Dead Honey Bees Research Articles

A semi-automated and high-throughput approach for the detection of honey bee viruses in bee samples.

Deformed wing virus (DWV) was first detected in dead honey bees in 1982 but has been in honey bees for at least 300 years. Due to its high prevalence and virulence, they have been linked with the ongoing decline in honey bee populations worldwide. A rapid, simple, semi-automated, high-throughput, and cost-effective method of screening colonies for viruses would benefit bee research and the beekeeping industry. Here we describe a semi-automated approach that combines an RNA-grade liquid homogenizer followed by magnetic bead capture for total virus nucleic acid extraction. We compare it to the more commonly applied nucleic acid column-based purification method and use qPCR plus Oxford Nanopore Technologies sequencing to evaluate the accuracy of analytical results for both methods. Our results showed high reproducibility and accuracy for both approaches. The semi-automated method described here allows for faster screening of viral loads in units of 96 samples at a time. We developed this method to monitor viral loads in honey bee colonies, but it could be easily applied for any PCR or genomic-based screening assays.

Microfungal flora of Apis mellifera anatoliaca (Hymenoptera: Apidae) and Varroa destructor (Mesostigmata: Varroidae) from the Eastern Black Sea Region and fungal vector capacity in honey bee colonies.

Honey bees have a great economic importance both in Turkey and in the world due to the products they produce and their contribution to pollination. For this reason, many microflora and microbiota studies have been conducted on bees. While these research were primarily focused on pathogen isolation, the ecological roles of non-pathogenic flora members and how they may be used are now being studied more extensively. Considering the importance of pathogens, the number of studies is expected to continue to increase. This study was carried out to determine the microfungal flora of the body surfaces and digestive tracts of dead honey bee (Apis mellifera anatoliaca) and Varroa destructor samples taken from different apiaries in the Eastern Black Sea Region of Turkey (Gümüşhane, Trabzon, Artvin and Ordu) in 2022. As a result of the study, a total of 11 different fungal species belonging to the genera Penicillium, Alternaria, Mucor, Trichoderma, Fusarium, Aspergillus and Verticillium were identified and the relationships of these fungi with bees were discussed based on the literature.

Visitation of Apis mellifera L. in Runner Bean (Phaseolus coccineus L.) and Its Exposure to Seasonal Agrochemicals in Agroecosystems

Plant species and abiotic factors including season appear to be the most important variables influencing the frequency of visits by honeybees (Apis mellifera L.). In the present study, we evaluated the activity of honeybee workers visiting runner bean (Phaseolus coccineus L.) local cultivar ‘Piękny Jaś’. The runner beans are widely cultivated in south-eastern Poland, and are an important forage plant for honeybees in agroecosystems. We aimed at a comprehensive monitoring of the health of colonies and symptoms in A. mellifera in response to acute exposure to pesticides. The most numerous visits of A. mellifera were observed at the highest flower opening of the runner bean. A very weak positive correlation was observed between the number of honeybees on P. coccineus, the number of visited flowers, the time spent per flower and air temperature. The visitation rates of honeybees were more frequent at mid-day and decreased after 15:00. Signs of poisoning were detected in two out of seven apiaries monitored for acute pesticide exposure symptoms on runner bean plantations. The analysis of dead honeybee samples revealed the acute exposure of honeybees to the imidacloprid (neonicotinoid) and chlorpyrifos (organophosphorus) insecticides, which are highly toxic and banned in the European Union. Hazard quotient (HQ) screening showed an elevated burden of imidacloprid and chlorpyrifos corresponding to 7.1% and 10% of the LDD50, respectively, most likely indicating bee poisoning due to chronic exposure to these substances with contaminated food. Noteworthy was the presence of three fungicides that could pose a risk of poisoning in honeybees.

Metagenomic analysis of viromes in honey bee colonies (Apis mellifera; Hymenoptera: Apidae) after mass disappearance in Korea.

