Honey Bee Parasite Research Articles

Assessing virulence of Varroa destructor mites from different honey bee management regimes

The mite Varroa destructor is an important honey bee parasite that causes substantial losses of honey bee colonies worldwide. Evolutionary theory suggests that the high densities at which honey bees are managed in large-scale beekeeping settings will likely select for mites with greater growth and virulence, thereby potentially explaining the major damage done by these mites. We tested this hypothesis by collecting mites from feral bee colonies, “lightly” managed colonies (those from small-scale sedentary operations), and “heavily” managed colonies (those from large-scale operations that move thousands of colonies across the US on a yearly basis). We established 8 apiaries, each consisting of 11 colonies from a standardized lightly managed bee background that were cleared of mites, and artificially infested each apiary with controlled numbers of mites from feral, lightly managed, or heavily managed bees or left uninoculated as negative control. We monitored the colonies for more than 2 years for mite levels, colony strength (adult bee population, brood coverage, and honey storage), and survival. As predicted by evolutionary theory, we found that colonies inoculated with mites from managed backgrounds had increased V. destructor mite levels relative to those with mites from feral colonies or negative controls. However, we did not see a difference between heavily and lightly managed colonies, and these higher mite burdens did not translate into greater virulence, as measured by reductions in colony strength and survival. Our results suggest that human management of honey bee colonies may favor the increased population growth rate of V. destructor, but that a range of potential confounders (including viral infections and genotype-by-genotype interactions) likely contribute to the relationship between mite reproduction and virulence.

Hydroxylation patterns associated with pheromone synthesis and composition in two honey bee subspecies Apis mellifera scutellata and A. m. capensis laying workers

Colony losses due to social parasitism in the form of reproductive workers of the Apis mellifera capensis clones results from the production of queen-like pheromonal signals coupled with ovarian activation in these socially parasitic honey bees. While the behavioral attributes of these social parasites have been described, their genetic attributes require more detailed exploration. Here, we investigate the production of mandibular gland pheromones in queenless workers of two sub-species of African honey bees; A. m. scutellata (low reproductive potential) and A. m. capensis clones (high reproductive potential). We used standard techniques in gas chromatography to assess the amounts of various pheromone components present, and qPCR to assess the expression of cytochrome P450 genes cyp6bd1 and cyp6as8, thought to be involved in the caste-dependent hydroxylation of acylated stearic acid in queens and workers, respectively. We found that, for both subspecies, the quality and quantity of the individual pheromone components vary with age, and that from the onset, A. m. capensis parasites make use of gene pathways typically upregulated in queens in achieving reproductive dominance. Due to the high production of 9-hydroxy-decenoic acid (9-HDA) the precursor to the queen substance 9-oxo-decenoic acid (9-ODA) in newly emerged capensis clones, we argue that clones are primed for parasitism upon emergence and develop into fully fledged parasites depending on the colony's social environment.

Prevalence and population genetics of the emerging honey bee pathogen DWV in Chinese apiculture

Honey bees are agriculturally important, both as pollinators and by providing products such as honey. The sustainability of beekeeping is at risk through factors of global change such as habitat loss, as well as through the spread of infectious diseases. In China and other parts of Asia, beekeepers rely both on native Apis cerana and non-native Apis mellifera, putting bee populations at particular risk of disease emergence from multi-host pathogens. Indeed, two important honey bee parasites have emerged from East Asian honey bees, the mite Varroa destructor and the microsporidian Nosema ceranae. As V. destructor vectors viral bee diseases, we investigated whether another key bee pathogen, Deformed Wing Virus (DWV), may also have originated in East Asian honey bee populations. We use a large-scale survey of apiaries across China to investigate the prevalence and seasonality of DWV in managed A. mellifera and A. cerana colonies, showing that DWV-A prevalence was higher in A. mellifera, with a seasonal spike in prevalence in autumn and winter. Using phylogenetic and population genetic approaches, we show that while China and East Asian DWV isolates show comparatively high levels of genetic diversity, these bee populations are not a source for the current global DWV epidemic.

