Insecticidal Control of Small Hive Beetle

Developing a ready-to-use product

by Dr Garry Levot

September 2007

RIRDC Publication No 07/146 RIRDC Project No DAN 216A

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© 2007 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 542 4 ISSN 1440-6845 Insecticidal Control of Small Hive Beetle: Developing a ready-to-use product Publication No. 07/146 Project No. DAN216A Pt II The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances.

While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication.

The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors.

The Commonwealth of Australia does not necessarily endorse the views in this publication.

This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165.

Researcher Contact Details Garry Levot Elizabeth Macarthur Agricultural Institute (NSW DPI) Woodbridge Road Menangle, NSW 2568 Phone: 02 4640 6376 Fax: 02 4640 6300 Email: garry.levot@dpi.nsw.gov.au

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6271 4100 Fax: 02 6271 4199 Email: rirdc@rirdc.gov.au. Web: http://www.rirdc.gov.au Published in September 2007 Printed by Union Offset

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Foreword

This project aimed to refine the prototype refuge trap developed in DAN 216A Part I into a safe and effective insecticide-based device that beekeepers could use for the control of Small Hive Beetles in hives. The Small Hive Beetle Harbourage created comprises a fipronil-treated fluted cardboard insert permanently sandwiched between a pair of custom-designed plastic mouldings that once assembled, allow beetles to enter but exclude bees. The harbourage was shown to be compatible with existing hive structure and bee keeping habits, easy to use, highly effective in reducing beetle infestations and safe to users, bees and honey. There are patents pending for the invention not only in Australia but also in the United States of America, Canada and New Zealand. However, a lack of commercial interest in registering the product and restricted access to fipronil for this purpose may delay the commercial availability of the device until the fipronil patents lapse. The commercialisation of the device is essential so that beekeepers have an effective means for control of Small Hive Beetles in hives and to discourage the use of 'home remedies' that could lead to contamination of honey and place the industry at risk. This project was funded from industry revenue which is matched by funds provided by the Australian Government. This report, an addition to RIRDC’s diverse range of over 1600 research publications, forms part of our Honeybee R&D program, which aims to improve understanding and management of bee diseases and pests and reduce production losses caused by pests and diseases. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/fullreports/index.html • purchases at www.rirdc.gov.au/eshop Peter O’Brien Managing Director Rural Industries Research and Development Corporation

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Acknowledgments My thanks go to Mr Steve Goray, Mrs Lyn White and Mr Pat Carroll for allowing use of their hives in the field trials and especially to Mr Nicholas Annand and Mr Bruce White for their willing participation with the conduct of the field trials. The contribution of Dr Michael Hornitzky to this project is gratefully acknowledged and I thank Mr John Ryan, NSW DPI Manager Commercialisation for advice on the production of the commercialisation dossier and patent applications.

Abbreviations APVMA - Australian Pesticides and Veterinary Medicines Authority (the regulatory authority responsible for the registration of pesticides in Australia). NSW - New South Wales NSW DPI - New South Wales Department of Primary Industries LC50 - Lethal Concentration to 50% of individuals tested

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Contents Foreword ..................................................................................................................................iii

Acknowledgments.................................................................................................................... iv

Abbreviations........................................................................................................................... iv

Executive Summary ................................................................................................................ vi

Introduction .............................................................................................................................. 1

Objective................................................................................................................................. 3

Materials and Methods ............................................................................................................ 4

Refining the harbourage ......................................................................................................... 4

Residue trial with the End-Use-Product ................................................................................. 4

Honey.................................................................................................................................. 4

Cardboard inserts ................................................................................................................ 4

Commercialisation.................................................................................................................. 5

Field trials with the End-Use-Product .................................................................................... 5

Residual efficacy of the harbourages...................................................................................... 5

Refining the harbourage ......................................................................................................... 6

Residue trial with the End-Use-Product ............................................................................... 11

Commercialisation................................................................................................................ 14

Field trials with the End-Use-Product .................................................................................. 14

Trial 1. Wilberforce .......................................................................................................... 17

Trial 2. South Maroota...................................................................................................... 20

Trial 3. North Richmond................................................................................................... 22

Residual efficacy of the harbourages.................................................................................... 24

Discussion................................................................................................................................ 25

References ............................................................................................................................... 27