After the nationwide, massive winter losses of honey bees in Korea during the winter of 2021, samplings were conducted from live honey bees in colonies and dead honey bees nearby colonies in the same bee-farms in six regions in Korea. Each sample was subjected to virome analysis using high-throughput sequencing technology. The number of viral reads was the lowest in the live honey bee group sample with 370,503 reads and the highest in the dead honey bee group sample with 42,659,622 reads. Viral contigs were matched with the viral genomes of the black queen cell virus, deformed wing virus, Israeli acute paralysis virus, and sacbrood virus, all of which have been previously reported in Korea. However, Apis rhabdovirus 5, bee macula-like virus, Varroa orthomyxovirus-1, Hubei partiti-like virus 34, Lake Sinai virus 2, 3, and 4, and the Ditton virus, were also discovered in this study, which are the first records in Korea. Plant viral sequences resembling those of Arabidopsis latent virus 1, and a novel viral sequence was also discovered. In the present study 55 complete viral genome sequences were identified. This study is the first virome analysis of domestic honey bees and provides the latest information on the diversity of honey bee viruses in Korea.

Determination of Pesticides in Dead Honeybees

Pesticide residues were analyzed in dead honeybees by gas chromatography-tandem mass spectrometry (GC-MS/MS). Sample preparation using QuEChERS was followed by purification on a mixture of PSA and C18 sorbents. The extraction efficiency of > 80% was obtained for all analytes and pesticides were quantified in the range of 5-1000 ng/g. This method was used for investigating incidents of honeybees poisoning by pesticides. Fipronil was detected in all of the investigated samples originating from different regions of Russia. Additionally, τ-fluvalinate was detected in three samples.

The Honey Bee: An Active Biosampler of Environmental Pollution and a Possible Warning Biomarker for Human Health

Member states of the European Union are required to ensure the initiation of monitoring programs to verify honey bee exposure to pesticides, where and as appropriate. Based on 620 samples of dead honey bees—42 of pollen, 183 of honey and 32 of vegetables—we highlighted the presence, as analyzed by liquid and gas chromatography coupled with tandem mass spectrometric detection, of many active substances, mainly tau-fluvalinate, piperonyl butoxide, chlorpyrifos and chlorpyrifos-methyl, permethrin and imidacloprid. Among the active substances found in analyzed matrices linked to honey bee killing incidents, 38 belong to hazard classes I and II, as methiocarb, methomyl, chlorpyrifos, cypermethrin and permethrin, thus representing a potential risk for human health. We have shown that, at different times between 2015 and 2020, during implementation of the Italian national guidelines for managing reports of bee colony mortality or depopulation associated with pesticide use, pesticide pollution events occurred that could raise concern for human health. Competent authorities could, as part of a One Health approach, exploit the information provided by existing reporting programs on honey bees and their products, in view of the close correlation to human health, animal health and ecosystem health.

The Nasonov gland pheromone as a potential source of death cue in Apis cerana

The ability to detect and remove dead adult bees is an essential part of honeybee colony fitness that prevents the spread of pathogens. Fatty acid olfactory cues stimulate undertaking behavior among different social species within Hymenoptera, but the chemicals responsible for the death cue in Apis cerana have not yet been identified. We explored the Nasonov gland as a potential source of these chemicals in A. cerana. Gas chromatography indicated that unlike A. mellifera, the A. cerana Nasonov gland does not contain any volatile terpenes, only fatty acids. As a bioassay, dead honeybees were rinsed free of their individual cuticular hydrocarbons via dichloromethane and two concentrations of oleic acid and a synthetic blend of the Nasonov pheromone in A. cerana were applied to the dummies. Results showed that oleic acid did not stimulate corpse removal in A. cerana. However, the synthetic pheromone blend of A. cerana Nasonov did stimulate removal.

Ratios of Stable Isotopes 13С/12С and 15N/14N in Samples of Dead Honey Bees and Beekeeping Products

Ratios of Stable Isotopes 13С/12С and 15N/14N in Samples of Dead Honey Bees and Beekeeping Products

Identification of Mesophylic Bacterial Flora in Deceased Worker Adults of Apis mellifera caucasia (Pollmann, 1889)

