Impact of copper and zinc oral chronic exposure on Carniolan honey bee survival and feeding preference.

Honey bees are important plant pollinators and honey producers. Contamination of the environment with metals can lead to a decline in honey bee populations. Copper (Cu) and zinc (Zn) salts are commonly used as fungicides and foliar fertilizers. In this study, we investigated the effects of 10-day chronic oral exposure to different concentrations of Cu (CuSO4) and Zn (ZnCl2) on survival and feeding rates of Carniolan honey bees in laboratory conditions. We found that mortality in honey bee workers increased in a concentration-dependent manner and that Cu (lethal concentration [LC50] = 66 mg/l) was more toxic than Zn (LC50 = 144 mg/l). There was no difference in the feeding rate of Cu-treated bees for the different concentrations tested, but the feeding rate decreased with the increase in Zn concentration. To determine feeding preference or avoidance for Cu and Zn, we conducted 2-choice 24-h feeding experiments. We demonstrated that honey bees preferred Zn-containing solutions compared to the control diet. A two-choice experiment with Cu showed a tendency for honey bees to be deterred by Cu at high concentrations; however, it was not statistically significant. In summary, our results suggest that honey bee workers may suffer adverse effects when exposed to ecologically relevant concentrations of Cu and Zn.

CSI Pollen: Diversity of Honey Bee Collected Pollen Studied by Citizen Scientists.

A diverse supply of pollen is an important factor for honey bee health, but information about the pollen diversity available to colonies at the landscape scale is largely missing. In this COLOSS study, beekeeper citizen scientists sampled and analyzed the diversity of pollen collected by honey bee colonies. As a simple measure of diversity, beekeepers determined the number of colors found in pollen samples that were collected in a coordinated and standardized way. Altogether, 750 beekeepers from 28 different regions from 24 countries participated in the two-year study and collected and analyzed almost 18,000 pollen samples. Pollen samples contained approximately six different colors in total throughout the sampling period, of which four colors were abundant. We ran generalized linear mixed models to test for possible effects of diverse factors such as collection, i.e., whether a minimum amount of pollen was collected or not, and habitat type on the number of colors found in pollen samples. To identify habitat effects on pollen diversity, beekeepers' descriptions of the surrounding landscape and CORINE land cover classes were investigated in two different models, which both showed that both the total number and the rare number of colors in pollen samples were positively affected by 'urban' habitats or 'artificial surfaces', respectively. This citizen science study underlines the importance of the habitat for pollen diversity for bees and suggests higher diversity in urban areas.

An Approximate GEMM Unit for Energy-Efficient Object Detection.

Edge computing brings artificial intelligence algorithms and graphics processing units closer to data sources, making autonomy and energy-efficient processing vital for their design. Approximate computing has emerged as a popular strategy for energy-efficient circuit design, where the challenge is to achieve the best tradeoff between design efficiency and accuracy. The essential operation in artificial intelligence algorithms is the general matrix multiplication (GEMM) operation comprised of matrix multiplication and accumulation. This paper presents an approximate general matrix multiplication (AGEMM) unit that employs approximate multipliers to perform matrix-matrix operations on four-by-four matrices given in sixteen-bit signed fixed-point format. The synthesis of the proposed AGEMM unit to the 45 nm Nangate Open Cell Library revealed that it consumed only up to 36% of the area and 25% of the energy required by the exact general matrix multiplication unit. The AGEMM unit is ideally suited to convolutional neural networks, which can adapt to the error induced in the computation. We evaluated the AGEMM units' usability for honeybee detection with the YOLOv4-tiny convolutional neural network. The results implied that we can deploy the AGEMM units in convolutional neural networks without noticeable performance degradation. Moreover, the AGEMM unit's employment can lead to more area- and energy-efficient convolutional neural network processing, which in turn could prolong sensors' and edge nodes' autonomy.

In vivo continuous three-dimensional magnetic resonance microscopy: a study of metamorphosis in Carniolan worker honey bees (Apis mellifera carnica).

