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Article

Effect of the Combined Use of Postbiotics and Oxalic Acid Against Varroa destructor Under Field Conditions

by
Eduardo José García-Vicente
1,2,
María Benito-Murcia
1,2,*,
María Martín
2,3,
Ana Pérez
1,2,
Noelia Hermosilla
2,
Salomé Martínez-Morcillo
3,
Juan Manuel Alonso
3,* and
David Risco
1
1
Department of Animal Medicine, Faculty of Veterinary, University of Extremadura, 10003 Cáceres, Spain
2
Neobéitar S.L. Av. de Alemania, 6 1ºB, 10001 Cáceres, Spain
3
Department of Animal Health, Faculty of Veterinary, University of Extremadura, 10003 Cáceres, Spain
*
Authors to whom correspondence should be addressed.
Agriculture 2025, 15(12), 1292; https://doi.org/10.3390/agriculture15121292
Submission received: 6 May 2025 / Revised: 13 June 2025 / Accepted: 13 June 2025 / Published: 15 June 2025
(This article belongs to the Special Issue Recent Advances in Bee Rearing and Production)
Browse Figures
Versions Notes

Abstract

Varroa destructor is a parasitic mite of honeybees that is considered one of the main problems in beekeeping today. The reduction in the efficacy of conventional miticides and the presence of residues in beehives highlight the need to investigate new organic products as alternative treatments. Postbiotic products have been shown to decrease mite viability in in vitro experiments. However, their use in beehives has not yet been assessed. The aim of this study was to test the efficacy of postbiotics and oxalic acid against V. destructor under field conditions. Two experiments were performed during the summer and autumn seasons, with four study groups: control (C), postbiotic (POS), oxalic acid (OX), and both combined (POX). Phoretic and brood V. destructor were determined at the beginning and end of the study to assess their evolution, as well as the percentage effectiveness of each treatment by registering the mite fall in beehive sanitary bottoms. Postbiotic alone did not show a significant effect on V. destructor under field conditions. However, the combined treatment of postbiotics and oxalic acid improved the results obtained with oxalic acid alone, resulting in greater effectiveness and reduction of phoretic and brood V. destructor.

Graphical Abstract

1. Introduction

Varroa destructor is a parasitic mite of honeybees (Apis mellifera) that is distributed worldwide [1,2]. It is considered one of the main problems for beekeeping today, causing great production and economic losses for beekeepers [3]. Adult females feed on the fat body and hemolymph of adult honeybees, and this stage of the V. destructor life cycle is traditionally known as the phoretic phase [4,5]. However, this term is not the most accurate because the mite not only uses adult bees for transport, and dispersal phase is proposed as a more accurate term [2]. When honeybee larvae are ready to pass to pupal period, they emit hormonal signals so that the worker bees seal the cell. These signals are recognized by female mites, which access the cell to breed [6,7,8]. Infestations by V. destructor in a colony can cause several injuries to bees, such as improper development, malformations, weakness, weight loss, and, ultimately, colony collapse [7,9]. Furthermore, V. destructor can act as a vector for some bee viruses, such as Deformed Wing Virus (DWV) [10] or Acute Bee Paralysis Virus (ABPV) [11], and even act in synergy with them [12,13].
The control of V. destructor in honeybee colonies can become very complex due to different factors. First, the fact that mites reproduce inside capped brood cells limits or even prevents their exposure to most treatments [8,14]. Moreover, climate change is influencing the population dynamics of V. destructor [8,15]. Thus, one consequence of climate change is fewer cold winters, without the interruption of queen egg laying during the cold season. This makes the management of V. destructor infestation more difficult, since the constant presence of brood allows greater reproduction of the mites, and decreases the efficiency of treatments [15,16,17,18].
Second, the effectiveness of conventional synthetic miticides traditionally used in beekeeping such as coumaphos, tau-fluvalinate and amitraz is decreasing owing to the appearance of resistant populations of mites [19,20,21,22,23]. Additionally, these types of compounds are capable of generating residues in beehives, with the consequent risk to human and honeybee health due to chronic exposure to these pesticides [24,25,26,27,28,29].
Other treatments based on organic compounds, such as oxalic acid, formic acid or essential oils from plants (thymol, oregano) are commonly used in rotation with synthetic miticides [8]. The development of resistant populations due to the use of these compounds has not been described, but their use is not free from issues. Most of them have very narrow application temperature ranges, and outside of them, these compounds can greatly decrease their effectiveness or increase their toxic effects and mortality in honeybees [30,31,32,33,34]. Moreover, the use of combined management practices are recommended to ensure the efficacy of all these synthetic and organic compounds, including application in brood absence or the use of drone brood frames and subsequent removal, but this is not always possible [14]. In Spain, and more specifically in the Extremadura region, has a strong beekeeping tradition, with almost 2000 beekeeping operations and more than 600,000 beehives registered in 2023, which makes an average of 300 beehives per operation [35]. This large number of beehives and the normal management required (transhumance, honey/pollen harvest, reproduction, feeding) make it very difficult to apply some of the management practices against V. destructor previously described.
For all these reasons, it is necessary to investigate and find alternative products of organic origin that do not generate resistant populations or residues and are effective against the mite and/or enhance the efficacy of other compounds. Some experiments have studied the in vitro effect of bioactive compounds such as probiotics and postbiotics based on lactic acid bacteria against V. destructor, obtaining very promising results [36,37]. In fact, the experiment prior to the current study showed that lactic acid bacteria isolated from natural microbiota of honeybees, in postbiotic format, decreased the viability of the mites, and the best results were obtained when it was applied combined with oxalic acid, with a greater effect than each compound separately [38]. However, the effect of such postbiotic under field conditions remains unknown.
The aim of this study is to continue with the previous in vitro experiment and evaluate the effectiveness of postbiotics against V. destructor under field conditions, both alone and in combination with oxalic acid.

