Honey Bee Subspecies Research Articles

Electron microscope study on workers antennae and sting lancets of three subspecies of honey bee Apis mellifera L. (Hymenoptera: Apidae) and its bearing on their phylogeny

Ultrastructure of antennae and sting of the honey bee workers in three subspecies of Apis mellifera; Egyptian A. m. lamarckii, Carniolan A. m. carnica and Italian A. m. ligustica was investigated. Twelve types of sensilla were recorded on the worker antennae; placodea, basiconica, trichodea types (A, B, C and D), ampullaceal, coeloconica, coelococapitular, non-innervated hair seta, campaniformia and sensilla chaetica. The latter is recorded for the first time on antennae of A. mellifera. The number and distribution of the various types of sensilla are recorded. Sensilla trichodea were found most frequently and were present on all the antennal segments. Sensilla placodea were present on the distal eight flagellomeres. Sensilla ampullaceal and sensilla coeloconica were present on the distal five while sensilla campaniformia were confined to the distal seven flagellomeres. The barbs carried on the lancets of sting differ in numbers among the workers of the three subspecies; 10, 9 and 7 in A. m. lamarckii, A. m. carnica and A. m. ligustica, respectively. Depending on morphometrical analysis, a dendrogram separated the Egyptian subspecies from the other two subspecies with a highly genetic distance of 0.25 between them. The Italian and the Carniolan subspecies were grouped into one main cluster with a genetic distance of 0.01 between them.

Revisiting the Iberian honey bee (Apis mellifera iberiensis) contact zone: maternal and genome-wide nuclear variations provide support for secondary contact from historical refugia.

Dissecting diversity patterns of organisms endemic to Iberia has been truly challenging for a variety of taxa, and the Iberian honey bee is no exception. Surveys of genetic variation in the Iberian honey bee are among the most extensive for any honey bee subspecies. From these, differential and complex patterns of diversity have emerged, which have yet to be fully resolved. Here, we used a genome-wide data set of 309 neutrally tested single nucleotide polymorphisms (SNPs), scattered across the 16 honey bee chromosomes, which were genotyped in 711 haploid males. These SNPs were analysed along with an intergenic locus of the mtDNA, to reveal historical patterns of population structure across the entire range of the Iberian honey bee. Overall, patterns of population structure inferred from nuclear loci by multiple clustering approaches and geographic cline analysis were consistent with two major clusters forming a well-defined cline that bisects Iberia along a northeastern-southwestern axis, a pattern that remarkably parallels that of the mtDNA. While a mechanism of primary intergradation or isolation by distance could explain the observed clinal variation, our results are more consistent with an alternative model of secondary contact between divergent populations previously isolated in glacial refugia, as proposed for a growing list of other Iberian taxa. Despite current intense honey bee management, human-mediated processes have seemingly played a minor role in shaping Iberian honey bee genetic structure. This study highlights the complexity of the Iberian honey bee patterns and reinforces the importance of Iberia as a reservoir of Apis mellifera diversity.

Grooming behavior by worker bees of various subspecies of honey bees to remove Varroa destructor mites

Grooming behavior by honey bees is regarded as an important trait that contributes to the defense against the parasitic mite Varroa destructor. We carried out an assay to investigate how effective autogrooming by honey bees is in removing V. destructor mites. We compared the behavior of the Carniolan bee (Apis mellifera carnica), represented by two lines: Kortówka and Dobra; the Caucasian bee (Apis mellifera caucasica) of the Woźnica line; the black bee (Apis mellifera mellifera) of the Augustowska line; and the crossbreed of two subspecies: Apis mellifera capensis × A. m. carnica. We found that most worker bees do not tolerate the mite on their bodies. The most intense reaction, manifested by strong motor activity, was observed in A. m. mellifera worker bees; as many as 98% of worker bees in this group made an attempt to remove mites. In the remaining groups, a response was observed in 86% for Caucasian bees to 89.3% for Carniolan bees, Dobra line. The attempts made by bees of different origins to remove mites differed in effectiveness, duration, and frequency. The highest percentage of removed parasites was noted in Caucasian bees (11%), and the lowest in Carniolan bees of the Kortówka line (1.5%). A. m. mellifera devoted the most time to the reaction to varroa on their body, i.e., an average of 64.7 s of 180 s of the experiment. In most trials, worker bees manifested at least three defensive reactions, and the highest number of such trials was observed in the black bee (117). We did not observe allogrooming in all bee groups or types. The results indicate that A. m. mellifera shows the strongest grooming behavior of all bees used in the experiment.

