Morphometric parameters (wing venation) of honeybee workers from 10 bee colonies of the Krasnoyarsk Krai (Yenisei population).
Abstract
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.
Keywords
- honeybee
- COI-COII locus
- microsatellites
- Nosema
- Siberia
1. Introduction
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 [4]. 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
2.1. Region
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.
2.2. Samples
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 [5]. 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. [7]. 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 Nosema
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 [11]). 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, standard units |
Hantel index, standard units |
Discoidal shift, % | ||||
---|---|---|---|---|---|---|---|---|
– | 0 | + | ||||||
Ostyatskoe | 1 |
2.00 |
1.61±0.04 |
0.892 |
0.795±0.011 | 100.0 | 0 | 0 |
2 |
1.74 |
1.51±0.02 |
0.912 |
0.849±0.012 | 83.3 | 16.7 | ||
3 |
1.74 |
1.51±0.03 |
0.883 |
0.837±0.008 | 83.3 | 16.7 | 0 | |
4 |
1.67 |
1.45±0.02 |
0.900 |
0.837±0.009 | 97.0 | 3.0 | 0 | |
5 |
1.79 |
1.46±0.03 |
0.923 |
0.842±0.010 | 87.0 | 13.0 | 0 | |
Kolmogorovo | 1 |
2.10 |
1.60±0.05 |
0.900 |
0.820±0.009 | 97.0 | 3.0 | 0 |
2 |
1.76 |
1.51±0.03 |
0.919 |
0.845±0.008 | 93.0 | 7.0 | 0 | |
3 |
1.86 |
1.56±0.04 |
0.985 |
0.810±0.011 | 97.0 | 3.0 | 0 | |
4 |
1.76 |
1.45±0.04 |
0.945 |
0.830±0.011 | 97.0 | 3.0 | 0 | |
Yaksha | 1 |
1.85 |
1.59±0.02 |
0.846 |
0.775±0.008 | 100.0 | 0 | 0 |
Standard for |
||||||||
I |
2.10 |
1.70 |
0.923 |
No data | No data | |||
II |
1.90 |
1.5 to 1.7 |
0.923 |
91–100 | 5–10 | 0.00 |
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 [6]. 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.
Geographical location | Bee colony, № |
Number of studied bees |
Sequence composition of the COI- COII mtDNA locus |
Cubital index, standard units |
Hantel index, standard units |
||||||
---|---|---|---|---|---|---|---|---|---|---|---|
region | District | Settlement | |||||||||
Tomsk region |
Tomsky | p. Zarechnyi | 1 | 30 | PQQQ |
2.23 |
1.66 | 0.216 |
0.932 |
0.826 | 0.052 |
s. Kurlek | 2 | 28 | PQQQ |
3.29 |
2.14 | 0.376 |
1.053 |
0.937 | 0.055 | ||
Zyryansky | s. Dubrovka | 3 | 30 | PQQ |
2.47 |
1.69 | 0.232 |
0.933 |
0.849 | 0.060 | |
Molchanovsky | s. Mogochino | 4 | 30 | PQQ |
2.56 |
1.92 | 0.290 |
1.000 |
0.879 | 0.055 | |
5 | 43 | PQQ |
2.00 |
1.73 | 0.181 |
0.926 |
0.821 | 0.038 | |||
Altai Krai |
Zmeinogorsky | Vicinity of c. Zmeinogorsk |
6 | 29 | PQQ |
2.00 |
1.55 | 0.232 |
0.967 |
0.858 | 0.062 |
7 | 30 | PQQQ |
2.50 |
1.80 | 0.245 |
0.984 |
0.845 | 0.059 | |||
Krasno- yarsk Krai |
Yeniseisky | p. Yaksha | 8 | 30 | PQQ |
1.85 |
1.59 | 0.132 |
0.846 |
0.775 | 0.044 |
Tomsk region |
Tomsky | s. Semiluzhki | 9 | 50 | Q |
3.64 |
2.51 | 0.374 |
1.210 |
1.050 | 0.047 |
s. Kurlek | 10 | 29 | Q |
2.29 |
1.66 | 0.220 |
0.965 |
0.878 | 0.060 | ||
p. Sinii Utes | 11 | 30 | Q |
2.87 |
2.37 | 0.334 |
1.053 |
0.931 | 0.065 | ||
Standart of breeds |
PQQ, PQQQ and other |
2.10 |
1.70 | – |
0.923 |
– | – | ||||
1.90 |
1.6 | – |
0.923 |
– | – | ||||||
Q |
3.00 |
2.7 | – | ≥ 0.925 |
– | – | |||||
Q |
2.30 |
2.0 | – | No data | – | – |
Geographical location | Bee colony, № |
Number of studied bees |
Sequence composition of the COI- COII mtDNA locus |
Discoidal shift, % |
||||
---|---|---|---|---|---|---|---|---|
region | District | Settlement | ||||||
– | 0 | + | ||||||
Tomsk region | Tomsky | p. Zarechnyi | 1 | 30 | PQQQ | 73.30 | 26.70 | 0.00 |
s. Kurlek | 2 | 28 | PQQQ | 32.10 | 53.60 | 10.70 | ||
Zyryansky | s. Dubrovka | 3 | 30 | PQQ | 73.33 | 26.67 | 0.00 | |
Molchanovsky | s. Mogochino | 4 | 30 | PQQ | 70.00 | 30.00 | 0.00 | |
5 | 43 | PQQ | 100.0 | 0.00 | 0.00 | |||
Altai Krai | Zmeinogorsky | Vicinity of c. Zmeinogorsk | 6 | 29 | PQQ | 94.00 | 6.00 | 0.00 |
7 | 30 | PQQQ | 46.70 | 46.70 | 6.60 | |||
Krasnoyarsk Krai | Yeniseisky | p. Yaksha | 8 | 30 | PQQ | 100.0 | 0.00 | 0.00 |
Southern breeds* | ||||||||
Tomsk region | Tomsky | s. Semiluzhki | 9 | 50 | Q | 4.00 | 20.00 | 76.00 |
