Larval Exposure to Parasitic Varroa destructor Mites Triggers Specific Immune Responses in Different Honey Bee Castes and Species

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Varroa Mite Infestations in Elevated Honey Bee Brood Cells: Effects of Context and Caste

The Varroa mite infestation level of honey bee, Apis mellifera, worker larvae reared in individual raised cells was 6-fold higher than in the adjacent six cells surrounding them; this differential infestation rate is similar to published values of higher mite infestations of drone cells compared to worker cells. Infestation levels in control cells were the same as in the surrounding cells. In contrast to infestation of these individually raised cells, Varroa mites invaded worker larvae in raised cells along the perimeter of a patch of raised cells (10 by 21 rows) 2.5 times more often than surrounding unraised cells, and similarly ca. 2.5 times more often than in the remaining raised cells (interior) of this patch. In similarly prepared frames of drone comb, Varroa mites invaded individually raised drone cells 3.3-fold more often than the adjacent surrounding cells and control cells. On the other hand, Varroa mites infested drone larvae in the interior of the raised-patch area as often as drones in raised cells along the perimeter of the raised-patch, and this rate was ca. 2.5-fold higher than for drone larvae in unraised cells surrounding the raised-patch and drone larvae in control cells. The higher levels of infestation of raised cells did not come at the expense of the surrounding cells, i.e., the infestation levels of the adjacent surrounding cells were the same as in control cells. For worker larvae, the increased number of mites invading individual raised cells and edge cells of the raised patch were proportional to the number of surrounding nonraised cells. The relationship between raised cell-edges, observations of mite walking behavior on comb surfaces, and larval-to-cell-rim distances are discussed in relation to their possible roles in eliciting mite invasion of honey bee larval cells and contrasted to the putative role of kairomones in larval-host location.     

Intensively Cultivated Landscape and Varroa Mite Infestation Are Associated with Reduced Honey Bee Nutritional State

As key pollinators, honey bees are crucial to many natural and agricultural ecosystems. An important factor in the health of honey bees is the availability of diverse floral resources. However, in many parts of the world, high-intensity agriculture could result in a reduction in honey bee forage. Previous studies have investigated how the landscape surrounding honey bee hives affects some aspects of honey bee health, but to our knowledge there have been no investigations of the effects of intensively cultivated landscapes on indicators of individual bee health such as nutritional physiology and pathogen loads. Furthermore, agricultural landscapes in different regions vary greatly in forage and land management, indicating a need for additional information on the relationship between honey bee health and landscape cultivation. Here, we add to this growing body of information by investigating differences in nutritional physiology between honey bees kept in areas of comparatively low and high cultivation in an area generally high agricultural intensity in the Midwestern United States. We focused on bees collected directly before winter, because overwintering stress poses one of the most serious problems for honey bees in temperate climates. We found that honey bees kept in areas of lower cultivation exhibited higher lipid levels than those kept in areas of high cultivation, but this effect was observed only in colonies that were free of Varroa mites. Furthermore, we found that the presence of mites was associated with lower lipid levels and higher titers of deformed wing virus (DWV), as well as a non-significant trend towards higher overwinter losses. Overall, these results show that mite infestation interacts with landscape, obscuring the effects of landscape alone and suggesting that the benefits of improved foraging landscape could be lost without adequate control of mite infestations.     

Effects of Brood Pheromone Modulated Brood Rearing Behaviors on Honey Bee (Apis mellifera L.) Colony Growth

A hallmark of eusociality is cooperative brood care. In most social insect systems brood rearing labor is divided between individuals working in the nest tending the queen and larvae, and foragers collecting food outside the nest. To place brood rearing division of labor within an evolutionary context, it is necessary to understand relationships between individuals in the nest engaged in brood care and colony growth in the honey bee. Here we examined responses of the queen, queen-worker interactions, and nursing behaviors to an increase in the brood rearing stimulus environment using brood pheromone. Colony pairs were derived from a single source and were headed by open-mated sister queens, for a total of four colony pairs. One colony of a pair was treated with 336 μg of brood pheromone, and the other a blank control. Queens in the brood pheromone treated colonies laid significantly more eggs, were fed longer, and were less idle compared to controls. Workers spent significantly more time cleaning cells in pheromone treatments. Increasing the brood rearing stimulus environment with the addition of brood pheromone significantly increased the tempo of brood rearing behaviors by bees working in the nest resulting in a significantly greater amount of brood reared.     

