Morphometric parameters (wing venation) of honeybee workers from 10 bee colonies of the Krasnoyarsk Krai (Yenisei population).
A comprehensive study of some populations of honeybee (332 colonies) in Siberia (Tomsk region, Krasnoyarsk Krai (Yenisei population), Altai) using morphometric and molecular genetic methods was conducted. Infestation of bees (132 colonies) by Nosema has also been studied. Three variants of the COI-COII mtDNA locus were registered: PQQ, PQQQ (typical for Apis m. mellifera), and Q (specific for southern races). It was established that 64% of bee colonies from the Tomsk region and all colonies studied from the Krasnoyarsk and the Altai territories originate from Apis m. mellifera on the maternal line. According to the morphometric study, the majority of bee colonies of the Tomsk region are hybrids; in some colonies the mismatch of morphometric and mtDNA data was observed. Moreover, the majority of bee colonies infected by Nosema were hybrids. Yenisei population may be considered as a unique Apis m. mellifera population. Microsatellite analysis (loci А008, Ap049, AC117, AC216, Ap243, H110, A024, A113) showed the specific distribution of genotypes and alleles for some loci in the bees, which differ by geographical location. Loci A024 and Ap049 are of considerable interest for further study as candidate markers for differentiation of subspecies; locus A008 can be considered informative for determining of different ecotypes of Apis m. mellifera.
- COI-COII locus
In Siberia, the honeybee was introduced about 230 years ago. It was the dark-colored forest bee
At present, one of the beekeeping problems in different countries is a massive bee hybridization, which leads to the reduction of the range of native subspecies, the formation of hybrids, and “deterioration” of the genotypic composition of honeybees. Hybrid populations are less adapted to environmental conditions that rapidly change during the year and are characterized by the higher morbidity and low immunity [1–3].
Introgressive hybridization modifies the genetic pool of local honeybee populations leading to the loss of their genetic identity . The process of hybridization of different subspecies of honeybee can cause the destruction of the established gene complexes, leading to decrease in adaptive properties of organisms and populations and the change in biological and economically significant indicators of bees. The observed widespread hybridization of honeybees and the formation of hybrid bees can certainly contribute to the spread of disease. The extent of hybridization, characteristics of hybrid bees, the study of genetic processes that occur during hybridization, and evaluation of the effects of hybridization are of considerable interest.
The goal of this study is the morphometric and molecular genetic (mtDNA and microsatellite analysis) characterization of honeybees in Siberia and the assessment of the infestation of bee colonies by
2. Materials and methods
Bees and bee colonies were investigated in three regions of Siberia: the Tomsk region, the Krasnoyarsk Krai, and the Altai Krai (Figure 1).
The Tomsk region is located in the geographic center of Siberia, in the southeastern part of the West Siberian Plain. The distance between the northern and southern boundaries of the meridian is about 600 kilometers; therefore, the climate of the southern and northern regions is markedly different. A climatic characteristic of the northern region is a more severe and prolonged winter season. Almost the entire territory of the region is within the taiga zone, where forests cover about 60% of the territory. The climate is temperate continental with considerable daily and annual amplitudes and long winters (5–6 months). The average annual temperature is –0.6 °C, while the average temperature in July is +18.1 °C and in January is 19.2 °C. The frost-free period is 100–105 days. Precipitation is 435 mm.
The Krasnoyarsk Krai is located in the Eastern Siberia. The climate is sharply continental, where 70% of the territory is occupied by forests.
The Altai Krai is located in the south-east of Western Siberia. The region contains almost all natural zones of Russia—the steppe and forest steppe, taiga, and mountains. The climate of the Altai Territory is highly heterogeneous because of various geographical conditions. Foothills have a temperate climate, the transition to continental.
The samples are obtained from different geographic parts (ecologically and climatically different districts) of the Tomsk region, including districts with a high beekeeping activity (the southern districts) or districts with a low apicultural activity (the northern districts), according to the local knowledge of specialists from the Society of Beekeepers. Honeybees from the apiaries of the Krasnoyarsk Krai and the Altai Krai were also investigated for comparison.
A total of 332 bee colonies (60 apiaries) from Siberia were investigated by morphometric (3043 honey bee workers) and molecular genetic methods (2073 bees by mtDNA analysis and from 252 to 515 bees by microsatellite analysis): 318 bee colonies from the Tomsk region; 10 colonies from the Krasnoyarsk Krai, and 5 colonies from the Altai Krai (Figure 1).