Three-dimensional (3D) magnetic resonance microscopy (MRM) is a modality of magnetic resonance imaging (MRI) optimized for the best resolution. Metamorphosis of the Carniolan worker honey bee (Apis mellifera carnica) was studied in vivo under controlled temperature and humidity conditions from sealed larvae until the emergence of an adult. The 3D images were analyzed by volume rendering and segmentation, enabling the analysis of the body, tracheal system and gastrointestinal tract through the time course of volume changes. Fat content sensitivity enabled the analysis of flight muscles transformation during the metamorphosis by the signal histogram and gray level co-occurrence matrix (GLCM). Although the transformation during metamorphosis is well known, MRM enables an alternative insight to this process, i.e. 3D in vivo, which has relatively high spatial and temporal resolutions. The developed methodology can easily be adapted for studying the metamorphosis of other insects or any other incremental biological process on a similar spatial and temporal scale.

Comparison of sublethal effects of natural acaricides carvacrol and thymol on honeybees.

The parasitic mite Varroa destructor is a threat to beekeeping colonies. Among naturally derived acaricides, the monoterpenoid essential oil compound thymol is used in beekeeping for varroa mite control, but adverse impacts on honeybees has been already documented. Carvacrol, another monoterpenoid, also has a high acaricidal potential and could thus be promising for regular use in beekeeping, but information is scarce regarding the effects of prolonged systemic administration of carvacrol on honeybees. In this study, we evaluate and compared the sublethal effects of long term consumption of carvacrol and thymol on Carnolian honeybee workers (Apis mellifera carnica). Survival and feeding rate were determined preliminary to assess sublethal concentrations. The sublethal effects were analysed by the activity of the acetylcholinesterase (AChE), enzyme involved in the control of neurotransmission, and the activity of detoxifying enzyme glutathione S-transferase (GST) in heads and thoraces. We found that, thymol and carvacrol, caused mortality only at the highest concentrations tested, 1% and 5% respectively. As demonstrated by others, both substances could be effective against varroa at concentrations ten times lower than those causing significant honeybee mortality. However, we demonstrated sublethal effects at the 0.05% carvacrol and thymol exposure concentrations evidenced as increased activity of AChE and GST in the honeybee heads. In conclusion, prolonged treatment with thymol and carvacrol affects bee nervous system and induce detoxification processes possibly resulting in a limited use for acaricidal purposes. We postulate that under the same chronic exposure conditions carvacrol and thymol will have similar sublethal effects on honeybees.

Different response of acetylcholinesterases in salt- and detergent-soluble fractions of honeybee haemolymph, head and thorax after exposure to diazinon.

Organophosphate pesticide diazinon is a specific inhibitor of acetylcholinesterase (AChE), which is a common neurotoxicity biomarker in environmental studies. In honeybees, AChE exists in two forms having different physiological roles, one existing as a soluble form and the other as membrane-bound. In most studies AChE activity has been analysed without paying considerable attention to different forms of AChE. In this study, we exposed honeybees Apis mellifera carnica for 10days to diazinon via oral exposure and analysed the total AChE activities in salt soluble (SS) and detergent soluble (DS) fractions. We assumed that SS fraction would preferentially contain the soluble AChE, but the DS fraction would contain only membrane AChE. On the contrary, our results showed that SS and DS fractions both contain soluble and membrane AChE and the latter has considerably higher activity. Despite this we obtained a differential response of AChE activity in SS and DS fractions when exposed to diazinon. The head/thorax AChE activity in DS fraction decreased, while the head/thorax AChE activity in SS fraction increased at sublethal concentrations. The AChE activity in honeybee hemolymph shown here for the first time is a salt soluble enzyme. Its activity remained unaltered after diazinon treatment. In conclusion, we provide evidence that varying results regarding AChE activity alterations upon stressor exposure are obtained when extracted through different procedures. In further environmental studies with honeybees this differential response of AChE activity should be given considerable attention because this affects the outcome of ecotoxicity study.

Immune related gene expression in worker honey bee (Apis mellifera carnica) pupae exposed to neonicotinoid thiamethoxam and Varroa mites (Varroa destructor).