Learning and Task Interference by Corpse-removal Specialists in Honey Bee Colonies

Undertakers are considered to be among the most specialized of pre-foraging honey bee (Apis mellifera L.) workers. In this study we examined a possible benefit and a cost of the corpse-rem oval specialty, the improvement in performance with experience, and interference by individuals attempting to perform the same task in the same location, respectively. Experienced bees removed corpses significantly faster than less experienced bees and also were less likely to drop corpses while exiting the hive (5.5% vs. 14.3% of attempts). Superior performance by experienced undertakers might occur as a consequence of learning, or by greater ability from the outset. Because active undertakers (≥ 3 corpse removals) did not improve with experience over their own careers, learning was not demonstrated. An extreme specialist, Yellow 54, removed a total of 114 corpses (33.8% of experimentally introduced dead bees) from the hive over a 13-day period. This is the longest recorded tenure of undertaking to date and demonstrates how a few individuals can dominate this task in a honey bee colony-Yellow 54 removed corpses significantly faster than other active bees, but she demonstrated no obvious improvement in performance over her undertaking career. This suggests the possibility that active undertakers were more talented than less active undertakers, irrespective of learning. When two undertakers worked together to remove a corpse from the hive, they took longer to complete the task than did single individuals. When multiple undertakers flew together from the hive, they were less likely to clear a nearby obstruction than single undertakers and were more likely to drop the corpse within 1 m of the hive. Thus, mutual interference exacted a measurable cost as a result of the undertaking specialization while learning provided few benefits.     

Bidirectional Transfer of RNAi between Honey Bee and Varroa destructor: Varroa Gene Silencing Reduces Varroa Population

The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.     

Phenotypic and Genetic Analyses of the Varroa Sensitive Hygienic Trait in Russian Honey Bee (Hymenoptera: Apidae) Colonies

Varroa destructor continues to threaten colonies of European honey bees. General hygiene, and more specific Varroa Sensitive Hygiene (VSH), provide resistance towards the Varroa mite in a number of stocks. In this study, 32 Russian (RHB) and 14 Italian honey bee colonies were assessed for the VSH trait using two different assays. Firstly, colonies were assessed using the standard VSH behavioural assay of the change in infestation of a highly infested donor comb after a one-week exposure. Secondly, the same colonies were assessed using an “actual brood removal assay” that measured the removal of brood in a section created within the donor combs as a potential alternative measure of hygiene towards Varroa-infested brood. All colonies were then analysed for the recently discovered VSH quantitative trait locus (QTL) to determine whether the genetic mechanisms were similar across different stocks. Based on the two assays, RHB colonies were consistently more hygienic toward Varroa-infested brood than Italian honey bee colonies. The actual number of brood cells removed in the defined section was negatively correlated with the Varroa infestations of the colonies (r² = 0.25). Only two (percentages of brood removed and reproductive foundress Varroa) out of nine phenotypic parameters showed significant associations with genotype distributions. However, the allele associated with each parameter was the opposite of that determined by VSH mapping. In this study, RHB colonies showed high levels of hygienic behaviour towards Varroa -infested brood. The genetic mechanisms are similar to those of the VSH stock, though the opposite allele associates in RHB, indicating a stable recombination event before the selection of the VSH stock. The measurement of brood removal is a simple, reliable alternative method of measuring hygienic behaviour towards Varroa mites, at least in RHB stock.     

Fine-Scale Linkage Mapping Reveals a Small Set of Candidate Genes Influencing Honey Bee Grooming Behavior in Response to Varroa Mites

