Non-reproducing Varroa jacobsoni Oud. in honey bee worker cells—status of mites or effect of brood cells?

The parasitic mite Varroa jacobsoni Oud. reproduces in sealed honey bee brood cells. Within worker cells a considerable fraction of the mites do not produce offspring. It is investigated whether variation in the ratio of cells without reproduction is caused by properties of the worker brood, or by the state of the mites entering cells. Pieces of brood comb were taken from colonies of 12 different bee lines and were placed simultaneously into highly infested colonies. Non-reproduction was independent of the origin of the brood pieces, indicating a minor role of a variation due to different brood origin. Between colonies used for infestation, however, it differed considerably. A comparison of the proportion of cells without reproduction when infested by one Varroa mite or when infested by two or three Varroa mites showed, that non-reproduction was mainly related to the state of the mites entering cells, and only to a minor degree to an influence of the brood cells. A high ratio of worker cells without reproduction was consistently reported in bee lines which survive the disease without treatment, and a high level of non-reproduction is thus regarded to be a key factor in breeding bees for high Varroa tolerance. The current results indicate, that differences in this trait are only to a minor degree related to differences between bee lines in the ability of the bee brood to induce oviposition. These differences seem rather to depend on other, unknown colony factors influencing the reproductive state of Varroa when they enter cells for reproduction.     

Behaviour of Varroa mites invading honey bee brood cells

Invasion behaviour of Varroa jacobsoni into honey bee brood cells was studied using an observation hive. The mites were carried close to a suitable brood cell by the bees. Subsequently, the mites moved from the bees to the rim of the cell, walked quickly inside, crawled between the larva and the cell wall, and moved onto the bottom of the cell. Varroa mites were never seen walking across the comb, and entering and leaving brood cells as has been described for Tropilaelaps clareae. Differences in invasion strategies between V. jacobsoni and T. clareae are discussed.     

Factors influencing host choice of the honey bee parasite Varroa jacobsoni Oud

Reproducing Varroa jacobsoni obtained from brood cells of Apis mellifera L. with 13–16 day old bees (pupae) and Varroa mites kept on adult bees for at least 8 days were simultaneously tested for their choice in three host types. Comparisons were made of attractiveness of Varroa jacobsoni to nurse bees, pollen foragers as to larvae from nearly capped brood cells. Host choices were observed in Petri dishes and in an Y-shaped olfactometer. Varroa jacobsoni obtained from capped brood cells showed a stronger preference for nurse bees in Petri dish simultaneous choice tests with pollen foragers or larvae than did mites which were previously kept on adult bees. In olfactometer simultaneous choice tests, the two mite test groups showed no clear difference in preferences for bees of different ages. The preference of Varroa jacobsoni for bees of different ages is therefore not only influenced by host factors but also by intrinsic factors in female mites that depend on the mite's reproductive stage.     

Distribution of Paenibacillus larvae spores inside honey bee colonies and its relevance for diagnosis

One of the most important factors affecting the development of honey bee colonies is infectious diseases such as American foulbrood (AFB) caused by the spore forming Gram-positive bacterium Paenibacillus larvae. Colony inspections for AFB clinical symptoms are time consuming. Moreover, diseased cells in the early stages of the infection may easily be overlooked. In this study, we investigated whether it is possible to determine the sanitary status of a colony based on analyses of different materials collected from the hive. We analysed 237 bee samples and 67 honey samples originating from 71 colonies situated in 13 apiaries with clinical AFB occurrences. We tested whether a difference in spore load among bees inside the whole hive exists and which sample material related to its location inside the hive was the most appropriate for an early AFB diagnosis based on the culture method. Results indicated that diagnostics based on analysis of honey samples and bees collected at the hive entrance are of limited value as only 86% and 83%, respectively, of samples from AFB-symptomatic colonies were positive. Analysis of bee samples collected from the brood nest, honey chamber, and edge frame allowed the detection of all colonies showing AFB clinical symptoms. Microbiological analysis showed that more than one quarter of samples collected from colonies without AFB clinical symptoms were positive for P. larvae. Based on these results, we recommend investigating colonies by testing bee samples from the brood nest, edge frame or honey chamber for P. larvae spores.     

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.     