2. Materials and Methods

2.1. Elaboration of Treatments

The postbiotic product was prepared for the different treatments as previously described [38]. Three species of lactic acid bacteria (isolated from natural microbiota of honeybees) were cultured in liquid MRS medium (Scharlab, Barcelona, Spain) at 37 °C for 48 h. Their concentration was adjusted to 109 CFU/mL, and they were mixed in equal proportions and inactivated at 80 °C for 1 h. The absence of live bacteria was studied by culturing them in MRS medium.
Four different treatments were prepared for the experiments, with a format and concentration similar to the commercial product based on oxalic acid Aluén CAP®, with some modifications. Briefly, cellulose strips of 400 × 25 × 1 mm were impregnated with four different solutions, depending on the treatment to be tested: 15 mL of glycerin (Quimipur S.L.U., Madrid, Spain) for control group; 10 mL of glycerin +5 g of oxalic acid 99.6% (Oxaquim S.A., Tarragona, Spain); 10 mL of glycerin +5 mL of postbiotic product; and 5 mL of glycerin +5 g of oxalic acid +5 mL of postbiotic for the combination of both products (quantities per strip). For this, glycerin was heated to 65 °C and mixed with the rest of additives for each treatment. Then, the strips were submerged in this solution and kept in contact with the products for one day in darkness at room temperature conditions (20–25 °C), and in a closed container, to ensure that the strips were correctly impregnated.

2.2. Experimental Design

Four study groups were established: control (C); oxalic acid (OX); postbiotic product (POS); oxalic acid and postbiotic combination (POX). The beehives included in the study (Layens type; 20 per group for the summer experiment and 10 per group for the autumn experiment) were in an experimental apiary in Cáceres province (Extremadura, Spain; LT: 39.4745175, LN: −6.3716761). This region is characterized by a Mediterranean climate, with warm and dry summers, and cold winters. The temperature varies between 1 °C and 34 °C, and the rainiest time is early autumn, with an average of 60 mm of rain [39].
The first monitoring of the beehives (M0) was conducted on day 0, when the Phoretic V. destructor (PVD) or infestation rate in adult bees was determined. For this, one sample of approximately 300 worker bees from three different locations inside the beehive was collected, taken to laboratory and preserved at −20 °C. This measure was used to distribute the beehives homogenously into four study groups, based on their infestation levels. At day 1, two fragments of frames with approximately 100 capped cells (20.25 cm2) on both sides were extracted from each beehive to evaluate Brood V. destructor (BVD) or the infestation rates inside the brood cells. Treatments were then applied to the colonies depending on their group. Thus, eight of the previously elaborated strips were placed in the inter-frame spaces with higher activity of bees and amount of brood, two per inter-frame space. PVC sheets were placed at the sanitary bottom of the beehives to determine mite fall during the experiment. Sheets were taken to the laboratory, and the number of mites was counted and recorded every two–three days, and a new sheet was placed in the beehives. The sheets were impregnated with Vaseline before their application to beehives to ensure fixation of fallen mites [40].
Two weeks after the treatments application, a second monitoring (M1) was carried out. In this case, in addition to determining the PVD and BVD, the number of worker bees and amount of brood in the beehives were estimated, using a previously described protocol [41]. Thus, these parameters were measured by calculating the percentage of frame surface occupied by each one, obtaining the number of adult bees and capped brood cells per beehive. Samples of PVD and BVD were collected at this point following the protocol previously described for M0.
After M1, amitraz treatment was applied to each beehive using the product Amicel varroa (Maymó, Barcelona, Spain). For this, the capped brood was uncapped with a special spiked roller to ensure the contact between the treatment and the entire mite population in the beehive, and amitraz was administered following the instructions of the manufacturer. For approximately two more weeks the fall of mites was determined following the same protocol with PVC sheets described for experimental treatments, until the falls were less than one mite per day.
Two different experiments were conducted: one at the beginning of summer (28 May–26 June) and one at the beginning of autumn (18 September–17 October) of 2024, both using the same experimental design to assess the effect of the experimental treatments in two different scenarios.