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Executive Summary What the report is about This report details the development, safety and effectiveness of an insecticide-treated refuge trap for the control of adult Small Hive Beetles in the hive. Who is the report targeted at? The report is written for beekeepers, advisors to the honeybee industry and the agrochemical industry. Background The Small Hive Beetle is an introduced pest of bee hives causing hive losses and reduced productivity for beekeepers on Australia's eastern seaboard. Beekeepers may use oil traps to kill beetles in hives or permethrin soil drenches to control larvae in the soil. Both are laborious, need regular attention and are of limited effectiveness. Aims/Objectives This project aimed to refine the insecticidal harbourage developed during DAN 216A Part I into a ready-to-use product for beekeepers. It was hoped that the device could be registered with the Australian Pesticides and Veterinary Medicines Authority (APVMA) and marketed commercially by a manufacturer. Methods used The design of the harbourage evolved from discussions between the Principal Investigator, specialist plastics fabricators, cardboard manufacturers and beekeepers. Safety of the device was tested in a honey residue trial conducted in accordance with APVMA Guideline No. 28 Residues in Honey. Field trials conducted under APVMA Permit PER9732 in co-operation with commercial beekeepers in western Sydney were used to demonstrate effectiveness in naturally-infested hives. Results/Key findings A device that excluded bees but which was actively sought out by adult beetles was developed. The design: ensured that the insecticide-treated fluted cardboard insert was inaccessible to bees; is suitable for use in current hive boxes; is user-friendly; and safe for beekeepers, their bees and honey. Compared to 'control' hives, beetle infestations in hives at three western Sydney apiaries were reduced by 90-99% over six weeks with no detrimental effects on hive productivity or bee health. Implications for relevant stakeholders These results demonstrate that use of this device in well-managed commercial hives provides effective, safe and economical insecticidal control of Small Hive Beetle. The favourable results of the honey residue trial demonstrated that use of the device did not compromise the safety of honey to consumers by leaving unacceptable insecticide residues. There is no other control strategy with comparable efficacy to the Small Hive Beetle Harbourage but currently no company or other body has shown any interest in registering and commercialising the device in Australia or elsewhere in the world where patent protection exists. An essential element of the device is the use of the insecticide fipronil. Existing formulations of fipronil combine outstanding contact efficacy against Small Hive Beetle with favourable physicochemical characteristics such as extremely low vapour pressure but until patents expire BASF holds exclusive rights over all uses of fipronil. At the time of writing BASF has stated that the use of fipronil as proposed is inconsistent with its product stewardship position. Recommendations That effort continues to ensure this product becomes commercially available for beekeepers. It may be necessary to identify a sufficiently attractive overseas market to make registration of such a specialist device an economically attractive proposition.

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Introduction The Small Hive Beetle, Aethina tumida Murray was detected at Richmond in western Sydney hives late in 2002 (Fletcher and Cook 2002). A. tumida is a native of southern Africa where it causes little concern to apiarists. Primary damage caused by Small Hive Beetle is through the activity of the larvae that feed on brood, pollen and honey causing it to ferment. Stored supers of honey or extracted comb are also susceptible to damage by larvae and adult beetles (Elzen et al. 1999). Small Hive Beetle has established in eastern Australia although initial foci were around Richmond in Sydney's west, Cowra and Stroud (Gillespie et al. 2003). The Consultative Committee on Emergency Animal Diseases concluded that the incursion of Small Hive Beetle into NSW and Queensland could not be eradicated or contained. With the assistance of its expert steering committee comprising representatives of the Commonwealth and affected State governments, the Rural Industries Research and Development Corporation and the Australian Honeybee Industry Council, Animal Health Australia developed a National Small Hive Beetle Management Plan (2003) that had as one of its aims to "reduce the impact on productivity, slow the spread of the Small Hive Beetle in Australia and minimise the damage in infested apiaries by identifying and implementing measures (chemical, non-chemical and management) that minimise the risk of harmful chemical residues" …... to control Small Hive Beetle. Insecticide use was considered one of the strategies that could be used against adult and larval beetles. In particular, insecticidal control of beetles inside the hive was the ambitious primary aim. In DAN 216A Part I we identified fipronil as being superior to several alternative insecticides by having the desirable physicochemical characteristics of extremely low vapour pressure (Colliot et al. 1992) and low water solubility as well as excellent contact efficacy against adult Small Hive Beetles (Levot and Haque 2006) and no repellent effects. The behaviour of the beetles in laboratory culture (Haque and Levot 2005) suggested that a refuge trap incorporating core-fluted cardboard might be devised for in-hive use. Harbourages comprised of fipronil-treated core-fluted cardboard covered with adhesive-backed 50 µm thick aluminium foil were tested in the laboratory (Levot and Haque 2006) and in a small number of NSW DPI research hives. The laboratory experiments demonstrated that over 98% of beetles placed inside sealed hive boxes of stored comb were killed by the harbourages (Levot and Haque 2006) but the trials in the research hives suggested that unless the open ends of the cardboard were protected by several layers of foil bees could attack the cardboard (Figure 1) and hives would be lost. Moreover, small residues of fipronil were detected in the honey produced by hives in which the prototype harbourage was present. Another prototype (Figure 2) with strengthened, overhanging foil edges was tested in twenty commercial hives at two locations under APVMA Permit PER8167. After one month, with the use of the treated harbourages at a Wilberforce apiary, the numbers of live adult beetles seen on the bottom boards and elsewhere in the hives indicated a mean reduction of 93% compared to pre-treatment counts. At an apiary located at Cranebrook the mean reduction in live beetle counts was 87% compared with untreated control hives. However, at the Cranebrook site one hive was killed when bees damaged the harbourage.

