ACTIVITIES OF THE VARROA CONTROL TASK FORCE, YEARS

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1 In partnership with ACTIVITIES OF THE VARROA CONTROL TASK FORCE, YEARS Introduction The Varroa Control Task Force of COLOSS is composed of Varroa destructor researchers, bee health experts, and extension workers. Current participation includes 78 members from more than 20 countries. The Varroa Control Task Force is co-ordinated by: Ales Agricultural Institute of Slovenia Hacquetova Ljubljana, Slovenia and Giovanni Istituto Zooprofilattico Sperimentale del Lazio e della Toscana "Mariano Aleandri" Via Appia Nuova Rome, Italy. Varroa Control members focus on developing and encouraging sustainable solutions for management of the ectoparasitic mite V. destructor, one of the most devastating threats to honey bees. Pivotal to the philosophy of the group is the adoption of Integrated Pest Management schemes. The group s specific aims, that will be achieved by organizing workshops to design common experiments, are to: study the impact of beekeeper management on V. destructor and honey bee populations; study the efficacy and safety of current beekeeper management products; improve current management options and to develop novel strategies; explore mechanisms of V. destructor tolerance and resistance in different honey bee populations, and to develop diagnostic methods and breeding procedures that improve natural tolerance to varroosis in honey bee populations (in collaboration with COLOSS Task Force RNSBB); provide recommendations for sustainable V. destructor management to beekeepers (in collaboration with COLOSS Core Project B-RAP). 1

2 Varroa Control Task Force is organized in different Working Groups: Working Group 1 WG 1: Infestation assessments Leaders: Marco Pietropaoli and Ole Kilpinen Goal: to compare currently used methods to assess the infestation levels of the colony (e.g. the soapy solution, powdered sugar, and natural mite fall methods). Working Group 2 - WG 2: Brood interruption Leaders: Ralph Buechler and Antonio Nanetti Goal: to compare V. destructor control methods using total removal of brood or temporary caging of queens combined with oxalic acid treatment. Working Group 3 - WG 3.Damage thresholds (TEMPORARY NOT ACTIVED) Leaders: Per Kryger and Cecilia Costa Goal: to determine the range of damage threshold levels across Europe, and to link these to environmental factors. Working Group 4 - WG 4: Formic acid management Leaders: Benjamin Dainat and Giovanni Formato Goal: to better understand the influence of intra-colony and environmental parameters on efficiency of formic acid treatments for V. destructor control, with the final aim to improve treatment application and to provide baseline data for a Varroa-weather prediction tool to determine the best moment for treatment. Working Group 5 WG 5: Assessment of new control methods Leader: Maja Ivana Smodis Skerl Goal: testing new frontiers (e.g. new products, like Aluen cap) to control Varroa destructor, considering the beekeepers priorities and realizing comparative studies. Working Group 6 WG6: Communication Leaders:Victoria Soroker and Flemming Vejsnaes Goal: to guarantee a strong communication concerning the output of the Varroa Control Task Force towards the beekeepers (by training, guidelines and books), and the B-RAP COLOSS project leaders. Moreover, WG 6 should give feedbacks from beekeepers to the WGs leaders of the Varroa Control Task Force, suggesting them new studies/ideas/corrections for future activities. 2

3 Working Group 1 WG 1: Infestation assessments ACTIVITIES Leaders: Marco Pietropaoli and Ole Kilpinen The Varroa mite infestation level can be assessed at an apiary level or at a colony level (e.g. in breeding programs with focus on varroa resistance or IPM strategies). Several studies have been conducted on the subject of demonstrating that a soapy solution will effectively dislodge the mites after 30 minutes of shaking (Rinderer et al., 2004). Also the use of icing sugar has also been shown to be effective (Fakimzadeh 2000; Macedo et al. 2002; Lee et al., 2010a; Lee et al., 2010b) and in recent years has become the preferred method of beekeepers and technicians as it gives an immediate response and does not require collection of bee samples. All these methods evaluate the number of phoretic mites. Natural mite mortality verified with the use of sticky sheets has been demonstrated effective but several conditions must be strictly respected to ensure reliability (Branco et al., 2006). Natural mite fall has been established by a formal Varroa working group (CA 3686, 1998) as a reference method in research to estimate Varroa infestation in a colony. Aims of this WG were to compare different methods to assess the Varroa mite infestation on honey bees and also to collect the bibliography relative to studies in which the effectiveness of these methods (icing sugar, soapy solution) is assessed. According to the decisions taken during the COLOSS Workshops on Varroa control strategies of Bled, Slovenia, (May 22nd - 23rd 2014) and Bologna, Italy, (December 2nd - 3rd 2014) we shared and defined with the COLOSS members (from May 2014 to May 2015) two different protocols to evaluate three different methods to assess the Varroa mite infestation: icing sugar technique, soapy solution wash and natural mite fall, relating them with the total number of mites inside the colonies, evaluated with a follow-up treatment. In order to reach the maximum number of possible participants, the protocol was published on the COLOSS website ( presented in COLOSS workshops and Conferences and sent to the mailing list of WG1. The participant s confirmation deadline was June Participants should meet the following requirements: use at least one apiary with at least 15 colonies (to be able to ensure final data from 10 colonies); if possible use 2-3 apiaries from same area (i.e. within 100 km from each other); apiaries should be well described (geographic coordinates, colony disposition) and testers should be noted; use only fully developed (productive) colonies (e.g. ten combs of bees and eight combs of brood in Dadant-Blatt hives); use boxes with screened bottom floor to evaluate mite fall; specify kind of box and type of bottom boards (i.e. Dadant-Blatt, 10 frames ); use a scale to weigh samples in the field and specify kind; 3