Comparison of diagnostic methods for detection of low infestation levels ofVarroa jacobsoni in honey-bee (Apis mellifera) colonies

Thirty-five honey-bee colonies, originally free fromVarroa jacobsoni (Oudemans) were monitored approximately every third week for the presence of the mite during 16 months following an initial introduction of five to eight adultVarroa females in early July. Investigations of hive debris detected the presence ofV. jacobsoni in 22 colonies (63%) within three months of the mite introduction. During the first winter period (October–April), mites were found in the hive debris of 13 colonies (37%). In terms of detectingVarroa during the summer in colonies with sealed brood, investigations of hive debris were more effective than sampling of brood. Brood sampling was more effective than sampling of live bees. In colonies without sealed brood, investigations of hive debris or of live bee samples seemed approximately equally efficient. The highest correlation between sampling methods was found between daily mite downfall and mites per live bee (r=0.81) in colonies with sealed brood. During the winter, investigations of dead bees and hive debris were approximately equally efficient in detectingVarroa.     

Does time spent on adult bees affect reproductive success of Varroa mites?

Reproduction ofVarroa jacobsoni Oudemans (Acari: Varroidae) and the number ofVarroa mites that were found dead on the bottom board of the hive, were studied in relation to the period the mites spent on adult honey bees,Apis mellifera L. (Hymenoptera: Apidae), prior to invasion into brood cells. The maximum period on adult bees was 23 days. To introduce mites, combs with emerging worker brood, heavily infested with mites, were placed into a colony and removed the next day. At the beginning of the first day following emergence from brood cells, 18% of the mites introduced into the colony was found on the bottom of the hive. Part of these mites may already have died inside the capped brood cells, and then fallen down after cleaning of cells by the bees. At the second and third day following emergence, respectively 4% and 2% of the mites on adult bees at the previous day was recovered on the bottom, whereas from the fourth day on only 0.6% of the mites on adult bees was recovered on the bottom per day. After invasion into brood cells, 8–12% of the mites did not produce any offspring. Of the mites that did reproduce, the total number of offspring was 4.0–4.4 per mite during one reproductive cycle, part of which may reach maturity resulting in 1.2–1.3 viable daughters, and 8–10% of the mites produced only male offspring. Reproduction was independent of the period the mites had spent on adult bees prior to invasion into brood cells.     

The possible role of Varroa destructor in the spreading of American foulbrood among apiaries

The aim of this investigation was to establish whether Varroa destructor can play a role in the transmission of Paenibacillus larvae larvae spores from infected to healthy bee colonies. Mites, collected from an Apis mellifera carnica colony heavily infected with American foulbrood and treated with Apistan, were suspended in distilled water and treated in three different ways:homogenizing, shaking and stirring, or sonication. The resulting fluid samples were transferred onto selective agar medium. All culture plates showed colonies that could be identified as P.l. larvae. In view of the numbers of spores they can carry, it is concluded that mites may transmit American foulbrood from infected to healthy bee colonies.     

Time-activity budgets and space structuring by the different life stages ofVarroa jacobsoni in capped brood of the honey bee,Apis mellifera

Varroa jacobsoni reproduces in honey bee brood cells. Here the behavioral activity and use of space by infestingVarroa females and progeny were quantified in transparent artificial brood cells. The time-activity budget of both infesting and developing mites converged toward a stable pattern which was established during the bee prepupal stage of the infesting mites and the protonymphal stage of mite progeny. The pattern was such that infesting females and offspring eventually divided their activity between the fecal accumulation on the cell wall, which served as the rendezvous site for newly molted individuals, and the feeding site prepared on the pupa by the foundress. Other parts of the cell wall were used for oviposition and molting, away from the fecal accumulation on which activity of mobile stages was concentrated. Space structuring and the time-activity budget inVarroa probably evolved to enhance the number of fertilized females produced within the capped brood, where space and time are limiting factors. These behavioral adaptations parallel those of other mite species which show group behavior within cavities.     

DNA extraction and PCR detection of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques

Summary Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and brings an alternative tool for P. larvae detection in natural samples.     

Vitellogenesis in Varroa jacobsoni,a parasite of honey bees

Reproduction in Varroa jacobsoni occurs only in cells of the capped honey bee brood. Female mites were sampled at different times after cell sealing and ovaries containing a vitellogenic oocyte of the first gonocycle were examined under an electron microscope. It was found that the cytoplasmic connection between the lyrate organ and the oocyte persists far into the vitellogenic growth phase. In addition, a large amount of yolk material is taken up from the haemolymph. All ultrastructural features characteristic of vitellogenesis, such as microvilli, coated pits, vesicles and growing yolk platelets, are present. If more than four Varroa females live in an overcrowded brood cell, they appear to be in stress conditions and their vitellogenic oocytes may become atretic. Alterations typical for oocyte degradation and oosorption were observed in such situations.     

