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Sustainable Control of Small Hive Beetle Through Targeting In-ground Stages by Robert Spooner-Hart July 2008 RIRDC Publication No 08/115 RIRDC Project No UWS-22A
Sustainable Control of Small Hive Beetle Through Targeting
In-ground Stages
by
Robert Spooner-Hart
July 2008
RIRDC Publication No 08/115 RIRDC Project No UWS-22A
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© 2008 Rural Industries Research and Development Corporation.
All rights reserved.
ISBN 1 74151 703 6 ISSN 1440-6845 Sustainable Control of Small Hive Beetle Through Targeting In-ground Stages Publication No. 08/115 Project No. UWS-22A 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 Associate Professor Robert Spooner-Hart Centre for Plant & Food Science University of Western Sydney Locked Bag 1797 Penrith South DC NSW 1797 Phone: 0245701429 Fax: 0245701103 Email: [email protected]
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: [email protected] Web: http://www.rirdc.gov.au Published in July 2008 by Canprint
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Foreword This project was targeted at controlling an important, recently introduced pest of honeybees, small hive beetle (SHB). It particularly focussed on the “out-of hive” stages of SHB, evaluating a number of commercial strains of insect pathogens (nematodes and fungi) as alternatives to insecticides. This research provided useful preliminary findings supporting the effectiveness of at least one commercial strain of nematodes, and also demonstrated that they had some residual activity after initial application. The research findings have implications for the Australian honeybee industry in its attempts to control SHB. Provided further field trial data confirm the results reported here, entomopathogenic nematode drenches could be applied to soil areas surrounding hives to control SHB in apiary sites, around honey houses and honey extraction sites. The use of nematodes rather than synthetic insecticides will also reduce the likelihood of contamination of soil and hive products, as well as reducing the development of insecticide resistance in SHB. 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 1800 research publications, forms part of our Honeybee R&D program, which aims to improve the productivity and profitability of the Australian beekeeping industry. 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 I acknowledge the following people who contributed to the work reported here. Dr Zamir Hossain, who was instrumental in conducting the research trials reported here. Oleg Nicetic, who provided advice on experimental design and undertook analyses of the data. Dr Albert Basta, who is the supervisor of the entomology bioassay laboratory.
Abbreviations APVMA Australian Pesticides and Veterinary Medicines Authority SHB Small Hive Beetle
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Contents Foreword ............................................................................................................................................... iii Acknowledgments................................................................................................................................. iv Abbreviations........................................................................................................................................ iv Executive Summary ............................................................................................................................. vi Introduction ........................................................................................................................................... 1 Objectives ............................................................................................................................................... 3 Laboratory bioassays using entomopathogenic nematodes............................................................... 4
Source of nematodes and small hive beetle larvae .............................................................................. 4 Petri dish bioassays ............................................................................................................................. 4
Heterorhabditis zealandica (strain X1)........................................................................................... 4 Heterorhabditis bacteriophora (strain NJ) ..................................................................................... 5 Steinernema feltiae (strain T319).................................................................................................... 5 Discussion and changes to methodology......................................................................................... 6
Bioassays using media in containers ................................................................................................... 6 Preliminary investigation using H. bacteriophora........................................................................... 6 Detailed bioassays with known numbers of nematodes .................................................................. 7 Investigation with large containers to more closely simulate field conditions.............................. 11
Longevity of nematode activity......................................................................................................... 12 Laboratory bioassays using the entomopathogenic fungus M. anisopliae...................................... 14
Source of Metarhizium anisopliae. ................................................................................................... 14 Petri dish bioassays ........................................................................................................................... 14 Bioassays using soil media................................................................................................................ 15
Discussion............................................................................................................................................. 16 Laboratory bioassays using entomopathogenic nematodes............................................................... 16
Estimate of possible rate of nematode application in the field...................................................... 17 Longevity of nematode activity.......................................................................................................... 17 Laboratory bioassays using entomopathogenic fungi ....................................................................... 17
Implications.......................................................................................................................................... 18 Recommendations ............................................................................................................................... 19
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Executive Summary What the report is about This report is targeted at the control of a major exotic pest of honeybees, small hive beetle (SHB), Aethina tumida. SHB is an important pest of European honeybees, especially away from its region of origin, Southern Africa. Since its discovery in NSW in 2002, it has spread along the eastern coast into commercial and feral hives. This project particularly targets the “out-of hive” stages of SHB, as the larvae will leave the hive to burrow and pupate in the soil. Who is the report targeted at? This report is targeted at the Australian beekeepers to assist them in control of SHB. Aims/Objectives The aims of this work were to conduct laboratory bioassays, using commercial strains of entomopathogenic nematodes Steinernema spp., Heterorhabditis spp. and the entomopathogenic fungus Metarhizium anisopliae, to assess their efficacy against controlling SHB larvae and pupae. Methods used A number of commercial strains of entomopathogenic (insect-attacking) nematodes and fungi were evaluated in the laboratory and under simulated field conditions to target the in-ground stages of SHB as alternatives to insecticides. Results/Key findings This research provided useful preliminary findings supporting the effectiveness of commercial strains of nematodes from the genus Heterorhabditis, in particular H. bacteriophora, to control SHB larvae as they burrowed into the soil and pupated. It also demonstrated that the high insecticidal activity of the nematodes lasted for at least seven days after their initial application to soil. However, the research also showed that currently commercially available strains of the fungus Metarhizium anisopliae were not effective in controlling larvae/pupae of SHB. Implications for relevant stakeholders The research findings have implications for the Australian honeybee industry in its attempts to control SHB. Provided field trial data confirm the results reported here, drenches of entomopathogenic nematodes can be applied to soil areas surrounding hives to control SHB in apiary sites. As registered strains of nematodes are already registered and available commercially, uptake of this technology should be quite rapid. The use of nematodes rather than synthetic insecticides should also reduce the likelihood of contamination of soil and hive products, reduce environmental impacts, and reduce the likelihood of insecticide resistance development in SHB. Recommendations It is recommended that fully replicated field trials be conducted against larvae and pupae of SHB using drenches of the nematode H. bacteriophora to treat soil surrounding infested hives. These trials should generate data to assist obtaining Australian Pesticides and Veterinary Medicines Authority (APVMA) registration for use in eastern Australian states.
