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Article

Evaluating the Toxicity of Known Western Honey Bee-Safe Insecticides in Controlling Small Hive Beetles (Aethina tumida)

by
Julia St. Amant
* and
Cameron J. Jack
Entomology and Nematology Department, University of Florida, Gainesville, FL 32611, USA
*
Author to whom correspondence should be addressed.
Diversity 2025, 17(4), 230; https://doi.org/10.3390/d17040230
Submission received: 25 February 2025 / Revised: 15 March 2025 / Accepted: 20 March 2025 / Published: 25 March 2025
(This article belongs to the Special Issue Integrated Pest Management and Its Role in Biodiversity Conservation)
Versions Notes

Abstract

Currently, there is no integrated pest management approach for controlling small hive beetles (Aethina tumida), a widespread honey bee (Apis mellifera) pest. To date, only hive trapping has shown any effectiveness in controlling the pest. In this study, we tested several possible active ingredients that have been shown previously to demonstrate low toxicity towards honey bees. To test their toxicities, we treated both SHBs and honey bees topically and exposed SHBs to these compounds orally via pollen. Coumaphos (industry standard), a solvent control (acetone), and a positive control (dimethoate) were used for comparisons. Thiacloprid (LD50 = 1.3 ng/SHB; LC50 = 12 µg/g pollen) was the most toxic active ingredient tested against SHBs both topically and through pollen. Topically, thiacloprid was 340× more toxic to SHBs than coumaphos (LD50 = 431 ng/SHB). However, acetamiprid (selectivity ratio = 152) was much more toxic to SHBs than to honey bees compared to thiacloprid (selectivity ratio = 3). These findings demonstrate the need to find other active ingredients other than coumaphos and that acetamiprid has the greatest potential to reduce SHB populations safely in a honey bee hive. Field research using acetamiprid should be conducted to explore possible sub-lethal effects on honey bees.

1. Introduction

Small hive beetles (SHBs), Aethina tumida, are a damaging honey bee (Apis mellifera) pest and are invasive to the United States [1] and many other countries around the world [2]. Small hive beetles damage honey bee hives by eating the wax, honey, pollen, and honey bee eggs [1,3]. Additionally, SHBs can cause fermentation of hive products (“slime”) due to their waste [4] and association with the yeast Kodamaea ohmeri [5]. The impacts of SHBs may lead a colony to abscond, leaving behind a slime filled hive [1]. They also have the potential to be biological vectors of honey bee viruses, as it has been demonstrated that SHBs can become infected with honey bee viruses when SHBs stimulate trophallaxis feeding from honey bee workers or via food-borne transmission from feeding on deceased bees [6]. Honey bees are unable to remove SHBs from the hive due to SHBs running and hiding in small areas [7]. Even when a honey bee gets the opportunity to attack, SHBs have a hard elytra shell that allows them to tuck their head and appendages safely underneath themselves [1,8]. Intervention with integrated pest management (IPM) techniques is often the best choice for beekeepers to reduce growing populations of SHB in a hive [1].
Currently, there are no effective in-hive chemical treatments for SHBs. Coumaphos is the only in-hive pesticide registered to reduce SHBs in the United States. However, SHBs have developed resistance to coumaphos, and it is no longer an effective way to reduce SHB population [9]. Alternatively, mechanical control of SHBs is conducted using a wide variety of baited traps with varying amounts of success [1]. Manual removal of SHBs by the beekeeper can be difficult due to SHBs having dark coloration and their tendency to avoid sunlight [7]. For new chemical treatments to be used for SHBs, chemical compounds need to be screened to determine their toxicity to SHBs and honey bees.
The compounds in this study were chosen based on previous research that demonstrated that they have a relatively low toxicity to honey bees compared to other pesticides [10,11,12]. Acetamiprid and thiacloprid are both first-generation (chloropyridinyl) neonicotinoids, which are a class of pesticides often used in crop protection [13]. Acetamiprid has been shown to be an effective treatment for SHBs while being relatively safe for honey bees [14]. Though thiacloprid has a higher toxicity to honey bees, it may be effective at killing SHBs at doses that are non-toxic to honey bees [11]. Their mode of action is to act as agonists on postsynaptic nicotinic acetylcholine receptors in insect nervous systems [13]. Chlorantraniliprole and flubendiamide are crop pesticides in the diamide class that affect the ryanodine receptor found in the neuromuscular cells of insects by decreasing calcium stores, which leads to involuntary muscle contraction, decrease in feeding, and eventual death [12,15]. However, chlorantraniliprole is less likely to bind to the ryanodine receptors in Hymenoptera due to the sequence difference in the diamide binding site for the receptors [12]. Similarly, the binding sites for flubendiamide have higher selectivity in honey bees that cause flubendiamide to be less toxic [15]. Propiconazole is a triazole fungicide that works as a 14α-demethylase inhibitor in fungi [16]. Although propiconazole has been shown to have low toxicity to honey bees, it can have synergistic effects with other compounds to become more toxic, such as with acetamiprid in Apis cerana and thiamethoxam in Apis mellifera [11,17,18].
To test these compounds, topical assays were performed to determine how toxic each compound is to honey bees and SHBs. Additionally, pollen assays were used with SHBs as an additional route of exposure. We wanted to find a compound that causes high mortality in SHBs at low doses and low mortality in honey bees at high doses.

