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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Honey bee colony winter loss rates for 35 countries participating in the COLOSS survey for winter 2018–2019, and the effects of a new queen on the risk of colony winter loss
    Gray, Alison ; Adjlane, Noureddine ; Arab, Alieza ; Ballis, Alexis ; Brusbardis, Valters ; Charrière, Jean Daniel ; Chlebo, Robert ; F. Coffey, Mary ; Cornelissen, A.C.M. ; Amaro da Costa, Cristina ; Brodschneider, Robert - \ 2020
    Journal of Apicultural Research (2020). - ISSN 0021-8839
    apis mellifera - Mortality - colony winter losses - queens - queen replacement - monitoring - surveys - beekeeping - citizen science
    This article presents managed honey bee colony loss rates over winter 2018/19 resulting from using the standardised COLOSS questionnaire in 35 countries (31 in Europe). In total, 28,629 beekeepers supplying valid loss data wintered 738,233 colonies, and reported 29,912 (4.1%, 95% confidence interval (CI) 4.0–4.1%) colonies with unsolvable queen problems, 79,146 (10.7%, 95% CI 10.5–10.9%) dead colonies after winter and 13,895 colonies (1.9%, 95% CI 1.8–2.0%) lost through natural disaster. This gave an overall colony winter loss rate of 16.7% (95% CI 16.4–16.9%), varying greatly between countries, from 5.8% to 32.0%. We modelled the risk of loss as a dead/empty colony or from unresolvable queen problems, and found that, overall, larger beekeeping operations with more than 150 colonies experienced significantly lower losses (p < 0.001), consistent with earlier studies. Additionally, beekeepers included in this survey who did not migrate their colonies at least once in 2018 had significantly lower losses than those migrating (p < 0.001). The percentage of new queens from 2018 in wintered colonies was also examined as a potential risk factor. The percentage of colonies going into winter with a new queen was estimated as 55.0% over all countries. Higher percentages of young queens corresponded to lower overall losses (excluding losses from natural disaster), but also lower losses from unresolvable queen problems, and lower losses from winter mortality (p < 0.001). Detailed results for each country and overall are given in a table, and a map shows relative risks of winter loss at regional level.
    Beehold : the colony of the honeybee (Apis mellifera L) as a bio-sampler for pollutants and plant pathogens
    Steen, J.J.M. van der - \ 2016
    Wageningen University. Promotor(en): Huub Rijnaarts, co-promotor(en): Tim Grotenhuis; Willem Jan de Kogel. - Wageningen : Wageningen University - ISBN 9789462577510 - 206
    apis mellifera - honey bees - honey bee colonies - biological indicators - sampling - instruments - pollution - pollutants - heavy metals - plant pathogenic bacteria - erwinia amylovora - erwinia pyrifoliae - analytical methods - apis mellifera - honingbijen - honingbijkolonies - biologische indicatoren - bemonsteren - instrumenten (meters) - verontreiniging - verontreinigende stoffen - zware metalen - plantenziekteverwekkende bacteriën - erwinia amylovora - erwinia pyrifoliae - analytische methoden

    Bio-sampling is a function of bio-indication. Bio-indication with honeybee colonies (Apis mellifera L) is where the research fields of environmental technology and apiculture overlap. The honeybees are samplers of the environment by collecting unintentionally and simultaneously, along with nectar, pollen, water and honeydew from the flowers or on the leaves, other matter (in bio-indication terms: target matter) and accumulating this in the colony. Collected target matter, in this thesis heavy metals, the plant pathogens Erwinia pyrifoliae and Erwinia amylovora and the soil pollutant γ-HCH, is collected from the colony by subsampling. Subsampling the honeybee colony is done by taking and killing bees from the hive (sacrificial) or by collecting target matter from the bee’s exterior without killing the bee (non-sacrificial). In environmental technology terms the application of the honeybee colony is a Passive Sampling Method (PSM). In this thesis the possibilities and restrictions of the PSM honeybee colony are explored.