s. Kurlek | 10 | 29 | Q | 72.40 | 27.60 | 0.00 | ||
p. Sinii Utes | 11 | 30 | Q | 6.70 | 76.70 | 16.70 | ||
Standart of breeds | PQQ, PQQQ and other |
– | – | – | ||||
91–100 | 5–10 | 0.00 | ||||||
Q | 0–5 | 0–20 | 80–100 | |||||
Q | 60–70 | 20–30 | 3–5 |
mtDNA | Variant PQQ | Variant Q | |||
---|---|---|---|---|---|
Number of studied bees, % | 44.44 | 55.56 | |||
Race | Southern race |
Southern race |
|||
The combination of features characteristic for different races |
3 parameters х1 + х2 + х3 |
5.6 | 7.4 | 7.4 | 0.0 |
2 parameters, total, including х1 + х2 х1 + х3 х2 + х3 |
18.5 1.9 3.7 13.0 |
13.0 1.9 11.1 0 |
33.3 1.9 0 31.5 |
14.8 11.1 3.7 0 |
|
1 parameter, total |
13.0 | 18.5 | 14.8 | 33.3 |
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 |
---|---|---|---|
152–152 | 0.006 | Р152=0.0311±0.0054 Р162=0.8049±0.0123 Р166=0.0010±0.0031 Р168=0.0010±0.0031 Р170=0.0213±0.0045 Р172=0.0243±0.0048 Р174=0.0825±0.0086 Р176=0.0029±0.0017 Р178=0.0262±0.0050 Р180=0.0039±0.0019 |
|
152–162 | 0.049 | ||
152–170 | 0.002 | ||
162–162 | 0.736 | ||
162–168 | 0.002 | ||
162–170 | 0.016 | ||
162–172 | 0.039 | ||
162–174 | 0.033 | ||
166–172 | 0.002 | ||
170–170 | 0.006 | ||
170–174 | 0.016 | ||
172–172 | 0.004 | ||
174–174 | 0.037 | ||
174–176 | 0.004 | ||
174–178 | 0.031 | ||
174–180 | 0.008 | ||
176–178 | 0.002 | ||
178–178 | 0.010 | ||
118–127 | 0.002 | Р118=0.0010±0.0001 Р121=0.0069±0.0025 Р127=0.6581±0.0149 Р130=0.1759±0.0120 Р139=0.1403±0.0109 Р142=0.0010±0.0001 Р152=0.0168±0.0040 |
|
121–127 | 0.002 | ||
121–130 | 0.006 | ||
121–139 | 0.006 | ||
127–127 | 0.529 | ||
127–130 | 0.187 | ||
127–139 | 0.053 | ||
127–152 | 0.019 | ||
130–130 | 0.055 | ||
130–139 | 0.045 | ||
130–152 | 0.002 | ||
139–139 | 0.081 | ||
139–152 | 0.013 | ||
142–152 | 0.002 | ||
152–152 | 0.002 | ||
175–175 | 0.008 | Р175=0.0910±0.0092 Р179=0.0879±0.0090 Р183=0.8211±0.0123 |
|
175–179 | 0.020 | ||
175–183 | 0.145 | ||
179–179 | 0.012 | ||
179–183 | 0.131 | ||
183–183 | 0.683 | ||
162–162 | 0.567 | Р162=0.7522±0.0167 Р166=0.0627±0.0093 Р170=0.1851±0.0150 |
|
162-166 | 0.116 | ||
162–170 | 0.254 | ||
166–166 | 0.003 | ||
166–170 | 0.003 | ||
170–170 | 0.057 | ||
Locus | Genotype | Frequency of genotype | Allelic frequency with an error |
---|---|---|---|
255–255 | 0.401 | Р255=0.5278±0.0222 Р263=0.3175±0.0207 Р269=0.0833±0.0123 Р272=0.0635±0.0109 Р275=0.0079±0.0039 |
|
255–263 | 0.167 | ||
255–269 | 0.056 | ||
255–272 | 0.028 | ||
255–275 | 0.004 | ||
263–263 | 0.175 | ||
263–269 | 0.075 | ||
263–272 | 0.040 | ||
263–275 | 0.004 | ||
269–269 | 0.004 | ||
269–272 | 0.028 | ||
272–272 | 0.012 | ||
272–275 | 0.008 | ||
94–94 | 0.344 | Р94=0.4736±0.0186 Р96=0.1014±0.0112 Р98=0.0375±0.0070 Р100=0.0194±0.0051 Р102=0.2097±0.0152 Р104=0.1528±0.0134 Р106=0.0056±0.0028 |
|
94–98 | 0.036 | ||
94–100 | 0.033 | ||
94–102 | 0.175 | ||
94–104 | 0.014 | ||
96–96 | 0.067 | ||
96–104 | 0.058 | ||
96–106 | 0.011 | ||
98–98 | 0.019 | ||
100–100 | 0.003 | ||
102–102 | 0.089 | ||
102–104 | 0.067 | ||
104–104 | 0.083 | ||
208–212 | 0.003 | Р208=0.0013±0.0013 Р210=0.0144±0.0043 Р212=0.2350±0.0153 Р214=0.0026±0.0018 Р218=0.5953±0.0177 Р220=0.1084±0.0112 Р222=0.0013±0.0013 Р224=0.0013±0.0013 Р226=0.0183±0.0048 Р228=0.0196±0.0050 Р232=0.0026±0.0018 |
|
210–210 | 0.003 | ||
210–218 | 0.021 | ||
210–220 | 0.003 | ||
212–212 | 0.177 | ||
212–214 | 0.005 | ||
212–218 | 0.078 | ||
212–220 | 0.013 | ||
212–222 | 0.003 | ||
212–226 | 0.005 | ||
212–228 | 0.003 | ||
212–232 | 0.005 | ||
218–218 | 0.475 | ||
218–220 | 0.117 | ||
218–226 | 0.021 | ||
218–228 | 0.003 | ||
220–220 | 0.018 | ||
220–224 | 0.003 | ||
220–226 | 0.010 | ||
220–228 | 0.034 | ||
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
Locus | Alleles (pb) |
Allelic frequency | |||||
---|---|---|---|---|---|---|---|
Russia | Europe** | ||||||
Krasnoyarsk Krai |
Tomsk region | Ural* (Bashkor tostan) |
Belgium (Chimay) | Sweden (Umea) | France (eight geographic areas) |
||
148 | 0.783 | 0.727 | 0.267–0.969 | ||||
152 | 0.006 | ||||||
154 | 0.897 | 0–0.083 | |||||
155 | 0–0.033 | ||||||