4 bottom board wire screen must be clean of propolis and debris; use the container indicated by the WG to collect the honeybee samples; realize a video of the sampling method. The material necessary for the protocol application were: 1. Icing sugar ml container 3. Jar (Dipl.-Ing. (FH) Harald Wössner, Julius-Leber-Strasse 12, Deisslingen, Tel.: +49 (0)7420 / , mail: harald.woessner@buero-hw.de) 4. Sieves 5. Dish washing soap 6. Sticky sheets for bottom boards 7. Kitchen / lab scale (accuracy min 1g) To realize the sample of bees (from the frame realize one sample of bees), the researcher/beekeeper should follow this procedure: - Open the beehive; - Realize a measure of the strength of the colony with the Liebefeld method (link); - Select from the upperbox or the broodnest one external honey frame covered with an amount of foragers bees sufficient to fill a 120ml container; specify exact position of sample collection respect the distance from the brood frames (i.e. 1st = first frame near brood); - Weigh the bees, in the field before the icing sugar method estimation. If weight lower than 40g add bees. To realize the icing sugar method estimation on each sample of bees (according to Macedo et al and Danish method and Kirchain) the researcher/beekeeper should follow this procedure: Add 35g of fresh icing sugar (approximately 2 tablespoons) into the jar; Quickly pour the bees from the 120ml container into the jar; Close the cap; Gently rotate the jar with hands in 60 seconds in order to cover the bees with icing sugar; Leave for 3 minutes the jar with bees in vertical position (cap up); Shake violently the content of the jar (also with sidewall knock) through the screen lid, into a sieve that do not permit the passage of varroa mites. Shake for at least a couple of minutes. Preserve the sample of bees freezing it in order to bring it to the laboratory and to check the accuracy of the method with a soapy solution wash (see point 2). After the realization of the sample and the estimation of the number of mites with the icing sugar technique, a soapy solution wash was realized to verify the residual number of mites with this procedure: 1. Prepare a soapy solution (max 5 ml of soap to one liter of water) 2. In laboratory 1 1 If the laboratory equipments are not available you can use a whisk or an automatic beater to shake the bees in the solution for 2 minutes 4

5 3. pour all bees to be tested into the beaker; 4. Add approximately 200 ml of soapy solution into the beaker; 5. Use a magnetic stirrer to shake the bees in the solution for 30 minutes at a speed of 900 rev/min; 6. Pour the content through two sieves (one to collect the bees and one to collect the Varroa mites); See type (link) 7. Subsequently perform a wash of bees left in the sieve with high pressure water to check the fall of other varroa mites remained between bees; 8. With the help of a lamp equipped with a magnifying glass, count the number of adult mites in the sieve; 9. Repeat the procedure until there are no more mites in the sieve; 10. Count the number of bees. The natural mite fall evaluation was realized counting the mites fallen during the 10 days before the icing sugar sampling placing sticky or oily label sheets on the bottom boards, replaced every 2 days (or adapted to local conditions). A pre-requisite was to take care to prevent access of varroa-eating arthropods in different ways: cutting grass, putting the beehives on specific supports (i.e. legs of stands dipped in jars with water and an oily substance) and do not leaving trays in place during season but insert them only at the moment of starting the trial. In order to obtain data about the total amount of varroa mites (phoretic and reproductive) inside the colonies and to correlate that data with the infestation level obtained with the samples, we proposed two different protocols to be realized in the mid summer, after the main honey flow. The first protocol, named short term protocol was shorter in duration and consisted in: the count of the natural mite fall for 10 days, the subsequent application of the icing sugar technique, the verification with the soapy solution wash of the residual mites and the evaluation of the total mite infestation (phoretic and reproductive) The second protocol was longest and consisted in the realization of two samples of bees to verify a possible correlation between the different infestation levels checked in the two samples and the acaricide efficacy of a formic acid treatment. Above are described the two protocols. Short term protocol From Day 0 to Day 10: Evaluate the natural mite fall (point 3) with sticky or oily label sheets on the bottom boards, replaced every 2 day (or adapted to local conditions) Day 10: realize the bee sampling (point 1 and 2). Cage the queen (API.MO.BRU. Italian cage - link). Realize an oxalic acid treatment. Realize Liebefeld estimation. 2 2 **Depending on beekeeper and researcher availability you can evaluate only the number of phoretic varroa mites: From Day 10 to Day 15: Evaluate the phoretic mite fall with sticky or oily label sheets on the bottom boards, replaced every 2 days (or adapted to local conditions) to evaluate the amount of phoretic varroa mites. In this case the protocol finish on day 15. (It will be called VERY SHORT-TERM PROTOCOL ) 5