    Bio-indication is a broad research field with one common factor: a living organism (bio) is applied to record an alteration of the environment (indication). The environment may be small such as a laboratory or big such as an ecosystem. Alterations in the organism may vary from detecting substances foreign to the body to mortality of the organism. In environmental technology the concept Source-Path-Receptor (SPR) is applied to map the route of a pollutant. It describes where in the environment the pollution is, how it moves through the environment and where it ends. This environment is the same environment of all living organisms, ergo also honeybees. Honeybees depend on flowers for their food. In the SPR concept, a flower can be a source, path or receptor. Along with collecting pollen, nectar, water and honeydew, target matter is collected by honeybees. Each honeybee functions as a micro-sampler of target matter in the environment, in this case the flower. Each honeybee is part of a honeybee colony and in fact the honeybee colony is the bio-sampler. The honeybee colony is a superorganism. The well-being of the colony prevails over the individual honeybee. Food collection is directed by the colony’s need. Foragers are directed to the most profitable food sources by the bee dance and food exchange (trophallaxis). The result of this feature is that mainly profitable sources are exploited and poor food sources less or not at all. During the active foraging period hundreds to thousands of flowers are visited daily. The nectar, pollen, water and honeydew plus the unintentionally collected target matter is accumulated in the honeybee colony. In order to obtain target matter the colony must be subsampled. This is done by picking bees from the hive-entrance (hive-entering bees) or inside the hive (in-hive bees) and processing them for analysis (sacrificial). This is the most commonly applied method. However, it is possible to subsample the colony without picking and processing the bees by collecting target matter from the hive-entering bee’s exterior (non-sacrificial). For non-sacrificial subsampling of the honeybee colony the Beehold device with the sampling part Beehold tube has been developed. The results of bio-indication with honeybee colonies are qualitative and indicative for follow up study (Chapter 1).

    Six bio-indication studies with honeybee colonies for bio-indication of heavy metals, the plant pathogens Erwinia pyrifoliae and Erwinia amylovora and the soil pollutant γ-HCH are presented. Chapter 2 describes how the concentration of eighteen heavy metals in honeybees fluctuate throughout the period of July, August and September (temporal) at the study sites: the city of Maastricht, the urban location with an electricity power plant in Buggenum and along the Nieuwe Waterweg at Hoek van Holland (spatial). A number of the metals have not been previously analysed in honeybees. To study whether honeybees can be used for bio-indication of air pollution, the concentrations of cadmium, vanadium and lead were compared to concentrations found in honeybees. The honeybee colonies were placed next to the air samplers. Only significant differences of metal concentrations in the ambient air also show in honeybees. This was the case with vanadium in ambient air and honeybees. The spatial and temporal differences of cadmium and lead were too futile to demonstrate a correspondence (Chapter 3). In a national surveillance study in 2008 the concentration of eighteen metals in honeybees has been analysed. The results showed a distinct regional pattern. Honeybees in the East of the Netherlands have higher concentrations of heavy metals compared to the bees in the West. Besides regional differences local differences were also recorded. An approximate description of the land use around 148 apiaries (> 50% agriculture, > 50% wooded area, > 50% urban area and mixed use) indicated the impact of land use on metal concentrations in honeybees. In areas with > 50% wood significantly higher concentrations of heavy metals were detected (Chapter 4). Subsampling of the honeybee colonies in Chapter 2, 3 and 4 was done sacrificially. In the studies presented in Chapter 5, 6, and 7 the honeybee colonies were subsampled non-sacrificially or simultaneously non-sacrificially and sacrificially. The plant pathogen E. pyrifoliae causes a flower infection in the strawberry cultivation in greenhouses. In greenhouse strawberry cultivation honeybees are applied for pollination. In Chapter 5 the combination pollination / bio-indication by honeybee colonies is studied. This proved to be a match. E. pyrifoliae could be detected on in-hive bees prior to any symptom of the infection in the flowers. In the Beehold tube, the bacterium was detected at the same time as the first tiny symptoms of the infection. In Chapter 5 the principles on which the Beehold tube is based are presented and discussed. The plant pathogen E. amylovora causes fireblight in orchards. The combination pollination / bio-indication has also been applied in this study performed in Austria in 2013. It is known that E. amylovora can be detected on honeybees prior to any symptom in the flower or on the fruit tree. A fireblight outbreak depends on flowering period, humidity and temperature. In 2013 no fireblight infection emerged in the orchards where the study was performed. Therefore, the bacterium could not be detected on the honeybees. γ-HCH (Lindane) is one of the soil pollutants in the Bitterfeld region in Saxony-Anhalt in Germany. It is the result of dumping industrial waste around the production locations. Although γ-HCH is bound to soil particles there is a flux to groundwater and surface water. Consequently, the pollution may end up in the sediments of the streambed and flood plains. The study objective was to investigate the hypothetic route of γ-HCH from polluted soil (source), via soil erosion and atmospheric deposition (route) to the receptor (flowering flowers) by detecting γ-HCH in the Beehold tube. Although on average over 17000 honeybees passed through the Beehold tube daily for a maximal period of 28 days, no γ-HCH has been detected. The pollen pattern in the Beehold tube revealed where the bees collected the food (Chapter 7).