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Introduction Small Hive Beetle (SHB) (Aethina tumida), is an important pest of beehives, especially away from its site of origin in Southern Africa; it is also present in northern Africa and USA. Since its discovery in Australia in 2002 in the Hawkesbury district of NSW, it has spread, particularly along the coastal regions and into other eastern states (Gillespie et al., 2003). Both adult and larval SHB feed on honey, pollen and brood in Apis mellifera hives (Neumann et al., 2001), but it is probably the larvae that cause the greatest amount of damage (Sanford, 1998), by burrowing into cells (Elzen et al., 1999), consuming bee brood and defecating in honey (Elzen, 2001). It has been reported to cause serious damage to some, including commercial, hives and is a quarantinable pest. Its detection and control, are thus of significant importance. Adult female SHB typically lay their eggs in irregular egg masses along crevices and cavities on or near comb and they hatch two to four days later, with larval development generally requiring ten to 21 days (Lundie, 1940). Fully developed larvae vacate beehives and migrate to the soil where they burrow and make smooth-walled earthen cells in which they pupate. The pupal period varies from 15 to 60 days but the majority of beetles emerge as adults after approximately 3-4 weeks (Lundie, 1940) Over 80% of beetle developmental stages found in sandy soil were located in the first 10 cm of the soil profile and within 30 cm from the hive entrance (Evans et al., 2000; Pettis & Shimanuki, 2000), indicating that wandering beetle larvae do not travel very far from the hive to pupate if soil conditions are suitable. Survival rate to adult emergence in moist soils is reported to be >90% (Ellis et al., 2004). SHB control can be targeted at reducing adult entry into hives, controlling adults or larvae in-hive by pesticides and/or traps, or targeting larvae and pupae in soil around hives. Projects recently funded by RIRDC’s Honeybee program have investigated SHB biology and use of temperature manipulation (DAN-215A) and insecticidal control (DAN-216A) as well as evaluation of in-hive traps. DAN-215A evaluated in-hive and soil insecticidal treatments, and concluded that the synthetic pyrethroid permethrin could be used as an effective soil treatment against larvae and pupae of SHB. Ground drenches of permethrin have been reported to be effective in the USA (Deleplane, 1998; Eischen et al., 1998; Hood, 2000), and a soil chemical treatment has been authorised (Anon, http//.www.maarec.cas.psu.edu/PDFs/Small_Hive_Beetle_-_PMP.pdf, accessed December 24 2007). However, less permethrin is used in Australia, largely because of the migratory nature of beekeeping and use of temporary apiary sites. Never-the-less, such soil pesticide applications have environmental and safety implications and can lead to development of pesticide resistance in target species. There remained a number of options for more sustainable control of SHB, particularly the use of entomopathogens (insect diseases) such as fungi and nematodes, for controlling SHB larvae and pupae in-ground. Although insect pathogens have been known for some time, their mass production and use for large scale control has been relatively recent. The first insect fungal pathogen, Beauveria bassiana, was discovered in 1835 (Längle, 2007), and the green muscardine fungus, Metarhizium anisopliae, was first proposed for insect biological control in 1879 (Steinkraus & Tugwell, 1997). Since then, a number of fungi have been developed commercially as biological control agents (Clarkson & Charanley, 1996). Australian scientists have been at the forefront of development of insect pathogens (see Carswell et al., 1998; Milner 2001; Cannard et al., 2002) and Australia has five registered fungal biopesticides based on M. anisopliae, with two of these, BioGreen® and BioCane® targeting soil-dwelling beetle larvae.
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Nematode pathogens of insects have been known since the 17th century, but it was not until the 1930s that serious consideration was given to using them for insect control (Smart, 1995). The entomopathogenic nematodes, particularly in the key genera of Steinernema and Heterorhabditis, are soil-dwelling and are thus most suited to control of in-ground and other cryptic pests (Gaugler 1981; Cant & Spooner-Hart, 1993). A number of commercial, formulated nematode products are available worldwide; including in Australia H. zealandica, H. bacteriophora and S. feltiae for control of several species of soil-dwelling insect larvae, including beetles (Llewellyn 2002). Several researchers in the USA have reported that SHB can be naturally attacked by entomopathogenic fungi (Ellis et al., 2004; Richards et al., 2005), and Muerrie et al. (2006) have documented M. anisopliae naturally attacking SHB in Southern Africa, as well as the susceptibility of adult SHB to this and other species of entomopathogens. The efficacy of the entomopathogenic nematodes Heterorhabditis megidis, Steinernema carpocapsae and S. riobrave against wandering SHB larvae has also been recently reported (Cabanillas & Elzen, 2006). Subject to their satisfactory efficacy, entomopathogenic fungi and nematodes could provide a safer, more environmentally acceptable alternative to insecticides for soil application against SHB. They would also reduce the likelihood of insecticide resistance development in SHB. M. anisopliae has been successfully evaluated in USA for in-hive control of varroa mite, Varroa jacobsoni, with no harmful effects on bees or colony production (Kanga et al., 2003).
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Objectives This project explored these issues through a series of objectives: 1. Conduct laboratory bioassays, using commercial strains of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis spp., and the entomopathogenic fungus Metarhizium anisopliae against larvae and pupae of small hive beetle (Aethina tumida). 2. Evaluate efficacy of these above treatments in simulated soil conditions. 3. Generate preliminary data for use of drenches of entomopathogens in field apiary sites. More detailed field investigations to generate data for registration would form part of a future project.
