Quantifying Abdominal Coloration of Worker Honey Bees.

The main drawback in using coloration to identify honey bee subspecies is the lack of knowledge regarding genetic background, subjectivity of coloration grading, and the effect of the environment. The aim of our study was to evaluate the effect of environmental temperature on the abdominal coloration of honey bee workers and to develop a tool for quantifying abdominal coloration. We obtained four frames of honey bee brood from two colonies and incubated them at two different temperatures (30 and 34 °C). One colony had workers exhibiting yellow marks on the abdomen, while the other did not. We collected hatched workers and photographed abdomens. Images were analyzed using custom-written R script to obtain vectors that summarize the coloration over the abdomen length in a single value-coloration index. We used UMAP to reduce the dimensions of the vectors and to develop a classification procedure with the support vector machine method. We tested the effect of brood origin and temperature on coloration index with ANOVA. UMAP did not distinguish individual abdomens according to experimental group. The trained classifier sufficiently separated abdomens incubated at different temperatures. We improved the performance by preprocessing data with UMAP. The differences among the mean coloration index values were not significant between the gray groups incubated at different temperatures nor between the yellow groups. However, the differences between the gray and yellow groups were significant, permitting options for application of our tool and the newly developed coloration index. Our results indicate that the environmental temperature in the selected range during development does not seem to impact honey bee coloration significantly. The developed color-recording protocol and statistical analysis provide useful tools for quantifying abdominal coloration in honey bees.

Integrated Pest Management Strategies to Control Varroa Mites and Their Effect on Viral Loads in Honey Bee Colonies.

Honey bee viruses in combination with varroa mite are very damaging for honey bee colonies worldwide. There are no effective methods to control the viral load in honey bee colonies except regular and effective control of mites. Integrated Pest Management strategies are required to effectively control mites with veterinary medicines based on organic compounds. We evaluated the effect of two brood interruption techniques, queen caging (QC) and trapping comb (TC), followed by an oxalic acid treatment, on the mite fall, colony strength, and viral load of Deformed Wing Virus (DWV) and Acute Bee Paralysis Virus (ABPV). In this paper, we report the data obtained in two experimental sites, in Slovenia and Italy, in terms of the varroacide efficacy, colony strength, and viral load. The number of adult bees after the adoption of the two techniques showed similar decreasing trends in both locations. The viral load of Acute Bee Paralysis Virus did not show any significant reduction after 25 days, reported as the number of Real-Time PCR cycles needed to detect the virus. The viral load of DWV also did not show a significant reduction after 25 days. The acaricidal efficacy of the applied protocols was high in both experimental groups and in both apiaries. Both the queen caging and trapping comb techniques, followed by an oxalic acid treatment, can be considered effective varroa treatment strategies, but further studies should be carried out to evaluate the long-term effects on viral loads to plan the Integrated Pest Management strategy with the right timing before wintering.

SIMplyBee: an R package to simulate honeybee populations and breeding programs.

BACKGROUND: The Western honeybee is an economically important species globally, but has been experiencing colony losses that lead to economical damage and decreased genetic variability. This situation is spurring additional interest in honeybee breeding and conservation programs. Stochastic simulators are essential tools for rapid and low-cost testing of breeding programs and methods, yet no existing simulator allows for a detailed simulation of honeybee populations. Here we describe SIMplyBee, a holistic simulator of honeybee populations and breeding programs. SIMplyBee is an R package and hence freely available for installation from CRAN http://cran.r-project.org/package=SIMplyBee . IMPLEMENTATION: SIMplyBee builds upon the stochastic simulator AlphaSimR that simulates individuals with their corresponding genomes and quantitative genetic values. To enable honeybee-specific simulations, we extended AlphaSimR by developing classes for global simulation parameters, SimParamBee, for a honeybee colony, Colony, and multiple colonies, MultiColony. We also developed functions to address major honeybee specificities: honeybee genome, haplodiploid inheritance, social organisation, complementary sex determination, polyandry, colony events, and quantitative genetics at the individual- and colony-levels. RESULTS: We describe its implementation for simulating a honeybee genome, creating a honeybee colony and its members, addressing haplodiploid inheritance and complementary sex determination, simulating colony events, creating and managing multiple colonies at the same time, and obtaining genomic data and honeybee quantitative genetics. Further documentation, available at http://www.SIMplyBee.info , provides details on these operations and describes additional operations related to genomics, quantitative genetics, and other functionalities. DISCUSSION: SIMplyBee is a holistic simulator of honeybee populations and breeding programs. It simulates individual honeybees with their genomes, colonies with colony events, and individual- and colony-level genetic and breeding values. Regarding the latter, SIMplyBee takes a user-defined function to combine individual- into colony-level values and hence allows for modeling any type of interaction within a colony. SIMplyBee provides a research platform for testing breeding and conservation strategies and their effect on future genetic gain and genetic variability. Future developments of SIMplyBee will focus on improving the simulation of honeybee genomes, optimizing the simulator's performance, and including spatial awareness in mating functions and phenotype simulation. We invite the honeybee genetics and breeding community to join us in the future development of SIMplyBee.

