Haplotypes of the tRNAleu-COII mtDNA Region in Russian Apis mellifera Populations.

Analysis of the mtDNA tRNAleu-COII locus is a widely used tool to establish belonging to a particular evolutionary lineage of Apis mellifera L. (lineages A, M, C, O, and Y). In Russia, most of the area was once inhabited by Apis mellifera mellifera from the M evolutionary lineage, but the introduction of bee subspecies from the southern regions of Russia (A. m. caucasica, A. m. carnica) and from abroad (A. m. carnica, A. m. ligustica) led to fragmentation of their native range. In this study, the results of assessing the haplotype number for the tRNAleu-COII locus of mtDNA in Russian Apis mellifera populations were presented. We analyzed 269 colonies from 19 regions of Russia. As a result, two evolutionary lineages were identified: the East European lineage C (26.4%) and the Northwestern European lineage M (73.6%). A total of 29 haplotypes were identified, 8 of them were already reported, and 21 were found to be novel. From the C lineage, haplotypes C1, C2, C2c, C2j, and C3 were predominant. All M lineage samples from Russia belong to the M17 and M4' haplogroups but have only minor variations in the form of nucleotide substitutions. An analysis of publications devoted to the tRNAleu-COII locus haplotypes, as well as an analysis of the available tRNAleu-COII sequences in GenBank, showed that there is still a problem with the haplotype nomenclature.

Creation of a Biobank of the Sperm of the Honey Bee Drones of Different Subspecies of Apis mellifera L.

The cryopreservation of gametes and embryos is an important element of biodiversity conservation. One species in need of conservation is the honey bee Apis mellifera L. Changing environmental factors, especially the anthropogenic factor, have led to a reduction in the numbers of this insect species. In this study, we provide an example of the creation of a biobank of honey bee drone sperm. For sperm cryopreservation, drones of the most common subspecies of honey bees common in Russia were selected. These were the dark forest bee, Apis mellifera mellifera, from the Republic of Bashkortostan, with three subspecies (A. m. carnica, A. m. carpatica, and A. m. caucasica) from the southern regions of Russia, as well as two breeding stocks, the Far Eastern bee and Prioksky bee. For subspecies identification, morphometric and genetic methods were used. The subspecies of the studied samples were confirmed via the analysis of the tRNAleu-COII locus of mitochondrial DNA and nine microsatellite markers of nuclear DNA. It was shown that bees of the Prioksky breeding stock belong to the subspecies A. m. caucasica based on phylogenetic analysis, and the Far Eastern breeding stock is a stable hybrid, descending on the maternal line from the evolutionary lineage C or O. The results of the morphometric analysis are consistent with the results of the genetic analysis. For the cryopreservation of sperm, we used a cryoprotectant solution with honey. As a result, the viability of frozen-thawed sperm decreased by 20.3% compared to fresh sperm, and overall motility decreased 25-fold. The measurement of the sperm concentration in the spermatheca of artificially inseminated queens showed that it varied from 0.22 to 4.4 million/µL. Therefore, the use of honey in sperm cryopreservation has great potential.

Genetic Structure of Apis cerana Populations from South Korea, Vietnam and the Russian Far East Based on Microsatellite and Mitochondrial DNA Polymorphism.

In this article, we present the results of the genetic analysis of Apis cerana samples from the Russian Far East, South Korea and Vietnam. An analysis of the polymorphism of seven microsatellite loci and an assessment of the haplotype diversity of the mtDNA tRNAleu-COII locus were performed. A fragment of about 431 bp in tRNAleu-COII was sequenced. The analysis showed the presence of 14 haplotypes, while the predominant haplotype was Japan1. Microsatellite data revealed two differentiated clusters. The first cluster contained tropical climate A. cerana samples from Vietnam, and the second cluster combined temperate climate A. cerana samples from the Russian Far East and South Korea.

The Current State of the Protected Apis mellifera mellifera Population in Russia: Hybridization and Nosematosis.

The Southern Urals of Russia are the habitat of one of the surviving populations of the dark forest bee-the Burzyan population of Apis mellifera mellifera. In this study, we present the results of the subspecies identification of bee colonies in the Altyn-Solok Nature Reserve in the Southern Ural Mountains using the intergenic mtDNA COI-COII locus and the assessment of the prevalence of nosematosis. Analysis of the mtDNA COI-COII intergenic locus in the studied sample showed that 30.4% of the colonies belong to the lineage C. The PCR diagnostics of nosematosis in 92 colonies selected from different sectors of the Altyn-Solok Nature Reserve showed that about half of the analyzed colonies were infected with Nosema apis. Nosema ceranae was found in eight colonies. Both of these factors can lead to the extinction of this population of the dark forest bee.

Improved Apis mellifera reference genome based on the alternative long-read-based assemblies.

Apis mellifera L., the western honey bee is a major crop pollinator that plays a key role in beekeeping and serves as an important model organism in social behavior studies. Recent efforts have improved on the quality of the honey bee reference genome and developed a chromosome-level assembly of 16 chromosomes, two of which are gapless. However, the rest suffer from 51 gaps, 160 unplaced/unlocalized scaffolds, and the lack of 2 distal telomeres. The gaps are located at the hard-to-assemble extended highly repetitive chromosomal regions that may contain functional genomic elements. Here, we use de novo re-assemblies from the most recent reference genome Amel_HAv_3.1 raw reads and other long-read-based assemblies (INRA_AMelMel_1.0, ASM1384120v1, and ASM1384124v1) of the honey bee genome to resolve 13 gaps, five unplaced/unlocalized scaffolds and, the lacking telomeres of the Amel_HAv_3.1. The total length of the resolved gaps is 848,747 bp. The accuracy of the corrected assembly was validated by mapping PacBio reads and performing gene annotation assessment. Comparative analysis suggests that the PacBio-reads-based assemblies of the honey bee genomes failed in the same highly repetitive extended regions of the chromosomes, especially on chromosome 10. To fully resolve these extended repetitive regions, further work using ultra-long Nanopore sequencing would be needed. Our updated assembly facilitates more accurate reference-guided scaffolding and marker/sequence mapping in honey bee genomics studies.