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Laboratory bioassays using entomopathogenic nematodes These investigations were conducted at the University of Western Sydney’s Hawkesbury campus entomology laboratory at Richmond, NSW. Three different species of nematodes which are commercially available in Australia (Heterorhabditis bacteriophora, Heterorhabditis zealandica and Steinernema feltiae) were tested to assess their efficacy as biological control agents against the wandering larvae (prepupal stage) and pupae of SHB. The test nematode species were selected on their commercial availability and following discussions with the producers of the nematode products. Experiments were conducted in three phases. Initial screening experiments were conducted using Petri dishes with moistened filter paper; this was followed by bioassays using moistened media in small specimen vials; and finally, promising candidates were assessed in larger vials over a range of nematode concentrations, to simulate field conditions. Source of nematodes and small hive beetle larvae The nematodes used in these investigations study were supplied by EcoGrow Australia Pty Ltd (PO Box 7657, Bondi Beach, NSW 2026). All nematodes from all batches were received in small round plastic food punnets (275 mL) in a medium of technical grade micro cellulose containing 25 million infective nematodes per punnet. SHB were reared in the laboratory at 25 (±1) °C using the method described by Haque and Levot (2004), with the modification that honey comb containing honeybee brood was used as the oviposition site. Cohorts of SHB were reared separately, ensuring sufficient individuals of similar age for bioassays. These sources of both test and target organisms were used for all of the experiments, unless otherwise stated. Petri dish bioassays Heterorhabditis zealandica (strain X1) Materials and methods Preparation of nematode suspensions The recommended application rate for a single pack containing 25 million nematodes was 100 m2 soil, applied in 90 litres of water. This field concentration was used as the standard for subsequent bioassays. As the mass of the punnet contents (cellulose and nematodes) was 66.8 g, 1.86 g of mixed punnet contents was added to 250 mL water to obtain a stock nematode suspension (10 X field concentration), which was then diluted down to the experimental concentrations. Experimental treatments were: (i) Recommended field concentration (ii) Double recommended field concentration (iii) Quadruple recommended field concentration (iv) Water only control. Application method and observations
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Pathogenicity of H. zealandica against wandering larvae (pre-pupae) of SHB was tested in Petri dishes (Ø 90 × h14 mm). A single filter paper (Whatman® No. 1, Ø 90 mm) was laid on the lid of each Petri dish. A 2.5 mL volume of water was found to be sufficient to moisten the filter paper to enable nematode infectivity. Therefore, 2.5mL of the respective suspension containing infective juvenile nematodes was pipetted onto each filter paper. Suspensions were gently shaken each time just prior to pipetting so that nematodes remained homogeneously mixed in the suspension. The control filter paper was treated with 2.5 mL of water only. Five SHB larvae were randomly selected from the culture, and placed in each Petri dish and the bottom of the Petri dish was placed on top to cover the filter paper that contained larvae. Each Petri dish was then sealed with Parafilm® (Pechiney Plastic Packaging, Menasha, WI 94952, USA) to prevent moisture loss. Each treatment was replicated ten times. Petri dishes were incubated in a dark growth cabinet at 25°C with 75% relative humidity. SHB larval mortality was recorded daily for five consecutive days. The experimental set up was retained in the incubator for up to ten weeks for further observation. Results No SHB larval mortality was recorded in this experiment. Larvae in all treated Petri dishes remained active more than three weeks and most larvae survived up to six weeks as the containers were sealed and sufficient moisture was retained within the dishes. Some larvae eventually pupated, ultimately emerging as adults. Heterorhabditis bacteriophora (strain NJ) Materials and methods The nematode used in this experiment was Heterorhabditis bacteriophora (strain NJ). Nematode-water suspensions (treatments) and application methods were same as for H. zealandica. Results Results were same as the previous experiment as no SHB larval mortality occurred, and most larvae survived more the six weeks. Steinernema feltiae (strain T319) Materials and methods The nematode used in this experiment was Steinernema feltiae (strain T319) and as the mass of the punnet contents was 66.73 g, 1.83 g of the mixed contents was added to 250 mL water to obtain a stock suspension. Nematode-water suspensions (treatments) and application methods were same as H. zealandica. Results Results were similar to the previous experiments and no SHB larvae died; and most survived more the six weeks.
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Discussion and changes to methodology No mortality in SHB larvae was recorded in any of the Petri dish bioassays with any of the nematode species tested, even at concentrations four times the recommended field rate. Activity of the nematodes in the punnets was subsequently tested by microscopic examination and it confirmed that nematodes were alive and active. However, discussions with Ecogrow Pty Ltd and colleagues confirmed that results from Petri dish bioassays can be variable, and can sometimes produce negative results despite subsequent success in the field (Grewal et al., 1999; G. Chouinard, pers. comm. 2006). It was therefore decided to abandon this type of bioassay in favour of ones using a soil or potting medium substrate. The fact that the wandering SHB larvae were able to survive >6 weeks without food, looking for a suitable pupation location confirms their resilience at this stage in their life cycle. Bioassays using media in containers As both SHB and entomopathogenic nematodes spend at least a portion of their lifecycles in soil, a series of bioassays were conducted using an organic/sand medium. This provided a suitable substrate in which the SHB larvae could burrow for pupation, as well as to provide conditions for nematode movement and survival. Nematodes are best suited to moist (but not heavy), poorly-drained soils. Thus, they require similar conditions to pupating SHB. Preliminary investigation using H. bacteriophora A preliminary investigation was conducted using the nematode and the nematode-water suspension was prepared as previously described in Section 1. Materials and methods A composted pinebark-based potting mix (Australian Native Landscapes) was air-dried, sieved with 2.00 mm mesh size (Endecotts Ltd. London, England) and mixed with medium white sand (1:1), then sterilized at 100°C for 3 hours and aerated prior to use. The mixed medium was used to fill plastic vials (Ø 44 × h 105 mm) which were uniformly compacted to a depth of 60 mm. The bottoms and lids of all vials had been previously cut out (Ø 10 mm) to facilitate air ventilation and a piece of nylon mesh was glued with a hot glue gun on the base and lid end to prevent escape of any larvae or subsequently emerging beetle from the vial. A 25 mL volume of water was found to approximate field capacity when mixed with the medium mix in vial. Therefore, 25 mL of the relevant nematode suspension was slowly pipetted to each vial so that the water could be absorbed by the medium mix. Control vials received water only. One hour after nematode application, ten randomly-selected wandering larvae were placed on the medium mix on top of each vial. Usually the larvae were observed to crawled and burrow into the soil immediately. Each treatment was replicated six times. Vials were kept in a dark growth cabinet at 25 °C with 75% relative humidity. Adult SHB emergence in each vial was counted after 48 days. All adults (dead/live) were removed from each vial and, where possible, cadavers were recovered and checked for presence of nematodes As a number of active nematodes were observed on the surface of the medium of all nematode-treated vials while taking the count, it was decided to assess whether this generation of nematodes were infective and efficacious. Consequently, ten fresh wandering larvae were released into the top of each vial. To ensure adequate moisture in the media for this second trial, 10 mL of distilled water was added to each of the vials. Adult emergence from this second larval release was recorded after 36 days.