156 | 0.053 | 0.133 | 0.227 | 0.017–0.300 | |||
157 | 0–0.050 | ||||||
158 | 0.053 | 0.023 | 0–0.117 | ||||
159 | 0–0.017 | ||||||
160 | 0.050 | 0–0.100 | |||||
162 | 1.000 | 0.912 | 0.033 | 0–0.034 | |||
164 | 0.023 | 0–0.020 | |||||
166 | 0.003 | 0–0.017 | |||||
170 | 0.003 | ||||||
172 | 0.032 | ||||||
174 | 0.044 | ||||||
No data | No data | ||||||
94 | 0.216 | 0.741 | |||||
96 | 0.358 | ||||||
98 | 0.132 | 0.896 | 0.804 | ||||
100 | 0.034 | 0.020 | |||||
102 | 0.025 | 0.227 | |||||
104 | 0.216 | 0.012 | |||||
106 | 0.020 | 0.104 | 0.130 | ||||
108 | 0.065 | ||||||
202 | 0.083 | 0.024 | 0.017–0.267 | ||||
204 | |||||||
208 | 0–0.017 | ||||||
210 | 0.021 | 0.009 | |||||
212 | 0.174 | 0–0.030 | |||||
214 | 0.006 | 0.033 | 0.010–0.500 | ||||
216 | 0.063 | 0–0.017 | |||||
218 | 0.898 | 0.540 | 0.865 | 0–0.020 | |||
220 | 0.081 | 0.183 | 0.042 | 0.833 | 0.857 | 0.433–0.810 | |
222 | 0.003 | 0.032 | 0.024 | 0–0.041 | |||
224 | 0.003 | 0.017 | 0.048 | 0–0.060 | |||
226 | 0.040 | 0.048 | 0–0.034 | ||||
228 | 0.043 | 0.017 | 0.017–0.071 | ||||
230 | 0–0.052 | ||||||
232 | 0–0.017 | ||||||
234 | 0.017 | 0–0.017 | |||||
236 | 0–0.020 | ||||||
238 | 0–0.017 | ||||||
240 | 0–0.010 | ||||||
Locus | Alleles (pb) | Allelic frequency | ||
---|---|---|---|---|
Siberia | Ural | |||
Krasnoyarsk Krai | Tomsk region | Bashkortostan | ||
118 | 0.005 | |||
121 | 0.005 | 0.003 | ||
123 | 0.917 | |||
127 | 0.810 | 0.711 | ||
130 | 0.138 | 0.249 | 0.063 | |
138 | 0.021 | |||
139 | 0.014 | 0.037 | ||
152 | 0.029 | |||
254 | 0.646 | |||
255 | 0.280 | 0.524 | ||
257 | 0.354 | |||
263 | 0.542 | 0.254 | ||
269 | 0.140 | 0.056 | ||
272 | 0.037 | 0.143 | ||
275 | 0.024 | |||
160 | 0.615 | |||
162 | 0.624 | 0.837 | ||
163 | 0.302 | |||
166 | 0.376 | 0.056 | ||
168 | 0.083 | |||
170 | 0.107 | |||
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 | |
---|---|---|---|---|
Tomsk region | PQQ/PQQQ | 162 | 0.71–1.00 | |
Krasnoyarsky Krai | PQQ | 162 | 1.00 | |
Ural (Bashkir population)1 | PQQ | 154 | 0.63–1.00 | |
Tomsk region2 | Q | 174 | 0.58–0.61 | |
Sochi area3 | Q | 158 | 0.88–1.00 | |
Europe4 | 148 | 0.27–0.97 | ||
Tomsk region | PQQ/PQQQ | 218 212 220 |
0.67–0.82 0.61 0.50 |
|
Krasnoyarsky Krai | PQQ | 218 | 0.85–0.95 | |
Ural (Bashkir population)1 | PQQ | 218 220 |
0.50–1.00 0.50 |
|
Tomsk region2 | Q | 212 | 0.94–1.00 | |
Sochi area3 | Q | 222 | 0.50 | |
Europe4 | 220 | 0.433–0.857 | ||
Tomsk region | PQQ/PQQQ | 94 102 |
0.60–0.90 0.54 |
|
Krasnoyarsky Krai | PQQ | 98 96 |
0.50 0.50–0.71 |
|
Ural (Bashkir population)1 | PQQ | 98 106 |
0.50–1.00 0.50 |
|
Tomsk region2 | Q | 104 | 0.65 | |
Sochi area3 | Q | 106 | 0.88–1.00 | |
Europe4 | 98 | >0.80 | ||
Tomsk region | PQQ/PQQQ | 127 130 |
0.62–0.92 0.77 |
|
Krasnoyarsky Krai | PQQ | 127 | 0.50–0.96 | |
Ural (Bashkir population)1 | PQQ | 129 130 |
0.50–1.00 1.00 |
|
Tomsk region2 | Q | 139 | 0.66–1.00 | |
Sochi area3 | Q | 139 | 1.00 |
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 [3]. 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 Nosema in Siberia
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
Perhaps,
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
№ colonies |
Sequence composition of the COI-COII mtDNA locus |
Morphometric parameters | ||||
---|---|---|---|---|---|---|
Cubital index, standard units |
Hantel index, standard units |
|||||
Lim: max |
M ± m | Lim: max |
M ± m | |||
1 | Q |
2.29 |
1.66 ± 0.04 |
0.965 |
0.878 ± 0.011 | |
2 | PQQQ |
3.29 |
2.14 ± 0.07 |
1.053 |
0.937 ± 0.010 | |
3 | Q |
2.11 |
1.70 ± 0.03 |
0.917 |
0.804 ± 0.011 | |
4 | PQQ |
2.80 |
1.78 ± 0.06 |
1.0 |
0.846 ± 0.013 | |
5 | Q |
2.82 |
1.90 ± 0.06 |
1.176 |
0.880 ± 0.018 | |
6 | PQQ/Q |
2.80 |
1.73 ± 0.06 |
1.0 |
0.834 ± 0.015 | |
7 | Q |
2.35 |
1.86 ± 0.04 |
1.057 |
0.885 ± 0.011 | |
Standard breeds (subspecies)** | ||||||
PQQ, PQQQ and other |
2.1 |
1.7 |
0.923 |
No data | ||
Q |
3.0 |
2.65 | ≥0.925 | No data |
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
4. Conclusion
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,
Acknowledgments
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 8.1.66.2015).