6 **See note From Day 10 to Day 40: Evaluate the mite fall with sticky or oily label sheets on the bottom boards, replaced every 2 days (or adapted to local conditions) to evaluate the infestation level Day 35: Release the queen. Realize an oxalic acid treatment 3 3 Prepare the oxalic acid solution in this way: use 1 liter of hot water (70-80 C) to dissolve 100 grams of oxalic acid pure crystals. Then add 1kg of sugar (commercial one) to the hot solution. Then wait until it's cold. With a syringe drop 5ml of the solution for each intercomb space occupied by bees. 6

7 Long term protocol In this case we will integrate our protocol inside that proposed in WG4 (Formic acid management) in order to evaluate the total amount of Varroa and also the possible correlation of infestation level with the acaricide efficacy of the treatment. From Day 0 to Day 10: Evaluate the natural mite fall (point 3) with sticky or oily label sheets on the bottom boards, replaced every 2 days (or adapted to local conditions) Day 10: realize the bee sampling (point 1 and 2). Realize the formic acid treatment (specification will be provided by WG4). Realize Liebefeld estimation. From Day 10 to Day 20: Evaluate the mite fall with sticky or oily label sheets on the bottom boards, replaced every 2 days (or adapted to local conditions) to evaluate the varroa killed by formic acid. Day 20: Remove the dispenser. Realize bee sampling (point 1 and 2). Realize Liebefeld estimation. From Day 20 to Day 32: Evaluate the mite fall with sticky or oily label sheets on the bottom boards, replaced every 2 days (or adapted to local conditions) to evaluate the varroa died within cells. Day 32: Cage the queen (API.MO.BRU. Italian cage - Realize Liebefeld estimation. From Day 32 to Day 62: Evaluate the mite fall with sticky or oily label sheets on the bottom boards, replaced every 2 days (or adapted to local conditions) to evaluate the residual varroa. Day 57: Release the queen. Realize an oxalic acid treatment The protocol is described in the figure above: 7

8 A total of 10 researchers, coming from 7 different countries participated to the trials. 25 apiaries (326 beehives) were used for the trials. A shared file to collect all the data obtained by the different field trials was published on the COLOSS Varroa Task Force webpage. Restricted access was provided to participants. A google document accessible to this link: dit?usp=sharing has been created and shared with the interested WG participants to collect all the references available in the literature and the results of the activities concerning the varroa infestation assessment. References I. ALIANO N.P., ELLIS M. (2005). A strategy for using the powdered sugar to reduce varroa population in honey bee colonies. J. Apicult. Res. 44, II. BAK, B., et al. Comparison of two methods of monitoring honeybee infestation with Varroa destructor mite. Annals of Warsaw University of Life Sciences-SGGW, Animal Science, 2009, 46: III. BRANCO, M. R., KIDD, N. A., & PICKARD, R. S. (2006). A comparative evaluation of sampling methods for Varroa destructor (Acari: Varroidae) population estimation. Apidologie, 37(4), 452. IV. DE JONG, D., ROMA, D. D. A., & GONCALVES, L. S. (1982). A comparative analysis of shaking solutions for the detection of Varroa jacobsoni on adult honeybees. Apidologie, 13(3), V. FAKIMZADEH K. (2000). Potential of super fine ground, plain white sugar dusting as an ecological tool for the control of varroaasis in the honey bee (Apis mellifera), Am. Bee J. 140, VI. FAKIMZADEH K. (2001). Effectiveness of confectioner sugar dusting to knock down Varroa destructor from adult honey bees in laboratory trials. Apidologie,32(2), VII. LEE, K. V., MOON, R. D., BURKNESS, E. C., HUTCHISON, W. D., & SPIVAK, M. (2010a). Practical sampling plans for Varroa destructor (Acari: Varroidae) in Apis 8

9 mellifera (Hymenoptera: Apidae) colonies and apiaries. Journal of economic entomology, 103(4), VIII. LEE, K., REUTER, G., & SPIVAK, M. (2010b). Standardized sampling plan to detect Varroa density in colonies and apiaries. American bee journal J Econ Entomol Aug;103(4): IX. MACEDO P.A., WU J., ELLIS M.D. (2002). Using inert dusts to detect and assess varroa infestations in honey bee colonies. J. Apicult. Res. 41, 3-7. X. RAMIREZ B.W. (1989). Can varroa mite be controlled with dust? Apiacta 24, 3-6. XI. RINDERER, T., DE GUZMAN, L., & SYLVESTER, H. A. (2004). Re-examination of the accuracy of a detergent solution for varroa mite detection. American bee journal. 9