2. Materials and Methods

2.1. Small Hive Beetle Collection

A colony of in vitro-reared SHB was reared following the methods of Stuhl (2023) at the University of Florida Honey Bee Research and Extension Laboratory (UF HBREL) [19]. Small hive beetles of both sexes were collected by hand, within one week of emerging, into plastic cups (Uline 266 mL Squat Crystal Clear Plastic Cups and Flat Lids) with small ventilation holes as described in Kleckner et al. (2022) [14]. Prior to collecting the SHBs, a mesh was hot glued onto the inside of the cups to cover the ventilation holes and prevent SHBs from escaping.

2.2. Honey Bee Collection

The methods used throughout this study followed the protocol described in St. Amant et al. (2024) [20]. From June to November 2022, frames of emerging adult worker honey bees, Apis mellifera, were collected at the UF HBREL (Entomology and Nematology Department, Gainesville, FL, USA) from twelve optimally managed hives. These hives were managed to keep Varroa destructor levels low and food resources high, and they have never been treated with the chemicals tested in this study. Frames of emerging brood were removed from the hives, and the adult workers were brushed off the frames. In less than 24 h after the frames were placed inside an incubation room at 32 °C, the newly emerged adult worker honey bees were mixed into a large bin. The honey bees were gently picked up by hand and placed into plastic cups (118.294 mL clear round wide-mouth plastic jar with white lid; ULINE S-9934) with small ventilation holes. In each cup, two 3 mL syringes with the tips removed were filled with sucrose solution (1:1 w/v) and hung through holes on the top of the cups. The cups were placed in an incubator (Binder Incubator (Camarillo, CA, USA, #BD400UL-120V)) at 34 °C with no humidity control, and the assays were completed the same day.

2.3. Topical Small Hive Beetle Assays

The following compounds were used in the three assays described in this paper: acetamiprid (Chem Services, Inc., West Chester, PA, USA; 99% purity), chlorantraniliprole (Chem Service Inc., West Chester, PA, USA; 98% purity), flubendiamide (Sigma-Aldrich, St. Louis, MO, USA; analytical standard), propiconazole (Supelco, St. Louis, MO, USA; analytical standard), and thiacloprid (Thermo Scientific, Fair Lawn, NJ, USA; 95%). For comparison within each assay, dimethoate (MedChemExpress, Monmouth Junction, NJ, USA; 99% purity) was used as the positive control, and acetone was used as the solvent control. Each solid compound was mixed into acetone, and the mixtures were further diluted with acetone to make each treatment concentration. Dilutions of ten from 1000 µg/mL were initially used for range finding to decide treatment groups for each compound. Each treatment group contained three cups of ten SHBs.
To anesthetize the SHBs for assays, a handheld CO2 tire inflator (Genuine Innovations Ultraflate Plus Inflator with 12 g Crosman Powerlet CO2 Cartridges, San Luis Obispo, CA, USA) was sprayed into each cup containing the SHBs for approximately three seconds. Then, 1 µL of each solution was topically dosed on the dorsal side of each SHB using a micropipette. To feed the SHB for the duration of the assay, a wick (Richmond 10.16 cm, Medium Braided Cotton Roll, Charlotte, NC, USA) was added to a 1.5 microcentrifuge tube containing sucrose solution (1:1 w/v) to feed the SHB while preventing them from drowning in sucrose solution. A desiccator (Fisherbrand Acrylic Desiccator Cabinets 45.7 cm 08-642-23C) containing a basin filled with water was placed inside of an incubator (Thermo Scientific Herather, IMH100 51028067 Bench Top Incubator, Lagenselbold, Germany). The cups were kept inside the desiccator at 34 °C without humidity control. Mortality was checked every 24 h for 72 h, and dead SHBs were removed using forceps at each time point.