    The application of the honeybee colony has pros and cons. Distinctive pros are many micro samplers, the extensive collection of matter (both food and target matter) and the accumulation in the colony. For successful bio-indication with honeybee colonies, determining factors are: the target matter, location of the target matter, distance between target matter and the honeybee colony, individual or pooled subsampling, the minimal sampling frequency and sample size, and sacrificial or non-sacrificial subsampling applied solely or in combination. Taking bees from a colony impacts upon the colony’s performance and consequently the passive sampling method. Based on a long-years’ experience and inter-collegial discussion it is stated that 3% of the forager bees (hive-entering) and 1.5% of the in-hive bees can be sampled safely without impacting upon the colony. This restriction does not apply when carrying out non-sacrificial subsampling of the honeybee colony (Chapter 8).

    Performing bio-indication with honeybee colonies has more applications than have been exploited so far. Further research can make a change. In particular I mention here the combination of pollination and bio-indication and the application of non-sacrificial subsampling solely or in combination with sacrificial subsampling.

    Everywhere Apiculture is practiced (all over the world except the polar areas) bio-indication with honeybee colonies can be applied in a simple, practical and low cost way.

    Spuitschade (2) - 25 jaar registratie in Nederland
    Jilesen, C. ; Driessen, T. ; Steen, J.J.M. van der; Blacquière, T. ; Scheer, H. van der - \ 2015
    Bijenhouden 9 (2015)6. - ISSN 1877-9786 - p. 20 - 21.
    bijenhouderij - apis mellifera - honingbijen - bijensterfte - pesticiden - inventarisaties - geschiedenis - monitoring - bijenziekten - onbedoelde effecten - beekeeping - apis mellifera - honey bees - bee mortality - pesticides - inventories - history - monitoring - bee diseases - nontarget effects
    In 1990 is in Nederland een werkgroep opgericht om jaarlijks gevallen van massale bijensterfte te inventariseren die volgens getroffen imkers veroorzaakt zijn door blootstelling aan gewasbeschermingsmiddelen (Oomen, 1992). De werkgroep jubileert dit jaar en heeft daarom haar bevindingen met spuitschade in de afgelopen 25 jaar samengevat
    Spuitschade (1) - 25 jaar registratie in Nederland
    Jilesen, C. ; Driessen, T. ; Steen, J.J.M. van der; Blacquière, T. ; Scheer, H. van der - \ 2015
    Bijenhouden 9 (2015)5. - ISSN 1877-9786 - p. 18 - 19.
    bijenhouderij - apis mellifera - honingbijen - bijensterfte - pesticiden - inventarisaties - geschiedenis - monitoring - beekeeping - apis mellifera - honey bees - bee mortality - pesticides - inventories - history - monitoring
    In 1990 is in Nederland een werkgroep opgericht om jaarlijks gevallen van massale bijensterfte te inventariseren die volgens getroffen imkers veroorzaakt zijn door blootstelling aan gewasbeschermingsmiddelen (Oomen, 1992). De werkgroep jubileert dit jaar en heeft daarom haar bevindingen met spuitschade in de afgelopen 25 jaar samengevat
    Factoren die het foerageergedrag van honingbijen bepalen (deel I); Dracht in Nederland (cultuurgewassen en wilde planten) (deel II)
    Steen, J.J.M. van der; Cornelissen, B. - \ 2015
    Wageningen : Plant Research International, Wageningen UR (Rapport 606) - 94
    apis mellifera - honingbijen - diergedrag - bestuivers (dieren) - dansen (bijen) - door bijen verzameld stuifmeel - seizoenen - drachtplanten - veldgewassen - vruchtbomen - openbaar groen - wegbermplanten - wilde planten - waarden - apis mellifera - honey bees - animal behaviour - pollinators - dances - bee-collected pollen - seasons - pollen plants - field crops - fruit trees - public green areas - roadside plants - wild plants - values
