Environmental and Colony-Related Factors Linked to Small Hive Beetle (Aethina tumida) Infestation in Apis mellifera

Abstract

The small hive beetle (SHB) was first detected in Italy in 2014 and remains confined to the regions of Calabria and Sicily (Italy). The environmental and colony-related factors favorable to the development of SHBs are widely studied, but mainly at the laboratory level; it is not yet clear whether these factors also apply in the field within apiaries in regions affected by SHBs. In 2022, we conducted a study in the province of Reggio Calabria, Italy, to investigate if these factors influence SHB infestation levels in honey bee colonies. Data were collected from 67 hives in late winter and 81 hives in autumn, inspecting each hive three times per season. Overall, SHB infestation levels were low (an average 0.83 SHB/hive). Our analysis revealed a significant relationship between the SHB infestation level and the following six factors: the number of combs covered by adult bees, the total number of combs, combs surveillance, the previous month’s infestation, sun exposure, and season. GLM analysis predicted a higher number of SHBs in colonies with fewer combs covered by adult bees (2.543), with a greater number of combs (1.877), with lower comb surveillance (0.935), with a higher SHB infestation level in the previous month (1.192), in shaded locations compared to sunny ones (0.207), and in autumn compared to late winter (0.258), with peak infestations in September. These findings provide insights to inform surveillance plans, optimise sentinel apiaries setup in SHB-free regions, and offer practical guidance for beekeepers on implementing biosecurity measures to minimise infestation levels and enhance early detection. Future research should examine whether these factors have similar effects in regions with higher SHB infestation rates.

1. Introduction

Aethina tumida Murray, commonly known as the small hive beetle, is a pest that poses a significant threat to honey bee colonies. Native to sub-Saharan Africa, this invasive species [1] is the causative agent of aethinosis, a notifiable honey bee disease at colony-level as classified by the World Organisation for Animal Health [2]. Adult SHBs are dark-coloured, flattened beetles measuring approximately 5 mm in length and 3 mm in width [2]. They are attracted by volatile substances emitted by honey hives [3], which they choose as a preferred site for feeding, oviposition, and completing their life cycle [3,4]. Both larval and adult stages of SHBs feed on honey, pollen, brood, and hive debris, causing varying degrees of damage to honey bee colonies. Severe infestations can lead to colony collapse or absconding [1,5,6,7]. SHB larvae burrow through combs and contaminate honey with their faeces, leading to honey fermentation [8,9]. European honey bee subspecies are generally more susceptible to SHB infestations than their African counterparts, primarily due to differences in hygiene behaviours [7]. Aethinosis poses significant economic challenges to the beekeeping industry, not only through direct damage to colonies, but also due to indirect costs associated with restrictive veterinary policy measures that need to be implemented in previously SHB-free regions [10]. In Italy, the SHB was first detected in 2014 in Gioia Tauro, in the southern province of Reggio Calabria [8], where it subsequently became established. Models of potential SHB dispersal, considering both local spread between apiaries and long-distance transportation, identified the Port of Gioia Tauro as the likely origin of the Italian introduction [11]. When the SHB was also detected in Sicily that same year, rapid and effective eradication measures implemented by the Ministry of Health successfully prevented the beetle from establishing a permanent population on the island [9] until October 2024, when it was detected again [12]. Although a decade has passed since its initial introduction, SHB distribution in Italy remains confined to Calabria and Sicily, representing the sole presence of the beetle in Europe. In areas affected by the SHB in Italy, unlike what has occurred in other countries, the SHB does not pose a threat to the survival of the beekeeping sector. This is likely due, in part, to the climatic and environmental conditions of Southern Italy, as well as the widespread adoption of traps and good beekeeping practices by beekeepers operating in the Italian regions impacted by the SHB [13]. Current surveillance efforts in the above-mentioned Italian regions include specific sanitary measures coordinated by the Ministry of Health and local veterinary public health authorities. These measures include active and passive monitoring of the beetle’s presence, restrictions on the movement of bees and beekeeping equipment, destruction of heavily infested hives, and training for beekeepers. Active monitoring involves the inspection of hives and the deployment of sentinel apiaries, which ensure early detection of the pest. These apiaries are strategically located in “at-risk” areas, such as regions still free from the SHB, but close to endemic zones, areas near ports or airports, or locations with a high density of apiaries. In 2022, the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana (Rome, Central Italy) conducted a citizen science study in collaboration with the Calabrian Beekeepers Association (FAI Calabria). This study investigated several factors potentially influencing SHB infestation levels in colonies, aiming to optimise existing management strategies. These include the adoption of specific biosecurity measures in beekeeping (BMBs) [14] against the SHB, and the proper placement and setup of sentinel apiaries to enhance detection of the beetle. To understand factors influencing SHB infestation, we conducted a study examining its association with nine environmental and colony-related variables. We specifically investigated whether stronger colonies exhibit a greater ability to resist the SHB and maintain lower infestation levels, and if brood or stored resources contribute to increased SHB attraction and infestation. We also explored the potential for queenless colonies to be less susceptible to the SHB due to the more aggressive behavior of workers in such conditions. Moreover, we aimed to determine if the climatic conditions found to favor SHB development in the laboratory are also relevant in the field across different seasons.