Comparison of qPCR and Morphological Methods For Detection of Acarapis Woodi in Honey Bee Samples

Abstract The honey bee tracheal mite Acarapis woodi is an internal obligate parasite of adult honey bees (Apis mellifera). The small size of the mites and location within the bee’s trachea create a challenge for diagnostic identification. Detailed assessment of low-level mite infestation involves microscopic examination of the bee’s tracheae, but this traditional diagnostic method takes a long time. In recent years, new molecular diagnostic techniques have been developed to identify A. woodi with the use of PCRs. The purpose of this study was to compare the diagnostic sensitivity of microscopic examination and qPCR method for the detection of A. woodi in honey bee samples. Thirty-six samples were analyzed with both methods and qPCR was more sensitive than the microscopic method to detect A. woodi. This work demonstrates the practical application of molecular technology as a support tool for surveys and contingency management and to provide robust surveillance data on the presence or absence of A. woodi in honey bee colonies.

봉변에서 특이 유전자 검출법에 의한 봉군 내 꿀벌가시응애류 (Tropilaelaps)의 정량적 검출

Rapid detection of Tropilaelaps, an external parasite of honeybees that lead to malformation of honeybee or colony collapse disorder, is becoming important. But it is very difficult to find with the naked eye of Tropilaelaps. In this study, we have developed a method to detect the specific gene of Tropilaelaps from the hive debris and to know the number of Tropilaelaps in the hive through Tropilaelaps-specific quantitative detection. Tropilaelaps-specific gene amplified in DNA extracted from hive debris by consecutive PCR (1st detection, 2nd nested PCR). It could detect 10SUP1/SUP molecules level of Tropilaelapsspecific gene and confirm the amplification of the Tropilaelaps-specific gene. It was possible to accurately quantify the number of Tropilaelaps from the hive debris sample, which is difficult to discriminate the presence of Tropilaelaps visually, through Tropilaelaps-specific detection. Under the microscope, Tropilaelaps was collected and quantitative detection of Tropilaelaps-specific genes was performed. It was possible to quantify the number of Tropilaelaps present in the hive through the molecules of the quantified Tropilaelaps-specific genes. We suggest that hive debris can represent as a micro-environment to hive and show that it can be a simpler and more accurate sample than using a parasitic host honeybee. We expect that hive debris should facilitate the monitoring of Tropilaelaps in hive.

Evaluación de la eficacia de cuatro plantas medicinales como fumigantes contra Varroa destrutor en Argelia

Varroa destructor es el parásito más dañino de la abeja melífera (Apis mellifera) y es una de las mayores amenazas para la apicultura en todo el mundo. En la actualidad se han utilizado diversos productos químicos y naturales. El objetivo del presente estudio es evaluar el efecto del humo de cuatro plantas medicinales (Rosmarinus officinalis,Thymus pallescens, Mentha viridus y Laurus nobilis) contra el ácaro varroa en veinticinco colonias de abejas melíferas argelinas. 50 g de cada planta seca se administró durante un mes utilizando ahumador de abejas . Los resultados han mostrado que todas las plantas mostraron actividad fumigante en diferentes grados contra el ácaro varroa. La mayor tasa de eficacia se obtuvo con el laurel y el romero, que alcanzaron más del 80% de la mortalidad, seguidos por el tomillo y la hierbabuena. El ANOVA mostró una diferencia significativa entre los tratamientos (P

The presence of a larval honey bee parasite, Ascosphaera apis, on flowers reduces pollinator visitation to several plant species