Honey bee (Apis mellifera) wing images: a tool for identification and conservation.

BACKGROUND: The honey bee (Apis mellifera) is an ecologically and economically important species that provides pollination services to natural and agricultural systems. The biodiversity of the honey bee in parts of its native range is endangered by migratory beekeeping and commercial breeding. In consequence, some honey bee populations that are well adapted to the local environment are threatened with extinction. A crucial step for the protection of honey bee biodiversity is reliable differentiation between native and nonnative bees. One of the methods that can be used for this is the geometric morphometrics of wings. This method is fast, is low cost, and does not require expensive equipment. Therefore, it can be easily used by both scientists and beekeepers. However, wing geometric morphometrics is challenging due to the lack of reference data that can be reliably used for comparisons between different geographic regions. FINDINGS: Here, we provide an unprecedented collection of 26,481 honey bee wing images representing 1,725 samples from 13 European countries. The wing images are accompanied by the coordinates of 19 landmarks and the geographic coordinates of the sampling locations. We present an R script that describes the workflow for analyzing the data and identifying an unknown sample. We compared the data with available reference samples for lineage and found general agreement with them. CONCLUSIONS: The extensive collection of wing images available on the Zenodo website can be used to identify the geographic origin of unknown samples and therefore assist in the monitoring and conservation of honey bee biodiversity in Europe.

Cutting corners: The impact of storage and DNA extraction on quality and quantity of DNA in honeybee (Apis mellifera) spermatheca.

The purpose of our study was to investigate methods of short-term storage that allow preservation, transport and retrieval of genetic information contained in honeybee queen's spermatheca. Genotyping of the honeybee colony requires well ahead planned sample collection, depending on the type of data to be acquired. Sampling and genotyping of spermatheca's content instead of individual offspring is timesaving, allowing answers to the questions related to patriline composition immediately after mating. Such procedure is also cheaper and less error prone. For preservation either Allprotect Tissue Reagent (Qiagen) or absolute ethanol were used. Conditions during transportation were simulated by keeping samples 6-8 days at room temperature. Six different storing conditions of spermathecas were tested, complemented with two DNA extraction methods. We have analysed the concentration of DNA, RNA, and proteins in DNA extracts. We also analysed how strongly the DNA is subjected to fragmentation (through amplification of genetic markers ANT2 and tRNAleu-COX2) and whether the quality of the extracted DNA is suitable for microsatellite (MS) analysis. Then, we tested the usage of spermatheca as a source of patriline composition in an experiment with three instrumentally inseminated virgin queens and performed MS analysis of the extracted DNA from each spermatheca, as well as queens' and drones' tissue. Our results show that median DNA concentration from spermathecas excised prior the storage, regardless of the storing condition and DNA extraction method, were generally lower than median DNA concentration obtained from spermathecas dissected from the whole queens after the storage. Despite the differences in DNA yield from the samples subjected to different storing conditions there was no significant effect of storage method or the DNA extraction method on the amplification success, although fewer samples stored in EtOH amplified successfully in comparison to ATR storing reagent. However, we recommend EtOH as a storing reagent due to its availability, low price, simplicity in usage in the field and in the laboratory, and capability of good preservation of the samples for DNA analysis during transport at room temperature.

The Carniolan Honeybee from Slovenia-A Complete and Annotated Mitochondrial Genome with Comparisons to Closely Related Apis mellifera Subspecies.

