Piotr Medrzycki

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

Summary 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.

Standard methods for toxicology research in Apis mellifera

Summary Modern agriculture often involves the use of pesticides to protect crops. These substances are harmful to target organisms (pests and pathogens). Nevertheless, they can also damage non-target animals, such as pollinators and entomophagous arthropods. It is obvious that the undesirable side effects of pesticides on the environment should be reduced to a minimum. Western honey bees (Apis mellifera) are very important organisms from an agricultural perspective and are vulnerable to pesticide-induced impacts. They contribute actively to the pollination of cultivated crops and wild vegetation, making food production possible. Of course, since Apis mellifera occupies the same ecological niche as many other species of pollinators, the loss of honey bees caused by environmental pollutants suggests that other insects may experience a similar outcome. Because pesticides can harm honey bees and other pollinators, it is important to register pesticides that are as selective as possible. In this manuscript, we describe a selection of methods used for studying pesticide toxicity/selectiveness towards Apis mellifera. These methods may be used in risk assessment schemes and in scientific research aimed to explain acute and chronic effects of any target compound on Apis mellifera.

Influence of brood rearing temperature on honey bee development and susceptibility to poisoning by pesticides

Summary Adult honey bees (Apis mellifera) usually maintain colony brood rearing temperature between 34–35°C by thermoregulation. The brood may, however, also be subjected to suboptimal temperature. Here we investigated whether a decrease of brood rearing temperature may have effects on larval mortality, adult emergence, longevity, morphology and susceptibility to poisoning by pesticides (dimethoate). Using the in vitro rearing protocol of Aupinel (2005), we were able for the first time to control the brood temperature not only during the pupal stage but also during the larval stage. Honey bee larvae were reared in vitro at 35°C (optimal) and 33°C (suboptimal) from 12 h after hatching for 15 days. Dimethoate was tested by ingestion either on 4-day old larvae or on 7-day old adults. Our results showed that lower rearing temperature had no significant effects on larval mortality and adult emergence, but adult bee mortality was strongly affected. Moreover, adult workers emerging at 33°C were significantly more susceptible to dimethoate. Larval LD50 (48 h) was, however, 28 times higher at 33°C than at 35°C. The striking differences between larvae and adults may be explained by differential larval metabolism at 33°C and resulting slower active ingredient absorption. We conclude that adult honey bees reared at even slightly suboptimal brood temperature may be more susceptible to pesticide poisoning and be characterised by reduced longevity. Thus, low temperature brood rearing could be another stress factor for colonies.

Amino acid content and nectar choice by forager honeybees (Apis mellifera L.)

Dual choice feeding tests were performed to determine a preference of forager honeybees for specific amino acids. Artificial nectar containing proline was preferred over those containing only sugars. Nectar containing alanine was preferred on the first day, but preference was no longer significant thereafter. On the contrary, a negative response was found for serine. When the bees were given the choice between two nectars enriched with different compounds, proline was preferred above both alanine and serine, and alanine above serine.

Synergistic mortality between a neonicotinoid insecticide and an ergosterol‐biosynthesis‐inhibiting fungicide in three bee species

BACKGROUND Neonicotinoid insecticides have been identified as an important factor contributing to bee diversity declines. Nonetheless, uncertainties remain about their impact under field conditions. Most studies have been conducted on Apis mellifera and tested single compounds. However, in agricultural environments, bees are often exposed to multiple pesticides. We explore the synergistic mortality between a neonicotinoid (clothianidin) and an ergosterol-biosynthesis-inhibiting fungicide (propiconazole) in three bee species (A. mellifera, Bombus terrestris, Osmia bicornis) following oral exposure in the laboratory. RESULTS We developed a new approach based on the binomial proportion test to analyse synergistic interactions. We estimated uptake of clothianidin per foraging bout in honey bees foraging on seed-coated rapeseed fields. We found significant synergistic mortality in all three bee species exposed to non-lethal doses of propiconazole and their respective LD10 of clothianidin. Significant synergism was only found at the first assessment times in A. mellifera (4 and 24 h) and B. terrestris (4 h), but persisted throughout the experiment (96 h) in O. bicornis. O. bicornis was also the most sensitive species to clothianidin. CONCLUSION Our results underscore the importance to test pesticide combinations likely to occur in agricultural environments, and to include several bee species in environmental risk assessment schemes.

Monitoring of Polycyclic Aromatic Hydrocarbons in Bees (Apis mellifera) and Honey in Urban Areas and Wildlife Reserves

The honeybee is a good biological indicator that quickly reflects chemical impairment of the environment by its high mortality and the presence of pollutants in its body or in beehive products. In this work the honeybee (Apis mellifera) and honey were used to detect the presence of polycyclic aromatic hydrocarbons (PAHs) in several areas with different degrees of environmental pollution. All sampling sites showed the presence of PAHs. Benzo(a)pyrene was never detected. Fluorene, phenanthrene, anthracene, fluoranthene, benz(a)anthracene, benzo(b)fluoranthene, and benzo(k)fluoranthene were the PAHs detected in bees, whereas the honey contained only phenanthrene, anthracene, and chrysene. Phenanthrene showed the highest mean values in honeybees and honey. Independent from the season and location the pattern of PAHs in honeybees and honey was dominated by the presence of the lowest molecular weight PAHs. Furthermore, the mean PAH concentrations in honey samples were lower than those reported in honeybees, and no positive correlation was found between the compounds detected in bees and those in honey.