10 Leaders: Ralph Buechler and Antonio Nanetti Working Group 2 - WG 2: Brood interruption ACTIVITIES The COLOSS - VARROA CONTROL TASKFORCE took step forward and developed two protocols for initiating an international study entitled: Assessment of alternative summer methods for Varroa control. The protocols (Brood interruption with queen caging and Brood removal) in details describe the methodology for implementation of a study for the seasons 2016 and SEASONAL BROOD INTERRUPTION STUDY PROTOCOL 2016/2017 INTRODUCTION In many regions, long reproductive seasons cause strong Varroa population increases which often result in colony losses. An artificial interruption of the seasonal brood cycle can be a useful option to reduce varroa infestation levels in due time. During broodless periods all mites are forced in a phoretic stage and can easily be trapped with open brood combs or killed by varroacides (f.e. oxalic acid), which were primarily developed for winter treatment. During the spring workshop in May 2016 on Unije, WG2 members decided to organize an international study during season 2016/2017 to estimate the effects on colony development and Varroa treatment efficacy of different brood interruption techniques and oxalic acid application methods under different environmental conditions. Basic descriptions of the working procedures for the following methods: I. Queen caging and treatment II. Total brood removal combined with trapping comb III. Classical trapping comb technique IV. Trapping comb technique simplified are appended to this protocol. In order to achieve comparable data for a common evaluation and scientific publication each participating partner needs to test at least 2 groups of 10 colonies each according to the following protocol. All data have to be reported until April 2017 and will be revised during a workshop in spring EXPERIMENTAL DESIGN Material - An apiary of at least 20 full size, naturally infested colonies. - Varroa control cages and excluder frames (see basic method descriptions). - Sticky sheets (sprayed with vegetable oil) for counting varroa mites on bottom board. - Apistan, Amitraz, Bayvarol or CheckMite strips for critical control treatment. 10

11 Colony conditions - Create two or several groups of at least ten colonies each. Data on colony strength (Liebefeld check) and mite infestation level of bees, as measured at the beginning of the experiment should be used to distribute the colonies to homogeneous groups. - Take field measures to minimize drift and reinvasion. - Remove or pre-treat colonies not included in the trial. - Limit colony handling as far as possible. Do not affect natural bee population grow and brood development. Treatment variants 1. Caging, OA trickling 4.2% Queens are caged for 25 days, 4,2 % oxalic acid solution (prepared with 10 g oxalic acid dihydrat g sucrose ml water) is administered thereafter by trickling, 5 ml/comb. This variant will be used as a standard on all involved apiaries. Choose one or more of the following treatment variants for comparison to this standard: 2. Caging, OA trickling Apibioxal Queens are caged for 25 days, Apibioxal solution according to label is administered thereafter by trickling, 5 ml/comb. 3. Caging, OA trickling Oxuvar Queens are caged for 25 days, Oxuvar solution according to label is administered thereafter by trickling, 5 ml/comb. 4. Caging, OA sublimation Queens are caged for 25 days, 2 gr Oxalic acid dihydrate crystals are administered thereafter by sublimation with the VARROX -vaporizer according to label instructions. 5. Total brood removal All brood combs are removed according to the described working procedure. 6. Classical trapping comb technique The queen is caged on single combs which are removed after sealing, over a day period, according to the described working procedure. Note the use of either drone or worker brood as trapping combs and the rhythm of comb change (4*7 versus 3*9 days) in the protocol. 7. Trapping comb technique simplified (1*20 days) The queen is caged on a single comb foundation, which is removed after a 20-days period. Transfer the queen in the nest. Take the comb foundation on which the queen was caged, count varroa mites in the sealed cells and destroy the comb. After 5 more days, realize the oxalic-acid treatment on the colony. 8. Local standard treatment for colonies with brood A local standard treatment for colonies with brood, like the use of formic acid or thymol, may be included as an extra group in the experiment. Critical treatment All colonies have to be treated with either Apistan, Apivar, Apitraz, Bayvarol or CheckMite strips according to label instructions. The product is chosen with regard to the legal local registration and the presence of resistant mites. 11