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Results No adult SHB emerged in any of the H. zealandica treated containers after the first larval release. In controls, >93% of the released larvae emerged as adults. However, most larvae from the second release emerged as adults in all treatments. In response to these results, more detailed investigations were conducted, with known numbers of all three species of nematodes: H. bacteriophora, H. zealandica and S. feltiae. Detailed bioassays with known numbers of nematodes Materials and methods Efficacy of the three nematode species H. bacteriophora, H. zealandica and S. feltiae against larvae/pupae of SHB was assessed by using a range of nematode suspensions: 0, 500, 1000, 1500 and 2000 nematodes/mL water. To achieve these nematode concentrations, 180 g of cellulose containing nematodes was mixed in 200 mL distilled water, which gave stock suspensions. These stock suspensions were then gently shaken for 30 minutes. From each nematode stock suspension 10 mL aliquots were added to another 90 mL of water to obtain 500 nematodes/mL. Concentrations of nematodes were confirmed by taking 20 μL samples from the suspension and counting under a stereomicroscope over a minimum of 30 samples. Nematode numbers were adjusted either by adding more stock suspension or by diluting further with distilled water, for each replicate. All other nematode treatment concentrations were prepared similarly using this method, with each replicate concentration being prepared and assessed separately. The medium mix was moistened and thoroughly mixed with distilled water up a level where it could be made into a clod when held in the palm and pressed tightly. The moistened mix was sampled, and its moisture content (θd) was assessed gravimetrically (Black, 1965) to be 53.8%. This moistened medium mix was used to fill each plastic vial (Ø 44 × h 105 mm) to a depth of 70 mm, and was compacted loosely but uniformly. The lids of all vials were cut out (Ø 10 mm) to facilitate air ventilation and a piece of nylon mesh was glued with hot glue gun to prevent escape of any larvae and later on any beetle from the vial. Each suspension was pipetted at the rate of 2 mL/vial. Therefore, each vial received 0, 1000, 2000, 3000 or 4000 nematodes. Care was taken to evenly distribute the suspension over the surface of the medium. After 30 minutes, ten wandering SHB larvae were released in the top of each vial, as previously described. Each treatment was replicated ten times. Assessment of adult SHB emergence commenced 27 days after the larvae were released, which was when the first beetles started to emerge, and they were removed from the vials immediately after counting. Counting was continued for another four days until all adults had emerged from all replicates of the water only (control) vials. If live nematodes were observed with a stereomicroscope on the surface of the medium mix after the completion of the assessment, a second batch of ten wandering larvae was released in each container, using the method described above. Recording of adult emergence from this second batch of larvae commenced 28 days after their release and followed the same methodology as the first. Analysis of data Data from the first set of larvae were analysed using SPSS® for WindowsTM version 14.0 (SPSS Inc. 2003). The assumption of normal distribution was checked using P-P plot and homogeneity of variance using Levene’s test of equality of error variances. ANOVA was used to compare the treatment means and Dunnett’s T3 test or Games-Howell test were used to determine differences between treatments, in the event of unequal error variances.
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Results H. bacteriophora There were significant differences in SHB larval/pupal mortality resulting from application of different concentrations of H. bacteriophora (F4, 36= 19.960, P < 0.001) (Figure 1). Dunnett’s T3 test showed that all rates applied were significantly more efficacious than the water only control. The 1500 nematodes/mL treatment (90% SHB mortality) was significantly more efficacious than the 500 nematodes/mL treatment (40% SHB mortality). However, it was not significantly superior to the other two concentrations.
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Figure 1. Mortality of small hive beetle larvae/pupae resulting from applications of different concentrations of H. bacteriophora, and compared to water only control. Data presented are means ± SE. Bars headed by the same letter are not significantly different (P < 0.05) by Dunnett’s T3 test. This experiment was repeated with a second batch of nematodes, for confirmation. These results are presented in Fig. 2. Again, there were significant differences in SHB larval/pupal mortality between treatments (F4, 36= 65.743, P < 0.001), and all nematode treatments recorded significantly higher mortality than the water only control. However, all treatment concentrations were not significantly different from each other, with lowest SHB mortality of 80% occurring in the 500 nematodes/mL treatment.