References
- 1.
De la Rúa P, Jaffé R, Dall’Olio R, Muñoz I, Serrano J. Biodiversity, conservation and current threats to European honey bees. Apidologie. 2009;40(3):263-284. DOI: 10.1051/apido/2009027 - 2.
Meixner MD, Costa C, Kryger P, Hatjina F, Bouga M, Ivanova E, Büchler R. Conserving diversity and vitality for honey bee breeding. Journal of Apicultural Research. 2010;49(1):85-92. DOI: 10. 3896/IBRA.1.49.1.12 - 3.
Meixner MD, Pinto MA, Bouga M, Kryger P, Ivanova E, Fuchs S. Standard methods for characterising subspecies and ecotypes of Apis mellifera . In: Dietemann V, Ellis JD, Neumann P, Editors. The COLOSS BEEBOOK, Volume I: Standard Methods forApis mellifera Research. Journal of Apicultural Research. 2013;52(4):1-28. DOI 10.3896/IBRA.1.52.4.05 - 4.
Büchler R, Costa C, Hatjina F, Andonov S, Meixner MD, Le Conte Y, Uzunov A, Berg S, Bienkowska M, Bouga M, Drazic M, Dyrba W, Kryger P, Panasiuk B, Pechhacker H, Petrov P, Kezić N, Korpela S, Wilde J. The influence of genetic origin and its interaction with environmental effects on the survival of Apis mellifera L. colonies in Europe. Journal of Apicultural Research. 2014;53(2):205-214. DOI: 10.3896/IBRA.1.53.2.03 - 5.
Nikonorov YM, Ben’kovskaya GV, Poskryakov AV, Nikolenko AG, Vakhitov VA. The use of the PCR technique for control of the pure-breeding of honeybee ( Apis mellifera mellifera L.) colonies from the Southern Urals. Russian Journal of Genetics. 1998;34(11):1344-1347. - 6.
Ostroverkhova NV, Konusova OL, Kucher AN, Pogorelov YL, Belykh EA, Vorotov AA. Population genetic structure of honey bee ( Apis mellifera L.) in the village of Leboter in Chainsky district of the Tomsk region. Tomsk State University Journal of Biology. 2013;1(21):161-172. - 7.
Solignac M, Vautrin D, Loiseau A, Mougel F, Baudry E. Five hundred and fifty microsatellite markers for the study of the honey bee ( Apis mellifera L.) genome. Molecular Ecology Notes. 2003;3:307-311. DOI: 10.1046/j.1471-8286.2003.00436.x - 8.
Fries I, Chauzat MP, Chen YP, Doublet V, Genersch E, Gisder S, Higes M, McMahon DP, Martín-Hernández R, Natsopoulou M, Paxton RJ, Tanner G, Webster TC, Williams GR. Standard methods for Nosema research. In: Dietemann V, Ellis JD, Neumann P, Editors. The COLOSS BEEBOOK, Volume II: Standard Methods forApis mellifera Pest and Pathogen Research. Journal of Apicultural Research. 2013;52(1):1-28. DOI: 10.3896/IBRA.1.52.1.14 - 9.
Martín-Hernández R, Meana A, Prieto L, Salvador AM, Garrido-Bailon E, Higes M. Outcome of colonization of Apis mellifera byNosema ceranae . Applied and Environmental Microbiology. 2007;73(20):6331-6338. DOI: 10.1128/aem.00270-07 - 10.
Hamiduzzaman MM, Guzman-Novoa E, Goodwin PH. A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera ). Journal of Invertebrate Pathology. 2010;105(2):151-155. DOI: 10.1016/j.jip.2010.06.001 - 11.
Ostroverkhova NV, Konusova OL, Kucher AN, Kireeva TN, Vorotov AA, Belikh EA. Genetic diversity of the locus COI-COII of mitochondrial DNA in honeybee populations ( Apis mellifera L.) from the Tomsk region. Russian Journal of Genetics. 2015;51(1):80-90. DOI: 10.1134/S102279541501010X - 12.
Cauia E, Usurelu D, Magdalena LM, Cimponeriu D, Apostol P, Siceanu A, Holban A, Gavrila L. Preliminary researches regarding the genetic and morphometric characterization of honeybee ( A. mellifera L.) from Romania. Scientific Papers Animal Science and Biotechnologies. 2008;41(2):278-286. - 13.
Estoup A, Solignac M, Harry M, Cornuet JM. Characterization of (GT), and (CT) microsatellites in two insect species: Apis mellifera andBombus terrestris . Nucleic Acids Research. 1993;21:1427-1431. - 14.