Next generation sequencing of Apis mellifera syriaca identifies genes for Varroa resistance and beneficial bee keeping traits.

Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons typical of the region. However, persistent honey bee imports of commercial breeder lines are endangering local honey bee population. This study reports the use of next-generation sequencing (NGS) technology to study the A. m. syriaca genome and to identify genetic factors possibly contributing toward mite resistance and other favorable traits. We obtained a total of 46.2 million raw reads by applying the NGS to sequence A. m. syriaca and used extensive bioinformatics approach to identify several candidate genes for Varroa mite resistance, behavioral and immune responses characteristic for these bees. As a part of characterizing the functional regulation of molecular genetic pathway, we have mapped the pathway genes potentially involved using information from Drosophila melanogaster and present possible functional changes implicated in responses to Varroa destructor mite infestation toward this. We performed in-depth functional annotation methods to identify ∼600 candidates that are relevant, genes involved in pathways such as microbial recognition and phagocytosis, peptidoglycan recognition protein family, Gram negative binding protein family, phagocytosis receptors, serpins, Toll signaling pathway, Imd pathway, Tnf, JAK-STAT and MAPK pathway, heamatopioesis and cellular response pathways, antiviral, RNAi pathway, stress factors, etc. were selected. Finally, we have cataloged function-specific polymorphisms between A. mellifera and A. m. syriaca that could give better understanding of varroa mite resistance mechanisms and assist in breeding. We have identified immune related embryonic development (Cactus, Relish, dorsal, Ank2, baz), Varroa hygiene (NorpA2, Zasp, LanA, gasp, impl3) and Varroa resistance (Pug, pcmt, elk, elf3-s10, Dscam2, Dhc64C, gro, futsch) functional variations genes between A. mellifera and A. m. syriaca that could be used to develop an effective molecular tool for bee conservation and breeding programs to improve locally adapted strains such as syriaca and utilize their advantageous traits for the benefit of apiculture industry.

Reduced SNP panels for genetic identification and introgression analysis in the dark honey bee (Apis mellifera mellifera).

Beekeeping activities, especially queen trading, have shaped the distribution of honey bee (Apis mellifera) subspecies in Europe, and have resulted in extensive introductions of two eastern European C-lineage subspecies (A. m. ligustica and A. m. carnica) into the native range of the M-lineage A. m. mellifera subspecies in Western Europe. As a consequence, replacement and gene flow between native and commercial populations have occurred at varying levels across western European populations. Genetic identification and introgression analysis using molecular markers is an important tool for management and conservation of honey bee subspecies. Previous studies have monitored introgression by using microsatellite, PCR-RFLP markers and most recently, high density assays using single nucleotide polymorphism (SNP) markers. While the latter are almost prohibitively expensive, the information gained to date can be exploited to create a reduced panel containing the most ancestry-informative markers (AIMs) for those purposes with very little loss of information. The objective of this study was to design reduced panels of AIMs to verify the origin of A. m. mellifera individuals and to provide accurate estimates of the level of C-lineage introgression into their genome. The discriminant power of the SNPs using a variety of metrics and approaches including the Weir & Cockerham’s FST, an FST-based outlier test, Delta, informativeness (In), and PCA was evaluated. This study shows that reduced AIMs panels assign individuals to the correct origin and calculates the admixture level with a high degree of accuracy. These panels provide an essential tool in Europe for genetic stock identification and estimation of admixture levels which can assist management strategies and monitor honey bee conservation programs.