2.4. Topical Honey Bee Assays

Honey bees were topically treated similarly to SHBs as described in Section 2.3. Each treatment group contained three cups of ten honey bees. The honey bees were anesthetized with CO2, and 1 µL of solution was pipetted on the dorsal side of their thorax. The treated honey bees were returned to their plastic cups and placed back into the incubator at 34 °C without humidity control. Mortality was checked every 24 h for 72 h, as per the Organisation for Economic Co-operation and Development (OECD) protocol for acute topical assay for honey bees [21]. Dead honey bees were removed using forceps at each time point.

2.5. Pollen Small Hive Beetle Assays

Pollen balls were made with 1 g of pollen patties by rolling them and wrapping them in plastic wrap. They were stored in a freezer until the start of the assays. The middle of the pollen ball was pushed down to create a well shape, and 50 µL of solution was pipetted into the well as described in Kleckner et al. (2022) [14]. The pollen was mixed by hand until the solution was homogenous throughout. The treated pollen was added to the cups containing SHBs, and then the cups were placed back into the desiccator at 34 °C without humidity control. The pollen remained in the cup for 72 h. Mortality was checked every 24 h for 72 h, and dead SHBs were removed using forceps at each time point.

2.6. Pollen Honey Bee Assays

Honey bees were topically treated similarly to SHBs described in Section 2.3. Pollen was treated with 50 µL of solution and mixed by hand. The pollen was placed in the cup with the newly emerged honey bees. The honey bees were also given sucrose solution (1:1 w/v) using the tipless syringes hanging in the cups. The pollen remained in the cup for 72 h and the cups were placed into an incubator at 34 °C. Mortality was checked every 24 h for 72 h, and dead honey bees were removed using forceps at each time point.

2.7. Statistical Analysis

Statistical analysis was performed using R version 4.2.1 (23 June 2022) “Funny-Looking Kid” using an R script for probit analysis of insects [22]. Each compound and assay type was analyzed separately to produce individual LD50/LC50 values using a 95% confidence interval. Mortality after 72 h, the number of SHBs/bees tested, and the treatment concentrations were included in the calculations. Assays that had a greater than 25% solvent control mortality or less than 90% positive control mortality were removed from the analysis. When the solvent control mortality was greater than 5%, Abbott’s correction was calculated by R [22]. To calculate the selectivity ratio for topical application, honey bee LD50 was divided by the SHB LD50 of each compound. The activity ratio for each compound was calculated by dividing the LD50/LC50 of coumaphos, which is the current industry standard, by the LD50/LC50 of the compound within each assay type [23]. Coumaphos LD50/LC50 were taken from St. Amant et al. (2024) for comparison with the LD50/LC50 found in this study, as coumaphos was evaluated using the same methods at the same time as this work, using the same cohort of small hive beetles [20].

3. Results

3.1. Topical Small Hive Beetle Assays

Thiacloprid had the lowest LD50 value (LD50 = 1.3 ng/SHB) and the highest toxicity of the compounds tested topically on SHBs in this study (Table 1). Acetamiprid (14 ng/SHB) and thiacloprid were 30× and 340× more toxic to SHB than coumaphos (LD50 = 431 ng/SHB), respectively. Coumaphos was 1.7× more toxic to SHBs than chlorantraniliprole (LD50 = 730 ng/SHB). Flubendiamide (LD50 ≥ 6000 ng/SHB) and propiconazole (LD50 ≥ 10,000 ng/SHB) were the least toxic compounds, and they would not produce a >50% SHB mortality rate from doses up to 6000 ng/SHB and 10,000 ng/SHB, respectively. SHBs treated topically with dimethoate (1000 ng/SHB), the positive control, had a 90% or greater mortality rate within 72 h of exposure.