Varroa destructor is one of the most common parasites of honey bee colonies and is considered as a possible co-factor for honey bee decline. At the same time, the use of pesticides in intensive agriculture is still the most effective method of pest control. There is limited information about the effects of pesticide exposure on parasitized honey bees. Larval ingestion of certain pesticides could have effects on honey bee immune defense mechanisms, development and metabolic pathways. Europe and America face the disturbing phenomenon of the disappearance of honey bee colonies, termed Colony Collapse Disorder (CCD). One reason discussed is the possible suppression of honey bee immune system as a consequence of prolonged exposure to chemicals. In this study, the effects of the neonicotinoid thiamethoxam on honey bee, Apis mellifera carnica, pupae infested with Varroa destructor mites were analyzed at the molecular level. Varroa-infested and non-infested honey bee colonies received protein cakes with or without thiamethoxam. Nurse bees used these cakes as a feed for developing larvae. Samples of white-eyed and brown-eyed pupae were collected. Expression of 17 immune-related genes was analyzed by real-time PCR. Relative gene expression in samples exposed only to Varroa or to thiamethoxam or simultaneously to both Varroa and thiamethoxam was compared. The impact from the consumption of thiamethoxam during the larval stage on honey bee immune related gene expression in Varroa-infested white-eyed pupae was reflected as down-regulation of spaetzle, AMPs abaecin and defensin-1 and up-regulation of lysozyme-2. In brown-eyed pupae up-regulation of PPOact, spaetzle, hopscotch and basket genes was detected. Moreover, we observed a major difference in immune response to Varroa infestation between white-eyed pupae and brown-eyed pupae. The majority of tested immune-related genes were upregulated only in brown-eyed pupae, while in white-eyed pupae they were downregulated.

Feeding Preference and Sub-chronic Effects of ZnO Nanomaterials in Honey Bees (Apis mellifera carnica).

The extensive production of zinc oxide (ZnO) nanomaterials (NMs) may result in high environmental zinc burdens. Honeybees need to have special concern due to their crucial role in pollination. Our previous study indicated that low concentrations of ZnO NMs, corresponding to 0.8 mg Zn/mL, have a neurotoxic potential for honeybees after a 10-day oral exposure. Present study was designed to investigate the effect of a short, dietary exposure of honeybees to ZnO NMs at concentrations 0.8-8 mg Zn/mL on consumption rate, food preference, and two enzymatic biomarkers-a stress-related glutathione S-transferase (GST) and the neurotoxicity biomarker acetylcholinesterase (AChE). Consumption rate showed a tendency toward a decrease feeding with the increasing concentrations of ZnO NMs. None of Zn NMs concentrations caused alterations in mortality rate and in the activities of brain GST and AChE. To investigate if there is an avoidance response against Zn presence in food, 24-h two-choice tests were performed with control sucrose diet versus sucrose suspensions with different concentrations of ZnO NMs added. We demonstrated that honeybees prefer ZnO NMs ZnO NMs containing suspensions, even at highest Zn concentrations tested, compared with the control diet. This indicates that they might be able to perceive the presence of ZnO NMs in sucrose solution. Because honeybees feed frequently the preference towards ZnO NMs might have a high impact on their survival when exposed to these NMs.

Prochloraz and coumaphos induce different gene expression patterns in three developmental stages of the Carniolan honey bee (Apis mellifera carnica Pollmann).

The Carniolan honey bee, Apis mellifera carnica, is a Slovenian autochthonous subspecies of honey bee. In recent years, the country has recorded an annual loss of bee colonies through mortality of up to 35%. One possible reason for such high mortality could be the exposure of honey bees to xenobiotic residues that have been found in honey bee and beehive products. Acaricides are applied by beekeepers to control varroosis, while the most abundant common agricultural chemicals found in honey bee and beehive products are fungicides, which may enter the system when applied to nearby flowering crops and fruit plants. Acaricides and fungicides are not intrinsically highly toxic to bees but their action in combination might lead to higher honey bee sensitivity or mortality. In the present study we investigated the molecular immune response of honey bee workers at different developmental stages (prepupa, white-eyed pupa, adult) exposed to the acaricide coumaphos and the fungicide prochloraz individually and in combination. Expression of 17 immune-related genes was examined by quantitative RT-PCR. In treated prepupae downregulation of most immune-related genes was observed in all treatments, while in adults upregulation of most of the genes was recorded. Our study shows for the first time that negative impacts of prochloraz and a combination of coumaphos and prochloraz differ among the different developmental stages of honey bees. The main effect of the xenobiotic combination was found to be upregulation of the antimicrobial peptide genes abaecin and defensin-1 in adult honey bees. Changes in immune-related gene expression could result in depressed immunity of honey bees and their increased susceptibility to various pathogens.