12 In the case of any pharmacoresistance a combined use of two of these drugs is recommended to ensure a high efficacy level. Timing Start all groups at the same date and synchronize also the critical treatment. The starting date depends very much on the local conditions. The honey harvest has to be finished before the treatment with OA on day 25. The handling of the colonies in the beginning of the experiment (caging of queens, total brood removal) is easier if there is some natural nectar flow available. DATA COLLECTION Colony performance At the start of the experiment, check all the colonies and measure adult bee population and brood area with the Liebefeld method. Repeat the measures about 4 weeks after the start of the critical treatment, pre- and post-wintering. In all colonies, take note of queen mortality/supersedure during the experiment. If a honey flow is likely to occur during the treatment, weight the colonies at the start of the experiment and weight again post-harvest. Mite infestation To realize homogeneous groups for the varroa infestation level, take a sample of about 500 bees (50-60 gr) from each hive to determine the bee infestation level at the beginning of the experiment by the washing or powder sugar method. Alternatively, count varroa natural falls for 2 weeks before starting the experiment. Insert sticky sheets on all hives at the beginning of the experiment. Count the mites in intervals of 3-4 days, if possible. However, some flexibility is given about the intervals to adapt to the local working conditions. The sticky boards are used until 2 weeks after removing the strips for the critical treatment. As an optional information all removed trapping combs can be checked for mite infestation by uncapping and washing out the brood. The mites can be separated from the brood by the use of honey double sieves. Requeening Especially for large enterprises it might be attractive to combine the caging technique with requeening. If you can do so within this experiment collect data on the practicability and efficacy (queen acceptance rate) for one of the following procedures: Kill the old queen instead of caging it and wait for supersedure by an emergency queen. Treat with OA 25 days after removing the old queen. Kill the old queen immediately or after some days of caging, 7-9 days later destroy any supersedure cells and introduce a mature queen cell. Treat with OA on day 25. Cage the old queen, remove it after 25 days and introduce a young mated queen in a shipment cage under candy closure to be released by the bees within the next 12 hours. At the same time treat with OA. Take note of the queen age As an optional information, take note of the queen age. The success of the last method probably depends very much on the physiological status of the young queen. If possible take young queens directly from their mating boxes and introduce them to the colonies within the same day. 12

13 Additional recordings Locations Measure the GPS coordinates and the altitude of the apiary. Environmental recordings f.e. on nectar and pollen flow are useful to frame the trial correctly and to compare results between apairies. Climatic conditions If possible, take continuous recording of external and internal temperature and relative humidity (f.e.: ibutton Hygrochron Temperature/Humidity Logger DS1923). Bee mortality Dead bee counts may be taken with traps placed at the hive entrance. Note any obvious mortality of brood or adult bees occurring during the experiment. Bee diseases Note any symptoms of brood or bee diseases occurring during the experiment. If it is possible to arrange a labor analysis, bee samples for virus and Nosema control should be taken in autumn. Residues If it is possible to arrange a residue analysis for oxalic acid, honey, bees and wooden frames may be sampled a few days after OA treatment and again about 8 weeks after OA treatment. Work load Make notes on the time effort for the application of the different treatment variants. Online database Alex Uzunov will develop and share an online database to collect all relevant data for a common evaluation of the experiment. 13

14 Calendar of main activities *): If possible, sticky sheets should be exchanged in 3-4 day intervals but there is some flexibility to adapt the timing to local conditions 14

15 APPENDIX: WORKING PROCEDURES I: Queen caging and treatment Use Var-Control cages, 5 x 7.5 cm, with queen excluder on both sides 4. Fix the cage in a comb. This frame can be left in the colony for re-use. Refer to the following pictures. Locate (1) and gently remove (2) the queen from the comb. Introduce in the cage (3), shut firmly (4) and leave untouched for 25 days. On day 25, unseal the cage (5) and reintroduce the comb into its slot (the queen finds her way out shortly). Administer the oxalic acid solution onto the combs (6). 1) Locate the queen. 2) Remove the queen. 3) Introduce in the cage. 4) Shut the cage. 5) On day 25: open the cap. 6) On day 25: oxalic acid administration. 4 The normal queen excluders used for supers (slots of 4.2 mm) occasionally let tiny queens pass through. These slots of 3.7 mm are safer: (a device for beekeepers not familiar with queen handling is shown as well). 15

16 II: Total brood removal combined with trapping comb (see also: Step 1 Open the colony, find the frames with brood (open and sealed), shake the bees from them (Fig. 1 & 2) and dispose them in a new hive body or take them home for melting. Make sure that the queen remains in the original hive. Note I: for establishing a new colony from those frames -, leave about 500 worker bees per comb for warming the brood. The brood combs from different colonies can be put together in a new hive on 1 3 supers (10-30 frames). Make sure that sufficient food combs are added to supply the bees for the next few weeks. The brood collector should be moved to a separate apiary to avoid robbery and mite exchange with the original hive. If no queen in added, the bees will rear an emergency queen. After days all the brood is gone and the bees can then easily be treated against Varroa. Note II: all brood from the colony should be removed. Step 2 Insert frames with build combs or foundations (Fig. 3) on the free places of removed frames. Leave free space for a single frame. Step 3 Insert a single trapping frame (Fig. 4) with all stages of open brood. If there are any sealed brood cells present which could emerge within the next 9 days they have to be destroyed. Note I: in case of intensive honey flow the honey suppers should remain on the hive. Step 4 After 7-9 days, open the colony and remove and count varroa mites in the sealed brood. After that, destroy the single trapping frame now with sealed brood and confined mites, add an empty comb or foundation to replace the trapping comb. Step 5 Without regular nectar flow (rainy weather f.e.) there is need for colony feeding with 2-3 liters/colony/week. 16

