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.

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.

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.

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.