Deformed wing virus associated with <Emphasis Type="Italic">Tropilaelaps mercedesae</Emphasis> infesting European honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Mites in the genus Tropilaelaps (Acari: Laelapidae) are ectoparasites of the brood of honey bees (Apis spp.). Different Tropilaelaps subspecies were originally described from Apis dorsata, but a host switch occurred to the Western honey bee, Apis mellifera, for which infestations can rapidly lead to colony death. Tropilaelaps is hence considered more dangerous to A. mellifera than the parasitic mite Varroa destructor. Honey bees are also infected by many different viruses, some of them associated with and vectored by V. destructor. In recent years, deformed wing virus (DWV) has become the most prevalent virus infection in honey bees associated with V. destructor. DWV is distributed world-wide, and found wherever the Varroa mite is found, although low levels of the virus can also be found in Varroa free colonies. The Varroa mite transmits viral particles when feeding on the haemolymph of pupae or adult bees. Both the Tropilaelaps mite and the Varroa mite feed on honey bee brood, but no observations of DWV in Tropilaelaps have so far been reported. In this study, quantitative real-time RT-PCR was used to show the presence of DWV in infested brood and Tropilaelaps mercedesae mites collected in China, and to demonstrate a close quantitative association between mite-infested pupae of A. mellifera and DWV infections. Phylogenetic analysis of the DWV sequences recovered from matching pupae and mites revealed considerable DWV sequence heterogeneity and polymorphism. These polymorphisms appeared to be associated with the individual brood cell, rather than with a particular host.     

Detection of Paenibacillus larvae subspecies larvae spores in naturally infected bee larvae and artificially contaminated honey by PCR

American foulbrood (AFB), a severe bacterial disease of honeybee brood, has recently been found in Uruguayan apiaries. Detection of the causative agent, Paenibacillus larvae subspecies larvae, is a very important concern in order to prevent disease dissemination and decrease of honey production. Since spores are the infective forms of this pathogen, in the present work we report the use of polymerase chain reaction (PCR) to detect P. l. subsp. larvae spores from in vitro cultures, larvae with clinical symptoms and experimentally contaminated honey. The set of primers was designed based on the published P. l. subsp. larvae 16S rRNA gene. Using this approach we could amplify the pathogen DNA and obtain a great sensitivity and a notable specificity. Detection limit for spore suspension was a 10^–2 dilution of template DNA obtained from 32 spores, as determined by plate count. For artificially contaminated honey, we could detect the PCR product at a 10^–3 dilution of template DNA obtained from 170 spores. In addition, when PCR conditions were set to improve specificity, we were able to amplify P. l. subsp. larvae DNA selectively and no cross-reactions were observed with a variety of related bacterial species, including P. l. subsp. pulvifaciens. Since spore detection is very important to confirm the presence of the disease, this method provides a reliable diagnosis of AFB from infected larvae and contaminated honey in a few hours.     

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.     

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

Aim: To develop a real‐time PCR‐based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood (AFB), the most harmful pathology of honeybee brood. Methods and Results: A real‐time PCR that allowed selective identification and quantification of P. larvae 16S rRNA sequence was developed. Using standard samples quantified by flow cytometry, detection limits of 37·5 vegetative cells ml^?1 and 10 spores ml^?1 were determined. Compared to spread plate method, this real‐time PCR‐based strategy allowed, in only 2 h, the detection of P. larvae in contaminated honeys. No false‐positive results were obtained. Moreover, its detection limit was 100 times lower than that of the culture method (2 vs 200 spores g^?1 of honey). Conclusion: A rapid, selective, with low detection limit, sensitive and specific method to detect and quantify vegetative cells and spores of P. larvae is now available. Significance and Impact of Study: In addition to honey samples, this real‐time PCR‐based strategy may be also applied to confirm AFB diagnosis in honeybee brood and to screen other apiary supplies and products (bees, pollen, wax), thus broadening the control of AFB spreading.     

Number of reproductive cycles of Varroa jacobsoni in honey-bee (Apis mellifera) colonies

Very little data exists concerning the number of reproductive cycles performed by individual Varroa mites. To understand the population dynamics of the Varroa mite it is necessary to know the number of fertile female offspring each Varroa female produces during her lifetime. The lifetime reproduction capacity of the mite consists of the mean number of fertile female offspring produced during each reproductive cycle multiplied by the mean number of cell passages. This paper describes an experimental design to estimate the number of reproductive cycles where mites are transferred to new mite-free colonies for reproduction in sealed brood cells. The data presented suggests that the mean number of reproductive cycles performed by the individual female mite is larger than previously accepted. Under optimal conditions, the mean number of reproductive cycles by Varroa females is probably greater than 1.5 but less than 2. Furthermore, the results show that the reproductive success of Varroa females going into cells to reproduce is not influenced by previous brood cycles.     