    Om een inschatting te kunnen maken van het risico dat honingbijen blootgesteld worden aan gewasbeschermingsmiddelen, andere stoffen zoals atmosferische depositie van fijnstof en organismen zoals plantpathogene microorganismen, is in opdracht van het Ministerie van EZ/Landbouw een samenvatting gemaakt van de informatie, beschikbaar over de aantrekkelijkheid van Nederlandse gewassen voor honingbijen (Apis mellifera). De opdracht is vorm gegeven in twee delen. Deel I is een beschrijving van het bijenvolk met de focus op het foerageergedrag, gevolgd door een beschrijving van factoren die het foerageergedrag bepalen, hoe de bijen hun omgeving exploreren en exploiteren en een lijst met kengetallen over het foerageren van honingbijen. Deel II geeft een overzicht van cultuurgewassen en wilde planten met bijbehorende waarden van nectar en stuifmeel voor honingbijen met bloeitijden en verwijzingen naar goede drachtplantenboeken. Hieronder zijn puntsgewijs relevante zaken gegeven die in het rapport verder uitgewerkt zijn. Honingbijen zijn voor hun voedsel (nectar en stuifmeel) volledig afhankelijk van planten. Het foerageergedrag en de voorkeur voor gewassen hangt af van de behoefte in het volk en de aantrekkelijkheid van het gewas als nectar- en stuifmeelbron. Het foerageergedrag wordt voortdurend aangepast aan de beschikbare dracht en de behoeften van het bijenvolk. Honingbijen leven in volken die variëren in grootte van ~7000 individuen in het voorjaar (maart) tot 20 000 à 30 000 in de zomer en weer afnemend in oktober. In het actieve foerageer- en broedseizoen is een derde tot een vierde deel foerageerster (haalbij). In de loop van een seizoen halen de bijen ten behoeve van het volk 25 kg water, 20 - 30 kg stuifmeel, 125 kg nectar en kleine hoeveelheden hars (propolis). Voor het halen van deze voedselcomponenten vliegen bijen tot 2 km voor water, tot 6 km voor stuifmeel en tot 12 à 13 km voor nectar. Meestal zullen de vluchten echter beperkt zijn tot 600-800 meter. De foerageerafstanden zijn in de zomer (juli – augustus) langer dan in het voorjaar (maart – mei). Met andere woorden, in het voorjaar wordt het voedsel in een kleiner gebied verzameld dan in de zomer. Het risico dat bijen aan een bespuiting zullen worden blootgesteld zou daarom na half juni hoger kunnen zijn dan in het voorjaar. Maar aan de andere kant zijn dan de meeste bespuitingen met insecticiden achter de rug. Het risico van blootstelling aan een insecticide is hoger in een gewas met een goed nectar- (hoeveelheid en suikerconcentratie) en stuifmeelaanbod. Foerageersters vliegen per dag gemiddeld 10 keer uit om voedsel te verzamelen, elke trip kan van een paar minuten tot een uur duren. Door communicatie via de bijendans en trophallaxis (voedseluitwisseling) wordt de keuze voor het benutten van een bepaalde dracht sterk gestuurd. Dat betekent dat bijen zich niet homogeen verdelen over het drachtgebied maar focussen op de meest profijtelijke drachten. Als gevolg daarvan is ‘geen bezoek’ en ‘veel bezoek’ in de verdeling meer vertegenwoordigd dan ‘een beetje bezoek’. Bijenvolken van een bijenstand verdelen zich niet allemaal gelijk over het drachtgebied; verschillende volken bezoeken deels verschillende en deels overlappende drachten. Hoewel de triggers en veelal de drempels bekend zijn, evenals de manier van foerageren, is het nog niet mogelijk precies te voorspellen hoe een volk zich verdeelt over meerdere velden. Omgekeerd is ook niet te voorspellen welk aandeel van verschillende volken op verschillende locaties in een bepaald veld mag worden verwacht. De nectar die binnengebracht wordt, wordt binnen enkele uren verdeeld over het volk; foerageersters gebruiken het als brandstof voor nieuwe foerageervluchten, het komt in het larvenvoedsel terecht en het meeste wordt opgeslagen. Vaste deeltjes zoals fijnstof en microbiële plantpathogenen verdelen zich snel over de bijen in het volk door fysiek contact
