Angela Köhler

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.

Honeybees and nectar nicotine: Deterrence and reduced survival versus potential health benefits

Secondary metabolites produced by plants for herbivore defence are often found in floral nectar, but their effect on the foraging behaviour and physiological performance of pollinators is largely unknown. Nicotine is highly toxic to most herbivores, and nicotine-based insecticides may contribute to current pollinator declines. We examined the effects of nectar nicotine on honeybee foraging choices and worker longevity. Free-flying honeybee (Apis mellifera scutellata) workers from six colonies were given a choice between multiple nicotine concentrations (0-1000 μM) in artificial nectar (0.15-0.63 M sucrose). The dose-dependent deterrent effect of nicotine was stronger in lower sugar concentrations, but even the highest nicotine concentrations did not completely repel honeybees, i.e., bees did not stop feeding on these diets. Nicotine in nectar acts as a partial repellent, which may keep pollinators moving between plants and enhance cross-pollination. In the second part of the study, newly emerged workers from 12 colonies were caged and fed one of four nicotine concentrations (0-300 μM) in 0.63 M sucrose for 21 days. Moderate (≤30 μM) nicotine concentrations had no significant detrimental effect, but high nicotine concentrations reduced the survival of caged workers and their nectar storage in the honey comb. In contrast, worker groups that survived poorly on sugar-only diets demonstrated increased survival on all nicotine diets. In the absence of alternative nectar sources, honeybees tolerate naturally occurring nectar nicotine concentrations; and low concentrations can even be beneficial to honeybees. However, high nicotine concentrations may have a detrimental effect on colony fitness.

3-β-d-Glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc) is an insect detoxification product of maize 1,4-benzoxazin-3-ones

In order to defend themselves against arthropod herbivores, maize plants produce 1,4-benzoxazin-3-ones (BXs), which are stored as weakly active glucosides in the vacuole. Upon tissue disruption, BXs come into contact with β-glucosidases, resulting in the release of active aglycones and their breakdown products. While some aglycones can be reglucosylated by specialist herbivores, little is known about how they detoxify BX breakdown products. Here we report on the structure of an N-glucoside, 3-β-d-glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc), purified from Spodoptera frugiperda faeces. In vitro assays showed that MBOA-N-Glc is formed enzymatically in the insect gut using the BX breakdown product 6-methoxy-2-benzoxazolinone (MBOA) as precursor. While Spodoptera littoralis and S. frugiperda caterpillars readily glucosylated MBOA, larvae of the European corn borer Ostrinia nubilalis were hardly able to process the molecule. Accordingly, Spodoptera caterpillar growth was unaffected by the presence of MBOA, while O. nubilalis growth was reduced. We conclude that glucosylation of MBOA is an important detoxification mechanism that helps insects tolerate maize BXs.

Simultaneous stressors: Interactive effects of an immune challenge and dietary toxin can be detrimental to honeybees

Recent large-scale mortality of honeybee colonies is believed to be caused by multiple interactions between diseases, parasites, pesticide exposure, and other stress factors. To test whether a dual challenge has an additive effect in reducing survival, we experimentally stimulated the immune system of caged Apis mellifera scutellata workers from six colonies by injecting saline or Escherichia coli lipopolysaccharides (LPS), and additionally fed them the alkaloid nicotine (0 μM, 3 μM and 300 μM in 0.63 M sucrose). Workers did not increase their sucrose intake to compensate for the immune system activation, and those injected with E. coli LPS decreased their intake on the highest nicotine concentration. In the single challenges, injection and high nicotine doses negatively affected survival. All injected worker groups showed reduced survival. Without nicotine, survival of the saline and E. coli LPS worker groups was similar, but survival of E. coli LPS-challenged workers dropped below that of the saline groups when additionally challenged by nicotine, with bees dying earlier at higher nicotine concentrations. In the dual challenge of saline injection and dietary nicotine, a reduced effect on survival was observed, with lower mortality than expected from the summed mortalities due to the single challenges. However, additive and synergistic effects on survival were observed in workers simultaneously challenged by E. coli LPS and nicotine, indicating that interactive effects of simultaneous pathogen exposure and dietary toxin are detrimental to honeybee fitness.

Within-plant distribution of 1,4-benzoxazin-3-ones contributes to herbivore niche differentiation in maize

Plant defences vary in space and time, which may translate into specific herbivore-foraging patterns and feeding niche differentiation. To date, little is known about the effect of secondary metabolite patterning on within-plant herbivore foraging. We investigated how variation in the major maize secondary metabolites, 1,4-benzoxazin-3-one derivatives (BXDs), affects the foraging behaviour of two leaf-chewing herbivores. BXD levels varied substantially within plants. Older leaves had higher levels of constitutive BXDs while younger leaves were consistently more inducible. These differences were observed independently of plant age, even though the concentrations of most BXDs declined markedly in older plants. Larvae of the well-adapted maize pest Spodoptera frugiperda preferred and grew better on young inducible leaves irrespective of plant age, while larvae of the generalist Spodoptera littoralis preferred and tended to grow better on old leaves. In BXD-free mutants, the differences in herbivore weight gain between old and young leaves were absent for both species, and leaf preferences of S. frugiperda were attenuated. In contrast, S. littoralis foraging patterns were not affected. In summary, our study shows that plant secondary metabolites differentially affect performance and foraging of adapted and non-adapted herbivores and thereby likely contribute to feeding niche differentiation.