Estoup A, Garnery L, Solignac M, Cornuet JM. Microsatellite variation in honey bee ( Apis mellifera L.) populations: Hierarchical genetic structure and test of the infinite allele and stepwise mutation models. Genetics. 1995;140:679-695. - 15.
Franck P, Garnery L, Solignac M, Cornuet JM. The origin of West European subspecies of honeybees ( Apis mellifera ): New insights from microsatellite and mitochondrial data. Evolution. 1998;52(4):1119-1134. DOI: 10.2307/2411242 - 16.
Garnery L, Franck P, Baudry E, Vautrin D, Cornuet JM, Solignac M. Genetic diversity of the West European honey bee ( Apis mellifera mellifera andA. m. iberica ). II. Microsatellite loci. Genetics Selection and Evolution. 1998;30(1):S49-S74. - 17.
Franck P, Garnery L, Solignac M, Cornuet JM. Molecular confirmation of a fourth lineage in honeybees from the Near East. Apidologie. 2000;31(2):167-180. DOI: 10.1051/apido:2000114 - 18.
Franck P, Garnery L, Loiseau A, Oldroyd BP, Hepbum HR, Solignac M, Cornuet JM. Genetic diversity of the honeybee in Africa: microsatellite and mitochondrial data. Heredity. 2001;86(4):420-430. - 19.
De la Rúa P, Galián J, Serrano J, Moritz RFA. Microsatellite analysis of non-migratory colonies of Apis mellifera iberica from South-eastern Spain. Journal of Zoological Systematics and Evolutionary Research. 2002;40(3):164-168. DOI: 10.1046/j.1439-0469.2002.00187.x - 20.
De la Rúa P, Galián J, Serrano J, Moritz RFA. Genetic structure of Balearic honeybee populations based on microsatellite polymorphism. Genetics Selection Evolution. 2003;35:339-350. DOI: 10.1051/gse:2003012 - 21.
De la Rúa P, Jimenez Y, Galián J, Serrano J. Evaluation of the biodiversity of honey bee ( Apis mellifera ) populations from Eastern Spain. Journal of Apicultural Research. 2004;43(4):162-166. DOI: 10.1080/00218839.2004.11101130 - 22.
Sušnik S, Kozmus P, Poklukar J, Meglic V. Molecular characterisation of indigenous Apis mellifera carnica in Slovenia. Apidologie. 2004;35(6):623-636. DOI: 10.1051/apido:2004061 - 23.
Jensen AB, Palmer KA, Boomsma JJ, Pedersen BoV. Varying degrees of Apis mellifera ligustica introgression in protected populations of the black honeybee,Apis mellifera mellifera , in Northwest Europe. Molecular Ecology. 2005;14(1):93-106. DOI: 10.1111/j.1365-294X.2004.02399.x - 24.
De la Rúa P, Galián J, Pedersen BoV, Serrano J. Molecular characterization and population structure of Apis mellifera from Madeira and the Azores. Apidologie. 2006;37(6):699-708. DOI: 10.1051/apido:2006044 - 25.
Kandemir I, Meixner MD, Ozkan A, Sheppard WS. Genetic characterization of honey bee ( Apis mellifera cypria ) populations in northern Cyprus. Apidologie. 2006;37(5):547-555. DOI: 10.1051/apido:2006029 - 26.
Bodur C, Kence M, Kence A. Genetic structure of honeybee, Apis mellifera L. (Hymenoptera: Apidae) populations of Turkey inferred from microsatellite analysis. Journal of Apicultural Research. 2007;46(1):50-56. DOI: 10.3896/IBRA.1.46.1.09 - 27.
Dall’Olio R, Marino A, Lodesani M, Moritz RFA. Genetic characterization of Italian honeybees, Apis mellifera ligustica , based on microsatellite DNA polymorphisms. Apidologie. 2007;38(2):207-217. DOI: 10.1051/apido:2006073 - 28.
Lattorff HM, Moritz RFA, Crewe RM, Solignac M. Control of reproductive dominance by the thelytoky gene in honeybees. Biology letters. 2007;3(3):292-295. DOI: 10.1098/rsbl.2007.0083 - 29.
Miguel I, Iriondo M, Garnery L, Sheppard WS, Estonba A. Gene flow within the M evolutionary lineage of Apis mellifera : role of the Pyrenees, isolation by distance and post-glacial re-colonization routes in the Western Europe. Apidologie. 2007;38(2):141-155. DOI: 10.1051/apido:2007007 - 30.
Solignac M, Mougel F, Vautrin D, Monnerot M, Cornuet JM. A third-generation microsatellite-based linkage map of the honey bee, Apis mellifera , and its comparison with the sequence-based physical map. Genome Biology. 2007;8:R66. DOI: 10.1186/gb-2007-8-4-r66 - 31.
Bourgeois L, Sylvester A, Danka R, Rinderer T. Comparison of microsatellite DNA diversity among commercial queen breeder stocks of Italian honey bees in the United States and Italy. Journal of Apicultural Research and Bee World. 2008;47(2):93-98. DOI: 10.3896/IBRA.1.47.2.01 - 32.
Moritz RFA, Dietemann V, Crewe R. Determining colony densities in wild honeybee populations ( Apis mellifera ) with linked microsatellite DNA markers. Journal of Insect Conservation. 2008;12(5):455-459. DOI: 10.1007/s10841-007-9078-5 - 33.
Hernández-García R, De la Rúa P, Serrano J. Mating frequency in Apis mellifera iberiensis queens. Journal of Apicultural Research. 2009;48(2):121-125. DOI: 10.3896/IBRA.1.48.2.06 - 34.
Muñoz I, Dall’Olio R, Lodesani M, De la Rúa P. Population genetic structure of coastal Croatian honey bees ( Apis mellifera carnica ). Apidologie. 2009;40(6):617-626. DOI: 10.1051/apido/2009041 - 35.
Soland-Reckeweg G, Heckel G, Neumann P, Excoffier L. Gene flow in admixed populations and implications for the conservation of the Western honeybee, Apis mellifera . Journal of Insect Conservation. 2009;13(3):317-328. DOI: 10.1007/s10841-008-9175-0 - 36.