3.2. Topical Honey Bee Assays

Thiacloprid had the lowest LD50 value (LD50 = 3.8 ng/honey bee) and was >1571× more toxic to honey bees than coumaphos (LD50 ≥ 6000 ng/honey bee) (Table 2). Acetamiprid (LD50 = 2182 ng/honey bee) and chlorantraniliprole (LD50 = 569 ng/honey bee) were 3× and 11× more toxic than coumaphos to honey bees, respectively. Flubendiamide (LD50 ≥ 10,000 ng/honey bee) and propiconazole (LD50 ≥ 10,000 ng/honey bee) did not produce a honey bee mortality rate >50%, with doses up to 10,000 ng/honey bee. In addition to calculating LD50 values, selectivity ratios were used to compare the toxicity of each compound on SHBs with their toxicity to honey bees. A selectivity ratio for flubendiamide and propiconazole compounds could not be calculated. Acetamiprid, coumaphos, and thiacloprid were 152×, 14×, and 3× more toxic to SHBs than to honey bees, respectively. Chlorantraniliprole was 1.3× more toxic to honey bees than to SHBs. Honey bees treated topically with dimethoate (1000 ng/SHB), the positive control, had a 90% or greater mortality rate within 72 h of exposure.

3.3. Pollen Small Hive Beetle Assays

Chlorantraniliprole (LC50 ≥ 125 µg/g pollen), flubendiamide (LC50 ≥ 300 µg/g pollen), and propiconazole (LC50 ≥ 500 µg/g pollen) did not produce SHB mortality >50% after 72 h of exposure to treated pollen (Table 3). Acetamiprid (LC50 = 0.78 µg/g pollen) had the lowest LC50 and was 226× more toxic to SHBs through pollen than coumaphos (LC50 = 175 µg/g pollen). Similarly, thiacloprid (LC50 = 12 µg/g pollen) was 14× more toxic to SHBs through pollen than coumaphos. SHBs treated through pollen with dimethoate (1000 ng/SHB), the positive control, had a 90% or greater mortality rate within 72 h of exposure.

3.4. Pollen Honey Bee Assays

The honey bees in the pollen assays did not consistently produce a >90% rate of mortality for the positive control, dimethoate. Adjustments were made to the pollen to increase honey bee interaction by adding powdered sugar to the inside and outside of the pollen. However, during the 72-h exposure period, the honey bees did not interact with or consume the pollen enough to produce reliable LC50 values. Therefore, pollen honey bee assays with these compounds were considered invalid.

