, breeding, genetics, instrumental insemination and queen rearing, pollination, molecular studies, statistics, toxicology and numerous other techniques

Thiacloprid–Nosema ceranae interactions in honey bees: Host survivorship but not parasite reproduction is dependent on pesticide dose

Interactions between stressors contribute to the recently reported increase in losses of honey bee colonies. Here we demonstrated that a synergistic effect on mortality by the low toxic, commonly used neonicotinoid thiacloprid and the nearly ubiquitous gut parasite Nosemaceranae is dependent on the pesticide dose. Furthermore, thiacloprid had a negative influence on N.ceranae reproduction. Our results highlight that interactions among honey bee health stressors can be dynamic and should be studied across a broader range of combinations.

Promoting Pollinating Insects in Intensive Agricultural Matrices: Field-Scale Experimental Manipulation of Hay-Meadow Mowing Regimes and Its Effects on Bees

Bees are a key component of biodiversity as they ensure a crucial ecosystem service: pollination. This ecosystem service is nowadays threatened, because bees suffer from agricultural intensification. Yet, bees rarely benefit from the measures established to promote biodiversity in farmland, such as agri-environment schemes (AES). We experimentally tested if the spatio-temporal modification of mowing regimes within extensively managed hay meadows, a widespread AES, can promote bees. We applied a randomized block design, replicated 12 times across the Swiss lowlands, that consisted of three different mowing treatments: 1) first cut not before 15 June (conventional regime for meadows within Swiss AES); 2) first cut not before 15 June, as treatment 1 but with 15% of area left uncut serving as a refuge; 3) first cut not before 15 July. Bees were collected with pan traps, twice during the vegetation season (before and after mowing). Wild bee abundance and species richness significantly increased in meadows where uncut refuges were left, in comparison to meadows without refuges: there was both an immediate (within year) and cumulative (from one year to the following) positive effect of the uncut refuge treatment. An immediate positive effect of delayed mowing was also evidenced in both wild bees and honey bees. Conventional AES could easily accommodate such a simple management prescription that promotes farmland biodiversity and is likely to enhance pollination services.

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

STRUCTURE OF CUPIENNIUS SALEI VENOM HYALURONIDASE Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. FUNCTION OF VENOM HYALURONIDASES Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.

MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation

Adaptations to new pollinators involve multiple floral traits, each requiring coordinated changes in multiple genes. Despite this genetic complexity, shifts in pollination syndromes have happened frequently during angiosperm evolution. Here we study the genetic basis of floral UV absorbance, a key trait for attracting nocturnal pollinators. In Petunia, mutations in a single gene, MYB-FL, explain two transitions in UV absorbance. A gain of UV absorbance in the transition from bee to moth pollination was determined by a cis-regulatory mutation, whereas a frameshift mutation caused subsequent loss of UV absorbance during the transition from moth to hummingbird pollination. The functional differences in MYB-FL provide insight into the process of speciation and clarify phylogenetic relationships between nascent species.

Potential for virus transfer between the honey bees Apis mellifera and A. cerana

Viruses seem to play a key role in European honey bee, Apis mellifera health, and have a much broader host spectrum than previously thought. Few studies have investigated interspecific virus transfer within the genus Apis. The introduction of A. mellifera into Asia exposed endemic Apis species to the risk of obtaining new viruses or viral strains and vice versa. To investigate the potential for host shifts, virus prevalence and sequences were monitored over three years in single and mixed-species apiaries hosting introduced A. mellifera and endemic Apis cerana. Deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), and sacbrood virus (SBV) were found, but not KBV, VDV-1, ABPV, or CBPV. Virus infections and prevalence were generally lower in A. cerana compared to A. mellifera, and varied over the years. The sequence data provided evidence for interspecific transfer of IAPV, BQCV, and DWV, but SBV strains seem to be species specific. Prevalence and sequence results taken together indicate that interspecific transfers of viruses are rare, even if honey bees are kept in close proximity. We discuss the pattern observed in the context host specificity and resistance. Our understanding of the extent of these exchanges is limited by a lack of knowledge on the mechanisms of adaptation of viruses to different hosts.