Apis mellifera, widely farmed around the world, is the most economically important species within the genus Apis. While the microbiota of live honey bees have been extensively examined, bacteria found in deceased honey bees (which might indicate infection or opportunistic pathogens) is in contrast poorly studied. Therefore, we decided to investigate the mesophilic bacterial flora of dead honey bees. So, in September 2013, dead adult worker honey bees were collected from 12 different cities, most of which were in the border provinces of Turkey. We identified bacterial isolates at the species level by using different morphological, biochemical, physical and molecular methods, in conjunction with molecular phylogenetic analysis. We constructed phylogenetic trees for isolated bacteria with the MEGA 6.0 program and neighbor-joining trees were reconstructed based on 16S rDNA gene sequences. The phylogenetic trees indicated that isolates DE003, DE007, DE011, DE001, DE019 and DE016, DE029 could be new members of the genera Erwinia, Acidovorax, Hydrogenophaga and Bacillus genus, respectively. In the bioassay study results, we observed that DE019 Hydrogenophaga sp. (64.7%) and DE004 Klebsiella grimontii (73.3%) had lethal effects on the honey bees. The other mortalities ranged from 10% to 25% (p>0.05), and according to a One-Way ANOVA analysis DE004 and DE019 significantly affect the A. mellifera caucasia in adult worker honey bees. This study is the first report of Hydrogenophaga as honey bee pathogen.

Gone with the Wind? Dead Apis mellifera Bees Washed Up on a Tropical Beach

It is very uncommon for thousands of dead honey bees to wash up on beaches (Taylor Jr, 1977). As a result of this, little is known on the mortality of flying insects along shorelines (Schaefer et a...

Foodborne Transmission and Clinical Symptoms of Honey Bee Viruses in Ants Lasius spp.

Emerging infectious diseases are often the products of host shifts, where a pathogen jumps from its original host to a novel species. Viruses in particular cross species barriers frequently. Acute bee paralysis virus (ABPV) and deformed wing virus (DWV) are viruses described in honey bees (Apis mellifera) with broad host ranges. Ants scavenging on dead honey bees may get infected with these viruses via foodborne transmission. However, the role of black garden ants, Lasius niger and Lasius platythorax, as alternative hosts of ABPV and DWV is not known and potential impacts of these viruses have not been addressed yet. In a laboratory feeding experiment, we show that L. niger can carry DWV and ABPV. However, negative-sense strand RNA, a token of virus replication, was only detected for ABPV. Therefore, additional L. niger colonies were tested for clinical symptoms of ABPV infections. Symptoms were detected at colony (fewer emerging workers) and individual level (impaired locomotion and movement speed). In a field survey, all L. platythorax samples carried ABPV, DWV-A and –B, as well as the negative-sense strand RNA of ABPV. These results show that L. niger and L. platythorax are alternative hosts of ABPV, possibly acting as a biological vector of ABPV and as a mechanical one for DWV. This is the first study showing the impact of honey bee viruses on ants. The common virus infections of ants in the field support possible negative consequences for ecosystem functioning due to host shifts.

Body position of dead honey bee workers could indicate the cause of death

Body position of dead honey bee workers could indicate the cause of death

A Survey from 2015 to 2019 to Investigate the Occurrence of Pesticide Residues in Dead Honeybees and Other Matrices Related to Honeybee Mortality Incidents in Italy

Honeybee health can be compromised not only by infectious and infesting diseases, but also by the acute or chronic action of certain pesticides. In recent years, there have been numerous reports of colony mortality by Italian beekeepers, but the investigations of these losses have been inconsistent, both in relation to the type of personnel involved (beekeepers, official veterinarians, members of the police force, etc.) and the procedures utilized. It was therefore deemed necessary to draw up national guidelines with the aim of standardizing sampling active ties. In this paper, we present the results of a survey carried out in Italy from 2015 to 2019, following these guidelines. Residues of 150 pesticides in 696 samples were analyzed by LC-MS/MS and GC-MS/MS. On average, 50% of the honeybee samples were positive for one or more pesticides with an average of 2 different pesticides per sample and a maximum of seven active ingredients, some of which had been banned in Europe or were not authorized in Italy. Insecticides were the most frequently detected, mainly belonging to the pyrethroid group (49%, above all tau-fluvalinate), followed by organophosphates (chlorpyrifos, 18%) and neonicotinoids (imidacloprid, 7%). This work provides further evidence of the possible relationship between complex pesticide exposure and honeybee mortality and/or depopulation of hives.