Honeybees prefer warmer nectar and less viscous nectar, regardless of sugar concentration

The internal temperature of flowers may be higher than air temperature, and warmer nectar could offer energetic advantages for honeybee thermoregulation, as well as being easier to drink owing to its lower viscosity. We investigated the responses of Apis mellifera scutellata (10 colonies) to warmed 10% w/w sucrose solutions, maintained at 20–35°C, independent of low air temperatures, and to 20% w/w sucrose solutions with the viscosity increased by the addition of the inert polysaccharide Tylose (up to the equivalent of 34.5% sucrose). Honeybee crop loads increased with nectar temperature, as did the total consumption of sucrose solutions over 2 h by all bees visiting the feeders. In addition, the preference of marked honeybees shifted towards higher nectar temperatures with successive feeder visits. Crop loads were inversely proportional to the viscosity of the artificial nectar, as was the total consumption of sucrose solutions over 2 h. Marked honeybees avoided higher nectar viscosities with successive feeder visits. Bees thus showed strong preferences for both warmer and less viscous nectar, independent of changes in its sugar concentration. Bees may benefit from foraging on nectars that are warmer than air temperature for two reasons that are not mutually exclusive: reduced thermoregulatory costs and faster ingestion times due to the lower viscosity.

Dilute bird nectars: viscosity constrains food intake by licking in a sunbird

Floral nectars of bird-pollinated plants are relatively dilute. One hypothesis proposed to explain this concerns the difficulty for birds of drinking nectar of high viscosity. We examined the effects of viscosity, separately from those of sugar concentration, on feeding by captive whitebellied sunbirds (Cinnyris talatala). Viscosities of artificial nectar (sucrose solutions ranging in concentration from 0.25 to 1.5 mol/l) were altered with Tylose, an inert polysaccharide. Food consumption was measured over 3 h, and lick frequency and duration were recorded using photodetection devices on feeding apertures too small for the bill but large enough for the extended tongue. Volumetric intake rates (ml/s) were inversely proportional to nectar viscosity, and were similar over the range of sucrose concentrations when viscosity was held constant. Sucrose intake rates (mg/s) remained the same on pure sucrose solutions, but they decreased with increasing viscosity at a constant sucrose concentration. Lick frequencies and tongue loads were reduced at high viscosities, and lick duration increased, which confirms that sunbirds take longer to ingest viscous solutions. Licking behavior was remarkably similar in birds feeding on different sucrose concentrations if viscosity was held constant. Nectar ingestion rate is determined by viscosity; however, total food intake is mainly modulated by sugar concentration. Similar effects of food viscosity have been observed in insects that suck nectar.

A new design for honey bee hoarding cages for laboratory experiments

BBSRC, Defra, NERC, the Scottish Government and the Wellcome Trust, under the Insect Pollinators Initiative (BBI000968/1)

Short-term energy regulation of whitebellied sunbirds (Nectarinia talatala): effects of food concentration on feeding frequency and duration.

SUMMARY Avian nectarivores show compensatory feeding by adjusting their volumetric intake in response to variation in nectar concentration. This study used an infrared photo-detection system to investigate the short-term feeding patterns of whitebellied sunbirds (Nectarinia talatala) consuming three different sucrose concentrations (10, 20 and 30% w/w). Sunbirds increased their feeding frequency on the most dilute diet, but there was no change in feeding duration. Thus, the increase in total time spent feeding on the dilute diet was due to the increased feeding frequency. No difference in short-term feeding patterns was found between the 20% and 30% diets. Total time spent feeding was extremely short on all diets (96-144 s in every hour). Birds maintained the same steady increase in body mass over the course of the day on all three diets. Daily rhythms in feeding patterns were evident, with longer feeding duration and lower feeding frequency in the early morning and evening than during the rest of the day. Because ingestion rates on a particular diet may vary through the day, caution must be exercised in using feeding duration as a surrogate for meal size. Individual birds varied greatly in their feeding patterns irrespective of diet concentration.

Antioxidant supplementation can reduce the survival costs of excess amino acid intake in honeybees.

Over-consuming amino acids is associated with reduced survival in many species, including honeybees. The mechanisms responsible for this are unclear but one possibility is that excessive intake of amino acids increases oxidative damage. If this is the case, antioxidant supplementation may help reduce the survival costs of high amino acid intake. We tested this hypothesis in African honeybees (Apis mellifera scutellata) using the major antioxidant in green tea, epigallocatechin-3-gallate (EGCG). We first determined the dose-range of EGCG that improved survival of caged honeybees fed sucrose solution. We then provided bees with eight diets that differed in their ratio of essential amino acids (EAA) to carbohydrate (C) (0:1, 1:250, 1:100, 1:75, 1:50, 1:25, 1:10, 1:5 EAA:C) and also in their EGCG dose (0.0 or 0.4 mM). We found that bees fed sucrose only solution survived better than bees fed EAA diets. Despite this, bees preferred a diet that contained intermediate ratios of EAA:C (ca. 1:25), which may represent the high demands for nitrogen of developing nurse bees. EGCG supplementation improved honeybee survival but only at an intermediate dose (0.3-0.5 mM) and in bees fed low EAA diets (1:250, 1:100 EAA:C). That EGCG counteracted the lifespan reducing effects of eating low EAA diets suggests that oxidative damage may be involved in the association between EAAs and lifespan in honeybees. However, that EGCG had no effect on survival in bees fed high EAA diets suggests that there are other physiological costs of over-consuming EAAs in honeybees.