Miguel I, Baylac M, Iriondo M, Manzano C, Garnery L, Estonba A. Both geometric morphometric and microsatellite data consistently support the differentiation of the Apis mellifera M evolutionary branch. Apidologie. 2010; 42:150-161. DOI: 10.1051/apido/2010048 - 37.
Canovas F, De la Rúa P, Serrano J, Galián J. Microsatellite variability reveals beekeeping influences on Iberian honeybee populations. Apidologie. 2011;42(3):235-251. DOI: 10.1007/s13592-011-0020-1 - 38.
Nikolova SR. Genetic variability of local Bulgarian honey bees Apis mellifera macedonica (rodopica) based on microsatellite DNA analysis. Journal of Apicultural Science. 2011;55(2):117-129. - 39.
Oleksa A, Chybicki I, Tofilski A, Burczyk J. Nuclear and mitochondrial patterns of introgression into native dark bees ( Apis mellifera mellifera) in Poland. Journal of Apicultural Research. 2011;50(2):116-129. DOI: 10.3896/IBRA.1.50.2.03 - 40.
Muñoz I, De la Rúa P. Temporal analysis of the genetic diversity in a honey bee mating area of an Island population (La Palma, Canary Islands, Spain). Journal of Apicultural Science. 2012;56(1):41-49. DOI: 10.2478/v10289-012-0005-y - 41.
Nedić N, Francis RM, Stanisavljević L, Pihler I, Kezić N, Bendixen C, Kryger P. Detecting population admixture in the honey bees of Serbia. Journal of Apicultural Research. 2014;53:303-313. DOI: 10.3896/ibra.1.53.2.12 - 42.
Uzunov A, Costa C, Panasiuk B, Meixner MD, Kryger P, Hatjina F, Bouga M, Andonov S, Bienkowska M, Le Conte Y, Wilde J, Gerula D, Kiprijanovska H, Filipi J, Petrov P, Ruottinen L, Pechhacker H, Berg S, Dyrba W, Ivanova E, Buchler R. Swarming, defensive and hygienic behaviour in honey bee colonies of different genetic origin in a pan-European experiment. Journal of Apicultural Research. 2014;53:248-260. DOI: 10.3896/IBRA.1.53.2.06 - 43.
Muñoz I, Henriques D, Johnston JS, Chávez-Galarza J, Kryger P, Pinto MA. Reduced SNP panels for genetic identification and introgression analysis in the dark honey bee ( Apis mellifera mellifera ). PLoS ONE. 2015;10(4):e0124365. DOI: 10.1371/journal.pone.0124365 - 44.
Nikolova SR, Bienkowska M, Gerula D, Ivanova EN. Microsatellite DNA polymorphism in selectively controlled Apis mellifera carnica andApis mellifera caucasica populations from Poland. Archives of Biological Sciences. 2015;67(3):889-894. DOI:10.2298/ABS141102048N - 45.
Techer MA, Clémencet J, Simiand C, Portlouis G, Reynaud B, Delatte H. Genetic diversity of the honeybee ( Apis mellifera L.) populations in the Seychelles archipelago. Insect Conservation and Diversity. 2016;9(1):13-26. DOI: 10.1111/icad.12138 - 46.
Ostroverkhova NV, Konusova OL, Kucher AN, Kireeva TN. Investigation of polyandry in honey bees ( Apis mellifera ) using microsatellites. Russian Journal of Zoology. 2016;95(3):307-313. DOI: 10.7868/S0044513416030119 - 47.
Ilyasov RA, Kosarev MN, Poskryakov AV, Sharipov AY, Nikolenko AG. New approach to the assessment of genetic potential of colonies of dark European bee Apis mellifera mellifera based on polymorphism of microsatellite loci. Biomiks. 2015;7(2):138-152. - 48.
Meixner MD, Büchler R, Costa C, Francis RM, Hatjina F, Kryger P, Uzunov A, Carreck NL. Honey bee genotypes and the environment. Journal of Apicultural Research. 2014;53(2):183-187. DOI: 10.3896/IBRA.1.53.2.01 - 49.
Hatjina F, Costa C, Büchler R, Uzunov A, Drazic M, Filipi J, Charistos L, Ruottinen L, Andonov S, Meixner MD, Bienkowska M, Dariusz G, Panasiuk B, Le Conte Y, Wilde J, Berg S, Bouga M, Dyrba W, Kiprijanovska H, Korpela S, Kryger P, Lodesani M, Pechhacker H, Petrov P, Kezic N. Population dynamics of European honey bee genotypes under different environmental conditions. Journal of Apicultural Research. 2014;53(2):233-247. DOI: 10.3896/IBRA.1.53.2.05 - 50.
Zander E. Tierische Parasiten als Krankheitserreger bei der Biene. Münchener Bienenzeitung. 1909;31:196-204. - 51.
Fries I, Feng F, daSilva A, Slemenda SB, Pieniazek NJ. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey beeApis cerana (Hymenoptera, Apidae). European Journal of Protistology. 1996;32(3):356-365. DOI:10.1016/s0932-4739(96)80059-9 - 52.
Higes M, Martin R, Meana A. Nosema ceranae , a new microsporidian parasite in honeybees in Europe. Journal of Invertebrate Pathology. 2006;92(2):93-95. DOI: 10.1016/j.jip.2006.02.005 - 53.
Huang WF, Jiang JH, Chen YW, Wang CH. A Nosema ceranae isolate from the honeybeeApis mellifera . Apidologie. 2007;38:30-37. DOI: 10.1051/apido:2006054 - 54.
Klee J, Besana AM, Genersch E, Gisder S, Nanetti A, Tam DQ, Chinh TX, Puerta F, Ruz JM, Kryger P, Message D, Hatjina F, Korpela S, Fries I, Paxton RJ. Widespread dispersal of the microsporidian Nosema ceranae , an emergent pathogen of the western honey bee,Apis mellifera . Journal of Invertebrate Pathology. 2007;96:1-10. DOI: 10.1016/j.jip.2007.02.014 - 55.
