Italian Honey Research Articles

HS-SPME-GC-MS Analysis of the Volatile Composition of Italian Honey for Its Characterization and Authentication Using the Genetic Algorithm

Honey’s chemical and sensory characteristics depend on several factors, including its botanical and geographic origins. The consumers’ increasing interest in monofloral honey and honey with a clear indication of geographic origin make these types of honey susceptible to fraud. The aim was to propose an original chemometric approach for honey’s botanical and geographic authentication purposes. The volatile fraction of almost 100 Italian honey samples (4 out of which are from Greece) from different regions and botanical origins was characterized using HS-SPME-GC-MS; the obtained data were combined for the first time with a genetic algorithm to provide a model for the simultaneous authentication of the botanical and geographic origins of the honey samples. A total of 212 volatile compounds were tentatively identified; strawberry tree honeys were those with the greatest total content (i.e., 4829.2 ng/g). A greater variability in the VOCs’ content was pointed out for botanical than for geographic origin. The genetic algorithm obtained a 100% correct classification for acacia and eucalyptus honeys, while worst results were achieved for honeydew (75%) and wildflower (60%) honeys; concerning geographic authentication, the best results were for Tuscany (92.7%). The original combination of HS-SPME-GC-MS analysis and a genetic algorithm is therefore proposed as a promising tool for honey authentication purposes.

Genomic diversity and population structure of Carniolan honey bee in its native habitat

BackgroundResearch into the genetic diversity of honey bee (Apis melliferaL.) populations has become increasingly significant in recent decades, primarily due to population declines attributed to human activities and climate change. As a species of great importance, breeding programs that leverage understanding of genomic diversity could offer solutions to mitigate these challenges. The objective of this study was to examine the genomic diversity and population structure of Carniolan honey bees (Apis mellifera carnica) using the Illumina SNP chip on a large honey bee sample collected from Central and South-Eastern European countries. The study also aims to offer recommendations for future breeding programs.ResultsOur analysis involved Discriminant Analysis of Principal Components (DAPC), heterozygosity, admixture analysis, fixation indices (FST), Neighbour-Joining tree, gene flow and Isolation-by-distance analysis. DAPC indicated distinct separation between the Carniolan and Italian honey bee (Apis mellifera ligustica) populations, whereas the admixture analysis revealed varying levels of gene flow and genetic admixture within the Carniolan honey bee populations, demonstrating closer relationships between specific geographic regions (confirmed by Isolation-by-distance analysis). Furthermore, the research of heterozygosity, genomic inbreeding, pairwise FST values, and Neighbour-Joining tree provided insights into the patterns of genetic differentiation and similarity among the populations of Carniolan honey bee within its natural habitat. We have observed genetic homogeneity of the Carniolan honey bee population when considered in a broader genetic/geographical context. However, the Carniolan honey bee has sufficient genetic diversity in its geographical home range that needs to be carefully monitored and maintained.ConclusionsThis study provides important insights into the genetic composition, differentiation, and relationships among Carniolan honey bee populations in Central and South-Eastern European countries. The findings are crucial for conservation efforts, breeding programs, and sustainable beekeeping practices. They emphasise the importance of considering genetic factors and population structure in the breeding and management of honey bees. By understanding these genetic relationships, we can develop strategies to preserve genetic diversity, improve breeding outcomes, and ensure the resilience of honey bee populations in the face of environmental changes and challenges. This knowledge can also inform policy makers and stakeholders on best practices to maintain healthy bee populations, which are vital for ecosystem services and agricultural productivity.

The Nutritional Year-Cycle of Italian Honey Bees (Apis mellifera ligustica) in a Southern Temperate Climate

Nutrition is a key aspect influencing honey bee health and overwintering. Since honey bee seasonality in southern temperate climates represents a significant research gap, this study conducted long-term monitoring of honey bees in the Mediterranean (Sassari, Italy). Specifically, individual weight, fat body, and size measurements (head, thorax, abdomen, and total body) were recorded monthly so to detect changes in the nutrient storage of worker bees during an annual cycle. Data were analysed according to sampling date, climate (temperature, precipitation, and daylength), and flower diversity and were conducted for nurse and forager bees separately. The nutritional honey bee year-cycle generally followed the nectar flow and showed two critical timepoints: summer and winter dearth. A short cessation of activities in late fall/early winter coupled with an increase in nutrient storage indicated the presence of winter bees. Precipitation was found to play an important role in honey bee nutrition in the study area through its impacts on colony demography and plants in particular illustrating how climate change could pose a threat to European honey bee populations in the future. These results provide valuable insights into the nutritional dynamics of Apis mellifera ligustica in the Mediterranean that could support management decisions to improve overwintering success and prevent unnecessary colony losses.