Metatranscriptomic analyses of honey bee colonies.

Honey bees face numerous biotic threats from viruses to bacteria, fungi, protists, and mites. Here we describe a thorough analysis of microbes harbored by worker honey bees collected from field colonies in geographically distinct regions of Turkey. Turkey is one of the World's most important centers of apiculture, harboring five subspecies of Apis mellifera L., approximately 20% of the honey bee subspecies in the world. We use deep ILLUMINA-based RNA sequencing to capture RNA species for the honey bee and a sampling of all non-endogenous species carried by bees. After trimming and mapping these reads to the honey bee genome, approximately 10% of the sequences (9–10 million reads per library) remained. These were then mapped to a curated set of public sequences containing ca. Sixty megabase-pairs of sequence representing known microbial species associated with honey bees. Levels of key honey bee pathogens were confirmed using quantitative PCR screens. We contrast microbial matches across different sites in Turkey, showing new country recordings of Lake Sinai virus, two Spiroplasma bacterium species, symbionts Candidatus Schmidhempelia bombi, Frischella perrara, Snodgrassella alvi, Gilliamella apicola, Lactobacillus spp.), neogregarines, and a trypanosome species. By using metagenomic analysis, this study also reveals deep molecular evidence for the presence of bacterial pathogens (Melissococcus plutonius, Paenibacillus larvae), Varroa destructor-1 virus, Sacbrood virus, and fungi. Despite this effort we did not detect KBV, SBPV, Tobacco ringspot virus, VdMLV (Varroa Macula like virus), Acarapis spp., Tropilaeleps spp. and Apocephalus (phorid fly). We discuss possible impacts of management practices and honey bee subspecies on microbial retinues. The described workflow and curated microbial database will be generally useful for microbial surveys of healthy and declining honey bees.

Draft genome sequence of the Algerian bee Apis mellifera intermissa.

Apis mellifera intermissa is the native honeybee subspecies of Algeria. A. m. intermissa occurs in Tunisia, Algeria and Morocco, between the Atlas and the Mediterranean and Atlantic coasts. This bee is very important due to its high ability to adapt to great variations in climatic conditions and due to its preferable cleaning behavior. Here we report the draft genome sequence of this honey bee, its Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JSUV00000000. The 240-Mb genome is being annotated and analyzed. Comparison with the genome of other Apis mellifera sub-species promises to yield insights into the evolution of adaptations to high temperature and resistance to Varroa parasite infestation.

New SNP markers of the honeybee vitellogenin gene (Vg) used for identification of subspecies Apis mellifera mellifera L.

Preservation of the gene pool of honeybee subspecies Apis mellifera mellifera is of vital importance for successful beekeeping development in the northern regions of Eurasia. An effective method of genotyping honeybee colonies used in modern science is the mapping of sites of single nucleotide polymorphism (SNP). The honeybee vitellogenin gene (Vg) encodes a protein that affects reproductive function, behavior, immunity, longevity, and social organization in the honeybee Apis mellifera and is therefore a topical research subject. The results of comparative analysis of honeybee Vg sequences show that there are 26 SNP sites that differentiate M and C evolutionary branches and can be used as markers in selective breeding, DNA-barcoding, and the creation of genetic passports for A. m. mellifera colonies.

Mandibular gland pheromone contents in workers and queens of Apis mellifera adansonii

Secretions from the mandibular glands of honeybees have been studied extensively, with those of queens dominated by ώ-9 fatty acids and ώ-10 fatty acids dominating those of non-laying workers. Apis mellifera adansonii (Latreille) is one of the widely distributed subspecies of African honeybees. However, its mandibular gland pheromones have not been analysed previously. Using gas chromatography, we analysed the composition of mandibular gland pheromones in workers and queens of A. mellifera adansonii from Nigeria. Qualitatively, workers and queens have similar pheromone profiles to those previously reported in other African subspecies of honeybees. We found 9-ODA and high amounts of its precursor 9-hydroxy-2 (E)-decenoic acid (9-HDA) in workers, thus showing that they produce queen-like signals under queen-right conditions. We also found geographic variation in the pheromone profiles and morphometric characters of these workers, suggesting different pheromone and morphoclusters from the different ecological and climatological regions inhabited by A. m. adansonii in Nigeria.