2.3. Assessing the Effect of Treatments

In order to evaluate the effect of the different treatments against mites, four parameters were analyzed during the experiment: Phoretic V. destructor, Brood V. destructor, Effectiveness with amitraz treatment (Ef A), and Effectiveness without using amitraz (Ef B).
  • Phoretic V. destructor (PVD): Worker bees collected at M0 and M1, were immersed in a 5% ethanol solution and shaken for 5 min, after which they were passed through a sieve to count the total number of bees and mites [42]. Thus, the phoretic infestation level was determined for each beehive and monitored and expressed as the number of mites per 100 bees or percentage of phoretic infestation. Moreover, the evolution of this parameter was calculated between M0 and M1 for each colony as the Variation (Δ) of PVD.
  • Brood V. destructor (BVD): Using the extracted fragments of the frames with capped brood at both monitoring times, the number of brood cells was recorded and the pupae and V. destructor mites were extracted from cells and counted (adult females and immature mites), to determine the level of infestation in brood or number of mites per 100 capped brood cells [43,44]. Moreover, the evolution of this parameter was calculated between M0 and M1 for each colony as the variation (Δ) of BVD.
  • Effectiveness with amitraz treatment (Ef A): To check the capability of the different products to reduce the mite population in the beehives, the efficacy of each treatment was calculated using the fall of mites in the sanitary bottoms during the treatment period and later during amitraz treatments, assuming that amitraz made all of the mites fall, providing an estimation of the total V. destructor population that would be in the beehive after the experimental treatments [40]. Thus, the formula used to calculate the effectiveness was:
E f f e c t i v e n e s s   A = E x p e r i m e n t a l   t r e a t m e n t   f a l l E x p e r i m e n t a l   t r e a t m e n t   f a l l + A m i t r a z   f a l l × 100
Experimental treatment fall = number of mites fallen in sanitary bottom during experimental treatments. Amitraz fall = number of mites fallen in sanitary bottom during amitraz treatments.
This is the traditional formula used to calculate the effectiveness of an experimental treatment against V. destructor under field conditions. It assumes that amitraz efficacy is 100%, but mostly available amitraz-based products show lower efficacies depending on the amount of brood in the beehive and the presence of resistant mite populations [20]. For this reason, a second measurement of effectiveness was calculated to provide accurate results.
  • Effectiveness without using amitraz (Ef B): Due to the recent records of resistant mite populations against amitraz [20,21], an alternative method based on the estimation of the total number of mites in the beehives after the experimental treatments was also conducted. For that, with the estimation of the number of adult bees and capped brood cells obtained from monitoring 1, as well as the infestation levels of PVD and BVD determined at the same point, the total mite population that would be in the beehive after experimental treatments was estimated, thus replacing the fall data with amitraz in the formula as follows:
T o t a l   P h o r e t i c   M i t e s   T P M = P V D 100 × N B
T o t a l   B r o o d   M i t e s   T B M = B V D 100 × N C
E f f e c t i v e n e s s   B = E x p e r i m e n t a l   t r e a t m e n t   f a l l E x p e r i m e n t a l   t r e a t m e n t   f a l l + T P M + T B M × 100
PVD = percentage of phoretic mite infestation measured in M1.
NB = estimation of the number of adult bees measured at M1.
BVD = percentage of mite infestation in capped brood cells measured at M1.
NC = estimation of the number of capped brood cells measured at M1.
Experimental treatment fall = number of mites fallen in sanitary bottom during experimental treatments.