Comparative assessment of food consumption, longevity, thermoregulation, and molecular health markers in mite-resistant and Italian honey bee stocks

Identifying traits for adaptation to different management and environmental regimes is key to maintaining robust honey bee populations under global climate change. We compared mite-resistant (Pol-line and Russian) and Italian honey bee stocks in variable-temperature cage experiments (200 bees per cage) with respect to food consumption, thermoregulation, gene expression, and lifespan, in 3 experiments over 2 years. The Italian stock bees consumed more syrup and pollen on average than the mite-resistant stocks, but the mite-resistant stocks maintained higher cluster temperatures and had median lifespans 8 days longer, consistent with the increased expression of vitellogenin relative to Italian stock. Model results indicated that, to maintain the same colony size as the mite-resistant stocks, Italian stock colonies would need about 13% more sealed brood to offset reduced worker lifespans. These differences among bee stocks likely influence colony-level productivity and health, and showed the importance of experimental replication.

Preliminary data on glyphosate, glufosinate, and metabolite contamination in Italian honey samples.

Glyphosate and glufosinate are among the most widely used pesticides in agriculture worldwide. Their extensive use leads to the presence of their residues on crops and in the surrounding environment. Beehives, bees, and apiculture products can represent potential sources for the accumulation of these substances and their metabolites, and the consequences for bee health, as well as the level of risk to human health from consuming contaminated food, are still unclear. Furthermore, information on the contamination levels of honey and other beehive products by these compounds remains poorly documented. This study is part of a broader research effort aimed at developing specific analytical methods for monitoring the level of these contaminants in bee products. The methodology employed enabled the acquisition of preliminary information concerning the levels of glyphosate and glufosinate contamination in honey samples obtained from various retailers in Italy to assess compliance with the limits established by Regulation 293/2013. The liquid chromatography tandem mass spectrometry analysis of the 30 honey samples revealed quantifiable levels of glyphosate in eight samples, with contamination ranging from 5.4 to 138.5 ng/g. Notably, one sample of the wild-flower type showed residue levels nearly three times the maximum residue limit. Additionally, trace levels of glyphosate contamination were detected in another ten samples. It is noteworthy that glufosinate and its metabolites were not detected in any of the analyzed samples within the established method's detection ranges.

The Varroa paradox: infestation levels and hygienic behavior in feral scutellata-hybrid and managed Apis mellifera ligustica honey bees

The Varroa destructor mite is a parasitic threat to managed and feral honey bee colonies around the world. Beekeepers use miticides to eliminate Varroa in commercial hives, but these chemicals can diminish bee health and increase miticide resistance. In contrast, feral honey bees have developed multiple ways to counteract mites without chemical treatment. We compared mite levels, grooming habits, and mite-biting behavior between feral Africanized honey bees (genomically verified Apis mellifera scutellata hybrids) and managed Italian honey bees (A. mellifera ligustica). Surprisingly, there was no difference in mite infestation levels between scutellata-hybrids and managed bees over one year despite the regular use of miticides in managed colonies. We also found no differences in the social immunity responses of the two groups, as measured by their hygienic habits (through worker brood pin-kill assays), self-grooming, and mite-biting behavior. However, we provide the first report that both scutellata-hybrids and managed honey bees bite off mite chemosensory forelegs, which the mites use to locate brood cells for reproduction, to a significantly greater degree than other legs (a twofold greater reduction in foreleg length relative to the most anterior legs). Such biting may impair mite reproduction.