Many floral traits have evolved through pollinator-mediated selection. In selecting flowers for forage, pollinators rely on multi-modal flower signals and innate preferences. However, pollinators are known for their flexible foraging behavior which varies with experience and numerous cues, such as the presence of competitors and floral resource availability. The shared use of flowers by many pollinators leads to high rate of parasite transmission among pollinators, and flowers can be seen as hot-spots of parasite dispersal. Throughout the animal kingdom, behavioral adaptations enable avoidance of parasite exposure. Bumblebees are known to avoid contaminated flowers, however nothing is known regarding the impact of parasite presence on overall pollinator visitation. In this study, we measured the effect of a larval honey bee parasite, the fungus Ascosphaera apis causing chalkbrood disease, on hymenopteran pollinator visitation of several plant species in a natural setting. Several contaminated flowering plant species suffered not only the subsequent reduction of visitation by honey bees but also the subsequent reduction of their overall pollinator visitation. Two plant species with similar flower architecture were the least impacted by the presence of the spores. This study highlights the negative impact of the presence of pollinator parasites for plants and its possible consequences for floral evolution.

New Record of the Small Hive Beetle, Aethina tumida, in Africanized Honey Bee Colonies in Costa Rica

The small hive beetle (SHB) Aethina tumida Murray (Insecta: Coleoptera: Nitidulidae) is a honey bee parasite native to sub-Saharan Africa (Hepburn & Radloff, 1998). It was first described by Murray...

A next generation sequencing approach for targeted Varroa destructor (Acari: Varroidae) mitochondrial DNA analysis based on honey derived environmental DNA.

Honey contains DNA from many different organisms that are part of hive micro-environmental niches and honey bee pathospheres. In this study, we recovered and sequenced mite mitochondrial DNA (mtDNA) from honey from different locations around the world (Europe, Asia, Africa, North and South America). DNA extracted from 17 honey samples was amplified with eight primer pairs targeting three mite mtDNA genes, obtaining 88 amplicons that were sequenced with an Ion Torrent sequencing platform. A bioinformatic pipeline compared produced reads with Varroa spp. mtDNA sequence entries available in GenBank and assigned them to different mitotypes. In all honey samples, the highest percentage of reads was attributed to the K1 lineage, including a few variants derived from it, in addition to J1 reads observed in the two South American samples and C1-1 reads obtained from the Chinese honey. This study opens new possibilities to analyse mite lineages and variants and monitor their geographical and temporal distribution, simplifying surveillance against this damaging honey bee parasite.

An improved method for testing invertebrate encapsulation response as shown in the honey bee

In 1882, Metchnikoff documented the encapsulation response (ER) of the invertebrate immune system. Since then, researchers have used Metchnikoff’s method to quantify immune function—and examine its relationship with ecological and behavioral factors—across various insect taxa. While scientists continue to uncover information regarding invertebrate immunity, behavioral ecology, and ecological immunology, the basics of Metchnikoff’s method have remained unchanged. All but two previous studies investigating insect immunity have used sterile or PBS-coated inducers, although we know that the immune system recognizes specific pathogens. To account for the specificity of the immune system, we modified Metchnikoff’s method and coated nylon monofilaments with pathogen-associated molecular patterns (PAMPs). Using honey bees (Apis mellifera), we examined ER using implants coated with PAMPs (“PAMPlants”) found on known honey bee parasites and pathogens. Lipopolysaccharide (LPS), peptidoglycan (PGN), and β-1, 3-glucan (B13G) PAMPlants mimicked an infection with Gram-negative bacteria, Gram-positive bacteria, and fungi, respectively. Our PAMPlants induced stronger responses than the control implants in both singly- (one PBS-coated or PAMP-coated implant) and doubly- (internal control; one PBS-coated and one PAMP-coated implant) implanted animals. In doubly-implanted individuals, there was a significant increase in response to B13G and LPS when compared with internal controls. The PGN and BSA did not differ from the internal controls in the doubly implanted individuals. These methods provide an improvement when exploring responses to specific pathogens and exploring topics within the field of invertebrate ecological immunity. When applied to social systems, these methods can be used to examine the evolution of disease resistance in societies.

Harman and norharman, metabolites of entomopathogenic fungus Conidiobolus coronatus (Entomopthorales), disorganize development of Galleria mellonella (Lepidoptera) and affect serotonin-regulating enzymes.