Oviposition by Female Plodia interpunctella (Lepidoptera: Pyralidae): Description and Time Budget Analysis of Behaviors in Laboratory Studies.

The oviposition behavior of the Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), a major insect pest of durable stored foods, was studied in small experimental arenas under laboratory conditions using videography, and a time budget analysis of its behaviors was documented. Resting gravid females typically became active shortly after the start of the scotophase. The characteristic behaviors exhibited by mated females prior to oviposition included antennal movement, grooming of antennae and mouth parts using the forelegs, walking or flying, and abdomen bending and dragging. Pre-oviposition behaviors such as antennal grooming and walking or flying were observed to alternate several times before females commenced the abdominal dragging behavior that preceded egg laying. Eggs were laid singly or sometimes in groups, either freely or stuck to food material. Gravid females showed little or no movement during the photophase; however, they actively flew and oviposited during the scotophase. Females allocated only a small portion of their time to oviposition while the rest of the time was spent away from food. Females oviposited on food material by making repeated visits, predominantly during the first four hours of the scotophase. Visits and time spent on food declined as the scotophase advanced.

Neurotoxic potential of ingested ZnO nanomaterials on bees.

The honey bee is among most important pollinators threatened by environmental pollution, pest control and potentially, by products of nanotechnologies. The aim of the current study was an analysis of the neurotoxic potential of ingested zinc oxide nanomaterials (ZnO NMs) or zinc ions (Zn(2+)) on honey bees. We analysed a variety of biomarkers, including metabolic impairment, feeding rate, and survival, as well as the activities of a stress-related enzyme glutathione S-transferase, and the neurotoxicity biomarker acetylcholinesterase. Acetylcholinesterase activity was found to be elevated in bees exposed to either of the tested substances. In addition, we observed increased feeding rate in the group treated with Zn(2+) but not with ZnO NMs or control group. The observed effects we relate primarily to Zn(2+) ions. Here we provide evidence that zinc ions either originating from Zn salt or Zn-based NPs have a neurotoxic potential and thus might contribute to colony survival.

The behavior and social communication of honey bees (Apis mellifera carnica Poll.) under the influence of alcohol.

In this study, the effects of ethanol on honey bee social communication and behavior within the hive were studied to further investigate the usefulness of honey bees as an ethanol-abuse model. Control (1.5 M sucrose) and experimental (1.5 M sucrose, 2.5% w/v ethanol) solutions were directly administered to individual forager bees via proboscis contact with glass capillary tubes. The duration, frequency, and proportion of time spent performing social and nonsocial behaviors were the dependent variables of interest. No differences in the relative frequency or proportion of time spent performing the target behaviors were observed. However, ethanol consumption significantly decreased bouts of walking, resting, and the duration of trophallactic (i.e., food-exchange) encounters. The results of this study suggest that a low dose of ethanol is sufficient to disrupt both social and nonsocial behaviors in honey bees. In view of these results, future behavioral-genetic investigations of honey bee social behavior are encouraged.

Ethanol levels in honeybee hemolymph resulting from alcohol ingestion.

Our previous work on a social insect model of ethanol-induced behavior focused on behavioral studies of honeybees (Apis mellifera L.). We now investigate the dependence of honeybee blood ethanol concentration on both the amount of ethanol consumed and time elapsed since ingestion. Blood ethanol level was determined using gas chromatograph using hemolymph taken from harnessed bees. Significantly increased levels of ethanol in honeybee hemolymph were detected within 15 min of feeding bees alcohol. Within 30 min, ethanol concentration increased 2.7 times. The concentration of ethanol ingested also had a significant effect on blood ethanol level. However, postfeeding times greater than 30 min did not significantly increase ethanol concentration in bee hemolymph. This study integrates with our behavioral data on the effect of ethanol on honeybees. Our laboratory and field experiments show a correlation between the time frame for behavioral changes and significant increases of blood ethanol levels shown in this study.