Honey bee colony losses in Italy

Franco Mutinelli, Cecilia Costa, Marco Lodesani, Alessandra Baggio, Piotr Medrzycki, Giovanni Formato and Claudio Porrini Istituto Zooprofilattico Sperimentale delle Venezie, National Reference Laboratory for Beekeeping, Viale dell’Universita’ 10, 35020 Legnaro (PD), Italy. Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unita di Ricerca di Apicoltura e Bachicoltura (CRA-API), Via di Saliceto 80, 40128 Bologna, Italy. Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova 1411, 00178 Roma, Italy. Dipartimento di Scienze e Tecnologie Agroambientali (DiSTA), Universita di Bologna, Viale G. Fanin 42, 40127 Bologna, Italy.

The Status of Honey Bee Health in Italy: Results from the Nationwide Bee Monitoring Network

In Italy a nation-wide monitoring network was established in 2009 in response to significant honey bee colony mortality reported during 2008. The network comprised of approximately 100 apiaries located across Italy. Colonies were sampled four times per year, in order to assess the health status and to collect samples for pathogen, chemical and pollen analyses. The prevalence of Nosema ceranae ranged, on average, from 47–69% in 2009 and from 30–60% in 2010, with strong seasonal variation. Virus prevalence was higher in 2010 than in 2009. The most widespread viruses were BQCV, DWV and SBV. The most frequent pesticides in all hive contents were organophosphates and pyrethroids such as coumaphos and tau-fluvalinate. Beeswax was the most frequently contaminated hive product, with 40% of samples positive and 13% having multiple residues, while 27% of bee-bread and 12% of honey bee samples were contaminated. Colony losses in 2009/10 were on average 19%, with no major differences between regions of Italy. In 2009, the presence of DWV in autumn was positively correlated with colony losses. Similarly, hive mortality was higher in BQCV infected colonies in the first and second visits of the year. In 2010, colony losses were significantly related to the presence of pesticides in honey bees during the second sampling period. Honey bee exposure to poisons in spring could have a negative impact at the colony level, contributing to increase colony mortality during the beekeeping season. In both 2009 and 2010, colony mortality rates were positively related to the percentage of agricultural land surrounding apiaries, supporting the importance of land use for honey bee health.

Effects of a neonicotinoid pesticide on thermoregulation of African honey bees (Apis mellifera scutellata)

Thiamethoxam is a widely used neonicotinoid pesticide that, as agonist of the nicotinic acetylcholine receptors, has been shown to elicit a variety of sublethal effects in honey bees. However, information concerning neonicotinoid effects on honey bee thermoregulation is lacking. Thermoregulation is an essential ability for the honey bee that guarantees the success of foraging and many in-hive tasks, especially brood rearing. We tested the effects of acute exposure to thiamethoxam (0.2, 1, 2ng/bee) on the thorax temperatures of foragers exposed to low (22°C) and high (33°C) temperature environments. Thiamethoxam significantly altered honey bee thorax temperature at all doses tested; the effects elicited varied depending on the environmental temperature and pesticide dose to which individuals were exposed. When bees were exposed to the high temperature environment, the high dose of thiamethoxam increased their thorax temperature 1-2h after exposure. When bees were exposed to the low temperature, the higher doses of the neonicotinoid reduced bee thorax temperatures 60-90min after treatment. In both experiments, the neonicotinoid decreased the temperature of bees the day following the exposure. After a cold shock (5min at 4°C), the two higher doses elicited a decrease of the thorax temperature, while the lower dose caused an increase, compared to the control. These alterations in thermoregulation caused by thiamethoxam may affect bee foraging activity and a variety of in-hive tasks, likely leading to negative consequences at the colony level. Our results shed light on sublethal effect of pesticides which our bees have to deal with.

Neonicotinoid pesticides and nutritional stress synergistically reduce survival in honey bees

The honey bee is a major pollinator whose health is of global concern. Declines in bee health are related to multiple factors, including resource quality and pesticide contamination. Intensive agricultural areas with crop monocultures potentially reduce the quality and quantity of available nutrients and expose bee foragers to pesticides. However, there is, to date, no evidence for synergistic effects between pesticides and nutritional stress in animals. The neonicotinoids clothianidin (CLO) and thiamethoxam (TMX) are common systemic pesticides that are used worldwide and found in nectar and pollen. We therefore tested if nutritional stress (limited access to nectar and access to nectar with low-sugar concentrations) and sublethal, field-realistic acute exposures to two neonicotinoids (CLO and TMX at 1/5 and 1/25 of LD50) could alter bee survival, food consumption and haemolymph sugar levels. Bee survival was synergistically reduced by the combination of poor nutrition and pesticide exposure (−50%). Nutritional and pesticide stressors reduced also food consumption (−48%) and haemolymph levels of glucose (−60%) and trehalose (−27%). Our results provide the first demonstration that field-realistic nutritional stress and pesticide exposure can synergistically interact and cause significant harm to animal survival. These findings have implications for current pesticide risk assessment and pollinator protection.