2. Materials and Methods

2.1. Study Design

In 2022, we conducted a study in the province of Reggio Calabria (Italy), where the small hive beetle (SHB) is endemic, to investigate environmental and honey bee colony factors associated with SHB infestation levels. We adopted a citizen science methodology [15,16], selecting six beekeepers from the local beekeeping association and training them on standardised protocols for hive inspection, data collection, and form completion to assess SHB presence. Before the beginning of this study, each beekeeper provided their National Beekeeping Register ID, a detailed map of their apiary, the apiary’s address, and its GIS coordinates. Beekeepers inspected their colonies over three consecutive weekends, spaced three weeks apart, during late winter (21–23 January, 11–13 February, and 4–6 March) and autumn (30 September–2 October, 21–23 October, and 4–6 November) of 2022. Autumn and late winter were selected as the inspection periods based on the empirical observations of participating beekeepers, who have been engaged in the management of SHB infestations for approximately a decade. According to their empirical experience, these seasons correspond to the highest infestation levels. The Calabria region exhibits higher average temperatures than other Italian regions throughout the year, including during the autumn and winter months. This climatic pattern is attributable to its geographical position in southern Italy and the mitigating influence of the Mediterranean Sea. The presence of the SHB was assessed during 431 hive inspections. During late winter, 67 hives across six apiaries were inspected three times each, totalling 197 inspections. In autumn, 81 hives from five apiaries were inspected three times each, totalling 234 inspections. During the autumn phase, all colonies from the late-winter phase were re-examined, except for Apiary 1, which was included only in the late-winter phase. Beekeepers carried out these inspections using the mobile divider method [17] and recorded the following contingent aspects for each hive: date, SHB infestation levels (number of adults and larvae per hive), sun exposure, queen presence, and comb characteristics (total number of combs, number of combs covered by adult bees, number of combs containing brood, and number of storage combs). Firstly, beekeepers evaluated the number of combs covered by adult bees and containing brood and storage, following the EurBeST protocol [18]. Colonies were opened from above and the number of combs occupied by bees was estimated without the use of smoke. A comb was considered occupied by adult bees if at least 70% of its surface was covered. Furthermore, each comb was inspected to determine whether it contained brood or stored resources. The number of SHBs detected inside the hive was evaluated as the number of SHBs present both behind the mobile divider device and in the remaining volume of the hive.

2.2. Statistical Analysis

Statistical analyses were performed using SPSS (IBM SPSS Statistics, version 27.0.1.0) and Microsoft Excel (Microsoft Office 2007). Data normality was assessed using Kolmogorov–Smirnov and Shapiro–Wilk tests. We assessed the dependent variable, the number of SHBs detected in the total volume of the hive throughout the study period. We evaluated the average number of SHBs/hive in each month to find the month and the season with higher infestation levels. We also evaluated associations between the SHB infestation level (number of SHBs/hive) and nine environmental and colony-related variables: SHB infestation level in the previous month, season, sun exposure, queen presence, and comb characteristics (total number of combs, number of combs covered by adult bees, number of combs containing brood, number of storage combs, and combs surveillance). Comb surveillance by adult bees was introduced as a secondary factor and defined as the ratio between the number of combs covered by adult bees and the total number of combs in the hive. This factor was classified as partial if not all combs were fully covered by adult bees, and as total if all combs were completely covered. The presence of at least one comb not occupied by adult bees allowed the definition of the surveillance as partial. Generalised linear models (GLMs) were employed to investigate factors showing significant relationships in chi-square tests. GLM analyses were used to identify underlying trends and assess effects on multiple variables concurrently, while accounting for potential interactions beyond main effects.

3. Results

No severe outbreaks (massive aethinosis) or the presence of SHB larvae were observed during the study period. A total of 379 adult SHBs were detected and killed. The average number of adult SHBs per hive across all inspections throughout the experiment was 0.83 (almost one adult per colony) (

Table 1. Number of hive inspections during each check, and the total. Total and average numbers of SHBs detected in apiaries monitored during late winter and autumn. The six apiaries selected for the late-winter phase included all apiaries involved in the autumn phase, except Apiary 1, which was included only in the late-winter phase.