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Figure 2. Mortality of small hive beetle larvae/pupae resulting from applications of different concentrations of H. bacteriophora, and compared to water only control, repeat experiment. Data presented are means ± SE. Bars headed by the same letter are not significantly different (P < 0.05) by Dunnett’s T3 test. H. zealandica In the initial experiment, there were significant differences in larval mortality resulting from the treatments (F4, 36= 8.101, P < 0.001) and all nematode treatments had significantly higher SHB larval/pupal mortality than the water only control (Fig. 3) (Games-Howell test). However, there were no differences in mortality between any of the treatments. The highest mortality (57%) was recorded in the 1000 nematodes/mL treatment. The results for the follow-up experiment with the second batch of larvae are presented in Fig 4. Again, significant differences in larval mortality occurred between treatments (F4, 36= 5.152, P < 0.002) were recorded and all nematode treatments had significantly higher SHB larval/pupal mortality than the water only control (Games-Howell test). Overall the second generation nematodes gave slightly higher SHB mortality, with the 1500 nematodes/mL treatment recording the highest mortality of only 64%.
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Figure 3. Mortality of small hive beetle larvae/pupae resulting from applications of different concentrations of H. zealandica, and compared to water only control. Data presented are means ± SE. Bars headed by the same letter are not significantly different (P < 0.05) by Games-Howell test.
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Figure 5. Mortality of small hive beetle larvae/pupae resulting from applications of different concentrations of H. zealandica, and compared to water only control. [After 2nd release] Data presented are means ± SE. Bars headed by the same letter are not significantly different (P < 0.05) by Games-Howell test.
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S. feltiae Data from the both the initial and follow-up are presented in Fig 5. There was no significant difference in larval mortality between any of the treatments, including the water only control, and the highest mortality did not exceed 6% (Figure 5).
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First experimentRepeat Experiment
Figure 5. Mortality of small hive beetle larvae/pupae resulting from applications of different concentrations of S. feltiae, and compared to water only control. There are no significant differences between any treatments. Investigation with large containers to more closely simulate field conditions In this investigation, larger cylinders (Ø 10 × h 50 cm) were used in place of the smaller vials, to enable SHB larvae to burrow deeper and to assess whether they could still be effectively located and infected by nematodes. As H. bacteriophora was shown to be the most efficacious in the small vial experiments, it was selected for this investigation. Medium mix was prepared as previously described. He cylinders were uniformly filled and loosely compacted to a depth of 40 cm. The nematode concentrations prepared were 0, 500, 1000 and 2000/mL. Each cylinder was treated with a surface application of 50 mL of the relevant suspension. Forty wandering SHB larvae were placed in each cylinder. A piece of nylon mesh was used to cover the top of the cylinder to facilitate air ventilation and a strong was rubber used to hold the mesh in place to prevent escape of larvae or emerging beetles. Each treatment was replicated 6 times. This study was conducted in a temperature control room at 25 °C but, relative humidity was not able to be maintained in this experiment. Adult SHB emergence was recorded from 27 days after release and continued until several days after all adults had emerged from the water-only cylinders. Data Analysis Data were analysed using SPSS® for WindowsTM version 14.0 (SPSS Inc. 2003). ANOVA was used to compare the treatment means and Ryan-Einot-Gabriel-Welsch Range test was used determine differences between means.
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Results The results for this investigation are presented in Fig. 6. There were significant differences in larval/pupal mortality resulting from the treatments (F3, 15= 167.103, P < 0.001). All nematode treatments had significantly higher SHB larval/pupal mortality than the water only control. The 2000 nematodes/mL treatment was significantly more efficacious than all other treatments (lower nematode concentrations), with a recorded SHB larval/pupal mortality >90%.
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Figure 6. Mortality of small hive beetle larvae/pupae resulting from applications of different concentrations of H. bacteriophora, and compared to water only control. Data presented are means ± SE. Bars headed by the same letter are not significantly different (P < 0.05) by Ryan-Einot-Gabriel-Welsch Range test. Longevity of nematode activity The longevity of activity of the entomopathogenic nematodes H. bacteriophora, H. zealandica and S. feltiae, against larvae/pupae of SHB (when applied to a suitable medium) was determined in the laboratory. The medium mix and vials were prepared using the methodology described in 2.2. In this experiment, however, only suspensions of one concentration, 500 nematodes/mL, were prepared for each of the three nematode species. Each vial received a 2 mL aliquot of suspension at approximately same time; therefore each vial received ~1000 infective juveniles. After inoculation, the vials were kept in a dark growth cabinet at 25 ± 0.5°C with 75% relative humidity. Water-only treated vials (controls) were prepared similarly and kept separately inside the incubator. A total of 120 vials were prepared, 30 each for the three nematode treatments and 30 for water-only controls. At predetermined intervals following inoculation (viz. 0, 2, 7, 14, 21 and 28 days), five vials from each treatment were opened and ten wandering SHB larvae were released, as previously described. Baited vials were returned back to the growth cabinet and kept there until the end of the experiment.
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Data analysis Data were analysed using analysis of variance (ANOVA) SPSS® for WindowsTM version 14.0 (SPSS Inc. 2003). The assumption of normal distribution was checked using P-P plot and homogeneity of variance using Levene’s test of equality of error variances. Data for all assessment times met assumptions of normal distribution after Ln(X+1) transformation but assumption of homogeneity of variance was not met. Therefore, treatment means were separated using Dunnett’s T3 test. Results There was a significant time (days after inoculation) x treatment interaction (F15, 96= 3.343, P < 0.001), hence differences between treatments were analysed for each assessment time. The results are presented in Fig 7.
-20
0
20
40
60
80
100
0 2 7 14 21 28
Days after inoculation
Smal
l hiv
e be
etle
larv
al %
mor
talit
y water onlyS. feltiaeH. zealandicaH. bacteriophora
Figure 7. Persistence of activity of nematode species H. bacteriophora, H. zealandica and S. feltiae over time, as measured by SHB larval/pupal mortality. Data presented are means ± SE. 0 days. There were significant differences between treatments with regard to mean SHB larval/pupal mortality (F3, 16= 10.090, P < 0.001), with H. bacteriophora, H. zealandica treatments superior to S. feltiae and the water only control. 2 days after inoculation. There were significant differences between treatments with regard to mean SHB larval/pupal mortality (F3, 16= 47.297, P < 0.001), with H. bacteriophora, H. zealandica treatments superior to S. feltiae and the water only control. 7 days after inoculation. There were significant differences between treatments with regard to mean SHB larval/pupal mortality (F3, 16= 5.605, P < 0.008) with the H. bacteriophora treatment being superior to all other treatments. 14 days after inoculation. There were no significant differences between treatments, although there was a strong trend towards significance (F3, 16= 3.195, P = 0.052). >14 days after inoculation. There were no significant differences between treatments at 21 days (F3,
16= 1.326, P = 0.301) and 28 days (F3, 16= 0.699, P < 0.566) after inoculation. Even from day 0 and in all subsequent observations the S. feltiae treatment was not significantly different from the water-only control. More than 98% SHB mortality was observed two days after inoculation by H. bacteriophora but it had declined to 62% seven days after inoculation.