Foodborne Transmission of Deformed Wing Virus to Ants (Myrmica rubra).

Virus host shifts occur frequently, but the whole range of host species and the actual transmission pathways are often poorly understood. Deformed wing virus (DWV), an RNA virus described from honeybees (Apis mellifera), has been shown to have a broad host range. Since ants are often scavenging on dead honeybees, foodborne transmission of these viruses may occur. However, the role of the ant Myrmica rubra as an alternative host is not known and foodborne transmission to ants has not been experimentally addressed yet. Here, we show with a 16-week feeding experiment that foodborne transmission enables DWV type-A and -B to infect M. rubra and that these ants may serve as a virus reservoir. However, the titers of both plus- and minus-sense viral RNA strands decreased over time. Since the ants were fed with highly virus-saturated honeybee pupae, this probably resulted in initial viral peaks, then approaching lower equilibrium titers in infected individuals later. Since DWV infections were also found in untreated field-collected M. rubra colonies, our results support the wide host range of DWV and further suggest foodborne transmission as a so far underestimated spread mechanism.

Neonicotinoids and fipronil concentrations in honeybees associated with pesticide use in Brazilian agricultural areas

Brazil leads global lists of honeybee colony losses in South America as well as pesticide use, according to a web-based survey (http://www.nobeenofood.com/beealert). In association with that survey, Africanized honeybee (Apis mellifera) samples were opportunistically collected when bee poisoning was apparently linked to pesticide use in crops. The objective was to determine concentrations of fipronil and neonicotinoids in live and dead honeybees, in areas where these compounds are widely used in agriculture. Pesticide residues in honeybees (54 live and 60 dead composite samples) were detected with mass spectrometry (UHPLC-MS/MS using QuEChERS methodology). Toxicological analyses in both matrices detected multiple contaminations with highest indices by fipronil with frequency of 55.3% and amplitude (0.7–23,539.7 ng/g), thiamethoxam 20.2% (0.6–13.6 ng/g), imidacloprid 3.5% (4.5–16.2 ng/g), nitenpyram 1.8% (3.8–7.4 ng/g), and thiacloprid 0.9% (1.6 ng/g). Neonicotinoids and fipronil residues had higher frequencies and amplitudes in honeybees collected near sugarcane plantations and orange orchards in northwest Sao Paulo state and other agro-industrial rural landscapes across the country dominated with fields of soybean, corn, and tropical fruit crops. These systemic pesticides were presumed to be primary mechanisms of honeybee colony losses in Brazil, according to a recently published 5-year survey by the same authors and reinforced by current analyses.

A two-year monitoring of pesticide hazard in-hive: High honey bee mortality rates during insecticide poisoning episodes in apiaries located near agricultural settings

Pesticide residues in beebread, live and dead honey bees, together with honey bee death rate were monitored from June 2016 to June 2018 in three apiaries, located near agricultural settings and in wildlands. Dead honey bees were only collected and analyzed when significant mortality episodes occurred and pesticide content in beeswax of each experimental apiary was evaluated at the beginning of the study. Samples were extracted by a modified QuEChERS procedure and screened for pesticides residues by liquid chromatography mass spectrometry (LC-MS/MS). Pesticide hazard in the samples was evaluated through the hazard quotient approach (HQ). Beebread was widely contaminated with coumaphos and amitraz degradate 2, 4-dimethylphenylformamide (DMF), miticides detected in 94 and 97% of samples respectively. However, insecticides sprayed during citrus bloom like chlorpyrifos (up to 167 ng g −1) and dimethoate (up to 34 ng g −1) were the main responsible of the relevant pesticide hazard in this matrix. Pesticide levels in live bees were mostly residual, and pesticide hazard was low. Beeswax of the apiaries, contaminated by miticides, revealed a low pesticide hazard to honey bee colonies. Acute mortality episodes occurred only in the two apiaries located near agricultural settings. Dead bees collected during these episodes revealed high levels (up to 2700 ng g −1) of chlorpyrifos, dimethoate, omethoate and imidacloprid. HQ calculated in dead bees exceeded up to 37 times the threshold value considered as elevated hazard to honey bee health.