An Insightful Molecular Analysis Reveals Foreign Honeybees Among Algerian Honeybee Populations (Apis mellifera L.)

This study assessed the genetic diversity of honeybees (Apis mellifera) in Algeria, in North Africa, using the molecular marker mtDNA COI-COII (Cytochrome Oxidase I and II). In total, five hundred eighty-two honeybee workers were sampled from 22 regions of the country. A PCR-RFLP (Polymerase Chain Reaction Restriction Fragment Length Polymorphism) analysis of the mtDNA samples distinguished the honeybee evolutionary lineages and mtDNA haplotypes from each region. Our data revealed the presence of three different honeybee lineages among the studied populations, comprising the African (A), North Mediterranean (C) and West Mediterranean (M) lineages. Eight different mtDNA haplotypes were recorded at various frequencies (A1, A2, A8, A9, A10, A13, C7 and M4). For the first time, our results identified a low genetic introgression (3.1%) of non-local mtDNA haplotypes (C7 and M4) among the local Algerian honeybees, most likely due to the import of foreign honeybees. Notably, the southern Algerian honeybee populations had lower haplotype diversity than the northern populations. Overall, the local North African honeybee subspecies A. m. intermissa and/or A. m.sahariensis seem to be remarkably dominant across northern Algeria.

MtDNA COI-COII marker and drone congregation area: an efficient method to establish and monitor honeybee (Apis mellifera L.) conservation centres.

Honeybee subspecies have been affected by human activities in Europe over the past few decades. One such example is the importation of nonlocal subspecies of bees which has had an adverse impact on the geographical repartition and subsequently on the genetic diversity of the black honeybee Apis mellifera mellifera. To restore the original diversity of this local honeybee subspecies, different conservation centres were set up in Europe. In this study, we established a black honeybee conservation centre Conservatoire de l'Abeille Noire d'Ile de France (CANIF) in the region of Ile-de-France, France. CANIF's honeybee colonies were intensively studied over a 3-year period. This study included a drone congregation area (DCA) located in the conservation centre. MtDNA COI-COII marker was used to evaluate the genetic diversity of CANIF's honeybee populations and the drones found and collected from the DCA. The same marker (mtDNA) was used to estimate the interactions and the haplotype frequency between CANIF's honeybee populations and 10 surrounding honeybee apiaries located outside of the CANIF. Our results indicate that the colonies of the conservation centre and the drones of the DCA show similar stable profiles compared to the surrounding populations with lower level of introgression. The mtDNA marker used on both DCA and colonies of the conservation centre seems to be an efficient approach to monitor and maintain the genetic diversity of the protected honeybee populations.

Genetic assessment of Algerian honeybee populations by microsatellite markers

The genetic diversity and structure of 414 honeybee workers from eight different populations in Algeria were analyzed using 14 polymorphic DNA microsatellite loci. The results showed that the honeybee populations were characterized by substantial genetic variation in terms of the average number of alleles and the degree of heterozygosity. Most populations were at Hardy–Weinberg equilibrium. Phylogenetic and population structure analyses confirmed the African origin of the studied Algerian populations and clustered them in a group distinct from evolutionary lineages West Mediterranean (M), North Mediterranean (C), and Oriental (O). Structure analyses revealed weak allelic introgression from both lineages M and C. High genetic variability was found within the Algerian populations. Two honeybee subspecies, Apis mellifera intermissa and Apis mellifera sahariensis, were present. However, to delimit the natural spread area of A. mellifera sahariensis, more samples from southern Algerian are needed.

Estimating introgression in Apis mellifera siciliana populations: are the conservation islands really effective?

