Potential costs of bacterial infection on storage protein gene expression and reproduction in queenless Apis mellifera worker bees on distinct dietary regimes

Insects are able to combat infection by initiating an efficient immune response that involves synthesizing antimicrobial peptides and a range of other defense molecules. These responses may be costly to the organism, resulting in it exploiting endogenous resources to maintain homeostasis or support defense to the detriment of other physiological needs. We used queenless worker bees on distinct dietary regimes that may alter hemolymph protein storage and ovary activation to investigate the physiological costs of infection with Serratia marcescens. The expression of the genes encoding the storage proteins vitellogenin and hexamerin 70a, the vitellogenin receptor, and vasa (which has a putative role in reproduction), was impaired in the infected bees. This impairment was mainly evident in the bees fed beebread, which caused significantly higher expression of these genes than did royal jelly or syrup, and this was confirmed at the vitellogenin and hexamerin 70a protein levels. Beebread was also the only diet that promoted ovary activation in the queenless bees, but this activation was significantly impaired by the infection. The expression of the genes encoding the storage proteins apolipophorins-I and -III and the lipophorin receptor was not altered by infection regardless the diet provided to the bees. Similarly, the storage of apolipophorin-I in the hemolymph was only slightly impaired by the infection, independently of the supplied diet. Taken together these results indicate that, infection demands a physiological cost from the transcription of specific protein storage-related genes and from the reproductive capacity. (C) 2012 Elsevier Ltd. All rights reserved.

A method for harvesting unfermented pollen from stingless bees (Hymenoptera, Apidae, Meliponini)

Pollen traps used for harvesting pollen from Apis mellifera do not work for stingless bees, as most species have small entrances and rapidly deposit large quantities of propolis at any barrier in front of the nest. Some stingless beekeepers harvest pollen by removing it directly from pollen pots, but this pollen is normally fermented and unpalatable. The aim of this study was to test a new method for harvesting pollen from stingless bee colonies before it begins to ferment. Colonies of Scaptotrigona depilis were removed and replaced by empty hives, which were occupied by the returning foragers and used for storing pollen and nectar. After one week, the pollen and honey were harvested directly from the storing pots and weighed. On average, the colonies produced 8.7 g of honey and 54.2 g of unfermented pollen (n = 10). This method is a viable option for harvesting unfermented pollen from stingless bees, especially with species that harvest large amounts of pollen. The unfermented pollen of S. depilis was well received in taste tests, receiving higher scores than fermented pollen, and similar scores to A. mellifera pollen, so could have great commercial possibilities. It is also a good method for studying the foraging of stingless bees because the amount of harvested food can be easily and precisely quantified.

Effects of pesticides on honey bees (Apis mellifera L.): study of a specific route of exposure and evaluation of biochemical-physiological changes in the assessment of the pesticides toxicity

In this study, some important aspects of the relationship between honey bees (Apis mellifera L.) and pesticides have been investigated. In the first part of the research, the effects of the exposure of honey bees to neonicotinoids and fipronil contaminated dusts were analyzed. In fact, considerable amounts of these pesticides, employed for maize seed dressing treatments, may be dispersed during the sowing operations, thus representing a way of intoxication for honey bees. In particular, a specific way of exposure to this pesticides formulation, the indirect contact, was taken into account. To this aim, we conducted different experimentations, in laboratory, in semi-field and in open field conditions in order to assess the effects on mortality, foraging behaviour, colony development and capacity of orientation. The real dispersal of contaminated dusts was previously assessed in specific filed trials. In the second part, the impact of various pesticides (chemical and biological) on honey bee biochemical-physiological changes, was evaluated. Different ways and durations of exposure to the tested products were also employed. Three experimentations were performed, combining Bt spores and deltamethrin, Bt spores and fipronil, difenoconazole and deltamethrin. Several important enzymes (GST, ALP, SOD, CAT, G6PDH, GAPDH) were selected in order to test the pesticides induced variations in their activity. In particular, these enzymes are involved in different pathways of detoxification, oxidative stress defence and energetic metabolism. The results showed a significant effect on mortality of neonicotinoids and fipronil contaminated dusts, both in laboratory and in semi-field trials. However, no effects were evidenced in honey bees orientation capacity. The analysis of different biochemical indicators highlighted some interesting physiological variations that can be linked to the pesticide exposure. We therefore stress the attention on the possibility of using such a methodology as a novel toxicity endpoint in environmental risk assessment.