    Risicoanalyse import ei- en spermacellen van honingbijen uit de Verenigde Staten en Canada
    Steen, J.J.M. van der; Cornelissen, B. - \ 2012
    Wageningen : Plant Research International Wageningen UR, Business Unit Biointeracties en Plantgezondheid (Rapport / Plant Research International 486)
    apis mellifera - sperma - honingbijen - bijenziekten - quarantaine - nederland - vs - canada - apis mellifera - semen - honey bees - bee diseases - quarantine - netherlands - usa - canada
    Nosema apis en Nosema ceranae. Achtergrondinformatie Nosema
    Steen, J.J.M. van der - \ 2010
    Wageningen : Plant Research International
    honingbijen - apis mellifera - nosema apis - bijenziekten - infecties - insectenpathogene protozoën - nosemaziekte - protozoëninfecties - honey bees - apis mellifera - nosema apis - bee diseases - infections - entomopathogenic protozoa - nosema disease - protozoal infections
    Nosema apis en Nosema ceranae komen beide voor in de volwassen honingbij Apis mellifera. Beide nosema’s worden zowel apart als samen in de honingbij gevonden. Tegenwoordig komt N. ceranae meer voor dan N. apis. Omdat in Noord Europa, de schadelijke gevolgen van de N. ceranae besmettingen min of meer overeen komen met die van N. apis worden bij de preventie en bestrijding de twee soorten vooralsnog gelijk behandeld. Wanneer gesproken wordt over nosema worden hiermee beide nosemasoorten samen bedoeld Achtergrondinformatie: Nosema apis en Nosema ceranae komen beide voor bij A. mellifera. De eerste beschrijvingen van de parasiet N. apis bij Apis mellifera en de gevolgen van deze parasitering zijn 100 jaar geleden beschreven door Zander [18]. Besmettingen van A. mellifera met N. ceranae zijn van een meer recente datum. Fries et al. [6] hebben N. ceranae beschreven. Dat N. ceranae voorkomt bij de Europese honingbij en N. apis ook de Aziatische honingbij kan infecteren weten we sinds een paar jaar. Higes et al. [9] ontdekten in 2006 als eerste het voorkomen van N. cerana in de Europese honingbij in Spanje. N. ceranae komt al langer, zeker sinds 1998, bij de Europese honingbij voor zonder dat dit opgemerkt was [13]. In Nederland heeft R. van der Zee in 2007 de eerste N. ceranae infectie op Terschelling aangetoond. Beide parasieten kunnen op een bijenstand tegelijk voorkomen zoals in Nederland aangetoond is bij de landelijke monitoring van PRI bijen in 2008. Hieruit bleek dat N. apis op 10% van de standen voorkomt, N. ceranae op 87% en een combinatie van beide nosemasoorten is vastgesteld op 6% van de 170 bemonsterde bijenstanden. Op een aantal standen werd geen nosema gevonden
    Duurzame varroa bestrijding bij honingbijen
    Blacquière, T. ; Steen, J.J.M. van der; Cornelissen, B. - \ 2007
    apidae - honingbijen - apis mellifera - bijenziekten - oxaalzuur - bestrijdingsmethoden - duurzaamheid (sustainability) - varroa destructor - apidae - honey bees - apis mellifera - bee diseases - oxalic acid - control methods - sustainability - varroa destructor
    Varroa destructor vormt een bedreiging voor het voortbestaan van de Europese honingbij (Apis mellifera). Zonder bestrijding van varroa gaan bijenvolken meestal dood. De huidige bestrijding van varroa door imkers is niet toereikend door het beperkte aantal momenten waarop gedurende het jaar bestreden kan worden. Mogelijkheden van bestrijding met oxaalzuur in het vroege bijenseizoen wordt daarom onderzocht
    Varroa destructor virus 1: a new picorna-like virus in Varroa mites as well as honey bees
    Ongus, J.R. - \ 2006
    Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Monique van Oers; Dick Peters. - Wageningen : - ISBN 9789085043638 - 126
    bijenhouderij - honingbijen - apis mellifera - varroa - mijten - virussen - picornaviridae - virusreplicatie - varroa destructor - genexpressieanalyse - beekeeping - honey bees - apis mellifera - varroa - mites - viruses - picornaviridae - viral replication - varroa destructor - genomics