17 Fig. 1 - Removal of frame with sealed brood Fig. 2 - Establishment of new colony Fig. 3 - Insertion of frames with foundation Fig. 4 - Trapping frame with open brood (centre position) and frames with foundation and food 17

18 III: Classical trapping comb technique (3*9 days) The classical trapping comb technique was already developed in the 1980ies by Maul et al (see: Papers trapping comb technique.pdf) and proofed to be a highly efficient biotechnical control measure which allows to fight Varroa without the use of any chemicals. The queen has to be caged for subsequent intervals of 3 * 9 days or 4 * 7 days on one empty drone or worker comb per interval. For this purpose simple excluder frames can be constructed by the use of 10 mm wooden laths and plastic or metal queen excluders which fit exactly to the combs frames (1). 1): Construction of excluder frames to be fixed on both sides of a standard drone or worker comb Before you start to search for the queen a trapping comb is prepared with a central hole and an excluder fixed on the backside (2). As soon as the queen is found put it carefully through the hole to the protected backside of the comb and place the second excluder frame on the front side of the comb. A thin wire can be used to tightly fix the excluder frames on both sides of the trapping comb. Check carefully for the fit of the excluders to be sure that there are no larger gaps at the edges of the combs through which the queen might escape. The trapping comb is returned to the central brood nest. After 9 (or 7) days the comb is taken out, opened and the queen is transferred to a second trapping comb in the same way as described above. The first trapping comb which should contain all stages of open brood is marked and put back to the brood nest. Another 9 (or 7) days later the first trapping comb is completely sealed. It will be removed and melted to kill the mites trapped in (3). 18

19 2) Empty comb with a central hole and queen excluder frames prepared to introduce the queen 3) After 7 9 days the trapping comb should contain all stages of open brood (left). After moving the queen to the next trapping comb, it remains in the brood nest until all brood is sealed (right). After 27 (3*9) resp. 28 (4*7) days the queen is released. At that time old comb s can easily be replaced by new combs or foundation-combs. The last trapping comb is removed after another 9 (or 7) days. Depending on the season, colonies may build some supersedure cells on the remaining open worker brood during the first trapping period of the queen. To avoid any risk for the old queen the colonies may be checked and any supersedure cells be destroyed during the next inspection (7-9 days after the start of the treatment). 19

20 IV. Trapping comb technique simplified (1*20 days) The queen is caged on a single comb foundation for 20 days (use excluder frames as described before). After 20 days the comb is taken out, opened and the queen is released to the nest. Take the comb foundation on which the queen was caged, count varroa mites in the sealed cells and destroy the comb. After 5 more days, realize the oxalic-acid treatment on the colony. 20

21 Working Group 4 - WG 4: Formic acid management ACTIVITIES Leaders: Benjamin Dainat and Giovanni Formato This WG studied different protocols to evaluate and compare acaricide efficacy and toxicity for the honey bees of formic-acid based treatments in different Countries. We have protocols adopted in field experiment of 2014 and 2015 (WG protocols) and protocols adopted subsequently to the decision taken in the Unije Workshop (WG protocols). WG PROTOCOLS Active compound to study: formic acid, in association with oxalic acid as a control treatment. Apiaries: Locations: The apiaries should be located at different altitudes (coastal plain, hill, mountain) or at least different climatic conditions (sunny place or close to a forest etc ). The aim is to cover a wide range of conditions in different countries to be able to compare the efficacy of the Varroa treatment. Each apiary: Minimum 5 hives/treatment type. Minimum 10 colonies per apiaries. The more the better. We maintain the number rather high because we want to use the Liebefeld methods ( to verify the toxicity on the honey bees to estimate colony strength and if treatment affect colony development and strength. 2 mandatory treatments: formic acid using the dispenser Nassenheider Evaporator Professional; MAQS Other dispensers (1 or 2 or more) of your choice, including locally used dispenser Type if apiary has enough colonies: Generally there are 2 principles with dispensers. The one with a sponge system, the one where the acid is contained in a bottle. For example in CH will be studied: FAM (local type, sponge system) and Liebig Dispenser (Bottle system). Please consider the different dispenser principle when selecting the dispenser of your choice. Ideally you would test the two different principles. Hives: Specify the type of hives that will be studied in your respective country. 21