Populations of honey bees in North America have been experiencing high annual colony mortality for 15–20 years. Many apicultural researchers believe that introduced parasites called Varroa mites (V. destructor) are the most important factor in colony deaths. One important resistance mechanism that limits mite population growth in colonies is the ability of some lines of honey bees to groom mites from their bodies. To search for genes influencing this trait, we used an Illumina Bead Station genotyping array to determine the genotypes of several hundred worker bees at over a thousand single-nucleotide polymorphisms in a family that was apparently segregating for alleles influencing this behavior. Linkage analyses provided a genetic map with 1,313 markers anchored to genome sequence. Genotypes were analyzed for association with grooming behavior, measured as the time that individual bees took to initiate grooming after mites were placed on their thoraces. Quantitative-trait-locus interval mapping identified a single chromosomal region that was significant at the chromosome-wide level (p<0.05) on chromosome 5 with a LOD score of 2.72. The 95% confidence interval for quantitative trait locus location contained only 27 genes (honey bee official gene annotation set 2) including Atlastin, Ataxin and Neurexin-1 (AmNrx1), which have potential neurodevelopmental and behavioral effects. Atlastin and Ataxin homologs are associated with neurological diseases in humans. AmNrx1 codes for a presynaptic protein with many alternatively spliced isoforms. Neurexin-1 influences the growth, maintenance and maturation of synapses in the brain, as well as the type of receptors most prominent within synapses. Neurexin-1 has also been associated with autism spectrum disorder and schizophrenia in humans, and self-grooming behavior in mice.     

Mating Frequencies of Honey Bee Queens (Apis mellifera L.) in a Population of Feral Colonies in the Northeastern United States

Across their introduced range in North America, populations of feral honey bee (Apis mellifera L.) colonies have supposedly declined in recent decades as a result of exotic parasites, most notably the ectoparasitic mite Varroa destructor. Nonetheless, recent studies have documented several wild populations of colonies that have persisted. The extreme polyandry of honey bee queens—and the increased intracolony genetic diversity it confers—has been attributed, in part, to improved disease resistance and may be a factor in the survival of these populations of feral colonies. We estimated the mating frequencies of queens in feral colonies in the Arnot Forest in New York State to determine if the level of polyandry of these queens is especially high and so might contribute to their survival success. We genotyped the worker offspring from 10 feral colonies in the Arnot Forest of upstate New York, as well as those from 20 managed colonies closest to this forest. We found no significant differences in mean mating frequency between the feral and managed queens, suggesting that queens in the remote, low-density population of colonies in the Arnot Forest are neither mate-limited nor adapted to mate at an especially high frequency. These findings support the hypothesis that the hyperpolyandry of honey bees has been shaped on an evolutionary timescale rather than on an ecological one.     

Fighting and Stinging Responses are Affected by a Dopamine Receptor Blocker Flupenthixol in Honey Bee Virgin Queens

Fighting and aggression are important tasks for self-preservation in animals. In honey bees, virgin queens fight against each other for survival in a monogynous colony. Because the virgin queens have higher levels of dopamine (DA) in the brain than do mated queens with low aggressiveness, DA may promote fighting and aggression behaviours of virgin queens. In the present study, we investigated the effect of DA on the fighting and stinging response of honey bee virgin queens. We injected two concentrations (10^?3 M and 10^?2 M) of DA and the DA receptor blocker flupenthixol into the abdomen of one-day-old virgin queens and observed fighting and stinging responses. DA injection did not affect fighting and stinging. Injections of 10^?3 M flupenthixol decreased the winning rate significantly, whereas 10^?2 M flupenthixol increased the winning rate, indicating the opposite effects on fighting responses depending on the degrees of blockade of DA signalling. In terms of the stinging response, 10^?2 M flupenthixol-injected virgin queens stung significantly more often than control and 10^?3 M flupenthixol-injected virgin queens. These results suggest an involvement of DA signalling in the regulation of fighting and aggression in virgin queens, although a blockade of DA does not always inhibit these behaviours.     

Lower Virus Infections in Varroa destructor-Infested and Uninfested Brood and Adult Honey Bees (Apis mellifera) of a Low Mite Population Growth Colony Compared to a High Mite Population Growth Colony

A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.     