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Figure 1. Damage to cardboard done by bees that forced entry into an early prototype harbourage.

Figure 2. Early prototype cardboard harbourage used in the preliminary field trials (2005).

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Two conclusions were drawn from the limited field work: the harbourages worked in that beetle numbers in the hives were drastically and rapidly reduced, and; a more robust covering for the fipronil-treated cardboard insert was required to protect bees and minimize the possibility of honey contamination. This became the focus of the current project. Objective The project had a single objective: • To refine the insecticidal harbourage developed during DAN 216A Part I into a ready-to-use

product for use by beekeepers that could be registered with the Australian Pesticides and Veterinary Medicines Authority and developed commercially by a manufacturer.

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Materials and Methods

Refining the harbourage Technical drawings of a two-piece rigid plastic harbourage shell thought to contain all the features deemed important to the safety of bees and to the efficient operation of the device were made by the Principal Investigator in August 2005. Important design features of the harbourage were:

• A two-piece rigid plastic design

• Tamperproof after assembly (no user access)

• Made such that there are precisely sized openings that allow beetles in but exclude bees

• Protective of C flute cardboard insert

• Designed such that the cardboard insert is positioned 8 mm in from openings

• Black

• Compatible with silicone adhesives

• Inexpensive

• Disposable in household garbage.

Discussions were held with two potential manufacturers. The cardboard insert material ('C' flute corrugated cardboard (Australian Corrugated Box Pty. Ltd., Wetherill Park, NSW; Product Code no. CO3 RL150,) was deemed to be ideal in terms of a refuge for the beetles and as a matrix for the insecticide. As a check that the plastic harbourage was as attractive to the beetles as the foil-covered versions, a piece of untreated cardboard was placed inside a final version of the harbourage as soon as it was delivered. The assembled harbourage was, in turn, placed inside a plastic box containing over 100 adult Small Hive Beetles. The box was inspected four hours later.

Residue trial with the End-Use-Product Honey Five research hives located at Belgenny Farm, Camden were used in a honey residue trial that commenced on 12 September 2006 when ambient temperatures and flowering favoured bee activity. In accordance with APVMA Guideline 28 Residues in honey (APVMA 2001) two central frames from the middle super of each hive were removed and brought to the laboratory. The cells on each frame were uncapped and the honey extracted using a manual bench-top extractor. Honey from each hive was bulked into a 10 L plastic drum and sub-samples were poured into labelled clean glass jars prior to being placed into frozen storage. Drawing pins were pushed into the frames so that they could be easily identified later and the frames were then placed back into the hives. One month later the same frames were again removed. In accordance with the Guidelines the honey extracted from the ten frames was bulked together. Five sub-samples of this post-treatment honey were transferred into labelled clean glass jars which were then placed into a freezer. The frozen sample jars were sent to AgriSolutions Australia Pty. Ltd. in Queensland as coded samples (i.e. the laboratory was blind to the sample history).

Cardboard inserts To quantify the amount of fipronil present in the cardboard used in the harbourages eight 5 x 5 cm samples (identified as Cardboard samples CS, CT, CU, CV, CW, CX, CY and CZ) were sent to AgriSolutions for individual analysis. CZ was an untreated 'control' piece of cardboard. Samples CS, CT, CU, CV and CW were pieces cut from the cardboard that had been inside the harbourages used for one month in the honey residue trial whereas CX and CY were recently treated cardboard.

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Commercialisation Until fipronil comes off-patent, BASF holds exclusive rights to its use. In anticipation of a change of circumstance a commercialisation dossier was produced in collaboration with Mr John Ryan, Commercialisation Manager (NSW DPI). This dossier was advertised in a call for Expressions of Interest in the national newspapers in February 2007. The Executive Summary and the covering letter calling for Expressions of Interest were also directly mailed to fifteen agrochemical companies. The dossier contained a thorough description of the features, safety and effectiveness of the harbourage. The closing date for applications was 13 March 2007.