22 For example, in Switzerland there are 2 types of hives mainly used: Swiss hive in beehouses and Dadant. Studies will be performed on this two different types hives. In different countries different hive types may be tested. Experimental groups: (5 hives/dispenser) The treatments and the dispensers will always be realized according to the manufacturer user manual. For the test, no supers! Acid formic is supposed to be used after harvesting anyway. Consumption of Liquid fornic acid should be measured: if there is rest in the bottle after the treatment, measure the rest. In MAQS manual, According to manufacturer, you can use supers. This can have consequences because of the increasing of formic acid levels in your honey. Therefore we will test MAQS without supers. It is also said you can remove the strips or leave it after 7days. In this trial we will REMOVE the strips after 7 DAYS of treatment! If you really want to realize test with supers, you should include in the study the consequences for the honey produced after the treatments, like: taste of honey, increasing in free acids equivalents and formic acid, hydroxymethylfurfural (HMF) values. Remark: It is recommended to feed the colonies before formic acid treatment. Treatments: 1) Nassenheider Evaporator Professional (about 10 each evaporator; provided with a bottle with visible filling level). Formic acid 60%. Dosage: 180 ml/treatment for 10 days treatment. 2) MAQS. 7 days of treatment, according to label indications. 3) Optional: Your own local dispenser (e.g. FAM dispenser: Formic Acid 60%; Liebig: Formic acid 85%; BLV in Italy). Monitoring of T and H : T and H will be monitored by one logger/apiary placed at 2 meters, in the shade, protected from the rain. Proposed logger model: Ibutton Model: DS 1923 (cost: Maximum about 70 a piece). You will need to buy as well a cable to read them: DS1402D-DR8 and an 1-Wire USB Adapter (cost: about 15 ). Or something with the same technical requirements /resolution (e.g. EL-USB-2+) We also decided to have one logger per colony. 22

23 After the experiment each logger should be recalibrated to check if they are still working properly. If there is deviation a correction coefficient should be applied to the collected data. Humidity and Temperature will be recorded every hour during the treatment time. The logger should be located in a way that it is protected from the bees, not too close from the dispenser, but still close enough to register realistic temperature. Here some picture to show where to put the logger. Figure 1: Protection of the logger (left). Plastic cap + net + caoutchouc ring + Ibutton (right) Figure 2: Location of the logger on the FAM 23

24 Figure 3: Location of the logger on the Nassenheider Pro Figure 4: Location of the logger on Liebig Figure 5: Location of the logger for MAQS 24

25 You can adopt 2 different protocols: 1. SHORT-TERM PROTOCOL Goals: a) verify the efficacy of the treatment; b) verify the toxicity of the treatment on the honey bees (adopting the Liebefeld method to estimate strength of the hives). Mite fall counts will be realized on bottom board every 2-3 days. To verify the survivals of varroa after formic acid treatments, you can use oxalic acid 5 after confinement of the queen in cages for 25 days (Fig. 6), or another treatment realized with 2 different chemicals administered (e.g. 2 strips of Apistan or CheckMite and 2 strips of Apivar in the centre of the colonies, three combs apart, in cross) To calculate the percentage of acaricide efficacy (AE) of formic acid in each hive, use the V T following formula: AE= 100, where V T is the total number of mites killed with V (T+Follow-up) the formic acid treatment and V (T+Follow-up) represents the total number of mites killed by the tested treatment and the oxalic acid or the chemicals. 5 Api-Bioxal according to label or bihydrate oxalic acid, sucrose and demineralized water 1:10:10 in weight. ATTENTION: the use of Oxuvar or Ecoxal leads to insufficient concentration. 25

26 Fig. 6: Examples of queen cages. Var Control -API.MO.BRU (left); bamboo wooden queen cages(right) Links for cages: If possible, at the beginning of the trial record the queen age. Record the queen mortality at the end of the formic acid treatment. 26

27 Example of short term protocol Each apiary: 8 hives (8 hives/group; 3 groups). For a total of 24 hives When: Summer Experimental groups: (8 hives/group) 1) Nassenheider Evaporator Professional (considering 10 days of treatment). 2) MAQS. (considering 7 days of treatment, according to label indications). 3) Untreated (control) In the Figure queen is caged for less than 25 days as described above: from day 22 to 45 and from day 19 to 42. Chemicals (Apistan, ChecMite ) can be used to perform final treatment, after formic acid application. In this case more accurate data will be obtained to calculate formic acid efficacy. 27