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Laboratory bioassays using the entomopathogenic fungus M. anisopliae
Source of Metarhizium anisopliae. Two commercially available M. anisopliae products, BioCane™ and Chafer Guard™ (previously called ‘BioGreen’) granules, which contained the naturally occurring soil fungi accordingly M. anisopliae (isolate FI – 1045) and M. anisopliae (strain F001) as the active organism were used in this studies. These products were supplied by Becker Underwood Australia, RMB 1084 Pacific Highway, Somersby, NSW 2250. These test products were selected on the basis of their commercial availability and following discussions with the producers of the products. Petri dish bioassays Initial bioassays Materials and methods The recommended field dose to cover 1m2 soil is 1 g. Therefore, aqueous suspensions of both fungi were prepared by suspending the granules of conidia in an aqueous solution of 0.05% w/v Tween 80® (Sigma Aldrich Pty Ltd). Experimental treatments were as follows: (i) 1/10 of the recommended field dose (ii) Field dose (iii) 10 times then field dose (iv) Water only control. Pathogenicity of BioCane™ Metarhizium anisopliae (isolate FI – 1045) and Chafer Guard™ (strain F001- formerly BioGreen®) against wandering larvae and pupae of SHB was tested in Petri dish (Ø 90 × h14 mm) bioassays. A single filter paper (Whatman® No. 1, Ø 90 mm) was laid on the lid of each Petri dish. A 2.0 mL volume of water was found to be sufficient to moisten the filter paper sufficiently to optimise conidia germination. Therefore, 1.0 mL of each respective suspension containing fungal spores together with 1 mL water, was pipetted onto each filter paper. Suspensions were gently shaken each time just prior to pipetting so that spores remained homogeneously mixed in the suspension. The control filter paper was treated with 2.0 mL of water only. Five SHB larvae were then randomly selected from the culture, and placed into each Petri dish and the bottom of the Petri dish was placed on top to cover the filter paper that contained the larvae. Each Petri dish was then sealed with Parafilm® (Pechiney Plastic Packaging, Menasha, WI 94952, USA) to prevent moisture loss. Each treatment was replicated ten times. Petri dishes were incubated in a dark growth cabinet at 25 °C with 75% relative humidity. SHB larval mortality was recorded daily for 14 consecutive days. The experimental set up was retained in the incubator for up to ten weeks for further observation. Results No SHB larval mortality was observed with either fungus treatment.
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Detailed bioassays with known numbers of spores BioGreen® and BioCane® Pathogenicity of BioCane™ M. anisopliae (isolate FI – 1045) and Chafer Guard™ M. anisopliae (strain F001) against larvae/pupae of SHB was assessed by using a range of suspensions with known numbers of conidia. The concentration of conidia was determined using a haemocytometer and the density was adjusted to 1 x 109 conidia/mL. Tenfold dilutions with 0.05% aqueous solution of Tween 80® were prepared to get 1 x 108, 1 x 107, 1 x 105, 1 x 103 and 1 x 102 conidia/mL. Petri dishes and filter papers were prepared same as initial experiment. In addition to the conidial suspensions, water only and aqueous of Tween 80® solution were as control treatments. The experiment was replicated five times. SHB larval mortality was recorded daily for 14 consecutive days. Repeat experiment The whole experiment was repeated with a separate batch of fungal spores for confirmation. Conidia viability test Viability tests of conidia were performed prior to conducting each experiment to ensure a minimum of 70% viability. To perform the viability test, two agar discs (2 mm thick) were placed on glass slides, and one drop of conidia suspension was placed on each agar disc; and the slides were incubated at 25 °C for 24 hours. Germinating (a) and non-germinating (b) spores were counted using an Olympus compound microscope at 400x magnification, based on 20 randomly chosen fields of view, and percentage germination (i.e. % spore viability) was calculated as: [a/(a+b)] x 100 = % germination. All suspensions tested had more than 70% spore viability. Results No SHB larval mortality was recorded in any of the treatments. Bioassays using soil media In these studies instead of Petri dishes and filter papers, plastic vials were filled with pinebark-based potting mix and white sand (1:1), as previously described for the nematode experiments in Section 2.2. Treatments were the same as described in the experiments in section 4.2. Each vial received 2 mL of the respective conidial suspensions, and each treatment was replicated six times. Adult SHB emergence was recorded as previously described. Results No SHB larval/pupal mortality was recorded in any of the treatments observed in this study.