Colony Failure Linked to Low Sperm Viability in Honey Bee (Apis mellifera) Queens and an Exploration of Potential Causative Factors

Queen health is closely linked to colony performance in honey bees as a single queen is normally responsible for all egg laying and brood production within the colony. In the U. S. in recent years, queens have been failing at a high rate; with 50% or greater of queens replaced in colonies within 6 months when historically a queen might live one to two years. This high rate of queen failure coincides with the high mortality rates of colonies in the US, some years with >50% of colonies dying. In the current study, surveys of sperm viability in US queens were made to determine if sperm viability plays a role in queen or colony failure. Wide variation was observed in sperm viability from four sets of queens removed from colonies that beekeepers rated as in good health (n = 12; average viability = 92%), were replacing as part of normal management (n = 28; 57%), or where rated as failing (n = 18 and 19; 54% and 55%). Two additional paired set of queens showed a statistically significant difference in viability between colonies rated by the beekeeper as failing or in good health from the same apiaries. Queens removed from colonies rated in good health averaged high viability (ca. 85%) while those rated as failing or in poor health had significantly lower viability (ca. 50%). Thus low sperm viability was indicative of, or linked to, colony performance. To explore the source of low sperm viability, six commercial queen breeders were surveyed and wide variation in viability (range 60–90%) was documented between breeders. This variability could originate from the drones the queens mate with or temperature extremes that queens are exposed to during shipment. The role of shipping temperature as a possible explanation for low sperm viability was explored. We documented that during shipment queens are exposed to temperature spikes (<8 and > 40°C) and these spikes can kill 50% or more of the sperm stored in queen spermathecae in live queens. Clearly low sperm viability is linked to colony performance and laboratory and field data provide evidence that temperature extremes are a potential causative factor.     

Infestation of Japanese Native Honey Bees by Tracheal Mite and Virus from Non-native European Honey Bees in Japan

Invasion of alien species has been shown to cause detrimental effects on habitats of native species. Insect pollinators represent such examples; the introduction of commercial bumble bee species for crop pollination has resulted in competition for an ecological niche with native species, genetic disturbance caused by mating with native species, and pathogen spillover to native species. The European honey bee, Apis mellifera, was first introduced into Japan for apiculture in 1877, and queen bees have been imported from several countries for many years. However, its effects on Japanese native honey bee, Apis cerana japonica, have never been addressed. We thus conducted the survey of honey bee viruses and Acarapis mites using both A. mellifera and A. c. japonica colonies to examine their infestation in native and non-native honey bee species in Japan. Honey bee viruses, Deformed wing virus (DWV), Black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), and Sacbrood virus (SBV), were found in both A. mellifera and A. c. japonica colonies; however, the infection frequency of viruses in A. c. japonica was lower than that in A. mellifera colonies. Based on the phylogenies of DWV, BQCV, and SBV isolates from A. mellifera and A. c. japonica, DWV and BQCV may infect both honey bee species; meanwhile, SBV has a clear species barrier. For the first time in Japan, tracheal mite (Acarapis woodi) was specifically found in the dead honey bees from collapsing A. c. japonica colonies. This paper thus provides further evidence that tracheal-mite-infested honey bee colonies can die during cool winters with no other disease present. These results demonstrate the infestation of native honey bees by parasite and pathogens of non-native honey bees that are traded globally.     

Impact of the Phoretic Phase on Reproduction and Damage Caused by Varroa destructor (Anderson and Trueman) to Its Host,the European Honey Bee (Apis mellifera L.)

Varroa destructor is a parasitic mite of the honeybee that causes thousands of colony losses worldwide. The parasite cycle is composed of a phoretic and a reproductive phase. During the former, mites stay on adult bees, mostly on nurses, to feed on hemolymph. During the latter, the parasites enter brood cells and reproduce. We investigated if the type of bees on which Varroa stays during the phoretic phase and if the duration of this stay influenced the reproductive success of the parasite and the damage caused to bees. For that purpose, we used an in vitro rearing method developed in our laboratory to assess egg laying rate and the presence and number of fully molted daughters. The expression level of two Varroa vitellogenin genes (VdVg1 and VdVg2), known to vary throughout reproduction, was also quantified. Results showed that the status of the bees or time spent during the phoretic phase impacts neither reproduction parameters nor the Varroa vitellogenin genes levels of expression. However, we correlated these parameters to the gene expression and demonstrated that daughters expressed the vitellogenin genes at lower levels than their mother. Regarding the damage to bees, the data indicated that a longer stay on adult bees during the phoretic phase resulted in more frequent physical deformity in newborn bees. We showed that those mites carry more viral loads of the Deformed Wing Virus and hence trigger more frequently overt infections. This study provides new perspectives towards a better understanding of the Varroa-honeybee interactions.     