Naturally occurring entomopathogenic fungi such as Conidiobolus coronatus are important regulatory factors of insect populations. GC-MS analysis of fungal cell-free filtrates showed that C. coronatus synthesizes two β- carboline alkaloids: harman and norharman. Significantly higher levels of both alkaloids are produced by C. coronatus in minimal postincubation medium than in rich medium. The beta-carboline alkaloids may have an effect on the nervous system of insects and their behavior. Harman and norharman were applied to Galleria mellonella larvae (a parasite of honeybees) either topically or mixed with food. Larvae received alkaloids in three concentrations: 750, 1000 or 1250 ppm. The effect on the survival and further development of larvae was examined. Both harman and norharman delayed pupation and adult eclosion, and inhibit total monoamine oxidase activity. In addition, they increased the serotonin concentration and decreased the monoamine oxidase A level in the heads of the moths. It is likely that the alkaloids were metabolized by the insects, as their effect wore off 24 hours after topical application. This is the first study to show that C. coronatus produces alkaloids. Its aim was to identify the actions of β-carboline alkaloids on insect development and serotonin-regulating enzymes. Knowledge of the potential role of harman and norharman in the process of fungal infection might lead to the development of more effective and environmentally-friendly means of controlling insect pests.

Genome-Wide Identification of Circular RNAs in Fungal Parasite Nosema ceranae.

Circular RNAs (circRNAs) are newly discovered endogenous non-coding RNAs (ncRNAs) that play key roles in microRNA function and transcriptional regulation. Though a large number of circRNAs had been identified in animals and plants, however, little is known regarding circRNAs in Nosema ceranae, a widespread fungal parasite of honeybee. In this study, using deep sequencing technology and bioinformatic analysis, we predicted 204 circRNAs from N. ceranae spore samples, including 174 exonic circRNAs and 30 intergenic circRNAs. In addition, the expression of seven N. ceranae circRNAs was confirmed by RT-PCR assay. Furthermore, regulation networks of circRNAs were constructed, and 15 circRNAs were found to act as sponges of the corresponding three miRNAs. GO categorization and pathway enrichment analysis suggested that the circRNAs are likely to play significant roles in N. ceranae spore. This is the first report of circRNAs generated by a microsporidia species. Our results provide novel insights into understanding the basic biology of N. ceranae.

First identification of long non-coding RNAs in fungal parasite Nosema ceranae

Nosema ceranae is a unicellular fungal parasite of honey bees and causes huge losses for apiculture. Until present, no study on N. ceranae long non-coding RNAs (lncRNAs) was documented. Here, we sequenced purified spores of N. ceranae using strand-specific library construction and high-throughput RNA sequencing technologies. In total, 83 novel lncRNAs were predicted from N. ceranae spore samples, including lncRNAs, long intergenic non-coding RNAs (lincRNAs), and sense lncRNAs. Moreover, these lncRNAs share similar characteristics with those identified in mammals and plants, such as shorter length and fewer exon number and transcript isoforms than protein-coding genes. Finally, the expression of 12 lncRNAs was confirmed with RT-PCR, confirming their true existence. To our knowledge, this is the first evidence of lncRNAs produced by a microsporidia species, offering novel insights into basic biology such as regulation of gene expression of this widespread taxonomic group.

The reduced brood nursing by mite-infested honey bees depends on their accelerated behavioral maturation

The parasitic mite Varroa destructor is regarded as the most important parasite of honey bees and plays a fundamental role in the decline of bee colonies observed in the last decade in the Northern hemisphere. Parasitization has a number of detrimental effects on bees, including reduced nursing, which can have important impacts on colony balance.In this work we investigated at the individual level the causes of this abnormal behavior and found that the reduced nursing activity in mite-infested workers is associated with impaired learning performance and a series of physiological traits that are typical of foragers, including reduced response to brood pheromone, limited development of hypopharyngeal glands and higher juvenile hormone titre in the haemolymph. Altogether our data confirm the premature transition to foraging already postulated based on previous genomics studies, from a physiological point of view.