2.4. Data Analyses

The strength parameters (number of bees and amount of brood at M1) and Phoretic and Brood V. destructor infestation levels at M0 were used to carry out mean comparisons among the experiments to identify whether summer and autumn experiments were performed under different scenarios or similar conditions for the beehives. Parametric statistical tests (Student’s t-test (T)) were used when the variables showed a normal distribution. Otherwise, nonparametric statistical tests (Wilcoxon test (V)) were performed.
The obtained variables relative to the levels of infestation and efficacy of the treatments were used to perform mean comparisons among study groups, using parametric (one-way ANOVA (F)) or nonparametric tests (Kruskal–Wallis test (K)) and, in either case, post-hoc tests were applied (Tukey’s HSD test or pairwise Wilcoxon test, using the “Bonferroni” adjustment method).
Furthermore, for variables with data from M0 and M1 (PVD and BVD), intra-group mean comparisons were performed using parametric or nonparametric statistical tests (paired Student’s t-test (T) or paired Wilcoxon test (V)), depending on the normality of the variables. To compare both techniques used to determine the efficacy of the studied treatments, Ef A and Ef B, correlation tests between both techniques were carried out, using Pearson’s correlation test (cor) or Spearman’s correlation test (rho), depending on the normal or non-normal distribution of the variable, as well as mean comparisons for each group in both summer and autumn experiments. Finally, mean comparisons of Δ of PVD and BVD, as well as Ef A and Ef B were performed between summer and autumn experiments to determine the possible influence of the seasonality, using the previously described statistical tests.
Beehives with V. destructor loads higher than 3000 mites and lower than 300 mites were excluded from statistical analyses (determined based on infestation levels and mite falls) [40]. Statistical analyses were performed using R v.4.4.1. Statistical significance was set at (p-values) < 0.05 and between 0.05 and 0.1 marginally significant.

3. Results

A total of 23 and 12 beehives were excluded from the analyses in the summer and autumn experiments, respectively, because they did not meet the criteria for the number of mites in the beehives, with a mite load above 3000 or below 300. Thus, in the summer experiment, the final sample sizes per group were as follows: 14 beehives in C, 12 in OX, 14 in POS, and 17 in POX. The final sample size for the autumn experiment was seven beehives in C, six in OX, nine in POS, and six in POX.

3.1. Infestation Levels

The levels of infestation by V. destructor at M0 and M1 for each study group are included in Table 1, as well as their variations between the two monitoring periods, for summer and autumn experiments. Generally, the changes in brood infestation levels in the experiment were more noticeable than those in PVD.
In the summer experiment, treatments that included oxalic acid showed a trend of decreasing both PVD and BVD levels, which was more noticeable in POX, while the C and POS groups showed the opposite trend. A marginally significant increase in PVD between M0 and M1 was detected in POS (V = 129; p-value = 0.059), and this group showed marginally higher infestation than the POX treatment for this parameter at the end of the experiment (K = 8.34; p-value = 0.092). Regarding BVD, the C group showed a significant increase in infestation level (V = 36; p-value = 0.008). Variations between M0 and M1 were not statistically significant between the groups (Figure 1).
Regarding intra-group comparisons in autumn, the same trends as those in summer were recorded, except for the OX treatment, which significantly increased its levels of PVD throughout the experiment (T = 7.16; p-value = 1.8 × 10−5), while there was a statistically significant reduction in BVD (V = 72; p-value = 0.007). Thus, C and POS significantly increased the PVD (T = 4.08; p-value = 0.001/T = 6.31; p-value = 7.8 × 10−6, respectively) and BVD (V = 105; p-value = 1.2 × 10−4/T = 7.46; p-value = 9.3 × 10−7, respectively), and POX significantly decreased PVD (V = 71; p-value = 0.009) and BVD (V = 76; p-value = 0.001) through the experiment.
In this case, regarding comparisons among groups, POX treatment resulted in significantly lower PVD than POS at the end of the experiment (K = 9.54; p-value = 0.017). For BVD, OX showed significantly lower infestation than C (K = 17.89; p-value = 0.028) and POS (K = 17.89; p-value = 0.005) at M1, like POX regarding the C (K = 17.89; p-value = 0.084) and POS (K = 17.89; p-value = 0.005). Moreover, the Δ of BVD through the experiment indicated statistically significant differences between the groups that reduced their mite infestation, OX (F = 4.06; p-value = 0.037) and POX (F = 4.06; p-value = 0.083), and C group, which notably increased it (Figure 1).

