The Mobile Divider Method: An Effective Strategy to Detect Small Hive Beetle (Aethina tumida) Adults in Honey Bee Colonies (Apis mellifera) in Calabria, Italy

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This study promotes the Mobile Divider (MD) method as an effective approach for concentrating adult Small Hive Beetles (SHBs) behind the Mobile Divider. These results indicate that the MD method is not only time-saving but could also be effective for SHB surveillance, early detection, and control.

Abstract

Aethinosis, the honey bee disease caused by small hive beetle, is listed in the Animal Health Law and requires mandatory surveillance and control measures. The Mobile Divider (MD) method has been proposed as a time-saving alternative to the official surveillance method outlined by the Ministry of Health (MoH). The current study aimed to evaluate the efficacy of the MD method in concentrating SHBs behind the MD and optimizing the number of SHBs detected during hive inspections, thereby improving the surveillance strategies required by European regulations and the WOAH. In late winter and autumn 2022, we conducted 431 hive inspections across six apiaries in the province of Reggio Calabria, Italy. A total of 379 adult SHBs were collected and killed; no larvae were detected. Using the MD method, 238 SHBs were found behind the MD, while 141 SHBs were found in the remaining volume of the hive. Chi-square analysis confirmed the effectiveness of the MD method, showing that the SHB distribution behind the MD and in the remaining volume of the hive was not random (p < 0.0005). Further studies are needed to assess the effectiveness and potential benefits of the MD method in regions with higher SHB infestation levels.

1. Introduction

Aethina tumida Murray, also known as the Small Hive Beetle (SHB), is a parasite native to sub-Saharan Africa [1] present in Italy since September 2014 [2]. A decade after its introduction in Italy, SHB remains confined to the Calabria region and Sicily. To date, the only areas in the European Union that have encountered SHB are the regions of Calabria and Sicily (Italy) and La Réunion (France), where it is still present [3], as well as Portugal, where it was successfully eradicated in 2004 [4]. Outside its endemic range, SHB is considered an invasive species due to the damage it causes to both managed honeybees and wild bees, as well as to the economies of affected countries [5]. Aethinosis adversely affects beekeeping both directly, through colony damage, and indirectly through the implementation of veterinary measures for monitoring and control [6]. SHB could be attracted to honey bee colonies due to a combination of factors: honey bee pheromones, pollen and honey fermentation products, and SHB sex pheromones [7,8,9]. These attractants influence SHB to select hives as preferred sites for feeding, reproduction, and completing its life cycle [10]. Both larval and adult stages of SHB damage honey bee colonies [11,12], but larvae are particularly destructive, tunneling through combs and causing honey fermentation [13]. SHB is a listed honey bee disease by both the European Union [14] and the WOAH [15], and its classification under categories D and E requires mandatory surveillance and control measures to prevent its spread to disease-free regions [16]. Control measures include regular inspections conducted by beekeepers to detect and kill SHB, the use of in-hive traps, and restrictions on the movement of honey bees, unprocessed apiculture by-products, apiculture products in honeycomb for human consumption, and beekeeping materials outside of Calabria region and Sicily [17]. Surveillance measures in Italy are implemented across the entire national territory, but they differ depending on whether the regions are free from SHB or already affected. In disease-free regions, official veterinarians conduct surveillance on a predefined number of apiaries throughout the year, based on both random selection and risk-based criteria. In affected regions, including both protection and surveillance zones, a greater number of apiaries are inspected compared to disease-free areas. Moreover, sentinel hives are established and monitored within the protection zones [18,19]. Beekeepers are also required to carry out passive surveillance within their apiaries to verify the absence of this parasite [16]. To date, regardless of the region, clinical inspections in apiaries are conducted according to the protocol recommended by the Italian Ministry of Health (MoH). This protocol for SHB detection includes examining the outer and inner covers, inspecting both sides of each comb, and placing the inspected combs in an empty hive. The bottom and corners of the hive are also checked. Finally, the combs are returned to their original positions. In addition, supers and the bottom board are visually inspected if present. During this process, both SHB larvae and adults can be visually recognized, collected, and killed. However, this protocol is time-consuming, as it requires careful inspection of both sides of each frame under adequate lighting [20]. Furthermore, finding SHBs is particularly challenging when many adult bees cover the combs, especially in Italy since the SHB population is low and SHB adults are more common than larvae. SHB adults are smaller and quicker than larvae, so they can easily hide themselves behind the adult bees or quickly fly away [5]. All these considerations highlight the need to develop new strategies to enhance both the effectiveness of detecting SHB adults within hives and the efficiency of the method in terms of speed and ease of execution.

The Mobile Divider (MD) is a divider placed between the last frame and the hive wall (Figure 1). The MD method is a protocol designed to detect SHBs within colonies during hive inspections.