Paxton RJ, Klee J, Korpela S, Fries I. Nosema ceranae has infectedApis mellifera in Europe since at least 1998 and may be more virulent thanNosema apis . Apidologie. 2007;38:558-565. DOI: 10.1051/apido:2007037 - 56.
Invernizzi C, Abud C, Tomasco IH, Harriet J, Ramallo G, Campá J, Katz H, Gardiol G, Mendoza Y. Presence of Nosema ceranae in honeybees (Apis mellifera ) in Uruguay. Journal of Invertebrate Pathology. 2009;101(2):150-153. DOI: 10.1016/j.jip.2009.03.006 - 57.
Paxton RJ. Does infection by Nosema ceranae cause “Colony Collapse Disorder” in honey bees (Apis mellifera )? Journal of Apicultural Research. 2010;49(1):80-84. DOI: 10.3896/IBRA.1.49.1.11 - 58.
Nabian C, Ahmadi K, Nazem Shirazi MH, Gerami Sadeghian A. First detection of Nosema ceranae , a microsporidian protozoa of European honeybees (Apis mellifera ) in Iran. Iranian Journal of Parasitology. 2011;6(3):89-95. - 59.
Chen Y, Evans JD, Smith IB, Pettis JS. Nosema ceranae is a long-present and wide-spread microsporidian infection of the European honey bee (Apis mellifera ) in the United States. Journal of Invertebrate Pathology. 2008;97:186-188. DOI: 10.1016/j.jip.2007.07.010 - 60.
Williams GR, Shafer ABA, Rogers REL, Shutler D, Stewart DT. First detection of Nosema ceranae , a microsporidian parasite of European honey bees (Apis mellifera ), in Canada and central USA. Journal of Invertebrate Pathology. 2008;97(2):189-192. DOI: 10.1016/j.jip.2007.08.005 - 61.
Calderón RA, Sanchez LA, Yaňez O, Fallas N. Presence of Nosema ceranae in Africanized honey bee colonies in Costa Rica. Journal of Apicultural Research. 2008;47:328-329. DOI: 10.3896/IBRA.1.47.4.18 - 62.
Giersch T, Berg T, Galea F, Hornitzky M. Nosema ceranae infects honey bees (Apis mellifera ) and contaminates honey in Australia. Apidologie. 2009;40:117-123. DOI: 10.1051/apido/2008065 - 63.
Higes M, Martín-Hernández R, Garrido-Bailón E, Botias C, Meana A. The presence of Nosema ceranae (Microsporidia) in North African honey bees (Apis mellifera intermissa ). Journal of Apicultural Research. 2009;48:217-219. DOI: 10.3896/ibra.1.48.3.12 - 64.
Zinatullina ZJ, Zhigileva ON, Ignatjeva AN, Tokarev YS. Nosemosis of honeybees on the apiaries in Russian [Internet]. 2012. Available from: http://www.apiworld.ru/khochu-vsye-znat/iii-mezhdunarodnyy-forum-pchelovodov-medovyy-mir/nozematoz-na-pasekakh-rossii/ (Accessed 2015-12-12). - 65.
Ostroverkhova NV, Konusova OL, Kucher AN, Simakova AV, Golubeva EP, Kireeva TN, Sharakhov IV. Infestation of honeybee ( Apis mellifera ) byNosema (Microsporidia) in the Tomsk region. Parasitology. 2016;50(3). - 66.
Ostroverkhova NV, Konusova OL, Pogorelov YL, Kireeva TN, Salik MY, Golubeva EP. The first case of detection of Nosema ceranae on the apiary of the Tomsk region. Pchelovodstvo. 2014;9:28-30. - 67.
Fries I, Martin R, Meana A, García-Palencia P, Higes M. Natural infections of Nosema ceranae in European honey bees. Journal of Apicultural Research. 2006;45:230-233. DOI: 10.3896/ibra.1.45.4.13 - 68.
Higes M, Martín-Hernández R, Botías C, Garrido-Bailón E, González-Porto AV, Barrios L, del Nozal MJ, Bernal JL, Jiménez JJ, García-Palencia P, Meana A. How natural infection by Nosema ceranae causes honeybee colony collapse. Environmental Microbiology. 2008;10:2659-2669. DOI: 10.1111/j.1462-2920.2008.01687.x - 69.
Chauzat MP, Higes M, Martín-Hernández R, Meana A, Cougoule N, Faucon JP. Presence of Nosema ceranae in French honey bee colonies. Journal of Apicultural Research. 2007;46(2):127-128. DOI:10.1080/00218839.2007.11101380 - 70.
Cox-Foster DL, Conlan S, Holmes EC, Palacios G, Evans JD, Moran NA, Quan P-L, Briese S, Hornig M, Geiser DM, Martinson V, VanEngelsdorp D, Kalkseitn AL, Drysdale L, Hui J, Zhai J, Cui L, Hutchison S, Simons JF, Egholm M, Pettis JS, Lipkin WI. A metagenomic survey of microbes in honey bee colony collapse disorder. Science. 2007;318:283-287. DOI: 10.1126/science.1146498 - 71.
Mulholland GE, Traver BE, Johnson NG, Fell RD. Individual variability of Nosema ceranae infections inApis mellifera colonies. Insects. 2012;3(4):1143-1155. DOI: 10.3390/insects3041143 - 72.
Botias C, Martín-Hernández R, Barrios L, Meana A, Higes M. Nosema spp. infection and its negative effects on honey bees (Apis mellifera iberiensis ) at the colony level. Veterinary Research. 2013;44(1):25. DOI: 10.1186/1297-9716-44-25 - 73.