3.2. Effectiveness

On the other hand, Table 2 shows the mean values for both effectiveness measurements by groups and experiments, Ef A and Ef B.
Firstly, in summer, the treatments were grouped based on their effectiveness into highly effective (OX/POX) and lowly effective (C/POS) treatments (Figure 2). Thus, for Ef A, the C group showed statistically significant differences with OX (K = 37.7; p-value = 6.2 × 10−4) and POX (K = 37.7; p-value = 6.1 × 10−4), as well as POS showed them with both OX (K = 37.7; p-value = 1 × 10−4) and POX (K = 37.7; p-value = 9.1 × 10−8). Regarding Ef B, only POX (87.59%) showed a marginally significant difference regarding C (17.54%) (K = 12.52; p-value = 0.084).
Secondly, Ef A was similar for all the treatments in autumn, including the C group, and did not show statistical differences among them. However, regarding Ef B, the POX treatment showed a significantly higher efficacy than C (K = 15.56; p-value = 0.007) and POS (K = 15.56; p-value = 0.002) (Figure 2).

3.3. Techniques of Effectiveness Measurements

To compare both techniques used to determine the efficacy of the studied treatments, Ef A and Ef B, correlations between them were detected for summer (rho = 0.66; p-value = 0.003) and autumn (rho = 0.616; p-value = 6.32 × 10−4) experiments, as well as in a global analysis (rho = 0.667; p-value = 4.26 × 10−7). Regarding mean comparisons by groups, Ef A showed significantly higher efficacy than Ef B in C and POS groups, both in summer (C: T = 2.99; p-value = 0.018/POS: T = 3.44; p-value = 0.009) and in autumn (C: T = 3.39; p-value = 0.01/POS: T = 3.57; p-value = 0.004). However, OX and POX treatments showed similar values for both effectiveness measures and in both experiments, except for POX in summer, where Ef B was significantly higher than Ef A (T = −2.44; p-value = 0.041).

3.4. Seasonality of Experiments

The mean values of strength parameters (bees and brood) and V. destructor infestation levels at M0 of both experiments are summarized in Table 3. All the parameters were statistically different between the summer and autumn experiments, with a higher number of bees (W = 462; p-value = 0.002) and amount of brood (W = 415; p-value = 3.38 × 10−4) in summer, as well as less infestation by V. destructor in this experiment (phoretic: W = 1479; p-value = 3.83 × 10−11/brood: W = 443; p-value = 1.8 × 10−5).
Finally, the comparisons between summer and autumn experiments showed a marginally higher Ef B in the summer experiment (W = 171; p-value = 0.07), as well as for the POX treatment (T = −2.98; p-value = 0.017). However, the OX group showed a significantly higher reduction in BVD in autumn (W = 1; p-value = 0.014), and a significant increase in Ef A and Ef B was recorded in the POS group (T = 3.06; p-value = 0.011; T = 2.14; p-value = 0.064, respectively). This information is provided on Supplementary Table S1.

