Meral Kence

Managed honey bee colony losses in Canada, China, Europe, Israel and Turkey, for the winters of 2008–9 and 2009–10

Summary In 2008 the COLOSS network was formed by honey bee experts from Europe and the USA. The primary objectives set by this scientific network were to explain and to prevent large scale losses of honey bee (Apis mellifera) colonies. In June 2008 COLOSS obtained four years support from the European Union from COST and was designated as COST Action FA0803—COLOSS (Prevention of honey bee Colony Losses). To enable the comparison of loss data between participating countries, a standardized COLOSS questionnaire was developed. Using this questionnaire information on honey bee losses has been collected over two years. Survey data presented in this study were gathered in 2009 from 12 countries and in 2010 from 24 countries. Mean honey bee losses in Europe varied widely, between 7–22% over the 2008–9 winter and between 7–30% over the 2009–10 winter. An important finding is that for all countries which participated in 2008–9, winter losses in 2009–10 were found to be substantially higher. In 2009–10, winter losses in South East Europe were at such a low level that the factors causing the losses in other parts of Europe were absent, or at a level which did not affect colony survival. The five provinces of China, which were included in 2009–10, showed very low mean (4%) A. mellifera winter losses. In six Canadian provinces, mean winter losses in 2010 varied between 16–25%, losses in Nova Scotia (40%) being exceptionally high. In most countries and in both monitoring years, hobbyist beekeepers (1–50 colonies) experienced higher losses than practitioners with intermediate beekeeping operations (51–500 colonies). This relationship between scale of beekeeping and extent of losses effect was also observed in 2009–10, but was less pronounced. In Belgium, Italy, the Netherlands and Poland, 2008–9 mean winter losses for beekeepers who reported ‘disappeared’ colonies were significantly higher compared to mean winter losses of beekeepers who did not report ‘disappeared’ colonies. Mean 2008–9 winter losses for those beekeepers in the Netherlands who reported symptoms similar to “Colony Collapse Disorder” (CCD), namely: 1. no dead bees in or surrounding the hive while; 2. capped brood was present, were significantly higher than mean winter losses for those beekeepers who reported ‘disappeared’ colonies without the presence of capped brood in the empty hives. In the winter of 2009–10 in the majority of participating countries, beekeepers who reported ‘disappeared’ colonies experienced higher winter losses compared with beekeepers, who experienced winter losses but did not report ‘disappeared’ colonies.

A review of methods for discrimination of honey bee populations as applied to European beekeeping

Summary Here, scientists from 19 European countries, most of them collaborating in Working Group 4: “Diversity and Vitality” of COST Action FA 0803 “Prevention of honey bee COlony LOSSes” (COLOSS), review the methodology applied in each country for discriminating between honey bee populations. Morphometric analyses (classical and geometric) and different molecular markers have been applied. Even if the approach has been similar, however, different methodologies regarding measurements, landmarks or molecular markers may have been used, as well as different statistical procedures. There is therefore the necessity to establish common methods in all countries in order to have results that can be directly compared. This is one of the goals of WG4 of the COLOSS project.

Mitochondrial DNA variation in honey bee (Apis mellifera L.) populations from Turkey

Summary We have studied mitochondrial (mt) DNA variation in 334 honey bee colonies from 7 different geographic regions of Turkey. We have evaluated Dra I restriction profiles of the CO-I CO-II intergenic region, Hinf-I and Taq-I restriction profiles of the CO-I gene and EcoR-I restriction profiles of the whole mtDNA. We obtained three different mtDNA patterns by EcoR-I digestion. The pattern typical for A. m. carnica/A. m. ligustica predominated throughout Turkey (327 colonies, 97.9%). We observed the pattern common in African subspecies only in Hatay province (6 colonies, 1.8%) and a previously unreported pattern in one colony from Balıkesir province (0.3%). Dra-I restriction analysis of the CO-I CO-II intergenic region yielded seven haplotypes. Haplotype 1 (TrDra-1) was the most common one found in Turkey, whereas haplotype 2 (TrDra-2) was widely distributed in Eastern Anatolia. Based on mitochondrial ND2 sequences taken from two samples collected in each region, bees from Hatay clustered with A. m. lamarckii and A. m. meda (morphological A and O lineages), while bees from central Anatolia clustered within the C morphological lineage group.

Molecular detection of Nosema ceranae and N. apis from Turkish honey bees

Polymerase chain reaction specific for the rDNA marker for Nosema ceranae and Nosema apis was conducted on 84 Apis mellifera samples collected from 20 provinces in Turkey. N. ceranae was detected from three samples from the provinces of Artvin, Hatay, and Muğla. N. apis was detected in samples from the provinces of Sivas, Izmir, Bitlis and Gaziantep. All of the positive samples were from honey bees belonging to the ‘C’ lineage of A. mellifera. DNA sequencing analysis of the N. ceranae samples revealed that there was no intraspecific variation in the 208 bp of the 16S SSU of N. ceranae from Turkey. A TCS analysis revealed that the 16S SSU genotype from Turkey is identical to N. ceranae DNA sequences from Europe, Australia, and the United States. TCS analysis also revealed that this genotype is the basal ancestral genotype among six N. ceranae genotypes. This is the first study to confirm that N. ceranae is present in honey bees from Turkey.