Field trials with the End-Use-Product A Category 23 (Application for a Research Permit) permit application was dispatched to the APVMA in November 2006 as soon as the results of the honey residue trial with the plastic harbourage were received. The application was based on the efficacy data collected during the 2005 field trials with the prototype harbourage and on the favourable insecticide residue data collected during the spring 2006 trials with the plastic harbourage. The application contained supplementary information under the headings Residues and Trade Considerations, Occupational Health and Safety and Target Species Efficacy and Safety. A draft label with Directions for Use was also provided. Field trials began shortly after the issue of the permit. On 1 March, previously purchased knife-cut cardboard inserts were treated with Regent® 200SC Insecticide (1.5 mL per litre of water). When the cards were dry and flat one hundred harbourages were assembled and sealed shut with 'plumbers' plastic pressure pipe cement (Type P Plumbers Mate PVC-U Pipe Cement). Co-operators in South Maroota, Wilberforce and North Richmond were enlisted into the trials. Pre-treatment hive inspections were conducted at each apiary (14-21 March 2007). At each site ten (usually) hives with suitable numbers of beetles were selected and Small Hive Beetle counts recorded during disassembly of the hives. A single harbourage was placed on the bottom board of each 'trial hive' in the apiary and, where possible, into each other hive at the apiary. At the South Maroota and North Richmond sites two hives acted as 'controls'. At the smaller apiary at Wilberforce a single hive acted as a control. In these hives a harbourage containing an untreated cardboard insert was placed on the bottom board. Four and six weeks after placement the numbers of live and dead beetles were recorded and all dead beetles removed. At each time point the percentage reduction in beetle numbers was calculated with, and without correction for changes in beetle numbers in the 'control' hives. Hence, for each site the absolute reduction in beetle numbers compared to the pre-treatment number was calculated and also, the reduction in beetle numbers relative to the 'controls'. Here the % reduction in live beetles was calculated using the Henderson and Tilton (1955) formula: 100 x (1 - (Ta/Tb x Cb/Tb) where Ta and Tb were the mean number of beetles seen in the treated hives post- and pre-treatment respectively and Ca and Cb were the mean number of beetles seen in the untreated hives post- and pre-treatment. After the six weeks post-treatment inspections the harbourages were removed, placed into individual labelled sealable plastic bags and brought back to the laboratory where they were de-constructed and the number of dead beetles inside recorded. With these numbers added, the total number of dead beetles found in each hive during the trial interval was calculated. Of course, this number did not include any dead beetles removed from the hives by the bees.

Residual efficacy of the harbourages A trial was undertaken to assess the shelf-life of the fipronil treated cardboard. Cardboard inserts were removed from four assembled harbourages that had been stored at ambient temperatures for 14 weeks. The harbourages were from the same treatment batch used in the field trials (see below). The cardboard had been treated on 1 March and the inserts were removed on 4 June 2007. Pieces of the cardboard (9 x 9 cm) were wrapped in 50 µm thick adhesive backed aluminium foil and placed inside the plastic containers (18 x 12 x 4 cm) with approximately 20 laboratory reared (Haque and Levot 2005) adult beetles. The containers were kept in an illuminated incubator run at 28oC. Mortality was assessed after 48 hours and LC50s calculated according to the probit method of Finney (1970).

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Results Refining the harbourage The technical drawings and the project in general were discussed with Low Volume Plastics Pty. Ltd. Villawood, NSW on 6 September 2005 when a quotation for producing a prototype was requested. Despite repeated contact, Low Volume Plastics failed to respond and were abandoned as potential fabricators/manufacturers. Discussions with an alternative supplier were initiated immediately and subsequently an order was placed with ProPlas Pty. Ltd. (19 October 2005) to provide a milled sample of the two-piece prototype as originally drawn. A plastic prototype was delivered in early December 2005 (Figure 3) but the dimensions of the beetle entrance were larger than specification and another prototype was ordered. This was not delivered until 27 April 2006 but again was unacceptable (Figure 4). In this version the gap entrance was too small for beetles to enter. On 20 June yet another revised prototype was delivered but again was not as per specification. Although the entrance gap was the correct size in this version, the height of the internal 'atrium' into which this gap opens was less than the height of the cardboard insert. This effectively prevented beetles from gaining access to half of the tunnels in the corrugated cardboard. At this point the issue of making the assembled harbourage 'tamperproof' was resolved when the designer suggested that the proposed clip locking device would be too brittle for an acrylic plastic device and that the only way of permanently closing the device would be to ultrasonically weld the halves together or to use a plastics' glue. The final design was delivered in mid-July 2006 (Figure 5). At the recommendation of Mr. Bruce White, a very experienced apiarist, small holes that could accept thin wire retrieval lanyards were added to the design (Figures 6, 7). Inclusion of a wire lanyard allows easy retrieval of the harbourage without the need to dismantle the hive. Details of the internal structure of the harbourage, including the locking tabs that hold the two halves firmly together, are shown in Figures 8-10. The acrylic plastic harbourage is rigid so that there is little distortion of the assembled device and yet very resistant to breakage. It satisfies all of the criteria set out in the specifications. The plastic harbourage was eagerly sought out by the adult beetles in the laboratory experiment. When checked four hours after placement all the beetles were inside the trap.