Apiary	Season	Number of Hive Inspections/Apiary During the First Control	Number of Hive Inspections/Apiary During the Second Control	Number of Hive Inspections/Apiary During the Third Control	Total Number of Hive Inspections/Apiary	Total Number of SHBs Detected in the Whole Apiary	Average Number (SHBs/Hive Inspection)
Apiary 1	Late winter	8	8	8	24	11	0.46
	Autumn	NA	NA	NA	NA	NA	NA
Apiary 2	Late winter	26	26	26	78	18	0.23
	Autumn	36	36	33	105	245	2.33
Apiary 3	Late winter	10	8	8	26	16	0.62
	Autumn	10	10	10	30	3	0.10
Apiary 4	Late winter	7	7	7	21	20	0.95
	Autumn	19	17	16	52	8	0.15
Apiary 5	Late winter	6	6	6	18	3	0.17
	Autumn	8	8	7	23	29	1.26
Apiary 6	Late winter	10	10	10	30	13	0.43
	Autumn	8	8	8	24	13	0.54
Total Apiaries	Both seasons	148	144	139	431	379	0.83

).

The dependent variable, the SHB number per hive, was not normally distributed and presented a positive asymmetric distribution with high variability (Kolmogorov–Smirnov test: Statistic = 0.328, d.f. = 431. sign. ≃ 0.000; Shapiro–Wilk test: Statistic = 0.568, d.f. = 431. sign. ≃ 0.000) (Figure 1).

Table 2. Descriptive statistics of variables investigated.

	Minimum in Each Colony	Maximum in Each Colony	Average (M)	Standard Deviation (SD)	Coefficient of Variation (CV = SD/M)
Total number of SHBs	0	12	0.88	1.73	1.96
Total number of combs	2	10	5.74	1.72	0.30
Number of combs covered by adult bees	1	10	5.27	1.66	0.31
Number of combs containing brood	0	7	2.93	1.30	0.44
Number of storage combs	0	10	2.32	1.22	0.53
Exposure (0 = Shadow; 1 = Sun)	0	1	0.74	0.44	0.60
Queen (0 = Orphan; 1 = Queen right)	0	1	0.95	0.21	0.22

presents descriptive statistics for all variables assessed during hive inspections, including total SHBs, sun exposure, queen presence, total number of combs, combs covered by adult bees, combs containing brood, and storage combs.

During the entire study period, the queen was found to be absent in only 20 out of 431 hive inspections (0.05%). The average number of SHBs per hive in the two seasons was higher in autumn (SHBs/hive = 1.26), peaking in September (SHBs/hive = 1.80) compared to late winter (SHBs/hive = 0.41).

Table 3. Average number of SHBs found per hive in different months and seasons.

Calendar Months	Late Winter			Autumn
	Jan	Feb	Mar	Sep	Oct	Nov	Average
Average number of SHBs/hive	0.46	0.28	0.49	1.80	1.16	0.81	0.83
	0.41			1.26

presents the combined average number of SHBs across all apiaries for each month and both seasons throughout the study period.

GLM analysis was conducted exclusively on factors found to be significantly associated with SHB infestation levels in the chi-squared test: the previous month’s infestation, season, total number of combs, number of combs covered by adult bees, comb surveillance, and sun exposure. The use of GLM was necessary because the dependent variable (number of SHBs/hive) had a non-normal distribution, and to create SHB forecasts contextual to multiple combinations of modalities assumed by explanatory variables. We found an acceptance range for the estimated parameters (“95% Confidence Interval”). The fit of the obtained model, estimated by the R² value, was not always very good (0.3 < R² ≤ 0.72 in most cases). However, this is attributable to the high variability in the number of SHBs/hive and the number of explanatory variables included in the model. To simplify the model, we decided to use the direct effects of the factors, limiting interactions to only some cases, which yielded a quite satisfactory result, with R² = 0.649. The GLM model was able to predict higher SHB infestation levels (1.192 units) when the SHB infestation level of the previous month was higher than average (SHBs/hive > 1). Furthermore, the model predicted a higher presence of SHBs (0.344 units) even when the previous month’s infestation level was average (SHB/hive = 1). The GLM analysis predicted a higher number of SHBs/hive associated with fewer combs covered by adult bees (2.543), a greater number of combs (1.877), lower comb surveillance (0.935), in autumn compared to late winter (0.258), and in shaded locations compared to sunny locations (0.207) (

Table 4. Summary of the dependence between the SHB infestation level and six factors investigated in the present study.

Factors to Be Compared with the SHB Infestation Level	∆-GLM Extremes Impact
SHB infestation level in the previous month	±1.192
Seasons	±0.258
Total number of combs	±1.877
Number of combs covered by adult bees	±2.543
Combs surveillance	±0.935
Exposure to sunlight	±0.207

).