Bee colonies from the Krasnoyarsk Krai were collected from the unique isolated Old Believers population, which existed for more than 60 years in forest without the importation of new honeybees.
Bee colonies from the Altai Krai have been collected in the apiary, located in the foothills.
Infestation of bee colonies by
2.3. Morphometric method
Morphometric parameters (wing venation), including the cubital index, the hantel index, and the discoidal shift, were studied (Figure 2).
2.4. mtDNA analysis
DNA isolation and polymerase chain reaction (PCR) was carried out according to standard techniques with some modifications [5,6]. To amplify the COI–COII mtDNA locus, the following sequences of primers were used: 3′-CACATTTAGAAATTCCATTA, 5′-ATAAATATGAATCATGTGGA . Amplification products were fractionated in 1.5% agarose gel, and the results were documented with the use of Gel-Doc XR+.
2.5. Microsatellite analysis
Variability of eight microsatellite loci was studied: А008 (=A8), Ap049, AC117, AC216, Ap243, H110, A024, and A113. PCR was performed using specific primers and reaction conditions according to Solignac et al. . Amplification products were analyzed with ABI Prism 3730 Genetic Analyser (Applied Biosystems, Inc., Foster City, CA) and GeneMapper Software (Applied Biosystems, Inc.). Two microliters of PCR products were mixed with GeneScan500-ROX size standards (Applied Biosystems, Inc.) and deionized formamide. Samples were run according to the manufacturer’s recommendations. These genetic parameters were calculated: allelic frequencies and standard error.
2.6. Infestation of honeybees by
From 10 to 70 bees were randomly selected from each bee colony and were examined for the presence of
After extraction, the samples were submitted to duplex-PCR [9,10]. The primer sequences utilized to amplify the 218-bp fragment corresponding to the 16S ribosomal gene of
In addition to the use of specific primers and fragment size to identify the species present, a selection of fragments (both
3. Results and discussion
Using the mtDNA analysis (locus COI-COII), we performed molecular genetic analysis of bee colonies (5–6 samples from each bee colony) to determine the origin of bee colony on the maternal line.
3.1. Genetic diversity of COI-COII mtDNA locus
An assessment of the genetic diversity of the COI-COII mtDNA locus in honeybee populations from the Tomsk region was conducted (see details in reference ). Three variants of the COI-COII mtDNA locus were registered: PQQ, PQQQ (typical for Middle Russian race), and Q (typical for southern races). We established that 64% of bee colonies on the maternal line originate from the Middle Russian race, 28% of colonies originate from southern subspecies, and 8% are mixed bee colonies. The southern parts of the Tomsk region (with a high beekeeping activity) show a higher genetic diversity of honeybees as compared with the northern regions, which are dominated by bee colonies (96%) and apiaries (73%) that are homogeneous for the genetic variant of locus COI-COII. The bee colonies derived from the Middle Russian breed were genetically heterogeneous for the COI-COII locus: the PQQ variant was registered in 86.1% of the total number of bee colonies of the Middle Russian race, PQQQ was registered in 9.4%, and another 4.5% of bee colonies showed the presence of individuals with both allele PQQ and allele PQQQ.
Based on the analysis of mtDNA (locus COI-COII), assessment of the genetic diversity of the honeybee in apiaries of the Tomsk region has shown that the genetic structure of bee populations in the Tomsk region is complex and mosaic, especially in the southern parts of the region (Figure 3). No large areas with an array of bees having a homogeneous genetic (race) composition and maternally originating from the Middle Russian race have been found; a few apiaries were revealed, in which all bees originated from the Middle Russian breed.
In the study of variability of the COI-COII mtDNA locus in honeybees from apiaries of the Krasnoyarsk Krai and the Altai Krai, two variants of the COI-COII locus specific for Middle Russian race were identified: only variant PQQ was registered in honeybees of Krasnoyarsk Krai (Yenisei population) and two variants (PQQ and PQQQ) were found in honeybees from the Altai Krai. No a variant Q specific for southern races of bee was detected.
Due to the fact that mtDNA analysis allows assessing only the maternal component in the genome of the honeybee, bee colonies were investigated by the morphometric analysis to identify the characteristics of both the maternal and paternal lines, and to assess the level of hybridization.
3.2. Morphometric study of honeybees
The results of the morphometric study of honeybees from examined regions of Siberia (the Tomsk region, the Krasnoyarsk Krai, and the Altai Krai) were different.