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Figure 3. The first plastic prototype harbourage.

Figure 4. The second plastic prototype harbourage.

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Figure 5. The final product assembled.

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Figure 6. Assembled harbourage with retrieval wire attached.

Figure 7. Assembled harbourage showing detail of retrieval wire attachment.

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Figure 8. Open harbourage showing location of cardboard insert.

Figure 9. Open harbourage showing location of cardboard insert and locking tabs.

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Figure 10. Open harbourage showing locking tabs.

Residue trial with the End-Use-Product The residue trial was conducted under ideal conditions for hive activity. This is reflected in the fact that the bees stored approximately 9.4 kg of honey in the ten 'trial frames' during the month that the harbourages were in the hives. AgriSolutions Australia Pty. Ltd. provided a Certificate of Analysis (Figure 11). As expected, the total fiprole content (i.e. the sum of fipronil and its three toxic metabolites MB 46513, MB 45950 and MB 46136) in each of the pre-treatment samples (Honey samples #1, #2, #3, #4 and #5) was below the Limit of Quantification (<1.0 µg kg-1). The mean total fiprole content in the bulked post-treatment samples (Honey samples #6, #7, #8. #9 and #10) were also below the Limit of Quantification. No residue of either fipronil or any of its toxic metabolites was detected in three of the post-treatment samples. Two samples contained MB 46136 at the limit of detection (maximum residue 1.1 µg kg-1) but no fipronil or either of the other toxic metabolites (Figure 11). Mean fipronil content in the recently treated cards was 27.0 ± 1.3 (SE) µg cm-2 while that in the one month old 'used' cards was 21.2 ± 1.7 cm-2. No fipronil or metabolites was detected in the untreated sample.

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Figure 11. Certificate of Analysis for the honey and treated cardboard.

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Commercialisation The call for Expressions of Interest failed to illicit any commercial interest from potential registrants. Until fipronil comes off-patent, BASF holds exclusive rights to its use. When patent protection has expired a product could be registered or a Permit for general use issued, but at present no commercial manufacturer/registrant exists. In the absence of a commercial manufacturer/registrant NSW DPI could apply for a general use permit and manufacture harbourages. Field trials with the End-Use-Product On 28 February 2007 a letter was received from the APVMA advised that our permit application had been successful and that Permit PER9732 (Figure 12) had been issued effective from 26 February 2007 until 25 February 2009. This permit allows NSW DPI to conduct trials of the plastic harbourage in commercial hives subject to the condition that use of the device is restricted to Dr Garry Levot, Dr Michael Hornitzky, Mr Nicholas Annand, NSW DPI employees and persons under the supervision of the NSW DPI employees listed above. Mean beetle numbers increased by several fold in the 'control' hives at each site. This suggests that population numbers were naturally increasing during the trial period thereby providing an excellent test of the effectiveness of the harbourages.

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Figure 12. APVMA Permit PER 9732.

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Trial 1. Wilberforce The Wilberforce apiary comprised 10 hives (Figure 14). Three hives were lost to American Foul Brood during the trial interval. Hence by the end of the trial the mean reduction in beetle numbers was calculated from only six treated hives. The control hive was located at the end of the row of hives and contained only two beetles at the pre-treatment inspection. Insecticide treated harbourages were placed into each of the other hives where pre-treatment beetle numbers ranged from two to over 100. Beetle numbers were monitored in each hive for the duration of the trial. Data for the pre-treatment and four and six weeks post-treatment inspections are presented in Tables 1 and 2. After four weeks the absolute mean number of live beetles had declined from about 36 to 12 (66% reduction) in the treated hives but had increased from two to 70 in the control hive. When allowance was made for the increase in beetle numbers seen in the control hives the mean percentage reduction in the treated hives was 99%. At six weeks after treatment the absolute mean number of live beetles in the treated hives was five. This suggests and absolute reduction of 86%. In the two hives where over 100 beetles were estimated to have resided pre-treatment, only six and seven beetles were seen at six weeks. When allowance was made for the increase in beetle numbers seen in the control hive at six weeks after treatment, the mean percentage reduction across all treated hives was 99%. At Wilberforce 62 dead beetles were removed from the eight treated hives at the four weeks post-treatment inspection, followed by a further 93 beetles from the six remaining hives at the six weeks post-treatment inspection. Another 260 dead beetles were removed from the harbourages retrieved from the six hives remaining at the end of the trial (total 415). Examples of what was found inside the deconstructed harbourages are shown in Figure 14. Figure 13. The Wilberforce apiary.

