Environmental DNA (eDNA) has been proposed as a powerful tool to detect and monitor cryptic, elusive, or invasive organisms. We recently demonstrated that honey constitutes an easily accessible source of eDNA. In this study, we extracted DNA from 102 honey samples (74 from Italy and 28 from 17 other countries of all continents) and tested the presence of DNA of nine honey bee pathogens and parasites (Paenibacillus larvae, Melissococcus plutonius, Nosema apis, Nosema ceranae, Ascosphaera apis,Lotmaria passim, Acarapis woodi, Varroa destructor, and Tropilaelaps spp.) using qualitative PCR assays. All honey samples contained DNA from V. destructor, confirming the widespread diffusion of this mite. None of the samples gave positive amplifications for N. apis, A. woodi, and Tropilaelaps spp. M. plutonius was detected in 87% of the samples, whereas the other pathogens were detected in 43% to 57% of all samples. The frequency of Italian samples positive for P. larvae was significantly lower (49%) than in all other countries (79%). The co-occurrence of positive samples for L. passim and A. apis with N. ceranae was significant. This study demonstrated that honey eDNA can be useful to establish monitoring tools to evaluate the sanitary status of honey bee populations.

Environmental DNA (eDNA) contained in honey derives from the organisms that directly and indirectly have been involved in the production process of this matrix and that have played a role in the hive ecosystems where the honey has been produced. In this study we set up PCR-based assays to detect the presence of DNA traces left in the honey by two damaging honey bee pests: the small hive beetle (Aethina tumida) and the greater wax moth (Galleria mellonella). DNA was extracted from 82 honey samples produced in Italy and amplified using two specific primer pairs that target the mitochondrial gene cytochrome oxidase I (COI) of A. tumida and two specific primer pairs that target the same gene in G. mellonella. The limit of detection was tested using sequential dilutions of the pest DNA. Only one honey sample produced in Calabria was positive for A. tumida whereas about 66% of all samples were positively amplified for G. mellonella. The use of honey eDNA could be important to establish early and effective measures to contain at the local (e.g., apiary) or regional scales these two damaging pests and, particularly for the small hive beetle, to prevent its widespread diffusion.

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.

Mora Romagnola is an autochthonous pig breed, raised in the north of Italy. Mono-breed pork products of this breed are part of important niche value chain that is intrinsically linked to the conservation of this local genetic resources that can only survive due to the premium price that these products can obtain on the market. However, the added value attracts fraudsters that unscrupulously sell mis-labelled Mora Romagnola products, causing consumer distrust that, in turn, undermines the conservation strategy of this breed. To monitor and better characterise this local breed, we phenotyped 826 Mora Romagnola pigs for three breed-specific traits. Then, we genotyped almost all living sows and boars registered to the Herd Book (n. = 357 animals) for polymorphisms in the MC1R and NR6A1 genes (affecting coat colour and vertebral number, respectively). The results were used to re-define the breed descriptors of the Mora Romagnala breed that included information on the allowed genotypes at these two genes. A few pigs that did not carry the allowed genotypes were excluded from its Herd Book. Finally, we evaluated the usefulness of these DNA markers to authenticate Mora Romagnola meat against meat derived from other 11 pig breeds and wild boars. To our knowledge, the Mora Romagnola Herd Book is one of the first examples that established a direct link between a genetic standard of a breed with the possibility to authenticate mono-breed products using DNA markers with the specific purpose to combat frauds and, indirectly, support the conservation of a livestock genetic resource.

The complementary sex determiner (csd) gene plays an essential role in the sex determination of Apis mellifera L. Females develop only if fertilized eggs have functional heterozygous genotypes at this gene whereas males, being haploids, are hemizygous. Two identical csd alleles produce non vital males. In light of the recent decline in honey bee populations, it is therefore important to monitor the allele variability at this gene. In this study, we tested the application of next generation semiconductor-based sequencing technology (Ion Torrent) coupled with environmental honey DNA as a source of honey bee genome information to retrieve massive sequencing data for the analysis of variability at the hypervariable region (HVR) of the csd gene. DNA was extracted from 12 honey samples collected from honeycombs directly retrieved from 12 different colonies. A specifically designed bioinformatic pipeline, applied to analyze a total of about 1.5 million reads, identified a total of 160 different csd alleles, 55% of which were novel. The average number of alleles per sample was compatible with the number of expected patrilines per colony, according to the mating behavior of the queens. Allele diversity at the csd could also provide information useful to reconstruct the history of the honey.