Analysis of the Italian honey market

In the areas of the European Union, which are traditionally considered honey producers, we are observing a decrease in nectar resources and a decrease in the number of bee colonies due to anthropogenic factors, the destructive influence of pesticides and other chemical compounds, the development of new parasitocenoses, climate changes and experimental phenomena accompanying these changes, according to FAO, the population bees decreased by 37 %. Due to climate changes, honey production is decreasing in Italy. Honey consumption in the European Union exceeds production with self-sufficiency by 60 %. In 2021, the leading honey suppliers were Ukraine (which supplies 30 % of EU imports) and China (almost 28 % of EU imports). Comparing the data with 2020, we can note the increase in imports from China by 30 %). Since 2015, the European Commission has been monitoring the European honey market, noting a significant percentage of fraud and falsification of honey sold in the EU. Among the imported batches of honey in the EU in 2021 and 2022, almost every second (46 %) is suspected of adulteration, according to the results of “From the Hives” Joint Research Center (JRC) of the European Commission. Honey for sale must comply with Directive UE 2001/110/CE. In the coordinated joint actions of the EU, called “Dagli alveari” by 15 EU member states, Switzerland, and the Netherlands, 320 samples of honey originating from 20 exporting countries were selected. The research aimed to establish the presence of sugar syrups of exogenous origin to increase the volume to obtain more profit. Out of 320 examined samples, 147 (46 %) turned out to be falsified; their indicators did not meet the Directive UE 2001/110/CE, and accordingly, they are not “natural honey”. Falsified batches of honey were imported from Ukraine and China (66 out of 89 is 74 %), and honey originating from Turkey (14 out of 15 is 93 %). Honey imported from Great Britain has the highest adulteration rate (10 out of 10, 100 %). In Italy, a comprehensive action plan has been introduced to prevent the falsification of honey. The new control plan should include a quantitative increase in samples for research and the use of more effective tests to detect falsification: nuclear magnetic resonance (RMN) and high-performance liquid chromatography (HPLC). Honey produced in Italy must have the inscription “Italian honey” on the label, while honey originating from the countries of the European Union must contain the inscription “mixture of honey originating from the EU countries”, indicating the names of the producing countries. If honey comes from non-EU countries, it should be written “a mixture of honeys from non-EU countries” with the name of the producer country added.

Exploratory comparison of flower visiting behavior and pollination ability of mason bees, bumble bees, and honey bees.

This study explored the flower visiting behaviors and pollination abilities of mason bees (Osmia excavata Alfken (Hymenoptera: Megachilidae)), bumble bees (Bombus terrestris (Linnaeus, 1758) (Hymenoptera: Apidae)), and Italian honey bees (Apis mellifera ligustica Spinola (Hymenoptera: Apidae)) in apple orchards in early spring in Jinan (located in the central region of Shandong) and Yantai (located in the Peninsula of Shandong). We compared the pollen collection patterns, flower visiting behavior, flying speed, and effects on apple pollination of the 3 types of bees. The frequencies of flower visits were significantly higher for mason bees (12.89/min in Jinan and 10.63/min in Yantai) than bumble bees and Italian honey bees in the 2 regions. The single flower residence times were significantly higher for Italian honey bees (8.22 s in Jinan and 9.43 s in Yantai), but Italian honey bees were most affected by the climate. The 3 bees differed significantly in terms of the amount of apple pollen collected and their effects on the fruit setting rate in apples (mason bees > bumble bees > Italian honey bees). The results showed that the mason bee was the most suitable pollinating species for spring apple orchards; Bumble bees were more suitable as alternative pollinators during cloudy and low temperatures; Italian honey bees were able to take advantage of their large number of worker bees in sunny and warm weather. Compared to individual bee species, a combination of 2 or 3 species of bees might be more advantageous in dealing with complex and variable weather conditions.

Fingerprinting Chemical Markers in the Mediterranean Orange Blossom Honey: UHPLC-HRMS Metabolomics Study Integrating Melissopalynological Analysis, GC-MS and HPLC-PDA-ESI/MS.

(1) Background: Citrus honey constitutes a unique monofloral honey characterized by a distinctive aroma and unique taste. The non-targeted chemical analysis can provide pivotal information on chemical markers that differentiate honey based on its geographical and botanical origin. (2) Methods: Within the PRIMA project "PLANT-B", a metabolomics workflow was established to unveil potential chemical markers of orange blossom honey produced in case study areas of Egypt, Italy, and Greece. In some of these areas, aromatic medicinal plants were cultivated to enhance biodiversity and attract pollinators. The non-targeted chemical analysis and metabolomics were conducted using ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS). (3) Results: Forty compounds were disclosed as potential chemical markers, enabling the differentiation of the three orange blossom honeys according to geographical origin. Italian honey showed a preponderance of flavonoids, while in Greek honey, terpenoids and iridoids were more abundant than flavonoids, except for hesperidin. In Egyptian honey, suberic acid and a fatty acid ester derivative emerged as chemical markers. New, for honey, furan derivatives were identified using GC-MS in Greek samples. (4) Conclusions: The application of UHPLC-HRMS metabolomics combined with an elaborate melissopalynological analysis managed to unveil several potential markers of Mediterranean citrus honey potentially associated with citrus crop varieties and the local indigenous flora.