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Table 1. Pre- and four weeks post-treatment numbers of Small Hive Beetles in hives treated with the harbourages. Hive location Pre-treatment

live beetle count1

4 weeks post-treatment live beetle count

Mean percentage

reduction in beetle count3

Corrected mean

percentage reduction in beetle count4

Wilberforce Control

2 70 +97.1

Wilberforce 1 47 7 Wilberforce 2 3 4 Wilberforce 3 5 17 Wilberforce 4 5 4 Wilberforce 5 24 7 Wilberforce 6 2 21 Wilberforce 7 100+2 28 Wilberforce 8 100+2 11 TOTAL (MEAN) 35.8 12.1 66.2 99.0 Table 2. Pre- and six weeks post-treatment numbers of live Small Hive Beetles in hives treated with the harbourages. Hive location Pre-treatment

live beetle count1

6 weeks post-treatment live beetle count

Mean percentage

reduction in beetle count3

Corrected mean

percentage reduction in beetle count4

Wilberforce Control

2 41 +95.1

Wilberforce 1 47 7 Wilberforce 2 3 2 Wilberforce 3 5 AFB Wilberforce 4 5 6 Wilberforce 5 24 3 Wilberforce 6 2 AFB Wilberforce 7 100+2 6 Wilberforce 8 100+2 7 TOTAL (MEAN) 35.8 5.2 85.5 99.3 Footnotes to Tables 1-6. 1. No. of live adult beetles seen on bottom boards and elsewhere in hive during hive inspection. 2. Assumed to be 100 for calculations. 3. % reduction in live beetles compared to pre-treatment numbers calculated as: 100 x (B-A/B) where B is the mean 'pre-treatment' live beetle count and A is the mean 'post-treatment' live beetle count. 4. % reduction in live beetles compared to pre-treatment numbers with allowance made for changes in beetle numbers in the 'control' hives: calculated as: 100 x (1 - (Ta/Ca x Cb/Tb) where Ta and Tb are the mean number of beetles seen in the treated hives post- and pre-treatment respectively and Ca and Cb are the mean number of beetles seen in the untreated hives post- and pre-treatment (Henderson and Tilton 1955).

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Figure 14. Dead beetles inside a deconstructed harbourage removed from a hive at the end of the field trial.

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Trial 2. South Maroota The apiary at South Maroota comprised 35 hives (Figure 15). Three hives with very small entrances were not used in the trial. The two control hives were located on the ends of two rows (distant end in Figure 15). The remaining 30 hives were treated by placing a single harbourage on the bottom board. The two control hives and ten treated hives were monitored for beetle numbers for the six weeks duration of the trial. All hives remained strong throughout the trial interval. Figure 15. The South Maroota apiary.

Data for the pre-treatment and four and six weeks post-treatment inspections are presented in Tables 3 and 4. Pre-treatment beetle counts ranged from five to 36 in the monitored hives. The mean pre-treatment beetle count in the control hives was seven. Four weeks after the harbourages were put in place the absolute mean number of live beetles in the treated hives had declined from 16 to one (94% reduction). By this time the mean number of beetles in the control hives had risen to 20. When allowance was made for this increase in the control hives the percentage reduction was 98%. At six weeks after treatment the absolute mean numbers of live beetles was reduced to three in the treated hives (80% reduction). Between four and six weeks after placement of the harbourages there was a slight decline in the mean number of beetles in the control hives but still a substantial increase over the pre-treatment levels. When allowance was made for the increase in beetle numbers seen in the control hives at six weeks after treatment the percentage reduction was 90%. At the four weeks post-treatment inspection 115 dead beetles were removed from the treated hives followed by a further 65 beetles at the six weeks post-treatment inspection. Another 251 dead beetles were removed from inside the harbourages retrieved from the hives at the end of the trial (total 431).

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Table 3. Pre- and 4 weeks post-treatment numbers of Small Hive Beetles in hives treated with the harbourages. Hive location Pre-treatment

live beetle count1

4 weeks post-treatment live beetle count

Mean percentage

reduction in beetle count3

Corrected mean

percentage reduction in beetle count4

South Maroota Control A

6 4

South Maroota Control B

8 36

Combined Mean

+65.0

South Maroota 1 5 0 South Maroota 2 8 0 South Maroota 3 8 1 South Maroota 4 8 0 South Maroota 5 16 0 South Maroota 6 29 1 South Maroota 7 5 3 South Maroota 8 23 1 South Maroota 9 36 4 South Maroota 10 23 0 TOTAL (MEAN) 16.1 1.0 93.8 97.8 Table 4. Pre- and 4 weeks post-treatment numbers of Small Hive Beetles in hives treated with the harbourages. Hive location Pre-treatment

live beetle count1

6 weeks post-treatment live beetle count

Mean percentage

reduction in beetle count3

Corrected mean

percentage reduction in beetle count4

South Maroota Control A

6 3

South Maroota Control B

8 27

Combined Mean

+53.3

South Maroota 1 5 0 South Maroota 2 8 0 South Maroota 3 8 2 South Maroota 4 8 1 South Maroota 5 16 5 South Maroota 6 29 4 South Maroota 7 5 1 South Maroota 8 23 1 South Maroota 9 36 19 South Maroota 10 23 0 TOTAL (MEAN) 16.1 3.3 79.5 90.4