According to the morphometric study, the majority of the studied bee colonies of the Tomsk region are hybrids between the Middle Russian race of bees and bees of southern origin (predominantly Carpathian race). Data on the distribution of subspecies and hybrids in the apiaries of the Tomsk region on the basis of cubital index are shown in Figure 4. Some of the apiaries, which cultivate the Middle Russian bees, were found in the northern and southern parts of the Tomsk region.
Bee colonies obtained from isolated apiaries of the Krasnoyarsk Krai are of considerable interest. The area with these isolated apiaries was not influenced by other subspecies of honeybee for many years, and all studied bees had only variant PQQ of the locus COI-COII mtDNA. However, when comparing the data of the morphometric study of bees from isolated apiaries with the Russian and European standards of the
|Geographical location: settlement||Bee colony, №||Cubital index,|
|Discoidal shift, %|
|Standard for |
|No data||No data|
|1.5 to 1.7|
The results of morphometric analysis confirmed the origin of bee colonies of Altai population from the Middle Russian race, but some influence of the southern races have been shown. For example, the parameter “Discoidal shift” deviates from the Russian breed standard: individuals with a positive value and zero of discoidal shift were found in bee colony No. 7 (Table 2).
If bee colonies from the Krasnoyarsk Krai were obtained from the territory distant from the center and located in sparsely populated areas, in the taiga, the bee colonies from the Altai Krai inhabit the territory, characterized by high development of beekeeping and a constant active importation of bees of different origins.
3.3. The accordance of morphometric parameters and data of mtDNA analysis in honeybees in Siberia
The results of the outward morphological characters-based diagnostics of honeybees (the cubital index, the hantel index, and the discoidal shift) received from 11 bee colonies differing in the variants of the COI-COII mtDNA locus are presented (Table 2). Only for 4 of the 11 bee colonies, a full compliance with the criteria of the breed according to the morphometric and mtDNA analysis (the three
These data are consistent with the results of the research of hybrid apiary, where for many years (over 30) the Middle Russian bee was bred, but the last 10 years, the southern races have been actively imported . More than 50% of individuals refer to the southern races according to mtDNA analysis (variant Q of the locus COI-COII; “southern” mitotype). But none of these individuals corresponded to the southern race according to morphometric analysis (Table 3). In 33% of cases, individuals with “southern” mitotype had two morphometric features characteristic to the Middle Russian race.
For bees, originating from the Middle Russian race (variant PQQ of the locus COI-COII), full compliance between mitotype and morphometric parameters was found in approximately 6% of the individuals. 18% of bees had mitotype and two morphometric parameters which specific to the Middle Russian bees.
This indicates a process of cross-breeding of Middle Russian and southern races on this apiary. However, the process of “ousting of genes” is derived differently for bees of different origin: for bees of Middle Russian race the process of “ousting of genes” is smaller in scale, as among individuals with variant PQQ a smaller percentage of bees with “southern” morphometric characters was registered in comparison with the same data shown for bees with “southern” mitotypes.
of the COI-
|Zmeinogorsky||Vicinity of c.|
|p. Sinii Utes||11||30||Q|
of the COI-
|Tomsk region||Tomsky||p. Zarechnyi||1||30||PQQQ||73.30||26.70||0.00|
|Altai Krai||Zmeinogorsky||Vicinity of c. Zmeinogorsk||6||29||PQQ||94.00||6.00||0.00|
|Krasnoyarsk Krai||Yeniseisky||p. Yaksha||8||30||PQQ||100.0||0.00||0.00|
|Tomsk region||Tomsky||s. Semiluzhki||9||50||Q||4.00||20.00||76.00|
|p. Sinii Utes||11||30||Q||6.70||76.70||16.70|
|Standart of breeds||PQQ, PQQQ|
|mtDNA||Variant PQQ||Variant Q|
|Number of studied bees, %||44.44||55.56|
|The combination of features|
characteristic for different races
х1 + х2 + х3
х1 + х2
х1 + х3
х2 + х3
Thus, the result of study of hybrid apiaries and bee colonies indicate, on the one hand, the importance and the necessity of a comprehensive approach to the exact characterization of honeybee races. On the other hand, the results are of scientific interest for the study of genetic processes during hybridization of different subspecies of honeybee and for analyzing the process of “ousting of genes” of one race by genes of other race. For example, hybridization between the Middle Russian bee and Carpathian bee is of interest because the races belong to different evolutionary branches.