Proteomic analysis of chemosensory organs in the honey bee parasite Varroa destructor: A comprehensive examination of the potential carriers for semiochemicals

We have performed a proteomic analysis on chemosensory organs of Varroa destructor, the honey bee mite, in order to identify putative soluble carriers for pheromones and other olfactory cues emitted by the host. In particular, we have analysed forelegs, mouthparts (palps, chelicera and hypostome) and the second pair of legs (as control tissue) in reproductive and phoretic stages of the Varroa life cycle. We identified 958 Varroa proteins, most of them common to the different organs and stages. Sequence analysis shows that four proteins can be assigned to the odorant-binding protein (OBP)-like class, which bear some similarity to insect OBPs, but so far have only been reported in some Chelicerata. In addition, we have detected the presence of two proteins belonging to the Niemann-Pick family, type C2 (NPC2), which have also been suggested as semiochemical carriers.Biological significance: The mite Varroa destructor is the major parasite of the honey bee and is responsible for great economical losses. The biochemical tools used by Varroa to detect semiochemicals produced by the host are still largely unknown. This work contributes to understand the molecular basis of olfaction in Varroa and, more generally, how detection of semiochemicals has evolved in terrestrial non-hexapod Arthropoda. Moreover, the identification of molecular carriers involved in olfaction can contribute to the development of control strategies for this important parasite.

Promising antimicrobial activity against the honey bee parasite Nosema ceranae by methanolic extracts from Chilean native plants and propolis

The gut parasite Nosema ceranae is one of the most prevalent honey bee microsporidia worldwide and its chemical management in American countries is mainly based on the antibiotic fumagillin. However, it has recently been reported that this drug has negative side effects in honey bees; therefore, non-harmful alternative treatments must be found. The effects of methanolic extracts (2, 4, 8 and 16%) from Chilean native plant leaves (Aristotelia chilensis, Ugni molinae, and Gevuina avellana) and propolis (Biobío (BB) and Los Ríos (LR) regions) on N. ceranae load, diet consumption and the survival of N. ceranae-infected honey bees were evaluated through oral intake. When bees were N. ceranae-infected and treated with extracts of A. chilensis (8%), U. molinae (2 and 8%) and propolis BB (8%) simultaneously, the N. ceranae load decreased significantly and the survival of infected bees also improved. On the other hand, when bees were first extracts-treated and then N. ceranae-infected, their diet consumption increased and all extracts significantly decreased the N. ceranae load in infected bees. However, only extracts from U. molinae (2 and 8%) and propolis LR (8%) maintained a higher survival rate in infected bees. Natural compounds were also identified; U. molinae and A. chilensis leaves had high concentrations of rutin and myricetin, whereas galangin and pinocembrin were found mostly in southern Chilean propolis (LR). This study constitutes the first report of the antiparasitic activity of propolis against N. ceranae in Apis mellifera and the antimicrobial activity of Chilean plant extracts against this honey bee parasite.

Seasonal Effects and the Impact of In-Hive Pesticide Treatments on Parasite, Pathogens, and Health of Honey Bees.

Honey bee, Apis mellifera (L.; Hymenoptera: Apidae), populations are in decline and their losses pose a serious threat for crop pollination and food production. The specific causes of these losses are believed to be multifactorial. Pesticides, parasites and pathogens, and nutritional deficiencies have been implicated in the losses due to their ability to exert energetic stress on bees. While our understanding of the role of these factors in honey bee colony losses has improved, there is still a lack of knowledge of how they impact the immune system of the honey bee. In this study, honey bee colonies were exposed to Fumagilin-B, Apistan (tau-fluvalinate), and chlorothalonil at field realistic levels. No significant effects of the antibiotic and two pesticides were observed on the levels of varroa mite, Nosema ceranae (Fries; Microsporidia: Nosematidae), black queen cell virus, deformed wing virus, or immunity as measured by phenoloxidase and glucose oxidase activity. Any effects on the parasites, pathogens, and immunity we observed appear to be due mainly to seasonal changes within the honey bee colonies. The results suggest that Fumagilin-B, Apistan, and chlorothalonil do not significantly impact the health of honey bee colonies, based on the factors analyzed and the concentration of chemicals tested.