Sublethal effects of a common neonicotinoid pesticide, thiamethoxam, on honey bees: impact on locomotion and thermoregulation

Neonicotinoids have been pointed to as a factor responsible for the increased honey bee colony losses in the last decades. Many studies have investigated the effects of the first marketed neonicotinoid, imidacloprid, while fewer have focused on thiamethoxam. One recent study showed that sublethal doses of thiamethoxam lead to colony failure by decreasing forager homing flight success. We thus decided to investigate the mechanism which caused this phenomenon. Our hypothesis was that this effect was caused by impairment of forager locomotion abilities. Therefore we tested the effects of sublethal acute and chronic exposures to thiamethoxam on forager walking (Chapter 2) and flight (Chapter 3) performances. The acute treatment (1.34 ng/bee) affected walking locomotion firstly triggering hyperactivity (30 min post-treatment) and then impairing motor functioning (60 min post-treatment). 2-day continuous exposures to thiamethoxam (32.5, 45 ppb) elicited fewer effects on walking locomotion, however both exposure modes elicited an increased positive phototaxis. Similarly, in flight experiments, the single dose (1.34 ng/bee) elicited hyperactivity shortly after intoxication (increased flight duration and distance), while longer and continuous exposures (32.5, 45 ppb) impaired forager motor functions (decreased flight duration, distance, velocity). It is known that flight muscles temperature needs to be precisely regulated by bees during flight. Therefore, we further hypothesized that the impaired flight performances of neonicotinoid intoxicated bees were caused also by thermoregulation anomalies. We tested the effects that acute thiamethoxam exposures (0.2, 1, 2 ng/bee) elicit on forager thorax temperature (Chapter 4). Foragers treated with high doses exhibited hyperthermia or hypothermia when respectively exposed to high or low environmental temperatures. In summary, we show that sublethal doses of thiamethoxam affected forager walking and flight locomotion, phototaxis and thermoregulation. We also display the intricate mode of action of thiamethoxam which triggered, at different extents, inverse sublethal effects in relation to time and dose.

The alternative Pharaoh approach: stingless bees mummify beetle parasites alive

Workers from social insect colonies use different defence strategies to combat invaders. Nevertheless, some parasitic species are able to bypass colony defences. In particular, some beetle nest invaders cannot be killed or removed by workers of social bees, thus creating the need for alternative social defence strategies to ensure colony survival. Here we show, using diagnostic radioentomology, that stingless bee workers (Trigona carbonaria) immediately mummify invading adult small hive beetles (Aethina tumida) alive by coating them with a mixture of resin, wax and mud, thereby preventing severe damage to the colony. In sharp contrast to the responses of honeybee and bumblebee colonies, the rapid live mummification strategy of T. carbonaria effectively prevents beetle advancements and removes their ability to reproduce. The convergent evolution of mummification in stingless bees and encapsulation in honeybees is another striking example of co-evolution between insect societies and their parasites.

Worldwide Diaspora of Aethina tumida (Coleoptera: Nitidulidae), a Nest Parasite of Honey Bees

Native to sub-Saharan Africa, Aethina tumida Murray (Coleoptera: Nitidulidae) is now an invasive pest of honey bee, Apis mellifera L., colonies in Australia and North America. Knowledge about the introduction (s) of this beetle from Africa into and among the current ranges will elucidate pest populations and invasion pathways and contribute to knowledge of how a parasite expands in new populations. We examined genetic variation in adult beetle samples from the United States, Australia, Canada, and Africa by sequencing a 912-base pair region of the mitochondrial DNA cytochrome c oxidase subunit I gene and screening 10 informative microsatellite loci. One Canadian introduction of small hive beetles can be traced to Australia, whereas the second introduction seems to have come from the United States. Beetles now resident in Australia were of a different African origin than were beetles in North America. North American beetles did not show covariance between two mitochondrial haplotypes and their microsatellite frequencies, suggesting that these beetles have a shared source despite having initial genetic structure within their introduced range. Excellent dispersal of beetles, aided in some cases by migratory beekeeping and the bee trade, seems to lead to panmixis in the introduced populations as well as in Africa.