Abstract Apis mellifera siciliana is a honey bee subspecies prone to be conserved. Conservation measures include maintaining colonies on different islands to serve as parental lines for outcrossing. Molecular diversity and genetic structure of honey bees from Sicily (main island) and Vulcano and Filicudi (conservation islands) were analysed with mitochondrial and microsatellite markers to assess the efficacy of the ongoing conservation effort. Samples from continental Italy and Sardinia island were included for comparison. All the samples of the conservation islands were included in the African (A) evolutionary lineage, while western and northern (M) and eastern (C) European evolutionary lineages were also detected in colonies from the main island (17.1% and 22% respectively), thus highlighting introgression at the mitochondrial level. Nuclear data from conservation and main island populations were compared to evaluate the proportion of admixed honey bees. A higher level of hybridization was found in the central and eastern areas of Sicily main island. Our data describe the efficacy of the conservation islands, and suggest where and how to manage mating stations to further improve the present conservation strategy by recovering additional residual genetic variability of A. m. siciliana. We also propose the establishment of a European network of conservation islands to preserve honey bee subspecies and to implement research studies of adaptation to peculiar climatic conditions.

Genetic reincarnation of workers as queens in the Eastern honeybee Apis cerana.

Thelytokous parthenogenesis, or the asexual production of female offspring, is rare in the animal kingdom, but relatively common in social Hymenoptera. However, in honeybees, it is only known to be ubiquitous in one subspecies of Apis mellifera, the Cape honeybee, A. mellifera capensis. Here we report the appearance of queen cells in two colonies of the Eastern honeybee Apis cerana that no longer contained a queen or queen-produced brood to rear queens from. A combination of microsatellite genotyping and the timing of the appearance of these individuals excluded the possibility that they had been laid by the original queen. Based on the genotypes of these individuals, thelytokous production by natal workers is the most parsimonious explanation for their existence. Thus, we present the first example of thelytoky in a honeybee outside A. mellifera. We discuss the evolutionary and ecological consequences of thelytoky in A. cerana, in particular the role thelytoky may play in the recent invasions by populations of this species.

Morphometric characterisation of the native Honeybee, Apis mellifera Linnaeus, 1758, of Saudi Arabia

The morphometry of native honeybees from Saudi Arabia was analysed and compared with 7 Apis mellifera subspecies, based on 198 colony samples from 36 locations. Twenty five standard morphological characters were evaluated, and samples were compared with seven reference honeybee subspecies (Apis mellifera carnica, A. m. ligustica, A. m. meda, A. m. syriaca, A. m. lamarckii, A. m. litorea and A. m. jemenitica) obtained from the Oberursel Data Bank (Institut für Bienenkunde, Frankfurt University, Germany). Results confirmed that samples from Saudi Arabia are very similar to samples from the subspecies A. m. jemenitica (Ruttner, 1967), previously described from Oman, Yemen and Saudi Arabia. Samples were well-separated from the other subspecies, but the distinction was less in relation to A. m. litorea. While locally kept bees were well-separated, samples from migratory beekeeping showed broader variation and were less clearly separated, indicating the influence of ingression and hybridization with introduced honeybee subspecies.

Wing geometric morphometrics and microsatellite analysis provide similar discrimination of honey bee subspecies

Identification of honey bee (Apis mellifera) subspecies is important for their protection. It is also used by queen breeders to maintain some breeding lines. In this study, we compared three methods of subspecies identification based on the following: 17 microsatellite loci, COI-COII mitotypes and geometric morphometrics of forewing venation. The methods were used to classify colonies and workers from a mixed population of A. m. mellifera and A. m. carnica. There was highly significant correlation between results obtained using the three methods. More than three quarters of colonies were classified to the same subspecies by all three methods. The agreement was highest between microsatellites and morphometrics. More than 90 % of colonies were classified to the same subspecies by the two methods. There was also relatively high agreement (75 %) between microsatellites and morphometrics when workers were classified as pure subspecies or hybrids. In particular, one pure subspecies was never misclassified as other pure subspecies. The results presented here show that morphometrics can be used for detection of hybrids between A. m. mellifera and A. m. carnica.