The complete mitochondrial genome of the Carniolan honeybee (Apis mellifera carnica) from Slovenia, a homeland of this subspecies, was acquired in two contigs from WGS data and annotated. The newly obtained mitochondrial genome is a circular closed loop of 16,447 bp. It comprises 37 genes (13 protein coding genes, 22 tRNA genes, and 2 rRNA genes) and an AT-rich control region. The order of the tRNA genes resembles the order characteristic of A. mellifera. The mitogenomic sequence of A. m. carnica from Slovenia contains 44 uniquely coded sites in comparison to the closely related subspecies A. m. ligustica and to A. m. carnica from Austria. Furthermore, 24 differences were recognised in comparison between A. m. carnica and A. m. ligustica subspecies. Among them, there are three SNPs that affect translation in the nd2, nd4, and cox2 genes, respectively. The phylogenetic placement of A. m. carnica from Slovenia within C lineage deviates from the expected position and changes the perspective on relationship between C and O lineages. The results of this study represent a valuable addition to the information available in the phylogenomic studies of A. mellifera-a pollinator species of worldwide importance. Such genomic information is essential for this local subspecies' conservation and preservation as well as its breeding and selection.

Non-Destructive Genotyping of Honeybee Queens to Support Selection and Breeding.

In traditional bee breeding, the honeybee queen is chosen for breeding based on the performance of the colony produced by its mother. However, we cannot be entirely certain that a specific queen will produce offspring with desirable traits until we observe the young queen's new colony. Collecting the queen's genetic material enables quick and reliable determination of the relevant information. We sampled exuviae, feces, and wingtips for DNA extraction to avoid fatally injuring the queen when using tissue samples. Quantity and purity of extracted DNA were measured. Two mitochondrial markers were used to determine the lineage affiliation and exclude possible contamination of DNA extracts with non-honeybee DNA. dCAPS (derived Cleaved Amplified Polymorphic Sequences) markers allowed detection of single nucleotide polymorphisms (SNPs) in nuclear DNA regions presumably associated with Varroa sensitive hygiene and set the example of successful development of genotyping protocol from non-destructive DNA sources. One of the logical future steps in honeybee breeding is introducing genomic selection and non-destructive sampling methods of genetic material may be the prerequisite for successful genotyping. Our results demonstrate that the extraction of DNA from feces and exuviae can be introduced into practice. The advantage of these two sources over wingtips is reducing the time window for processing the samples, thus enabling genotyping directly after the queen's emergence.

Neurostatistical approach to toxicological testing in honeybees.

We present method that detects changes in mortality as a consequence of application of a chemical/pharmacological agent. Often is the evaluation of consequential mortality impaired by natural mortality, especially in organisms with high turn of generations, like honeybees. Investigators in the field of olfactory neurophysiology are faced with similar situation in which olfactory receptor neuron is spontaneously active. This activity makes it difficult to evaluate the onset or the offset of the response to a stimulus. The investigators have bridged this issue by developing the detection of response via change of slope in cumulative firing rate of the olfactory receptor neuron. We have adjusted this method to the requirements of toxicological test and used in the analysis lithium toxicity. The method has several steps in which the absolute mortality counts are converted in cumulative sum of dead bees, followed with a normalization of cumulative sum to the last sampling point before the application of the lithium. The changes of the normalized curve from time point to time point are calculated as slope, which is then statistically compared between groups. The method was validated on unrelated dataset, giving an estimation of the duration of agents' effect.• Contain between 1 and 3 bullet points highlighting the customization rather than the steps of the procedure.○ The method enables to evaluate the toxicological effect of the tested substance by overcoming the issues related to natural mortality and difference between tested biological systems, here specifically demonstrating its utility and effectiveness in evaluating the impact of lithium on honeybee colonies mortality rate.○ Simple set up of conditions for toxicological testing in honeybee colonies is described.○ Shorten the window to exclude the history since there is no refractory period is known within normal mortality rate.

Effects on Some Therapeutical, Biochemical, and Immunological Parameters of Honey Bee (Apis mellifera) Exposed to Probiotic Treatments, in Field and Laboratory Conditions.

Several negative factors contribute to a decline in the number of insect pollinators. As a novel approach in therapy, we hypothesize that the EM® for bees could potentially have an important therapeutic and immunomodulatory effect on honey bee colonies. The aim of our study was to evaluate its impact on honey bees at the individual and colony level. This is the first appliance of the commercial probiotic mix EM® PROBIOTIC FOR BEES in honey bees as economically important social insects. The sugar syrup with 10% of probiotic was administered by spraying or feeding the honey bee colonies in the field conditions, in order to evaluate the infection levels with spores of Nosema spp. and colonies' strength. Moreover, in laboratory-controlled conditions, in the hoarding cages, adult workers have been fed with sugar syrup supplemented with 2.5, 5, and 10% of EM® for bees for biochemical and immunological analyses of hemolymph, and with 5 and 10% for measuring the size of hypopharyngeal glands. It was found that following the EM® for bees administration the Nosema spp. spore counts in colonies were significantly reduced, and colonies' strength was increased. The results at the individual level showed significant positive physiological changes in treated groups of adult bees, revealing at the same time a higher mortality rate when feeding sugar syrup supplemented with the probiotic.