Lithium chloride effectively kills the honey bee parasite Varroa destructor by a systemic mode of action

Honey bees are increasingly important in the pollination of crops and wild plants. Recent reports of the weakening and periodical high losses of managed honey bee colonies have alarmed beekeeper, farmers and scientists. Infestations with the ectoparasitic mite Varroa destructor in combination with its associated viruses have been identified as a crucial driver of these health problems. Although yearly treatments are required to prevent collapses of honey bee colonies, the number of effective acaricides is small and no new active compounds have been registered in the past 25 years. RNAi-based methods were proposed recently as a promising new tool. However, the application of these methods according to published protocols has led to a surprising discovery. Here, we show that the lithium chloride that was used to precipitate RNA and other lithium compounds is highly effective at killing Varroa mites when fed to host bees at low millimolar concentrations. Experiments with caged bees and brood-free artificial swarms consisting of a queen and several thousand bees clearly demonstrate the potential of lithium as miticidal agent with good tolerability in worker bees providing a promising basis for the development of an effective and easy-to-apply control method for mite treatment.

Apiarists’ Awareness and Responses to Honey Bee Colony Parasite and Pathogen Infections in Kenya

Globally, honey bee (Apis mellifera L. ) is the most domesticated bee species due to their ability to produce honey of commercial value, wax, pollen, royal jelly and propolis. They also facilitate pollination processes of flowering plants. However, there has been an increase in reported incidences of hive and colony infection by honey bee parasites and pathogens, contributing to colony collapse disorder. This study explored the beekeeper’s approaches to hive infestation by bee parasites and pathogens in Kenya. Using a questionnaire, 78 individual beekeepers and 15 beekeeping groups in eleven different Counties in Kenya were surveyed between 2012 and 2013, and their ability to identify and manage honey bee pathogens and parasites was determined. Majority of the farmers (95%) said they experienced challenges with pests. Ants were the pests mostly mentioned by the apiarists (90%), followed by small hive beetles (32%), wax moths (28%) and pirate wasps (18%). Only one respondent mentioned Varroa mites and no respondent mentioned any of the pathogens transmitted by these parasites. Most (90%) management practices apiarists mentioned targeted controlling ants. Farmers had no idea of how to control other pest and/or parasites, in spite of their presence as was confirmed by this study that all colonies inspected in all sites were infested with Varroa mites. Majority (93%) of the apiarist harvested honey at night when visibility is poor and hence could not see other pests and/or pathogens. Apiarist’ need education on bee husbandry in order to contain honey bee parasites and pathogens to improve their awareness on the same and to manage colony collapse disorders. They need to embrace colony inspection and honey harvesting during the day when sufficient lighting is available for detection of parasites and pathogens.

Nosemosis control in European honey bees, Apis mellifera, by silencing the gene encoding Nosema ceranae polar tube protein 3.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene and is conserved in a wide range of eukaryotic organisms. The RNAi mechanism has provided unique opportunities for combating honey bee diseases caused by various parasites and pathogens. Nosema ceranae is a microsporidian parasite of European honey bees, Apis mellifera, and has been associated with honey bee colony losses in some regions of the world. Here we explored the possibility of silencing the expression of a N. ceranae putative virulence factor encoding polar tube protein 3 (ptp3) which is involved in host cell invasion as a therapeutic strategy for controlling Nosema parasites in honey bees. Our studies showed that the oral ingestion of a dsRNA corresponding to the sequences of N. ceranae ptp3 could effectively suppress the expression of the ptp3 gene in N. ceranae-infected bees and reduce Nosema load. In addition to the knockdown of ptp3 gene expression, ingestion of ptp3-dsRNA also led to improved innate immunity in bees infected with N. ceranae along with an improvement in physiological performance and lifespan compared with untreated control bees. These results strongly suggest that RNAi-based therapeutics hold real promise for the effective treatment of honey bee diseases in the future, and warrant further investigation.