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Trial 3. North Richmond The apiary at North Richmond comprised 44 hives in two rows (Figure 16). The control hives were located at the end of one row (distant end of right hand row in Figure 16) and four hives in from the opposite end of the other row. Harbourages were placed into all other hives, however, only the two control hives and 10 treated hives were monitored. Pre-treatment beetle numbers in the monitored hives ranged from 12 to 65 (Tables 5 and 6). All hives remained in excellent condition throughout the six weeks trial interval. Figure 16. The North Richmond apiary.

Data for the pre-treatment and four and six weeks post-treatment inspections are presented in Tables 5 and 6. After four weeks the absolute mean number of live beetles in the treated hives declined from 33 to seven (79% reduction) whereas numbers increased by two to three-fold in the control hives. When allowance was made for this increase the mean percentage reduction was 92%. At six weeks after treatment the absolute mean numbers of live beetles was reduced to only three (90% reduction). The mean number of beetles in the control hives continued to increase during this time. When allowance was made for this increase the mean percentage reduction was 97%. At North Richmond 132 dead beetles were removed from the treated hives at the four weeks post-treatment inspection, followed by a further 21 beetles at the six weeks post-treatment inspection. Another 146 dead beetles were removed from the harbourages retrieved from the hives at the end of the trial (total 299).

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Table 5. Pre- and four weeks post-treatment numbers of Small Hive Beetles in hives treated with the harbourages. Hive location Pre-treatment

live beetle count1

4 weeks post-treatment live beetle count

Mean percentage

reduction in beetle count3

Corrected mean

percentage reduction in beetle count4

North Richmond Control A

16 37

North Richmond Control B

45 133

Combined Mean

+64.1

North Richmond 1 30 0 North Richmond 2 48 4 North Richmond 3 22 13 North Richmond 4 28 9 North Richmond 5 33 6 North Richmond 6 65 30 North Richmond 7 12 1 North Richmond 8 38 4 North Richmond 9 31 2 North Richmond 10 24 1 TOTAL (MEAN) 33.1 7 78.9 92.4 Table 6. Pre- and six weeks post-treatment numbers of Small Hive Beetles in hives treated with the harbourages. Hive location Pre-treatment

live beetle count1

6 weeks post-treatment live beetle count

Mean percentage

reduction in beetle count3

Corrected mean

percentage reduction in beetle count4

North Richmond Control A

16 52

North Richmond Control B

45 129

Combined Mean

+66.3

North Richmond 1 30 0 North Richmond 2 48 0 North Richmond 3 22 9 North Richmond 4 28 3 North Richmond 5 33 4 North Richmond 6 65 9 North Richmond 7 12 0 North Richmond 8 38 5 North Richmond 9 31 2 North Richmond 10 24 1 TOTAL (MEAN) 33.1 3.3 90.0 96.6

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Residual efficacy of the harbourages The cardboard removed from the 14 weeks old harbourages that had been stored at room temperature caused 100% mortality of beetles when tested in the laboratory bioassay. Control mortality was 7% (Table 7). Table 7. Effectiveness of treated cardboard from 14 weeks old harbourages against Small Hive Beetle adults.