Pyrrolizidine Alkaloids from Monofloral and Multifloral Italian Honey

Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by plants as a self-defense against insects. After bioactivation in the liver, some PAs can cause acute or chronic toxicity in humans. The aim of this study was to determine the presence of PAs in 121 samples of monofloral and multifloral honey from three different Italian regions (Friuli-Venezia Giulia, Marche and Calabria) to meet the European Food Safety Authority (EFSA) suggestion. An in-house liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was validated according to European Union Reference Laboratory (EURL) performance criteria. This method allowed the detection and quantification of 35 PAs. Of the 121 honey samples, 38 (31%), mostly from Calabria, contained PAs. The total content of the PAs ranged from 0.9 µg/kg to 33.1 µg/kg. In particular, echimidine was the most prevalent PA. A rapid human exposure assessment to PAs in honey and a risk characterization was performed using the EFSA RACE tool. The assessment highlighted a potential health concern only for toddlers who frequently consume elevated quantities of honey. This study showed a low presence of PAs in Italian honey; however, the importance of continuously monitoring these compounds is stressed, along with the suggestion that the relevant authorities establish maximum limits to guarantee support for producers and consumer safety.

Reproductive quality in drones bred from Caucasian (A. m. caucasica) and Italian (A. m. ligustica) honey bee colonies

Reproductive quality in drones bred from Caucasian (A. m. caucasica) and Italian (A. m. ligustica) honey bee colonies

Characterization of Botanical Origin of Italian Honey by Carbohydrate Composition and Volatile Organic Compounds (VOCs).

Honey is a natural sweetener constituted by numerous macro- and micronutrients. Carbohydrates are the most representative, with glucose and fructose being the most abundant. Minor honey components like volatile organic compounds (VOCs), minerals, vitamins, amino acids are able to confer honey-specific properties and are useful to characterize and differentiate between honey varieties according to the botanical origin. The present work describes the chemical characterization of honeys of different botanical origin (multifloral, acacia, apple–dandelion, rhododendron, honeydew, and chestnut) produced and collected by beekeepers in the Trentino Alto-Adige region (Italy). Melissopalynological analysis was conducted to verify the botanical origin of samples and determine the frequency of different pollen families. The carbohydrate composition (fourteen sugars) and the profile of VOCs were evaluated permitting to investigate the relationship between pollen composition and the chemical profile of honey. Statistical analysis, particularly partial least squares discriminant analysis (PLS-DA), demonstrates the importance of classifying honey botanical origin on the basis of effective pollen composition, which directly influences honey’s biochemistry, in order to correctly define properties and value of honeys.

Italian Consumer Preferences for Eucalyptus Honey: An Exploratory Study

The growing concern for environmental issues has underlined the need to promote sustainable consumption and production. Taking into consideration the three pillars of sustainability, honey should be seen as an important food from a sustainability perspective. Among honey varieties, the eucalyptus one is becoming increasingly popular with people for its aroma and the plant’s therapeutic properties. However, the beekeeping sector in Italy does not yet have sufficient knowledge and understanding of consumer needs with a view to increasing earnings. This paper aimed to analyze the drivers that make people pick eucalyptus honey and tries to investigate which extrinsic and intrinsic quality attributes affect consumer behavior. Data came from an online survey of 403 Italian honey consumers. An ordered Probit model was applied. The results show that consumers consider the taste, viscosity, therapeutic properties, brand reputation, variety, geographical indication, Italian origin, and organic certification of honey as the most important factors that drive the consumption of eucalyptus honey. The findings of the study should help the beekeepers and provide them with the right tools of communication, such as quality or sustainability labeling, thereby increasing their competitiveness.

A multivariate statistical approach to identify the factors influencing the grayanotoxin content of Italian Rhododendron honey

This work represents a step in the identification of hazards associated with the consumption of Italian Rhododendron honey, which could contain grayanotoxins, which can be found in plant belonging to genera of the Ericaceae family. An LC-MS/MS method was developed and validated to quantify Grayanotoxin I (GTX I) in 125 Italian Rhododendron honeys collected from 2017 to 2019. Sensitivity and linearity were demonstrated in the range 10–1000 μg kg−1, and a LOQ value of 10 μg kg−1 was obtained. Absolute recovery ranged from 88.2% to 99.3% and no matrix effect was measured. GTX I was found in the 30.4% of the samples, and the concentrations ranged from 12.3 to 103.5 μg kg−1. Multivariate statistical approaches were applied to investigate the relationship between the presence/absence of GTX I and different honey features (e.g. environmental factors, quality aspects). The main factors affecting the presence of GTX I resulted the environment, province, altitude and the purity of botanical origin. The mean GTX I level detected in the ‘positive’ Italian Rhododendron honeys was 119–668 times lower than GTX content found in mad honeys, which caused human intoxication, so the probability of an acute toxic syndrome, in this scenario, is remote.