4. Discussion

Our study focused on evaluating the toxicity of pesticides on SHBs that have shown in previous studies to have low toxicity to honey bees. Topical and pollen SHB assays and topical honey bee assays were performed using acetamiprid, chlorantraniliprole, flubendiamide, propiconazole, and thiacloprid. Among these pesticides, acetamiprid had the largest selectivity ratio, which shows that it is far more toxic to SHBs than to honey bees through topical exposure. In contrast, the industry standard, coumaphos, has a selectivity ratio that is about 11× smaller than the selectivity ratio of acetamiprid [20]. Although coumaphos was relatively non-toxic to honey bees through acute exposure, it takes a higher dose to kill SHBs with coumaphos compared to some of the other compounds tested. Coumaphos has been shown to be far more toxic to honey bees through chronic exposure than through acute exposure due to the compound building up over time [24]. Coumaphos is also highly toxic to feeding larvae [25]. Therefore, if a high dose of coumaphos is needed to kill SHBs in a hive, then there is a higher risk for chronic effects on both adults and larvae as coumaphos builds up in parts of the hive, such as the bee bread [26].
Chlorantraniliprole had a higher toxicity to honey bees than to SHBs and was unable to kill more than 50% of the adult SHBs that were exposed to it through pollen. Flubendiamide and propiconazole were relatively non-toxic to honey bees through topical exposure. They were also relatively non-toxic to SHBs for both topical and pollen exposure routes. Both chlorantraniliprole and flubendiamide are typically used to target immature lepidopteran or coleopteran pests [27,28]. Here we demonstrated their inability to control SHB adults; however, chlorantraniliprole’s use on SHB larvae has demonstrated noteworthy potential. Hackmeyer et al. (2023) found that mixing chlorantraniliprole into pollen patties almost entirely prevented SHB larvae development [29]. Thus, these compounds have no potential as an SHB adulticide but can possibly be useful as an SHB larvicide or preventative measure.
Thiacloprid was the most toxic compound that was tested on SHBs topically. However, it was also the most toxic compound to honey bees. This makes thiacloprid an unfit candidate for use in a hive because the dose needed to effectively kill SHBs is too close to the dose that would kill honey bees. Based on these results, acetamiprid is the only compound in this study that has the potential to be an effective and safe in-hive chemical treatment for SHBs.
A selectivity ratio for SHB and honey bee pollen assays was not able to be conducted due to unsuccessful honey bee pollen assays. The honey bees in this study seem to avoid ingesting the pollen, which led to low mortality rates in our positive control treatment groups. The addition of sugar to the pollen did not increase honey bee pollen ingestion. Additionally, we tried to encourage protein consumption by introducing honey bee brood pheromone to the cups during the pollen assays [30]. However, the brood pheromone did not increase pollen consumption.
In the future, it would be beneficial to complete acute and chronic honey bee oral assays on acetamiprid and coumaphos. The impacts of acetamiprid on honey bee larvae should also be determined, as it is important to understand the short-term and long-term impacts of compounds before introducing them in a hive. Following those tests, semi-field tests using acetamiprid should be conducted to determine its safety within honey bee colonies and effectiveness at reducing SHB populations within a hive.

5. Conclusions

Overall, acetamiprid is the most promising compound tested in this study to reduce small hive beetle populations in a hive. This is due to its high toxicity to SHBs and low toxicity to honey bees. Chlorantraniliprole, thiacloprid, flubendiamide, and propiconazole are not effective options for adult SHB treatment, despite their low toxicity to honey bees reported in previous studies. Additional compounds should be screened to find compounds that are safe for honey bees and toxic to SHBs, as well as testing multiple exposure routes of acetamiprid to SHBs and honey bees. Still, we contend that chemical control should be used in conjunction with other integrated approaches and only as a last resort once other methods are insufficient at reducing pest populations on their own.