4. Discussion

To the best of our knowledge, this is the first experiment carried out using postbiotics as a measure to control V. destructor in beehives under field conditions, as well as in combination with oxalic acid. The results obtained in this study showed that the application of postbiotics through cellulose strips did not seem to produce effects against the mite but enhanced the action of oxalic acid when both were applied together.
In the first place, the differences in strength and levels of V. destructor infestation between the two experiments, as well as the obvious differences in weather conditions, underline the fact that summer and autumn experiments are not replicas but different scenarios. The similar results obtained in both experiments despite the different conditions underline the robustness and repeatability of the results.
Generally, two types of treatments were recorded: those that decreased PVD and BVD and had high Ef A and Ef B, OX, and POX; and C and POS groups, in which infestation levels increased throughout the experiments and had low effectiveness. Thus, treatments including oxalic acid proved to be effective against the mite. This was an expected result because oxalic acid is a usual treatment against V. destructor that is commonly employed in beekeeping and has been previously proven effective [30,31,33]. The recorded efficacies in previous studies show a wide range, approximately between 37% and 99%, but comparisons among the different studies are too complex owing to the use of different doses, application methods, or types of beehives, as well as different weather conditions and seasonality [30,31,45,46,47].
Otherwise, although the postbiotic product was able to reduce the viability of the mites in laboratory bioassays [38], this was not reflected under field conditions. This viability reduction, although statistically significant, was slight compared to that of other products such as oxalic acid, and together with a possible lower exposure of the mites to the product inside the beehive, since the direct contact of mites with the strips is less than in laboratory trials, could be the reason for this lack of effectiveness. Nevertheless, the combined product based on postbiotic and oxalic acid was the best treatment against V. destructor, just like it happened in the previous laboratory bioassays regarding each product separately [38]. POX treatment produced the largest reductions in PVD and BVD in both experiments (except for BVD in autumn, when OX was the most effective treatment), and had significantly better Ef B (87.6%) in summer and Ef A (66.4%) and Ef B (61.8%) in autumn, which was the only group that showed significant differences in Ef B regarding in the C group. This synergistic effect recorded for the combined product may be due to the great reduction in pH by oxalic acid [8,14] combined with the production of lactic acid and other metabolites such as hydrogen peroxide, bacteriocins, or biosurfactants by the lactic acid bacteria from postbiotics [48,49,50]. To obtain the expected effect of the postbiotics by themselves against V. destructor, more studies are being performed with different methods of application (different material strips, drip, or vaporization), and techniques to concentrate the postbiotic product as well as some of the previously mentioned metabolites, to understand the action mechanisms and to increase mite exposure.
Other findings of the study were that despite the positive and significant correlation between both techniques of effectiveness measurement detected, some study groups showed statistically significant differences between Ef A and Ef B. Thus, a relatively high Ef A for the C group was observed in both summer (38.01%) and autumn (53.65%) experiments, despite registering increases in all PVD (summer: +0.37%; autumn: +2.01%) and BVD (summer: +3.32%; autumn: +10.9%) infestation levels through the experiments. Similarly, this occurred for the POS treatment, with 25.26% Ef A in summer, when its PVD increased by 1.2% and its brood infestation level increased by 1.55%, and 49.15% Ef A in autumn, when it increased mite infestation throughout the experiment (Phoretic: +0.68%; Brood: +5.56%). Moreover, both C and POS showed statistically significantly lower Ef B than Ef A in both experiments. The fact that Ef A was calculated using the fallen mites by the subsequent amitraz treatment could be responsible for the abnormally high efficacy results, since the possible presence of resistant populations of V. destructor to this compound could reduce the effectiveness of the aforementioned treatment, thus not eliminating the total of the remaining mites and overestimating the Ef A of all groups, but to a greater extent in both C and POS, as these groups would have larger population of mites after the treatments [19,20,21,22,23]. Ef B showed lower values for both treatments, more similar to those detected in other studies for control groups which varied between 3.5% and 39% [30,31,47]. However, Ef B was significantly higher than Ef A in summer only for POX group. These differences could be a problem when using the technique described in the literature to test new treatments against V. destructor [40], since there are currently no treatments with sufficiently high efficacy and no resistant mite populations [19,20,21,22,23]. The proposed methodology for calculating efficacy by determining infestation levels after experimental treatment and estimating the bee population and the number of brood cells could provide a useful solution to the problem described above. However, this hypothesis needs to be studied by testing the possible resistance of the mites of the beehives included in the experiment.
Regarding comparisons among the different seasons, a generally better Ef B was detected in summer experiment, as well as for POX treatment, increasing its efficacy by approximately 25% in autumn. For the rest of the parameters, variable results were recorded for this group, with higher Ef A in summer too, but with a better reduction of PVD and BVD in autumn, although without significant differences. The OX group showed a greater reduction of BVD in autumn, but this could have been due to a very high initial level of infestation in this experiment. The remaining parameters showed better results in summer and, in fact, PVD decreased in this experiment while it increased during the autumn one for OX. This scenario has been registered previously, with lower efficacy records in autumn, for oxalic acid applied in the beehives by contact strips [30,51]. This may be due to the lower temperatures during this season, resulting in less activity and mobility of the honeybees inside the beehive, with the consequent less contact and spread of the product [30]. Finally, the POS group showed significantly higher Ef A and Ef B in autumn, increasing both measures approximately 50% in this experiment regarding the summer. The fact that this is the first report of the effect of postbiotic products such as treatment against V. destructor under field conditions did not allow comparisons with results of other experiments in different seasons, weather conditions, or application methods. To better understand this increase in the effectiveness of postbiotics in autumn, more studies are needed with a deeper evaluation of the mechanism by which postbiotics interfere with mites and act in synergy with oxalic acid under different conditions.
In summary, the results obtained in this study showed higher reductions in V. destructor infestation levels and effectiveness for the treatments that included oxalic acid, keeping in mind that their application was in the presence of brood in the beehives. While the postbiotic product did not show a clear effect against the mite by itself, its combination with oxalic acid was the treatment with the greatest efficacy, acting in synergy with it and enhancing its effect against V. destructor, especially in summer. Thus, the use of postbiotics could be an important tool to include in the fight against the mite, improving the effectiveness of other organic compounds. The inclusion of postbiotics in normal beekeeping management practices would be an easy-to-implement measure that does not entail high production costs or great labor efforts for beekeepers, improves the results of treatments against the mite, and does not generate residues in the beehive or in the resistant mite populations. Nevertheless, more studies are needed to evaluate the relationship of the postbiotics with other organic treatments and under different field conditions, as well as for a better understanding of postbiotic composition and how it interacts with the mites.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15121292/s1.