For such studies, microsatellite loci are the most informative molecular genetic markers. Microsatellite markers can be useful for the study of genetic structure of different honeybee populations and bee colonies, evaluation of genetic diversity and introgressive hybridization, differentiation of different subspecies (ecotypes), the establishment of evolutionary relationships and adaptive features of four evolutionary branches (A, M, C, and O), mapping quantitative trait loci (QTL), and search of genetic markers associated with economically significant characteristics [3,7,13–46].
Characterization of the allele spectrum of microsatellite loci and analysis of their variability in subspecies, colonies, and individuals in the honeybee populations is the initial stage of any of the above research.
3.4. Microsatellite analysis
Variability of eight microsatellite loci (А008 (=A8), Ap049, AC117, AC216, Ap243, H110, A024, and A113) in honeybee from Siberian region was studied. Seven loci were polymorphic and only for AC216 locus one homozygous genotype was registered in all the studied bees (allele 91 bp). For each locus, the range and frequency of genotypes and alleles were determined (Table 4).
|Locus||Genotype||Frequency of genotype||Allelic frequency|
with an error
|Locus||Genotype||Frequency of genotype||Allelic frequency|
with an error
Microsatellite loci differed in variability: the minimum number of alleles was detected for loci AC117 and H110 (3 alleles) and the maximum number of alleles was registered for loci A008 (10 alleles) and A113 (11 alleles). At the same time, for six of the seven polymorphic loci (except locus A024), one major allele with a frequency of more than 0.5 (from 0.5278 for allele “255”of locus Ap243 to 0.8211 for allele “183” of locus AC117) was registered regardless of the number of detected alleles.
To identify the features of honeybee from different geographical areas, the comparative analysis of the variability of the studied loci was carried out for the bees of
|Belgium (Chimay)||Sweden (Umea)||France (eight|
|No data||No data|
|Locus||Alleles (pb)||Allelic frequency|
|Krasnoyarsk Krai||Tomsk region||Bashkortostan|
Siberian populations (Tomsk region and Krasnoyarsk Krai) are closest in spectrum and allele frequencies of most studied loci (A008, Ap049, A113, Ap243, H110). The Ural population located to the west of Siberian region differs from Siberia for some loci: for locus A008 differences were registered in the spectrum of alleles, for the locus A024—in the frequency of alleles, for the loci Ap049 and Ap243—in both the spectrum and frequency of alleles. It is remarkable that the Ural population has a greater similarity in the spectrum of alleles of loci A024 and A008 to European populations.
The differences in the spectrum of alleles and the frequency of allele registration for locus A008 were revealed in honeybees of Siberia, the Ural, and European populations. For honeybees of the Ural and Europe, shorter alleles of locus A008 were predominant (154 bp and 148 bp, respectively), whereas for bees from Siberia allele “162” was the most specific. Probably this locus should be considered as a marker related to geographic and environmental conditions (specific adaptation to local conditions) [1,3,48,49] because the different populations of dark-colored forest bee (European, Ural, and Siberian populations) were compared in this study.
For some loci, for example A113, allelic spectrum overlaps, but the frequency of the alleles was different in honeybees of different populations. Different factors of population dynamics (such as founder effect, genetic drift, natural selection) can be causes of this phenomenon.
Thus, it is shown that for some loci the specific distribution of genotypes and alleles were detected in the bees, which differ by geographical location. Further research is needed and the expansion of gene-geographic studies of honeybee is relevant.
To assess the informativeness of studied loci for the differentiation of different subspecies of honeybee, the comparison of the spectrum of predominant alleles in bees of different evolutional branches (M and C) and from different geographical localization was conducted (Table 7). Comparison of the data on the variability of microsatellite loci studied in bees of different origin and different geographical location allows making some conclusions and adjustments with respect to informativeness of these loci as markers for differentiation of subspecies of honeybee.
For locus A008, the differences in the spectrum of the most common alleles are registered between the
For locus A113 clear differences in length of the most frequently detected allele were not detected both among bees of a common origin and between bees belonging to different races. Probably this locus cannot be considered informative for determining of the subspecies.