Infestation Rates and Tiller Morphology Effects by the Switchgrass Moth on Six Cultivars of Switchgrass

Switchgrass (Panicum virgatum L.) is a potential biomass crop for native species-based biofuel systems in North America. A recently identified pest of switchgrass, the switchgrass moth, Blastobasis repartella (Dietz) (Lepidoptera: Coleophoridae), feeds in the basal above-ground internodes and below-ground in the proaxis and rhizomes, causing premature tiller and rhizome loss. Our goal was to determine genetic and temporal variation among six upland cultivars for frequency of tiller infestation by larvae of the switchgrass moth in mature stands in the northern Great Plains and if variation in biomass production was associated with variation in frequency of infestation. Data were collected in 2011 and 2012 for tiller density, biomass, frequency of infestation, number of leaves per healthy and infested tiller, and weights of healthy and infested tillers. Differences were found among cultivars for tiller density, biomass yield, and numbers of leaves per healthy and infested tillers. ‘Summer’, ‘Sunburst’, ‘Pathfinder’, and ‘Cave-In-Rock’ were the highest yielding cultivars. Mean frequency of infestation was different between 2011 (6.7 %) and 2012 (9.6 %). Infested tillers had one less collared leaf than healthy tillers. The weights of healthy tillers were ca. 3× those of infested tillers in both years, suggesting an impact on biomass accumulation and economic value. Levels of infestation were similar for all six cultivars, indicating no feeding preference by the switchgrass moth larva among genetically diverse cultivars of switchgrass. Regression of biomass yield on frequency of infestation showed negative linear relationships for ‘Carthage’ and ‘Kentucky 1625’.     

A Mathematical Model of Forager Loss in Honeybee Colonies Infested with <Emphasis Type="Italic">Varroa destructor</Emphasis> and the Acute Bee Paralysis Virus

We incorporate a mathematical model of Varroa destructor and the Acute Bee Paralysis Virus with an existing model for a honeybee colony, in which the bee population is divided into hive bees and forager bees based on tasks performed in the colony. The model is a system of five ordinary differential equations with dependent variables: uninfected hive bees, uninfected forager bees, infected hive bees, virus-free mites and virus-carrying mites. The interplay between forager loss and disease infestation is studied. We study the stability of the disease-free equilibrium of the bee-mite-virus model and observe that the disease cannot be fought off in the absence of varroacide treatment. However, the disease-free equilibrium can be stable if the treatment is strong enough and also if the virus-carrying mites become virus-free at a rate faster than the mite birth rate. The critical forager loss due to homing failure, above which the colony fails, is calculated using simulation experiments for disease-free, treated and untreated mite-infested, and treated virus-infested colonies. A virus-infested colony without varroacide treatment fails regardless of the forager mortality rate.     

Genetic Differentiation of the Giant Honey Bee (Apis dorsata) in Thailand Analyzed by Mitochondrial Genes and Microsatellites

Genetic diversity and population differentiation of the giant honey bee (Apis dorsata) in Thailand were examined. Six PCR-RFLP mitotypes were generated from digestion of the COI-COII, Cytb-tRNA^ser, ATPase6-8, and lrRNA genes with Dra I and Hin fI. Low genetic diversity (h=0.074, π=0.032%) and a lack of genetic population differentiation between A. dorsata originating from geographically different regions were observed from mtDNA polymorphisms (P > 0.05). In contrast, microsatellite (A14, A24, and A88) polymorphisms revealed a relatively high level of genetic diversity in A. dorsata (H _o=0.68–0.74, average number of alleles per locus=6.0–9.0). Both A24 and A88 indicated significant population differentiation between bees from the north-to-central region (north, northeast, and central regions), peninsular Thailand, and Samui Island.     

Improved Technique for Identification and Enumeration of Methanogenic Bacterial Colonies on Roll Tubes by Epifluorescence Microscopy

Methanogenic fluorescent colonies can be clearly identified on roll tubes by using an epifluorescence microscope equipped with a × 2 objective. Methanogenic and nonmethanogenic colonies could be counted in roll tubes prepared from methanogenic enrichment cultures. Late-developing colonies appearing after 25 days of incubation were mainly methanogenic.