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.     

大蜂螨对一些气味物质的趋性

狄斯瓦螨Varroa destructor Anderson & Trueman是意大利蜜蜂Apis mellifera Spinola的主要外寄生螨。雌成螨在幼虫巢房封盖前不久侵入幼虫巢房,并开始繁殖为害。从雌成螨在一个很短的时间内进入蜜蜂幼虫巢房,以及雄蜂幼虫巢房蜂螨的寄生率明显高于工蜂幼虫巢房的现象,表明蜜蜂幼虫体表一些信息素(semiochemicals)可能起着重要的引诱作用。作者对与大蜂螨相关的19种气味物质进行筛选,并对封盖前工蜂幼虫和雄蜂幼虫表皮挥发物进行气谱及气-质联谱测定。结果表明:雄蜂6龄幼虫对大蜂螨的引诱作用显著高于丁香水等10种气味物质。工蜂和雄蜂末龄幼虫体表挥发物的共有组份是9-二十三烯(C23H46),但它在雄蜂幼虫中所占的比例要明显高于工蜂幼虫。工蜂幼虫的特有主要组分是十八烷(C18H38)和9-甲基十九烷(C19H40);而雄蜂幼虫的特有主要组分是二十五烷(C25H52)和二十三烷(C23H48)。     

Biology and control of Varroa destructor

The ectoparasitic honey bee mite Varroa destructor was originally confined to the Eastern honey bee Apis cerana. After a shift to the new host Apis mellifera during the first half of the last century, the parasite dispersed world wide and is currently considered the major threat for apiculture. The damage caused by Varroosis is thought to be a crucial driver for the periodical colony losses in Europe and the USA and regular Varroa treatments are essential in these countries. Therefore, Varroa research not only deals with a fascinating host–parasite relationship but also has a responsibility to find sustainable solutions for the beekeeping.This review provides a survey of the current knowledge in the main fields of Varroa research including the biology of the mite, damage to the host, host tolerance, tolerance breeding and Varroa treatment. We first present a general view on the functional morphology and on the biology of the Varroa mite with special emphasis on host–parasite interactions during reproduction of the female mite. The pathology section describes host damage at the individual and colony level including the problem of transmission of secondary infections by the mite. Knowledge of both the biology and the pathology of Varroa mites is essential for understanding possible tolerance mechanisms in the honey bee host. We comment on the few examples of natural tolerance in A. mellifera and evaluate recent approaches to the selection of Varroa tolerant honey bees. Finally, an extensive listing and critical evaluation of chemical and biological methods of Varroa treatments is given.This compilation of present-day knowledge on Varroa honey bee interactions emphasizes that we are still far from a solution for Varroa infestation and that, therefore, further research on mite biology, tolerance breeding, and Varroa treatment is urgently needed.     

Nosema ceranae Can Infect Honey Bee Larvae and Reduces Subsequent Adult Longevity

Nosema ceranae causes a widespread disease that reduces honey bee health but is only thought to infect adult honey bees, not larvae, a critical life stage. We reared honey bee (Apis mellifera) larvae in vitro and provide the first demonstration that N. ceranae can infect larvae and decrease subsequent adult longevity. We exposed three-day-old larvae to a single dose of 40,000 (40K), 10,000 (10K), zero (control), or 40K autoclaved (control) N. ceranae spores in larval food. Spores developed intracellularly in midgut cells at the pre-pupal stage (8 days after egg hatching) of 41% of bees exposed as larvae. We counted the number of N. ceranae spores in dissected bee midguts of pre-pupae and, in a separate group, upon adult death. Pre-pupae exposed to the 10K or 40K spore treatments as larvae had significantly elevated spore counts as compared to controls. Adults exposed as larvae had significantly elevated spore counts as compared to controls. Larval spore exposure decreased longevity: a 40K treatment decreased the age by which 75% of adult bees died by 28%. Unexpectedly, the low dose (10K) led to significantly greater infection (1.3 fold more spores and 1.5 fold more infected bees) than the high dose (40K) upon adult death. Differential immune activation may be involved if the higher dose triggered a stronger larval immune response that resulted in fewer adult spores but imposed a cost, reducing lifespan. The impact of N. ceranae on honey bee larval development and the larvae of naturally infected colonies therefore deserve further study.     