Loci A024 and Ap049 are of considerable interest for further study as candidate markers for inclusion in the diagnostic panel, differentiating subspecies. So, in general, for the locus A024 the majority of bees and bee colonies
|Geographical location||Sequence composition|
of the COI-COII
mtDNA locus (breed)
|Predominant allele||Allelic frequency|
|Ural (Bashkir population)1||PQQ||154||0.63–1.00|
|Ural (Bashkir population)1||PQQ||218|
|Ural (Bashkir population)1||PQQ||98|
|Ural (Bashkir population)1||PQQ||129|
In order to determine the subspecies status of an individual honeybee, a honeybee colony, or a honeybee population, it is important to compare allelic counts and genotypes across different studies. However, no standard reference material, such as a standard allelic ladder, is available for honeybees . In addition, the spectrums of analyzed microsatellite markers often do nоt overlap and primary data on the allele spectrum and allele frequencies are not always presented in publications. In general, the present stage of the study of variability of microsatellite loci in
3.5. Infestation of honeybees by
Importation of races of southern origin to the territory of Siberia, where the Middle Russian breed for a long time lived, on the one hand, led to a massive hybridization of bees, a loss of purebred, decreased immunity, and increased incidence of bees. On the other hand, the import of bee families from other areas (the European part of Russia, Uzbekistan), disadvantageous in the epidemiological situation, led to the spread of diseases that have not previously registered in the territory of Siberia.
This situation was evaluated for nosemosis: the distribution
Nosemosis is a parasitic disease of adult honeybees (
The geographical distribution of
For the period of 2012–2015, a screening study of 132 bee colonies from 68 apiaries of Siberia for the presence of
The samples of 28 bee colonies from 33 infected colonies (84.8%) from 19 apiaries were positive by PCR using
The studied bees from apiaries of Krasnoyarsk Krai and Altai Krai were not infected with
Reports on the impact of
Currently, several reasons for the widespread presence of the parasite
It is assumed that the level of infestation in honeybees can be associated with the race and the origin (local or non-local) of the bees. Some differences in the resistance to
To determine if the infection incidence of bees by
of the COI-COII
|M ± m||Lim:|
|M ± m|
|1.66 ± 0.04|
|0.878 ± 0.011|
|2.14 ± 0.07|
|0.937 ± 0.010|
|1.70 ± 0.03|
|0.804 ± 0.011|
|1.78 ± 0.06|
|0.846 ± 0.013|
|1.90 ± 0.06|
|0.880 ± 0.018|
|1.73 ± 0.06|
|0.834 ± 0.015|
|1.86 ± 0.04|
|0.885 ± 0.011|
|Standard breeds (subspecies)**|
|PQQ, PQQQ and other|
For comparison, the assessment of the origin of the bee colonies not infected with
At present, the cold climate is considered as one of the limiting factors of
The different prevalence of
In our research, the majority of bee colonies infected by
This study of honeybees in Siberia shows the need for a comprehensive approach to the study of various aspects of the honeybee, such as differentiation of subspecies, the role of environmental (geographical) factors in the formation of the genetic diversity of bees, and the incidence of bees.
The primary task of the study of the genetic diversity of honeybees is to determine their subspecies composition. When performing gene-geographical research, it is important to consider the assessment of adaptive and selective significance of genetic markers. This is also important for the planning and conducting of works having applied nature.
Along with exterior characters used for a long time to identify the breed of honeybees, molecular genetic techniques are actively applied. However, in connection with the high level of hybridization of bees, when about one-third of bee colonies show an imbalance between genetic and morphometric parameters, and in some cases, their complete mismatch occurs, a comprehensive analysis of the bees is necessary.
The presence of hybrid forms in an area where the genetic diversity is studied, on the one hand, creates unfavorable background for conservation of gene pools of unique subspecies (for example, dark-colored forest bee), on the other hand, makes it difficult to search for adaptively significant and economically valuable traits (possible distortion of results and their interpretation). Therefore, it should be taken into account in conducting such studies. The above data also indicate that only the exterior or just genetic traits may be insufficient to determine the origin of bees and only the simultaneous analysis of morphometric parameters and data on the variability of locus COI-COII of mtDNA allow to evaluate the breed and cases of hybridization objectively.
In the conditions of widespread crossbreeding of bees, genetic methods to control the purity of bee colonies must also be improved. Research in this direction is carried out by international and Russian researchers [43,47,90]. Therefore, on the basis of extensive research carried out on the territory of Eastern Europe (search of informative markers was conducted among more than 1,000 SNP using five different analytical methods), five panels, consisting of 48, 96, 144, 192, and 284 markers informative for determining the ancestral origin of species have been developed. The authors propose to use the results of this study to identify and evaluate the impurity of C-lines (in particular,
This study was supported by the Russian Foundation for Basic Research (research grant No 13-04-98116-r-siberia-а) and by the Tomsk State University Academic D.I. Mendeleev Fund Program in 2015 (research grant No 18.104.22.1685).
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