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Discussion Laboratory bioassays using entomopathogenic nematodes No mortality in SHB larvae was recorded in any of the Petri dish bioassays with any of the nematode species tested, even at concentrations four times the recommended field rate. Activity of the nematodes in the punnets was subsequently tested by microscopic examination and it confirmed that nematodes were alive and active. However, discussions with Ecogrow Pty Ltd and colleagues confirmed that results from Petri dish bioassays can be variable, and can sometimes produce negative results despite subsequent success in the field (G. Chouinard, pers. comm. 2006). It was decided to abandon this type of bioassay in favour of ones using a soil or potting medium substrate. The fact that the wandering SHB larvae were able to survive >6 weeks without food, looking for a suitable pupation location confirms their resilience at this stage in their life cycle. The bioassays using an organic potting mix/sand media in plastic vials were effective in assessing the relative efficacy of the three nematode species tested, as well as the optimal dose of nematodes for control of SHB larvae/pupae. The media mix was also suitable for SHB pupation, with >90% adult emergence, a figure consistent with previously published reports in moist sandy soil (Ellis et al. 2004) The most efficacious of the three nematode species tested was H, bacteriophora, followed by H. zealandica. H, bacteriophora bioassays recorded >90% SHB mortality, when ≥ 1000 nematodes were applied per small vial, and as low as 40% at 500 nematodes/vial. H. zealandica applied at ≥ 1000 nematodes/vial recorded < 60% SHB mortality. S. feltiae performed the poorest of the three species, with a maximum of only 6% SHB mortality. Interestingly, when a second cohort of SHB wandering larvae were placed in the H. zealandica vials at the completion of the investigation, when nematodes were noted to be present, a similar SHB mortality was achieved. This also occurred with H. bacteriophora, although these data are not presented here. These results suggest that nematode generations subsequent to the initial application (i.e. emerging infective juveniles living in soil) may also be effective in controlling SHB. Reasons for the difference in efficacy between the three nematode species may have been a result of their host preference, their suitability for the experimental conditions, or their behaviour as predators. The two most efficacious species were in the genus Heterorhabditis, a taxon known to search out prey (i.e. “hunters”), whereas Steinernema spp. are regarded as “ambush” predators (Grewal et al., 1999) While it may be argued that the small vial bioassays did not, because of their dimensions, adequately represent field conditions, never-the-less they were able to identify the less efficacious nematode treatments. The vials were only 10cm deep and only container 8 cm of media mix, which only enabled the SHB larvae the opportunity to bury to this depth and provided the nematodes with a limited challenge in their search for and infection of larvae/pupae. In fact, this should have therefore provided suitable conditions for ambush-type nematodes such as S. carpocapse. Bioassays, using only H. bacteriophora, tested whether these nematodes would be efficacious under conditions where SHB larvae could bury themselves much deeper, and thus be more likely to avoid detection and infection. These investigations confirmed that H. bacteriophora was still capable of causing >90% mortality in SHB larvae/pupae, but indicated that this was only achieved at the highest nematode dose applied (viz. 2000 nematodes/container). Lower nematode doses resulted in significantly lower mortality. Thus, it appears that H. bacteriophora would be likely to be efficacious under similar field conditions.
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Estimate of possible rate of nematode application in the field The recommended rate of application of the nematode drenches in the field is 25x106 nematodes (1 punnet)/100 m2. This equates to a rate of 25 nematodes/cm2. In the investigation with large containers (Ø 10 cm), the dose which achieved >90% mortality was 2000 nematodes. This equates to 25.4 nematodes/cm2, essentially the same as the recommended field rate of application for other pest species. While this is encouraging, particularly because this bioassay was intended to more closely represent field conditions, it should be noted that frequently, higher doses of insecticidal treatments are needed to achieve field results than those required in the laboratory. Longevity of nematode activity All three species of nematode, H. bacteriophora, H. zealandica and S. feltiae, were tested for longevity of activity, although it was again confirmed that the latter species had no efficacy against SHB. Significant activity by the heterorhabditids was maintained for seven days, and this trend, although not significant, continued for a further seven days (until day 14 after inoculation). This implies that burrowing SHB larvae may be, at least in part, controlled for seven or more days after soil drench applications of nematodes, especially H. bacteriophora. Laboratory bioassays using entomopathogenic fungi No SHB larval/pupal mortality was recorded in either Petri dish bioassays, nor in bioassays using potting mix medium, at any of the spore concentrations tested. This implies that SHB was not a suitable host for the strains of M. anisopliae tested, particularly given the positive results from the nematode investigations. There may be more suitable strains of M. anisopliae which would be more efficacious, but the objective of this project (after discussions with industry and RIRDC) was to test only currently commercially available and registered products. Thus, except one standard laboratory strain for initial comparative purposes, we did not screen any non-commercial strains of M. anisopliae.
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Implications • These research findings have implications for the Australian honeybee industry in its attempts
to control SHB in a sustainable and economic way. • Provided field trial data confirm the results reported here, entomopathogenic nematode
drenches of H. bacteriophora could be applied to soil areas surrounding hives to control SHB in apiary sites.
• As this is a currently available, commercial, registered formulation containing the nematode
H. bacteriophora, uptake of this technology by the Australian honeybee industry should be quite rapid.
• The dose of nematodes which achieved >90% SHB mortality in laboratory bioassays was
similar to that recommended for field application of the nematode product against other pests. This indicates that, if this dose is confirmed by field trial data, this product is likely to be economic to apply in apiaries.
• The use of nematodes rather than synthetic insecticides should also reduce the likelihood of
contamination of soil and hive products, as well as reducing the likelihood of insecticide resistance development in SHB and would provide a useful alternative or adjunct to pesticides, for soil treatment.
• It should be noted that the work reported here was only preliminary in nature, and over a
period of approximately 15 months generated data in laboratory investigations which, if positive, could be tested later in the field.
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Recommendations
• It is recommended that fully replicated field trials be conducted against larvae and pupae of SHB using drenches of the nematode H. bacteriophora to treat soil surrounding infested hives. These trials should generate data to assist in obtaining APVMA registration for use in eastern Australian states.
• It is also recommended that in future bioassays with nematodes, soil or potting mix methods
be used in preference to filter paper/Petri dish methods.
• The honeybee industry may consider funding further research to screen for more efficacious strains of M. anisopliae against SHB. It should, however, be noted that international reports have not been highly encouraging, and also any new strain identified will require registration prior to its commercial use in Australia.
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References Black CA 1965. Methods of soil analysis: Part I Physical and mineralogical properties.