Author Contributions

Conceptualization, E.J.G.-V., M.B.-M. and D.R.; methodology, E.J.G.-V., M.B.-M., M.M., A.P., N.H. and S.M.-M.; formal analysis, E.J.G.-V.; writing—original draft preparation, E.J.G.-V.; writing—review and editing, M.B.-M., M.M., A.P., N.H., S.M.-M., J.M.A. and D.R.; supervision, D.R.; project administration, D.R. and J.M.A.; funding acquisition, D.R. and M.B.-M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the “NEOTEC program” [SNEO-20222326] and the Industrial PhD program [DIN2021-012212], financed by the CDTI of the Ministerio de Ciencia, Innovación y Universidades, Gobierno de España.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Acknowledgments

The authors wish to thank María Bravo Santillana and Ingulados S.L. for the advice offered on the management of the bioactive compounds.

Conflicts of Interest

The authors Eduardo J. García-Vicente, María Benito-Murcia, María Martín, Ana Pérez, and Noelia Hermosilla were employed by the company Neobéitar S.L. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CControl group
POSGroup treated with the postbiotic
OXGroup treated with oxalic acid
POXGroup treated with a postbiotic and oxalic acid combination
PVDPhoretic V. destructor
BVDBrood V. destructor
TPMTotal phoretic mites
TBMTotal mites in brood cells
NBNumber of bees
NCNumber of capped brood cells
Ef AEffectiveness A calculated with amitraz treatment fall
Ef BEffectiveness B calculated without amitraz treatment fall