4. Discussion

This study investigated factors influencing SHB infestation levels in apiaries within the endemic area of Calabria. The SHB infestation levels detected in Calabria were low, with an absence of larvae and an average number of 0.83 SHBs per hive throughout the experiment, contrasting with patterns observed elsewhere [19]. The absence of larvae observed in our study is consistent with the low SHB infestation level encountered in the apiaries under investigation. The limited adult SHB population consequently resulted in a reduced number of ovipositing females, thereby diminishing the potential for larval hatching. Furthermore, a low quantity of eggs or larvae within the bee colonies can be readily managed by the bees’ hygienic behavior, enabling robust colonies to effectively remove them prior to their development [20]. The number of autumnal inspections (n = 234) in our study was higher than late-winter inspections (n = 197) due to the inclusion of additional hives to increase the sample size. Our study revealed a clear seasonal pattern of SHB infestations, with a significantly higher prevalence in autumn compared to late winter, which peaked in September. This variation may be attributed to SHB life cycle requirements, as larvae require humidity and elevated temperatures to complete their life cycle [7], and eggs hatch optimally at higher temperatures [6,21]. These conditions appear more favourable in Calabria during autumn than in late winter. Additionally, an increase in food resources after the main honey harvest may create an environment conducive to SHB reproduction. SHBs are also known to be more active during summer, potentially producing up to five generations [6] before retreating to the hive as temperatures decrease [22]. Specific environmental conditions during the autumn months may play a role in the increased infestation rates observed and should be investigated further. A significant relationship was identified between adult SHB infestation levels detected in a given month and the preceding month’s infestation, indicating that an elevated presence of SHBs in a month is associated with a higher infestation level in the following month. This pattern could be attributed to the attraction of adult SHBs to volatile compounds produced by the interaction of the yeast Kodamaea ohmeri with pollen within the hive [23,24]. This yeast, introduced into the hive by all SHB life stages, causes fermentation [25,26] and increases ethanol levels, enhancing the attractiveness of hive products for SHBs [27,28]. However, because larval faeces are the primary drivers of fermentation [29] and larvae were absent in our study, the observed attraction likely stemmed from other factors, such as the beetle’s sex pheromones. In Coleoptera, sex pheromones generally attract both sexes, not just the opposite sex, thereby increasing the number of beetles drawn to the hive [30]. This creates a positive feedback loop that amplifies the likelihood of infestation. We found a high positive correlation between SHB infestation levels and the total number of combs in a hive. Conversely, infestation levels decreased as the number of combs covered by adult bees increased. Hives with more combs, but fewer combs covered by adult bees, provide SHBs with more undisturbed spaces to lay eggs and inhabit the hive, as a greater number of honey and pollen cells remain available to supply the SHBs, uncontrolled by the bees [31]. Conversely, a high number of adult bees covering the combs could hinder the visual detection of SHBs, potentially leading to an underestimation of the actual infestation levels. Partial surveillance, where at least one comb was not covered by adult bees, increased SHB infestation levels, suggesting that incomplete bee surveillance facilitates SHB proliferation. Although SHBs are attracted to worker bee pheromones, such as isopentyl acetate (an Apis mellifera alarm pheromone) [28,32], the presence of a high number of frames covered by adult bees is favourable for a low level of infestation, as adult bees easily detect and attack beetles [33]. This finding suggests that a larger adult bee population, typical of stronger colonies, is more effective at defending the hive against SHB infestations and reducing colony damage [5,6]. As confirmed by previous studies [5,6,34], exposure to sunlight was associated with lower SHB infestation, showing SHBs’ preference for shaded environments and suggesting that strategically placing hives in sunny areas may effectively reduce the SHB infestation risk [35]. SHB infestation levels were not correlated with the number of combs containing brood or storage combs. Evidence suggests that SHBs are not initially drawn to fresh honey or pollen, but fermentation renders them attractive [19,32]. Finally, no significant impact of queen presence on SHB infestation levels was observed, likely due to the limited number of queenless hives (0.05% of all inspections). A review of the existing literature also revealed no prior evidence of a relationship between queen presence and SHB infestation levels.

5. Conclusions

Our study found that while SHB infestations in Calabria are generally limited, they have a higher incidence in autumn, peaking in September, and are influenced by environmental and colony-related factors. Environmental variables like shading increase vulnerability to SHBs, underlining the importance of positioning sentinel apiaries for surveillance activities in shaded areas. Colony-related factors like the previous month’s infestation level, a high number of combs in the colony, and a low number of combs covered by adult bees (consequently a low surveillance) increase this risk as well. These outcomes permit better planning and conducting of inspections, which are crucial for effective SHB management in Calabria and Sicily. Further research is needed to investigate the specific factors contributing to the observed seasonal variation in SHB populations, including potential relationships with environmental variables such as temperature and humidity. Future studies should also explore whether these environmental and colony-related variables have similar influences on SHB infestation levels in regions with higher infestation rates. Additionally, the impact of specific beekeeping practices, such as hive management strategies, the use of traps, and the application of integrated pest management (IPM) strategies, on SHB infestation levels should be further explored.