Treatment No. live beetles No. dead beetles Total no. beetles exposed

% mortality

Control (rep. A) 18 3 21

Control (rep. B) 20 0 20

Combined 38 3 41 7.3

Treated (rep. A) 0 21 21

Treated (rep. B) 0 24 24

Treated (rep. C) 0 22 22

Treated (rep. D) 0 21 21

Combined 0 88 88 100

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Discussion The ultimate version of the Small Hive Beetle Harbourage has all the attributes identified as being essential to the development of a safe, effective device for the control of adult Small Hive Beetles in the hive. It comprises a two-piece rigid, black, acrylic plastic design. Once assembled, the harbourage is robust and safe to handle and effectively tamperproof. Any attempt to open the assembled harbourage would result in obvious and malicious damage. The acrylic plastic is compatible with silicone adhesives that can be used to anchor the harbourages to uneven bottom boards. Once clipped together and sealed by either glue or ultrasonic welding the plastic shell is protective of the cardboard insert which is held in position 8 mm in from the end openings by flanges in the plastic. The dimensions of the opening slots allow beetles to enter and exclude bees. Beyond the thresholds of the slots but some distance in front of the cardboard, the gap between the two halves of the harbourage increases to the full height of the cardboard insert, thereby creating an atrium from which beetles may enter either the 'top' or 'bottom' corrugations of the cardboard insert. The development of the final design of the protective plastic shell of the harbourage was meticulous and culminated in the production by Pro-Plas Pty. Ltd. of injection moulds that can be used for mass-production. Similarly, the selection of knife-cut pieces of the standard C-flute corrugated cardboard manufactured by Australian Corrugated Box Co. Pty. Ltd. for the insert provided an excellent refuge for the beetles and an easily treated substrate to retain the insecticide. The End-Use-Product should be economical for beekeepers. The cost will depend on commercial decisions made by a registrant/manufacturer. Insecticide cost would be minimal but labour costs to treat the cardboard, assemble the harbourages and, if necessary, attach the retrieval wire would add to the overall cost and, in turn, to the price of the devices. What was not achieved was finding a potential registrant willing to commercialise the invention. This is a great pity considering the plight of beekeepers not only in Australia but in other parts of the world where Small Hive Beetle is impacting negatively on the productivity and profitability of commercial bee keeping. Failure to attract commercial interest in the Small Hive Beetle harbourage and lack of access to fipronil at least until patents expire, are likely to impact on the success of this project. The design of the plastic harbourage combined with the extremely low vapour pressure of fipronil combine to make the likelihood of fipronil residues in honey extremely low and the risk to bees negligible. The APVMA was sufficiently satisfied that the device was safe to bees and honey to issue the Research Permit for its use in commercial hives (Figure 12). In anticipation of an international market for the device NSW DPI sought patent protection for the device beyond Australia in the United States of America, Canada and New Zealand. Notwithstanding the potential overseas market, results of a survey (J. Rhodes, unpublished data) of 645 registered NSW beekeepers (response rate was 323 (49.4%)) suggested that 107 out of 142 respondents to the question "If an in-hive adult Small Hive Beetle control device using pesticide as a control agent became available to beekeepers, would you use the device in your hives?" would use the device. It was impossible to suggest a likely cost for the device but this would, of course, also influence beekeepers. Potential uptake of the device in Queensland is likely to be similar to that in NSW. The Small Hive Beetle Harbourages were convenient to use. For our purpose it was necessary to dissemble the monitored hives to obtain beetle counts but for the remaining hives the harbourages could be 'posted' through the front hive entrance slot and reclaimed by pulling the retrieval wire. In this regard they were extremely easy to use. There would always be an advantage in opening the hive to scrape clean the bottom board and position the harbourage for optimal performance but incorporation of the retrieval wire means that there can be no excuse for not removing the harbourages when control has been established or when cool weather lessens the threat of Small Hive Beetle infestation. Analysis of the cardboard inserts from harbourages that had been used in hives for one month indicated that mean levels of fipronil dropped very slightly over the period. Moreover, at the time of writing laboratory trials to determine the shelf-life of harbourages were continuing but

26

effectiveness of 14 weeks old harbourages remained optimal (Table 7). Service life for the harbourage of at least six months is anticipated but further work on stability is required before shelf-life and service life are determined. The Small Hive Beetle harbourages performed extremely well in field trials conducted at three locations in western Sydney. The trials were conducted during March and April 2007 when beetle numbers were naturally increasing at each trial site. These were excellent conditions to evaluate the effectiveness of the harbourages. Six weeks after placement of the harbourages the mean number of beetles in the treated hives ranged from three to five compared to pre-treatment means of 16-36. This equals a reduction of 80-90%. Only two hives contained in excess of 100 beetles prior to treatment. Both were at the Wilberforce location. By the end of the trial these hives each contained seven or fewer beetles (Table 2). Across the three sites beetles could not be found in six of the 26 monitored treated hives that remained at the completion of the trials and 19 hives contained five or fewer beetles. At the same time beetle numbers increased several fold in the control hives suggesting true reductions in beetle numbers of 90-99%. At the Wilberforce, South Maroota and North Richmond sites respectively 415, 431 and 299 dead beetles were removed from the treated hives during the four and six weeks inspections or retrieved from the deconstructed harbourages at the conclusion of the trials. These numbers do not include the unknown number of dead beetles ejected from the hives by the bees. This number can be considerable with records for one single hive suggesting 110 beetles were ejected (and collected in an oil tray positioned in front of the entrance) over 23 days during April/May 2007 (B. White, unpublished data). These trials have demonstrated that the harbourages are capable of achieving excellent control of Small Hive Beetles in the hive. They have been shown to be very user-friendly and compatible with existing hive structure and entrances. Use of the harbourages did not lead to residues in honey and no ill effects were noticeable in the bees or in productivity. A commercial manufacturer is needed to take this invention to the waiting markets both in Australia and overseas. In the absence of a commercial registrant at that time, an application to APVMA for a General Use Permit will be needed and an alternative manufacturing and marketing strategy developed.

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