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 tRNA(leu)-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

Evaluating the potential for mating control in honey bee breeding in three SE European countries (preliminary results)

The study was conducted to explore alternatives for mating control as a part of honey bee breeding programs for genetic improvement & conservation of local honey bee (Apis mellifera) populations in Croatia (HR), Macedonia (MK) and Slovenia (SI). We observed nuptial flights of 87 virgin queens (30 in HR, 35 in MK and 22 in SI) on potential sites. Mating success was related to the presence of drone producing colonies, but even at locations chosen for their isolation from known apiaries, mating success of 70% was achieved. On average, queens performed nuptial flights on 1.8 days in HR, 1.6 to 4.3 days in MK and 1.6 to 2.4 days in SI. Unsuccessful nuptial flights were two/threefold shorter than successful flights

Standard methods for direct observation of honey bee (Apis mellifera L.) nuptial flights

Honey bees (Apis mellifera) have a peculiar and complex reproductive biology, with queens being polyandrous and mating with several drones during one or more mating (nuptial) flights in so-called drone congregation areas. Observing the virgin queens' and drones' flight behaviour provides data to understand and interpret a portion of the honey bees' complex reproductive process. Observing the behaviour of the virgin queens on the hive entrance also serves to estimate the distance from the mating place or potential drone congregation areas (DCAs) as well as to detect the presence of airborne drones in the area.In this paper, we provide a detailed description of the methodology used for observing queens' and drones' flights during the period of expected mating. In addition, we provide information about required equipment, tools as well as step by step description of the observation and recordkeeping process

Three pillars of Varroa control

The beekeeping sector is facing many challenges. One of the greatest is maintaining healthy colonies that produce high-quality products without any residues of veterinary medicines and with low environmental impact. The main enemy is the ectoparasitic mite Varroa destructor, the most significant honeybee pest and a key factor in high colony losses worldwide. In the previous four decades, three pillars of Varroa control have crystallized to be essential for sustainable management: apitechnical measures, chemical treatments, and resistant stocks of honey bees. In the long term, the latter is probably the most sustainable as it is a step to self-sustaining populations of feral and managed colonies. We recognize the significance of progress in knowledge of all three pillars to conquer Varroa and of their successful usage in accordance with local and global conditions and capabilities. In this review, we present a possible integration of the components of the three pillars of Varroa control strategies in the light of sustainable beekeeping and provide their linkage to the production of high-quality and safe honeybee products and maintaining healthy colonies.European Research Area on Sustainable Animal Production Systems (ERA-Net SusAn); European UnionEuropean Commission [696231]This work was prepared in the context of the B-PRACTICE project funding from the European Research Area on Sustainable Animal Production Systems (ERA-Net SusAn), co-funded under the European Union's Horizon 2020 Research and Innovation Programme (www.era-susan.eu) under Grant Agreement No. 696231. Funding parties on the national level are presented in the table below

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