Author Contributions

Conceptualization, J.S.A. and C.J.J.; methodology, J.S.A. and C.J.J.; validation, J.S.A. and C.J.J.; formal analysis, J.S.A.; investigation, J.S.A.; resources, C.J.J.; data curation, J.S.A.; writing—original draft preparation, J.S.A.; writing—review and editing, J.S.A. and C.J.J.; visualization, J.S.A.; supervision, C.J.J.; project administration, C.J.J.; funding acquisition, C.J.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Florida Department of Agriculture and Consumer Services (AWD12534) and the USDA National Institute of Food and Agriculture Multistate Project (NC1173).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We gratefully acknowledge Jordan Buisch (University of Florida, Honey Bee. Research and Extension Laboratory, HBREL) for their help in the laboratory. Additionally, we thank. Hannah Gurland (HBREL) for their help rearing small hive beetles.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. 72 h post-treatment lethal dose (LD50) values and confidence intervals for SHBs acute topical toxicity generated in R. The activity ratio (AR) is included to compare each test compound to the industry standard (AR = LD50 of SHBs with coumaphos/LD50 of SHBs with the test compound). * = Coumaphos LD50 values were taken from St. Amant et al. (2024) [20].
Table 1. 72 h post-treatment lethal dose (LD50) values and confidence intervals for SHBs acute topical toxicity generated in R. The activity ratio (AR) is included to compare each test compound to the industry standard (AR = LD50 of SHBs with coumaphos/LD50 of SHBs with the test compound). * = Coumaphos LD50 values were taken from St. Amant et al. (2024) [20].
CompoundSHBs (n)SHBs LD50 (95% CI)AR
Coumaphos *270431 ng/SHB
(394.0–437.49)		--
Acetamiprid24014 ng/SHB
(11.34–17.41)		30
Chlorantraniliprole270730 ng/SHB
(395.61–1325.75)		0.6
Flubendiamide300>6000 ng/SHB<0.07
Propiconazole240>10,000 ng/SHB<0.04
Thiacloprid2401.3 ng/SHB
(0.15–9.0)		340
Table 2. 72 h post-treatment lethal dose (LD50) values and confident intervals for honey bee acute topical toxicity generated in R. The activity ratio (AR) is included to compare each test compound to the industry standard (AR = LD50 of honey bees with coumaphos/LD50 of honey bees with the test compound). The selectivity ratio (SR) is the topical LD50 of honey bees divided by the topical LD50 of small hive beetles (reported in Table 1) to compare each test compound [23]. * = Coumaphos LD50 values were taken from St. Amant et al. (2024) [20].
Table 2. 72 h post-treatment lethal dose (LD50) values and confident intervals for honey bee acute topical toxicity generated in R. The activity ratio (AR) is included to compare each test compound to the industry standard (AR = LD50 of honey bees with coumaphos/LD50 of honey bees with the test compound). The selectivity ratio (SR) is the topical LD50 of honey bees divided by the topical LD50 of small hive beetles (reported in Table 1) to compare each test compound [23]. * = Coumaphos LD50 values were taken from St. Amant et al. (2024) [20].
CompoundHoney Bees (n)Honey Bees LD50 (95% CI)ARSR
Coumaphos *210>6000 ng/honey bee-->14
Acetamiprid2402182 ng/honey bee
(1519.72–3261.48)		>3152
Chlorantraniliprole210569 ng/honey bee
(371.6–741.24)		>110.8
Flubendiamide180>10,000 ng/honey bee>0.6--
Propiconazole180>10,000 ng/honey bee>0.6--
Thiacloprid4203.8 ng/honey bee
(0.20–84.57)		>15713
Table 3. 72 h post-treatment lethal concentration (LC50) values and confidence intervals for the toxicity of compounds through pollen generated in R. The activity ratio (AR) is included to compare each test compound to the industry standard (AR = LC50 of SHBs with coumaphos/LC50 of SHBs with the test compound). * = Coumaphos LC50 values were taken from St. Amant et al. (2024) [20].
Table 3. 72 h post-treatment lethal concentration (LC50) values and confidence intervals for the toxicity of compounds through pollen generated in R. The activity ratio (AR) is included to compare each test compound to the industry standard (AR = LC50 of SHBs with coumaphos/LC50 of SHBs with the test compound). * = Coumaphos LC50 values were taken from St. Amant et al. (2024) [20].
CompoundSHBs (n)SHBs LC50 (95% CI)AR
Coumaphos *362175 µg/g pollen
(108.75–457.12)		--
Acetamiprid2680.78 µg/g pollen
(0.2–1.92)		226
Chlorantraniliprole390>125 µg/g pollen<1.4
Flubendiamide210>300 µg/g pollen<0.6
Propiconazole90>500 µg/g pollen<0.4
Thiacloprid24012 µg/g pollen
(5.5–49.78)		14
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St. Amant, J.; Jack, C.J. Evaluating the Toxicity of Known Western Honey Bee-Safe Insecticides in Controlling Small Hive Beetles (Aethina tumida). Diversity 2025, 17, 230. https://doi.org/10.3390/d17040230

AMA Style

St. Amant J, Jack CJ. Evaluating the Toxicity of Known Western Honey Bee-Safe Insecticides in Controlling Small Hive Beetles (Aethina tumida). Diversity. 2025; 17(4):230. https://doi.org/10.3390/d17040230

Chicago/Turabian Style

St. Amant, Julia, and Cameron J. Jack. 2025. "Evaluating the Toxicity of Known Western Honey Bee-Safe Insecticides in Controlling Small Hive Beetles (Aethina tumida)" Diversity 17, no. 4: 230. https://doi.org/10.3390/d17040230

APA Style

St. Amant, J., & Jack, C. J. (2025). Evaluating the Toxicity of Known Western Honey Bee-Safe Insecticides in Controlling Small Hive Beetles (Aethina tumida). Diversity, 17(4), 230. https://doi.org/10.3390/d17040230

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