Effects of Infection on Honey Bee Population Dynamics: A Model

We propose a model that combines the dynamics of the spread of disease within a bee colony with the underlying demographic dynamics of the colony to determine the ultimate fate of the colony under different scenarios. The model suggests that key factors in the survival or collapse of a honey bee colony in the face of an infection are the rate of transmission of the infection and the disease-induced death rate. An increase in the disease-induced death rate, which can be thought of as an increase in the severity of the disease, may actually help the colony overcome the disease and survive through winter. By contrast, an increase in the transmission rate, which means that bees are being infected at an earlier age, has a drastic deleterious effect. Another important finding relates to the timing of infection in relation to the onset of winter, indicating that in a time interval of approximately 20 days before the onset of winter the colony is most affected by the onset of infection. The results suggest further that the age of recruitment of hive bees to foraging duties is a good early marker for the survival or collapse of a honey bee colony in the face of infection, which is consistent with experimental evidence but the model provides insight into the underlying mechanisms. The most important result of the study is a clear distinction between an exposure of the honey bee colony to an environmental hazard such as pesticides or insecticides, or an exposure to an infectious disease. The results indicate unequivocally that in the scenarios that we have examined, and perhaps more generally, an infectious disease is far more hazardous to the survival of a bee colony than an environmental hazard that causes an equal death rate in foraging bees.     

Interaction between Varroa destructor and imidacloprid reduces flight capacity of honeybees

Current high losses of honeybees seriously threaten crop pollination. Whereas parasite exposure is acknowledged as an important cause of these losses, the role of insecticides is controversial. Parasites and neonicotinoid insecticides reduce homing success of foragers (e.g. by reduced orientation), but it is unknown whether they negatively affect flight capacity. We investigated how exposing colonies to the parasitic mite Varroa destructor and the neonicotinoid insecticide imidacloprid affect flight capacity of foragers. Flight distance, time and speed of foragers were measured in flight mills to assess the relative and interactive effects of high V. destructor load and a field-realistic, chronic sub-lethal dose of imidacloprid. Foragers from colonies exposed to high levels of V. destructor flew shorter distances, with a larger effect when also exposed to imidacloprid. Bee body mass partly explained our results as bees were heavier when exposed to these stressors, possibly due to an earlier onset of foraging. Our findings contribute to understanding of interacting stressors that can explain colony losses. Reduced flight capacity decreases the food-collecting ability of honeybees and may hamper the use of precocious foraging as a coping mechanism during colony (nutritional) stress. Ineffective coping mechanisms may lead to destructive cascading effects and subsequent colony collapse.     

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.     

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.     

Prevalence and Seasonal Variations of Six Bee Viruses in Apis mellifera L. and Varroa destructor Mite Populations in France

A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.     

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.     

Honey Bee Colonies Headed by Hyperpolyandrous Queens Have Improved Brood Rearing Efficiency and Lower Infestation Rates of Parasitic Varroa Mites

A honey bee queen mates on wing with an average of 12 males and stores their sperm to produce progeny of mixed paternity. The degree of a queen’s polyandry is positively associated with measures of her colony’s fitness, and observed distributions of mating number are evolutionary optima balancing risks of mating flights against benefits to the colony. Effective mating numbers as high as 40 have been documented, begging the question of the upper bounds of this behavior that can be expected to confer colony benefit. In this study we used instrumental insemination to create three classes of queens with exaggerated range of polyandry– 15, 30, or 60 drones. Colonies headed by queens inseminated with 30 or 60 drones produced more brood per bee and had a lower proportion of samples positive for Varroa destructor mites than colonies whose queens were inseminated with 15 drones, suggesting benefits of polyandry at rates higher than those normally obtaining in nature. Our results are consistent with two hypotheses that posit conditions that reward such high expressions of polyandry: (1) a queen may mate with many males in order to promote beneficial non-additive genetic interactions among subfamilies, and (2) a queen may mate with many males in order to capture a large number of rare alleles that regulate resistance to pathogens and parasites in a breeding population. Our results are unique for identifying the highest levels of polyandry yet detected that confer colony-level benefit and for showing a benefit of polyandry in particular toward the parasitic mite V. destructor.     

Small RNAs, RNAi and the Inheritance of Gene Silencing in Caenorhabditis elegans

Invasive nucleic acids such as transposons and viruses usually exhibit aberrant characteristics, e.g., unpaired DNA or abnormal double-stranded RNA. Organisms employ a variety of strategies to defend themselves by distinguishing self and nonself substances and disabling these invasive nucleic acids. Furthermore, they have developed ways to remember this exposure to invaders and transmit the experience to their descendants. The mechanism underlying this inheritance has remained elusive. Recent research has shed light on the initiation and maintenance of RNA-mediated inherited gene silencing. Small regulatory RNAs play a variety of crucial roles in organisms, including gene regulation, developmental timing, antiviral defense, and genome integrity, via a process termed as RNA interference (RNAi). Recent research has revealed that small RNAs and the RNAi machinery are engaged in establishing and promoting transgenerational gene silencing. Small RNAs direct the RNAi and chromatin modification machinery to the cognate nucleic acids to regulate gene expression and epigenetic alterations. Notably, these acquired small RNAs and epigenetic changes persist and are transmitted from parents to offspring for multiple generations. Thus, RNAi is a vital determinant of the inheritance of gene silencing and acts as a driving force of evolution.     