Declines of managed honey bees and beekeepers in Europe

Growing evidence indicates that European managed honey bees are in decline, but information for Europe remains patchy and localized. Here we compile data from 18 European countries to assess trends in the number of honey bee colonies and beekeepers between 1965 and 2005. We found consistent declines in colony numbers in central European countries and some increases in Mediterranean countries. Beekeeper numbers have declined in all of the European countries examined. Our data support the view that honey bees are in decline at least in some regions, which is probably closely linked to the decreasing number of beekeepers. Our data on colony numbers and beekeepers must, however, be interpreted with caution due to different approaches and socioeconomic factors in the various countries, thereby limiting their comparability. We therefore make specific recommendations for standardized methodologies to be adopted at the national and global level to assist in the future monitoring of honey bees.

Localization of Deformed Wing Virus (DWV) in the Brains of Apis mellifera (European Honey Bees)

The purpose of the current research project is to design a successful in-situ hybridization to identify regions within the brains of honeybees where DWV replicates. The localization of the virus in the brains of the bees can draw a connection between CCDand DWV.In conclusion, these results demonstrate that in bees infected with DWV the virus replicates actively in very important regions of the brain, including neuropils that are responsible for vision and olfaction. This means that the virus could adversely affect the vision and olfaction of the honeybees making it difficult for bees to behave normally.

Floral scents affect the distribution of hive bees around dancers

Floral scents are important information cues used to organize foraging-related tasks in honeybees. The waggle dance, apart from encoding spatial information about food sources, might facilitate the transfer of olfactory information by increasing the dissipation of volatiles brought back by successful foragers. By assuming that food scents are more intensive on specific body parts of returning foragers, i.e., the posterior legs of pollen foragers and mouthparts of nectar foragers, we quantified the interactions between hive mates and foragers during dances advertising different types of food sources. For natural sources, a higher proportion of hive mates contacted the hind legs of pollen dancers (where the pollen loads were located) with their heads compared to non-pollen dancers. On the other hand, the proportion of head-to-head contacts was higher for non-pollen foragers during the waggle runs. When the food scent was manipulated, dancers collecting scented sugar solution had a higher proportion of head-to-head contacts and a lower proportion around their hind legs compared to dancers collecting unscented solution. The presence of food odors did not affect in-hive behaviors of dancers, but it increased the number of trophallaxes in-between waggle runs (i.e., during circle phases). These results suggest that the honeybee dance facilitates the olfactory information transfer between incoming foragers and hive mates, and we propose that excitatory displays in other social insect species serve the same purpose. While recent empirical and theoretical findings suggested that the colony level foraging benefits of the spatial information encoded in the waggle dance vary seasonally and with habitats, the role of the dance as a compound signal not only indicating the presence of a profitable resource but also amplifying the information transfer regarding floral odors may be important under any ecological circumstances.

Learning Paradigms Exemplified by Virtual Experiments with Honey Bees

Learning is based on rules that can be elucidated by behavioural experiments. This article focuses on virtual experiments, in which non-associative learning (habituation, sensitization) and principles of associative learning (contiguity, inhibitory learning, generalization, overshadowing, positive and negative patterning) can be examined using 'virtual' honey bees in PER (Proboscis Reaction Extension) conditioning experiments. Users can develop experimental designs, simulate and document the experiments and find explanations and suggestions for the analysis of the learning experiments. The virtual experiments are based on video sequences and data from actual learning experiments. The bees' responses are determined by probability-based learning profiles.

Sex-Specific Differences in Pathogen Susceptibility in Honey Bees (Apis mellifera)

Sex-related differences in susceptibility to pathogens are a common phenomenon in animals. In the eusocial Hymenoptera the two female castes, workers and queens, are diploid and males are haploid. The haploid susceptibility hypothesis predicts that haploid males are more susceptible to pathogen infections compared to females. Here we test this hypothesis using adult male (drone) and female (worker) honey bees (Apis mellifera), inoculated with the gut endoparasite Nosema ceranae and/or black queen cell virus (BQCV). These pathogens were chosen due to previously reported synergistic interactions between Nosema apis and BQCV. Our data do not support synergistic interactions between N. ceranae and BQCV and also suggest that BQCV has limited effect on both drone and worker health, regardless of the infection level. However, the data clearly show that, despite lower levels of N. ceranae spores in drones than in workers, Nosema-infected drones had both a higher mortality and a lower body mass than non-infected drones, across all treatment groups, while the mortality and body mass of worker bees were largely unaffected by N. ceranae infection, suggesting that drones are more susceptible to this pathogen than workers. In conclusion, the data reveal considerable sex-specific differences in pathogen susceptibility in honey bees and highlight the importance of ultimate measures for determining susceptibility, such as mortality and body quality, rather than mere infection levels