Lithium contamination of honeybee products and its accumulation in brood as a consequence of anti-varroa treatment.

Varroa destructor, the primary honeybee pathogen, is kept in check by various chemical compounds which may enter the human diet through honeybee products. Lithium is an emerging varroa control substance, and we investigated its accumulation in honey, bee bread, brood and adults along with the mortality of bees. Increased lithium concentrations were detected in workers, fed individually once per os with 10 µL of 25 mM LiCl in sucrose solution (6.50-40.10 mg/kg) or had the same solution available ad libitum (39.25-266.00 mg/kg). A three-day treatment of honeybee colonies with 25 mM LiCl in 1L/day sucrose solution increased lithium concentrations in five-day-old larvae, honey, and bee bread: up to 45.0, 1.2, and 47.0 mg/kg, respectively. Lithium concentrations peaked three days post-treatment in both larvae and honey and increased worker mortality was observed. The control colonies exhibited lithium concentrations below the limit of quantification (0.5 mg/kg). Prudence in lithium use is advised.

On the spot: utilization of directional cues in vibrational communication of a stink bug.

Although vibrational signalling is among the most ancient and common forms of communication, many fundamental aspects of this communication channel are still poorly understood. Here, we studied mechanisms underlying orientation towards the source of vibrational signals in the stink bug Nezara viridula (Hemiptera, Pentatomidae), where female vibrational song enables male to locate her on the bean plant. At the junction between the main stem and the leaf stalks, male placed his legs on different sides of the branching and orientation at the branching point was not random. Analyses of signal transmission revealed that only a time delay between the arrival of vibrational wave to receptors located in the legs stretched across the branching was a reliable directional cue underlying orientation, since, unexpectedly, the signal amplitude at the branching point was often higher on the stalk away from the female. The plant and the position of the vibrational source on the plant were the most important factors influencing the unpredictability of the amplitude cue. Determined time delays as short as 0.5 ms resulted in marked changes in interneuron activity and the decision model suggests that the behavioural threshold is in the range between 0.3 and 0.5 ms.

A single mechanism driving both inactivation and adaptation in rapidly adapting currents of DRG neurons?

Rapidly adapting (RA) currents expressed in dorsal root ganglia somatosensory neurons reduce their amplitude in response to prolonged and/or repeated mechanical stimulation. Both inactivation of mechanotransducer channels and adaptation of the force acting on the channels have been suggested to independently decrease RA currents. However, these two mechanisms have similar kinetics and dependence on calcium and voltage. These experimental findings suggest that a single mechanism might underlie both. We constructed a simple Hodgkin-Huxley-type model with a single gating variable driving both inactivation and adaptation to test this hypothesis. Predictions of the model successfully describe key features of mechanical activation as well as inactivation, adaptation, and recovery. The model thus supports the possibility of a single mechanism driving inactivation and adaptation in RA currents. On its own, the model can be integrated into higher-order models of touch receptors because of its accurate simulation of RA currents.

Dynamic dendritic compartmentalization underlies stimulus-specific adaptation in an insect neuron.

In many neural systems, repeated stimulation leads to stimulus-specific adaptation (SSA), with responses to repeated signals being reduced while responses to novel stimuli remain unaffected. The underlying mechanisms of SSA remain mostly hypothetical. One hypothesis is that dendritic processes generate SSA. Evidence for such a mechanism was recently described in an insect auditory interneuron (TN-1 in Neoconocephalus triops). Afferents, tuned to different frequencies, connect with different parts of the TN-1 dendrite. The specific adaptation of these inputs relies on calcium and sodium accumulation within the dendrite, with calcium having a transient and sodium a tonic effect. Using imaging techniques, we tested here whether the accumulation of these ions remained limited to the stimulated parts of the dendrite. Stimulation with a fast pulse rate, which results in strong adaptation, elicited a transient dendritic calcium signal. In contrast, the sodium signal was tonic, remaining high during the fast pulse rate stimulus. These time courses followed the predictions from the previous pharmacological experiments. The peak positions of the calcium and sodium signals differed with the carrier frequency of the stimulus; at 15 kHz, peak locations were significantly more rostral than at 40 kHz. This matched the predictions made from neuroanatomical data. Our findings confirm that excitatory postsynaptic potentials rather than spiking cause the increase of dendritic calcium and sodium concentrations and that these increases remain limited to the stimulated parts of the dendrite. This supports the hypothesis of "dynamic dendritic compartmentalization" underlying SSA in this auditory interneuron.