    Varroa destructor mite is an ectoparasite of the honey bee Apis mellifera. This species was recently differentiated from Varroa jacobsoni species which infests the Asian bee Apis cerana. Varroa mites feed entirely on the bee's haemolymph and have been associated with the spread of a number of viruses. Since the mites were first observed in Java, Indonesia in 1904, they have been reported in most regions of the world except Australia and the equatorial regions of Africa. V. destructor severely affects and threatens the survival of A. mellifera. The mite was spread to ?. mellifera colonies in other areas by migratory practices of bee keepers and by drifting swarms. The survival of bee colonies attacked by these mites is threatened since the mites weaken the colony if left untreated. Honey bees are important pollinators in nature and in the agricultural and horticultural industries. Bee keepers can also earn from the sale of honey, bee wax and propolis. At this moment, there is no absolute method available for controlling the mite. The methods currently employed use chemicals which contaminate honey and other bee products, and mites are developing resistance to some of the chemicals used. The economic impact of mite infestation makes it necessary to investigate alternative options for control.

    At the onset of the research described in this thesis, 27 nm picorna-like virus particles were observed in mite tissue apparently going through an infection cycle. The identity of this virus was unknown and it was unclear if it was infectious to the mite only or to the bee as well. The aim of this research was to isolate this virus from the mite, characterise its genome in detail and study its behaviour in mite and bee populations with the intention to determine its potential as a biological control agent against the Varroa mite.

    Electron microscopic examinations showed para-crystaüine aggregates of virus particles in the cytoplasm of mite tissue. The virus particles were purified from mites collected from the Wageningen University apiary and used to raise rabbit polyclonal antibodies. The antibody was applied to locate the virus in tissue sections of mites in an immunohistology examination which revealed that the virus was abundantly located in the tissues of the lower digestive tract (Chapter 2). The virus was not detected in salivary glands, indicating that this virus is not transmitted via these glands.

    In the next phase RNA was isolated from these virus particles and the viral genome was fully sequenced (Chapter 3). The virus has a single-stranded, positive-sense genome which is polyadenylated at the 3' terminus and can serve directly as messenger RNA. The genome has a length of 10,112 nucleotides (without the poly-A tail) and one large open reading frame (ORF) encoding a polyprotein of 2893 amino acids. The structural proteins are located in the N-terminal half and the non-structural proteins on the C-termina! half of the polyproiein, and are produced by autoproteolytic cleavage. The ORF is flanked on either side by nontranslated regions (NTRs). Phylogenetic analysis of its RNA-dependent RNA polymerase in a comparison with those of related viruses in the GenBank database revealed that this virus shows high sequence similarity to members of the genus Iflavirus. The genome organisation is also highly comparable to iflaviruses and clearly distinct from that of dicistroviruses. Since the genome sequence of this virus had not been previously reported, the virus was named Varroa destructor virus 1 (VDV-I) after the mite from which it was first isolated. VDV-I is most closely related to Deformed wing virus (DWV) which was isolated from honey bees with wing abnormalities. VDV-I and DWV have 84% genome and 95% polyprotein identity.

    To confirm that VDV-I is able to replicate in the mite, primers were designed to detect specifically the negative-sense RNA strand, which only occurs as replication intermediate, and hence provides evidence for viral replication. With these primers cDNA was synthesised and was further amplified by PCR. Two sets of specific primers were made to distinguish between and detect either VDV-I or DWV. In a similar way, primers, specific for the positive-sense strand, were used to detect the genomes of both VDV-I and DWV in the mites. These experiments showed that both VDV-I and DWV were replicating in the Varrao mite (Chapter 4). These findings are in good agreement with the observation of para-crystalline aggregates in electron microscopy images, which also supports the replication of both viruses in the cytoplasm of mite cells (Chapter 2).

    In Chapter 5, the structural proteins of the isolated virus were examined by SDS-PAGE and it was observed that the two largest proteins (VPl and VP2) were present in relatively equal amounts In the virus. These proteins were N-terminally sequenced to reveal the amino acids that determined the proteolytic cleavage sites. VP2 was mapped directly N-terminal to the non-structural helicase, while VPl was located immediately upstream of VP2. Through Western blot analysis it was demonstrated that only VPl reacted very strongly to the antiserum prepared against the virus. In a next experiment the structural proteins were expressed individually fused to glutathione S-transferase. The resulting fusion proteins were tested in Western blot analysis using the antiserum against purified virus. This study revealed that VP1 was the only structural protein which was recognised, implying that this protein probably covered the entire surface of the virus particle, hiding the other structural proteins beneath, or that the other structural proteins were not immunogenic. The viral 3C-like protease was also expressed as a fusion protein and used to raise antibodies, which efficiently detected the protease polypeptide in a control experiment. This antiserum might be used to detect viral replication using an antibody-based method such as ELISA, or to localise replication in the mite body using histochemical methods in addition to molecular techniques.

    So far there is no report on the structure and function of the 5' nontranslated region (5' NTR) of the RNA of iflaviruses. One of the aims of this investigation was to predict whether conserved secondary structures occurred in the 5' NTR of four iflaviruses (Chapter 6). The predictions revealed two types of structures. VDV-I and DWV have long 5' NTRs with complex structures that resemble those of enteroviruses (Picornavtridae), particularly that of Poliovirus. Perina nuda picorna-Iike virus (PnPV) and Ectropis obliqua picorna-iike virus (EoPV) have shorter 5' NTRs with simpler structures that are unique and do not resemble any 5' NTR structures among picornaviruses. The translation of the ORF of genome organisation is also highly comparable to iflaviruses and clearly distinct from that of dicistroviruses. Since the genome sequence of this virus had not been previously reported, the virus was named Varroa destructor virus 1 (VDV-I) after the mite from which it was first isolated. VDV-I is most closely related to Deformed wing virus (DWV) which was isolated from honey bees with wing abnormalities. VDV-I and DWV have 84% genome and 95% polyprotein identity.