Clostridium botulinum spores in European honey bees from Serbia

A total of 61 honey bees from different regions of the Republic of Serbia were analyzed for Clostridium botulinum (C. botulinum) spores. The microbiological methods and molecular methods (multiplex PCR/mPCR and PCR method) were utilized to examine multiple subunits of each honey bees samples. The C. botulinum spores in PCR-positive samples were estimated by the most probable number method (MPN). The presence of C. botulinum spores, by applying mPCR and PCR methods, was detected in 1 of the 61 honey bees (1.64%). Using MPN method, the number of spores in positive sample was 110/kg. Detection of C. botulinum spores directly from untreated honey bees, without prior enrichment, is impossible by applying PCR. Using conventional microbiological methods, detection of C. botulinum spores in dead honey bees is not possible without preenrichment. Therefore, conventional, microbiological methods are not suitable for the detection of C. botulinum spores in honey bees. In order to detect C. botulinum spores in honey bees using PCR methods, due to the small and/or unequal distribution of spores in the samples, it is desirable to use multiple subunits/replicates for each sample examined.

First report on the presence of Megaselia rufipes (Meigen, 1804) and Megaselia praeacuta (Schmitz, 1919) in honey bee colonies in the Republic of Macedonia

The presence of the two species Megaselia rufipes (Meigen, 1804) and Megaselia praeacuta (Schmitz, 1919) (Diptera: Phoridae) was recorded from samples of dead honey bee colonies under laboratory conditions during 2017. The material was taken from an apiary in Bitola Region where the winter losses of honey bee colonies in 2016/2017 season were estimated at 97%. This was the first record of M. rufipes in the fauna of the Republic of Macedonia. Another species that has previously been recorded as a leaf miner and pest on chickpeas is M. praeacuta, but this was the first time when the species was recorded on honey bee colonies. Both species emerged from dead honey bees.

The digestive index as a benchmark to quantify the digestive capabilities of honey bees (Apis mellifera)

An easy method to obtain the digestive index as a benchmark number for measuring the digestion capabilities of Apis mellifera is developed and presented in this paper. The aim of this method is to provide an entity that allows to quantifying digestion efficiency of the western honey bee. This will enable a fundamental understanding of digestive illnesses in bee hives as well as a general insight into the biological mechanisms into the digestive apparatus. Although the methodology presented in this work is based on experimental data of honey bees it can, in principle, be applied to other insects as well, provided that the necessary experimental data can be made available. A small number of seven bees is used to provide an example of how to calculate the digestive index. The bowels were extracted from these seven dead honey bees and their mass in fresh and dried condition was used for the determination of the digestive index. The mathematical model for the calculation of the digestive index is described. Thus, it is demonstrated that the digestion capability of honey bees can be quantified by the simple method outlined in this paper.

Metabolisation of thiamethoxam (a neonicotinoid pesticide) and interaction with the Chronic bee paralysis virus in honeybees

Pathogens and pesticides are likely to co-occur in honeybee hives, but much remains to be investigated regarding their potential interactions. Here, we first investigated the metabolisation kinetics of thiamethoxam in chronically fed honeybees. We show that thiamethoxam, at a dose of 0.25ng/bee/day, is quickly and effectively metabolised into clothianidin, throughout a 20day exposure period. Using a similar chronic exposure to pesticide, we then studied, in a separate experiment, the impact of thiamethoxam and Chronic bee paralysis virus (CBPV) co-exposure in honeybees. The honeybees were exposed to the virus by contact, mimicking the natural transmission route in the hive. We demonstrate that a high dose of thiamethoxam (5.0ng/bee/day) can cause a synergistic increase in mortality in co-exposed honeybees after 8 to 10days of exposure, with no increase in viral loads. At a lower dose (2.5ng/bee/day), there was no synergistic increase of mortality, but viral loads were significantly higher in naturally dead honeybees, compared with sacrificed honeybees exposed to the same conditions. These results show that the interactions between pathogens and pesticides in honeybees can be complex: increasing pesticide doses may not necessarily be linked to a rise in viral loads, suggesting that honeybee tolerance to the viral infection might change with pesticide exposure.