Climate rather than geography separates two European honeybee subspecies

Both climatic and geographical factors play an important role for the biogeographical distribution of species. The Carpathian mountain ridge has been suggested as a natural geographical divide between the two honeybee subspecies Apis mellifera carnica and A. m. macedonica. We sampled one worker from one colony each at 138 traditional apiaries located across the Carpathians spanning from the Hungarian plains to the Danube delta. All samples were sequenced at the mitochondrial tRNA(Leu)-cox2 intergenic region and genotyped at twelve microsatellite loci. The Carpathians had only limited impact on the biogeography because both subspecies were abundant on either side of the mountain ridge. In contrast, subspecies differentiation strongly correlated with the various temperature zones in Romania. A. m. carnica is more abundant in regions with the mean average temperature below 9 °C, whereas A. m. macedonica honeybees are more frequent in regions with mean temperatures above 9 °C. This range selection may have impact on the future biogeography in the light of anticipated global climatic changes.

Cheating workers with large activated ovaries avoid risky foraging

Eusocial insects undoubtedly evolved from solitary ancestors, but how this occurred is not well established. The Ground Plan hypothesis suggests that gene networks that once regulated the oviposition and foraging phases of an ancestral solitary insect’s life cycle have been co-opted to establish the queen–worker dimorphism in extant eusocial insects; queens permanently express genes that were once expressed during the oviposition phase, whereas workers express genes that were once associated with foraging. An extension of the Ground Plan hypothesis, the Reproductive Ground Plan-forager hypothesis, proposes that foraging specialization by worker honey bees for either pollen or nectar is controlled by the same reproductive gene networks. According to the Reproductive Ground Plan-forager hypothesis, workers with more ovarioles forage early in life and specialize in pollen collection. Here we find that among workers of a highly reproductive honey bee subspecies, Apis mellifera capensis, there is a positive correlation between ovariole number and age at onset of foraging, and no association between ovariole number and foraging preference, thus contradicting key aspects of the Reproductive Ground Plan-forager hypothesis. We also find a negative association between ovariole number and ovary activation, suggesting that high ovariole number is not directly related to reproductive potential as previously assumed.

Study the correlation of morphological and production traits of honey bee (Apis mellifera L.) (Review

There are several morphological differences among the honey bee subspecies, which proofs that they adapted different way to the environmental factors of the given area. Morphological observations are not the only way to separate subspecies, it is possible on their genetic speciality as well. The authors’s aim in this present study was to examine morphological characteristics of subspecies, furthermore looking for correlation between morphological parameters and honey yield. According to their results, among the most important parameters considered by Hungarian breeders, in the case of the native Apismelliferacarnica subspecies, the proboscis length could be the indirect tool for honey-productivity focused selection.

A parent-of-origin effect on honeybee worker ovary size

Apis mellifera capensis is unique among honeybees in that unmated workers can produce pseudo-clonal female offspring via thelytokous parthenogenesis. Workers use this ability to compete among themselves and with their queen to be the mother of new queens. Males could therefore enhance their reproductive success by imprinting genes that enhance fertility in their daughter workers. This possibility sets the scene for intragenomic conflict between queens and drones over worker reproductive traits. Here, we show a strong parent-of-origin effect for ovary size (number of ovarioles) in reciprocal crosses between two honeybee subspecies, A. m. capensis and Apis mellifera scutellata. In this cross, workers with an A. m. capensis father had 30% more ovarioles than genotypically matched workers with an A. m. scutellata father. Other traits we measured (worker weight at emergence and the presence/absence of a spermatheca) are influenced more by rearing conditions than by parent-of-origin effects. Our study is the first to show a strong epigenetic (or, less likely, cytoplasmic maternal) effect for a reproductive trait in the honeybee and suggests that a search for parent-of-origin effects in other social insects may be fruitful.