American Society of Agronomy, Madison, Wisconsin, USA. Cabanillas HE, Elzen PJ 2006. Infectivity of entomopathogenic nematodes (Steinernematidae and
Heterorhabditidae) against the small hive beetle Aethina tumida (Coleoptera: Nitidulidae). Journal of Apicultural Research 45:1, 49-50.
Cannard M, Spooner-Hart RN, Milner RJ 2002. Pathogenicity of water and oil based suspensions of Metarhizium anisopliae (Metshnikoff) Sorokin and Beauveria bassiana (Balsamo) Vuillemin to citrus mealybug Planococcus citri (Risso) (Hemiptera: Pseudococcidae). General and Applied Entomology 31, 75-79.
Cant R, Spooner-Hart RN 1993. Parasitism of potato tuber moth Phthorimaea operculella (Zeller) (Lepidoptera: Gelichiidae) by entomopathogenic nematodes of the genera Heterorhabditis and Steinernema (Nematoda). In Corey SA, Daly DJ, Milne WM (eds) Pest Control and Sustainable Agriculture, CSIRO Canberra.
Carswell I, Spooner-Hart RN, Milner RJ 1998. The laboratory efficacy of the entomogenous fungi Metarhizium anisopliae and Beauveria bassiana against two dipteran species, Musca domestica and Bactrocera tryoni. Australian Journal of Entomology 37:3 281-284.
Clarkson JM, Charnley AK 1996. New insights into the mechanisms of fungal pathogenesis in insects. Trends in Microbiology 4: 197-203.
Deleplane KS 1998. The small hive beetle, Aethina tumida, in the southeast. American Bee Journal 138: 884-885.
Eischen FA, Baxter JR, Elzen PJ, Westervelt D, Wilson WT 1998. Is the small hive beetle a serious pest of US honeybees? American Bee Journal 138: 882-883.
Ellis JD, Rong IH, Hill MP, Hepburn HR, Elzen PJ 2004. The susceptibility of small hive beetle (Aethina tumida Murray) to fungal pathogens. American Bee Journal 144:6 486-488.
Elzen PJ, Baxter JR, Westervelt D, Randall C, Cutts L, Wilson WT, Eishen FA, Delaplane KS, Hopkins DL 1999. Status of the small hive beetle. Bee Culture 127, 28-29.
Elzen PJ, Baxter JR, Neumann P, Solbrig A, Pirk C, Hepburn HR, Westervelt D, Randall C. 2001. Behaviour of African and European subspecies of Apis mellifera toward the small hive beetle, Aethina tumida, Journal of Apicultural Research 40, 40-41.
Evans JD, Pettis JS, Shimanuki H. 2000. Mitochondrial DNA relationships in an emergent pest of honeybees: Aethina tumida (Coleoptera: Nititulidae) from the United States and Africa. Annals of the Entomological Society of America, 93:3, 415-420.
Gaugler R 1981, Biological potential of neoaplectanid nematodes. Journal of Nematology 15, 241-249.
Gillespie P, Staples J, King C, Fletcher MJ, Dominiak BC 2003. Small hive beetle Aethina tumida (Murray) (Coleoptera: Nititulidae) in New South Wales. General and Applied Entomology 5-7.
Grewal PS, Converse V, Georgis R. 1999. Influence of production and bioassay methods on infectivity of two ambush foragers (Nematoda: Steinernematidae). Journal of Invertebrate Pathology 73:1, 40-44.
Haque, NMM. and Levot, GW. (2004). An improved method for laboratory rearing of the Small Hive Beetle, Aethina tumida Murray (Coleoptera: Nitidulidae). General and Applied Entomology 34: 29-31.
21
Hood WM 2000. Overview of the small hive beetle Aethina tumida, in North America. Bee World 81, 129-137.
Kanga, LH, James RR, Jones WA 2003. Field trials using the fungal pathogen, Metarhizium anisopliae (Deutermycetes: Hyphomycete) to control the ectoparasitic mite, Varroa destructor (Acari: Varroidae) in honey bee colonies. Journal of Economic Entomology 96:4, 1091-1099
Längle T 2007 Beauveria bassiana (Bals.-Criv.) Vuill. – A biocontrol agent with more than 100 years of history of safe use http://www.rebeca-net.de/downloads/Beauveria%20bassiana.pdf (accessed December 24, 2007).
Llewellyn R (ed.) 2002. The good bug book. Bugs for Bugs, Mundubbera, Qld, Australia.
Lundie AE 1940. The small hive beetle, Aethina tumida. South African Department of Agriculture and Forestry, Entomology Bulletin 220. 30 pp.
Milner RJ 2001. Current status of Metarhizium for insect control in Australia. Biocontrol News and Information 21, 47N-50N.
Muerrie TM, Neumann P, Dames JF, Hepburn HR, Hill MP 2006. Susceptibility of adult Aethina tumida (Coleoptera: Nititulidae) to entomopathogenic fungi. Journal of Economic Entomology 99:1, 1-6.
Neumann P, Hepburn R, Elzen PJ, Baxter JR 2001. Laboratory rearing of small hive beetles Aethina tumida (Coleoptera: Nititulidae). Journal of Apicultural Research 40, 111-112.
Pettis JS, Shimanuki H 2000. Observations on the small hive beetle Aethina tumida Murray, in the United States. American Bee Journal 140, 152-155.
Richards CS, Hill MP, Dames JF 2005. The susceptibility of small hive beetle (Aethina tumida Murray) pupae to Aspergillus niger (van Tieghem) and A. flavus (Link:Grey). American Bee Journal, 145-751.
Sanford MT 1998. A new beehive pest in the U.S. Bee Culture 126, 24-26.
Smart GC 1995. Entomopathogenic nematodes for the biological control of insects. Journal of Nematology. 27:45, 529-533.
Steinkraus DC, Tugwell NP 1997. Beauveria bassiana (Deuteromycetes: Moniliales) effects on Lygus lineolaris (Hemiptera: Miridae). Journal of Entomological Science 32, 79-90.