Comparison between two thymol formulations in the control of Varroa destructor: effectiveness, persistence, and residues

An apiary trial on the use of two acaricide formulations (gel-Apiguard and vermiculite and Api Life VAR) in the control of Varroa destructor (Anderson & Trueman) was conducted in summer 2001 in Sardinia (Italy). The main goals were 1) to determine their effectiveness against V. destructor, taking into account natural mite mortality in control hives; and simultaneously 2) to determine the persistence of both formulations and residues in honey and wax, by using a new extraction method. Both thymol formulations, after the treatments, reduced significantly the levels of mite infestations of adult bees and sealed brood, but their efficacy, expressed as percentage of mortality, was lower for both products (Api Life VAR 74.8 +/- 13.1 and 81.3 +/- 15.5, Apiguard 90.4 +/- 8.3 and 95.5 +/- 8.7 for sealed brood and adult bees, respectively) than the efficacy previously obtained with the same products in other experimental conditions. Moreover, a considerable colony-to-colony variability was recorded, and a significant negative effect of the thymol treatments on colony development was observed. During 2 wk of treatment, the bees removed nearly 95% of all the applied product (gel or vermiculite). Residues found in honey collected from the nest varied from 0.12 to 4.03 mg/kg for Api Life VAR and from 0.40 to 8.80 mg/kg for Apiguard. The residues were relatively higher in wax (Api Life VAR = 21.6 +/- 13.0; Apiguard = 147.7 +/- 188.9) than in honey, because thymol is a fat-soluble ingredient.     

Population growth of Varroa destructor (Acari: Varroidae) in commercial honey bee colonies treated with beta plant acids

Varroa (Varroa destuctor Anderson and Trueman) populations in honey bee (Apis mellifera L.) colonies might be kept at low levels by well-timed miticide applications. HopGuard^® (HG) that contains beta plant acids as the active ingredient was used to reduce mite populations. Schedules for applications of the miticide that could maintain low mite levels were tested in hives started from either package bees or splits of larger colonies. The schedules were developed based on defined parameters for efficacy of the miticide and predictions of varroa population growth generated from a mathematical model of honey bee colony–varroa population dynamics. Colonies started from package bees and treated with HG in the package only or with subsequent HG treatments in the summer had 1.2–2.1 mites per 100 bees in August. Untreated controls averaged significantly more mites than treated colonies (3.3 mites per 100 bees). By October, mite populations ranged from 6.3 to 15.0 mites per 100 bees with the lowest mite numbers in colonies treated with HG in August. HG applications in colonies started from splits in April reduced mite populations to 0.12 mites per 100 bees. In September, the treated colonies had significantly fewer mites than the untreated controls. Subsequent HG applications in September that lasted for 3 weeks reduced mite populations to levels in November that were significantly lower than in colonies that were untreated or had an HG treatment that lasted for 1 week. The model accurately predicted colony population growth and varroa levels until the fall when varroa populations measured in colonies established from package bees or splits were much greater than predicted. Possible explanations for the differences between actual and predicted mite populations are discussed.     

Down-Regulation of Honey Bee IRS Gene Biases Behavior toward Food Rich in Protein

Food choice and eating behavior affect health and longevity. Large-scale research efforts aim to understand the molecular and social/behavioral mechanisms of energy homeostasis, body weight, and food intake. Honey bees (Apis mellifera) could provide a model for these studies since individuals vary in food-related behavior and social factors can be controlled. Here, we examine a potential role of peripheral insulin receptor substrate (IRS) expression in honey bee foraging behavior. IRS is central to cellular nutrient sensing through transduction of insulin/insulin-like signals (IIS). By reducing peripheral IRS gene expression and IRS protein amount with the use of RNA interference (RNAi), we demonstrate that IRS influences foraging choice in two standard strains selected for different food-hoarding behavior. Compared with controls, IRS knockdowns bias their foraging effort toward protein (pollen) rather than toward carbohydrate (nectar) sources. Through control experiments, we establish that IRS does not influence the bees'' sucrose sensory response, a modality that is generally associated with food-related behavior and specifically correlated with the foraging preference of honey bees. These results reveal a new affector pathway of honey bee social foraging, and suggest that IRS expressed in peripheral tissue can modulate an insect''s foraging choice between protein and carbohydrate sources.