References

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Agriculture 15 01292 g001
Figure 1. Mean Δ of Phoretic and Brood V. destructor for each study group in the summer and autumn experiments. Values that showed significant (p-values < 0.05) or marginal differences (p-values between 0.05 and 0.1) among groups were marked with the opposite group. Error bars show the standard deviations.
Figure 1. Mean Δ of Phoretic and Brood V. destructor for each study group in the summer and autumn experiments. Values that showed significant (p-values < 0.05) or marginal differences (p-values between 0.05 and 0.1) among groups were marked with the opposite group. Error bars show the standard deviations.
Agriculture 15 01292 g001
Agriculture 15 01292 g002
Figure 2. Mean of Effectiveness A (Ef A, using amitraz after experimental treatments) and B (Ef B, without using amitraz). p-values < 0.05 were considered statistically significant, and between 0.05 and 0.1 were considered marginally significant. Values that showed significant or marginal differences among groups were marked with the opposite group. Error bars show the standard deviations.
Figure 2. Mean of Effectiveness A (Ef A, using amitraz after experimental treatments) and B (Ef B, without using amitraz). p-values < 0.05 were considered statistically significant, and between 0.05 and 0.1 were considered marginally significant. Values that showed significant or marginal differences among groups were marked with the opposite group. Error bars show the standard deviations.
Agriculture 15 01292 g002
Table 1. Means of the variables related to levels of infestation by V. destructor determined at M0 and M1, as well as the variation throughout the experiments, in the summer and autumn experiments, with their associated p-values of the inter (“Bonferroni” adjustment method) and intra-group mean comparisons. Statistical significance was set at p-values < 0.05, and between 0.05 and 0.1 were considered marginally significant. Values that showed significant or marginal differences among groups are marked with the opposite group, and values marked with * indicate differences between M0 and M1 in the same study group. This information is provided on Supplementary Table S1.
Table 1. Means of the variables related to levels of infestation by V. destructor determined at M0 and M1, as well as the variation throughout the experiments, in the summer and autumn experiments, with their associated p-values of the inter (“Bonferroni” adjustment method) and intra-group mean comparisons. Statistical significance was set at p-values < 0.05, and between 0.05 and 0.1 were considered marginally significant. Values that showed significant or marginal differences among groups are marked with the opposite group, and values marked with * indicate differences between M0 and M1 in the same study group. This information is provided on Supplementary Table S1.
Summer
Means of Phoretic V. destructorMeans of Brood V. destructor
TreatmentM0M1p-ValueΔM0M1p-ValueΔ
C (N = 14)0.81.170.1190.370.85 *4.17 *0.0083.32
OX (N = 12)0.650.620.812−0.031.951.360.527−0.59
POS (N = 14)0.66 *1.86POX *0.0591.21.853.40.131.55
POX (N = 17)0.970.2POS0.67−0.772.440.840.308−1.6
p-value0.9240.04 0.210.880.16 0.21
Autumn
Means of Phoretic V. destructorMeans of Brood V. destructor
TreatmentM0M1p-ValueΔM0M1p-ValueΔ
C (N = 7)7.07 *9.08 *0.0012.016.81 *17.71OX/POX *1.2 × 10−410.9OX/POX
OX (N = 6)4.49 *4.62 *1.8 × 10−50.1314.95 *2.87C/POS *0.007−12.07C
POS (N = 9)9.65 *10.33POX *7.8 × 10−60.6814.78 *20.34OX/POX *9.3 × 10−75.56
POX (N = 6)4.19 *1.98POS *0.009−2.2112.7 *3.63C/POS *0.001−9.07C
p-value0.210.023 0.5140.2624.6 × 10−4 0.018
Table 2. Mean values of percentage effectiveness A (Ef A, using amitraz after experimental treatments) and B (Ef B, without amitraz). Statistical significance was set at <0.05, and p-values between 0.05 and 0.1 were considered marginally significant. Values that showed significant or marginal differences among the groups were marked with the opposite group.
Table 2. Mean values of percentage effectiveness A (Ef A, using amitraz after experimental treatments) and B (Ef B, without amitraz). Statistical significance was set at <0.05, and p-values between 0.05 and 0.1 were considered marginally significant. Values that showed significant or marginal differences among the groups were marked with the opposite group.
Summer
GroupEf A (%)Ef B (%)
C (N = 14)38.01OX/POX17.54POX
OX (N = 12)76.61C/POS81.97
POS (N = 14)25.26OX/POX10.8
POX (N = 17)67.21C/POS87.59C
p-value3.3 × 10−80.006
Autumn
GroupEf A (%)Ef B (%)
C (N = 7)53.6515.46POX
OX (N = 6)63.5961.1
POS (N = 9)49.1520.57POX
POX (N = 6)66.4461.82C/POS
p-value0.5870.001
Table 3. Means of strength parameters (bees as the number of worker bees and brood as the surface occupied by capped brood cells (cm2)) and Phoretic and Brood V. destructor infestation levels at M0 (%). Values marked with * indicate significant differences among experiments.
Table 3. Means of strength parameters (bees as the number of worker bees and brood as the surface occupied by capped brood cells (cm2)) and Phoretic and Brood V. destructor infestation levels at M0 (%). Values marked with * indicate significant differences among experiments.
ExperimentBeesBroodPhoretic V. destructor M0 (%)Brood V. destructor M0 (%)
Summer (N = 57)7610 *15,556 *0.786 *1.852 *
Autumn (N = 28)4844 *5849 *6.728 *12.377 *
p-value0.0023.38 × 10−43.83 × 10−111.8 × 10−5
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MDPI and ACS Style

García-Vicente, E.J.; Benito-Murcia, M.; Martín, M.; Pérez, A.; Hermosilla, N.; Martínez-Morcillo, S.; Alonso, J.M.; Risco, D. Effect of the Combined Use of Postbiotics and Oxalic Acid Against Varroa destructor Under Field Conditions. Agriculture 2025, 15, 1292. https://doi.org/10.3390/agriculture15121292

AMA Style

García-Vicente EJ, Benito-Murcia M, Martín M, Pérez A, Hermosilla N, Martínez-Morcillo S, Alonso JM, Risco D. Effect of the Combined Use of Postbiotics and Oxalic Acid Against Varroa destructor Under Field Conditions. Agriculture. 2025; 15(12):1292. https://doi.org/10.3390/agriculture15121292

Chicago/Turabian Style

García-Vicente, Eduardo José, María Benito-Murcia, María Martín, Ana Pérez, Noelia Hermosilla, Salomé Martínez-Morcillo, Juan Manuel Alonso, and David Risco. 2025. "Effect of the Combined Use of Postbiotics and Oxalic Acid Against Varroa destructor Under Field Conditions" Agriculture 15, no. 12: 1292. https://doi.org/10.3390/agriculture15121292

APA Style

García-Vicente, E. J., Benito-Murcia, M., Martín, M., Pérez, A., Hermosilla, N., Martínez-Morcillo, S., Alonso, J. M., & Risco, D. (2025). Effect of the Combined Use of Postbiotics and Oxalic Acid Against Varroa destructor Under Field Conditions. Agriculture, 15(12), 1292. https://doi.org/10.3390/agriculture15121292

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