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29 2. LONG-TERM PROTOCOL To verify: a) the toxicity of the treatment for the honey bees (using the Liebefeld methods) and b) the efficacy of the treatment checking the varroa survivals in Autumn/Winter time. In this case there are more variables: e.g. seasonal variability) and should be used more apiaries.control treatments: oxalic acid trickling in absence of brood generally November-December in central Europe climate conditions. If you are in an area with no brood stop in Winter, queen caging with your usual method is recommended for an oxalic treatment in November- December. Varroa infestation level: To measure Varroa treatment fall and natural mite fall we will count the Varroa on bottom board every 2-3 days. To protect against ants, we will use paper towels impregnated with oil (canola or sunflower oil) to be changed after each counting. Between last formic acid treatment and the control oxalic acid treatment Varroa should be counted once a week (if not possible at least twice a month) to monitor any Varroa reinvasion. Flight entrance should be reduced, any feeding with sugar syrup during day prohibited in order to reduce risk of robbery. Liebefelder method: To measure colony strength and development. Just before first treatment, after the treatment, before wintering (in Switzerland mid-october), and in spring (in Switzerland end of March beginning of April). Here is the method described: You can do the online exercise to train yourself. In the field for the population measurement by taking a computer with excel (Excel files are provided in separate files) or by printing the population measurement sheet (provided separately in DOC format). You can enter the data then back to the computer. Collection of the data 29

30 All the collected data will be gathered in a common excel file. Like that we all have the data in the same format and ready to use for statistics. This data are the population measurement data, T and H, Varroa and the remarks. When to start? Beekeeping season Weather forecast: by Stefan. This is planned in a later stage. 30

31 Example of Long term protocol Time table plan References Guideline on veterinary medicinal products controlling Varroa destructor parasitosis in bees.; European Medicines Agency - EMA (2010). Available online (last accessed ): pdf Imdorf A, Charrière JD, Kilchenmann V, Bogdanov S, Fluri P (2003) Alternative strategy in central Europe for the control of Varroa destructor in honey bee colonies. Apiacta 38: Technical guidelines for the evaluation of treatments for control of varroa mites in honey bee colonies; European working Group for Integrated Varroa Control. Available online (last accessed ): A statistical analysis of the data obtained in the field trials realized by the different Institutes that worked according to the short term protocol and to the long term protocol will be performed. 31

32 WG PROTOCOLS This 2nd protocol was written to compare the efficacy of formic acid in presence and absence of brood. Aims: 1. The absence of brood will change the in-hive temperature and humidity and will give the opportunity to better understand the influence of internal and external conditions on efficacy of the formic acid treatment. However, we cannot exclude the fact that variations in efficiency would be due to a different behaviour of workers due to the presence of brood, other than the simple production of heat. Minimum number of colonies for each experiment is 12 (6 with brood, 6 without brood). 2. Evaluation of efficacy of flash formic acid treatment without brood compared to oxalic acid will provide baseline data for potential new application method for flash formic acid treatment. Minimum number of colonies for each experiment is 12 (6 with oxalic acid, 6 with formic acid). Note: loggers use is according to the dispenser tests: No brood experiment N. 1 32

33 Brood is added to one of the two groups that are otherwise treated in the same way. Added brood is removed before emergence in order not to increase the amount of varroa in the test colonies. The alternative manipulation of removing brood in one of the groups is not suitable since it would lead to the removal of most varroa and bias the evaluation of efficiency. No brood experiment N. 2 33

34 Working Group 5 WG 5: Assessment of new control methods Leader: Maja Ivana Smodis Skerl ACTIVITIES AND PERSPECTIVES Goal: testing new frontiers to control Varroa destructor, considering the beekeepers priorities and realizing comparative studies. This Working Group has recently been formed, in the Varroa Control Task Force Workshop held in Unije (Croatia) on 19th and 20th of May The activities that are willing to be realized are: - consider beekeeper s priorities (interest for new effective varroa control treatments leaving no residues in bee products)/collaboration with Working Group 6; - to realize field trials among Varroa control TF members Countries to compare and evaluate different new methods verifying efficacy against varroa and toxicity for honey bees. Special interest will be given to new Varroa control products (e.g. AluenCap from Argentina oxalic acid based product; Varroa kill from Austria based on hyperthermia, etc.); - to define common protocols amongvarroa control TF members Countries to test the different products; - to contact the wholesale dealer to get the product for field trials and understand new trends. 34

35 Working Group 6 WG6: Communication ACTIVITIES AND PERSPECTIVES Leaders:Victoria Soroker and Flemming Vejsnaes Goal: To guarantee a strong communication concerning the output of the Varroa Control Task Force towards the beekeepers (by training, guidelines and books), and the B-RAP COLOSS project leaders. Moreover, WG 6 should give feedbacks from beekeepers to the WGs leaders of the Varroa Control Task Force, suggesting them new studies/ideas/corrections for future activities. This Working Group has recently been formed, in the Varroa Control Task Force Workshop held in Unije (Croatia) on 19th and 20th of May As this group is very new we are still in the process of formulating the ways of cooperation with different working group and information exchange with the beekeepers. We expect that B-rap group of COLOSS (Flemming Vejsnaes is the Co-chair of this group) will be the main cooperator especially since its mission is bridging research and practice. The below diagram is how we expect our relationship and focus to be in the nearest future. The other partnership we were thinking of was a monitoring group. As using the questionnaire platform, we could get info and feedback on commonly used and new Varroa control practices. 35