Goblirsch M, Huang ZY, Spivak M. Physiological and behavioral changes in honey bees ( Apis mellifera ) induced byNosema ceranae infection. PLoS ONE. 2013;8(3):e58165. DOI: 10.1371/journal.pone.0058165 - 74.
Francis RM, Amiri E, Meixner MD, Kryger P, Gajda A, Andonov S, Uzunov A, Topolska G, Charistos L, Costa C, Berg S, Bienkowska M, Bouga M, Büchler R, Dyrba W, Hatjina F, Ivanova E, Kezic N, Korpela S, Le Conte Y, Panasiuk B, Pechhacker H, Tsoktouridis G, Wilde J. Effect of genotype and environment on parasite and pathogen levels in one apiary – a case study. Journal of Apicultural Research. 2014;53(2):230-232. DOI: 10.3896/IBRA.1.53.2.14 - 75.
Meixner MD, Francis RM, Gajda A, Kryger P, Andonov S, Uzunov A, Topolska G, Costa C, Amiri E, Berg S, Bienkowska M, Bouga M, Büchler R, Dyrba W, Gurgulova K, Hatjina F, Ivanova E, Janes M, Kezic N, Korpela S, Le Conte Y, Panasiuk B, Pechhacker H, Tsoktouridis G, Vaccari G, Wilde J. Occurrence of parasites and pathogens in honey bee colonies used in a European genotype-environment interactions experiment. Journal of Apicultural Research. 2014;53(2):215-229. DOI: 10.3896/IBRA.1.53.2.04 - 76.
Milbrath MO, van Tran T, Huang WF, Solter LF, Tarpy DR, Lawrence F, Huang ZY. Comparative virulence and competition between Nosema apis andNosema ceranae in honey bees (Apis mellifera ). Journal of Invertebrate Pathology. 2015;125:9-15. DOI: 10.1016/j.jip.2014.12.006 - 77.
Fries I. Infectivity and multiplication of Nosema apis Z. in the ventriculus of the honey bee. Apidologie. 1988;19:319-328. DOI: 10.1051/apido:19880310 - 78.
Chen YP, Evans JD, Murphy C, Gutell R, Zuker M, Gundensen-Rindal D, Pettis JS. Morphological, molecular, and phylogenetic characterization of Nosema ceranae , a microsporidian parasite isolated from the European honey bee,Apis mellifera . Journal of Eukaryotic Microbiology. 2009;56:142-147. DOI: 10.1111/j.1550-7408.2008.00374.x - 79.
Higes M, Meana A, Bartolomé C, Botías C, Martín-Hernández R. Nosema ceranae (Microsporida), a controversial 21st century honey bee pathogen. Environmental Microbiology Reports. 2013;5(1):17-29. DOI:10.1111/1758-2229.12024 - 80.
Genersch E. Honey bee pathology: current threats to honey bees and beekeeping. Applied Microbiology and Biotechnology. 2010;87:87-97. DOI: 10.1007/s00253-010-2573-8 - 81.
Fries I. Nosema ceranae in European honey bees (Apis mellifera ). Journal of Invertebrate Pathology. 2010;103:S73-S79. DOI: 10.1016/j.jip.2009.06.017 - 82.
Gisder S, Hedtke K, Möckel N, Frielitz MC, Linde A, Genersch E. Five-year cohort study of Nosema spp. in Germany: does climate shape virulence and assertiveness ofNosema ceranae ? Applied and Environmental Microbiology. 2010;76(9):3032-3038. DOI: 10.1128/AEM.03097-09 - 83.
Higes M, García-Palencia P, Botias C, Meana A, Martín-Hernández R. The differential development of microsporidia infecting worker honey bee ( Apis mellifera ) at increasing incubation temperature. Environmental Microbiology Reports. 2010;2(6):745-748. DOI: 10.1111/j.1758-2229.2010.00170.x. - 84.
Sánchez Collado JG, Higes M, Barrio L, Martín-Hernández R. Flow cytometry analysis of Nosema species to assess spore viability and longevity. Parasitology Research. 2014;113(5):1695-1701. DOI:10.1007/s00436-014-3814-z - 85.
Van der Zee R, Gómez-Moracho T, Pisa L, Sagastume S, García-Palencia P, Maside X, Bartolomé C, Martín-Hernández R, Higes M. Virulence and polar tube protein genetic diversity of Nosema ceranae (Microsporidia) field isolates from Northern and Southern Europe in honeybees (Apis mellifera iberiensis ). Environmental Microbiology Reports. 2014;6(4):401-413. DOI: 10.1111/1758-2229.12133 - 86.
Kharitonov NN. Breeding of honeybees that are resistant to disease. Pchelovodstvo. 2006;7:14-16. - 87.
Bourgeois AL, Rinderer TE, Sylvester HA, Holloway B, Oldroyd BP. Patterns of Apis mellifera infestation byNosema ceranae support the parasite hypothesis for the evolution of extreme polyandry in eusocial insects. Apidologie. 2012;43(5):539-548. DOI: 10.1007/s13592-012-0121-5 - 88.
Chauzat MP, Laddomada A. Foreword. In: Dietemann V, Ellis JD, Neumann P, Editors. The COLOSS BEEBOOK, Volume II: Standard Methods for Apis mellifera Pest and Pathogen Research. Journal of Apicultural Research. 2013;52(4):1-2. DOI: 10.3896/IBRA.1.52.4.17 - 89.
Fenoy S, Rueda C, Higes M, Martín-Hernández R, del Aguila C. High-level resistance of Nosema ceranae , a parasite of the honeybee, to temperature and desiccation. Applied and Environmental Microbiology. 2009;75(21):6886-6889. DOI: 10.1128/AEM.01025-09 - 90.
Ostroverkhova NV, Konusova OL, Kucher AN, Kireeva TN, Baghirov RT-o. Characterization of the genetic diversity of honey bees ( Apis mellifera L.) in Tomsk population using mtDNA and microsatellite markers. A.I. Kurentsov’s Annual Memorial Meetings. 2015;XXVI:227-240.