Development of early infestations by the miteVarroa jacobsoni in honey-bee (Apis mellifera) colonies in cold climates

The development of an infestation by five to eight introduced adult females ofVarroa jacobsoni Oud. in 35 honey-bee (Apis mellifera L.) colonies was monitored for 16 months with no outside source of infestation. Calculations on the size of the mite populations were based on collection of debris, samples of bees and brood, and estimates of number of bees and broodcells during the summer. In the winter, only dead bees and debris were collected. Samples were taken at 3-week intervals. Data indicated that the mite population probably could increase more than 100 times within one summer, and more than ten times between years, in a climate with a brood-rearing period of less than five months. A large variation in mite population increase existed between colonies. The winter mortality of mites that die with the host or drop from the winter cluster has a large influence on the population dynamics of the mite. Data also indicated that the simple method of counting mites in hive debris is a useful parameter for monitoring the population development ofVarroa in colonies with hatching brood.     

Further observations on the correlation between attractiveness of honey bee brood cells toVarroa jacobsoni and the distance from larva to cell rim

Varroa jacobsoni Oudemans (Acari: Varroidae) was studied with respect to invasion into different types of honeybee,Apis mellifera L., brood cells. Different cell types were obtained by shortening and elongating of cells, grafting worker larvae into drone cells andvice versa. The type of cell strongly affected the number of mites per cell, and the attractive period of the cells to the mites. The type of cell also affected the distance from larva to cell rim preceding cell capping. When this distance was larger in comparison to control cells of the same age, the attractive period of the brood cells was shorter andvice versa. Since in all cell types the distance from larva to cell rim continuously decreased preceding cell capping, this negative correlation is in agreement with the hypothesis that there is a critical larva-rim distance under which brood cells are attractive to mites. Then, the length of the attractive period of brood cells depends on the moment this critical distance is reached. The distribution of mites over different cell types in turn results from differences in the attractive period.     

Reproduction of Varroa destructor in worker brood of Africanized honey bees (Apis mellifera)

Reproduction and population growth of Varroa destructor was studied in ten naturally infested, Africanized honeybee (AHB) (Apis mellifera) colonies in Yucatan, Mexico. Between February 1997 and January 1998 monthly records of the amount of pollen, honey, sealed worker and drone brood were recorded. In addition, mite infestation levels of adult bees and worker brood and the fecundity of the mites reproducing in worker cells were determined. The mean number of sealed worker brood cells (10,070 ± 1,790) remained fairly constant over the experimental period in each colony. However, the presence and amount of sealed drone brood was very variable. One colony had drone brood for 10 months and another for only 1 month. Both the mean infestation level of worker brood (18.1 ± 8.4%) and adult bees (3.5 ± 1.3%) remained fairly constant over the study period and did not increase rapidly as is normally observed in European honey bees. In fact, the estimated mean number of mites fell from 3,500 in February 1997 to 2,380 in January 1998. In May 2000 the mean mite population in the study colonies was still only 1,821 mites. The fertility level of mites in this study was much higher (83–96%) than in AHB in Brazil(25–57%), and similar to that found in EHB (76–94%). Mite fertility remained high throughout the entire study and was not influenced by the amount of pollen, honey or worker brood in the colonies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of a lactic acid treatment during winter in temperate climate upon Varroa jacobsoni Oud. and the bee (Apis mellifera L.) colony

Three groups of bee colonies were treated with lactic acid, the pesticide Perizin or lactic acid and Perizin in order to validate the applicability of lactic acid in Varroa mite control. The lactic acid treatment was conducted during winter. Eight ml of lactic acid (15%) per comb side were applied with a dosage gun. The treatment was highly efficient and 94.2%–99.8% of the mites in a colony were killed. Due to precise dosage the lactic acid treatment caused less bee mortality than a treatment with the pesticide Perizin. A lactic acid treatment at-0.2°C caused bee mortality comparable to a Perizin treatment. The number of queen losses after lactic acid treatment and after Perizin treatment was comparable. The number of bees, the size of the brood area, the amount of stored honey and Nosema infestation rates were not significantly different in lactic acid treated colonies and Perizin treated colonies in spring after treatment.