    To confirm that VDV-I is able to replicate in the mite, primers were designed to detect specifically the negative-sense RNA strand, which only occurs as replication intermediate, and hence provides evidence for viral replication. With these primers cDNA was synthesised and was further amplified by PCR. Two sets of specific primers were made to distinguish between and detect either VDV-I or DWV. In a similar way, primers, specific for the positive-sense strand, were used to detect the genomes of both VDV-I and DWV in the mites. These experiments showed that both VDV-I and DWV were replicating in the Varrao mite (Chapter 4). These findings are in good agreement with the observation of para-crystalline aggregates in electron microscopy images, which also supports the replication of both viruses in the cytoplasm of mite cells (Chapter 2).

    In Chapter 5, the structural proteins of the isolated virus were examined by SDS-PAGE and it was observed that the two largest proteins (VPl and VP2) were present in relatively equal amounts In the virus. These proteins were N-terminally sequenced to reveal the amino acids that determined the proteolytic cleavage sites. VP2 was mapped directly N-terminal to the non-structural helicase, while VPl was located immediately upstream of VP2. Through Western blot analysis it was demonstrated that only VPl reacted very strongly to the antiserum prepared against the virus. In a next experiment the structural proteins were expressed individually fused to glutathione S-transferase. The resulting fusion proteins were tested in Western blot analysis using the antiserum against purified virus. This study revealed that VP1 was the only structural protein which was recognised, implying that this protein probably covered the entire surface of the virus particle, hiding the other structural proteins beneath, or that the other structural proteins were not immunogenic. The viral 3C-like protease was also expressed as a fusion protein and used to raise antibodies, which efficiently detected the protease polypeptide in a control experiment. This antiserum might be used to detect viral replication using an antibody-based method such as ELISA, or to localise replication in the mite body using histochemical methods in addition to molecular techniques.

    So far there is no report on the structure and function of the 5' nontranslated region (5' NTR) of the RNA of iflaviruses. One of the aims of this investigation was to predict whether conserved secondary structures occurred in the 5' NTR of four iflaviruses (Chapter 6). The predictions revealed two types of structures. VDV-I and DWV have long 5' NTRs with complex structures that resemble those of enteroviruses (Picornavtridae), particularly that of Poliovirus. Perina nuda picorna-Iike virus (PnPV) and Ectropis obliqua picorna-iike virus (EoPV) have shorter 5' NTRs with simpler structures that are unique and do not resemble any 5' NTR structures among picornaviruses. The translation of the ORF of picorna(-like) viruses in general is initiated by the recognition of one or two internal ribosome entry sites (IRES). The cap-dependent mechanism of translation initiation employed by most cellular mRNAs is not used by these viruses. The IRES activity in the 5' NTRs of iflaviruses had not yet been determined experimentally. Therefore, IRES activity in the 5' NTR of VDV-I was examined. This research was also aimed at identifying a permissive cell line with the ability to support VDV-I IRES function, which is a prerequisite for the translation of the polyprotein ORF, and which might potentially support viral replication. The activity of the IRES of VDV-I was investigated by cloning the 5' NTR between two reporter genes: enhanced green fluorescent protein (EGFP) and firefly luciferase (Fluc). EGFP was cloned directly downstream of the OpIE2 promoter, which is activated by cellular factors, and is translated via a cap-dependent mechanism. The translation of Fluc was dependent on IRES activity in the 5'NTR of VDV-I in the respective cell lines. The presence of the 5' NTR of VDV-I greatly improved the expression levels of the second reporter gene (Fluc) in Lymantria dispar Ld652Y cells, showing that the 5' NTR of VDV-I contains a functional IRES element. This IRES element was active in a host specific manner since it showed much lower activity in Spodoptera frugiperda Sf21 cells and no activity in Drosophila melanogaster S2 cells.