Evaluation of gamma radiation–induced genotoxicity in the workers of Italian honey bees, Apis mellifera ligustica, using single cell gel electrophoresis (comet) assay

The environment exposed to radionuclide faces detrimental effects of ionizing radiations and these radiations can damage DNA by inducing genotoxicity in living organisms. The gamma radiation induced genotoxicity was evaluated by employing Single cell gel electrophoresis (comet) assay in the foragers of Apis mellifera ligustica by exposing to different doses (1 to 60 Gy) of gamma radiation. DNA damage was measured as the percentage of comet tail DNA. The results showed a dose dependent increase and post irradiation time (1-24 h) dependent decrease in % tail DNA level in bee foragers. A significant increase in DNA damage was observed in all the samples and the extent of damage was proportional to the dose of gamma radiation received by the bees except in those treated with the lower doses (1, 5 and 10 Gy) of gamma radiation. The highest amount of DNA damage was recorded at 1 h post-irradiation which decreased gradually with time, that is, at 3 h and 6 h post-irradiation. However, there were no significant differences in the % tail DNA of bees of 12 h and 24 h post irradiation. The present study shows gamma radiation induced DNA damage in foragers of A. m. ligustica and also the comet assay is a suitable technique for detection of gamma radiation induced DNA damage.

Detection and evaluation of saccharide adulteration in Italian honey by NMR spectroscopy

The detection of honey adulteration is still an open question, and in these last year's particularly challenging due to the very limited production. This illegal practice is attracting producers worldwide, mostly due to the relevant economic gain available and to the lack of an official analytical method able to detect adulterations, particularly syrups. In the present study the use of both targeted and untargeted NMR spectroscopy have been applied to reveal inulin, corn/malt syrup and invert sugar syrup addition. Untargeted NMR data of pure and artificially adulterated samples in the range of 10–40% were statistical analyzed and OPLS models were used to predict samples spiked with the saccharide adulterants, resulting in very good predictions of the adulteration percentage. Targeted NMR data were concurrently evaluated, allowing to predict the adulteration percentage on the basis of calibration curves. Both these approaches revealed their robust capability in detecting and estimating the percentage of adulteration in the three different botanical origins of Italian honeys, like chestnut, polyfloral and acacia, thus suggesting the potential applicability of this approach to other botanical origins.

Consumers’ attitude towards honey consumption for its health benefits: first insights from an econometric approach

PurposeAs is well known, there are several aspects that characterise honey consumption and the reasons for purchasing it. Despite this, little attention has been paid to the drivers that move consumers towards the use of honey for its health benefits and therapeutic properties. The aim of this study is to define which drivers move the consumption of honey for its health benefits.Design/methodology/approachThe study, conducted on 640 Italian honey consumers, applied inferential tests (Chi-square) and an econometric model (logit regression), and showed that about 66% of the respondents stated that they consume honey for its health properties.FindingsThe main drivers of honey consumption are both among the intrinsic (Colour) and extrinsic (Origin and Organic certification) attributes of the product. What also emerges is that the propensity to consume honey for health purposes is influenced by the consumer characteristics and habits, such as Age cohort, Gender, BMI and Large retail buyer. Moreover, we observed that consumption is influenced by BMI but not by lifestyle characteristics such as sport and diet.Originality/valueThis study could be a support tool for policymakers who are interested in promoting good nutrition and improving public health, since there is great interest in the functional properties of foods and the need to enhance the value of products, while at the same time ensuring consumer protection.