A general odorant background affects the coding of pheromone stimulus intermittency in specialist olfactory receptor neurones.

In nature the aerial trace of pheromone used by male moths to find a female appears as a train of discontinuous pulses separated by gaps among a complex odorant background constituted of plant volatiles. We investigated the effect of such background odor on behavior and coding of temporal parameters of pheromone pulse trains in the pheromone olfactory receptor neurons of Spodoptera littoralis. Effects of linalool background were tested by measuring walking behavior towards a source of pheromone. While velocity and orientation index did drop when linalool was turned on, both parameters recovered back to pre-background values after 40 s with linalool still present. Photo-ionization detector was used to characterize pulse delivery by our stimulator. The photo-ionization detector signal reached 71% of maximum amplitude at 50 ms pulses and followed the stimulus period at repetition rates up to 10 pulses/s. However, at high pulse rates the concentration of the odorant did not return to base level during inter-pulse intervals. Linalool decreased the intensity and shortened the response of receptor neurons to pulses. High contrast (>10 dB) in firing rate between pulses and inter-pulse intervals was observed for 1 and 4 pulses/s, both with and without background. Significantly more neurons followed the 4 pulses/s pattern when delivered over linalool; at the same time the information content was preserved almost to the control values. Rapid recovery of behavior shows that change of perceived intensity is more important than absolute stimulus intensity. While decreasing the response intensity, background odor preserved the temporal parameters of the specific signal.

Reproductive strategy of the Nearctic leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae).

Mating behaviour of Scaphoideus titanus Ball, the vector of the grapevine disease Flavescence dorée, was investigated in order to determine the role of substrate-borne vibrational signals in intra-specific communication and pair formation. Vibrational signals were recorded from grapevine leaves with a laser vibrometer. Signalling activity of single males changed throughout the day and the peak in activity was associated with twilight and early night when 'call and fly' behaviour was observed. Pair formation began with the spontaneous emission of male signals. The male calling signal consisted of a single series of pulses, partially accompanied with a 'rumble'. The male courtship phrase consisted of four consecutive sections characterized by two sound elements, pulse and 'buzz'. Female vibrational signals were emitted only in response to male signals. The female response was a single pulse that closely resembled male pulses and was inserted between pulses within the male signals. All recorded vibrational signals of S. titanus have a dominant frequency below 900 Hz. A unique feature of vibrational communication in S. titanus is well-developed intrasexual competition; males may use alternative tactics, in the form of disturbance signals, or silently approach duetting females (satellite behaviour). While the male-female duet appears to be essential for successful localization of females and copulation, it is also vulnerable to, and easily disrupted by, alternative tactics like masking.

Morphology and physiology of vibratory interneurons in the thoracic ganglia of the southern green stinkbug Nezara viridula (L.).

The central processing mechanisms of vibratory signals in small plant-dwelling insects that rely primarily on substrate-borne vibratory communication are still largely unknown. To elucidate the neural mechanisms involved in vibratory signaling, the vibration-sensitive interneurons in thoracic ganglia of the southern green stinkbug, Nezara viridula, were investigated electrophysiologically by single-cell recordings and staining. Ten types of interneurons were described and divided into four categories, based on their gross morphology. The cell body of the L-shaped CG-AC neurons is located in the metathoracic neuromere of the central ganglion, and the axon ascends contralaterally. This group comprises five types of neurons differing in their fine structure and functional properties. CG-AB neurons are dorsal unpaired median (DUM) neurons with cell bodies in the mesothoracic neuromere of the central ganglion and two axons that ascend bilaterally into the prothoracic ganglion. Group CG-L includes three types of local neurons limited to the central ganglion. With ipsilateral dendritic arborizations and contralateral axonal branching, their gross morphology is similar to that of cricket omega cells. Interneuron PTG-DC, with the cell body in the prothoracic ganglion (PTG) and a contralaterally descending axon, conveys information received by the sensory organs of the front contralateral leg to the neuropil regions of the ipsilateral middle and hind legs. Based on their frequency tuning and acceleration sensitivity, the vibratory interneurons fall into two groups: the low-frequency units are tuned to 50 Hz and the middle frequency units to 200 Hz, with their acceleration thresholds at 10(-1) m/s(2) and 5 x 10(-3) m/s(2), respectively. Their function is discussed with relevance to the vibratory communication of N. viridula.