Genetic structure of honeybee, Apis mellifera L. (Hymenoptera :Apidae) populations of Turkey inferred from microsatellite analysis

Summary We analyzed the genetic structure of II honeybee (Apis mellifera L) populations from Turkey and one from Cyprus using 9 microsatellite marker loci. Heterozygosity levels, mean number of alleles per population, number of diagnostic alleles, and pairwise FST values were computed for the populations studied. Heterozygosity levels were found to range between 0.54 and 0.68. We detected high levels of genetic divergence among the populations based on pairwise FST values; 50 of 66 pairwise FST values were significant. The presence of a large number of rare alleles and highly differentiated populations of honeybees are consistent with Anatolias role as a genetic center for Middle Eastern honeybees. We suggest that certain precautions should be taken to limit introduction of foreign subspecies to preserve native genetic resources.

Determination of Malathion and Diazinon Resistance by Sequencing the MdαE7 Gene from Guatemala, Colombia, Manhattan, and Thailand Housefly (Musca domestica L.) Strains

Organophosphate (OP) insecticides (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of αE7 gene, which is a member of α-esterase gene cluster, is probably playing a role in detoxyfication of the xenobiotic esters. In parathion/diazinon resistant M. domestica species Gly137 to Asp substitution was found in the active center of the product of αE7 gene. In malathion (an OP) resistant M. domestica strains Trp251 to Ser substitution was identified in the active center of the MdαE7. In our research, to understand the allelic diversity of the MdαE7, the gene was partially sequenced from four different housefly strains from different localities (Guatemala, Manhattan (USA), Colombia (USA), and Thailand). It was found out that; in Thailand strain one allele has Cys residue at the position of 251, the other allele contains a Trp for the same site. In Colombia strain, one allele has Asp137, the other allele contains a Gly residue at this point. The Manhattan and Guatemala strains have Asp137 and Trp251 residues on their both alleles at these two different positions.

The Genetic Basis of Malathion Resistance in Housefly ( Musca domestica L.) Strains From Turkey

Organophosphate insecticide (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of MdαE7 gene is probably playing a role in detoxification of xenobiotic esters. In our research, we have isolated, cloned and sequenced the MdαE7 gene from five different Turkish housefly strains. High doses of malathion (600 μg/fly) were applied in a laboratory environment for one year to Ceyhan1, Ceyhan2, Adana, and Ankara strains while no insecticide treatment was performed in the laboratory to Kirazli strain. Trp251 → Ser substitution was found in the product of MdαE7 gene in all malathion-resistant and Kirazli stocks. In addition, we checked the malathion carboxylesterase (MCE), percent remaining activities in acetylcholinesterase (AChE), glutathion-S-transferase (GST), and general esterase activities in all five strains used in this study. In comparing with universal standard sensitive control WHO, a high level of MCE and GST activities were observed while lower level of general esterase activities was detected in the tested strains. In addition, a higher percent remaining activities in AChE than WHO susceptible strain were observed in all malathion-resistant strains.

Proboscis Conditioning Experiments with Honeybees, Apis Mellifera Caucasica, with Butyric Acid and DEET Mixture as Conditioned and Unconditioned Stimuli

Abstract Three experiments are described investigating whether olfactory repellents DEET and butyric acid can support the classical conditioning of proboscis extension in the honeybee, Apis mellifera caucasica (Hymenoptera: Apidae). In the first experiment DEET and butyric acid readily led to standard acquisition and extinction effects, which are comparable to the use of cinnamon as a conditioned stimulus. These results demonstrate that the odor of DEET or butyric acid is not intrinsically repellent to honey bees. In a second experiment, with DEET and butyric acid mixed with sucrose as an unconditioned stimulus, proboscis conditioning was not established. After several trials, few animals responded to the unconditioned stimulus. These results demonstrate that these chemicals are gustatory repellents when in direct contact. In the last experiment a conditioned suppression paradigm was used. Exposing animals to butyric acid or DEET when the proboscis was extended by direct sucrose stimulation or by learning revealed that retraction of the proboscis was similar to another novel odor, lavender, and in all cases greatest when the animal was not permitted to feed. These results again demonstrate that DEET or butyric acid are not olfactory repellents, and in addition, conditioned suppression is influenced by feeding state of the bee.

Scientific note: colony losses survey in Turkey and causes of bee deaths

There have been unexpected and alarmingcolony losses in different regions of the world in thepast few years (reviewed in Oldroyd, 2007; EFSA,2008; see also vanEngelsdorp et al., 2008). Concur-rent with those in the US, high colony losses alsowerereportedinTurkey(Girayetal.,2007). Healthof honey bees in Turkey is of general importance,both because of native honey bee biodiversity (seeBodur et al., 2007; reviewed in Kence, 2006)anda high number of domestic colonies (>5 million;FAOSTAT, 2008), which is second only to China.We investigated the extent and causes of losses inTurkey using a questionnaire study, which was con-ducted in 2007 and covered the three previous years.This report includes analyses of 288 question-naires reporting on 35597 colonies and, in contrastwith a preliminary report (88 questionnaires; Girayet al., 2007), provides a broad representation ofgeographical regions, a better estimate of regionalcolony losses, and novel insights such as on colonycollapse disorder (CCD) symptoms and losses.Questionnaires were collected through an aca-demic and trade journal for beekeeping (U. BeeJ.), local beekeeping organizations, their field repre-sentatives, the internet, and Turkish mail. Returnedquestionnaires (see supplemental information S1)included names and contacts of beekeepers. The re-sults were coded and analyzed by different authorsCorresponding author: T. Giray,tgiray2@yahoo.com*Manuscript editor: David TarpyOnline material is available at:http://www.apidologie.orgto maintain blindness and confidentiality. Statisticalanalyses were performed using JMP

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 Worlds 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.