This method involves inspecting the outer and inner covers of the hive, examining and removing each comb starting from the side opposite the MD, and transferring the inspected combs to an empty hive. Slow movements are used to encourage SHBs to concentrate on the remaining frames. When three combs and the divider remain, these are slowly moved to the empty side of the hive, and the divider, the space behind it, the corners, walls, and the bottom of the hive are inspected. Finally, the method includes observing the contents of the bottom board if present. The MD method was previous described as a time-saving method, saving 3 min and 44 s for every inspection compared to the classical inspection method by the MoH [20]. The aim of this study was to evaluate the efficacy of the MD method in concentrating SHBs behind the MD and improving SHB surveillance and early detection. In detail, early detection is crucial to hinder the spread of SHB to new areas where it is not yet present and to control the SHB infestation level within apiaries before significant damage occurs.

2. Materials and Methods

A citizen science study was conducted in the province of Reggio Calabria, Italy, during 2022. Six beekeepers from the local beekeepers’ association were involved and trained on uniform protocols for hive inspections and data collection to assess the presence of SHB. Beekeepers were selected based on their extensive experience in managing SHB infestations. They inspected their colonies over three consecutive weekends in late winter 2022 (21–23 January, 11–13 February, and 4–6 March) and autumn 2022 (30 September–2 October, 21–23 October, and 4–6 November), with a three-week interval between each inspection. The MD device was placed on the left side of each hive between the last frame and the left wall at the beginning of the study. The device remained in the same position throughout the study and was only removed during inspections to count the number of SHB present behind it. During each inspection, beekeepers evaluated the number of SHBs present inside the hive using the MD method [20]. They began by inspecting the outer and inner covers of the hive then removed each comb from the side opposite the MD and transferred them to an empty hive, leaving the last three combs and the MD inside the original hive. Each removed frame was carefully inspected on both sides before being moved to the empty hive to count the number of SHBs present. Next, the beekeepers slowly moved the last three combs to the empty side of the hive and inspected each frame on both sides to count any SHBs present. After completing these evaluations, they extracted the MD device from the hive, examined it along with the space behind it, and recorded the number of SHBs found. Lastly, the beekeepers observed the content of the bottom board if present and completed a document separately recording the number of SHBs found behind the Mobile Divider and those found in the remaining volume of the hive. The number of SHBs in the remaining volume of the hive was calculated by adding the SHBs detected on both sides of the inspected frames before moving the MD device and inspecting the area behind it. Figure 2 provides a descriptive diagram of the method used in this study. We evaluated the efficacy of the MD method in concentrating SHBs by comparing the number of SHBs detected behind the MD device with those found in the remaining volume of the hive. Data were analyzed using IBM SPSS Statistics version 27.0.1.0 and Microsoft Excel 2007. The Kolmogorov–Smirnov and Shapiro–Wilk tests were used to assess the normality of data distributions. Non-parametric tests, such as the χ² analysis, have been applied on the number of SHBs detected by the two methods. We also calculated the percentage of SHBs found behind the MD relative to the total number of SHBs captured in the hive. In addition, we assessed the mean number of SHBs captured per hive per inspection to analyze the seasonal trend of SHB captures in late winter and autumn.

3. Results

The presence of SHB in hives was investigated over a six-month period, including three months in late winter and three months in autumn. A total of 431 inspections were carried out: 197 inspections of up to 67 beehives across six apiaries in late winter, and 234 inspections of up to 81 beehives across five apiaries in autumn (

Table 1. Number of SHBs detected and number of hives inspected during each inspection.

	Late Winter				Autumn
	First Inspection (January 21–23)	Second Inspection (February 11–13)	Third Inspection (March 4–6)	Fourth Inspection (September 30–October 2)	Fifth Inspection (October 21–23)	Sixth Inspection (November 4–6)
Total number of SHBs detected	31	18	32	146	92	60
Number of hives inspected	67	65	65	81	79	74

). Some colonies could not be inspected three times during the same season due to colony death. A total of 379 SHBs were collected and killed during the inspections. No larvae have been detected.

Table 2. Number of SHBs detected during each inspection in each apiary involved in this study, as well as the total number of SHBs detected across all inspections in each apiary. In this table, the number of SHBs found in each apiary during each inspection is separated into those detected behind the Mobile Divider and those found in the remaining volume of the hive.