Reduced ability of ethanol drinkers for social communication in honeybees (Apis mellifera carnica Poll.).

Foraging behavior was evaluated in honeybees trained to fly to a feeder containing sucrose only, 1% ethanol, 5% ethanol, or 10% ethanol. The results indicated that exposure to ethanol disrupted several types of honeybee social behavior within the hive. Consumption of ethanol at the feeding site reduced waggle dance activity in foraging bees and increased occurrence of tremble dance, food exchange, and self-cleaning behavior. These ethanol-induced changes in behavior may reflect effects on the central nervous system similar to the previously observed effects of food poisoning with sublethal doses of insecticides.

Behavioral response in the terrestrial isopod Porcellio scaber (Crustacea) offered a choice of uncontaminated and cadmium-contaminated food.

The objective of this study was to find out whether Porcellio scaber discriminates against Cd-contaminated food. The foraging behavior in animals offered uncontaminated and Cd-contaminated food simultaneously was quantified for 48-h employing computer-aided video tracking. To see whether the isopods' selection of less contaminated food could diminish the influence Cd on food consumption, growth, metal assimilation, moulting and mortality, Cd-dosed food (20, 45, 200 and 450 mg kg(-1) dry weight) was offered together with untreated food for 3 weeks. Data from the video tracking experiments revealed that animals visited Cd-dosed food as often as untreated food, but spent much less time near Cd-dosed food. Discrimination against Cd-contaminated food increased with previous experience with contaminated food and/or with increased Cd body burden. In 3 weeks exposure uncontaminated food preference rose with time of exposure and cadmium concentration in food and reached a maximal preference ratio of 65% (untreated food): 35% (Cd-dosed food). The decreased consumption of Cd-dosed food was compensated by the increased consumption of control food. Cadmium body burden increased with time of exposure and cadmium concentration in food consumed, while the influence of Cd on food consumption, growth and moulting was diminished.

Development of an ethanol model using social insects: III. Preferences for ethanol solutions.

Experiments are designed to assess whether free-flying honey bees have an aversion to an ethanol solution when given a choice between targets containing an ethanol solution in sucrose or sucrose only. Animals given a choice between a 1% ethanol solution and sucrose only show no aversion to the ethanol solution either in acquisition or extinction. Honey bees given a choice between a 5% ethanol solution and sucrose only show no differences in the initial choice of targets but some ees do switch over to the sucrose-only target. Performance during extinction indicates that bees landed on the previously reinforced sucrose-only target more than the target previously containing the 5% ethanol solution. An experiment in which bees were given a single 5%, ethanol target showed that of 20 bees, 11 returned for the entire 12 trials of the experiment. All bees returned at least 6 times to the 5% ethanol target. Additional experiments were run on harnessed foragers in a palatability study of alcoholic beverages consumed by humans. The results of the palatability experiment indicate that in general, bees prefer more sweet drinks with less alcohol.

Behavioural response in paired food choice experiments with Oniscus asellus (Crustacea, Isopoda) as an indicator of different food quality.

This paper describes a study of behavioural response in terrestrial isopod Oniscus asellus when offered two food pellets of different quality. One group had a choice of sterilised food and food pellets covered with mould. The other group had a choice of uncontaminated and cadmium-dosed food. During the behavioural test, the animals were monitored by a video camera and each visit to food pellets and time spent around it was counted. The results show that animals spent significantly less time near sterilised and cadmium-dosed food than with uncontaminated or mould-covered food. Discrimination between offered food pellets showed that avoidance behaviour can be used as an indicator of different food quality.

Variation in JH synthesis rate in mature honeybees and its possible role in reprogramming of hypopharyngeal gland function.

Juvenile hormone (JH) synthesis was higher in followers than in dancers, which suggests that higher JH levels in followers might be an internal motivational stimulus to induce them to leave me hive to search for food. The positive correlation of in vitro JH synthesis rate and α-glucosidase activity in the hypopharyngeal gland suggests that JH is involved in the reprogramming of the hypopharyngeal gland from producing larval food (immature adults) to production of α-glucosidase in mature adults.