Variation in Flowering Behaviour and Honey Bee Visitation on Eucalyptus Clones

With the adoption of clonal forestry by the farmers on large tracts in north-western India, the reports of negligible flowering in Eucalyptus clonal plantations need to be analysed. This study reports the results from the experimental trial involving 14 clones replicated thrice in randomized block design with plot size of 5 trees. Floral buds appeared from June and continued up to July. Profuse flowering was recorded only on three clones i.e., PE-7, PE-8 and PE-9. On the other hand, no floral buds could be seen on C-72, C-413, PE-12 and PE-14. All the four honey bee species i.e., Apis mellifera L. (Italian honey bee), A. dorsata F. (rock bee), A. cerana indica F. (Indian honey bee) and A. florea (little bee) visited the flowers. The frequency of their visit was maximum (7.3/ 2 min) during the afternoon (1 pm) and the lowest (4.9) in the evening. The maximum honey bee visitation was observed on clone PE-9.

Multidisciplinary analysis of Italian Alpine wildflower honey reveals criticalities, diversity and value

Wildflower honeys produced in mountain grasslands are an expression of the biodiversity of these fragile habitats. Despite its importance, the botanical origin of honey is often defined without performing formal analysis. The aim of the study was to characterize six wildflower mountain honeys produced in the Italian Alps with different analytic techniques (SPME–GC–MS, HPLC-Orbitrap, cicatrizing and antioxidant activity) alongside melissopalynological analysis and botanical definition of the production area. Even though the apiaries were in mountain grasslands rich in Alpine herbaceous species, the honey could be defined as rhododendron/raspberry unifloral or raspberry and rhododendron bifloral while the honey produced at the lowest altitude differed due to the presence of linden, heather and chestnut. The non-compliance of the honey could be due to habitat (meadows and pastures) fragmentation, but also to specific compounds involved in the plant–insect relationship, such as kynurenic acid, present in a high quantity in the sample rich in chestnut pollen. 255 volatile compounds were detected as well as some well-known markers of specific botanic essences, in particular chestnut, linden and heather, also responsible for most of the differences in aroma profiling. A high correlation between nicotinaldehyde content and percentage of raspberry pollen (r = 0.853, p < 0.05) was found. Phenolic acid and hydroxy-fatty acid were predominant in the chestnut pollen dominant honey, which presented the highest antioxidant activity and the lowest cicatrizing activity, while the flavonoid fraction was accentuated in one sample (rhododendron pollen prevalent), that was also the one with the highest effect on wound closure, although all samples had similar cicatrizing effects apart from the chestnut pollen dominant honey (lowest cicatrizing activity). Our study highlighted the difficulty of producing mountain wildflower honey and the importance of a thorough characterization of this product, also to encourage its production and valorisation.

Distribution of the Main Apis mellifera Mitochondrial DNA Lineages in Italy Assessed Using an Environmental DNA Approach.

Simple SummaryThe conservation of the genetic diversity of the native honey bee subspecies is a hot topic in many European countries. Mitochondrial DNA (mtDNA) analyses can provide some information that is useful to monitor the genetic integrity of Apis mellifera populations. A preliminary distribution of the main honey bee mitotypes in Italy was obtained more than 20 years ago. In this study we obtained an updated and more detailed distribution map of the main groups of honey bee mitotypes using an unconventional method that exploits the information derived from the environmental DNA contained in the honey. The results were quite different from the picture taken two decades ago. The African mtDNA lineage was spread all over Italy and not only in Sicily, where it is mainly attached to the A. m. siciliana subspecies, and where it was identified in the previous investigation. A reduction in the frequency of the M lineage was also evident, and, on the other hand, a counterbalanced increase in the C mitotypes was observed in regions all over. The obtained results provided an updated distribution map of the A, C and M groups of mitotypes in Italy, which could be a starting point to design appropriate conservation programs for native honey bee subspecies.Growing interest has been emerging on the need to monitor the genetic integrity of the European Apis mellifera subspecies that could be threatened by the human-mediated dispersion of non-native populations and lines. Mitochondrial DNA (mtDNA) lineages can provide useful information for this purpose. In this study, we took advantage of the environmental DNA (eDNA) contained in the honey, which can be analyzed to detect the main groups of mitotypes of the honey bees that produced it. In this study, we applied this eDNA to produce a distribution map all over the Italian peninsula and the two major islands (Sicily and Sardinia) of the following three honey bee mtDNA lineages: A, C and M. A total of 607 georeferenced honey samples, produced in all Italian regions, was analyzed to detect these lineages. The A lineage was widespread in Sicily, as expected, considering that A. m. siciliana carries the African lineage. Surprisingly, this lineage was also reported in about 14% of all other samples produced in almost all continental regions, and in Sardinia. The applied method obtained an updated distribution map of honey bee mtDNA lineages that could be useful to design policies for the conservation of Italian honey bee genetic resources.