		N° SHBs Detected During Each Inspection						N° SHBs Detected Across All Inspections
		Late winter			Autumn
		First inspection (January 21–23)	Second inspection (February 11–13)	Third inspection (March 4–6)	Fourth inspection (September 30–October 2)	Fifth inspection (October 21–23)	Sixth inspection (November 4–6)
Apiary 1	Behind MD	2	1	1	NA	NA	NA	4
	In the remaining volume of the hive	5	0	2	NA	NA	NA	7
Apiary 2	Behind MD	1	1	0	9	9	8	28
	In the remaining volume of the hive	0	1	0	0	3	0	4
Apiary 3	Behind MD	7	2	4	87	38	33	171
	In the remaining volume of the hive	2	3	0	44	35	8	92
Apiary 4	Behind MD	4	1	11	3	2	3	24
	In the remaining volume of the hive	1	1	2	0	0	0	4
Apiary 5	Behind MD	0	0	0	0	0	1	1
	In the remaining volume of the hive	6	4	3	2	5	5	25
Apiary 6	Behind MD	0	3	6	0	0	1	10
	In the remaining volume of the hive	3	1	3	1	0	1	9

provides a detailed breakdown of the number of SHBs detected both behind the MD and in the remaining volume of the hive during each inspection across all six apiaries.

Figure 3 shows the trend in the total number of SHBs detected throughout the sampling period, without distinguishing between each apiary. It also illustrates the distribution of SHBs found behind the MD and in the remaining volume of the hive. A peak in SHB presence—with 1.8 SHBs detected per hive—was observed during the fourth inspection (September 30–October 2), and the majority of the SHBs detected (99 out of 146; 68%) were located behind the MD.

Both the total number of SHBs found within the beehive—including those found behind the MD and in the remaining volume of the hive—and the number of SHBs found exclusively behind the MD exhibited a positive asymmetric distribution with high variability, deviating from a normal distribution (Figure 4).

Considering all the inspections of the study across all the apiaries, we found a total of 238 SHBs (63%) behind the MD device and 141 SHBs (37%) in the remaining volume of the hive (Figure 5).

Chi-square analysis revealed a statistically significant difference in SHB presence behind the MD and in the remaining volume of the hive (χ² = 24.83, df = 1, p < 0.0005), confirming a non-random distribution of SHBs. A total of 238 out of 379 SHBs were found behind the MD. This value is well beyond the upper limit of the acceptance interval of 3 σ (160.3 ≤ n ≤ 218.7), allowing a stricter degree of reliability of 99.7%.

4. Discussion

In this study, we found a statistically significant difference in SHB presence between the area behind the MD and the remaining volume of the hive, highlighting the effectiveness of the MD method in concentrating SHBs behind the MD and optimizing the number of SHBs detected during hive inspections. These results can be attributed to the natural behavior of adult SHBs, which tend to escape from light [5]. As SHBs flee from light and beekeeper manipulation, they initially concentrate on the remaining frames before ultimately hiding behind the Mobile Divider device, which remains inside the hive. We should consider that the MD divider occupies a significantly smaller volume within the hive, suggesting that the actual difference in SHB abundance between the MD and other parts of the hive might be even greater. The absence of larvae during the inspection is consistent with other data on SHB infestation in Calabria, which are generally characterized by low infestation levels and the absence of larval stages [21].

Currently, the ‘gold standard’ diagnostic method for detecting the presence of SHBs in honey bee colonies, with 100% efficacy, remains the destruction of the infested colony, followed by visual inspection involving dissection of each frame [5]. However, this method cannot be applied systematically, as it is primarily intended for experimental purposes. As suggested by the Italian Ministry of Health, visual inspection of each frame is commonly used, but this method has proven to be time-consuming [20,22] in comparison to the MD method presented here. The efficacy of the MD method in concentrating SHBs behind the Mobile Divider can be compared to other diagnostic strategies, such as the use of corrugated plastic strips on the bottom board of each hive. In our study, the MD method showed an efficacy of 63%, detecting 63% of the total SHBs identified through full visual inspection of the colonies. This is higher than the approximately 30% efficacy reported for diagnostic strips [23,24]. Another study evaluated the efficacy of bottom board traps and reported an efficacy of 43.03 ± 27.02% [25]. However, since that study compared trap-detected SHBs with those found through post-mortem colony dissection, direct comparison with the MD method is not applicable.

During autumn 2022, the number of SHBs caught per inspection—conducted at three-week intervals—was higher than in late winter 2022 (Figure 3). However, even at the peak of SHB presence during the fourth inspection (1.8 SHBs/hive), the number of SHBs detected per hive remained low throughout both the seasons. Therefore, a three-week inspection frequency can be considered appropriate for both autumn and winter, ensuring that adult SHBs are effectively and promptly detected and removed. Our results regarding the efficacy of the MD method in concentrating SHBs, enhancing surveillance strategies, and supporting early detection are promising both in regions where SHB is already present and in regions where it has not yet been detected. This method may be particularly useful because, by concentrating SHBs behind the MD device, it allows for the detection of their presence in an apiary even when only a few adults are present and the infestation level is still very low. However, capturing and removing the detected SHBs presents some limitations, as there is no locking system to restrain them. As a result, while SHBs can be easily spotted, their removal and elimination depend on the beekeeper’s skill in catching them. These findings were obtained in the Calabria region, where SHB infestation levels are low. Further studies should explore the factors influencing the MD method’s efficacy and evaluate its performance in regions with higher SHB infestation levels.