    The transmission ofVDV-1 between the mite V. destructor and the honey bee A. mellifera was surveyed in comparison with DWV (Chapter 7). To determine the spread of these viruses in mites (adults, nymphs and eggs) and bees (eggs, larvae, pupae and adults), the antiserum raised against purified virus was used in ELISA analyses. Infections by VDV-1 or DWV could not be distinguished using this immunology technique due to the high similarity (97%) in the immunodominant protein VPl. The proportion of Varroa mites (71%) infected with VDV-I and/or DWV at the Wageningen University apiary was slightly higher than that of bees (65%). Vertical transmission to the next generation was established in the mite population since virus was detected in mite eggs using a dot-blot immunoassay. Virus could not be detected in bee eggs, implying that no vertical transmission occurred in this species. Subsequently, nested RT-PCR was used to distinguish VDV-I from DWV and with this technique both viruses could be detected even in the same individual. Eighty eight percent of the mites had VDV-I, 19% of the mites were co-infected with VDV-I and DWV, and no mites with only DWV infection were detected. Seventy nine percent of the adult bees had VDV-I, 26% of the adult bees were co-infected with VDV-I and DWV and there was no DWV only infection in the adult bees either, in the specimens tested. The conclusion from this experiment is that VDV-I and DWV are able to co-exist in an individual mite or bee and can replicate in both organisms. A limited survey of these viruses among mites from different regions of mainland Europe indicated that the two viruses exist together in hives across this part of the world.

    The research described in this thesis compared two closely related viruses able to infect both the mite and bee. One of these viruses (VDV-I) is new. In this study the pathogenicity ofVDV-1 in mites and bees was not analysed in detail, but so far clear symptoms have not been found in the mite or the bee that could be attributed to VDV-I infection. In these studies, DWV also did not result in clear symptoms in mites and bees. Due to the results obtained, VDV-1 is not a prime candidate for biological control of the V. destructor mite.

    Stimulating natural supersedure of honeybee queens, Apis mellifera
    Hendriksma, H.P. ; Calis, J.N.M. ; Boot, W.J. - \ 2004
    Proceedings of the Netherlands Entomological Society meeting 15 (2004). - ISSN 1874-9542 - p. 29 - 33.
    apis mellifera - bijenkoninginnen - verdringing - koninginnencellen - koninginnenteelt - apis mellifera - queen honey bees - supersedure - queen cells - queen rearing
    When a honeybee queen starts to fail, she is often superseded by a young queen that takes over reproduction inside the colony. Natural supersedure in winter leads to an unfertilised young queen and colony loss. To reduce these losses we tried to stimulate supersedure of queens earlier in the season. In 50 colonies we introduced queen cells with one-day-old larvae and capped queen cells. Although many larvae were fed initially, few of them were reared to mature queens and none of the cases resulted in supersedure. This suggests that supersedure cannot be evoked by artificially bypassing the initial phases of the process.
    Van Varroa jacobsoni naar Varroa destructor
    Calis, Johan - \ 2002
    Bijen : maandblad voor imkers 11 (2002)10. - ISSN 0926-3357 - p. 282 - 283.
    honingbijen - bijenhouderij - varroa - varroa jacobsoni - mijten - apis mellifera - apis cerana - voortplanting - voortplantingsgedrag - gastheer parasiet relaties - mijtenbestrijding - bijenziekten - insectenplagen - diergedrag - natuurlijke selectie - evolutie - varroa destructor - honey bees - beekeeping - varroa - varroa jacobsoni - mites - apis mellifera - apis cerana - reproduction - reproductive behaviour - host parasite relationships - mite control - bee diseases - insect pests - animal behaviour - natural selection - evolution - varroa destructor
    Resultaten van onderzoek naar de verschillende voortplantingsstrategieën van Varroa jacobsoni en Varroa destructor in respectievelijk de Oosterse honigbij (Apis cerana) en de Westerse honingbij (Apis mellifera). Het hygiënisch gedrag van de Oosterse honingbij (leegruimen van aangetaste werksterbroedcellen) verklaart het grotendeels gescheiden voorkomen van de twee verschillende mijtensoorten in de verschillende bijensoorten (natuurlijke selectie). Dit biedt aanknopingspunten voor de zoektocht naar een meer varroatolerante Westerse honingbij
    Bestuiving van komkommer (Cucumis sativa) door hommels (Bombus terrestris) en honingbijen (Apis mellifera)
    Eijnde, J. van den - \ 1991
    Hilvarenbeek : Landelijk Proefbedrijf voor Insektenbestuiving en Bijenhouderij 'Ambrosiushoeve' - 12
    komkommers - cucumis sativus - bestuiving - honingbijen - apis mellifera - bombus terrestris - nederland - cucumbers - pollination - honey bees - netherlands
    Bestuiving van aardbeien onder glas door hommels en honingbijen
    Eijnde, J. van den - \ 1991
    Hilvarenbeek : Landelijk Proefbedrijf voor Insektenbestuiving en Bijenhouderij 'Ambrosiushoeve' - 31
    aardbeien - fragaria - bestuiving - apis mellifera - honingbijen - strawberries - pollination - honey bees
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